Regulation of mitochondrial function during spermatogenesis and sperm maturation

Abstract

With the recent increase in the incidence of male infertility, greater attention is being paid to male reproductive health. The causes of male infertility are complex, and damage occurring during any process from spermatogenesis to fertilization can affect sperm quantity and quality of the sperm. Mitochondria are the power sources of cells and help regulate cellular homeostasis and physiological function. Mitochondria play a crucial role in male reproduction. Mitochondria undergo dynamic changes during spermatogenesis, sperm maturation, and fertilization. Mitochondrial dynamics and mitophagy help regulate the structure and function of mitochondria by meeting the cellular energy requirements of sperm during reproduction and reducing levels of damaged mitochondrial DNA (mtDNA); the elimination of excess mtDNA during fertilization prevents the spread of genetic mutations. Stable mitochondrial function ensures the smooth occurrence and maturation of sperm, maintaining male fertility. Externally induced mitochondrial dysfunction can lead to an inadequate energy supply, oxidative stress, cellular apoptosis, and abnormal sperm structure formation, which can lead to male infertility. In this article, the mechanism through which mitochondrial dysfunction affects the entire process of male reproduction, from spermatogonial stem cell division to final fertilization, and leads to infertility is discussed in chronological order. This article explores potential therapeutic targets for improving male fertility through therapies targeting mitochondrial function to provide a reference for subsequent research and more precise treatment directions.

Graphical abstract

Introduction

From spermatogonia to fertilization: the developmental journey and functional maturation of the male gamete

Infertility can be primary or secondary; however, this distinction remains controversial [1]. It has become a major global health challenge with an incidence of approximately 15%, of which approximately 40% of cases are attributable to male factors [2, 3]. The occurrence and maturation of normal sperm are complex and tightly regulated processes, and their smooth progression is a prerequisite for fertility. The male reproductive process involves spermatogenesis in the seminiferous tubules of the testes, sperm maturation in the epididymis, and fertilization in the female reproductive tract [4]. Abnormal sperm development can lead to changes in sperm morphology or a decrease in sperm count, thereby affecting male fertility and causing infertility [5].

Core functions of mitochondria

Energy metabolism center: coordination of ATP synthesis and glycolysis

The core of mitochondrial energy metabolism is the production of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS), which involves the coupling of redox processes, proton translocation, and ATP synthesis [6].

Glycolysis converts glucose to pyruvate, which is then converted to acetyl-coenzyme A (Acetyl-CoA), which then enters the tricarboxylic acid (TCA) cycle to produce oxaloacetate and electron carriers (nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2)) [7]. The TCA cycle transfers electrons to the electron transport chain (ETC) through an electron carrier, which ultimately transfers them to oxygen [8, 9]. TCA cycle disruption and enhanced glycolysis impair ATP production, leading to elevated reactive oxygen species (ROS) levels and subsequent abnormal apoptosis during spermatogenesis [10]. Glycolysis occurs under anaerobic or hypoxic conditions, in which glucose or glycogen is broken down into pyruvate, producing lactic acid without undergoing the TCA cycle [11]. This pathway has a lower production capacity than OXPHOS, but does not require oxygen and generates fewer ROS, making it less prone to oxidative damage [12].

Mitochondrial quality control systems

Mitochondrial quality control is crucial for maintaining the stability of the cellular environment and its functions, including mitochondrial dynamics, mitophagy, and mitochondrial biogenesis. Biogenesis is primarily regulated by mitochondrial DNA (mtDNA) and nuclear genes (nDNA) [13]. Mitochondrial quality control involves various proteins located within and outside the mitochondrion [14]. Recent studies have shown that mitochondrial dynamics and mitophagy play important roles in maintaining male fertility. As this has become a prominent research topic, this article elaborates on these two aspects of mitochondrial quality control.

Mitochondrial dynamics: fusion and fission

Mitochondrial dynamics refers to the process through which mitochondria undergo continuous changes between tubular and fragmented forms through fission and fusion, thereby altering their structure to adapt to various environmental stimuli and respond to various cellular demands [15, 16].

Mitochondrial fusion maintains the normal function of the tubular mitochondrial network (whose formation depends on mitochondrial fusion proteins and is commonly observed in cells with high energy demands) and also plays a crucial role in embryonic development [17, 18]. The mitofusin 1 (MFN1) and mitofusin 2 (MFN2) proteins on the outer mitochondrial membrane have a transmembrane domain. Their N-terminal guanosine triphosphatase (GTPase) and heptad repeat 1 (HR1) domains are located in the cytoplasm, while their C-terminal HR2 domains are located between the inner and outer mitochondrial membranes [19]. Conformational changes in the GTPase domain of MFN during GTP binding and hydrolysis lead to oligomerization, thereby promoting docking and fusion of two mitochondria at the outer mitochondrial membrane (OMM) [20, 21].

Mitochondrial fission regulates the cell cycle, promotes equal distribution of mtDNA, and removes damaged mitochondria, aiding in quality control [22–24]. After dynamin-related protein 1 (DRP1) is recruited to the OMM by mitochondrial fission factor (MFF), mitochondrial dynamics protein (MID) 49, MID 51, and mitochondrial fission protein 1 (FIS1), it undergoes oligomerization to form a circular structure, thereby enhancing preexisting mitochondrial contractions [25]. Thereafter, DRP1 hydrolyzes GTP and recruits dynamin 2 (DNM2) to the contraction site of the mitochondria to complete the mitochondrial fission process [26].

Mitophagy

Mitophagy is the process through which cells engulf and eliminate defective or redundant mitochondria through autophagic mechanisms, thereby maintaining mitochondrial and intracellular homeostasis. This process can be categorized into two types: ubiquitin- and receptor-mediated mitophagy. Ubiquitin-mediated mitophagy includes phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)/PARKIN and other ubiquitin-mediated pathways. Receptor-mediated mitophagy pathways include Bcl-2/adenovirus E1B 19-kDa interacting protein (BNIP3)-, B cell lymphoma protein-2 (Bcl-2)-, FUN14 domain containing 1 (FUNDC1)-mediated mitophagy, and lipid-mediated mitophagy. Among these, ubiquitin-mediated mitophagy plays a central role [27].

Characteristics and maternal inheritance of mitochondrial DNA

MtDNA, also known as the mitochondrial genome, is located in the mitochondrial matrix and is structurally similar to bacterial DNA as a double-stranded loop consisting of 16,569 base pairs, which is packaged into a nucleus-like high-level structure by proteins such as mitochondrial transcription factor A (TFAM) [28, 29]. MtDNA encodes 13 protein subunits of the OXPHOS system, 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs) that play a role in mitochondrial translation [30]. Thus, mtDNA plays a role in, among other things, mitochondrial respiratory energy metabolism, the assembly and function of the OXPHOS system, and ATP synthesis [31, 32].

Morphological remodeling and functional evolution of mitochondria in the male reproductive system

Mitochondrial morphological remodeling

Mitochondria are semi-autonomous organelles in cells that rely on quality control mechanisms, including mitochondrial dynamics and mitophagy, as well as the stability of mtDNA, to regulate quantity and morphology and participate in energy production and metabolism [33, 34]. Mitochondria are primarily located in the midpiece of the sperm and maintain normal sperm development and function by producing ATP and ROS, thereby ensuring male fertility [35]. A close relationship exists between mitochondria and sperm, and mitochondrial activity is positively correlated with sperm function [36–39].

During the development of male germ cells in Rattus norvegicus (rat), differentiated spermatogonia have expanded mitochondrial cristae and higher mitochondrial activity levels than undifferentiated spermatogonia, which have oval-shaped and layered cristae to meet the energy requirements for differentiation and mitosis in rats [40]. Mitochondrial elongation and an increase in quantity occur in spermatocytes during the zygotene and pachytene phases. In the middle and late phases of the pachytene stage, mitochondria aggregate in spermatocytes and the space inside the mitochondrial cristae is swollen, appearing round and dense. Mitochondria no longer aggregate, and mitochondrial cristae no longer expand during the diplotene stage or in secondary spermatocytes [41]. In mature and testicular sperm, the middle section of the mitochondria forms a spiral array in the middle segment [40, 42]. Because of the semi-autonomous nature of mitochondria and their maternal inheritance pattern, sperm mitochondria are degraded after fertilization, whereas those in the oocyte revert to their normal morphology [43, 44].

The functional evolution of mitochondria

Mitochondrial function undergoes dynamic changes during the reproductive process. This manifests as changes in mitochondrial energy metabolism, alterations in mitochondrial fusion and fission, activation of mitophagy, and a decrease in mtDNA copy number.

Energy metabolism reprogramming: a paradigm shift from proliferative metabolism to energy supply for exercise

There is a shift in the dominance of these two energy metabolism processes during male reproduction [45]. During sperm formation, a gradual transition in Mus musculus (mouse) from glycolysis in spermatogonial stem cells (SSCs) andspermatogonia to OXPHOS occurs in spermatocytes and sperm in mouse [46, 47]. During the transition from spermatocytes to sperm, OXPHOS activity gradually increases and is primarily used for cell proliferation and differentiation [37]. In spermatocytes, the enzymatic activity of the glycolysis and pentose phosphate pathways decreases, whereas TCA cycle enzyme activity increases [48].

However, in mature sperm, glycolysis occurs at a higher rate and subsequently serves as the primary source of energy [48].

During sperm capacitation, mitochondria are activated, the mitochondrial membrane potential (MMP) increases in a time-dependent manner, glucose uptake increases, and glycolysis and OXPHOS are enhanced [49, 50]. To support sperm motility and the generation of appropriate ROS, changes in energy metabolism processes may be affected by differences in ATP demand and environmental conditions [48]. During the acrosome reaction and fusion with the ovum, the energy metabolism of sperm continues to be enhanced to ensure smooth penetration of the zona pellucida and binding to the ovum membrane, reaching its peak during fertilization to fuel sperm movement and fusion with the ovum [51].

Quality control: dynamics and mitophagy cooperatively shape functional units

Dynamics shift and stability

Mitochondrial dynamics are involved in male reproductive processes via various mechanisms. During spermatogenesis in mouse, mitochondria undergo frequent fission and fusion to maintain mitochondrial quality and promote ATP production and the formation of related structures to ensure the smooth progression of spermatogenesis. Mitochondrial fusion increases mitochondrial content, elongates mitochondria, upregulates OXPHOS, and promotes mitosis and meiosis [46, 52, 53]. Furthermore, mitochondrial fission participates in maintaining the numbers of SSCs and spermatogonial cells in Drosophila (fruit fly) [54]. Mitochondrial fission induces the mitochondria to return to an intermediate state and form a spiral structure that promotes sperm formation [55, 56].

During sperm capacitation and motility, mitochondrial fusion is increased to maintain efficient energy metabolism. During the acrosome reaction, both mitochondrial fusion and fission increase; however, mitochondrial fusion was predominant [57]. During sperm formation, a gradual transition in Mus musculus (mouse) from glycolysis in spermatogonial stem cells (SSCs) andspermatogonia to OXPHOS occurs in spermatocytes and sperm [58].

Precise regulation of mitophagy

During spermatogenesis, mitophagy sustains mitochondrial number and structural stability, adapts to external changes to support cell fission and differentiation, and participates in the shedding of excess mitochondria and acrosome biogenesis [59, 60].

During sperm maturation, mitophagy is involved in the removal of abnormal mitochondria to safeguard sperm viability but also helps maintain ROS homeostasis [61].

Mitochondria also play a role in promoting sperm motility [62]. Mitophagy is activated during fertilization and induces selective degradation of sperm-derived mitochondria to ensure exclusive maternal mtDNA inheritance [63]. Freezing of sperm increases intracellular ROS production, leading to oxidative damage [64]. In line with this, Gallardo Bolanos et al. studied the cryopreservation of Equus caballus (horse) sperm and determined that light chain 3B (LC3B)-I/II protein expression was significantly upregulated in sperm stored at 5 °C for 1–5 days, with LC3B levels showing a positive correlation with cryopreservation duration [65].

On the basis of these findings, autophagy likely protects spermatozoa in the early stages by removing abnormal mitochondria through mitophagy, thereby sustaining their viability.

Precise control of genetic material: mtDNA copy number reduction and genetic bottleneck

During spermatogenesis, the mtDNA copy number gradually declines. In mature spermatocytes, mtDNA molecules may increase in response to higher ATP demand, providing energy reserves for sperm motility [66, 67].

During fertilization, sperm mitochondria are selectively degraded and diluted by the mtDNA of oocytes, resulting in a sharp decline in the amount of mtDNA, which may prevent the spread of DNA mutations to offspring and reduce ROS that increase during fertilization [68–71]. As illustrated in Fig. 1 and Table 1, this entire process of mitochondrial regulation is tightly orchestrated across the distinct stages of male reproduction, from spermatogenesis in the testis to sperm maturation in the epididymis and ultimately fertilization in the female genital tract, with coordinated shifts in energy metabolism, quality control, and mtDNA dynamics.

Fig. 1.

Regulation of mitochondrial function during spermatogenesis and sperm maturation. a The representative stages of male reproduction, including spermatogenesis, sperm maturation, and fertilization. b Dynamic shifts in energy metabolism pathways during the development of male germ cells: During the differentiation of SSCs into spermatogonia, energy metabolism is primarily driven by glycolysis. As spermatogonia develop into primary spermatocytes, TCA cycle activity increases and energy metabolism shifts to be primarily reliant on OXPHOS to meet the rising energy demands. During sperm maturation, glycolysis once again becomes the main source of energy; during fertilization, glycolysis and oxidative phosphorylation work synergistically to ensure an adequate energy supply. Spermatogonial stem cells, SSCs; tricarboxylic acid, TCA; oxidative phosphorylation, OXPHOS. c Mitochondrial quality control mechanisms during the development of male germ cells: During spermatogenesis, mitochondrial fission helps maintain the quantity of SSCs and spermatogonia, while mitochondrial fusion promotes germ cell development by enhancing OXPHOS. During the sperm maturation stage, mitochondrial dynamics shift toward fusion; during fertilization, mitochondrial fusion is further enhanced to sustain efficient energy metabolism. Mitophagy is increased during spermatogenesis and sperm maturation to eliminate dysfunctional mitochondria and limit ROS levels; during fertilization, mitophagy is initially transiently restricted to support sperm motility and is subsequently activated to eliminate paternal mitochondria. Spermatogonial stem cells, SSCs; oxidative phosphorylation, OXPHOS; reactive oxygen species, ROS. d Dynamic changes in mtDNA copy number during the development of male germ cells.: The mtDNA copy number gradually decreases during spermatogenesis, may slightly increase during sperm maturation to support motility, and is selectively degraded after fertilization to prevent the transmission of paternal mitochondrial DNA. Mitochondrial DNA, mtDNA

Table 1.

Dynamic changes in mitochondrial function during the male reproductive process

Sperm mitochondria	Spermatogenesis			Sperm maturation	Fertilization
	Mitosis	Meiosis	Spermiogenesis		Sperm capacitation and motility	Acrosome reaction	Sperm–ovum fusion
Energy metabolism (OXPHOS and glycolysis)	Mainly glycolysis	Mainly low OXPHOS	Mainly high OXPHOS	Mainly glycolysis	Mainly glycolysis	Both increase	Both increase
Dynamics (fusion and fission)	Low (mainly fission)	Increased (gradually turned to mitochondrial fusion)	Increase (mainly fusion)	Stability (mainly fusion)	Stability (mainly fusion)	Stability (mainly fusion)	Stability (mainly fission)
Mitophagy	Lower	Increased	Increased	Moderate	Restricted	Increased	Significantly increased
MtDNA (copy number)	Reduced	Reduced	Reduced	Increased	Stable	Reduced	Reduced

Furthermore, existing studies have found that mtDNA copy number may also influence normal cellular signaling processes by affecting nDNA methylation, potentially leading to disease [72]. Notably, there is a tissue-specific methylation region at exon 2 of the mtDNA polymerase γ catalytic subunit (PolgA), where DNA methylation of exon 2 negatively regulates mtDNA copy number [73].

The repetitive telomeres sequences at the ends of chromosomes play an important role in spermatogenesis. Because sperm are highly energy-dependent, mitochondria serve as key regulatory factors that may influence the smooth progression of spermatogenesis by affecting the normal maintenance of telomere function. For example, mitochondrial dysfunction induces oxidative damage, making telomeres more vulnerable to ROS, which disrupts telomere maintenance and impedes telomerase activity, ultimately accelerating telomere attrition [74, 75]. Furthermore, studies have shown that telomere length is closely linked to mtDNA copy number, mitochondrial function, and sperm quality [76, 77]. This indicates that DNA methylation or telomere length may interact with mtDNA to regulate nuclear gene and mitochondrial genome expression.

Overall, mitochondrial dysfunction can lead to impaired sperm production, abnormal sperm maturation, and impaired fertilization, resulting in reduction in sperm count, structural abnormalities, and lower fertilization rates, which can affect male fertility.

Stage-specific regulation of mitochondrial function during spermatogenesis

Spermatogenesis can be divided into three stages: mitosis, meiosis, and spermiogenesis. During mitosis, SSCs undergo multiple mitotic cycles to produce undifferentiated (type A spermatogonia) and differentiated spermatogonia (type B spermatogonia). Undifferentiated spermatogonia are capable of self-renewal and give rise to differentiated spermatogonia. Differentiated spermatogonia undergo DNA replication, chromosome alignment at the cell equator, segregation to opposite poles, and cytokinesis, thereby producing two primary spermatocytes. This completes mitosis, after which meiosis is initiated [78].

The second stage is meiosis. Primary spermatocytes rapidly enter the first meiotic fission, chromosome duplication, association, exchange, and reorganization, separate homologous chromosomes into distinct combinations, and ultimately divide to form two secondary spermatocytes with chromosome numbers that are not halved; the secondary spermatocytes rapidly complete the second meiotic fission to form two round spermatids with halved chromosome numbers. The first prophase of meiosis can be further divided into leptotene, zygotene, pachytene, diplotene, and diakinesis [79].

The third stage is spermiogenesis. In this stage of spermatogenesis, round sperm cells differentiate into tadpole-shaped elongated spermatozoa, the nucleus condenses into the sperm head, the Golgi apparatus differentiates into the acrosome, the centriole microtubule differentiates into the axoneme of the sperm tail, the mitochondria differentiate into the mitochondrial sheath, and residual cytoplasm is shed, thus completing spermiogenesis [55, 80–82].

Regulation of mitochondrial function during mitosis of spermatogonial stem cells

Mitochondrial energy metabolism and glycolysis

SSCs predominantly adopt glycolysis as their primary metabolic pathway, maintaining elevated glycolytic activity within spermatogonia [12, 47]. This metabolic preference is presumably modulated by the local microenvironment, which functions to restrict excessive ROS accumulation and thereby mitigate the occurrence of DNA damage [46]. However, because of an increase in energy demand, retinoic acid signal transduction in spermatogonia leads to a gradual decline in glycolysis and an increase in mitochondrial respiratory function [83, 84]. Compared with undifferentiated spermatogonia, the ratio of NAD⁺/NADH in spermatogonia is low, ATP and ROS production is increased, OXPHOS is enhanced, and the basic oxygen consumption rate is sharply increased [83]. Stability of energy metabolism is closely related to the smooth progression of mitosis.

Studies have shown that glycolysis is involved in the regulation of mitotic processes. For example, the expression levels of genes associated with the positive regulation of glycolysis and those encoding key glycolytic enzymes were higher in the SSCs of the P53 knockout mouse than in those of the control mouse, which in turn increased glycolytic activity and promoted the pluripotent transformation of SSCs [85, 86]. After treatment of mouse cells with the glycolytic inhibitor clonamide, glucose-6-phosphate (G6P) formation was reduced, inhibiting spermatogonia differentiation and decreasing the expression of glial cell line-derived neurotrophic factor (GDNF) family receptor alpha 1 (GFRα1) and inhibitor of DNA-binding 4 (ID4), along with a reduction in the number and colony size of spermatogonia [83]. Similar results were obtained after treatment with the OXPHOS inhibitor rotenone/antimycin A [83].

Moreover, studies have shown that OXPHOS is involved in the regulation of mitosis. For example, the basal nuclear protein 1 (BNC1) is primarily expressed in germ cells and is involved in spermatogenesis [87]. Mouse experiments have shown that knockdown of Bnc1 leads to downregulation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)/silent mating type information regulation 2 homolog 1 (SIRT1)/Forkhead Box O3 (FOXO3) signaling, which in turn leads to mitochondrial dysfunction, decreased mitochondrial membrane potential, elevated ROS production, and increased expression of cytochrome c (Cyt-c), resulting in spermatogonial apoptosis and the failure of sperm production [88]. By knocking down E4f1 in mouse germ cells, Yan et al. reported that E4F1 deletion led to abnormal mitochondrial morphology and activity defects in spermatogonia, with reduced mitochondrial membrane potential for OXPHOS, reduced ROS levels, and fatty acid accumulation, which impeded the balance between SSC self-renewal and differentiation [89]. In addition, in this model, the percentage of germ cells in the S and M phases of the mitotic cell cycle in the testis decreased, most notably affecting G1/S phase arrest [89]. Similarly, Moison et al. studied clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 induced by doxycycline to eliminate E4F1 expression in the Homo sapiens osteosarcoma U2OS cell line and revealed that the deletion of E4F1 led to G2 phase cell stagnation and delayed S phase progression, hindering the normal progression of mitosis [90].

Mitochondrial quality control

Mitochondrial dynamics

The stability of mitochondrial dynamics, including the balance between mitochondrial fission and fusion, maintains the normal progression of mitosis.

Mouse studies have shown that deletion of MFN1 or MFN2 results in reduced mitochondrial content, fragmentation, the appearance of heterogeneous mitochondria, downregulation of OXPHOS, and severe depletion of differentiated spermatogonia [46]. Similarly, other studies have shown that the deletion of MFN2 enhances mitochondrial oxidative stress (OS) and promotes chromosomal instability and mitotic arrest in HeLa cells [91, 92].

In Drosophila melanogaster studies have shown that the loss of Drp1 in early germ cells destroys mitochondrial fission, increases ROS levels in germ cells, promotes the expression of the epidermal growth factor (EGF) ligand Spitz, and induces the phosphorylated extracellular signal-regulated kinase (pERK) in germ cells adjacent to cyst cells to activate EGF receptor (EGFR) signaling. This causes the loss of germ stem cells (GSCs) in fruit fly testes and the premature differentiation of early spermatogonia, resulting in a decrease in their numbers [54]. In addition, Drp1 small interfering RNA (siRNA) was used to transfect HeLa cells to reduce the expression level of Drp1. The results showed that mitochondrial fission and inhibited fragmentation led to incomplete cytoplasmic fission during mitosis and an uneven distribution of mitochondria in daughter cells [22]. In vitro-cultured HCT116 cells were treated with mdivi-1 to inhibit DRP1 expression, which resulted in severe chromosome dislocation during mitosis [93]. Zhang et al. studied mouse spermatogonia overexpressing mitochondrial MFN1, MFN2, and DRP1 and showed that the expression of the differentiation marker genes KIT proto-oncogene, receptor tyrosine kinase (Kit), and stimulated by retinoic acid 8 (Stra8) significantly increased after retinoic acid (RA)-induced differentiation of the overexpressing cells, indicating that the enhancement of mitochondrial fusion and fission is beneficial for spermatogonial differentiation [94].

Mitophagy

Appropriate mitophagy alleviates damage, whereas abnormalities in mitophagy aggravates damage and affects normal progression of mitosis.

Feng et al. treated the freezing medium of Capra aegagrus hircus (goat) SSC with melatonin and determined that melatonin significantly inhibited the expression of pro-apoptotic proteins BCL2 associated X, apoptosis regulator (Bax), B-cell leukemia/lymphoma (Bcl-1), LC3-I, and LC3-II and increased the expression of anti-apoptotic proteins Bcl-2, Bcl-XL, and receptor protein (P62). Melatonin inhibits mitochondrial Cyt-c release into the cytoplasm by regulating Bax/Bcl-2 expression, thereby preventing caspase-3 activation, reducing autophagosome formation, and suppressing apoptosis in SSCs [95]. In the goat heat stress (HS) model, melatonin was administered at low concentrations. The mitochondrial fusion protein level of SSCs was upregulated, whereas mitochondrial fission-related proteins, mitophagy-related proteins, and P62 levels were downregulated, along with a reduction in ROS levels. This inhibited the activation of the PINK1/PARKIN pathway, restored mitophagy flux and mitochondrial homeostasis, and inhibited SSC apoptosis [96]. However, Liu et al. used mouse spermatogonial cells (GC-1 cells) exposed to nickel nanoparticles (Ni-NPs) and determined that excessive expression of PINK1 and PARKIN promoted mitophagy, increased the expression of Bax, caspase-9, and caspase-3 proteins, and decreased the expression of Bcl-2, resulting in an increase in the Bax/Bcl-2 ratio, which in turn led to apoptosis. Therefore, PINK1/PARKIN-mediated mitophagy was impaired in male spermatogonial reproductive toxicity, which accelerated apoptosis [97]. These regulatory mechanisms are schematically summarized in Fig. 2, which illustrates how mitochondrial dynamics, mitophagy, and apoptotic signaling collectively govern spermatogonial survival and function.

Fig. 2.

Mechanisms of mitochondrial metabolism and quality control in the regulation during mitosis of SSCs. a Abnormal energy metabolism during mitosis of SSCs: Bnc1 deficiency inhibits the CREB/SIRT1/FOXO3 pathway, resulting in elevated levels of ROS and Cyt-c, as well as reduced MMP levels, which leads to decreased OXPHOS activity, thereby inducing spermatogonial apoptosis and blocking mitosis. Similarly, E4f1 deficiency reduces OXPHOS activity and impairs the self-renewal and differentiation of SSCs, thereby inhibiting mitosis. Spermatogonial stem cells, SSCs; basal nuclear protein 1, BNC1; reactive oxygen species, ROS; cytochrome c, Cyt-c; oxidative phosphorylation, OXPHOS. b Imbalance in mitochondria quality control during mitosis of spermatogonial stem cells: Abnormal mitochondrial dynamics and dysregulated autophagy disrupt mitochondrial homeostasis. Loss of MFN1/2 impairs mitochondrial OXPHOS function, enhances OS, and obstructs mitosis. In addition, the deletion of Drp1 disrupts mitochondrial fission, which by elevating ROS levels, activates the EGFR pathway and causes premature differentiation of spermatogonia, thereby blocking mitosis. Meanwhile, in the Ni-NP treatment model, the expression of PINK1 and PARKIN was upregulated, promoting the expression of Bax and caspase-3/9 while inhibiting Bcl-2 expression, thereby facilitating mitophagy and hindering the normal progression of mitosis. Mitofusin 1/2, MFN1/2; oxidative phosphorylation, OXPHOS; oxidative stress, OS; dynamin-related protein 1, DRP1; reactive oxygen species, ROS; PTEN-induced putative kinase 1, PINK1

Regulation of mitochondrial function during meiosis in spermatocytes

When primary spermatocytes undergo meiosis to form spermatids, mitochondrial cristae become more dilated, and mitochondria elongate and aggregate more than during the early stages of spermatogenesis. In addition, mitochondrial aggregates form more concentrated mitochondria clusters [98]. This suggests that mitochondrial activity is further enhanced at this stage to meet the high energy demands of spermatocytes.

Mitochondrial energy metabolism and glycolysis

Sertoli cells form the blood–testis barrier (BTB) that divides the seminiferous epithelium into basal and luminal compartments [99]. The differentiated spermatogonia produce spermatocytes that traverse the BTB and complete meiosis [100]. This may result from changes in the microenvironment, energy demand, and expression of mitochondrial pyruvate carrier 1 (MPC1) in spermatocytes [46]. In spermatocytes, the ATP pathway shifts from glucose utilization to lactic acid and pyruvate mainly through OXPHOS [101].

Mitochondrial energy metabolism plays a critical role in spermatocyte meiosis [45]. Abnormalities in the proteins related to mitochondrial structure and function can affect mitochondrial energy metabolism and inhibit meiosis. In mouse lacking testis-specific adenine nucleotide translocase 4 (Ant4), the cell respiration level decreased, the ATP supply was blocked, and synaptonemal complex protein 3 (SYCP3) dot staining and γH2AX foci were observed. These observations indicated impaired progression from the leptotene to zygotene stage and arrest at meiotic prophase I, leading to elevated apoptosis level and the absence of spermatids and mature spermatozoa within the seminiferous tubules [102, 103]. Caseinolytic protease P (ClpP) and caseinolytic protease X (ClpX) help maintain mitochondrial integrity and function [104, 105]. Guo et al. demonstrated through studies on mouse germ cells with conditional knockout of ClpP/ClpX that spermatocytes exhibited alterations in mitochondrial quantity and morphology, reduced mitochondrial membrane potential, diminished recovery capacity of ROS levels, and altered expression of the genes encoding respiratory chain complexes I and IV. These changes affected the energy supply during meiosis [106, 107].

Fluoride inhibits spermatogenesis by affecting the fusion of mitochondria and endoplasmic reticulum. In an NaF-treated mouse spermatocyte line (GC-2spd), Guo et al. found that mitochondria and endoplasmic reticulum were over-colocalized, mitochondrial-associated endoplasmic reticulum membrane (MAM) formation was increased, Ca²⁺ in the cytoplasm and mitochondria of spermatocytes was disordered, mitochondrial membrane potential was significantly reduced, ATP supply was reduced, and the expression of Bax, Cyt-c, and caspase-3 was upregulated. Notably, the expression of B-cell lymphoma 2 (Bcl-2) was significantly downregulated, leading to apoptosis of spermatocytes [108].

In addition, in some diseases, sperm meiosis can be blocked by affecting mitochondrial energy metabolism. For example, in the rat model of testicular ischemia–reperfusion injury, a decreased NAD/NADH ratio, overexpression of uncoupling protein 2 (UCP2), mitochondrial dysfunction, and elevated Cyt-c levels were observed, promoting germ cell apoptosis and leading to impaired spermatogenesis in spermatocytes and a deficiency in sperm cells [109]. Similarly, in varicocele-induced rat testes, decreased blood flow, reduced adenine nucleotide concentration, impaired energy metabolism, and decreased ATP production resulted in a reduction in spermatocytes and sperm cell numbers, ultimately impairing spermatogenesis [110, 111].

Notably, when oxidative phosphorylation is inhibited, spermatocyte energy metabolism shifts toward glycolysis; however, the low energy production under such conditions fails to meet cellular demands, potentially leading to meiotic arrest. In the mouse model of Rsrc1-161 aa deletion, lactate dehydrogenase (LDH) activity increased, pyruvate dehydrogenase (PDH) activity decreased, the expression of OXPHOS-related proteins (such as Cytochrome c oxidase subunit I (COX1), NADH:ubiquinone oxidoreductase subunit A9 (NDUFA9), and ubiquinol–cytochrome c reductase, Rieske iron–sulfur polypeptide 1 (UQCRFS1)) decreased, and the basal lactate excretion rate and lactate level increased significantly. These findings suggest that reduced OXPHOS, compensatory upregulation of glycolysis, elevated ATP levels, and increased ROS production collectively disrupt mitochondrial energy metabolism, leading to meiosis I arrest with persistent metaphase residuals and subsequent apoptosis. This cellular stress ultimately leads to the accumulation of spermatocytes and a reduction in round spermatid numbers [112].

Mitochondrial quality control

Mitochondrial dynamics

Mitochondrial dynamics are critical for maintaining normal meiotic progression [46, 113, 114]. The Mfn1-deficient mouse model exhibits abnormal meiosis, with the accumulation of primary spermatocytes and reduced formation of secondary spermatocytes. Moreover, the deletion of Mfn1 or Mfn2 results in the severe depletion of germ cells in the mouse model [46]. In a mouse experiment, Mfn2 knockout led to testicular seminiferous tubule atrophy, decreased spermatocyte numbers during the middle and late stages of the first wave of peak spermatogenesis, and vacuolization [113]. Therefore, mitochondrial fusion plays an important role in meiosis, particularly in energy metabolism.

Compared with the wild-type mouse model, the Mfn1 knockout mouse model exhibited significantly reduced testis size and scarce germ cells in the seminiferous tubules, and no sperm were observed in the epididymis. Further studies revealed that Mfn1 knockout resulted in increased mitochondrial fission, leading to the appearance of thinner and longer cristae and elevated activity of respiratory enzymes, cytochrome c oxidase (COX), and NADH dehydrogenase, whereas nuclear DNA-encoded succinate dehydrogenase (SDH) was unaffected, and mitochondrial metabolic dysfunction blocked spermatogonial formation [52]. Similarly, in the Mfn1 and Mfn2 double-knockout mouse model, spermatocytes showed mitochondrial fragmentation and reduced mitochondrial content, abnormal cristae, and lower numbers and activity of OXPHOS subunits (NdufB6 and MTCOI), resulting in obstruction from the leptotene to the pachytene stage, inhibiting spermatogenesis, and leading to a significant decrease in sperm [46].

Similar results were observed in cell models. Varuzhanyan et al.’s spermatogenic cell-specific knockout mouse experiment showed that Mfn1 depletion caused mitochondrial fragmentation and OXPHOS downregulation, resulting in spermatocyte accumulation in the leptotene and zygotene stages, meiosis I arrest, and reduced sperm cell production [46]. Other studies have reported that Mfn2-knockout spermatocytes in the mouse model exhibited mitochondrial aggregation, elevated ROS levels, and disrupted respiratory chains, which led to impaired mitochondrial OXPHOS and lipid metabolism, abnormal meiotic processes, and ultimately inhibited spermatogenesis [114]. Presenilin-associated rhomboid-like protein (PARL) is an inner mitochondrial membrane serine protease involved in mitochondrial fusion [115]. A Parl-deficient mouse model showed a decrease in the expression of glutathione peroxidase 4 (GPX4) and coenzyme Q, which led to abnormal mitochondrial structure in spermatocytes. This subsequently induced spermatocyte arrest and severe vacuolar degeneration, and spermatocyte death, resulting in nearly complete meiosis I arrest [116].

Mitophagy

PINK1/PARKIN-mediated mitophagy abnormalities under exogenous stimulation have been shown to accelerate spermatocyte apoptosis and affect meiosis.

In short-photoperiod-stimulated Microtus ochrogaster (prairie vole), testis weight and testicular germ cells decreased, mature sperm decreased, and the expression of mitophagy-related genes Pink, Prkn, Tomm, and Lc3 in spermatocytes increased, whereas the level of lysosomal-associated membrane glycoprotein 2 (LAMP2) did not change significantly. The reduction of mitochondrial cristae was accompanied by mitochondrial swelling, indicated impaired mitophagy, which inhibited spermatocyte development and promoted apoptosis [117]. Liu et al. treated a mouse spermatocyte cell line (GC-2) with polystyrene microplastics (PS-MPS) and found that the relative protein expression levels of PINK1 and PARKIN were significantly elevated compared with those in the control group and that the PINK1/PARKIN pathway was activated, which then triggered mitophagy and ultimately promoted spermatocyte apoptosis [118]. In the mouse testicular injury model treated with triptolide (TP), GC-2 cell mitochondria exhibited atrophy, cristae disarray, significantly reduced MMP levels, enhanced OS, an activated mitophagy pathway, a significantly upregulated Bax/Bcl-2 ratio, increased caspase-3 protein expression, enhanced mitophagy, and spermatocyte apoptosis [119]. Li et al. and Zhang et al. performed similar experiments using NaF and CdCl₂, respectively. Mouse spermatocytes were exposed to NaF and CdCl₂, and both experiments showed increased caspase-3 and caspase-9 expression, elevated LC3-I and LC3-II levels, and decreased P62 expression, leading to enhanced apoptosis and autophagy [120, 121]. Mitophagy process involving LC3 and P62 may play an important role in accelerating apoptosis and autophagy in spermatocytes stimulated with NaF and CdCl₂.

Characteristics and inheritance of mtDNA

Abnormal mtDNA levels can cause mitochondrial respiratory dysfunction, leading to meiotic arrest and enhanced spermatocyte apoptosis. In addition, especially during prophase I of meiosis, spermatogonia carrying pathogenic mtDNA may accumulate large amounts of mutant mtDNA in spermatocytes, while maintaining normal mitosis, inducing mitochondrial respiratory dysfunction and decreasing OXPHOS activity [122]. Owing to the high energy demand of spermatocytes during meiosis, especially during prophase I, this low ATP level may cause abnormal synapse formation and meiotic arrest [46, 103].

Mutations and damage to mtDNA lead to mitochondrial dysfunction, which affects meiotic processes. In the mouse model with high levels of pathogenic deletion (4696 bp) in mtDNA, COX activity was found to be deficient, one of the respiratory chain complexes was abnormal, and low OXPHOS activity was observed, resulting in stagnation between the zygotene and pachytene phases, inhibition of meiosis and apoptosis, and a decrease in sperm count, which in turn caused oligozoospermia [122]. Similarly, in the mouse model with a high mutant mtDNA load, meiosis was arrested and apoptosis initiation was enhanced in spermatocytes, whereas spermatocytes with a low mutant load were able to complete meiosis; however, most spermatozoa had reduced COX activity and showed abnormal mid-phase and nuclear fractions, indicating that the spermatogenesis was aberrant [122]. In addition, compared with the controls, the testicular spermatocytes of the high-fat diet (HFD) mouse model showed elevated malondialdehyde (MDA) and glutathione (GSH) levels, increased lipid peroxidation, and induced OS, which led to mtDNA damage, reduced relative amplification of mtDNA fragments, reduced copy number, more instability, and more lesions, as well as higher expression of the DNA repair-related proteins 8‐oxoguanine DNA glycosylase 1 (OGG1) and apurinic/apyrimidinic endonuclease 1 (APE1). This leads to a decrease in mitochondrial function and turnover rate, inhibits spermatogenesis, and causes testicular damage [123]. Jiang et al. studied an mtDNA mutant male sterile the mouse model and modulated their mtDNA copy number by controlling the expression of TFAM. Decreased mtDNA copy number reduced COX activity and impaired mitochondrial morphology and OXPHOS, resulting in the obstruction of sperm meiosis and the loss of spermatocytes and round sperm cells, whereas an increase in the mtDNA copy number significantly improved and restored normal sperm function. They believe that an increase in the mtDNA copy number can increase the likelihood of encoding sufficient functional gene products, thereby promoting mitochondrial activity. This may be due to the unchanged proportion of mutated mtDNA (heterogeneity level) [124].

In addition, mtDNA damage in the Sertoli cells is involved in the regulation of meiotic processes. Spermatocytes initiate meiosis by crossing the blood–testis barrier and are fueled by lactate. Pyruvate secreted by Sertoli cells is a key substrate for OXPHOS to support the meiotic process [125]. According to Kujoth et al., the accumulation of mtDNA mutations accelerates aging in animals. The testes of a 10-month-old mtDNA mutant mouse model showed severe damage to the seminiferous tubules and the exhaustion of germ cells [126]. This may result from decreased mtDNA expression in the Sertoli cells, increased OS inducing apoptosis of the Sertoli cells, and the lack of an energy source in spermatocytes, leading to meiotic arrest [127]. Figure 3 illustrates the relevant mechanisms regulating mitochondrial function in spermatocytes during meiosis.

Fig. 3.

Multidimensional regulation of mitochondria during spermatocyte meiosis. a Abnormal energy metabolism during spermatocyte meiosis: Deficiency of ClpP/ClpX or knockout of Ant4 or Rsrc1–161 aa leads to decreased expression of respiratory chain components such as COX1, NDUFA9, and UQCRFS1; under testicular ischemia–reperfusion conditions, a reduced NAD/NADH ratio and UCP2 overexpression also impair mitochondrial respiratory chain function. These changes collectively impair OXPHOS, reduce ATP production, and induce compensatory enhancement of glycolysis, accompanied by ROS accumulation and mitochondrial dysfunction, ultimately hindering spermatocyte meiosis and inducing apoptosis. Cyclooxygenase 1, COX; oxidative phosphorylation, OXPHOS; adenosine triphosphate, ATP; reactive oxygen species, ROS. b Imbalance in mitochondria quality control during spermatocyte meiosis: Abnormal mitochondrial dynamics and dysregulated mitophagy synergistically disrupt mitochondrial homeostasis. Knockout of Mfn1/2 leads to abnormal mitochondrial morphology, aggregation, and respiratory chain dysfunction, thereby inhibiting OXPHOS and blocking the meiotic process. Exposure to sodium fluoride or cadmium chloride induces abnormal expression of caspase-3/caspase-9 and LC3-I/LC3-II. In the SP treatment model, disruption of the PINK1/PARKIN signaling pathway, accompanied by mitochondrial swelling, disturbed mitophagy balance, accelerated spermatocyte apoptosis, and impaired spermatogenesis, was observed. Mitofusin 1/2, MFN1/2; oxidative phosphorylation, OXPHOS; PTEN-induced putative kinase 1, PINK1. c Disruption of mtDNA homeostasis during spermatocyte meiosis: mtDNA deletion (4696 bp) or downregulation of TFAM leads to a reduction in mtDNA copy number, causing loss of COX activity and abnormalities in the respiratory chain structure, thereby inhibiting OXPHOS and resulting in meiotic arrest. Under HFD conditions, MDA and GSH levels increase, lipid peroxidation is enhanced, further damaging mtDNA and its repair process, leading to mtDNA loss and decreased mitochondrial turnover, ultimately causing testicular injury and inhibiting spermatogenesis. Mitochondrial DNA, mtDNA; transcription factor A, TFAM; cyclooxygenase 1, COX; oxidative phosphorylation, OXPHOS; high-fat diet, HFD; malondialdehyde, MDA; glutathione, GSH; Short-photoperiod, SP

Mechanism of mitochondrial function in sperm formation

During sperm formation in the rat model, condensed mitochondria gradually develop more curled cristae, and dense mitochondria appear as semilunar cristae, showing an intermediate state [41]. Some mitochondria move toward the flagellum, whereas others aggregate. The flagellar mitochondria elongate and assume a helical shape around the flagellum, forming a tubular structure that constitutes the middle segment of the sperm [41]. These mitochondria tightly wrap around the axon, forming a compact mitochondrial sheath that powers sperm motility [66, 128].

Mitochondrial energy metabolism and glycolysis

Mitochondrial energy metabolism is closely associated with spermiogenesis. During this process, OXPHOS serves as the main energy-generating pathway [37], providing most of the ATP required for the sperm tail and acrosome, which is crucial for the successful maturation of sperm [129].

Microtubule protein polymerization-promoting protein 2 (TPPP2) is involved in sperm formation and plays an important role in the formation and sperm cell morphology [130]. Zhu et al. found through mouse experiments that TPPP2 deficiency leads to a lack of mitochondrial inner membrane cristae. Compared with the control group, the expression of the components in the ETC complex located in the mitochondrial inner membrane was reduced, impairing mitochondrial energy production and causing severe sperm dysfunction [131]. This may explain the observed oligoasthenospermia phenotype. Glycerol kinase-like 1 (Gykl1) and glycerol kinase 2 (Gk2) specifically target mitochondria in round and elongated spermatids and cooperate with phospholipase D family member 6 (Pld6) to induce mitochondrial aggregation in cells [132]. In the mouse model lacking Gykl1 or Gk2, ATP regulation is disrupted, resulting in mitochondrial sheath defects and sperm tail damage. The deletion of Gykl1 or Gk2 dramatically alters the mitochondrial morphology and dynamics during spermatogenesis, resulting in sperm defects that may induce asthenoteratozoospermia [132].

Mitochondrial quality control

Mitochondrial dynamics

Mitochondrial fusion and fission are necessary in mitochondrial recombination during spermiogenesis [133–135].

Armadillo repeat-containing 12 (ARMC12) is an evolutionarily conserved testis-enriched protein involved in mitochondrial fusion and can aggregate mitochondria to regulate mitochondrial dynamics [128, 136]. Shimada et al. observed abnormal mitochondrial expansion in mouse sperm by knocking out Armc12 in a male mouse experiment, thereby disrupting mitochondrial winding and affecting mitochondrial sheath formation. In fruit fly sperm, deletion of drp1 led to abnormal aggregation of mitochondria in spermatocytes and affected the distribution of mitochondria during meiosis, resulting in the loss of mitochondria in round sperm cells, further affecting sperm formation [133]. In addition, the number of sperm in Mfn2-deficient mouse model decreased, and most were abnormally mature [114]. Moreover, the mitochondrial function of spermatocytes in Mfn2-deficient mouse was impaired owing to mitochondrial aggregation and increased ROS levels. In the model of Mfn2-deficient mouse testis treated with γH2A.X and SYCP3 antibody, γH2A.X in Mfn2-deficient spermatocytes was primarily retained on autosomes compared with the wild-type group, which may be due to abnormalities in the meiotic process from the zygotene to pachytene stage [114].

Abnormalities in mitochondrial dynamics-related proteins can lead to an imbalance in mitochondrial dynamics resulting in asthenospermia and round sperm. Genetic ablation of the Mff-deficient mouse model revealed that MFF is an essential component of mitochondrial sheath tissue in sperm cells [55]. In sperm cells, after meiosis, Mff-mediated acute mitochondrial fragmentation produces small mitochondrial spheres that are arranged vertically on axons in the middle segment [137]. These mitochondria are elongated and tightly wrapped around the axons in a coordinated manner, forming a compact mitochondrial sheath that provides power for sperm motility [138, 139]. Grigor et al. determined that, in the round sperm of the MFF-deficient mouse model, the mitochondria had obvious central contractions and could not be divided. The mitochondrial sheath was disconnected in elongated sperm cells, and the mitochondria were highly swollen. The activity of respiratory chain complex IV decreased, which led to a decrease in sperm motility and induced asthenospermia [55, 134]. Similarly, studies have shown that mitochondrial defects may occur in round sperm cells of the Fis1 gene knockout mouse model because of abnormal mitochondrial contraction [135]. Defects in sperm cells and acrosomal transport during early stages prevent normal maturation, leading to globozoospermia.

Mitophagy

Sperm formation is closely associated with mitophagy. Appropriate mitophagy maintains the smooth progress of sperm formation, and its disruption leads to the obstruction of the sperm formation process and the production of abnormal sperm. Studies have shown that knockout of mitophagy-related genes can lead to impaired mitophagy and affect sperm formation. Fis1 is a mitochondrial dynamics gene that regulates mitochondrial morphology and mitophagy during sperm cell maturation [135]. This study found, that in the sperm cells of the male mouse model lacking Fis1, mitophagy defects, abnormal mitochondrial accumulation, and severe morphological abnormalities led to physiological defects in sperm cells. Further studies have shown that the mouse model lacking Fis1 exhibits spermatogenesis arrest during the development of round sperm cells [135]. Abnormal mitophagy can induce oligozoospermia, azoospermia, teratozoospermia, globozoospermia, and asthenospermia. For example, F-box-only protein 7 (FBXO7) is associated with mitophagy [140]. In an Fbxo7-knockout mouse experiment, the PINK1/PARKIN-dependent mitophagy process was inhibited, resulting in failure of the sperm cytoplasm to be correctly remodeled and eliminated; mitochondrial rearrangement was inhibited, resulting in blocked sperm formation and teratozoospermia [141]. In addition, caspase-2 is activated, resulting in the death of germ cells and almost no mature sperm in the epididymal lumen, affecting fertility [142]. Luo et al. found that neuregulin receptor degradation protein-1 (NRDP1) can promote the ubiquitination of mitophagy-related PARKIN, and PARKIN interacts with the autophagic protein SIP [143, 144]. NRDP1 deficiency leads to elevated levels of PARKIN and autophagy substrate p62 in sperm, while reducing the levels of pro-autophagic protein SIP and autophagy marker LC3-II. These changes are accompanied by suppressed autophagy, disorganized mitochondrial arrangement in round spermatids, abnormal expression of acrosome-associated proteins, and defects in acrosome formation, ultimately resulting in globozoospermia [145]. One study found that autophagy-related protein 7 (ATG7) is expressed in round and elongated sperm cells [60]. LC3 recruitment is inhibited in the Atg7 knockout mouse model, LAMP2 expression is lower, autolysosome formation is impaired, sperm cytoplasmic clearance and mitochondrial rearrangement are inhibited, and PDLIM1 accumulates owing to impaired clearance, disrupting the assembly of sperm flagella and resulting in blocked sperm formation, reduced sperm motility, and asthenospermia [60, 146].

Characteristics and inheritance of mtDNA

A decrease or mutation in the mtDNA copy number can lead to abnormal sperm formation.

Nakada et al. determined that, in the mutant mouse model with pathogenic mtDNA accumulation, mitochondrial respiratory defects occurred during sperm formation, which in turn induced meiotic arrest and apoptosis of sperm cells [122]. In addition, TFAM is necessary to maintain mtDNA and initiates the transcription process to produce RNA primers required for mtDNA replication. It is one of the main regulatory factors of mtDNA copy number [147]. Previous studies in mouse, rat, and human have consistently found that the TFAM protein level is significantly downregulated during spermatogenesis, and that this downregulation is accompanied by a decrease in the mtDNA copy number to one-tenth of its initial value [147, 148]. Timothy et al. tripled the mtDNA copy number in mouse sperm by directly deleting one copy of Tfam, which is contrary to the rapid amplification of mtDNA copy number during oogenesis, and may be caused by the mutation of mtDNA in sperm [149]. These results indicate that the TFAM level directly affects the mtDNA copy number, and its regulatory mechanism is considered to be the downregulation of mtTFA protein levels during spermatogenesis, which leads to a corresponding decrease in mtDNA copy number. The change in TFAM levels in mitochondria was likely due to the presence of testis-specific TFAM messenger RNA (mRNA) subtypes [69]. Therefore, the regulation of TFAM and mtDNA copy numbers during spermatogenesis is a basic mechanism that ensures normal sperm formation. Downregulation of mtDNA copy number is essential for normal sperm function.

In addition, during spermatogenesis, the promoter regions of nuclear genes related to mitochondria tend to remain hypomethylated or demethylated, ensuring proper mitochondrial function and maintaining sperm function and motility [72]. Compared with the wild type, the Tdrkh-knockout mouse model exhibits defects in Piwi-interacting RNA (piRNA) biogenesis in the testes, which disrupts piRNA-specific DNA methylation patterns, leading to hypomethylation of the CpG regions in the Line1 transposon promoter. This results in upregulation of Line1 proteins, causing homologous recombination failure and severe DNA damage, ultimately arresting spermatogenesis at the meiotic stage [150, 151]. Glycerol-3-phosphate acyltransferase 2 (GPAT2) is a MiLi-binding protein located on the outer mitochondrial membrane [152]. Shiromoto and colleagues found that, in germline stem cells with low GPAT2 expression, the number of MiLi-bound piRNAs was significantly reduced, while GPAT2 mutants could rescue piRNA function [153].

Regulation of mitochondrial function in sperm maturation

Elongated spermatids in the epididymis undergo further refinement in structure, metabolism, function, and other related aspects, thereby acquiring motility and fertilizing capacity. They are maintained in quantity and stored in the tail of the epididymis and prepare for the process of fertilization via sperm maturation [154, 155]. Sperm maturation mainly involves the migration and detachment of cytoplasmic droplets at the top of the sperm, a decrease in sperm membrane fluidity, an increase in permeability, and changes in the nucleus [156]. At the end of sperm cell maturation, excess mitochondria are eliminated into residual bodies and degraded through heterophagy in Sertoli cells [157]. Studies have shown that mitochondrial transcription (MT) abundance and sperm membrane potential are higher in the epididymal tail than in the epididymal body, indicating that mitochondrial function increases and mitochondria are gradually activated during sperm maturation [158, 159].

Moreover, sperm maturation occurs in the epididymis, and immature sperm cells have been found in the ejaculations of patients with cryptorchidism, indicating that sperm maturation is blocked [61]. Thus, the structure and function of the epididymis is related to sperm maturation.

Mitochondrial energy metabolism and glycolysis

During sperm maturation in the epididymis, the activity of glycolysis-related enzymes gradually increases, and energy metabolism is altered again and maintained until fertilization [160, 161]. This may be due to the need to prevent excessive ROS from damaging the sperm membrane and DNA, because glycolysis is mainly enriched in the main segment of sperm tail due to the lack of mitochondria and OXPHOS in the midpiece cannot meet the requirements for flagellar movement, because the rapid rate of energy production through glycolysis matches the functional requirements of sperm, or because of an increase in the number and activity of glycolysis-related enzymes [129, 162–165]. Furthermore, ATP produced by OXPHOS promotes sperm maturation, and moderate ROS production is also involved in sperm signal transduction and metabolic regulation. Therefore, these two metabolic forms are essential for ensuring the smooth progress of sperm maturation in the epididymis [35].

Oppong et al. studied sperm from the mouse model with a specific knockout of Glycerol-3-phosphate (Gro3p) phosphatase (G3pp) and found that MMP was increased, ROS expression was upregulated, OS was enhanced, and a small number of mitochondria showed morphological abnormalities such as mitochondrial dislocation and fibrous sheath loss. Sperm showed morphological abnormalities with flagellar folding and reduced sperm function, resulting in decreased sperm motility and impaired sperm capacitation [166]. Wang et al. found that, in the mouse model, a deficiency of thioredoxin-glutathione reductase (TXNRD3), which is abundant at the mitochondrial sheath formation site, leads to an imbalance in the redox regulation of mitochondrial structural proteins during capacitation, resulting in mitochondrial ultrastructural defects. Mitochondrial condensation, cristae collapse, and loss of MMP control affect ATP production thereby inhibit sperm maturation and impair sperm morphology and motility, which may induce asthenospermia [167]. In addition, fatty acid β-oxidation (FAO), a key link in mitochondrial energy metabolism, can also participate in energy production during sperm maturation [168]. Solute carrier family 22 member 14 (SLC22A14) is a transport protein located in the inner mitochondrial membrane of the midpiece of sperm and is involved in regulating the transport of riboflavin. In the Slc22a14-knockout mouse model, dysfunction of this protein impaired the transport of riboflavin to the mitochondrial matrix, resulting in the downregulation of FAO and OXPHOS and inadequate ATP production, thus affecting sperm motility and quality [169].

Excessive ROS production affects sperm maturation. Park et al. determined the differences in protein expression and showed that, during epididymal sperm maturation, sperm mitochondrial proteins in low-vigor Bos taurus (bulls) lacked modification compared with high-vigor bulls [170]. Irregular modification of mitochondrial proteins may lead to excessive ROS production, early sperm activation, depletion of sperm energy, and reduced activity. Excessive ROS production in sperm can cause lipid peroxidation and DNA damage, thereby affecting male fertility [171]. In addition, excessive ROS can reduce the intracellular ATP concentration, resulting in a lower flagellar beat frequency and loss of sperm motility [172, 173].

Mitochondrial quality control

Mitophagy

A close association exists between sperm maturation and mitophagy. Cryptorchidism is associated with impaired germ cell maturation, and immature sperm cells exist in the ejaculate [174, 175]. The abnormal temperature environment in patients with cryptorchidism leads to mitochondrial damage in sperm cells and activates mitophagy. There are numerous autophagosomes in the mitochondria of cryptorchidism sperm cells, which increases their survival rate [61, 176]. Deep sequencing of the miRNA library of extracellular vesicles (EVs) in seminal plasma (SPEVs) isolated from Gallus gallus (chicken) has shown that mitophagy is potentially related to sperm maturation [177]. Mitophagy may maintain the vitality of cryptorchid sperm, even if it is poor [61, 62].

Characteristics and inheritance of mtDNA

MtDNA plays a critical role in sperm maturation, and its copy number mutations and abnormalities obstruct sperm maturation. In 1999, Holyoake found that the mtDNA adenosine triphosphatase 6 (ATPase6) gene T8821A mutation may be associated with sperm maturation and may lead to male infertility [178]. Figure 4 shows the relevant regulatory mechanisms of mitochondrial function during sperm formation and maturation.

Fig. 4.

The regulation mechanism of mitochondrial function during sperm formation and maturation. a Mitochondrial energy metabolism during sperm formation: In Tppp2-deficient mouse model, loss of mitochondrial cristae severely impairs spermatogenesis. Deficiency of Gykl1 or Gk2 blocks mitochondrial aggregation and impairs mitochondrial sheath formation, thereby inhibiting spermiogenesis. Microtubule protein polymerization-promoting protein 2, TPPP2; glycerol kinase-like 1, Gykl1; glycerol kinase 2, Gk2. b Mitochondrial quality control during sperm formation: Fis1 knockout leads to the failure of the contraction ring to break, and the development of the acrosome is blocked, which in turn causes globozoospermia. Meanwhile, Mff knockout causes excessive mitochondrial fusion to form a huge structure, leading to a series of effects such as mitochondrial sheath separation, eventually leading to asthenospermia. Abnormal PARKIN ubiquitination in the NRDP-deficient mouse model impaired mitophagy and abnormal acrosome development, eventually leading to globozoospermia. The recruitment of LC3 and the expression of LAMP2 in the sperm of the Atg7 knockout mouse model were inhibited, and the formation of autophagic lysosomes was blocked, eventually leading to asthenospermia. Fission protein 1, FIS1; mitochondrial fission factor, MFF; neuregulin receptor degradation protein-1, NRDP1; light chain 3, LC3; lysosomal-associated membrane glycoprotein 2, LAMP2; autophagy-related gene 7, ATG7. c Mitochondrial genetic information during spermatogenesis: In the sperm of pathogenic mtDNA mutant mouse model, mitochondrial respiratory function is defective. Mitochondrial DNA, mtDNA. d Mitochondrial genetic information during sperm formation: TXNRD3 deficiency leads to decreased MMP and ETC disorder and SLC22A14 deficiency inhibits fatty acid β-oxidation, both of which lead to decreased ATP synthesis and impaired sperm motility. Mitochondrial membrane potential, MMP; electron transport chain, ETC; solute carrier family 22 member 14, SLC22A14; adenosine triphosphate, ATP. e Mitochondrial quality control during sperm maturation: Heat shock induces mitochondrial membrane damage; an abnormal increase in ROS triggers OS, triggers mitochondrial swelling and apoptosis, and reduces the number of functional sperm. Reactive oxygen species, ROS; oxidative stress, OS. f Mitochondrial genetic information during sperm maturation: mtDNA T8821C mutation causes abnormal ATP synthase subunits, loss of OXPHOS function, and sperm stagnation during the immature stage due to interruption of energy metabolism. Oxidative phosphorylation, OXPHOS; mitochondrial DNA, mtDNA; adenosine triphosphate, ATP

Regulation of mitochondrial function in fertilization

After ejaculation in the female genital tract, sperm migrate to the ampulla of the fallopian tube. During this migration, the sperm pass through and are capacitated in the female genital tract, which enhances their motility and confers the ability to fertilize an oocyte [179]. Many capacitated sperm pass through the cumulus cells around the ovum and release acrosin to dissociate the follicular cells. Subsequently, some sperm can directly contact the zona pellucida, bind to the zona pellucida proteins, and release acrosin to dissolve the zona pellucida [180]. Subsequently, sperm fuses with the oocyte plasma membrane, triggering the cortical reaction. The cortical granules of the oocyte release their contents, which change the structure of the zona pellucida, thus preventing other sperm from crossing the zona pellucida and ensuring monospermic fertilization [181]. Concurrently, the oocyte completes its second meiosis to form a female pronucleus, and the second polar body is squeezed out. The sperm head is depolymerized, and the male pronucleus, which is larger than the original, is re-formed. The two pronuclei come together to fuse and form the fertilized ovum [182–184].

Regulation of mitochondrial function during sperm capacitation

Mitochondrial energy metabolism and glycolysis

Spermatozoa provide ATP for flagellar movement to drive sperm motility, with energy generated mainly through OXPHOS in the midpiece of the tail and glycolysis in the primary fiber sheath. Glycolysis is the first energy metabolism process [163, 185]. Sperm with higher motility have elevated mitochondrial membrane potential and bioenergy metabolism [186]. Therefore, abnormal mitochondrial energy metabolism affects ATP production and inhibits sperm motility [163].

The capacitation process and motility of human sperm are closely related to the MMP level and respiratory chain activity [187, 188]. Furthermore, Cedíková et al. discovered that, in contrast to normal sperm, complex I activity in the mitochondria of sperm from men with asthenozoospermia was decreased. This reduction in complex I activity led to increased proton leakage, subsequently causing reduced OXPHOS efficiency. Consequently, ATP production decreased, potentially resulting in reduced sperm motility [189]. Obesity can lead to decreased sperm energy production and the inhibition of sperm motility [190]. Jia et al. found that sperm ATP content negatively correlated with an increase in body mass index (BMI) in all patients, and overweight/obese men exhibited reduced MMP and low-quality sperm [190]. Moreover, their findings indicated that the mouse testicular antioxidant enzymes, including GPX, catalase, and superoxide dismutase (SOD), were reduced. Concurrently, serum MDA levels increased significantly. These changes trigger elevated ROS generation, reduced MMP, and decreased ATP synthesis. Ultimately, these alterations lead to reduced sperm motility [190]. Gong et al. found that exposure to 42 °C (HS) for 6 h significantly decreased COX4 and HSP70 protein levels in boar sperm; MMP, and complex I and IV activities were also reduced, thereby inhibiting OXPHOS and reducing ATP production, which in turn led to a significant decrease in sperm progressive motility [191].

In addition, the expression levels of glycolysis-related factors GAPDHS, PGK2, and LDHC in sperm are closely related to sperm motility. The expression levels of these enzymes are lower in elderly men than in young men, which may be the cause of asthenospermia [192]. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is also involved in glycolysis. Miki et al. discovered that, in Gapds-knockout male mouse sperm, although mitochondrial oxygen consumption remained unchanged, ATP content was reduced. This reduction led to a decrease in sperm motility, causing the sperm to move at a slower pace [185]. In addition, in vitro experiments with human sperm cells showed that, after exogenous pyruvate treatment, the intracellular ATP level increased, resulting in an increase in sperm motility. Furthermore, they further found that inhibition of the ETC did not affect the experimental results and [¹³]C-labeled pyruvate was converted into lactic acid, further, confirming that energy metabolism during sperm motility is dominated by glycolysis [193].

Fatty acid metabolism is also closely related to sperm energy and motility [169]. SLC22A14 is a member of the solute carrier family of transporters and is closely associated with male fertility [194]. Kuang et al. detected increased ROS levels, decreased ATP production, and the accumulation of free fatty acids (FFA) and triglycerides (TAG) in Slc22a14-deficient sperm motility [169]. Further research revealed that flavin function was impaired, resulting in a decline in MMP and suppression of OXPHOS. In addition, FAO was blocked, and the levels of acylcarnitine and its metabolites in the TCA cycle were significantly reduced. Abnormal sperm energy metabolism reduced ATP production and decreased athletic ability [169]. Furthermore, glycolysis and lactate production in sperm were enhanced. However, this increase did not counterbalance the decrease in ATP production. This result further confirms the harmonious cooperation between the two metabolic processes during sperm motility.

Mitochondrial quality control

Mitochondrial dynamics

Mitochondrial dynamics are involved in the maintenance of sperm motility, and imbalances in mitochondrial fusion and fission can lead to impaired sperm motility.

Compared with the control group, the expression of MFN2 protein in the sperm of patients with asthenospermia was lower, resulting in abnormal mitochondrial morphology, thereby causing a lower level of MMP, which in turn affects sperm motility [195]. Similarly, Varuzhanyan et al. ascertained that, in an Mf'f mutation experiment, the sperm motility of the mutant mouse model was significantly lower than that of normal mouse model [55]. In study using male mouse with Sirt1 heterozygous mutation, it was noted that, compared with wild-type control, after 2 h of capacitation, sperm from the mutant mouse model were overactivated, the percentage of highly activated sperm decreased, sperm capacitation was inhibited, and the fertilization rate decreased [196]. In addition, studies have shown that mitochondrial superoxide levels are high, indicating mitochondrial dysfunction. SIRT1 can inhibit the expression of mitochondrial fission proteins and increase the expression of mitochondrial fusion proteins to regulate mitochondrial dynamics [196]. ARMC12 regulates mitochondrial dynamics [128]. Sperm motility in the mouse model lacking Armc12 decreased, with a significant reduction in the average path velocity, linear velocity, and curvilinear velocity of sperm [128].

Mitophagy

Sperm motility is closely associated with mitophagy. Mitophagy-related proteins such as LC3, P62, M-TOR, and PINK1 have been detected in human sperm [62]. Activation of mitophagy significantly enhances sperm motility, whereas its inhibiting mitophagy reduces sperm motility. This may be because mitophagy can remove damaged mitochondria to maintain homeostasis and metabolism in sperm. When mitophagy is inhibited, mitochondrial dysfunction leads to a decreased ATP concentration and high ROS production, resulting in low sperm motility [62].

Accumulation of PINK1, translocase of the outer membrane 20 (TOM20), and LC3-II in human sperm stimulated by chloroquine leads to the inhibition of mitophagy, accumulation of damaged mitochondria, and a decrease in ATP and intracellular calcium concentrations, consequently affecting sperm motility, which also leads to a significant increase in caspase-3/7 activation and promotes apoptosis [62]. ATG7 is an E1-like activating enzyme that plays an important role in the two ubiquitin-like coupling systems of autophagy [197]. Given that sperm motility depends on the energy provided by the mitochondria, studies have shown that the lack of Atg7 in rat sperm results in incorrect mitochondrial rearrangement, and sperm motility depends on the energy provided by the mitochondria, which is one of the reasons for the sharp decline in sperm motility in mutant mouse [60, 198]. Further findings indicated that ornidazole (ORN) treatment leads to ROS overproduction and mitochondrial injury in rat sperm. In contrast, Qiangjing tablets improved sperm motility by upregulating the liver kinase B1 (LKB1)/5′-adenosine monophosphate-activated protein kinase (AMPK)/Unc-51 like autophagy activating kinase 1 (ULK1) pathway, suppressing oxidative damage, enhancing mitophagy and ubiquitination, and reducing mitochondrial ultrastructural abnormalities [199, 200].

Characteristics and inheritance of mtDNA

Mutations, deletions, and increased copy numbers of mtDNA can cause abnormalities in genes essential for mitochondrial energy production, ultimately affecting sperm energy supply and motility.

COXII (m.8021 G/A) and tRNA (His) (m.12187 C > A) mutations have been identified in infertile men with asthenospermia [201]. This may lead to decreased sperm motility by affecting the OXPHOS of gene-related sequences. This study further revealed that deletions in mtDNA (4977 bp and 7436 bp regions) could result in the loss of genes critical for OXPHOS [202]. Collectively, inhibition of mitochondrial OXPHOS leads to decreased ATP production, which affects sperm motility [203, 204].

Faja et al. determined that the mtDNA copy number in semen samples from patients with asthenospermia was higher than that in normal human semen samples, and was negatively correlated with sperm concentration, total sperm count, and total active sperm count [205]. Darr et al. analyzed sperm samples from American quartet horses. Their study demonstrated that an elevation in the mtDNA copy number correlated with a reduction in the overall sperm motility of these horses [206]. Guo et al. found that the mitochondrial and mtDNA contents of motile sperm were lower than those of less motile sperm. However, the level of mtDNA-encoded protein, which plays a key role in OXPHOS, was higher, and the mitochondrial transmembrane potential and mitochondrial respiratory chain complex I activity were also higher, which may explain this result [207].

Acrosome reaction and fertilization and mitochondrial function

Mitochondrial energy metabolism and glycolysis

Mitochondrial energy metabolism and glycolysis are essential for energy supply during the sperm acrosome reaction. Administration of ATP induces sperm acrosome exocytosis, and extracellular ATP can mediate this reaction, through an increase in intracellular calcium concentration [208, 209]. In human sperm treated with ATP-containing dodecanoylphosphatidylcholine (PC12) liposomes, the ATP content in the control group decreased significantly, whereas the ATP content in the experimental group did not change significantly, and the percentage of acrosome-reactive sperm increased [210].

In an MMP dissipation experiment of human sperm induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), ROS production increased, ATP content decreased, and apical protein activity and acrosome reaction decreased compared with untreated controls [211]. Furthermore, studies have confirmed that MMP is positively correlated with acrosome reactions and fertilization rates [212, 213]. During sperm maturation, mitochondria are enriched in the middle of the sperm and begin to gradually restructure as sperm capacitation progresses [214]. OXPHOS is an energy source for the excessive activation of sperm motility during capacitation, providing the necessary energy for the acrosome reaction [215]. Furthermore, the levels of PKA and phosphotyrosine decreased, ATP levels significantly decreased, and the acrosome reaction was inhibited in the sperm of mouse model stimulated with a high-dose phthalate combination [216]. After PCB126 treatment of human sperm, phosphotyrosine (P-TYR) and lysine glutarylation (Kglu) protein levels decreased, malondialdehyde levels and ROS production increased, mitochondrial membrane potential decreased, mitochondrial respiration was inhibited, ATP production decreased, and mitochondrial function was damaged, resulting in a significant decrease in the spontaneous acrosome reaction of capacitated sperm [217].

Glycolysis is also involved in acrosome reactions [218]. The percentage of spontaneous and induced acrosome exocytosis did not change in the experiment on Sus scrofa (boar) sperm treated with glucose, but in the presence of peroxide, glucose could induce acrosome exocytosis of sperm [218]. However, Williams et al. reported that human sperm exhibit a lower acrosome reaction without glycolysis [219]. These different results may be due to differences in the cell models. Similarly, in goat sperm treated with an adenosine 5′-monophosphate-activated protein kinase (AMPK) inhibitor, the AMPK activity value was lower, the signal of phosphorylated AMPK decreased, the lactate level and LDH activity decreased, glycolysis was inhibited, membrane potential decreased, ATP production decreased, and the acrosome reaction was inhibited [220]. Moreover, 2-deoxy-d-glucose (DOG) can inhibit glycolysis through pyruvate-or lactic acid-induced movement without inhibiting ATP synthesis in the mitochondria. Mukai et al. ascertained that, compared with sperm activated only with pyruvate or lactic acid, the flagella amplitude and beating frequency of sperm exposed to DOG decreased sharply, and the ATP content in the presence of DOG decreased significantly [161]. In summary, glycolysis plays an important role in fertilization.

Concomitantly, mitochondrial OXPHOS and glycolysis are coordinated in the acrosome reaction. Glycogen synthase kinase-3 (GSK3) is involved in energy metabolism, in which GSK3α/β is expressed in sperm and its abundance increases during transport along the epididymis and is localized around the sperm acrosome [221, 222]. In goat sperm treated with CHIR99021 (which can promote GSK3α/β phosphorylation), the findings indicated that the amounts of phosphorylated-Ser21-GSK3α and phosphorylated-Ser9-GSK3β were elevated. This elevation triggered an increase in LDH, MDH, and SDH activity. Subsequently, the mitochondrial membrane potential was augmented, thereby boosting ATP synthesis. Finally, both the percentage of sperm undergoing the acrosome reaction and the overall quantity of sperm bound to the zona pellucida are increased [222].

Mitochondrial quality control

Mitochondrial dynamics

Mitochondria maintain a constant structure and number through continuous fusion and fission, thereby affecting the production of ATP and ROS, which are necessary for normal acrosome reactions [211]. However, the specific mechanism through which mitochondrial dynamics affect the acrosome reaction remains unknown, although some results indicate a potential correlation between the two. Starovlah et al. used the acrosome reaction inducer progesterone to change the transcription profile of mitochondrial dynamics markers. The transcription levels of the mitochondrial fusion-related markers MFN1/2 and OPA1 and the mitochondrial fission marker DRP1 increased, indicating that the regulation of mitochondrial dynamics may be directly related to the acrosome reaction [57].

In addition, mitochondrial dynamics are involved in mitochondrial clearance during fertilization. Mitophagy is involved in the degradation of sperm-derived mitochondria. Fragmented mitochondria caused by mitochondrial fission promote mitophagy, whereas fusion has an inhibitory effect [223]. Therefore, mitochondrial fission in sperm promotes sperm-derived mitochondrial degradation, whereas mitochondrial fusion inhibits this process [58]. Research in Caenorhabditis elegans (nematode worm) embryos showed that, compared with fertilization with wild-type sperm, sperm-derived mitochondrial clusters in embryos fertilized by drp-1-deficient sperm persisted longer. In contrast, those in embryos fertilized by fzo-1-deficient sperm did not last as long [58].

Mitophagy

Mitophagy/lysosomal and ubiquitin–proteasome pathways are involved in the degradation of sperm-derived mitochondria. In the mouse model, LC3 and ubiquitin are located in the middle of the sperm 3 h after fertilization and are co-localized with mitochondria. Sperm-derived mitochondria, LC3, and ubiquitin levels are reduced in embryos after the morula stage [63, 224, 225].

In Parkin and Mitochondrial E3 ubiquitin ligase 1 (Mul1)-knockdown mouse embryos, mitochondrial ubiquitination was reduced and mitophagy was inhibited, causing defects in sperm-derived mitochondrial degradation in early mouse embryos and resulting in retention [226]. Fibronectin type III domain-containing protein 1 (FNDC-1) is a mitophagy receptor expressed in sperm of the nematode worm; paternal mtDNA is largely undetectable in wild-type larvae, whereas the degradation rate of sperm-derived mitochondria in the offspring of fndc-1 mutant males is delayed, resulting in mtDNA detection [68]. Similarly, prohibitin 1/prohibitin 2 (PHB1/PHB2) binds to LC3-II and participates in mitophagy. However, sperm-derived mitochondria accumulate in phb-2-knockdown paternal (nematode worm) [227].

However, other studies have shown that mitophagy is not involved in sperm-derived mitochondria clearance after fertilization. LC3 protein is recruited into sperm mitochondria during fertilization and then disappears before the four-cell stage embryo. This study used allele-specific polymerase chain reaction (PCR) and restriction enzyme analysis to detect the number of mtDNAs in different germ cells and found that the mtDNA of the most likely fertilized sperm had long been eliminated before fertilization [228].

Characteristics and inheritance of mtDNA

Normal mtDNA is essential for fertilization. Mitochondrial DNA copy number and mutations are negatively correlated with fertilization rates [229, 230]. In the sperm of infertile men with leukocytospermia, the Cyt B and NADH5 levels are low, leading to respiratory chain disorders and mitochondrial dysfunction, increased ROS production, and high mtDNA levels. Excessive ROS leads to a decrease in the stability or reactivity of CatSper in the sperm plasma membrane, and sperm activity and acrosome reactions [231]. Similarly, in AgNP-treated mouse sperm experiments, dichlorofluorescein (DCF)-positive signals increased, promoting ROS production and inducing OS, which resulted in an increased mtDNA copy number, mitochondrial dysfunction, and a significant decrease in the proportion of CD46-positive sperm. This reduced the sperm acrosome reaction, increasing the percentage of unfertilized oocytes and causing a significant decrease in the blastocyst formation rate [232]. Figure 5 summarizes the coordinated regulation of mitochondrial function during fertilization.

Fig. 5.

The regulation mechanism of mitochondrial function during fertilization. a Abnormal energy metabolism during sperm capacitation and motility: After high-temperature treatment, the expression of COX4 and HSP70 proteins decreased, MMP was reduced, and the activities of respiratory chain complexes I and IV were inhibited, leading to decreased OXPHOS, which in turn reduces ATP production and sperm motility. HFD reduced SOD and GPX activity while excessively increasing ROS and MDA expression, resulting in decreased ATP and impaired sperm motility. GAPDHS deficiency leads to inhibition of glycolysis, whereas SLC22A14 deficiency results in impaired fatty acid transport and β-oxidation, with a compensatory increase in glycolysis and elevated lactate production, along with inhibited activities of respiratory chain complexes I and II and a compromised TCA cycle. Both scenarios lead to limited ATP generation, affecting sperm motility. Cyclooxygenase 1, COX; heat shock protein 60, HSP; mitochondrial membrane potential, MMP; oxidative phosphorylation, OXPHOS; adenosine triphosphate, ATP; high-fat diet, HFD; glutathione peroxidase, GPX; superoxide dismutase, SOD; malondialdehyde, MDA; reactive oxygen species, ROS; solute carrier family 22 member 14, SLC22A14; tricarboxylic acid, TCA. b Imbalance in mitochondria quality control during sperm capacitation and motility: Abnormal mitochondrial dynamics and dysregulated mitophagy synergistically disrupt mitochondrial homeostasis. In patients with oligozoospermia, the expression of MFN2 protein in sperm is reduced. Additionally, both MFF gene knockout and reduced MFN2 expression lead to mitochondrial dynamics disorder, inhibiting mitochondrial fission and the activity of respiratory chain complex IV. Meanwhile, SIRT1 deficiency causes DRP1-mediated abnormal mitochondrial fission, leading to increased ROS levels, both of which result in decreased ATP synthesis and impaired sperm motility. Chloroquine treatment induces abnormal accumulation of PINK1 and LC3-II, while ORN stimulation increases ROS levels and obstructs the LKB1/AMPK/ULK1 signaling pathway. Both conditions impair mitophagy and affect mitochondrial function, thereby inhibiting ATP production and suppressing sperm motility. Mitofusin 2, MFN2; mitochondrial fission factor, MFF; dynamin-related protein 1, DRP1; reactive oxygen species, ROS; adenosine triphosphate, ATP. c Disruption of mtDNA homeostasis during sperm capacitation and motility: Mutations in COXⅡ and tRNA (His) affect the expression of OXPHOS-related proteins, while 4977 bp and 7436 bp deletions lead to the loss of complex I and complex IV genes. Meanwhile, oligozoospermia results in increased mtDNA copy number and reduced energy allocation efficiency, both of which decrease ATP synthesis and inhibit sperm motility. Mitochondrial DNA, mtDNA; oxidative phosphorylation, OXPHOS; adenosine triphosphate, ATP. d Abnormal energy metabolism during acrosome reaction and gamete fusion: CCCP induces a decrease in P-Tyr and KGLU protein levels, which leads to reduced MMP, increased ROS, and decreased ATP, resulting in a weakened acrosome reaction. In addition, reduced AMPK activity leads to decreased lactate and LDH activity, inhibited glycolysis, decreased membrane potential, and reduced ATP production, thereby suppressing the acrosome reaction. Mitochondrial membrane potential, MMP; reactive oxygen species, ROS; adenosine triphosphate, ATP; adenosine 5′-monophosphate-activated protein kinase, AMPK. e Mitochondrial quality control imbalance during acrosome reaction and gamete fusion: Knockout of Parkin and Mul1 genes or deletion of Fndc and Phb genes leads to reduced mitochondrial ubiquitination levels and inhibition of mitophagy, resulting in defective degradation of paternal mitochondria. In addition, deletion of the Drp1 gene affects paternal mitochondrial degradation by inhibiting mitochondrial fission. Mitochondrial E3 ubiquitin ligase 1, MUL1; fibronectin type III domain-containing protein, FNDC; prohibitin, PHB; dynamin-related protein 1, DRP1. f Disruption of mtDNA homeostasis during acrosome reaction and gamete fusion: In infertile men, decreased levels of Cyt B and NADH5 in sperm, dysfunction of the respiratory chain, and increased ROS lead to reduced CatSper stability in the sperm membrane, thereby inhibiting sperm motility and the acrosome reaction. Additionally, excessive ROS can also increase mtDNA copy number and inhibit the acrosome reaction. Mitochondrial DNA, mtDNA; reactive oxygen species, ROS; cytochrome B, Cyt B; reactive oxygen species, ROS

Summary and outlook

From spermatogenesis to fertilization, the role of mitochondria has been confirmed in several studies. Mitochondria maintain the cellular energy supply through functions including oxidative metabolism, mtDNA, mitochondrial dynamics, and mitophagy and participate in the formation of important sperm structures, removal of damaged mitochondria, protection of sperm homeostasis, inhibition of OS, inflammation, sperm damage, and apoptosis. However, there remain areas worthy of further study and discussion. Throughout the process of male reproduction, the number and structure of mitochondria as well as their functions change, resulting in changes in energy metabolism, adjustments in mitochondrial dynamics, decreases in mtDNA copy number, and changes in mitophagy. However, the specific causes and effects of these changes in each process, whether external stimuli can hinder the reproductive process by affecting these changes, and the underlying pathological mechanisms have not previously been analyzed or summarized. This paper summarizes the dynamic changes in mitochondria during the male reproductive process, from spermatogenesis to fertilization (Table 1), and the causes and effects of these changes. In addition, under external stimulation, abnormal mitochondrial function-related genes or in the male reproductive system itself can lead to mitochondrial dysfunction, aggravate male-related diseases (varicocele, cryptorchidism, testicular torsion, etc.), inhibit the male reproductive process, decrease the sperm count and motility, and induce male infertility.

Although current research on mitochondrial dysfunction and male infertility is relatively comprehensive, most studies have involved in vitro cell experiments and low-level biological models. Study populations and models are limited and inadequate, and the experimental results may not be able to be confirmed in mammalian and human studies. Therefore, future studies should include more comprehensive, diverse, and higher-level research models, as well as a broader range of external stimuli, to improve and enrich our understanding of the mechanism of mitochondrial function in male infertility and to facilitate translation into clinical practice.

On the basis of the dynamic changes in mitochondrial morphology and function during male reproduction, the focus of treatment at each stage differs. Therefore, we have listed the currently targeted drugs for mitochondrial function in the treatment of male infertility in Table 2, to provide a reference for more precise treatments. In addition, there are methods for repairing mutations in mtDNA and restoring male fertility through gene editing tools such as transcription activator-like effector nucleases (TALEN) and CRISPR-Cas9. Effective methods also exist for the accurate regulation of ROS, calcium, and potassium ion homeostasis. The above analysis shows that OS inhibited the male reproductive process and reduces male fertility [233]. The precise regulation of calcium signaling may also play a role in infertility treatment. Regulation of mitochondrial calcium plays an important role in germ cells. It can regulate energy metabolism to maintain ATP production, control ROS levels, and maintain intracellular calcium balance [234]. After H₂O₂ treatment of human sperm in vitro, mitochondrial function was impaired, intracellular Ca₂₊ levels increased, and sperm motility decreased [233]. Stimulation with pyrphenine (PDB) and etoxazole disrupts mitochondrial and calcium homeostasis and induces testicular toxicity and male reproductive abnormalities [235, 236]. In addition, potassium channels appear to be involved in the maintenance of male fertility. In the sperm of male patients with infertility with the potassium channel subfamily U1 (KCNU1, also known as SLO3) mutation, the mRNA and protein levels of SLO3 decreased, the mitochondrial membrane potential decreased, and the levels of acrosome marker glycoprotein and mitochondrial sheath protein heat shock protein 60 (HSP60) decreased, resulting in acrosome dysplasia and sperm motility defects [237]. Notably, perturbation of mitochondrial homeostasis, whether through excessive fission or fusion, is detrimental to germ cells. Therefore, any therapeutic strategy aimed at modulating mitochondrial dynamics must be contingent upon a precise assessment of its pathological state [16].

Table 2.

Targeted therapy for mitochondrial function

Animal/cell model	Drugs/physiological conditions	Targeting mitochondrial function	Stages of male reproduction	Result	Reference (DOI/PMID)
Wild ground squirrel	Breeding season	Mitophagy activation	Spermatogenesis	Testicular OS is inhibited Increased sperm count	[238]
Spermatogenic cells in varicocele (VC) liver Qi stagnation (LQS) rat	Tongjingling (TJL)	Energy metabolism	Spermatogenesis	ROS decreased, MMP increased in spermatogenic cells	[239]
Triptolide (TP)-induced testicular injury in mouse	MitoQ	Mitochondrial dynamics	Spermatogenesis	Relieve blood–testis barrier dysfunction	[240]
BNC1 truncated mutant mouse	Nicotinamide nucleoside or metformin	Energy metabolism	Spermatogenesis	Improved the structure of seminiferous tubules, inhibited testicular cell apoptosis	[88]
Idiopathic asthenozoospermia (iOAZS) rat	Xianlu Oral Liquid (XL)	Energy metabolism	Spermatogenesis	Improve the disruption of seminiferous tubules, reduce spermatogenic cell apoptosis and OS	[241]
Human sperm	Rapamycin	Mitophagy activation	Sperm motility	Promoted ATP production and increased intracellular calcium concentration	[62]
Frozen human sperm	Elamipretide	Oxidative stress	Sperm motility and acrosome reaction	Sperm motility and antioxidant activity were improved, spontaneous acrosome reaction was reduced, and inducible acrosome reaction was increased	[242]
Sperm of homozygous Immp2l mutant mouse	SkQ1	Oxidative stress	Sperm motility	Germ cell apoptosis decreased, GPX4 expression increased	[243]
γ-Irradiation-induced testicular injury in rat	MitoQ	Mitochondrial dynamics	Sperm motility	Upregulated mitochondrial dynamic proteins, improved OS	[244]
Freeze–thaw chicken semen	Mito-TEMPO	Oxidative stress	Sperm motility, acrosome reaction, and fertilization	Total sperm motility, acrosome integrity and motility were improved, and the fertilization rate was increased	[245]

However, owing to the close relationship and mutual influence between mitochondrial functions, targeted therapy with a single mechanism seems to have a limited effect; thus, multitarget treatment strategies may provide more effective therapeutic outcomes, by comprehensively inhibiting ROS, enhancing mitophagy, maintaining mitochondrial dynamics stability, and regulating energy metabolism. Better and more accurate regulation of mitochondria can lead a comprehensive improvement in sperm quality and reproductive health. In addition, the establishment of a mitochondria-related biomarker library may detect and diagnose infertility early as well as facilitate dynamic monitoring of the therapeutic effect to allow us to treat the disease more continuously, comprehensively, and accurately, and improve the cure rate of infertility. Targeted therapeutic strategies for restoring mitochondrial function are summarized in Fig. 6 and Table 2.

Fig. 6.

Mitochondrial function repair treatment strategy. a Targeting mtDNA mutations with CRISPR-Cas9 to restore sperm function: Design a specific Cas9–sgRNA complex to bind and cleave mutated mtDNA, while introducing a wild-type mtDNA template to guide cellular repair and correct the mutation, thereby treating sperm disorders caused by mtDNA mutations. Mitochondrial DNA, mtDNA. b CoQ 10-mediated reduction of ROS enhances sperm function: CoQ 10 promotes the conversion of ROS to H₂O₂ by enhancing GPX activity and works synergistically with the GSH system to eliminate ROS; it simultaneously optimizes the ETC, reduces electron leakage and ROS generation, alleviates ROS-induced damage to mitochondrial membranes, inhibits cell apoptosis and mtDNA mutations, ultimately improving sperm quality. Coenzyme Q10, CoQ10; reactive oxygen species, ROS; glutathione peroxidase, GPX; glutathione, GSH; electron transport chain, ETC. c QJT promotes mitophagy via the LKB1–AMPK–ULK pathway to treat asthenospermia: QJT can activate the LKB1/AMPK/ULK1 pathway, upregulate the expression of LKB1, AMPKα, Beclin-1, p-ULK1, PHB, and LC3-II, and promote mitophagy, thereby reducing intracellular ROS levels and OS, enhancing sperm motility, and subsequently improving oligozoospermia. Qiangjing tablets, QJT; liver kinase B1, LKB1; adenosine 5′-monophosphate-activated protein kinase, AMPK; Unc-51 like autophagy activating kinase 1, ULK1; reactive oxygen species, ROS; oxidative stress, OS

Author contributions

Kai Meng, Qin Qin, Fei Gao, and Jinxiang Yuan conceptualized this research and provided resources. Kai Meng, Ziming Zhu, Haocheng Jia, Yingying Feng, Jingwen Feng, Yanlin Shen, Wenjia Jiang, and Chenyan Liu wrote the first draft of the manuscript. Fei Gao and Jinxiang Yuan were responsible for reviewing and editing. All authors have read and agreed to the published version of the manuscript.

Funding

The authors acknowledge Shandong Provincial Natural Science Foundation (ZR2025MS1440), the Incubation Fund for High-level Scientific Research Projects in Jining Medical University (JYGC2023KJ003), and College Students’ Innovation Training Program of Jining Medical University (cx2024007z; cx2025138).

Data availability

No datasets were generated or analyzed during the current study.

Declarations

Competing interests

The authors declare no competing interests.