The impact of animal models on translating male infertility research from bench to bedside: a narrative review

Abstract

Male infertility (MI) represents a complex clinical issue affecting millions of couples worldwide. Its diverse etiologies and incompletely elucidated mechanisms limit the development of effective therapeutic strategies. Animal models, serving as a bridge between basic research and clinical practice, play an indispensable role in simulating the pathophysiological processes of human MI, deciphering key underlying mechanisms, and screening and evaluating potential therapeutic agents. This review systematically summarizes the various animal models employed in MI research, including chemically induced models (e.g., using tripterygium glycosides or cyclophosphamide), endocrine-disrupting agent-induced models (e.g., using Bisphenol A (BPA) or finasteride), and physically induced models (e.g., via heat stress or experimental varicocele). We focus on elucidating the core pathological mechanisms these models recapitulate, encompassing oxidative stress, inflammatory responses, novel cell death pathways such as apoptosis, pyroptosis, and ferroptosis, as well as endocrine disruption of the hypothalamic-pituitary–gonadal (HPG) axis. Through an in-depth analysis of the characteristics of each model, associated signaling pathways-including the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, and the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome, and identified pharmacological targets, this article aims to provide insights into the complex etiology of human MI and offer a crucial theoretical foundation and experimental framework for the development and translation of novel mechanism-based therapeutics.

Male infertility (MI) refers to a reproductive system disorder where a couple, having cohabited for over one year with normal sexual activity and without using contraception, fails to conceive after excluding female-related factors [1]. According to statistics, approximately 200 million people worldwide face fertility challenges, with around 50% of these cases attributed to male factors [2]. Along with the accelerated pace of modern life as well as the ongoing advancement of social sectors, the incidence of MI has been on the rise year by year and exhibits a tendency to involve younger age groups [2, 3]. It has now become a significant issue impacting the healthy development of society [2, 3]. Despite significant advances in reproductive medicine, particularly in assisted reproductive technologies, the complex etiology and incompletely elucidated molecular mechanisms of MI have limited the development of targeted and effective therapeutic strategies.

Animal models serve as a crucial bridge connecting basic research with clinical disease diagnosis and treatment, playing an indispensable role in deeply investigating the pathogenesis of MI, screening potential therapeutic agents, and evaluating novel treatment strategies. In recent years, researchers have established various animal models to simulate key pathological features of human MI. These models have not only provided critical experimental evidence for elucidating the complex pathogenic mechanisms of MI but have also laid an important foundation for identifying potential pharmacological targets and evaluating the efficacy and safety of candidate drugs. Although animal models are widely used in MI research, comprehensive and systematic analyses of their mechanistic basis and translational relevance remain lacking. Existing literature predominantly focuses on single model [4–6] types or isolated mechanisms [7–9], lacking systematic integration and comparative analysis of core pathological mechanisms, key signaling pathways, and potential therapeutic targets across different models.

Based on these research gaps, this review aims to systematically summarize commonly used animal models in MI research, with a particular focus on their core pathological mechanisms. Through integrative analysis of modeling methods, pathological features, molecular mechanisms, and potential therapeutic targets across different models, we seek to construct a comprehensive analytical framework. On this basis, we further discuss the advantages and limitations of existing models, identify current research deficiencies, and propose future directions for translational research. This review aims to provide a theoretical foundation and experimental reference for understanding the complex etiology of MI, promoting mechanism-based drug development, and facilitating the translation from basic research to clinical applications.

Common animal models of MI

To date, a variety of animal models for Male Infertility (MI) have been established. Based on the inducing factors, these models can be categorized into three main types: chemically induced models (e.g., models induced by tripterygium glycosides, gossypol acetic acid, cyclophosphamide, adenine, busulfan, or ornidazole), endocrine-induced models (e.g., models induced by Bisphenol A (BPA), 5α-reductase inhibitors, or phthalates), and physically induced models (e.g., models of heat stress injury, radiation injury, or infertility related to varicocele) (Fig. 1).

Fig. 1.

Common types of MI models

Chemically induced models

Tripterygium wilfordii multiglycosides (GTW)-induced model

Derived from the traditional Chinese medicine Tripterygium wilfordii, Tripterygium wilfordii multiglycosides (GTW) represents a key bioactive ingredient. Due to its high incidence of reproductive toxicity, GTW has been used as a model drug to induce animal infertility;it serves as a classic model for studying human idiopathic oligoasthenospermia [10].Research findings show that GTW undermines blood-testis barrier (BTB) integrity by boosting the levels of key pro-inflammatory cytokines (interleukin-6 (IL-6), interleukin-17A (IL-17A), interleukin-1α (IL-1α), and tumor necrosis factor-α (TNF-α)) in testicular tissue [11], leading to epididymal sperm deformities, a decrease in sperm density and motility, and other sperm-related abnormalities [11]. Other studies have demonstrated that GTW impairs sperm quality in rats through multiple mechanisms: Firstly, it induces oxidative stress that lowers the activity of superoxide dismutase (SOD) and catalase (CAT), diminishes the total antioxidant capacity in testicular and epididymal tissues, elevates malondialdehyde (MDA) content, and inhibits the nuclear factor erythroid 2-related factor 2 (Nrf2) -mediated antioxidant pathway [12]. Secondly, it regulates apoptosis-related proteins, thereby promoting spermatogenic cell apoptosis. Thirdly, it disrupts the morphology of testicular and epididymal tissues, resulting in seminiferous tubule atrophy and disorganized arrangement of spermatogenic cells; ultimately, this reduces sperm count and motility and induces oligospermia and asthenospermia [12].In a separate study, Hang et al [13]. administered GTW to sexually mature male Sprague–Dawley (SD) rats (8 weeks, 260 ± 10 g)via gavage at a dose of 40 mg/kg/d for 28 days. Their findings revealed significant reductions in the organ coefficients of the testes and epididymides, as well as marked decreases in sperm concentration and motility. Histological examination demonstrated irregular seminiferous tubule structure, expanded interstitial spaces, atrophic and disorganized tubules with luminal narrowing, and cytoplasmic vacuolization in Sertoli cells. Serum analysis indicated significant decreases in the levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T). From a molecular perspective, phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (p-Akt), and the anti-apoptotic B-cell lymphoma/leukemia-2 (Bcl-2) exhibited notably reduced protein and mRNA expression. In contrast, the pro-apoptotic protein Bcl-2-associated X protein (Bax) and its corresponding mRNA showed a significant upregulation. Liu, Zhao, and their colleagues investigated the mechanism of testicular injury induced by tripterygium glycosides at the genetic and pathway levels. The results showed that the mRNA expression levels of Estrogen Receptor 1 (ESR1) and Epidermal Growth Factor Receptor (EGFR) were significantly upregulated in the tripterygium glycosides group, indicating that these genes play a critical role in the mechanism underlying testicular injury. KEGG pathway analysis showed that genes with differential expression were predominantly implicated in pathways related to spermatogenic dysfunction, including the PI3K/Akt signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, GTP-binding protein (Ras) signaling pathway, and cyclic adenosine monophosphate (cAMP) signaling pathway [14, 15]. Therefore, this model provides a reliable preclinical platform for testing the efficacy of antioxidants and anti-inflammatory drugs.

Gossypol acetate-induced model

Gossypol serves as a highly toxic crystalline polyphenolic substance, predominantly distributed in the seeds of cotton plants [16]. This agent exerts a direct effect on testicular tissue and is capable of triggering azoospermia or oligospermia. Research findings have shown that the toxic impacts of gossypol on male reproductive performance vary among different species [17], for example, it increases sperm abnormalities in bulls, diminishes sperm quantity and motility in rats, and hastens spermatocyte degeneration in hamsters. Notably, this toxicity exhibits dose-dependent, time-dependent, and even reversible properties [18]. Furthermore, gossypol exerts its toxic effects through two key pathways. To begin with, gossypol induces oxidative stress (OS) via enhancing the generation of reactive oxygen species (ROS) and lipid peroxides, which in turn compromises the permeability of sperm plasma membranes, ATPase activity, and glucose transport capacity. Next, it elicits Leydig cell degeneration, resulting in a marked decrease in serum testosterone (T) levels [19]. Saleh et al.’s research [19] demonstrated that sexually mature male SD rats (10–12 weeks, 100–130 g) were administered intraperitoneal injections of gossypol acetate at a dosage of 5 mg/kg over 16 consecutive days. Findings indicated that, in comparison to the control group, the model group exhibited elevated levels of nitric oxide (NO) and lipid peroxides in both serum and testicular tissue, accompanied by reduced serum glutathione concentrations. Additionally, levels of TNF-α, interleukin (IL)−1β, IL-6, and interleukin-18 (IL-18) in testicular tissue were markedly increased, whereas the levels of interleukin-12 (IL-12) were decreased. These findings confirm that gossypol impairs spermatogenesis and sperm function by inducing OS and inflammatory responses. Therefore, the gossypol acetate-induced model not only serves as a powerful tool for elucidating the central role of the interplay between oxidative stress and inflammation in spermatogenic impairment, but also provides a stable and mechanistically well-defined preclinical platform for screening potential therapeutic agents aimed at improving the testicular microenvironment.

Cyclophosphamide-induced model

Cyclophosphamide (CP) is a common chemotherapeutic agent known to induce testicular damage [20], and it is widely used in andrological research to establish animal models of oligospermia. This agent acts rapidly, offering a short-term and stable approach for modeling. Notably, CP can effectively simulate the pathological processes of testicular injury, including OS, lipid peroxidation (LPO), apoptosis, and histopathological changes [21]. More precisely, CP not only diminishes the enzymatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) in rat testicular tissue—thus triggering oxidative stress (OS)—but also elevates the expression level of Bax and reduces that of Bcl-2 [22]. Studies have indicated that CP exposure can trigger specific inflammatory responses via the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and nuclear factor kappa-B (NF-κB) pathways. This form of activation results in the activation of cysteinyl aspartate specific proteinase 1 (caspase-1), enhanced secretion of interleukin-1β (IL-1β) as well as IL-18, and ultimately the initiation of inflammatory cascades [23]. Additionally, CP has been documented to compromise spermatogonial stem cell proliferation and differentiation, trigger spermatocyte apoptosis, impair the genetic material in germ cells, as well as reduce spermatogonia production [24–26]. CP can be administered via oral gavage or intraperitoneal injection. The latter route is particularly rapid and convenient, enabling the establishment of spermatogenic dysfunction models within 1–2 weeks. In a study by Adana et al. [27] adolescent male Wistar rats (6 weeks, 100–110 g) were administered CP at a dosage of 20 mg/kg through daily intragastric administration over a consecutive 21-day period. Results showed that CP compromised both the functional and structural integrity of rat testes, inhibited spermatogenesis at various developmental stages, damaged Leydig cells and proliferating cell nuclear antigen (PCNA), reduced the number and motility of testicular sperm, and ultimately caused infertility in male rats.

Adenine-induced model

Adenine (6-aminopurine) is categorized as a nitrogenous heterocyclic compound of the purine class and is regarded as an ideal agent for establishing oligoasthenospermia models. Its underlying mechanism may involve the following process: the xanthine oxidase (XOD)-mediated reaction generates excessive free radicals, which overwhelm the testicular antioxidant system. This results in dysregulation between ROS generation and elimination in the testicular microenvironment, eventually triggering testicular impairment and disrupting spermatogenic processes [28]. Research findings have indicated that adenine is capable of lowering serum T levels, suppressing the biosynthesis of steroid hormones, as well as downregulating the mRNA expression associated with endothelin-1 in testicular tissue [28]. In a study conducted by Yang et al. [28], sexually mature male SD rats (8 weeks, 180–220 g) in the model group were administered adenine via gavage at a dose of 20 mg/100 g body weight for 28 consecutive days. Results showed significant pathological changes in the rat testes after adenine treatment. Specifically, the protein expression levels of neuropeptide Y (NPY) and relaxin family peptide receptor 1 (RXFP1) were increased, whereas the expression of proopiomelanocortin (POMC) in the hypothalamus, and luteinizing hormone/choriogonadotropin receptor (LHCGR) and insulin-like 3 (INSL3) in the testes were decreased. Such research outcomes suggest that adenine is capable of disturbing the expression of proteins and genes associated with reproduction across the hypothalamic-pituitary–testicular (HPT) axis. This disruption may further cause endocrine hormone imbalances and gonadal dysfunction, thereby reducing sperm production.

Busulfan-induced model

Busulfan (1,4-butanediol dimethanesulfonate, BU) serves as a cytotoxic bifunctional alkylating agent exhibiting remarkable testicular toxic effects. It is capable of triggering oligospermia, azoospermia, and testicular atrophy in males [29], and has been identified as among the most optimal tools for constructing models of oligospermia and azoospermia [30, 31]. In adult testes, BU mainly exerts toxic actions on G1-phase cells; and it brings about infertility primarily via eradicating mitotic-phase spermatogonia by means of apoptosis, autophagy, and ferroptosis [32–35]. Busulfan damages spermatogenic cells via OS and disrupts intercellular junctions in Sertoli cells [36]. The core mechanism accounting for BU-induced MI entails DNA alkylation—this phenomenon suppresses cellular division and eradicates spermatogenic cells across different developmental phases, exerting a notable influence on spermatogonial stem cells [5]. BU exerts its cytotoxic actions via the generation of DNA-DNA crosslinks, DNA–protein crosslinks, as well as single-strand DNA breaks [29]. Halimeh et al. [37] explored the optimal methods and appropriate dosages for establishing busulfan-induced azoospermia models in adolescent male Wistar rats (6–8 weeks, 150 g). Their results showed that, compared with intraperitoneal injection, low-dose busulfan (5 mg/kg) administered via the intratesticular route represents a relatively non-invasive and safe method. This method can generate rat azoospermia models while minimizing toxicity to vital organs.

Ornidazole-induced model

Ornidazole (ORN) is an antimicrobial agent known to induce testicular toxicity in rats [38], and studies have indicated its ability to diminish sperm quality, lead to male infertility, thereby serving as a tool for constructing rat asthenospermia models. Tian et al. [39] found that ornidazole specifically induces OS in the epididymis, disrupts sperm structure, and inhibits sperm maturation. When ornidazole acts on the epididymis, the primary source of free radicals is an increase in ROS. Excessive amounts of ROS induce lipid peroxidation, thereby resulting in increased MDA levels as well as subsequent cellular impairment. Jin et al. [40] established a rat model of idiopathic oligoasthenospermia (iOAZS) via intragastric administration of ORN at a dose of 400 mg/kg body weight for 14 consecutive days in sexually mature male SD rats (180–200 g). Results showed that the iOAZS rats exhibited decreased total antioxidant capacity, reduced activities of GPx and SOD in testicular tissues. Additionally, these rats had elevated levels of hydrogen peroxide and MDA, as well as increased protein expression of 4-hydroxynonenal (4-HNE) in the testes.

Endocrine factor-induced models

BPA-induced model

BPA ranks among the most extensively researched endocrine-disrupting chemicals (EDCs) linked to reproductive dysfunction and impaired spermatogenesis [41]. It can impact the HPG axis, triggering endocrine dysfunction, cellular redox imbalance, and mitochondrial dysfunction, which consequently lead to developmental alterations in the structure and function of the testes, manifesting as abnormal sperm parameters [42]. A study by Grami [43] in sexually mature male Wistar rats (10 weeks, 200–250 g) has shown that the estrogenic activity of BPA, even at low concentrations, is capable of compromising the functional integrity of the HPG axis. Furthermore, low concentrations of BPA directly affect Leydig cells: it inhibits their proliferation and disrupts normal steroidogenesis, thereby reducing testosterone (T) levels and resulting in decreased sperm count, motility, and normal morphology [44]. Genomic studies reveal that BPA compromises the genomic DNA integrity of sperm, induces DNA fragmentation, and suppresses repair mechanisms, leading to aberrant transmission of genetic information. It disrupts epigenetic modifications by abnormally regulating histone acetylation and the expression of DNA methyltransferases, thereby affecting the activity of reproductive-related genes. BPA brings about meiotic dysfunction via compromising the repair of DNA double-strand breaks, along with downregulating genes associated with meiosis, ultimately leading to germ cell stagnation. Furthermore, it selectively suppresses genes involved in steroid synthesis and Sertoli cell function, ultimately disrupting the spermatogenic microenvironment and causing oligoasthenospermia [45].

5α-Reductase inhibitor-induced model

Finasteride is a potently selective type II 5α-reductase inhibitor (5ARI) that functions by inhibiting the transformation of T to dihydrotestosterone (DHT) [46]. Investigations have demonstrated that finasteride can induce an increase in ROS generation in the testes and seminal fluid, enhance the expression levels of pro-apoptotic factors Bax and cysteinyl aspartate specific proteinase 3 (caspase-3), and diminish the expression of the anti-apoptotic Bcl-2 gene. Through these effects, it enhances apoptotic pathways, thereby inducing detrimental impacts on male reproduction [47]. Shalaby et al. [48] developed a rat infertility model through oral gavage of finasteride at a dosage of 5 mg/kg/day for 8 weeks in sexually mature male Wistar albino rats (8–10 weeks, 180–200 g). Results revealed that the model rats had significantly decreased serum DHT levels, increased MDA levels, and substantially reduced activities of GPx, SOD, and catalase (CAT). Histological analysis revealed degenerative alterations in the seminiferous epithelium of certain seminiferous tubules. Kiran et al. [49] further demonstrated that sexually mature male SD rats (10–12 weeks, 300–320 g) treated with finasteride at 1 mg/kg/day via oral gavage for 90 days exhibited reduced spermatogenic cell density, increased apoptotic index, elevated markers of endoplasmic reticulum stress and apoptosis, and impaired fertility.

Phthalate-induced model

Toxicological investigations have verified that di(2-ethylhexyl) phthalate (DEHP) imposes notable toxic impacts on the male reproductive system [50]. Specifically, DEHP functions through its metabolites to disturb the balance of the redox enzyme and antioxidant system, trigger OS within testicular tissues, and ultimately induce structural anomalies in seminiferous tubules, spermatocyte dysfunction, and impairment of Sertoli cells [51, 52]. Azam et al. [53] documented that following oral administration of DEHP to sexually mature male NMRI mice (4–5 weeks, 24–26 g) at a dosage of 2 g/kg/day for 14 consecutive days, the subsequent alterations were noted: a marked reduction in testicular weight; diminished counts of spermatogonia, primary spermatocytes, early spermatids, and late spermatids; and impaired sperm motility (including progressive motility). Additionally, serum T levels declined, while serum LH and FSH levels rose. These changes were linked to increased oxidative stress and strengthened inflammatory reactions within testicular tissue samples.

Physical factor-induced models

Heat stress injury-induced model

Research findings have demonstrated that heat stress is linked to suppressed testicular hormone levels and increased OS [54, 55]. Jeremy et al. revealed that heat stress is capable of triggering germ cell apoptosis through upregulating the expression of Bax and active caspase-3, and downregulating the expression of the anti-apoptotic Bcl-2 protein [56]. Scrotal thermal therapy constitutes a valid approach to establish azoospermia animal models, with merits including stability, simplicity, cost-effectiveness, and low mortality [57]. Increased scrotal temperature triggers testicular heat stress, and this has been demonstrated to fully suppress spermatogenesis and additionally elevate OS [54, 55]. Notably, heat stress likewise compromises the HPG axis, resulting in decreased circulating concentrations of estrogen, FSH, LH, and T in rat models [58]. Jeremy et al. [56] additionally verified that heat stress triggers germ cell apoptosis in sexually mature male Wistar albino rats (120 ± 5 g) via the above-described pathway—namely, increasing the expression levels of active caspase-3 and Bax and decreasing those of Bcl-2. They successfully established an azoospermia model by subjecting male rats to consecutive heat exposure at 43 °C, a temperature threshold known to trigger selective germ cell damage. Sanaz et al. [59] documented that, in sexually mature male NMRI mice (6–8 weeks, 25–30 g) subjected to chronic scrotal hyperthermia (43 °C for 20 min every other day for 5 weeks, followed by 35 days of recovery), the heat treatment group demonstrated a notable decrease in sperm count and testicular mass. Meanwhile, this group presented a significant elevation in the levels of ROS, MDA, oxidized glutathione (GSSG), and apoptotic cells within testicular tissue samples. These results suggest that chronic scrotal heat treatment may impair spermatogenesis by inducing OS, consistent with the well-documented link between heat stress and oxidative damage to testicular cells. Gan et al. [60] observed that testicular atrophy in sexually mature male ICR mice (3 months, 33.83 ± 2.19 g) subjected to scrotal heat exposure (43 °C water bath for 25 min) progressed most rapidly from days 3 to 7 post-heat stress, with the progression rate slowing from days 7 to 10. This time-dependent pattern may be associated with the spermatogenic epithelial cycle of mice, indicating that a single heat stress event may primarily damage specific phases of the spermatogenic epithelium (e.g., stages 1–4 and 10–12, as observed in rodent studies).

Radiation damage-induced model

Radiation may be divided into ionizing and non-ionizing forms of radiation (IR and non-IR) [61], with ionizing radiation being particularly capable of damaging human cells, tissues, and organs. Owing to their elevated proliferative capacity, the testes rank among the body’s most radiation-sensitive organs [61]. Various studies have indicated that radiation imposes adverse effects on spermatogenesis, sperm quality indices, male sex hormones, testicular tissue structural integrity, and testicular antioxidant defense systems. These detrimental effects are mediated by reduced antioxidant capacity, induced OS, triggered inflammation, and promoted apoptosis—all of which collectively contribute to testicular damage [62]. Radiation-induced injury models can be divided into two types: whole-body irradiation models and localized testicular irradiation models. While whole-body X-ray irradiation may exert negative impacts on multiple systems in model animals [63], targeted testicular irradiation directly mirrors radiation-induced toxic impacts on the reproductive system [64]. Although high dose rate (HDR) radiation during whole-body exposure typically causes more severe damage to multiple organs, Bae et al. [65] observed an opposite trend in testicular tissue in adolescent male Balb/c mice (4–5 weeks, 20–25 g): low dose rate radiation caused more pronounced harm to testicular tissue and spermatogenic processes than high dose rate radiation exposure. This finding holds significant implications for radiation protection, particularly in scenarios such as medical occupational exposure and environmental low-dose radiation.

Varicocele-induced model

Research findings suggest that varicocele (VC) is linked to the compromise of multiple biological pathways within testicular tissue, encompassing heat stress, OS, inflammation, nitrosative stress, and endocrine disruption [66]. In VC animal model research, rat models are the primary choice for simulating VC and its pathological effects [67]. Currently, the most common method involves inducing VC in rats via partial ligation of the left renal vein [68]. Research shows that in sexually mature male SD rats (7 weeks, 200–230 g) with experimentally induced varicocele via partial ligation of the left renal vein, the VC model rats exhibit thinning and atrophy of testicular epithelial cells, disorganized arrangement of germ cells, a reduction in the average diameter of seminiferous tubules, and lower Johnsen scores [69]. In addition to partial ligation of the left renal vein, complete ligation of the common iliac vein’s spermatic vein branch via surgical microscopy is capable of notably enhancing the success rate and stability of the VC model in rats [67]. Beyond elucidating the exact pathophysiological mechanisms of VC, VC animal models also serve as optimal platforms for developing new non-surgical and minimally invasive treatment strategies [67].

Comparative analysis of MI models

The selection of appropriate animal models is crucial for elucidating the pathogenic mechanisms of male infertility and evaluating potential therapeutic interventions (Table 1). Based on the inducing factors, chemically induced, endocrine-disrupting agent-induced, and physically induced models each exhibit distinct advantages and limitations, and their suitability varies depending on the specific research objectives. Currently, chemically induced and endocrine-disrupting agent-induced models are the most widely used. Chemically induced models offer the advantages of mature technology, low cost, short modeling cycles, and extensive literature support for their reproducibility and stability, making them irreplaceable for high-throughput drug screening and preliminary toxicity evaluation. However, their drawbacks are also notable: the sources, dosages, and administration protocols of modeling agents have not been standardized, limiting comparability between studies; moreover, non-specific toxicity to systemic organs such as the liver and kidneys may interfere with the pharmacokinetics of test compounds, thereby reducing the accuracy and reliability of efficacy evaluations. Endocrine-disrupting agent-induced models are characterized by disrupting the HPG axis to establish MI models, accurately simulating spermatogenic disorders caused by human reproductive endocrine disturbances, and serving as an ideal platform for studying the effects of environmental endocrine disruptors and evaluating hormone-based therapeutics. However, these models typically require longer exposure periods and exhibit significant species differences, necessitating caution in clinical translation. Among physically induced models, heat stress and radiation injury models feature controlled localized reproductive damage, making them suitable for studying regenerative repair mechanisms after acute injury and short-term drug intervention effects. However, the difficulty in standardizing injury parameters and the tendency for reproductive function to recover easily limit their application in long-term efficacy evaluation. The other category is surgical models, such as those for varicocele and cryptorchidism, which have clearly defined etiologies and target organs, offering high clinical relevance and being particularly suitable for evaluating treatment strategies combining surgery with adjunctive pharmacotherapy. In summary, no single model can fully recapitulate the complex etiology of human male infertility. The choice of model should be guided by the specific research question, the required mechanistic depth, and the intended translational outcomes. When appropriate, a combination of multiple models may provide the most comprehensive insights into the multifactorial pathogenesis of male infertility.

Table 1.

Key characteristics of animal models for male infertility

Model Type	Model Name	Animal Strain	Age (weeks)	Weight (g)	Reproductive Status	Reference
Chemical	GTW-Induced	SD rat	8	260 ± 10	Sexually mature	[13]
	Gossypol Acetate-Induced	SD rat	10–12	100–130	Sexually mature	[19]
	CP-Induced	Wistar rat	6	100–110	Adolescent	[27]
	Adenine-Induced	SD rat	8	180–220	Sexually mature	[28]
	BU-Induced	Wistar rat	6–8	150	Adolescent	[37]
	ORN-Induced	SD rat	Not specified	180–200	Sexually mature	[40]
Endocrine	BPA-Induced	Wistar rat	10	200–250	Sexually mature	[43]
	5α-Reductase Inhibitor-Induced	Wistar albino rat	8–10	180–200	Sexually mature	[48]
	5α-Reductase Inhibitor)-Induced	SD rat	10–12	300–320	Sexually mature	[49]
	DEHP-Induced	NMRI mouse	4–5	24–26	Sexually mature	[53]
Physical	Heat Stress-Induced	Wistar albino rat	Not specified	120 ± 5	Sexually mature	[56]
	Heat Stress-Induced	NMRI mouse	6–8	25–30	Sexually mature	[59]
	Heat Stress-Induced	ICR mouse	3 months	33.83 ± 2.19	Sexually mature	[60]
	Radiation-Induced (low dose rate)	Balb/c mouse	4–5	20–25	Adolescent	[65]
	VC-Induced	SD rat	7	200–230	Sexually mature	[69]

Reproductive status was determined based on the age and weight reported in the original studies. “Sexually mature” indicates animals were reported as adult or at an age consistent with sexual maturity for the species/strain; “Adolescent” indicates animals were at a juvenile or peripubertal stage

Non-animal models of infertility

Cell experiments

Although animal models play a critical role in screening substances that impact male reproductive capacity, trustworthy in vitro models are equally indispensable for pinpointing cellular targets and intracellular signaling pathways that mediate chemical toxicity within the male reproductive system. Testicular Sertoli cell cultivation models are primarily divided into two classes: primary cultures of Sertoli cells and immortalized Sertoli cell lines. Primary cell cultures closely resemble the in vivo properties of target tissues, thus earning the title of the "gold standard" for in vitro models. However, they have short lifespans and cannot be passaged long-term. Immortalized cell lines are more readily available and capable of continuous division in vitro, but they gradually lose Sertoli cell-specific functions during prolonged cultivation. Thus, immortalized cells are suitable for preliminary screening experiments, while primary cultures remain the preferred choice for studying the pathological mechanisms of MI—owing to their better simulation of the in vivo functions of Sertoli cells [70]. The pathogenesis of MI involves the coordination of multiple cell types, including germ cells, Sertoli cells, and Leydig cells. Single-cell models cannot fully replicate this complex pathological process. Consequently, stem cells represent an important direction for the future development of cell models. Stem cell models do not rely solely on "cell replacement"; instead, they synergistically repair damaged testicular function through the dual mechanisms of "direct differentiation and regeneration" and "indirect microenvironment regulation," covering the core pathological aspects of MI [71].This approach provides more reliable tools for mechanistic studies and drug screening in male infertility.

Omics studies

Metabolomics, proteomics, and genomics provide crucial support for deciphering the pathological mechanisms of MI, identifying diagnostic markers, and exploring intervention targets. Metabolomics has detected a series of metabolite alterations associated with reproductive dysfunction in samples such as semen, plasma, and urine, offering new perspectives for the early diagnosis of MI and novel targets for disease treatment [72]. In rats with non-obstructive azoospermia (NOA), elevated levels of dihydroceramides (DhCers) and dihydrosphingosine (DhSph) were detected in urine. Research indicates that sphingolipid metabolism disorders are linked to sperm membrane dysfunction and structural alterations during epididymal sperm maturation, primarily affecting sperm motility [73]. Proteomics focuses on changes in protein expression profiles in testicular and epididymal tissues, sperm cells, and seminal plasma. Barrachina et al. [74] discovered that epididymis-derived proteins crucial for sperm maturation (such as SLC27A2 and EDDM3B) exhibit abnormalities in epididymal epithelial cells (e.g., downregulation, disrupted regional distribution) or impaired transport to sperm via epididymosomes (e.g., defective epididymosome formation, reduced binding capacity to sperm). These disruptions may prevent sperm from acquiring proteins essential for maturation, leading to insufficient motility, loss of fertilization capacity, and ultimately infertility. This approach addresses the limitations of conventional semen analysis (e.g., assessment of sperm motility and morphology) at the molecular mechanism level, advancing the diagnosis of male infertility from phenotypic evaluation to molecular mechanism characterization. Within the realm of genomics, genetic variations linked to infertility susceptibility have been detected via genomic screening conducted on infertile populations and animal experimental models. Gene-edited mouse models have confirmed that mutations in genes such as SYCP3 (chr12q23.1) [75] and TEX11 (Xq13.1) [76] can directly lead to meiotic arrest. Gene technology facilitates the screening of genetic targets and provides a potential avenue for treating male infertility [77].Current research is advancing toward establishing comprehensive animal model evaluation systems, developing integrated multi-omics analytical approaches, and constructing large-scale cohorts. Through enhanced interdisciplinary collaboration and the translation of basic research to clinical practice, these efforts are expected to achieve breakthroughs in elucidating the genetic mechanisms of male infertility and advancing its precision diagnosis and treatment [78].

Experimental techniques for infertility

Multiple experimental techniques have been described to further understand the mechanisms associated with MI (Fig. 2). To establish a heat stress model, the rat testes are immersed in a constant-temperature water bath set at 43 °C. Relevant parameters are then recorded [79]. However, some studies adopt subjective assessment methods to detect sperm motility, which may introduce observer bias. In localized testicular irradiation, the scrotal area is first secured with ligatures and then fixed using a paraffin mold. This ensures uniform distribution of the radiation dose to the target organs. Irradiation is subsequently delivered using a radiation source, with the dose predetermined based on the dose–response relationship between radiation exposure and the effects on reproductive tissues [64]. For the establishment of the VC model, male rats are subjected to anesthesia, and a midline abdominal incision is performed to reveal the left kidney, adrenal gland, renal vein, and the left spermatic vein that empties into the ipsilateral renal vein. Distal from the spermatic vein junction, the region posterior to the left renal vein undergoes blunt dissection. A silk suture is placed around this section of the left renal vein, and a rigid probe is placed parallel to the renal vein surface. The suture is tightened over the probe then secured; after removing the probe, the vein expands within the constricted area. This narrowing procedure increases venous pressure distal to the stenosis. Theoretically, this pressure is transmitted retrogradely to the spermatic vein, subsequently inducing VC [80].

Fig. 2.

Experimental techniques and evaluation of MI models

H&E staining is the most fundamental histological technique for observing the overall structure and cellular morphology of testicular tissues. The degree of histopathological damage is assessed using the Johnsen scoring system: 100 seminiferous tubules are randomly selected and scored on a scale of 0 to 10 based on the number of layers and quantity of spermatogenic cells, with final results expressed as a percentage of the total score [81]. The meiotic index (MI) is a key histological parameter for evaluating testicular spermatogenic function, as it reflects the efficiency of meiotic completion. Calculation is achieved by ascertaining the proportion of round spermatids relative to primary spermatocytes [82]. Immunohistochemistry employs specific antibodies capable of binding to tissue antigens to localize and identify the distribution and expression of specific proteins (e.g., Bax, Bcl-2) [83], inflammatory factors (e.g., IL-1β, IL-6), and other pro-inflammatory mediators such as TNF-α within testicular tissue samples [84]. Acridine orange (AO) staining leverages the differential fluorescence emission properties of DNA and RNA to evaluate sperm DNA damage [85]. The TUNEL assay, originally developed to detect somatic cell DNA damage, has been adapted for sperm analysis. By detecting sperm fluorescence intensity via flow cytometry or fluorescence microscopy, it enables precise quantification of the proportion of cells with DNA damage [86]. Western blotting ascertains the expression quantities of specific proteins within testicular tissue samples, while qRT-PCR quantifies specific mRNA expression levels. Electron microscopy techniques—particularly transmission electron microscopy and scanning electron microscopy—offer higher resolution than light microscopy, making them suitable for observing ultrastructural details of testicular and epididymal tissues. Enzyme-linked immunosorbent assay (ELISA) serves as a high-sensitivity biochemical method designed for quantitatively determining specific proteins or other bioactive molecules within animal serum samples and tissue fluid specimens. In infertility research, ELISA is commonly used to measure serum testosterone levels, inflammatory factors, and OS markers [87]. Furthermore, the feasibility of gene recombination technology in animal experiments has been confirmed [88], indicating favorable potential for practical use in the future investigations pertaining to infertility research.

Research advances in mechanisms and treatments of infertility animal models

In the field of MI, molecular biology aids in clarifying the associated signaling pathways and pathogenic mechanisms (Fig. 3). Based on the literature, we summarize the common mechanisms of different types of ED models in Table  2 and provide a brief overview of the modeling characteristics and commonly used evaluation metrics.

Fig. 3.

MI-related mechanisms (Created in FigDraw.). Abbreviations: Keap 1:Kelch-like ECH-associated protein 1; Nrf2:Nuclear factor erythroid 2-related factor 2; ROS:Reactive Oxygen Species; ARE:Antioxidant Response Element; NLRP3:NOD-like receptor pyrin domain-containing protein 3; TNF-α:Tumor Necrosis Factor-alpha; SOD:Superoxide Dismutase; GPX4:Glutathione Peroxidase 4; IL-6:Interleukin-6; IL1β:Interleukin-1 beta; COX-2:Cytochrome c Oxidase Subunit 2; N-GSDMD:N-terminal Gasdermin D; GSDMD:Gasdermin D; Bax:BCL2-Associated X Protein; Cyt c:Cytochrome c; Apaf-1:Apoptotic Protease-Activating Factor 1; caspase-9:Cysteine-Asspartic Acid Protease 9; caspase-3:Cysteine-Asspartic Acid Protease 3

Table 2.

Mechanisms associated with various types of MI models

Animal models	Methods and characteristics	Related mechanisms	References
Chemical factor-induced models	Tripterygium Glycosides-Induced Model	1.Promotion of Apoptosis: Downregulation of the PI3K/Akt signaling pathway disrupts the Bcl-2/Bax ratio, leading to germ cell apoptosis 2.Disruption of the HPG Axis: Reduces serum levels of LH, FSH, and testosterone	[10–15]
	Gossypol Acetate-Induced Model	1.Induction of OS: Promotes the generation of ROS and lipid peroxides, impairing sperm plasma membrane permeability, ATPase activity, and glucose transport 2.Inflammatory response induction: Activates pro-inflammatory factor release and suppresses anti-inflammatory factors, damaging spermatogenesis and sperm function 3.Induction of Leydig cell degeneration, reducing testosterone synthesis	[16–19]
	Cyclophosphamide-induced model	1.Induction of OS: Decreases antioxidant enzyme activity, increases oxidative damage products, and disrupts the testicular oxidative balance 2.Promotion of Apoptosis: Alters the Bcl-2/Bax ratio, activates the mitochondrial apoptosis pathway, and impairs spermatogonial stem cell proliferation and differentiation 3.Activation of Inflammatory Response: Activates the NLRP3/NF-κB axis, promotes pro-inflammatory factor release, and damages germ cell genetic material	[20–27]
	Adenine-induced model	1.Induction of OS: Mediates excessive free radical generation, surpassing testicular antioxidant capacity and causing testicular tissue damage 2.Disruption of the HPG axis: Downregulates reproduction-related genes, leading to endocrine disruption and steroid synthesis inhibition	[28]
	Busulfan-induced model	1.Induction of OS 2.Apoptosis 3.Ferroptosis, Autophagy	[29–37]
	Ornidazole-induced model	1.Induction of epididymal OS: Increases ROS generation, triggers lipid peroxidation, and impairs sperm membrane structure 2.Inhibition of sperm maturation: Disrupts sperm structural integrity, reducing sperm motility and fertilizing capacity	[38–40]
Endocrine factor-induced model	Bisphenol A-induced model	1.Disruption of the HPG axis: Mimics estrogenic effects, disrupts hormonal homeostasis, and diminishes the regulatory effects of LH/FSH on the testes 2.Impairment of Leydig cells: Suppresses their proliferation, disrupts steroidogenic pathways, and reduces testosterone production 3.Induction of OS and mitochondrial dysfunction: Disturbs cellular redox balance, adversely affecting testicular development and spermatogenesis	[41–45]
	5α-reductase inhibitor-induced model	1.OS: Increases testicular and seminal ROS, decreases antioxidant enzyme activity, and damages spermatogenic cells 2.Promotion of apoptosis: Activates endoplasmic reticulum stress and apoptotic pathways, increasing spermatogenic cell apoptosis 3.Hormonal imbalance: DHT deficiency leads to abnormalities in the spermatogenic microenvironment, inhibiting spermatogenic cell proliferation and reducing fertility	[46–49]
	Phthalate-induced model	1.OS: Metabolites disrupt the redox enzyme-antioxidant system homeostasis, increase ROS, and damage seminiferous tubule structure 2.Cellular damage: Causes spermatocyte dysfunction and Sertoli cell injury, reducing sperm production 3.Feedback activation of the HPG axis: Decreased testosterone triggers compensatory rises in LH and FSH, yet fails to reverse spermatogenic impairment	[50–53]
Physical factor-induced model	Heat stress-induced injury model	1.Induction of OS: Hyperthermia increases ROS generation, disrupts testicular oxidative balance, and impairs spermatogenesis 2.Promotion of germ cell apoptosis: Upregulates pro-apoptotic molecules, downregulates anti-apoptotic molecules, and activates the mitochondrial apoptotic pathway 3.Suppression of the HPG axis: Reduces gonadotropin and sex hormone levels, inhibiting spermatogenesis 4.Damage to the spermatogenic epithelial cycle: Disrupts the rhythm of the seminiferous epithelium, leading to spermatogenic arrest	[54–60]
	Radiation-induced injury model	1.OS: Decreases antioxidant enzyme activity, increases ROS, and damages germ cells and tissues 2.Inflammatory activation: Stimulates the release of inflammatory factors, exacerbating testicular damage 3.Apoptosis: Induces germ cell apoptosis and depletes the spermatogonial stem cell pool	[61–65]
	Varicocele-induced model	1.OS: Excessive ROS generation damages germ cells 2.Inflammatory activation: Release of pro-inflammatory factors disrupts the spermatogenic microenvironment 3.Endocrine disruption: Interferes with hormone synthesis and regulation, further aggravating spermatogenic impairment	[66–69]

Oxidative stress

OS is a critical factor contributing to sperm dysfunction [89, 90]. Spermatozoa are highly susceptible to OS owing to their distinctive features, including elevated polyunsaturated fatty acid (PUFA) levels in their cell membranes, restricted cytoplasmic volume, and inadequate antioxidant defense mechanisms. Furthermore, their DNA repair ability is notably weaker compared to somatic cells, which further enhances their vulnerability to oxidative harm [91–93]. Elevated ROS can trigger Nrf2 activation, and this transcription factor assists cells in lowering ROS levels to counteract OS by inducing the expression of antioxidant genes. Furthermore, Nrf2 is also capable of blocking the activation of the NF-κB/NLRP3 axis inflammasome through the inhibition of NF-κB activation [23]. Nrf2-driven gene transcription reduces the expression of NLRP3 inflammasome components (caspase-1, IL-1β, and IL-18), thus modulating inflammatory reactions [94]. ROS can also trigger signaling cascades like the NF-κB pathway [95], which initiates cellular inflammatory responses and promotes apoptosis [96].

Signal pathway

Nrf2 serves as a core regulator of various antioxidant enzymes, possessing the capacity to regulate cellular redox equilibrium and detect OS conditions [97].It also exerts effects in anti-inflammatory reactions, anti-apoptotic pathways, and sustaining cellular redox homeostasis [98, 99]. Under physiological conditions, Nrf2 remains bound to its cytoplasmic inhibitory protein Keap-1, staying in an inactive state. When exposed to ROS, Keap-1 undergoes oxidation or covalent modification and dissociates from Nrf2. The free Nrf2 subsequently translocates into the cell nucleus, binds to the Antioxidant Response Element (ARE) [100], induces the expression of diverse antioxidant enzymes, and promotes the synthesis of SOD. This cascade regulates LPO levels, thereby reducing LPO accumulation in sperm, enhancing cellular antioxidant capacity, and alleviating reproductive damage [101, 102]. In the development of drug targets, Nrf2 activators have become a key focus. For instance, sulforaphane (SFN) binds to Keap1 to alleviate its inhibitory action on Nrf2, facilitating Nrf2 nuclear translocation and increasing the expression of antioxidant genes. Animal studies have confirmed that SFN can ameliorate cyclophosphamide-induced spermatogenic damage in male mice. Additionally, novel Nrf2 modulators (such as bardoxolone methyl derivatives) can enhance Nrf2 stability while improving testicular OS and reducing systemic side effects, demonstrating potential for clinical application.

Ferroptosis is a type of cellular demise regulated by iron metabolic processes and lipid peroxidation, and it is intimately linked to the pathogenesis of MI and impaired spermatogenesis. Research findings suggest that ROS accumulation serves as the main contributor to ferroptosis-related MI. Reduced expression of heat shock factor 1 (HSF1), kelch domain-containing protein 3 (KLHDC3), and GPX4 might be associated with ferroptosis occurring in NOA. GPX4, a selenium-dependent antioxidant enzyme, is essential for counteracting lipid peroxides and fulfills a critical function in inhibiting ferroptosis [103]. Nrf2 has been recognized as a core regulator of the antioxidant defense system, capable of activating GPX4 to mitigate OS. Furthermore, ferroptosis inhibitors or natural extracts are able to alleviate MI through ferroptosis suppression, underscoring the significance of ferroptosis targeting as a promising therapeutic strategy for male infertility.

Inflammation

OS and inflammation are closely interconnected [94].This complex converts pro-caspase-1 into active caspase-1; the latter then processes pro-IL-1β and pro-IL-18 into their biologically active forms (IL-1β and IL-18), thereby amplifying inflammatory responses and promoting excessive OS. This cascade ultimately causes sperm DNA damage and impairs sperm maturation, quality, and function [104, 105]. Elevated levels of ROS in semen have been demonstrated to induce inflammatory responses [106]. Studies have identified spermatocytes and spermatids as the primary target cells of inflammatory damage. Inflammation can reduce the lipid content of the sperm flagellar membrane, increasing membrane rigidity and decreasing sperm motility—these changes further lead to sperm agglutination and asthenospermia. Additionally, inflammation can terminate spermatogenesis and inhibit sperm maturation [107]. NF-κB is a key transcription factor that regulates inflammatory responses by controlling the expression of cytokines such as TNF-α, IL-6, and cyclooxygenase-2 (COX-2) [94]. Research by Kaya et al. [108] confirmed that NF-κB acts as the primary mediator of the link between inflammatory cytokines and sperm parameters. Excessive ROS production activates NF-κB and upregulates the expression of the NLRP3 inflammasome. The NLRP3 inflammasome serves as a key regulatory pathway for coordinating inflammatory responses in organisms [109] —this structure constitutes a multisubunit protein assembly assembled from NLRP3 and an apoptosis-linked speck-like protein with a caspase recruitment domain (ASC). In cells, it exerts a pivotal effect by identifying pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) that appear during infection or cellular stress responses [110]. The complex mediates the conversion of pro-caspase-1 to active caspase-1, and this active protease proceeds to process pro-IL-1β and pro-IL-18 into their biologically active counterparts (IL-1β and IL-18), ultimately augmenting inflammatory responses and promoting excessive OS. This cascade ultimately causes sperm DNA damage, which in turn negatively affects sperm maturation, inherent quality, and normal function [111, 112].

Pyroptosis is a pro-inflammatory modality of regulated cell death mediated by proteins of the gasdermin family (e.g., gasdermin D (GSDMD)). This process is defined by plasma membrane pore generation, cytokine secretion (e.g., IL-1β), and osmotic lysis, while fulfilling a dual function in testicular immune defense and pathological inflammatory responses. Both clinical and preclinical investigations demonstrate that pyroptosis serves as a signature of spermatogenic impairment [113]. Under baseline physiological states, pyroptosis facilitates the elimination of infected or damaged cells, thereby preserving immune equilibrium [114]. However, aberrant activation of the NLRP3 inflammasome, driven by OS or mitochondrial dysfunction-impairs spermatogenic processes via proteolytic processing of caspase-1. These proteases enzymes process GSDMD to form membrane pores, which then facilitate the release of pro-inflammatory cytokines (IL-1β, IL-18) and cellular debris [115, 116]. Research demonstrates that the NLRP3/GSDMD axis constitutes the central cascade in testicular damage driven by pyroptosis, with caspase induction and mitochondrial impairment serving as primary drivers. Of note, pyroptosis has come to serve as a critical hub linking inflammation, OS, and environmental toxins to MI [113]. Targeting pyroptosis pathways holds therapeutic potential: this strategy has shown efficacy in preclinical studies, though it still requires clinical validation. Future research should focus on two key areas: engineering cell-selective delivery platforms for pyroptosis inhibitors, and investigating combinatorial therapeutic regimens that target both OS and mitochondrial integrity.

Apoptosis

In MI, semen OS, sperm DNA impairment, and apoptotic processes are mutually intertwined, constituting a closely integrated pathogenic molecular pathway [117]. OS not only perturbs intracellular homeostatic balance but also triggers cell death in spermatogenic cells [118, 119]. Notably, DNA impairment driven by OS initiates programmed cell death (PCD)—a process also termed apoptosis—which serves a key function in normal spermatogenesis [120]. This process sustains cellular equilibrium by clearing surplus germ cells. This ensures the germ cell population matches the supportive capacity of Sertoli cells, thereby maintaining an appropriate number of germ cells [121]. Apoptosis occurs via two pathways: the extrinsic (death receptor) pathway and the intrinsic (mitochondrial) pathway. These two routes exhibit cross-talk and mutual regulation [122], with the mitochondria-driven intrinsic pathway acting as the primary apoptotic signaling cascade in male germ cells. Bcl-2 and Bax act as core regulatory factors in mitochondria-driven apoptotic processes [123, 124], and their expression is modulated by inflammatory cytokines and OS. The ratio of these two proteins is a fundamental determinant of cell fate. Bcl-2—an anti-apoptotic protein—blocks caspase activation through two mechanisms: either by suppressing the release of cytochrome c (CytC) and apoptosis-inducing factor from mitochondria to the cytosolic compartment, or by sequestering caspases. In contrast, the pro-apoptotic protein Bax is typically present in both the cytoplasm and mitochondria [125]. Notably, elevated expression of proteins belonging to the Bcl-2 family enhances cellular survival, whereas Bax overexpression induces cellular demise.

Apoptosis of testicular cells is governed by a range of factors and genes; among these, the Caspase pathway serves a vital function in regulating Leydig cell apoptotic events [126]. Members of the Caspase family reside in cells as inert zymogens, and may be activated through mitochondria-dependent pathways [127]. Once activated, the Caspase pathway triggers irreversible apoptotic processes. As a core protein within the mitochondrial pathway, Caspase-9 is triggered by CytC—its activation is especially vital for launching the full intrinsic apoptotic cascade [128]. When CytC is released from mitochondria, it associates with apoptotic protease-activating factor-1 (Apaf-1) and activates Apaf-1 under ATP-sufficient conditions. The activated Apaf-1 then binds to pro-Caspase-9 and activates it through proteolytic cleavage. Subsequently, this active Caspase-9 cleaves pro-Caspase-3 into functional Caspase-3 tetramers, ultimately prompting apoptotic cell death [129]. Bax can trigger the release of mitochondrial CytC, whereas Bcl-2 suppresses this CytC release from mitochondria as well as the activation of the caspase-3 signaling cascade.

Exosomes are lipid bilayer vesicles (30–120 nm in diameter) that have shown therapeutic benefits in managing diverse diseases [130, 131]. These vesicles carry crucial components from their parent cells, including proteins, lipids, mRNA, DNA, and non-coding RNAs [132–135]. This enables them to regulate intercellular communication and participate in physiological and pathological processes such as cell proliferation, migration, apoptosis, RNA transport, immune responses, and tissue repair [136–140]. Studies indicate that exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs) boost the proliferation and motility of GC-1 spermatogonial cells, inhibit ROS production and cell apoptosis, and exert a key function in repairing damaged tissues [141]. The DNA and RNAs (e.g., mRNA, microRNA, lncRNA, circRNA) sequestered in such exosomes can regulate a diverse array of cellular signaling cascades, such as those associated with cell proliferation, apoptosis, angiogenesis, and immune regulation. Through modulating the physiological status and functional properties of target cells, such exosomes facilitate wound repair progression and tissue regeneration processes. A study [141] demonstrated that hUCMSC-derived exosomes not only inhibit apoptotic processes to protect the sperm population in testicular tissues but also alleviate OS and prevent spermatogonial cell damage—thereby enhancing tissue regeneration outcomes. Additional studies show these exosomes can accelerate the recovery of spermatogenic function in mouse testes, drive structural restoration of seminiferous tubules, and boost the proliferative and migratory potential of spermatogenic cells. Overall, exosomes originating from human umbilical cord mesenchymal stem cells provide a novel strategy for addressing traumatic lesions and degenerative disorders affecting the male reproductive tract.

Steroids

In mammals, spermatogenesis is tightly regulated by multiple hormones, including gonadotropin-releasing hormone, gonadotropins, and gonadal steroids [56]. Disruptions in steroid hormone metabolism directly impair the hormonal microenvironment required for spermatogenesis. Testosterone, the principal steroid hormone in the testes, serves a pivotal function in male reproductive system development, germ cell differentiation, the sustainment of normal sperm motility, and the facilitation of sperm development and maturation [142]. Synthesized and secreted by Leydig cells [143], approximately 95% of testosterone biosynthesis is controlled by the HPG axis. Within this system, the hypothalamus produces gonadotropin-releasing hormone (GnRH), which induces the pituitary gland to release FSH and LH. FSH acts on the seminiferous tubules of the testes, promoting the proliferation and division of spermatogenic cells; LH exerts a regulatory effect on Leydig cells to trigger T secretion. These two hormones work synergistically to drive spermatogenesis [144]. Reduced levels of LH and FSH might result in developmental abnormalities in Leydig cells while compromising spermatogenesis. Additionally, FSH can bind with high specificity to follicle-stimulating hormone receptor (FSHR) on Sertoli cells, thereby promoting Sertoli cell proliferation and supporting spermatogenesis [145]. Studies indicate that OS can disrupt the HPG axis through testicular tissue damage, thus diminishing the concentrations of FSH, LH, and T [146, 147].

Recent research has demonstrated that hydrogen sulfide (H₂S) serves a pivotal function in testosterone biosynthesis—a role governed by LH secretion [148]. In vivo experiments indicate that H₂S facilitates steroidogenesis within Leydig cells, while also upregulating the expression of genes linked to testicular testosterone biosynthesis (e.g., steroidogenic acute regulatory protein (StAR), cytochrome P450 17A1 (p450c17), 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 side-chain cleavage enzyme (P450scc)) [149]. Moreover, H₂S exerts antioxidant properties by boosting the activity levels of SOD and glutathione peroxidase (GSH-Px) [150]. Future research may identify specific protein targets of persulfidation; a deeper understanding of this mechanism will be crucial for pinpointing key therapeutic targets and developing more precise, effective drugs.

Summary

A variety of animal models have been used in infertility research, each with distinct characteristics in terms of effectiveness and limitations. Stable and reproducible animal models of infertility are essential prerequisites for drug screening and evaluation. On one hand, these models allow researchers to directly observe the therapeutic effects of drugs via administration routes such as injection or oral gavage. This approach not only visually demonstrates improvements in key sperm parameters (e.g., count, motility, and morphology) but also enables monitoring of testicular histopathological repair and changes in serum hormone levels (including T, FSH, and LH). During this process, multiple technical methods can be used for auxiliary assessment: observing seminiferous tubule structure via H&E staining, detecting germ cell apoptosis through TUNEL assay, and quantitatively analyzing inflammatory factors and OS markers using ELISA. These methods provide a preliminary evaluation of drug efficacy for MI. Building on this foundation, researchers further use molecular and cellular biology techniques to investigate the mechanisms of drug action in depth. These approaches not only facilitate in-depth exploration of the pathogenesis of different types of infertility but also enable preliminary drug screening based on relevant molecular targets, thereby guiding subsequent research directions. Meanwhile, long-term and short-term toxicity studies based on these models allow monitoring of drug effects on animal body weight, vital organ function, and blood biochemical parameters—thus enabling comprehensive assessment of drug safety.Additionally, non-animal models contribute to predicting MI etiology and exploring both well-established and novel molecular mechanisms. Advances in various experimental techniques have provided important support for the discovery and validation of drug mechanisms of action.

OS, inflammation, apoptosis, and steroid hormone metabolic disorders are core pathological mechanisms in male infertility, and these mechanisms can interact synergistically. Currently, therapeutic targets for MI primarily focus on OS and steroid hormone metabolic disorders, while research on novel cell death mechanisms (e.g., ferroptosis and pyroptosis) is still in its early stages. Further clarification is needed regarding three key aspects: the differential roles of the NLRP3/GSDMD axis in infertility of varying etiologies; the specific molecular mechanisms by which GPX4 regulates ferroptosis; and the precise protein substrates and regulatory networks involved in H₂S persulfidation. More precise mechanistic insights will enhance the druggability of these targets. Concurrently, efforts should be made to strengthen translational research on target intervention strategies, including optimizing the delivery routes and targeted delivery systems for candidate drugs (e.g., Nrf2 activators and pyroptosis inhibitors). Exploring the feasibility of using stem cell exosomes as drug carriers or direct therapeutic agents will promote their transition from basic research to clinical applications, providing more solid theoretical and experimental foundations for achieving personalized and precise treatment of MI.

Authors’ contributions

Shengnan Li: Writing-original draft. Boxian Gao: Writing-original draft. Tengfei Chen: Writing -review and editing. Hongyan Chen: Writing-review and editing. Chongfu Zhong: Writing-review and editing.

Funding

General Program of the National Natural Science Foundation of China (NSFC), Shandong Key Laboratory of Traditional Chinese Medicine Efficacy and Mechanism.

Grant/Award Number:82374455,PKL2024C23.

Data availability

No data was used for the research described in the article.

Declarations

Ethics approval and consent to participate

None.

Competing interests

The authors declare no competing interests.