Unique Physiological Mechanisms of Sperm Storage and Prolonged Sperm Survival in Hen Oviducts: A Review

Abstract

Poultry sperm survive for extended periods within sperm storage tubules (SSTs) located at the uterovaginal junction (UVJ) of the oviduct, and their survival period is directly correlated with fertility. In this review, the mechanisms underlying sperm longevity in SSTs are discussed, with a focus on recent discoveries related to the functions of SSTs and UVJ tissue. In particular, the possible role of fatty acids and exosomes secreted by SST cells in sperm survival are discussed. Subsequently, the importance of gonadal steroid receptors in maintaining the integrity and function of the SSTs is described. Additionally, the role of the local immune system in protecting sperm from infection and facilitating the selection of high-quality sperm is discussed. Disruption of these SST functions may result in reduced fertility. This review provides updated information on the mechanisms that enable prolonged sperm survival in SSTs.

Introduction

In laying hens, reproductive organs, such as the ovary and oviduct, develop exclusively on the left side of the abdominal cavity. The oviduct consists of five segments arranged from the cephalic to caudal ends: the infundibulum, magnum, isthmus, uterus, and vagina. The outer perivitelline layer and chalaza surrounding the ovum are formed in the infundibulum, whereas the albumen layer, eggshell membrane, and eggshell are formed in the magnum, isthmus, and uterus, respectively. The vagina opens into the cloaca along with the urinary duct and colorectum. The uterovaginal junction (UVJ) is located between the uterus and vagina. When hens are naturally copulated or artificially inseminated, sperm ascend the oviduct and encounter the ovum in the infundibulum, where fertilization occurs.

Poultry hens exhibit a unique reproductive trait in which sperm survive in the oviduct for a prolonged period. The duration of fertile egg laying following a single copulation or insemination reflects the length of time that sperm remain viable in the oviduct. The reported duration of fertile egg laying is 2–3 weeks in chickens, 10–15 weeks in turkeys, and 1–2 weeks in Japanese quail[1]. The duration of sperm survival in the oviduct differs among animal species. Some reptiles and fishes store fertile sperm for more than 1–2 years, whereas most mammals store sperm for shorter periods (2–5 days in many species including humans, but up to 6 months in bats)[2,3].

Preservation of sperm in the oviduct may be beneficial for the reproduction of animals with limited mating opportunities. Sperm have been identified in the infundibulum and UVJ of hens; however, the primary site of sperm storage in the oviduct is the sperm storage tubules (SSTs) in the UVJ[4,5]. It is hypothesized that good quality sperm are selected in the vagina, and only approximately 1% of sperm enter the SSTs in chickens[6]. This process may involve the exclusion of sperm from the vagina by ciliary movement that transports debris toward the cloaca, as well as an immune response to sperm[7]. Since the expression of inflammatory cytokines increases in the vagina after insemination, the immune response by inflammatory cytokines may also be one of the factors responsible for reducing and eliminating low-quality sperm in the vagina[8].

Although the mechanism by which sperm survive for a prolonged period in SSTs has not yet been established, understanding this process is important to improve fertility. SSTs are formed by the invagination of the UVJ surface epithelium; the type of epithelium differs between the UVJ and SSTs[7]. The UVJ mucosal surface is lined with ciliated pseudostratified epithelium, similar to other segments of the oviduct. In contrast, SSTs are lined with a single layer of cuboidal cells with developed microvilli and rich in lipid droplets and lysosomes[7,9]. However, the roles of these cellular characteristics in sperm survival remain unclear.

During storage in SSTs, sperm motility may be suppressed to avoid energy loss in physiological and high-calcium environments. Although numerous studies have been conducted, the factors involved in sperm survival within SSTs have not yet been explored. Current studies are using modern biological techniques, including transcriptome analysis, to investigate this issue[10,11,12].

Sperm formed in the testis are transported through the male genital tract toward the cloaca and after natural copulation or artificial insemination, they travel through the vagina to enter SSTs in the oviduct. During travel through the male and female genital tracts, sperm are exposed to non-sterile environments. Thus, they rely on the immune defenses of genital organs and sperm to defend themselves against infectious pathogens. Furthermore, sperm must evade the female anti-sperm immune response to survive in the genital tract and SSTs. Reports show that repeated insemination causes destruction of SSTs, an increase in lymphocytes in the UVJ, and a decline in fertility[13,14]. A recent study reported the expression of anti-inflammatory cytokines, particularly transforming growth factor (TGF)-β, which regulate the immune response in the UVJ during sperm storage in healthy hens[15].

In this minireview, the mechanisms that enable sperm survival in SSTs for prolonged periods in hens are discussed. Because the duration of sperm survival in the oviduct is closely associated with fertility, understanding these mechanisms is essential to improve fertility and reproductive performance. Specifically, factors that may enable sperm to survive for a prolonged period in SSTs, immunological events, and their regulation in relation to sperm survival are discussed.

Possible factors that may enable sperm to survive for a prolonged period in SSTs

The lipid and fatty acid composition of sperm correlate with sperm motility in chickens[16]. SST cells are rich in lipid droplets; thus, sperm function may be affected by lipid components or fatty acids derived from these cells. Unsaturated fatty acids in the lipid fractions of spermatozoa play a role in maintaining the high membrane fluidity and flexibility required for sperm motility and oocyte fusion[17]. Various cells secrete exosomes that may contain and transfer substances, such as proteins, nucleic acids, and fatty acids to other cells to regulate cellular functions[18,19,20,21]. Thus, it is possible that exosomes secreted by SST cells regulate sperm viability.

Lipases and fatty acids in SSTs

Histochemical studies show that SST cells in the UVJ contain lipid droplets[22,23] and exhibit acid phosphatase activity[24]. Brady et al.[11] report that sperm metabolism is quiescent during in vivo storage; however, it has also been hypothesized that sperm metabolize fatty acids derived from SST cells to perform basic cell functions.

We have previously reported the expression of genes encoding endothelial lipase, lipase H, adipose triglyceride lipase (ATGL), and lipoprotein lipase in the UVJ mucosa; however, only ATGL is observed in SST cells isolated by laser microdissection[25]. ATGL expression in SST cells was significantly upregulated after artificial insemination, suggesting that ATGL activity increased in response to the presence of sperm in the SST. Five fatty acids, myristic (C14), palmitic (C16), stearic (C18), oleic (C18:1n9), and linoleic acid (C18:2n6), have been identified in UVJ mucosal tissues containing SSTs, and the viability of cultured sperm is improved by adding 1 mM oleic acid or linoleic acid[25]. Based on these studies, we hypothesize that lipids in SST cells may be degraded by ATGL, and that fatty acids, including oleic and linoleic acid, may be released into the SST lumen to support sperm survival.

Possible role of SST exosomes in sperm survival

Various cells, including epithelial cells in the urinary duct[26] and intestine[27], and B cells[28], secrete vesicles of different sizes into the extracellular space. Depending on their origin or presumed function, these vesicles are termed exosomes, microvesicles, or apoptotic blebs[29,30]. They may carry and transfer substances that regulate cellular functions, such as active enzymes[18], nucleic acids, and fatty acids[19,20,21].

Gilbert et al.[24] have demonstrated acid phosphatase activity in the apical cytoplasm of SST cells. Because acid phosphatase activity is often associated with the presence of lysosomes in the cytoplasm, higher levels of activity in SST cells suggest that these cells are rich in lysosomes. Electron microscopy has revealed the presence of lysosomes in the apical region of some SST cells[23]. Lysosomes play a role in regulating exosome biogenesis from late endosomes (multivesicular bodies) and exosome release depends on lysosomal exocytosis[31,32,33]. Thus, SST cells may actively synthesize exosomes because of the inclusion of lysosomes in their cytoplasm.

Bakst and Bauchen[34] report that microvillus blebs (MvBs) containing lipid material are shed from SST cells and observed on the apical tips of SSTs where they interact with resident sperm. The authors suggested that these MvBs serve as a lipid delivery system for the basic cellular metabolism of sperm. These MvBs fuse with the plasmalemma of sperm residing in the SST lumen, suggesting that MvB content may augment sperm survival in the SST.

In the mammalian male reproductive system, Arienti et al.[35] report that prostasomes, which are membranous vesicles approximately 150–200 nm in diameter, are secreted by the prostate glands into human semen. The authors demonstrated that lipids were transferred from prostasomes to the sperm membrane.

Huang et al.[36] have identified CD63-positive exosome-like substances in SST cells and the UVJ mucosal epithelium. Therefore, CD63 may be used as an exosomal marker. The CD63 protein tended to appear in the SST lumen, where inseminated sperm were stored, suggesting that exosomes were secreted by SST cells into the lumen during sperm storage. Huang et al.[36] have isolated exosomes from the culture media of the vaginal mucosa and UVJ by ultracentrifugation. The viability of sperm incubated with vaginal exosomes significantly decreased, whereas that of sperm incubated with UVJ exosomes was unaffected. Exosomes carry multiple active enzymes[37], mRNAs, miRNAs[38], and free fatty acids[39]. Exosomes also deliver specific combinations of signals to target cells and influence normal and pathological processes[40]. Exosomes then bind to target cells via fusion and influence host cells to perform certain functions. Exosomes from vaginal cells are thought to produce substances that reduce sperm viability. However, exosomes from UVJ cells, including SST cells, are not harmful to sperm, but may transfer substances, such as fatty acids derived from SST cells, that support sperm survival. Further studies are necessary to determine whether substances, such as fatty acids, delivered by UVJ exosomes support sperm survival.

Progesterone and estrogen receptor expression in SST cells

Gonadal steroids are the primary factors that regulate the growth, differentiation, and function of oviductal cells. Therefore, the production and activity of these hormones must be tightly regulated. Immunoreactive progesterone receptors (PRs) and estrogen receptor α (ERα) have been localized in the nuclei of SST cells in laying hens[41]. Injection of immature chicks with diethylstilbestrol (an estrogenic chemical) induces oviductal growth, accompanied by SST structure development in association with induction of PR and ERα expression[41]. Thus, these data suggest that estrogen participates in the development of SST structures and induction of gonadal steroid receptors. SST cells also express membrane progestin receptors[42] and nuclear PRs[41,42]. The nuclear PRs and ERα likely control the function of SST cells. However, the specific functions of the steroid receptors remain unknown. Ito et al.[42] report that progesterone stimulates the release of resident sperm from the SST in Japanese quail and that the release of sperm may occur via membrane progestin receptor-mediated signal transduction. Khillare et al.[43] report that in SSTs, genes encoding sex steroid receptors (PR, ERα and ERβ) and gonadotropin receptors (luteinizing hormone receptor and follicle-stimulating hormone receptor) are expressed, with expression peaking at weeks 5 and 6, respectively. The authors suggested that gonadotropin receptors might also play a role in the development of SSTs in the UVJ during sexual maturation. However, studies confirming the role of gonadotropins in SST development have not yet been conducted.

Frequently repeated artificial insemination may cause destruction and abnormalities in SST function, and an anti-sperm immune response may occur in these SSTs. In these birds, sperm cannot be retained within SSTs, resulting in a decline in fertility[40]. The expression of ERα mRNA in the UVJ is significantly decreased after frequently repeated artificial insemination. The decreased estrogen stimuli in SST cells in the UVJ due to ERα reduction may lead to destruction of SSTs, which may cause reduced fertility in these birds[44]. Thus, a reduction in steroid hormone activity, including receptor expression, may be involved in fertility decline.

Immune defense of sperm against infection

Defense against pathogenic agents in reproductive organs and gametes is essential for normal reproductive activities. Immune cells associated with the adaptive and innate immune systems are localized in the male chicken reproductive tract[45]. Within the innate immune system, pattern recognition receptors, including toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, initiate immune responses. The expression of cytokines and antimicrobial peptides is a key feature of the innate immune response[46]. Das et al.[47] report the expression of TLRs and avian β-defensins (AvBDs), an antimicrobial peptide, in rooster sperm. The authors identified the polymerase chain reaction products of 7 TLRs among 8 TLRs in ejaculated sperm and showed the expression of 9 out of 14 AvBDs. The expression of several AvBDs was upregulated in sperm exposed to lipopolysaccharide (LPS; a TLR4 ligand) and Pam3CSK4 (a synthetic triacylated lipopeptide; a TLR2 ligand), suggesting that chicken sperm have an innate defense mechanism against infection. This innate immune response may contribute to sperm protection in the male and female genital tracts. In a study focusing on AvBD3 in chicken sperm, immunoreactive AvBD3 molecules have been identified in elongated spermatids and sperm in the seminiferous tubules, as well as in ejaculated sperm[48]. The authors identified mRNA expression of AvBD3 in the testicular tissue, but not in ejaculated sperm, suggesting that AvBD3 was synthesized by late-stage spermatids in the testes and that the protein was retained even after ejaculation. Furthermore, AvBD3 was retained in sperm stored in SSTs, suggesting that it plays a role in protecting sperm from infection and enabling survival in the oviduct. However, diversity in AvBD expression may exist among chicken strains and individuals. Further studies are necessary to examine whether other antimicrobial peptides are synthesized and retained in sperm for defense during storage in the oviduct.

Oviductal mucosal defenses affecting spermsurvival

It is hypothesized that good-quality sperm are selected for better fertility in the oviduct. The vagina is thought to be the site of sperm selection, and only 1% of the sperm are observed within the SST 24 h after intravaginal insemination in chickens[7]. After insemination, the expression of the proinflammatory cytokines including interleukin-1β and lipopolysaccharide-induced tumor necrosis factor is increased, leading to sperm degradation in the chicken vagina[8]. Another study has revealed that the localization and number of adaptive immune cells, including CD3+ T cells, immunoglobulin (Ig)A+ B cells, and IgY+ B cells, changes in response to the presence of sperm, suggesting that the adaptive immune system of the oviduct plays a role in the sperm response[49]. However, reports suggest that the immune response may be suppressed in the UVJ to ensure sperm survival in SSTs[15,50]. Atikuzzaman et al.[51] report that immune response genes are downregulated in the UVJ of mated hens, which may favor the survival of sperm in the SST. The authors also demonstrated that sperm deposition caused a shift in expression of genes involved in pH-regulatory functions in the UVJ, which may regulate the local pH to ensure sperm survival or motility. Lactic acid has been suggested to promote sperm quiescence in the SSTs of Japanese quail[52]. Das et al.[15] report that TGF-β expression is increased in the UVJ in the presence of sperm, suggesting that TGF-β may help protect sperm in the SST by suppressing the immune response. Thus, these data suggest that an immune response involving inflammatory cytokines is triggered by sperm, and only good-quality sperm are selected from the vagina to enter the SST in the UVJ. The immune response may be suppressed by the factors from UVJ tissues to ensure sperm survival in the SST.

The addition of exosomes isolated from vaginal cell cultures reduces the viability of cultured sperm[36], suggesting that exosomes participate in the fate of sperm in the vagina. However, further in vivo studies are needed to confirm this, such as ultrastructural examination showing the degradation of sperm associated with exosomes in the vagina.

Declining fertility is a major concern for poultry breeders. Despite increasing demand for both eggs and broilers, projections suggest that hatchability rates may decrease to approximately 60% by 2050 without corrective action[53]. We previously reported that in hens whose fertility was reduced by frequent artificial insemination, SSTs appeared swollen with thinning of the epithelium. Additionally, the frequency of antigen-presenting cells and CD4+ and CD8+ T cells is significantly increased in these hens[14]. Kosonsiriluk et al.[54] also report that in turkey hens that undergo artificial insemination three times in the first week and once per week until 27 weeks, the local immune response that is suppressed during peak laying (8 weeks after the first artificial insemination) increases at the late laying stage (27 weeks after the first artificial insemination) in the UVJ. Furthermore, in hens where repeated insemination leads to SST degeneration and reduced fertility, the expression of ERα, which may play a role in maintaining SST cell integrity and function, is significantly decreased in the UVJ[44]. These findings suggest that repeated insemination should be optimized to prevent immunological damage and abnormalities in SST function in association with estrogen receptor reduction, so that sperm may be retained in the SST, resulting in better fertility.

Concluding remarks

This review discussed the mechanisms by which poultry sperm survive in SSTs for prolonged periods, with a focus on recent discoveries regarding the functions of SST cells and UVJ tissues. One possible substance provided by SST cells to resident sperm for survival may be unsaturated fatty acids, including oleic and linoleic acids (Fig. 1). Exosomes secreted by SST cells may play a role in transferring substances, including fatty acids, to sperm in the SST (Fig. 1). The immune response may be suppressed in the UVJ during storage of sperm in the SST, whereas innate immune molecules, such as AvBDs, may play a role in the defense of sperm against pathogenic agents. A key factor in fertility decline may be the loss of SST function. The decline in ER expression associated with SST cell dysfunction should also be considered as a contributing factor. Future studies should address dysfunction in both SSTs and UVJ tissues, as these are important to improve fertility in poultry.

Fig. 1.

  Possible role of fatty acids and exosomes secreted by sperm storage tubule (SST) cells in sperm survival.

In SST cells, lipids are digested by lipases to produce oleic and linoleic acids. These substances may be secreted into SSTs and used by sperm for metabolism. Exosomes from vaginal cells may increase sperm death, whereas those from SST cells may not affect sperm survival. Exosomes from SST cells containing fatty acids, such as oleic and linoleic acid, may contribute to sperm survival.