Zearalenone induces oxidative stress and autophagy in goat Sertoli cells.

Zearalenone (ZEA), one of the non-steroidal estrogen mycotoxin, can cause male reproductive damage and genotoxicity in mammals. Testicular oxidative injury is an important factor causing male sterility. Testicular Sertoli cells are essential for spermatogenesis and male fertility. At present, the mechanism of oxidative injury in dairy goat Sertoli cells after exposure to ZEA remains unclear. This study explored the effects of ZEA on oxidative stress and autophagy in dairy goat Sertoli cells. It was found that treatment of primary Sertoli cells with 25, 50 and 100 µmol/L ZEA for 24 h can promote ROS production, decrease cell viability, antioxidant enzyme activity and mitochondrial membrane potential, induce caspase-dependent cell apoptosis and autophagy activity. ZEA-induced autophagy was confirmed by LC3-I/LC3-II transformation. More importantly, N-acetylcysteine (NAC) pretreatment can remarkably inhibit ZEA-induced oxidative stress, apoptosis and autophagy in Sertoli cells by eliminating ROS. In conclusion, this study indicates that ZEA induces oxidative stress and autophagy in dairy goat Sertoli cells by promoting ROS production.

FSH-inhibited autophagy protects against oxidative stress in goat Sertoli cells through p62-Nrf2 pathway.

Oxidative stress is a common cause of male infertility. Sertoli cells are one of the target cells of oxidative injury, which leads to impaired testicular function. Follicle-stimulating hormone (FSH) is critical in Sertoli cell function. However, the role of FSH in the response of goat Sertoli cells to H2O2-induced oxidative stress has not been studied yet. To investigate this response, we established an oxidative stress model using goat Sertoli cells. FSH pretreatment significantly enhanced the decreased cell viability (p < 0.05) caused by oxidative injury and inhibited autophagic flux. FSH significantly increased p62 mRNA and protein levels (p < 0.01). Further investigations revealed that FSH also increased the expression level and nuclear translocation of Nrf2 in Sertoli cells (p < 0.01), which resulted in increased antioxidant enzyme activity (p < 0.05). In contrast, treatment with siNrf2 and sip62 abolished this protective effect of FSH. These findings suggest that FSH protects Sertoli cells against oxidative stress via the p62-Nrf2 pathway, and that p62 accumulation maintains persistent activation of Nrf2. Thus, p62 and Nrf2 are required for FSH-mediated protective role in H2O2-induced Sertoli cell injury. The findings reveal new mechanisms by which FSH protects against oxidative injury in goat Sertoli cells.

Single-cell transcriptomics reveals male germ cells and Sertoli cells developmental patterns in dairy goats.

Spermatogenesis holds considerable promise for human-assisted reproduction and livestock breeding based on stem cells. It occurs in seminiferous tubules within the testis, which mainly comprise male germ cells and Sertoli cells. While the developmental progression of male germ cells and Sertoli cells has been widely reported in mice, much less is known in other large animal species, including dairy goats. In this study, we present the data of single cell RNA sequencing (scRNA-seq) for 25,373 cells from 45 (pre-puberty), 90 (puberty), and 180-day-old (post-puberty) dairy goat testes. We aimed to identify genes that are associated with key developmental events in male germ cells and Sertoli cells. We examined the development of spermatogenic cells and seminiferous tubules from 15, 30, 45, 60, 75, 90, 180, and 240-day-old buck goat testes. scRNA-seq clustering analysis of testicular cells from pre-puberty, puberty, and post-puberty goat testes revealed several cell types, including cell populations with characteristics of spermatogonia, early spermatocytes, spermatocytes, spermatids, Sertoli cells, Leydig cells, macrophages, and endothelial cells. We mapped the timeline for male germ cells development from spermatogonia to spermatids and identified gene signatures that define spermatogenic cell populations, such as AMH, SOHLH1, INHA, and ACTA2. Importantly, using immunofluorescence staining for different marker proteins (UCHL1, C-KIT, VASA, SOX9, AMH, and PCNA), we explored the proliferative activity and development of male germ cells and Sertoli cells. Moreover, we identified the expression patterns of potential key genes associated with the niche-related key pathways in male germ cells of dairy goats, including testosterone, retinoic acid, PDGF, FGF, and WNT pathways. In summary, our study systematically investigated the elaborate male germ cells and Sertoli cells developmental patterns in dairy goats that have so far remained largely unknown. This information represents a valuable resource for the establishment of goat male reproductive stem cells lines, induction of germ cell differentiation in vitro, and the exploration of sequential cell fate transition for spermatogenesis and testicular development at single-cell resolution.

Recent advances in isolation, identification, and culture of mammalian spermatogonial stem cells.

Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells (SSCs). SSCs, the only male reproductive stem cells that transmit genetic material to subsequent generations, possess an inherent self-renewal ability, which allows the maintenance of a steady stem cell pool. SSCs eventually differentiate to produce sperm. However, in an in vitro culture system, SSCs can be induced to differentiate into various types of germ cells. Rodent SSCs are well defined, and a culture system has been successfully established for them. In contrast, available information on the biomolecular markers and a culture system for livestock SSCs is limited. This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process, the biology and niche of SSCs, the isolation and culture systems of SSCs, and the biomolecular markers and identification of SSCs. This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals, enhancement of human male fertility, reproductive medicine, and protection of endangered species.

FSH inhibits autophagy and lysosomal biogenesis to regulate protein degradation in cultured goat Sertoli cells.

Although the follicle-stimulating hormone (FSH) plays a vital role in male reproduction, the molecular relationships among FSH, autophagy, and the secretory function of Sertoli cells remain largely undetermined. In this study, we sought to investigate the effects of FSH on dairy goat Sertoli cell autophagy and the role of autophagy in protein clearance. FSH treatment of primary Sertoli cells was found to enhance the expression level of LC3-II, reduce p62 degradation and the number of lysosomes, and downregulate the levels of LAMP2 protein and lysosomal gene mRNAs. Further analyses revealed that starvation-induced autophagy promotes the translocation of transcription factor EB (TFEB) from the cytoplasm to nucleus and its binding to the promoter region of LAMP2, whereas FSH suppresses the nuclear translocation of TFEB. Moreover, we found that the FSH-mediated inhibition of autophagy extends the biological half-lives of androgen-binding protein (ABP), glial-derived neurotrophic factor (GDNF), and stem cell factor (SCF) and promotes the secretion of these proteins. Collectively, these observations indicate that FSH inhibits autophagy by reducing lysosomal biogenesis, which is associated with the suppression of TFEB nuclear translocation via activation of the PI3K/Akt/mTOR pathway, thereby extending the biological half-lives and enhancing the expression of ABP, GDNF, and SCF in dairy goat Sertoli cells.

Changes in histology, protein expression, and autophagy in dairy goat testes during nonbreeding season†.

Seasonal reproduction contributes to increased chances of offspring survival in some animals. Dairy goats are seasonal breeding mammals. In this study, adult male Guanzhong dairy goats (10-12 months old) were used. Testis size, semen quality, hormone level, apoptosis of germ cells, and autophagy of Sertoli cells were analyzed in dairy goats during the breeding (October) and nonbreeding (April) seasons. We found that, during the nonbreeding season for dairy goats, semen quality, follicle-stimulating hormone (FSH) levels, and testosterone levels were reduced, and the number of apoptotic germ cells increased. The proliferation with decrease activity of germ cells in dairy goat during the nonbreeding season was significantly affected. However, the testis size did not change seasonally. Interestingly, Sertoli cell autophagy was more active during the nonbreeding season. The expression levels of FSH receptor, wilms tumor 1, androgen binding protein, glial cell derived neurotrophic factor, and stem cell factor decreased in dairy goats during the nonbreeding season. In summary, our results indicate that spermatogenesis in dairy goats during the nonbreeding season was not completely arrested. In addition, germ cell apoptosis and the morphology of Sertoli cells considerably changed in dairy goats during the nonbreeding season. Sertoli cell autophagy is involved in the seasonal regulation of spermatogenesis in dairy goats. These findings provide key insights into the fertility and spermatogenesis of seasonal breeding animals.

TLR7/8 signalling affects X-sperm motility via the GSK3 α/ß-hexokinase pathway for the efficient production of sexed dairy goat embryos.

BACKGROUND: Goat milk is very similar to human milk in terms of its abundant nutrients and ease of digestion. To derive greater economic benefit, farmers require more female offspring (does); however, the buck-to-doe offspring sex ratio is approximately 50%. At present, artificial insemination after the separation of X/Y sperm using flow cytometry is the primary means of controlling the sex of livestock offspring. However, flow cytometry has not been successfully utilised for the separation of X/Y sperm aimed at sexing control in dairy goats. RESULTS: In this study, a novel, simple goat sperm sexing technology that activates the toll-like receptor 7/8 (TLR7/8), thereby inhibiting X-sperm motility, was investigated. Our results showed that the TLR7/8 coding goat X-chromosome was expressed in approximately 50% of round spermatids in the testis and sperm, as measured from cross-sections of the epididymis and ejaculate, respectively. Importantly, TLR7/8 was located at the tail of the X-sperm. Upon TLR7/8 activation, phosphorylated forms of glycogen synthase kinase α/ß (GSK3 α/ß) and nuclear factor kappa-B (NF-κB) were detected in the X-sperm, causing reduced mitochondrial activity, ATP levels, and sperm motility. High-motility Y-sperm segregated to the upper layer and the low-motility X-sperm, to the lower layer. Following in vitro fertilisation using the TLR7/8-activated sperm from the lower layer, 80.52 ± 6.75% of the embryos were XX females. The TLR7/8-activated sperm were subsequently used for in vivo embryo production via the superovulatory response; nine embryos were collected from the uterus of two does that conceived. Eight of these were XX embryos, and one was an XY embryo. CONCLUSIONS: Our study reveals a novel TLR7/8 signalling mechanism that affects X-sperm motility via the GSK3 α/ß-hexokinase pathway; this technique could be used to facilitate the efficient production of sexed dairy goat embryos.

Rosmarinic acid improves boar sperm quality, antioxidant capacity and energy metabolism at 17°C via AMPK activation.

Boar sperm are susceptible to oxidative damage caused by reactive oxygen species (ROS) during storage. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an important therapeutic target, because it is a cellular metabolism energy sensor and key signalling kinase in spermatozoa. We evaluated the effects of rosmarinic acid (RA), an antioxidant, on boar sperm during liquid storage to determine whether it protects boar sperm via AMPK activation. Boar ejaculates were diluted with Modena extender with different concentrations of RA and stored at 17°C for 9 days. Sperm quality parameters, antioxidant capacity, energy metabolism, AMPK phosphorylation and fertility were analysed. Compared with the control, 40 µmol/L significantly improved sperm motility, plasma membrane integrity and acrosome integrity (p < .05). The effective storage time of boar sperm was up to 9 days. On the third and seventh days, the sperm with RA exhibited increased total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, adenosine triphosphate (ATP) content, mitochondrial membrane potential (ΔΨm) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, whereas malondialdehyde (MDA) content was significantly decreased (p < .05). Western blot showed that RA, as well as AICAR (AMPK activator), promoted AMPK phosphorylation, whereas Compound C (AMPK inhibitor) inhibited this effect. The sperm-zona pellucida binding experiment showed that 40 µmol/L RA increased the number of sperm attached to the zona pellucida (p < .05). These findings suggest meaningful methods for improved preservation of boar sperm in vitro and provide new insights into the mechanism by which RA protects sperm cells from oxidative damage via AMPK activation.

Platelet-derived growth factor-BB and epidermal growth factor promote dairy goat spermatogonial stem cells proliferation via Ras/ERK1/2 signaling pathway.

Spermatogonial stem cells (SSCs) have been used for the production of transgenic animals and for the recovery of male fertility. However, the proliferation of SSCs in vitro is still immature, and the mechanisms and pathways involved in the proliferation of SSCs are not clear. Here, the effects of platelet-derived growth factor-BB (PDGF-BB) and epidermal growth factor (EGF) on the proliferation of dairy goat SSCs in vitro were detected. The results showed that 20 ng/ml PDGF-BB or 25 ng/ml EGF was the optimum concentration, and that the BCL2 in the experimental groups was significantly higher than that in the control (P < 0.05), while BAX and BAD were dramatically downregulated (P < 0.05). The pERK1/2 in the experimental groups was about 3-5 times higher than that in the control. After the specific MEK1/2 inhibitor was added, BCL2 was reduced significantly (P < 0.001), while BAX and BAD were upregulated (P < 0.001). The expression of pERK1/2 decreased by 10%-30%. We speculated that these two growth factors may be mediated through the Ras/ERK1/2 signaling pathway to regulate the expression of pERK1/2 protein, and thus enhance the resistance of SSCs to apoptosis. However, further studies are needed to verify this hypothesis.

Melatonin Protects Goat Spermatogonial Stem Cells against Oxidative Damage during Cryopreservation by Improving Antioxidant Capacity and Inhibiting Mitochondrial Apoptosis Pathway.

Spermatogonial stem cells (SSCs) are the only adult stem cells that pass genes to the next generation and can be used in assisted reproductive technology and stem cell therapy. SSC cryopreservation is an important method for the preservation of immature male fertility. However, freezing increases the production of intracellular reactive oxygen species (ROS) and causes oxidative damage to SSCs. The aim of this study was to investigate the effect of melatonin on goat SSCs during cryopreservation and to explore its protective mechanism. We obtained SSCs from dairy goat testes by two-step enzymatic digestion and differential plating. The SSCs were cryopreserved with freezing media containing different melatonin concentrations. The results showed that 10-6 M of melatonin increased significantly the viability, total antioxidant capacity (T-AOC), and mitochondrial membrane potential of frozen-thawed SSCs, while it reduced significantly the ROS level and malondialdehyde (MDA) content (P < 0.05). Further analysis was performed by western blotting, flow cytometry, and transmission electron microscopy (TEM). Melatonin improved significantly the enzyme activity and protein expression of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (P < 0.05), thereby activating the antioxidant defense system of SSCs. Furthermore, melatonin inhibited significantly the expression of proapoptotic protein (Bax) and increased the expression of antiapoptotic proteins (Bcl-2 and Bcl-XL) (P < 0.05). The mitochondrial apoptosis pathway analysis showed that the addition of melatonin reduced significantly the mitochondrial swelling and vacuolation, and inhibited the release of cytochrome C from mitochondria into the cytoplasm, thereby preventing the activation of caspase-3 (P < 0.05) and inhibiting SSC apoptosis. In addition, melatonin reduced significantly the autophagosome formation and regulated the expression of autophagy-related proteins (LC3-I, LC3-II, P62, Beclin1, and ATG7) (P < 0.05), thereby reversing the freeze-induced excessive autophagy. In summary, melatonin protected goat SSCs during cryopreservation via antioxidant, antiapoptotic, and autophagic regulation.

Trehalose protects testicular tissue of dairy goat upon cryopreservation.

The aim of this study was to investigate whether the addition of trehalose to cryomedia reduces cellular damage and improves gene expression in cryopreserved dairy goat testicular tissues. Testicular tissues were cryopreserved in cryomedia without or with trehalose at a concentration of 5%, 10%, 15%, 20% or 25%. Cryopreserved testicular tissues were analysed for TUNEL-positive cell number, expression of BAX, BCL-2, CREM, BOULE and HSP70-2. Isolated Leydig cells from cryopreserved tissue were cultured, and spent medium was evaluated for testosterone level. The results showed that though the TUNEL-positive cell number increased in cryopreserved testicular tissues, the presence of trehalose reduced apoptotic cell number significantly. Quantitative real-time polymerase chain reaction results showed that although the expression of BAX was upregulated following cryopreservation, the presence of trehalose downregulates it in cryopreserved testicular tissues. Expression of BCL-2, CREM, BOULE and HSP70-2 was downregulated following cryopreservation but the presence of trehalose significantly upregulated their expression in cryopreserved testicular tissues. Leydig cells isolated from testicular tissues cryopreserved with trehalose produced higher testosterone than the one without it (control). These results suggest that trehalose has a protective role in cryopreservation of dairy goat testicular tissue, and the most suitable trehalose concentration for cryopreservation is 15%.

Effects of sulfanilamide on boar sperm quality, bacterial composition, and fertility during liquid storage at 17°C.

Sulfanilamide (SA) is an effective broad-spectrum antibacterial agent in human and veterinary medicine. The purpose of this study was to evaluate the effects of SA on boar sperm quality during liquid storage at 17°C and determine the optimal concentration of SA and its effects on bacterial growth, microbial composition, and maternal fertility. Boar ejaculates were diluted with a basic extender, containing different concentrations of SA, and stored in a 17°C incubator for 6 days. The sperm motility, plasma membrane integrity, and acrosome integrity were measured daily. The results showed that when the concentration of SA was 0.02 g/L, the sperm quality parameters were significantly higher than those of all other treatment groups (p < .05). We also monitored the bacterial growth and compared the differences in the microbial species between the 0.02 g/L SA group and the control by 16S rDNA sequencing. The results revealed that some bacteria, such as Staphylococcus and Pseudomonas, were considerably lower in the 0.02 g/L SA group than in the control group (p < .05). In addition, preserved semen was used for artificial insemination, and results showed that 0.02 g/L SA group had a higher litter size, and its pregnancy rate was 92.5%.

Effects of elevated ambient temperature and local testicular heating on the expressions of heat shock protein 70 and androgen receptor in boar testes.

Heat stress damaged spermatogenesis and semen quality, however, the exact molecular mechanism is not clear. The objective of this study was to determine the effects of elevated ambient temperature and local testicular heating on the expressions of heat shock protein 70 and androgen receptor in boar testes. A growing body of evidence demonstrated that germ cell apoptosis can be aggravated by heat stress and androgen deprivation, and at normal temperature, withdrawal of androgen led to germ cell apoptosis. There were no reports that heat stress damaged spermatogenesis has relationship with androgen. In this study, adult boars (Landrace, n = 9) were used and randomly divided into: control group (CON), 20-27 °C; environmental hyperthermia group (EH), 37-40 °C, 3 h/d 42 d; and local testicular heating group (LTH), 42 °C 1 h. After heat treatments, all boars were castrated and the testes were harvested. qRT-PCR and Western Blot results showed that the mRNA and protein levels of heat shock protein 70 and androgen receptor were significantly increased after heat treatments. Immunohistochemistry results showed that heat stress caused a redistribution of heat shock protein 70 from the cytoplasm to the nucleus, and androgen receptor was mainly expressed in Sertoli cells. These results indicated that heat stress promoted the inhibition of heat shock protein 70 on the androgen receptor, suggesting that the possible mechanism of heat stress damaged spermatogenesis and semen quality was that heat stress reduced the sensitivity of testicular cells to androgen by up-regulating heat shock proteins.

Expression of glucose transporter 8 (GLUT8) in spermatogenesis of adult boar testes.

The present study was designed to investigate the glucose transporter 8 (GLUT8) expression and localization in adult boar testis. Localization and expression of GLUT8 were conducted with Western Blotting, immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) methods GLUT8 protein and mRNA were expressed in the boar testes. The results of Western Blotting analysis showed specificity of the antibody for protein of boar testes. The immunohistochemistry results showed that GLUT8 protein mainly localized in spermatocytes, round spermatids and elongated spermatids of the seminiferous tubules in the adult boar testes. And the GLUT8 expression persists during eight stages of boar spermatogenesis. GLUT8 may mainly provide glucose for the later stage of germ cell differentiation in adluminal compartment in adult boar testes. These results suggested that GLUT8 is important for the spermatogenesis in the adult boar testes.

Germ cell apoptosis and expression of Bcl-2 and Bax in porcine testis under normal and heat stress conditions.

The aim of this study was to examine whether an elevated ambient temperature (37-40°C) had an effect on the apoptosis of germ cells and the expression of Bcl-2 and Bax in porcine testis. Six boars were used. Three boars were subjected to an elevated ambient temperature (37-40°C, 7days, 3h per day) as a heat stress (HS) group. The other 3 boars were kept in a room temperature house (20-27°C) as a control group. All boars were castrated and the testes were harvested. TUNEL assay was used for the detection of apoptotic cells. Immunohistochemistry, Western blotting and quantitative real-time PCR were used to analyze protein and mRNA levels of Bcl-2 and Bax in response to heat treatment. The results showed that apoptotic signals increased under heat stress conditions compared with the control (P<0.01), and the cell types most affected by heat treatment were spermatocytes and spermatids. In both the control and experimental groups, Bcl-2 was expressed in the cytoplasm and nucleus of spermatogonia, spermatocytes and differentiating spermatids and Bcl-2 preferentially localized close to the seminiferous tubule's luminal surface in late spermatocytes and spermatids. Compared with the control group, the expression levels of Bcl-2 protein and mRNA significantly increased in heat treatment group, while the expression levels of Bax protein and mRNA did not show significant changes between the control and experimental group. Low to moderate Bax immunoreactivity staining was observed in all kinds of germ cells in the control group. Strong staining was observed in spermatogonia, and low to moderate Bax staining was observed in spermatocytes and spermatids. A redistribution of Bax from a cytoplasmic to perinuclear or nuclear localization could be observed in the spermatogonia, spermatocytes and spermatids obtained in the heat treated group. These results showed that elevated ambient temperatures induced germ cell apoptosis. In response to heat stress, the expression of Bcl-2 increased and a redistribution of Bax from a cytoplasmic to a perinuclear or nuclear localization. This indicates that Bcl-2 and Bax may be involved in regulation of germ cell apoptosis induced by heat stress in boars.