Sertoli Cell Proliferation Research Articles

Estradiol ameliorates metformin-inhibited Sertoli cell proliferation via AMPK/TSC2/mTOR signaling pathway

Metformin is a commonly used for treating type 2 diabetes and it acts on a variety of organs including the male reproductive system. 17β-estradiol plays an important role in Sertoli cell (SC) proliferation which determines the germ cell development and spermatogenesis. The aim of this study is to investigate the effect of metformin on immature chicken SC proliferation and the potential mechanisms by which 17β-estradiol regulate this process. Results showed that metformin significantly inhibited SC proliferation, whereas 17β-estradiol weakened the inhibitory effects of metformin on SC viability, cell growth, and cell cycle progression. SC proliferation-inhibiting effect of metformin exposure was regulated by decreasing adenosine triphosphate level and respiratory enzyme activity in the mitochondria; this process was possibly mediated by the adenosine monophosphate-activated protein kinase (AMPK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) signaling pathway, which was regulated by the down-expressed miR-1764 and by the decreased antioxidant enzyme activity and excessive reactive oxygen species generation. In addition, SCs transfected with the miR-1764 agomir led to an improvement of proliferation capacity through down-regulating AMPKα2 level, which further decreased TSC2 expression and induced mTOR activation. However, the anti-proliferative effect of miR-1764 antagomir can be alleviated by 17β-estradiol treatment via the up-expression of miR-1764 in transfected SCs. Our findings suggest appropriate dose of exogenous 17β-estradiol treatment can ameliorate the inhibitory effect of metformin on SC proliferation via the regulation of AMPK/TSC2/mTOR signaling pathway, this might reduce the risk of poor male fertility caused by the abuse of anti-diabetic agents.

MiRNA-34c Regulates Bovine Sertoli Cell Proliferation, Gene Expression, and Apoptosis by Targeting the AXL Gene.

Simple SummaryFertility is one of the essential reproduction traits of bulls, and accurate prediction of fertility potential using a semen sample from a donor bull for artificial insemination is crucial to achieve consistently high reproductive efficiency. Somatic cells, such as Sertoli cells and Leydig cells, are important in testis formation and provide a nurturing and regulatory environment for spermatogenesis. Furthermore, it was suggested that non-coding RNAs, such as microRNAs, long non-coding RNAs, circular RNAs, and Piwi-interacting RNA, function as important regulators of gene expression at post-transcriptional level in spermatogenesis. In this study, microRNA-34c was verified to specifically regulate the AXL gene by targeting a sequence in the 3’ UTR; miRNA-34c can also influence the proliferation, apoptosis, and relative abundance of the transcript of male-reproduction-related genes. Therefore, microRNA-34c can be considered an essential regulator in the process of bull spermatogenesis. These results identify a key microRNA and functional genes in the process of cattle male reproduction, providing useful information for future marker-assisted selection of bulls with excellent sperm quality.MicroRNAs (miRNAs) play significant roles in mammalian spermatogenesis. Sertoli cells can provide a stable microenvironment and nutritional factors for germ cells, thus playing a vital role in spermatogenesis. However, few studies elucidate the regulation of bovine testicular Sertoli cells by miRNAs. Here, we have reported that miRNA-34c (miR-34c) regulates proliferation, apoptosis, and relative transcripts abundance gene in bovine Sertoli cells. In bovine Sertoli cells, overexpression of miR-34c inhibited proliferation and relative abundance of gene transcripts while promoting apoptosis of Sertoli cells, and the effects were the opposite when miR-34c was knocked down. Receptor tyrosine kinase (AXL) was identified as a direct target gene of miR-34c in Sertoli cells, validated by analysis of the relative abundance of AXL transcript and dual-luciferase reporter assay. The relative abundance of the transcript of genes related to male reproduction in Sertoli cells was changed after the AXL gene was overexpressed, as demonstrated by the RT2 Profiler PCR Array results. In summary, miR-34c specifically regulated the AXL gene by targeting a sequence in the 3′-UTR, which could influence proliferation, apoptosis, and relative abundance of the transcript of male reproduction-related genes. Therefore, miR-34c could be considered an essential regulator in the process of bull spermatogenesis.

P–115 Supplementation of healthy Sertoli cells into culture media containing follicle stimulating hormone (FSH)/testosterone (T) has no advantage in germ cell maturation

Abstract Study question In nonobstructive azoospermia (NOA) cases, whether supplementation of healthy Sertoli cells (SCs) has an effect on spermatogenic differentiation in culture medium containing FSH/T. Summary answer Expression of Crem and Acrosin increased significantly in both medium with FSH/T and medium with additional healthy SCs but there was no difference between them What is known already In NOA the induction of spermatogonial stem cells (SSCs) proliferation and differentiation has been demonstrated using different culture systems. SCs have vital roles in the regulation of spermatogenesis. Hormonal control of spermatogenesis is through FSH and T activity on SCs. Growth factors secreted by SCs via FSH, stimulate proliferation and colonization of SSCs. Although germ cells do not express androgen receptors, FSH receptors are localized on spermatogonia. It is not clear whether native SCs are sufficient for FSH/T added to the culture medium to be effective in induction of spermatogenesis, and whether supplementation of healthy SCs will increase this activity. Study design, size, duration 34 NOA and 12 obstructive azoospermia (OA) cases were included. Testicular tissue samples were taken with testicular sperm extraction (TESE) in the study and control groups. In a group of fertile cases, healthy Sertoli cells were identified and purified and then cryopreserved. Tissue samples of each case prepared in standard DMEM/F12 medium were processed in 2 separate environments containing FSH/T and FSH/T plus thawed healthy SCs for 7 days. Participants/materials, setting, methods The characterization of healthy SCs isolated from fertile cases was done by flow cytometry (FC) and immunohistochemistry using antibodies specific for GATA4 and vimentin. FITC-conjugated annexin V/PI staining and MTT assay were performed to compare the viability and proliferation of SCs before and after freezing. FC was used to measure the 7th day levels of specific markers expressed in spermatogonia (Vasa), meiotic cells (Crem) and post-meiotic cells (Protamine–2 and Acrosin). Main results and the role of chance In Annexin V staining, no difference was found in percentages of live and apoptotic SCs, and MTT exhibited that cryopreservation didn’t inhibite the SCs proliferation compared to the pre-freezing state. Vasa and Acrosin basal levels were found to be lower in infertile patients compared to the control group (8.2% vs. 30.6% and 12.8% vs. 30.5%, p < 0.05). Compared to day 0 measurements, on the 7th day in FSH/T environment, Crem level increased by 58.8% and Acrosin level increased by 195.5% (p < 0.05). Similarly, in medium supplemented with healthy SCs, by day 7, the Crem and Acrosin levels were increased to 92.2% and 204.8%, respectively (p < 0.05). Although Vasa and Protamine levels increased in both groups, they did not reach a significant level. No significant difference was found between the 7th day increase rates of Crem, Vasa, Acrosin and Protamine–2 in either FSH/T-containing medium or in medium additionally supplemented with healthy SCs (58.8% vs. 92.2%, 120.6% vs. 79.4%, 195.5% vs. 204.8% and 232.3% vs. 198.4%, respectively, p > 0.05). Our results suggest that freezing-thawing process would not impair the viability and proliferation of SCs, and adding healthy SCs to the culture medium to correct impaired gene expression does not have an advantage over FSH/T. Limitations, reasons for caution The 7-day culture period we determined might be not sufficient for spermatogenic differentiation completion. This period could be extended in order to see further morphological differentiation may need. Wider implications of the findings: The failure of the culture media containing FSH/T to show the expected effectiveness could be thought to be due to the SCs’ inadequate response to these hormones. Therefore, healthy SCs supplementation would be needed, but this could pose ethical issues. Our findings show that it is not necessary. Trial registration number 214S532

MiR-126 Controls the Apoptosis and Proliferation of Immature Porcine Sertoli Cells by Targeting the PIK3R2 Gene through the PI3K/AKT Signaling Pathway.

Simple SummaryMicroRNAs (miRNAs) have been reported with potential regulatory roles in spermatogenesis. In the present study, we demonstrated that miR-126 can stimulate cell proliferation and restrain the apoptosis of immature porcine Sertoli cells by targeting the PIK3R2 gene. Through this process, miR-126 further activates the PI3K/AKT signaling pathway. These results indicated that miR-126, PIK3R2, and the PI3K/AKT signaling pathway might play pivotal regulatory roles in porcine spermatogenesis by deciding the destiny of immature Sertoli cells.The quantity of Sertoli cells in the adult testis decides the daily gamete formation, and accumulating evidence indicates that epigenetic factors regulate the proliferation of Sertoli cells. Research on the function and regulatory mechanism of microRNAs (miRNAs) in Sertoli cells has not been comprehensive yet, especially on domestic animals. In this article, we report that miR-126 controls the proliferation and apoptosis of immature porcine Sertoli cells based on previous studies. Our results confirmed that miR-126 elevation promotes cell cycle progression, cell proliferation and represses cell apoptosis; on the contrary, the inhibitory effects of miR-126 result in the opposite. The phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) gene, a member of the PI3K family, was verified as a direct target of miR-126 using the dual-luciferase reporter analysis. miR-126 negatively regulated the mRNA and protein expression level of PIK3R2 in immature porcine Sertoli cells. siRNA-induced PIK3R2 inhibition caused similar effects as miR-126 overexpression and eliminated the influences of miR-126 knockdown in immature porcine Sertoli cells. In addition, both miR-126 overexpression and PIK3R2 inhibition elevated the phosphorylation of PI3K and AKT, whereas the miR-126 knockdown demonstrated the contrary result. In short, miR-126 controls the proliferation and apoptosis of immature porcine Sertoli cells by targeting the PIK3R2 gene through the PI3K/AKT signaling pathway. The research supplies a theoretical and practical foundation for exploring the functional parts of miR-126 in swine sperm by defining the destiny of immature Sertoli cells.

The Molecular Mechanism of Sex Hormones on Sertoli Cell Development and Proliferation

Sustaining and maintaining the intricate process of spermatogenesis is liable upon hormones and growth factors acting through endocrine and paracrine pathways. The Sertoli cells (SCs) are the major somatic cells present in the seminiferous tubules and are considered to be the main regulators of spermatogenesis. As each Sertoli cell supports a specific number of germ cells, thus, the final number of Sertoli cells determines the sperm production capacity. Similarly, sex hormones are also major regulators of spermatogenesis and they can determine the proliferation of Sertoli cells. In the present review, we have critically and comprehensively discussed the role of sex hormones and some other factors that are involved in Sertoli cell proliferation, differentiation and maturation. Furthermore, we have also presented a model of Sertoli cell development based upon the recent advancement in the field of reproduction. Hence, our review article provides a general overview regarding the sex hormonal pathways governing Sertoli cell proliferation and development.

MiR-191 promotes the porcine immature Sertoli cell proliferation by targeting the BDNF gene through activating the PI3K/AKT signaling pathway.

The number of Sertoli cells in the testis is a major regulator on the sperm production capacity. MicroRNAs (miRNAs) participate in regulating the proliferation and apoptosis of porcine immature Sertoli cells. However, the functions and mechanisms of action of most identified miRNAs in porcine Sertoli cells remain largely unknown. In the present study, based on our previous results from an EdU-based high-content screening assay, we further studied the mechanism of action of miR-191 on the proliferation and apoptosis of porcine immature Sertoli cells through flow cytometry, Western blotting, and dual-luciferase activity analyses. The results demonstrated that overexpression of miR-191 promoted cell cycle progression from G1 phase to the S and G2 phases, enhanced cell proliferation, and inhibited apoptosis in the porcine immature Sertoli cells, whereasmiR-191 inhibition resulted in the opposite effects. The results from a luciferase reporter assay showed that miR-191 directly targeted the 3'-UTR of theBDNF gene. BDNF knockdown also promoted cell cycle progression to the S phase, cell proliferation and inhibited cell apoptosis, which were consistent with the effects of the miR-191overexpression. A co-transfection experiment showed that BDNF knockdown abolished the effects of miR-191 inhibition. Furthermore, both miR-191 overexpression and BDNFinhibition elevated the phosphorylation of PI3K and AKT, the key components of the PI3K/AKT signaling pathway, whereas BDNFinhibition offset the effects of the miR-191 knockdown. Overall, these data indicated that miR-191 promotes cell proliferation and inhibits apoptosis in porcine immature Sertoli cells by targeting theBDNF gene through activating the PI3K/AKT signaling pathway. This study provides a novel scientific basis for further investigation on the biological functions of miR-191 on porcine spermatogenesis.

MicroRNA-125a-5p modulates the proliferation and apoptosis of TM4 Sertoli cells by targeting RAB3D and regulating the PI3K/AKT signaling pathway.

Sertoli cells provide protection and nutrition for developing sperm. Each stage of sperm development occurs on the surface of Sertoli cells. MicroRNA (MiR)-125a-5p is involved in male reproduction. The current research aimed to probe the role of miR-125a-5p in Sertoli cell function. Functionally, miR-125a-5p knockdown facilitated Sertoli cell proliferation, while miR-125a-5p overexpression suppressed Sertoli cell proliferation, as evidenced by 5-ethynyl-20-deoxyuridine incorporation assay. Additionally, miR-125a-5p knockdown inhibited Sertoli cell apoptosis, while miR-125a-5p upregulation facilitated Sertoli cell apoptosis, as evidenced by flow cytometry analysis. Computationally, we identified four predicted mRNA targets of miR-125a-5p. Based on the results of luciferase reporter assay, miR-125a-5p was confirmed to bind to the predicted sequence in the Ras-related protein Rab-3D (RAB3D) 3'UTR. Rescue experiments showed that miR-125a-5p suppressed the proliferative ability of TM4 Sertoli cells and facilitated their apoptosis by targeting RAB3D. Finally, our data confirmed that miR-125a-5p and RAB3D modulated activation of the PI3K/AKT pathway. In conclusion, our data showed that miR-125a-5p regulated Sertoli cell proliferation and apoptosis by targeting RAB3D and regulating the PI3K/AKT pathway.

Effect of Wuzi Yanzong Pills on Sertoli cells and blood-testis barrier in heat-stressed rats based on Akt signalling pathway.

The blood-testis barrier (BTB) of Sertoli cells (SCs) is an important biological barrier that maintains spermatogenesis and provides a favourable microenvironment for spermatogenesis. However, heat stress can directly damage the BTB structural proteins of testicular SCs, leading to dyszoospermia. Wuzi Yanzong Pills (WYP) is a traditional Chinese medicine formula used to treat male reproductive diseases. However, whether WYP could ameliorate heat stress injury in primary SCs extracted from rat testes and BTB proteins remains unknown. Here, treatment with WYP (low, medium and high dose) increased the SC viability and the proliferation of cell antigen Ki67 significantly. Additionally, it promoted SC maturation, which presented in the form of increased androgen receptors (ARs) and decreased cytokeratin 18 (CK-18) in three WYP dose groups. WYP upregulated BTB proteins such as zonula occludens 1 (ZO-1) and occludin across all WYP groups and decreased phosphorylated Akt (p-Akt) in the middle and high-dose groups; however, ZO-1 and occludin recovery were reduced with the presence of Akt inhibitor in WYP groups. WYP improved SC viability and proliferation, and ameliorated dedifferentiation and BTB-proteins damaged by heat stress via Akt signalling. The findings present theoretical support for the effects of WYP in the management of dyszoospermia and male infertility.

Three-dimensional structure of testis cords in mice and rats.

Testis cord elongation and coiling, which occur in the final stage of testis formation, have been attributed to Sertoli cell proliferation; however, the underlying mechanisms remain unclear. The aim of the present study was to clarify the precise three-dimensional structure of testis cords in the final stage of testis formation in mice and rats. We reconstructed whole testis cords in the final stage of testis formation in mice (on embryonic days 15.5 and 18.5) and rats (on embryonic days 16.5 and 19.5) using serial paraffin sections and high-performance three-dimensional reconstruction software. Detailed morphometric parameters were calculated for three-dimensionally reconstructed testis cords in six mouse and rat testes each. The mean numbers of testis cords in mice and rats were 12.7 and 27.8, respectively. The mean number of branching points per testis cord was 1.52 in mice, whereas it was only 0.30 in rats. In contrast, the mean ratio of the inner cords, that is, cords not in contact with the tunica albuginea, was 23.0% in rats, whereas it was only 6.5% in mice. In both species, the cords on the cranial side coiled more strongly than those on the caudal side, consistent with the greater expansion of the testis volume on the caudal side. All cords formed right-handed helices from the rete testis side. The present results suggest that testis cords undergo anastomosis at a higher frequency in mice than in rats and that the coiling of testis cords proceeds from the cranial to caudal side of the testis in both species.

Chlorogenic Acid Ameliorates Damage Induced by Fluorene-9-Bisphenol in Porcine Sertoli Cells.

4,4′-(9-Fluorenylidene) diphenol (BPFL, also known as BHPF and fluorene-9-bisphenol) is a novel bisphenol A substitute that is used in the plastics industry as an organic synthesis intermediate and is a potential endocrine disruptor. However, the deleterious effects of BPFL on porcine Sertoli cells (SCs) and the possible underlying mechanisms are still unclear. Chlorogenic acid (CA) is a free radical scavenger in the cellular antioxidant system that prevents oxidative damage and apoptosis. In the present research, we found that BPFL induced impairments in porcine SCs in a dose-dependent manner and that CA protected porcine SCs against BPFL exposure-induced impairments. Cell viability, proliferation and apoptosis assay results revealed that BPFL exposure could inhibit porcine SC proliferation and induce apoptosis, while CA supplementation ameliorated the effects of BPFL. Further analysis revealed that BPFL exposure induced oxidative stress, mitochondrial membrane potential dysfunction and DNA damage accumulation. Transcriptome analysis and further real-time quantitative PCR and Western blot results showed that BPFL exposure induced endoplasmic reticulum stress and apoptosis. Supplementation with CA dramatically ameliorated these phenotypes in BPFL-exposed porcine SCs. Overall, the present research reveals the possible underlying mechanisms by which BPFL exposure induced impairments and CA supplementation protected against these impairments in porcine SCs.

Hsa-miR-100-3p Controls the Proliferation, DNA Synthesis, and Apoptosis of Human Sertoli Cells by Binding to SGK3.

Human Sertoli cell is required for completing normal spermatogenesis, and significantly, it has important applications in reproduction and regenerative medicine because of its great plasticity. Nevertheless, the molecular mechanisms underlying the fate decisions of human Sertoli cells remain to be clarified. Here, we have demonstrated the expression, function, and mechanism of Homo sapiens-microRNA (hsa-miR)-100-3p in human Sertoli cells. We revealed that miR-100-3p was expressed at a higher level in human Sertoli cells by 10% fetal bovine serum (FBS) than 0.5% FBS. MiR-100-3p mimics enhanced the DNA synthesis and the proliferation of human Sertoli cells, as indicated by 5-ethynyl-2′-deoxyuridine (EdU) and Cell Counting Kit-8 (CCK-8) assays. Flow cytometry showed that miR-100-3p mimics reduced the apoptosis of human Sertoli cells, and notably, we predicted and further identified serum/glucocorticoid regulated kinase family member 3 (SGK3) as a direct target of MiR-100-3p. SGK3 silencing increased the proliferation and decreased the apoptosis of human Sertoli cells, while SGK3 siRNA 3 assumed a similar role to miR-100-3p mimics in human Sertoli cells. Collectively, our study indicates that miR-100-3p regulates the fate decisions of human Sertoli cells by binding to SGK3. This study is of great significance, since it provides the novel epigenetic regulator for the proliferation and apoptosis of human Sertoli cells and it may offer a new clue for gene therapy of male infertility.

α-Linolenic acid induced TM4 Sertoli cells proliferation and enhanced total antioxidant capacity

The present study was undertaken to elucidate the direct ALA effects on mice TM4 Sertoli cells proliferation in vitro. Our results showed that TM4 cells viability was significantly stimulated by ALA (p < 0.05). The 50 μM ALA increased the concentration of total antioxidant capacity, induced the mitochondrial membrane hyperpolarized, and markedly decreased the number of apoptosis cells (p < 0.05). ALA also up-regulated G2/Mitotic-specific cyclin-B1 gene and apoptosis suppressive gene Bcl2 expression (p < 0.05). In conclusion, those results indicated that ALA could increase TM4 Sertoli cells antioxidant capacity, induced the mitochondrial membrane hyperpolarized, inhibited cells apoptosis and stimulated TM4 Sertoli cells proliferation in vitro.

Neonatal maternal separation increases the number of p27-positive Sertoli cells in prepuberty

Neonatal maternal separation (NMS) is an experimental model for early life stress, which affects the growth and development of various organs, resulting in adverse health effects in humans and animals. In our previous study, we demonstrated that NMS [(0.5-, 1-, 2-h/day NMS, from postnatal day (PND) 1–10] induced morphological changes to the male reproductive system, including decreased Sertoli cell numbers in mouse testes at PND 70. To clarify the mechanism by which NMS decreases Sertoli cell numbers, we evaluated the effects of NMS on mouse testes at PNDs 10 and 16. At PND 10, the Sertoli cell number was not significantly different among experimental groups; however, it decreased in 0.5- and 2-h/day NMS mice at PND 16. The termination of Sertoli cell proliferation in prepuberty can be induced by p27, a cyclin-dependent kinase inhibitor. At PND 10, we observed an increase in the number of p27-positive Sertoli cells in 2-h/day NMS mice. The seminiferous tubule diameters decreased significantly in 1- and 2-h/day NMS mice, and the relative interstitial area increased in 2-h/day NMS mice. Serum corticosterone level significantly increased, and serum testosterone level significantly decreased in the 2-h/day NMS mice. At PND 16, the tubule diameters and height of seminiferous epithelium were significantly higher in 0.5- and 2-h/day NMS mice. Our results suggest that NMS disturbs serum corticosterone and testosterone levels and increases the number of p27-positive Sertoli cells at PND 10, resulting in a decrease in the number of Sertoli cells at PND 16.

Influence of freezing techniques and glycerol-based cryoprotectant combinations on the survival of testicular tissues from adult collared peccaries

The objective of the study was to evaluate the effects of different cryopreservation techniques including glycerol-based cryoprotectant combinations on the structure and viability of testicular tissues from adult collared peccaries. Tissue biopsies (3.0 mm³) from 5 different individuals were allocated to 10 different groups: fresh control; slow freezing (SF), conventional vitrification (CV), or solid-surface vitrification (SSV); each of them using three different combinations of cryoprotectants [dimethyl sulfoxide (DMSO) + ethylene glycol (EG); DMSO + Glycerol; and EG + Glycerol]. After thawing/warming, samples were evaluated for histomorphology, viability, proliferative capacity potential, and DNA integrity. Most effective preservation of testicular histomorphology was achieved using SF and CV with DMSO + EG. However, the use of glycerol-based cryoprotectant combinations increased the occurrence of tubular cell swelling, tubular cell loss and shrinkage from the basal membrane. Cell viability was comparable among cryopreservation methods and cryoprotectant combinations. Regarding cell proliferative capacity, the use of SF with EG + Glycerol and SSV with DMSO + Glycerol impaired the conservation of spermatogonia proliferative potential compared to other treatments. Moreover, CV with DMSO + EG was better than SF with EG + Glycerol for Sertoli cell proliferation potential. Regarding DNA integrity, less damage occurred when using SF with DMSO + EG while more fragmentations were observed when using CV with EG + Glycerol or DMSO + Glycerol as well as SSV with EG + Glycerol or DMSO + Glycerol. In sum, SF and CV appeared to be the most suitable methods for the cryopreservation of adult peccary testicular tissues. Additionally, the use of glycerol-based cryoprotectant combinations did not improve testicular tissues preservation with DMSO + EG being the most efficient option.

Glycine alleviates fluoride‐induced oxidative stress, apoptosis and senescence in a porcine testicular Sertoli cell line

Glycine is a well-known free radical scavenger in the cellular antioxidant system that prevents oxidative damage and apoptosis. Excessive fluoride exposure is associated with multiple types of cellular damage in humans and animals. The objective of the present study was to investigate the protective effects of glycine on sodium fluoride (NaF) exposure and the possible underlying mechanisms in a porcine testicular Sertoli cell line model. Cellular viability and proliferation were examined following NaF exposure and glycine supplementation, and glycine dramatically ameliorated the decreases in NaF-induced porcine testicular Sertoli cell viability and proliferation. Further investigations revealed that glycine decreased NaF-induced intracellular reactive oxygen species production, DNA fragment accumulation and the apoptosis incidence in the porcine testicular Sertoli cell line; in addition, glycine improved mitochondrial function and ATP production. Notably, results of the SPiDER-β-Gal analysis suggested that glycine alleviated NaF-induced cellular senescence and downregulated P53, P21, HMGA2 and P16INK4a gene expression in the porcine testicular Sertoli cell line. Collectively, the beneficial effects of glycine alleviate NaF-induced oxidative stress, apoptosis and senescence, and together with our previous findings, support the hypothesis that glycine plays an important role in protecting against NaF exposure-induced impairments in the porcine testicular Sertoli cell line.

Postnatal metformin treatment alters rat Sertoli cell proliferation and daily sperm production.

The direct correlation between Sertoli cell number and sperm production capacity highlights the importance of deciphering external factors that modify Sertoli cell proliferation. A growing body of evidence in vitro suggests that metformin, the main pharmacological agent for type 2 diabetes treatment in children, exerts anti-proliferative effects on Sertoli cells. The aims of this study were to investigate the effect of metformin administration during postnatal period on Sertoli cell proliferation and on cell cycle regulators expression and to analyze the impact of this treatment on the sperm production capacity in adulthood. Sprague Dawley rat pups were randomly divided into two groups: MET (receiving daily 200mg/kg metformin, from Pnd3 to Pnd7 inclusive) and control (receiving vehicle). BrdU incorporation was measured to assess proliferation. Gene expression analyses were performed in Sertoli cells isolated from animals of both groups. Daily sperm production and sperm parameters were measured in adult male rats (Pnd90) that received neonatal treatment. MET group exhibited a significant decrease in BrdU incorporation in Sertoli cells. Concordantly, MET group showed a reduction in cyclin D1 and E2 expression and an increase in p21 expression in Sertoli cells. In addition, metformin-treated animals displayed lower values of daily sperm production on Pnd90. These results suggest that metformin treatment may lead to a decrease in Sertoli cell proliferation, a concomitant altered expression of cell cycle regulators and ultimately, a reduction in daily sperm production in adult animals.

Thyroid hormones, Sertoli cell proliferation and differentiation in progenies from carbamazepine-treated rat dams during pregnancy and lactation.

Carbamazepine (CBZ) is used in the control of seizure and affective disorders, causing hypothyroidism. Thyroid hormones regulate the Sertoli cell proliferation and differentiation. Clinical aspects must be considered since epileptic fertile women need to continuously use CBZ during pregnancy and lactation. This study aimed to evaluate the effects of CBZ on testis development of rat offspring from dams treated during pregnancy/lactation. Rat dams received CBZ (20mgkg-1 day-1 ) or vehicle by intra-peritoneal route during gestation and lactation. Progenies were euthanised at 4, 14, 41, 63 and 93-days post-partum (dpp) for the evaluation of T3, T4 and TSH plasma total levels. Testicular cross sections were submitted to anti-Ki67, anti-PCNA, anti-p27kip1 and anti-transferrin immunolabelling for the evaluation of Sertoli cells. There was a significant reduction in p27kip1 -positive Sertoli cell numerical densities and an increase in TSH level at 14 dpp. CBZ exposure affected the volume density of transferrin-positive immunolabelling at 63 dpp. These results suggest that CBZ may cause a dysregulation of the controller system of thyroid hormones homeostasis leading to an increase in the proliferation rate at the neonatal phase and a differentiation delay of the Sertoli cell, culminating in an altered function at late puberty. The occurrence of hypothyroidism cannot be completely discarded.

Molecular cloning, characterization, and function analysis of the AMH gene in Yak (Bos grunniens) Sertoli cells

Sertoli cells (SCs) are important testicular somatic cells that carry out various functions in spermatogenesis. Understanding the biological mechanisms underlying SC development may facilitate the understanding of animal reproduction. Anti-Mullerian hormone (AMH) is a dimeric glycoprotein produced by SCs and plays essential roles in spermatogenesis. In this study, we cloned the coding sequence of the yak AMH, predicated the structure of AMH protein, analyzed AMH expression in the testis at different stages, and studied the functions of AMH in yak SCs. The open reading frame (ORF) of the yak AMH contained 1728 bp and encoded 575 amino acids. Structural analysis revealed that the yak AMH protein had a highly conserved transforming growth factor-β (TGF-β) domain. The mRNA expression level for the AMH gene in yak testis increased significantly from the fetal stage to calf stage, then decreased with the increase of age. The highest expression was found in calf stage. Cell proliferation was depressed in AMH-deficient SCs. Expression of several genes involved in SC proliferation and development, including PCNA, BCL-2, BAX, CASP3, AR and AMHR2 were altered after knockdown of AMH. Also, three SC-secreted factors essential for spermatogenesis, SCF, GDNF and ABP, were repressed at the transcription level after AMH knockdown in yak SCs. Moreover, supplementation with exogenous AMH protein partially rescued SC proliferation, and the expression of PCNA, BCL-2, AR and AMHR2 after AMH gene interference. This research provided theoretical basis for understanding the mechanism by which AMH regulates yak spermatogenesis and might give new insights in improving yak reproductive performance in the future.

AR regulates porcine immature Sertoli cell growth via binding to RNF4 and miR-124a.

Sertoli cells are the only somatic cells in the seminiferous epithelium which directly contact with germ cells. Sertoli cells exhibit polarized alignment at the basal membrane of seminiferous tubules to maintain the microenvironment for growth and development of germ cells, and therefore play a crucial role in spermatogenesis. Androgens exert their action through androgen receptor (AR) and AR signalling in the testis is essential for maintenance of spermatogonial numbers, blood-testis barrier integrity, completion of meiosis, adhesion of spermatids and spermiation. In the present study, we demonstrated that AR gene could promote the proliferation of immature porcine Sertoli cells (ST cells) and the cell cycle procession, and accelerate the transition from G1 phase into S phase in ST cells. Meanwhile, miR-124a could affect the proliferation and cell cycle procession of ST cells by targeting 3'-UTR of AR gene. Furthermore, AR bound to the RNF4 via AR DNA-binding domain (DBD) and we verified that RNF4 was necessary for AR to regulate the growth of ST cells. Above all, this study suggests that AR regulates ST cell growth via binding to RNF4 and miR-124a, which may help us to further understand the function of AR in spermatogenesis.

Changes in testicular gene expression following reduced estradiol synthesis: A complex pathway to increased porcine Sertoli cell proliferation

Porcine Sertoli cell number including number present at puberty is increased if testicular estradiol synthesis is reduced during the neonatal interval. Evaluating the changes in gene expression during the crucial interval of suppressed estradiol that leads to the increased Sertoli cell population will increase our understanding of Sertoli cell biology but this evaluation first required a more precise determination of the critical interval for treatment and timing of a detectable response. Previously, reduced testicular estrogens from 1 week of age were accompanied by increased Sertoli cell number at 6.5 weeks of age but the age at which Sertoli cell numbers were initially increased was unknown, one of the current objectives. Additional experiments were designed to further delineate the essential timing of treatment for the Sertoli cell response. Finally, changes in gene expression induced by the reduced estradiol synthesis were evaluated to elucidate molecular mechanisms. Experimental design typically consisted of one member of littermate pairs of boars treated with the aromatase inhibitor, letrozole, beginning at 1 week of age and the remaining member treated with canola oil vehicle. Weekly treatments continued through 5 weeks of age or tissue collection, whichever came first. Increases in Sertoli cell numbers were not detectable prior to 6.5 weeks of age and persistent treatment through 5 weeks of age was required to induce the increase in Sertoli cell numbers. This increase resulted from prolonging the first interval of Sertoli cell proliferation in the treated animals. Few genes exhibited dramatically altered transcription and similarities in pathway analysis or principal modified genes were quite limited in 2, 3, and 5-week-old boars. The critical timing and prolonged treatment required and the sequential changes in gene expression suggest a complex mechanism is involved in this model of increased proliferation of Sertoli cells.