Heat exposure promotes apoptosis and pyroptosis in Sertoli cells

Heat stress is an important influence on the male reproductive organs.Therefore,the effects of heat stress on genes or pathways related to the reproductive system of male mice were experimentally explored in this paper to further determine the effects of heat stimulation on mammals.Herein,models of heat-exposed mouse testicular tissue and heatexcited cells were successfully established.Many scorched vesicles were found after heat excitation of testis supporting cells,testicular mesenchymal(TM4)cells.Western blot,in situ terminal deoxynucleotide transferase dUTP Nick end labeling(TUNEL)and transmission electron microscopy showed that membrane rupture,mitochondrial damage and autophagic vesicles occurred in TM4 cells after thermal excitation.The N-segment fragment of the associated protein shear was increased,and the TUNEL result was positive.In conclusion,thermal excitation induced apoptosis and scorch death in TM4 cells.Thus,the Hippo pathway and apoptosis-related pathway were significantly enriched after heat stimulation in mouse testis,and the scorch death effect in TM4 cells was induced by heat excitation.

The effects of hormone-mediated PI3K/AKT signaling on spermatogenesis in Sertoli cells

The phosphoinositide-3-kinase/Akt(PI3K/AKT)signaling pathway is crucial for Sertoli cell development and completing spermatogenesis.Its main role is to promote proliferation and inhibit apoptosis.Many factors activate the PI3K/AKT pathway,like hormones,such as follicle stimulating hormone(FSH),androgen,estrogen,insulin to name a few.Many of these factors have receptors inside or on the surface of Sertoli cells(SCs).This review summarizes how these hormones directly regulate the PI3K/AKT signaling pathway in SCs,which in turn affects SC proliferation and differentiation.Further,hormone-mediated PI3K/AKT signaling also stimulates SC secretion,which is essential for germ cell development,suggesting an indirect role of PI3K/AKT signaling during spermatogenesis.These functions include promoting spermatogonia proliferation and differentiation,meiosis of spermatocytes,sperm maturation,and their release.This review also provides potential hints for clinically treating male infertility issues like cryptorchidism and Sertoli cell-only syndrome.

Effect of 43℃ treatment on expression of heat shock proteins 105, 70 and 60 in cultured monkey Sertoli cells

Aim： To examine the possible effect of heat treatment on expression of heat shock proteins （Hsps） 105, 70, and 60 in primary monkey Sertoli cells and to evaluate the possible signal pathways. Methods： Western blot analysis, realtime polymerase chain reaction （PCR）, and confocal immunohistochemistry were used to analyze mRNA and protein levels of the Hsps in response to 43~C treatment of Sertoli cells isolated from pubertal monkey testes. Results： Staining with Hoechst 33342 indicated Sertoli cells did not undergo apoptosis after heat treatment. Hspl05 was expressed in cytoplasm of untreated Sertoli cells. Both Hspl05 mRNA and protein levels were increased approximately 20-fold compared to those of the untreated controls at 12 h after heat treatment. Untreated Sertoli cells did not express Hsp70, but heat stress induced its expression in the cell cytoplasm. The time-course of changes in Hsp70 was similar to that of Hsp105. In contrast to Hsp105 and Hsp70, the change in Hsp60 expression was much less obvious. The protein level between 12 h and 48 h after heat treatment was only approximately 1.5-fold that of the untreated control. Extracellular regulated kinase （ERK） 1/2 inhibitor （U0126） or phosphoinositide kinase-3 （PI3K） inhibitor （LY294002） could partially block the response of Hspl05 and Hsp70 induced by heat treatment. Conclusion： These results indicate that the heat-induced expression of the three types of Hsp in monkey Sertoli cells might be regulated by ERK and/or PI3K signal pathways, but the profile of their expression is different, suggesting that they might have different regulatory functions in Sertoli cells.

dentification of testosterone-/androgen receptor-regulated genes in mouse Sertoli cells

Androgen and androgen receptor （AR） play important roles in male spermatogenesis and fertility, yet detailed androgen/AR signals in Sertoli cells remain unclear. To identify AR target genes in Sertoli cells, we analyzed the gene expression profiles of testis between mice lacking AR in Sertoli cells （S-AR-/y） and their littermate wild-type （WT） mice. Digital gene expression analysis identified 2276 genes downregulated and 2865 genes upregulated in the S-AR-/y mice testis compared to WT ones. To further nail down the difference within Sertoli cells, we first constructed Sertoli cell line TM4 with stably transfected AR （named as TM4/AR） and found androgens failed to transactivate AR in Sertoli TM4 and TM4/AR cells. Interestingly, additional transient transfection of AR-cDNA resulted in significant androgen responsiveness with TM4/AR cells showing 10 times more androgen sensitivity than TM4 cells. In the condition where maximal androgen response was demonstrated, we then analyzed gene expression and found the expression levels of 2313 genes were changed more than twofold by transient transfection of AR-cDNA in the presence of testosterone. Among these genes, 603 androgen-/ AR-regulated genes, including 164 upregulated and 439 downregulated, were found in both S-AR-/y mice testis and TM4/AR cells. Using informatics analysis, the gene ontology was applied to analyze these androgen-/AR-regulated genes to predict the potential roles of androgen/AR in the process of spermatogenesis. Together, using gene analysis in both S-AR-/y mice testis and TM4/AR cells may help us to better understand the androeen/AR signals in Sertoli cells and their influences in spermatogenesis.

FSH Promoting Proliferation of Calf Sertoli Cells Through Wnt/β-catenin Signaling Pathway with CDC25B Being Involved

The aim of this experiment was to investigate whether FSH could regulate the proliferation of calf Sertoli cells and the relationship with CDC25 B through Wnt/β-catenin signaling pathway. The experimental method was to culture Sertoli cells with different concentrations of FSH(40, 80, 120 and 150 ng · mL) and to treat the cultured cells with XAV939(β-catenin inhibitor) and to detect the expression of CDC25 B, CDC2, C-MYC and β-catenin. The results of the experiment showed that FSH could promote the proliferation of cells and its effect was good at 80 ng · mLconcentration. FSH could promote the expression of CDC25 B, CDC2, C-MYC and β-catenin. The expression of C-MYC and β-catenin in group FSH+XAV939 was lower than that in group FSH. There was no difference in mRNA expression of CDC25 B in group FSH and group FSH+XAV939. The protein expression of CDC25 B in group FSH+XAV939 was lower than that in group FSH. The conclusion was that FSH could promote the proliferation of calf Sertoli cells through Wnt/β-catenin signaling pathway. CDC25 B was upregulated by FSH and CDC2 could promote the proliferation of calf Sertoli cells. The protein level of CDC25 B was affected by Wnt/β-catenin signaling pathway.

Effects of Lycium barbarum polysaccharides on the proliferation and differentiation of primary Sertoli cells in young rats

Objective:Sertoli cells(SCs)provide physical support and material supply for germ cells and participate in the formation of blood-testis barrier.The number of SCs is directly proportional to the number of germ cells.And mature SCs ensure the growth of germ cells and the production of sperm.In this study,we explored the effect and underlying mechanism of Lycium barbarum polysaccharides(LBP)on primary SCs in young rats.Methods:Primary SCs were isolated from the testis of 20-day old rats.The cells were then treated with different concentrations of LBP.Immunocytochemistry was used to detect the expression of Ki67 and the androgen receptor(AR),and western blotting was used to detect the expression of cytokeratin-18(CK-18),AR and phosphorylated Akt(Ser473)in SCs.Results:The number of SCs increased significantly after LBP treatment,and the 100 mg/mL.LBP group had 14%more cells than the control group.The expression of Ki67 in LBP treated groups also increased significantly.LBP inhibited the expression of cytokeratin 18 in SCs.Besides,LBP increased the expression of AR on SCs and promoted the activation of Akt at the ser473 phosphorylation site.Conclusion:LBP promotes the proliferation of immature SCs in young rats and also accelerates their differentiation and maturation.This seems to be associated with activation of the Akt signaling pathway via up-regulation of AR.

Effect of Ureaplasma Urealyticum Infection on the Interleukin-1 Secretion in Rat Sertoli Cells

Objective To investigate the effect of Ureaplasma Urealyticum(UU) on the expression of IL 1 secretion in rat Sertoli cells Material & Methods Isolated rat Sertoli cells were infected by living UU, UU supernatants, inactivated UU of different dosage respectively. IL 1 secretion by rat Sertoli cells is determined by using group t test and F test. Results UU infection, both living UU and supernatant without UU, could inhibit the IL 1 secretion in rats Sertoli cells (P<0.01). Conclusion The infection of Sertoli cells by UU inhibit the biological function of the Sertoli cells in rat.

Activation of extracellular signal-related kinases 1 and 2 in Sertoli cells in experimentally cryptorchid rhesus monkeys

Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. Methods： Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. Results： The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 （CK18）, a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Condusion： The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.

Upregulation of macrophage migration inhibitory factor and calgizzarin by androgen in TM4 mouse Sertoli cells

Aim： To identify proteins induced by androgen in Sertoli cells during spermatogenesis. Methods： We analyzed protein profiles in TM4 Sertoli cells treated with dihydrotestosterone （DHT） using surface enhanced laser desorption ionization time-of-flight mass spectrometry （SELDI-TOF-MS）. Results： We found increases in the expression of a 5.0-kDa protein at 15 min, an 11.3-kDa protein at 24 h and 4.3 kDa, 5.7 kDa, 5.8 kDa, 9.95 kDa and 9.98 kDa proteins at 48 h after the treatment. In contrast, the expression of 6.3 kDa and 8.6 kDa proteins decreased at 30 min, and 4.9 kDa, 5.0 kDa, 12.4 kDa and 19.8 kDa proteins at 48 h after the treatment. The ll.3-kDa protein was identified as macrophage migration inhibitory factor （MIF） known to having various functions. The 9.98-kDa protein was identified as calgizzarin related to calcium channels. The timing of their expression suggests that MIF and calgizzarin are involved in late regulation of spermatogenesis in Sertoli cells by androgen. Conclusion： MIF and calgizzarin are two important androgen-responsive proteins produced by Sertoli cells and they might play a role in regulating spermatogenesis.

Profiling of miRNAs in porcine Sertoli cells

Background: Sertoli cells(SCs) create a specialized environment to support and dictate spermatogenesis.MicroRNAs(miRNAs), a kind of ~ 22 nt small noncoding RNAs, have been reported to be highly abundant in mouse SCs and play critical roles in spermatogenesis. However, the miRNAs of porcine SCs remain largely unknown.Methods: We isolated porcine SCs and conducted small RNA sequencing. By comparing miRNAs in germ cells, we systematically analyzed the miRNA expression pattern of porcine SCs. We screened the highly enriched SC miRNAs and predicted their functions by Gene Ontology analysis. The dual luciferase assay was used to elucidate the regulation of tumor necrosis factor receptor(TNFR)-associated factor 3(TRAF3) by ssc-miR-149.Results: The analysis showed that 18 miRNAs were highly expressed in SCs and 15 miRNAs were highly expressed in germ cells. These miRNAs were predicted to mediate SC and germ cell functions. In addition, ssc-miR-149 played critical roles in SCs by targeting TRAF3.Conclusion: Our findings provide novel insights into the miRNA expression pattern and their regulatory roles of porcine SCs.

CDC25B Involved in Proliferation of Sertoli Cells of New Born Calves Through FSH and Possibly Being Key Regulating Factor

The effects of FSH on the proliferation of sertoli cells of new born calves were studied in order to provide some data for theoretical research and practical use of spermatogenesis in vitro. Different concentrations of FSH （0, 0.01, 0.02, 0.04, and 0.08 IU· mL^-1） were taken to treat bovine sertoli cells in vitro culture, the number of sertoli cells and the expression of seven genes were determined at 6, 12 and 24 h after FSH treatments. FSH could significantly promote the proliferation of in vitro cultured sertoli cells. FSH had no significant effects on the expression of CDC25A and could significantly improve the expression of CDC25B. 0.04 IU· mL^-1 and 0.08 IU· mL^-1 FSH treatments decreased the expression of CDC25C at 12 h. 0.08 IU· mL^-1 FSH treatment decreased the expression of CDC25C at 24 h. 0.04 IU. mLI FSH could significantly decrease the expression of GSK-3β and improve the expression of β-catenin at 6, 12 and 24 h. 0.02, 0.04 and 0.08 IU· mL^-1 FSH treatments enhanced the expressions of CYCLIND1 and C-MYC. In conclusion, FSH promoted the proliferation of sertoli cells and 0.04 IU· mL^-1 FSH concentration could significantly promote the proliferation of in vitro cultured sertoli cells. FSH promoted the proliferation of sertoli cells by CDC25B and WNT/ β-eatenin and CDC25B might be the key regulator to the proliferating rate of sertoli cells of bovine calf.

Disruption of ectoplasmic specializations between Sertoli cells and maturing spermatids by anti-nectin-2 and anti-nectin-3 antibodies

Aim： To understand the biological functions of the ectoplasmic specializations between Sertoli cells and maturing spermatids in seminiferous epithelia. Methods： In order to disrupt the function of the ectoplasmic specializations, nectin-2, which is expressed at the specialization, was neutralized with anti-nectin-2 antibody micro-injected into the lumen of the mouse seminiferous tubule. Anti-nectin-3 antibody was also micro-injected into the lumen in order to neutralize nectin-3, which is expressed at the specialization. Results： The actin filaments at the specialization disappeared, and exfoliation of maturing spermatids was observed by electron microscopy. Conclusion： Nectin-2 was neutralized by anti-nectin-2 antibody and nectin-3 was neutralized by anti-nectin-3 antibody, respectively. Inactivated nectin-2 and nectin-3 disrupted the nectin-afadin-actin system, and finally the actin filaments disappeared. As a result, the specialization lost the holding function and detachment of spermatids was observed. One of the functions of the specialization seems to be to hold maturing spermatids until spermiation.

17β-Estradiol Regulates SKP2 Expression in Cultured Immature Boar Sertoli Cells Mainly via Estrogen Receptor β,cAMP-PKA and ERK1/2

Estrogen plays an important role in regulating testicular Sertoli cell number. Furthermore, S-phase kinase-associated protein 2 （SKP2） plays a central role in mammalian cell cycle progression. The objective of this study was to determine whether 17β-estradiol can regulate the expression of SKP2, and the Sertoli cell cycle, via estrogen receptor β （ERβ）, the cyclic adenosine monophosphate （cAMP）-protein kinase A （PKA） and extracellular signal-regulated kinase （ERK1/2） pathway. When cultured immature boar Sertoli cells were treated with 17β-estradiol, a time-dependent increase in SKP2 mRNA and protein level was observed by real-time PCR and Western blot, and 17β-estradiol activity peaked at 30 min. Treatment with ICI182780 and ERβ antagonist reduced 17β-estradiol-induced expression of SKP2 and proliferating cell nuclear antigen （PCNA）, while increasing the protein concentration of p27kip1. However, the effect of ERa antagonist on these parameters was lower than that of ICI 182780 and ERβ. Forskolin had a similar effect as 17β-estradiol on the expression of SKP2, PCNA and p27kip1, Rp-cAMP, H-89 and U0126 treatment reduced 17β-estradiol-induced changes, while H-89 also inhibited ERK1/2 activation. Therefore, 17β-estradiol mainly regulates SKP2 mRNA and protein expression via ERβ-cAMP-PKA and ERK1/2 activation. SKP2 and PCNA expression were positively correlated, while increased SKP2 expression likely resulted in p27kip1 degradation.

Influence of FSH Treatment on Expression of CDC25A, TSSK3 and P53 in Vitro Cultured Sertoli Cells of Calf

CDC25A, TSSK3 and P53 expressions in vitro in cultured sertoli cells after FSH treatment were studied in order to provide some data for further researches of spermatogenesis. Different concentrations of FSH（0, 0.01, 0.02, 0.04, and 0.08 IU ·m L^-1） were used to treat sertoli cells cultured in vitro. The expression of CDC25 A, TSSK3 and P53 was determined by real-time-PCR at 6 h,12 h and 24 h after FSH treatment of sertoli cells. The results showed that FSH had no significant effect on expression of CDC25A（p〈0.05）, could significantly improve the expression of TSSK3 and P53（p0.05）, and had no significant effect on expression of CDC25 A in sertoli cells, but it could significantly improve the expression of TSSK3. CDC25 A was likely to play a role in other signaling pathways in sertoli cells. Within the range of certain concentration of FSH, TSSK3 in sertoli cells had the highest expression at about 24 h. TSSK3 protein produced in sertoli cells was likely to play an important role in substrate-level phosphorylationbe in meiosis and mitosis of spermatogenic cells. FSH could promote P53 expression and the highest expression was at about 12 h, and P53 might control the division of spermatogenic cells as well as sertoli cells.

Effects of mature Sertoli cells on allogeneic islets cocultured in vitro

Objective： To set up a method for isolation and culture of mature Sertoli cells and to estimate their effects on allogeneic islets cocultured in vitro. Methods： Adult SD rat testicular Sertoli cells were prepared successfully by three-step enzyme digestion. Then they were cocultured respectively with allogeneic islets and activated Wistar rat splenocytes. 24-hour cumulative insulin release and glucose-stimulated insulin secretion test were performed to detect islet function between pure islets culture group and coculture group. Splenocyte proliferation activity was determined by MTT colorimetry assay to observe the inhibition effect of Sertoli cells in different densities. Result： Firstly, in pure islet culture group, the 24-hour cumulative insulin release was gradually decreased in 21-day culture time. Compared to day 3, this change was significant on day 7 （P 〈 0.05） and on day 10,14,21 （P 〈 0.01）. In contrast, in coculture group, compared to day 3, the 24-hour cumulative insulin release was increased significantly on day 7 （P 〈 0.01 ）, and then gradually decreased on day 10 and 14, but still higher than that of day 3. It was on day 21 that it began to decrease compared to day 3 （P 〈 0.05）. During the culture time in vitro, the 24-hour cumulative insulin release of islet coculture group was significantly higher than that of pure islets culture group （P 〈 0.01）. In the case of stimulation index（SI）, there was a similar tendency as insulin release in the two groups. Secondly, mature Sertoli cells（1×10^6/mL） pretreated by 15 grays irradiation could decrease proliferation activity of activated splenocytes compared to that of control group （P 〈 0.01 ）. This inhibition effect was dose-dependent. Conclusion： Mature Sertoli cells can improve the function and prolong the survival of islet cells cultured in vitro. They can also provide an immune protection to islet cells. The approach described above might be applicable to human islet transplantation as soon as if it is also valid in large animal models.

Low XIST expression in Sertoli cells of Klinefelter syndrome patients causes high susceptibility of these cells to an extra X chromosome

Klinefelter syndrome(KS)is the most common genetic cause of human male infertility.However,the effect of the extra X chromosome on different testicular cell types remains poorly understood.Here,we profiled testicular single-cell transcriptomes from three KS patients and normal karyotype control individuals.Among the different somatic cells,Sertoli cells showed the greatest transcriptome changes in KS patients.Further analysis showed that X-inactive-specific transcript(XIST),a key factor that inactivates one X chromosome in female mammals,was widely expressed in each testicular somatic cell type but not in Sertoli cells.The loss of XIST in Sertoli cells leads to an increased level of X chromosome genes,and further disrupts their transcription pattern and cellular function.This phenomenon was not detected in other somatic cells such as Leydig cells and vascular endothelial cells.These results proposed a new mechanism to explain why testicular atrophy in KS patients is heterogeneous with loss of seminiferous tubules but interstitial hyperplasia.Our study provides a theoretical basis for subsequent research and related treatment of KS by identifying Sertoli cell-specific X chromosome inactivation failure.

Isolation and culture of adult Sertoli cells and their effects on the function of co-cultured allogeneic islets in vitro

Background Globally, 180 million people suffer from diabetes mellitus. Islet transplantation is believed to be an almost ideal therapy for insulin-dependent patients. How to maintain the viability and the function of isolated human islets is a challenge in clinical practice. Sertoli cells are considered ＇ nurse cells＇ in the seminiferous tubules and have been used in cell graft protocols for neurodegenerative diseases and diabetes in many studies. Many researchers have used immature murine testes as the primarily source of Sertoli cells in islet transplantation because they are easily purified. Mature human Sertoli cells have been seldom investigated. In the present study, we developed a method for the isolation and culture of Sertoli cells derived from adult human testes, and investigated their effects on the function of allogeneic islets when they were cultured together in vitro. Methods Adult Sertoli cells were prepared successfully by two-step enzyme digestion with trypsin, collagenase and hyaluronidase. They were identified by morphological characteristics and their activity was determined by MTT colorimetry over a 28-day culture time in vitro. A glucose-stimulated insulin secretion test was performed to detect the effects of Sertoli cells on allogeneic islets＇ function when they were co-cultured for 21 days in vitro. Results In cultured cells, mature human Sertoli cells accounted for more than 90% of total cells. The activity of Sertoli cells reached 95 % and they remained highly cytoactive for a long time in vitro （P〉0. 05 ）. Compared with the islets cultured alone, the co-cultured islets with allogeneic Sertoli cells maintained higher sensitivity to glucose stimulation for the duration of the experiment （P〈0.01 ）. Conclusions A method of isolation and culture of Sertoli cells from adult testes has been established. Sertoli cells could enhance allogeneic islets＇ function when they were co-cultured in vitro. They could be a helper cell in islet transplantation.

NODAL secreted by male germ cells regulates the proliferation and function of human Sertoli cells from obstructive azoospermia and nonobstructive azoospermia patients

This study was designed to explore the regulatory effects of male germ cell secreting factor NODAL on Sertoli cell fate decisions from obstructive azoospermia （OA） and nonobstructive azoospermia （NOA） patients. Human Sertoli cells and male germ cells were isolated using two-step enzymatic digestion and SATPUT from testes of azoospermia patients. Expression of NODAL and its multiple receptors in human Sertoli cells and male germ cells were characterized by reverse transcription-polymerase chain reaction （RT-PCRI and immunochemistry. Human recombinant NODAL and its receptor inhibitor SB431542 were employed to probe their effect on the proliferation of Sertoli cells using the CCK-8 assay. Quantitative PCR and Western blots were utilized to assess the expression of Sertoli cell functional genes and proteins. NODAL was found to be expressed in male germ cells but not in Sertoli cells, whereas its receptors ALK4, ALK7, and ACTR-IIB were detected in Sertoli cells and germ cells, suggesting that NODAL plays a regulatory role in Sertoli cells and germ cells via a paracrine and autocrine pathway, respectively. Human recombinant NODAL could promote the proliferation of human Sertoli cells. The expression of cell cycle regulators, including CYCLIN A, CYCLIN D1 and CYCLIN E, was not remarkably affected by NODAL signaling. NODAL enhanced the expression of essential growth factors, including GDNF, SCF, and BMP4, whereas SB431542 decreased their levels. There was not homogeneity of genes changes by NODAL treatment in Sertoli cells from OA and Sertoli cell-only syndrome （SCO） patients. Collectively, this study demonstrates that NODAL produced by human male germ cells regulates proliferation and numerous gene expression of Sertoli cells.

Comparative Potency of Seven Compounds Isolated from Tripterygium Wilfordii on Cultred Rat Leydig Cells and Sertoli Cells

The damaging effect of seven compounds isolated from Triplelygium Wilfordii Hook.F were tested on the rat Leydig cells and Sertoli cells in culture. The reslllts showed thai 10 μg/ ml orthosphenic acid (TW2), 10μg/ ml 39,22α -dihydroxy-△12 ursen- 30-oic acid(TW5), 5.0 μg/ ml 39,22a--dihydroxy-△12-oleallen--29-oic acid(TW6), 5.0 μg/ ml salaspermic acid(TW7), 0.5μg/ ml celastrol(TW27) alld 1.25μg/ ml polpunonic acid (TW28) Ivere all detrimental to Leydig cells. and Sertoli cells. Holvever, 30 μg/ ml wilforlide A (TW1) did not affect the viability of Leydig cells and Sertoli cells significantly.The most toxic compounds was TW27, and the leasl,TW1.These results suggested that the-COOH at the C-20 site of these triterpenoids may be the functional chemical group responsible for the damaging effect on the Leydig cells and Sertoli. cells.

A Chinese Herbal Formula,Wuzi Yanzong Pill(五子衍宗丸),Improves Spermatogenesis by Modulating the Secretory Function of Sertoli Cells

Objective： To evaluate the effects of the Chinese herbal formula Wuzi Yanzong Pill （五子衍宗丸, WYP） on the spermatogenesis and specific secretory functions of Sertoli cells in rat model and to investigate the underlying mechanism. Me^ods： Five groups of male Sprague-Dawley rats including the control group, the model group, the low-dose WYP group, the medium-dose WYP group and the high-dose WYP group （5 in each group） were treated daily with vehicle, multiglycosides of Tripterygium wilfordii Hook f （GTW） either alone （20 mg/kg） or followed by WYP （0.5, 1.0, or 2.0 g/kg daily）, respectively for 30 days. Serum levels of follicle-stimulating hormone （FSH）, inhibin B （INHB） and testosterone （r） were evaluated using enzyme-linked immunosorbent assay. Androgen- binding protein （ABP） gene expression and transferrin （TF） protein expression in testis tissue specimens of all rats were determined using real-time reverse transcriptase polymerase chain reaction and Western blotting analysis, respectively. Histopathological alterations in the testis were determined using Johnsen＇s score. Results： The toxicity of GTW towards Sertoli cell secretory functions and spermatogenesis was accompanied by increased serum FSH concentrations and decreased INHB and T concentrations. Upregulated ABP mRNA levels, and decreased TF protein expression and Johnsen＇s scores were detected in the model group compared with the control group （P〈0.05 or P〈0.01）. Oral high-dose WYP administrations to GTW-treated rats effectively alleviated all of the GTW- induced changes in specific secretory functions of Sertoli cells （ABP, INHB and TF）. Furthermore, serum T level and Johnsen＇s score of the testis increased greatly compared with the model group （P〈0.01）. Conclusion： WYP has the ability to improve the spermatogenesis, possibly through modulating the secretory proteins expression of Sertoli cells.