Final Sertoli Cell Number Research Articles

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.

Molecular Mechanisms and Signaling Pathways Involved in Sertoli Cell Proliferation.

Sertoli cells are somatic cells present in seminiferous tubules which have essential roles in regulating spermatogenesis. Considering that each Sertoli cell is able to support a limited number of germ cells, the final number of Sertoli cells reached during the proliferative period determines sperm production capacity. Only immature Sertoli cells, which have not established the blood-testis barrier, proliferate. A number of hormonal cues regulate Sertoli cell proliferation. Among them, FSH, the insulin family of growth factors, activin, and cytokines action must be highlighted. It has been demonstrated that cAMP/PKA, ERK1/2, PI3K/Akt, and mTORC1/p70SK6 pathways are the main signal transduction pathways involved in Sertoli cell proliferation. Additionally, c-Myc and hypoxia inducible factor are transcription factors which participate in the induction by FSH of various genes of relevance in cell cycle progression. Cessation of proliferation is a pre-requisite to Sertoli cell maturation accompanied by the establishment of the blood-testis barrier. With respect to this barrier, the participation of androgens, estrogens, thyroid hormones, retinoic acid and opioids has been reported. Additionally, two central enzymes that are involved in sensing cell energy status have been associated with the suppression of Sertoli cell proliferation, namely AMPK and Sirtuin 1 (SIRT1). Among the molecular mechanisms involved in the cessation of proliferation and in the maturation of Sertoli cells, it is worth mentioning the up-regulation of the cell cycle inhibitors p21Cip1, p27Kip, and p19INK4, and of the gap junction protein connexin 43. A decrease in Sertoli cell proliferation due to administration of certain therapeutic drugs and exposure to xenobiotic agents before puberty has been experimentally demonstrated. This review focuses on the hormones, locally produced factors, signal transduction pathways, and molecular mechanisms controlling Sertoli cell proliferation and maturation. The comprehension of how the final number of Sertoli cells in adulthood is established constitutes a pre-requisite to understand the underlying causes responsible for the progressive decrease in sperm production that has been observed during the last 50 years in humans.

HIF involvement in the regulation of rat Sertoli cell proliferation by FSH

The final number of Sertoli cells reached during the proliferative periods determines sperm production capacity in adulthood. It is well known that FSH increases the rate of proliferation of Sertoli cells; however, little is known about the transcription factors that are activated by the hormone in order to regulate Sertoli cell proliferation. On the other hand, Hypoxia Inducible Factors (HIFs) are master regulators of cell growth. HIFs are dimers of HIF-β and HIF-α subunits. Considering that HIF-β is constitutively expressed, HIF transcriptional activity is regulated through the abundance of HIF-α subunits. To date, three HIF-α isoforms have been described. The association of the different HIF-α subunits with HIF-β subunit constitutes three active transcription factors —HIF-1, HIF-2 and HIF-3— which interact with consensus hypoxia-response elements in the promoter region of target genes. Hypoxia has been classically considered the main stimulus that increases HIF transcriptional activity, however, regulation by hormones under normoxic conditions was also demonstrated. The aim of this work has been to investigate whether HIFs participate in the regulation of rat Sertoli cell proliferation by FSH. Sertoli cells obtained from 8-day old rats were cultured in the absence or presence of FSH. It has been observed that FSH increases HIF transcriptional activity and HIF-2α mRNA levels without modifying either HIF-1α or HIF-3α expression. Incubations with FSH have been also performed in the absence or presence of a pharmacological agent that promotes HIF-α subunit degradation, LW6. It has been observed that LW6 inhibits the FSH effect on proliferation, CCND1 expression and c-Myc transcriptional activity. Altogether, these results suggest that HIFs might be involved in the regulation of Sertoli cell proliferation by FSH.

Signal transduction pathways in FSH regulation of rat Sertoli cell proliferation

The final number of Sertoli cells reached during the proliferative periods determines sperm production capacity in adulthood. It is well known that FSH is the major Sertoli cell mitogen; however, little is known about the signal transduction pathways that regulate the proliferation of Sertoli cells. The hypothesis of this investigation was that FSH regulates proliferation through a PI3K/Akt/mTORC1 pathway, and additionally, AMPK-dependent mechanisms counteract FSH proliferative effects. The present study was performed in 8-day-old rat Sertoli cell cultures. The results presented herein show that FSH, in addition to increasing p-Akt, p-mTOR, and p-p70S6K levels, increases p-PRAS40 levels, probably contributing to improving mTORC1 signaling. Furthermore, the decrease in FSH-stimulated p-Akt, p-mTOR, p-p70S6K, and p-PRAS40 levels in the presence of wortmannin emphasizes the participation of PI3K in FSH signaling. Additionally, the inhibition of FSH-stimulated Sertoli cell proliferation by the effect of wortmannin and rapamycin point to the relevance of the PI3K/Akt/mTORC1 signaling pathway in the mitotic activity of FSH. On the other hand, by activating AMPK, several interesting observations were made. Activation of AMPK produced an increase in Raptor phosphorylation, a decrease in p70S6K phosphorylation, and a decrease in FSH-stimulated Sertoli cell proliferation. The decrease in FSH-stimulated cell proliferation was accompanied by an increased expression of the cyclin-dependent kinase inhibitors (CDKIs) p19INK4d, p21Cip1, and p27Kip1. In summary, it is concluded that FSH regulates Sertoli cell proliferation with the participation of a PI3K/Akt/mTORC1 pathway and that AMPK activation may be involved in the detention of proliferation by, at least in part, a decrease in mTORC1 signaling and an increase in CDKI expression.

Upstream Stimulatory Factor Induces Nr5a1 and Shbg Gene Expression During the Onset of Rat Sertoli Cell Differentiation1

Within the testis, each Sertoli cell can support a finite number of developing germ cells. During development, the cessation of Sertoli cell proliferation and the onset of differentiation establish the final number of Sertoli cells and, thus, the total number of sperm that can be produced. The upstream stimulatory factors 1 and 2 (USF1 and USF2, respectively) differentially regulate numerous Sertoli cell genes during differentiation. To identify genes that are activated by USF proteins during differentiation, studies were conducted in Sertoli cells isolated from 5- and 11-day-old rats, representing proliferating and differentiating cells, respectively. Usf1 mRNA and USF1 protein levels were increased between 5 and 11 days after birth. In vitro studies revealed that USF1 and USF2 DNA-binding activity also increased at 11 days for the promoters of four potential target genes, Fshr, Gata4, Nr5a1, and Shbg. Chromatin immunoprecipitation assays confirmed that USF recruitment increased in vivo between 5 and 11 days after birth at the Fshr, Gata4, and Nr5a1 promoters. Expression of Nr5a1 and Shbg, but not of Fshr or Gata4, mRNAs was elevated in 11-day-old Sertoli cells compared with 5-day-old Sertoli cells. Transient transfection of USF1 and USF2 expression vectors up-regulated Nr5a1 and Shbg promoter activity. RNA interference assays demonstrated that USF1 and USF2 contribute to Nr5a1 and Shbg expression in differentiating cells. Together, these data indicate that increased USF levels induce the expression of Nr5a1 and Shbg during the differentiation of Sertoli cells, whereas Fshr and Gata4 expression is not altered by USF proteins during differentiation.

Prenatal Plus Postnatal Exposure to Di(n-Butyl) Phthalate and/or Flutamide Markedly Reduces Final Sertoli Cell Number in the Rat

Androgens may be important regulators of Sertoli cell (SC) proliferation perinatally, with implications for the testicular dysgenesis syndrome (TDS) hypothesis. Fetal exposure of rats to 500 mg/kg . d di(n-butyl) phthalate (DBP) reduces fetal testosterone production and SC number at birth, but SC number recovers to normal by postnatal d (Pnd)25. It is unclear when and how SC proliferation is affected prenatally by DBP exposure or when and how postnatal compensation occurs. This study addressed these questions and investigated whether continued maternal exposure to DBP or to flutamide from Pnd1-Pnd15 could prevent SC number compensation, because this would have implications for how sperm counts might be lowered in TDS. DBP exposure attenuated SC proliferation by 7-18% throughout embryonic d (e)15.5-e21.5 (P < 0.05 at e21.5). After birth, SC proliferation increased significantly (>1.5-fold) between Pnd6 and Pnd10 in prenatally DBP-exposed animals, explaining the compensation. Continued maternal administration of DBP after birth attenuated (19% reduction) SC number compensation at Pnd25 and maternal administration of flutamide (100 mg/kg . d) to prenatally DBP-exposed animals was even more effective (42% reduction), suggesting the postnatal compensatory increase in SC proliferation after prenatal DBP exposure is androgen dependent. SC maturation (Pnd25) was unaffected, based on analysis of expression of key proteins, but lumen formation/expansion was attenuated in parallel with treatment-induced reduction in SC number. Our results provide further evidence that perinatal SC proliferation is androgen dependent and, importantly, show that similar exposure of mothers to antiandrogenic chemicals before birth and during lactation reduces final SC number, with implications for the origin of low sperm counts in TDS.

USF1/2 Transcription Factor DNA-Binding Activity Is Induced During Rat Sertoli Cell Differentiation1

Each Sertoli cell can support a finite number of developing germ cells. During development of the testis, the cessation of Sertoli cell proliferation and the onset of differentiation determine the final number of Sertoli cells and, hence, the number of sperm that can be produced. We hypothesize that the transition from proliferation to differentiation is facilitated by E-box transcription factors that induce the expression of differentiation-promoting genes. The relative activities of E-box proteins were studied in primary Sertoli cells isolated from 5-, 11-, and 20-day-old rats, representing proliferating, differentiating, and differentiated cells, respectively. E-box DNA-binding activity is almost undetectable 5 days after birth but peaks with initiation of differentiation 11 days after birth and remains elevated. Upstream stimulatory factors 1 and 2 (USF1 and USF2) were found to be the predominant E-box proteins present within DNA-protein complexes formed after incubating E-box-containing probes with nuclear extracts from developing Sertoli cells. The known potentiator of Sertoli cell differentiation, thyroxine, increases USF DNA-binding activity in Sertoli cells before differentiation (5-day-old Sertoli cells) but not after differentiation is initiated (11- and 20-day-old Sertoli cells). The developmental-specific increase in USF1 and USF2 DNA-binding activity may facilitate the switch from proliferation to differentiation and, thus, determine the ultimate number of Sertoli cells present within the testes and the upper limit of fertility.

Sertoli cell proliferation in the fetal and neonatal rat testis: A continuous phenomenon?

Sertoli cell population kinetics, as evidenced by semi-quantitative immunolabeling for proliferating cell nuclear antigen (PCNA) and Ki-67, in developing Wistar rat male gonads of embryos and neonates [14.5 days post conception (dpc)-7 days post partum (dpp)], was investigated. Throughout the examined period a gradual increase of immunolabeled Sertoli cell number, associated with intense mitotic activity, was observed. PCNA labeling index of Sertoli cells increased from 66.67 (at 14.5 dpc) to 89.74 (at 18.5 dpc) and then dropped to 75.24 (at 20.5 dpc). At birth, the percentage of PCNA immunoreactive Sertoli cells reached 98.70% and remained high thereafter, attaining a peak value of 99.90% at 7 dpp. The percentage of Ki-67 immunoreactive Sertoli cells in the fetal testis increased from E14.5 (43.95%) to E20.5 (77.40%). The proliferation rate did not alter considerably in the neonatal testis until 5 dpp. At this point, a significant increase of the Ki-67 labeling index was observed and a peak value of 95.76% was reached at 7 dpp. The pattern of Sertoli cell proliferation with age and the establishment of the final Sertoli cell number in vivo established in the present study was compared to the results from earlier investigations reported in the literature and the observed fluctuation of dividing cell numbers, associated with immunolabeling results throughout the examined period, complements and extends existing data. An appraisal of the timing of Sertoli cell proliferation in other species, namely mouse and man, is presented. The current investigation may be useful in evaluating the potential influence of factors interfering with normal mitotic activity of Sertoli cells, including cell selection mechanisms, such as apoptosis, senescence, DNA repair and hormonal/paracrine growth modulation.

Evidence that androgens and oestrogens, as well as follicle-stimulating hormone, can alter Sertoli cell number in the neonatal rat

Neonatal treatment of male rats with diethylstilboestrol (DES) or a gonadotrophin-releasing hormone antagonist (GnRHa) reduces final Sertoli cell number, an effect presumed to occur via suppression of follicle-stimulating hormone (FSH). As both treatments also suppress androgen action, we investigated whether androgens and oestrogens might affect Sertoli cell nuclear volume/number in the rat using single or combined treatments that differentially affected FSH, testosterone and oestrogen (DES) levels. Neonatal treatment with flutamide (50 mg/ kg) significantly reduced Sertoli cell nuclear volume/ number per testis and blood inhibin-B levels at day 18, despite elevating FSH levels; this treatment also exacerbated the reduction in Sertoli cell nuclear volume per testis induced by treatment with 0.1 mug DES without affecting FSH levels. Treatment with 0.1 mug DES on its own also reduced Sertoli cell nuclear volume per testis without affecting FSH/testosterone levels, but co-administration of 0.1 mug DES+GnRHa, which suppressed FSH and testosterone levels, resulted in a markedly greater effect. Treatment with GnRHa alone or 10 mug DES alone grossly suppressed FSH and testosterone levels and reduced Sertoli cell nuclear volume/number per testis by approximately 60%, but co-administration of the two treatments had no greater effect than either alone. Co-administration of testosterone esters with 10 mug DES partially prevented the 10 mug DES-induced reduction in Sertoli cell nuclear volume per testis, and normalized FSH levels. In all treatment groups, plasma levels of inhibin-B paralleled changes in Sertoli cell nuclear volume/number per testis, but treatments that suppressed FSH levels (GnRHa, 10 mug DES) caused a proportionately greater reduction (approximately 90%) in inhibin-B levels than in Sertoli cell nuclear volume/number (50-60%). Germ cell volume per unit Sertoli cell was reduced in animals treated with 10 mug DES alone or in those treated with 0.1 mug DES plus either flutmaide or GnRHa, but otherwise remained relatively constant between treatment groups. It is concluded that, in the neonatal rat, (1) endogenous androgens, as well as FSH, play a physiological role in increasing Sertoli cell number, (2) exogenous oestrogen exposure can decrease Sertoli cell number without altering FSH levels, (3) these effects probably share a common pathway and (4) blood inhibin-B provides a robust indicator of change in Sertoli cell number.

In vitro proliferation of ICR mouse sertoli cells supplemented with recombinant h-FSH (rh-FSH) at different concentrations

Objective: Sertoli cells are important nurse cells in the seminiferous tubules for male germ cell growth and differentiation into spermatozoa. In contrast to the germ cells, Sertoli cells have receptors for follicle stimulating hormone, which are the main hormonal regulators of spermatogenesis. Hormonal control of spermatogenesis involves control of the maturation and supporting activities of Sertoli cells. Impairment of Sertoli cell function has severe consequences for spermatogenesis. In adult testes, the final number of Sertoli cells correlates with the increase of germ cell production and total sperm output. In this study, we have cultured Sertoli cells for studying the growth kinetics of these cells in culture condition in vitro. Design: With the aim of setting up a proliferation assay to investigate the control of Sertoli cell division, we used an easy and rapid technique, based on the use of selective lectin binding which allows to obtain functional Sertoli cells. Materials/Methods: Datura stramonium agglutin (DSA)-coated dish were used to rapidly single Sertoli cells from immature mouse testis. Isolated Sertoli cells were cultured for 17 days in Eagle’s minimal essential medium (EMEM) with supplements according treatment groups. The proliferative activity of Sertoli cells to FSH responsiveness was examined at each different concentration( 0, 37.5, 75, 150, 300 and 600 mIU/ml) of recombinant h-FSH (rh-FSH). The cell purity of Sertoli cell cultured was examined by immunocytochemical procedure using mouse monoclonal anticytokeratin Pan. Results: The rh-FSH dose of 37.5 mIU/ml and 75 mIU/ml showed the dose dependent increase of Sertoli cells. The supplemented rh-FSH was found to stimulate Sertoli cell proliferation in culture condition. But, the proliferation of cells in rh-FSH dose of 300 mIU/ml and 600 mIU/ml were not showed in this study. Hyper-dose of rh-FSH was showed detrimental effect of the Sertoli cell proliferation. Conclusions: The Sertoli cell culture using immunocytochemistry and DSA-coated dish is useful tool in studies of growth kinetics and cell proliferation in vitro. Also the optimal dose of rh-FSH was important factor for Sertoli cell proliferation.

Inhibin B levels in plasma of the male rat from birth to adulthood: effect of experimental manipulation of Sertoli cell number.

Sertoli cells undergo important changes in their number and function at different ages in the rat and may be the primary source of circulating inhibin B. The aims of this study were 1) to establish the profile of inhibin B levels from birth to adulthood in normal rats and 2) to identify whether experimental manipulation of Sertoli cell numbers was able to alter this profile. Levels of inhibin B, measured by a specific two-site assay, increased fivefold in normal Wistar rats between day 3 and days 10-15, plateaued, and then declined in late puberty to reach adult levels which were approximately 60% of those observed on days 10-15. The increase in inhibin B levels in the neonatal period coincided with the period of Sertoli cell multiplication as indicated by incorporation of bromodeoxyuridine. Neonatal treatment of rats with a GnRH antagonist (GnRHa) reduced Sertoli cell number and adult testis weight by 48% and significantly reduced plasma levels of inhibin B at all ages through to adulthood. Induction of neonatal hypothyroidism in Sprague-Dawley rats by administration of propylthiouracil (PTU) up to day 25 of age increased final testis weight by 41% (indicative of increased Sertoli cell numbers) and resulted in elevation of plasma levels of inhibin B at all ages beyond 7 days of age. The degree of change in inhibin B levels in adult rats in the two experimental treatment groups was approximately proportional to the change in final testis weight. Plasma follicle-stimulating hormone (FSH) showed changes opposite to inhibin B, with levels being lowered in PTU-treated rats and elevated (beyond day 25) in GnRHa-treated animals. The present results suggest that final Sertoli cell number per testis exerts an important effect on the circulating level of inhibin B (and FSH) in the rat. These findings are compared to the emerging data for the human male.

High neonatal triiodothyronine levels reduce the period of Sertoli cell proliferation and accelerate tubular lumen formation in the rat testis, and increase serum inhibin levels.

T3 was injected daily in newborn rats from birth to 16 days of age. Control rats received daily injections of vehicle during the same period. The proliferative activity of the Sertoli cells was studied by means of bromodeoxyuridine incorporation, and tubular lumen formation and nuclear size were taken as markers of Sertoli cell differentiation. T3 treatment strongly reduced the proliferative activity of Sertoli cells from day 7 on, and on day 12, proliferation of Sertoli cells had ceased, while in control rats proliferating Sertoli cells were observed up to day 16. As a result of the reduced Sertoli cell proliferation, the final Sertoli cell number per testis at 23 days of age was reduced by 50% from 38 +/- 1 x 10(6) in control rats to 19 +/- 1 x 10(6) in T3-treated rats. Lumen formation in seminiferous tubules of T3-treated rats began at 12 days of age, while in controls lumen formation was first observed at 16 days. The area of the Sertoli cell nuclei was somewhat larger in T3-treated rats on day 16, but not at any other age examined. Body and testis weights in adult rats at 100 days of age were reduced by 46% and 48% of control values, respectively. The high neonatal T3 levels reduced serum levels of TSH on days 7 and 9, but not at any other age examined. FSH levels were reduced in T3-injected rats on days 5 and 7 and increased on day 23, after cessation of treatment. Immunoreactive inhibin-alpha levels were increased on days 5-9 and reduced on days 16 and 23. These findings indicate that T3 stimulates the production of immunoreactive inhibin by Sertoli cells, but also of bioactive inhibin, as indicated by the reduced FSH levels. It is concluded that the levels of thyroid hormones early in life are important for the terminal differentiation of Sertoli cells and, therefore, for determining adult testis size. The data indicate that this might be a direct effect of T3 on Sertoli cells.