Primary Culture and Identification of Rat Testicular Sertoli Cells in Vitro and Expression of Inhibin Inside the Cells
0
Citation
0
Reference
20
Related Paper
Abstract:
Objective:To establish a primary culture method of rat testicular Sertoli cells and study the expressions of inhibin in Sertoli cells.Methods:Sertoli cells aged 18-22 days were separated and cultured from rat testes and the biological characteristics of Sertoli cells were observed,and the expressions of inhibin α,βA and βB subunits in Sertoli cells were detected by means of immunohistochemistry.Results:Sertoli cells in rat testicles were successfully cultured in vitro in the present study and the degree of purity in rat Sertoli cells was more than 90% of the total cells.Sertoli cells in vitro were positive for Inhibin α,βA and βB subunits.Conclusion:In vitro culture method of rat Sertoli cells was successfully established.Sertoli cells in vitro were positive for inhibin α,βA and βB subunits and rat testicular Sertoli cells were capable of producing inhibin and proven to be the major source of inhibin.Keywords:
Testicle
FGF9
Cite
Cell type
Cite
Citations (118)
Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an α and a βB subunit. In adult testes, the cellular site of production is still controversial, and it was hypothesized that germ cells contribute to inhibin B production. To determine which cell types in the testes may produce inhibin B, the immunohistochemical localization of the two subunits of inhibin B were examined in adult testicular biopsies with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only (SCO) tubules. Moreover, using in situ hybridization with mRNA probes, the mRNA expression patterns of inhibin α and inhibin/activin βB subunits have been investigated. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin α subunit and expressed inhibin α subunit mRNA. Using inhibin βB subunit immunoserum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids but not in Sertoli cells. Inhibin βB subunit mRNA expression was intense in germ cells from spermatogonia to round spermatids and in Sertoli cells in these testes. In testes with SCO, high inhibin βB subunit mRNA labeling density was observed in both Sertoli cells and Leydig cells, whereas βB subunit immunostaining was negative for Sertoli cells and faintly positive for Leydig cells. These results agree with the recent opinion that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells.
Immunostaining
Testicle
Cite
Citations (83)
Data from several experimental approaches strongly suggest that Sertoli cells exert a paracrine control of the two main testicular functions, androgen secretion and spermatogenesis. Further evidence supporting this role of Sertoli cells was obtained by coculture of Sertoli cells with other testicular cells. Coculture of pig or rat Sertoli cells with pig Leydig cells produces an increase in the hCG receptor number and an increase in the steroidogenic activity of Leydig cells. Pretreatment with FSH further increases the values of these two parameters. These biochemical changes were associated with ultrastructural changes in Leydig cells. The effects of Sertoli cells on Leydig cells depend upon the ratio of the two cells and on the substrate in which the cells are cultured. Moreover, Leydig cells produce an increase in the FSH receptor number and in the FSH stimulation of plasminogen activator production by Sertoli cells. Coculture of rat or pig Sertoli cells with rat germ cells, induces an increase in the RNA and DNA biosynthetic activities of germ cells. Most of the stimulatory effects seemed to be mediated by diffusible factors, secreted by Sertoli cells, but full expression of the stimulatory action was observed when germ cells were in contact with other cells. In this coculture system, a fraction of rat germ cells containing mainly mature forms of spermatocytes inhibited rat Sertoli cell RNA and DNA synthesis, but had no effect on pig Sertoli cells. On the contrary, a fraction of rat germ cells richer in spermatogonias and preleptotene spermatocytes, stimulated rat Sertoli cell DNA synthesis but was without effect on pig Sertoli cells. These results clearly show that the stimulatory effects of Sertoli cells on Leydig and on germ cells which are not species specific are mediated mainly by diffusible factors, the secretion of which is regulates by FSH.
Cite
Citations (21)
Contents Formalin‐fixed, paraffin‐embedded sections from 32 canine pairs of testes were immunohistochemically examined for Inhibin‐α (INHα). Samples were subdivided into two groups (group 1, neonates; group 2, puppies and adults) and results statistically compared. Inhibin‐α was significantly expressed only in Sertoli cells of neonatal testes, while in Leydig cells it was expressed without significant difference between groups. These results suggest that, in dogs, INHα expression switches from Sertoli to Leydig cells during testicular maturation and that, in adult, Leydig cells represent the main source of INHα.
Cite
Citations (13)
Methods for the isolation and culture of enriched populations of Sertoli cells from 20-60 day old rats are described. The identity of the Sertoli cells was verified by bright light and electron microscopy. Freshly isolated Sertoli cells specifically bound follicle stimulating hormone (FSH) but not luteinizing hormone (LH) and responded to FSH stimulation with dramatic increase in cyclic AMP level. Isolated Sertoli cells, maintained in culture for 11 days, showed no evidence of proliferation but retained their characteristic ultrastructural features and FSH binding ability. Incubation of cultured cells with FSH resulted in a significant stimulation of cyclic AMP and androgen binding protein (ABP). Since the freshly isolated or cultured cells were predominantly (greater than 80%) Sertoli cells, these results provide direct evidence that the Sertoli cells represent a primary target site for FSH activity in the testes. The culture method also provides a valuable in vitro model for the study of chronic effects of various agents on the Sertoli cell.
Isolation
Cite
Citations (208)
To simplify the method for separation and cultivation of rat testicular Sertoli cells with high viability, quantity and expression efficiency.Testicular Sertoli cells from 2 to 3-week-old male Wistar rats were prepared by digestion with collagenase, trypsin and DNase and cultured together with active lymphocytes to observe their killing effect against lymphocytes. After cell culture for 72 h, the Sertoli cells were morphologically observed by different means and identified with transmission electron microscope. Fas ligand and follicle-stimulating hormone receptor (FSHR) were examined immunohistochemically to identify testicular Sertoli cells. SABC method was used for labeling the Fas ligand on the testicular Sertoli cells.The viability of the isolated and cultured Sertoli cells was more than 90%, and in in vitro culture, Sertoli cells, which expressed the Fas ligand, could kill the active lymphocytes.This method improves the efficiency in acquisition of rat testicular Sertoli cells expressing Fas ligand, which are believed to be a potential donor for co-transplantation with parathyroid cells to offer immune privilege.
Immune privilege
Testicle
FGF9
Cite
Citations (2)
Sertoli cell monolayers were prepared from 30‐day‐old rat testes and cultured for 7 days to eliminate contaminant germ cells. Some of these monolayers were co‐cultured with a spermatogenic cell preparation enriched in pachytene spermatocytes and round spermatids (fraction 3 from a Percoll gradient), isolated from 30‐day‐old rat testes. After co‐culture for 4 to 48 h, germ cells were removed. RNA synthetic activity in rat Sertoli cell cultures alone was 216 800 ± 66 480 dpm [ 3 ]uridine.2h −1 .10 6 cells −1 (mean ± SD) compared to 98 390 ± 23 595 in rat Sertoli cells which had been co‐cultured with germ cells of fraction 3 for 24 h ( P < 0.01). By contrast, RNA synthesis in Sertoli cell monolayers prepared from immature pigs were unaffected by co‐culture with rat germ cells. A similar inhibitory effect of germ cells was observed in rat Sertoli cells stimulated with FSH or testosterone. Culture medium, conditioned by 20 h culture of a fresh preparation of rat spermatogenic cells of fraction 3, was active in inducing the inhibitory effect on RNA synthesis in rat Sertoli cells. Co‐culture of rat Sertoli cells with germ cells of this fraction also decreased the incorporative of [ 3 H]thymidine into DNA in rat Sertoli cells, from 9061 ± 3339 to 4766 ± 526 dpm.2h −1 .10 6 cells −1 ( P < 0.01), but no such change was found in pig Sertoli cells. A different spermatogenic cell preparation, partially deprived of pachytene spermatocytes (fraction 5), stimulated rat Sertoli cell DNA synthesis (Sertoli alone 7833 ± 2550, Sertoli cells which had been in co‐culture with germ cells of fraction 5, 13 300 ± 2279 dpm.2h −1 .10 6 cells −1 , P < 0.05). These inhibitory actions of some germ cells on Sertoli cells were observed together with the previously reported simultaneous stimulatory effect of Sertoli cells on germ cells. These Sertoli cell‐germ cell interactions of detected in culture may represent regulatory influences operating in vivo.
Testicle
Cite
Citations (10)
Having recently demonstrated highly vectorial and FSH-stimulated inhibin secretion by immature rat Sertoli cells in vitro, we wished to determine if vectorial secretion of inhibin was also characteristic of primate Sertoli cells. By adapting techniques for isolation of Sertoli cells from testes of the immature rat and cynomolgus monkey. Sertoli cells were isolated from immature baboon testes. Sertoli cells were then plated onto matrix-impregnated porous filters and cultured in twin chamber assemblies in fully defined, supplemented HEPES-buffered Eagles medium. Inhibin was measured in conditioned culture media by an heterologous RIA validated for primate inhibins. Throughout 28 days of culture immunoreactive inhibin was readily detectable in the upper chambers whereas inhibin was undetectable or just detectable in the lower chambers. The median ratio of upper to lower chamber inhibin content was 15.3 under basal conditions rising to 41 under FSH stimulation. Inhibin secretion into the upper chamber was increased 2.5 +/- 0.4 times by stimulation with ovine FSH (100 ng/ml). We conclude that immature Sertoli cells from a nonhuman primate demonstrate FSH-responsive and highly vectorial secretion of inhibin almost exclusively into the upper chamber. These data suggest that during maturation mammalian Sertoli cells secrete inhibin vectorially mainly from the apical surface of the cell towards the seminiferous tubular lumen. The predominance of inhibin secretion into the seminiferous tubule during testicular maturation suggests that inhibin may have an important paracrine or autocrine role in the developmental biology of spermiogenesis.
Seminiferous tubule
Testicle
Cite
Citations (19)
ABSTRACT The influence of in-vitro conditions on the production of inhibin by Sertoli cells from 21-day-old normal and prenatally irradiated rat testes was studied by measuring inhibin activity in culture media, using the suppression of the release of FSH from cultured rat pituitary cells. Sertoli cells secreted inhibin-like activity during at least 21 days of culture, and cells cultured at 37 °C produced significantly more inhibin than those cultured at 32 °C. The presence of fetal calf serum had no significant effect on inhibin production at 32 °C, while at 37 °C the production was decreased. The presence of ovine FSH stimulated inhibin secretion, while inhibin concentrations in Sertoli cell culture media were decreased after the addition of testosterone. Testosterone, added together with ovine FSH, suppressed inhibin secretion when compared with the levels found in the presence of FSH alone. The presence of spermatogenic cells decreased the release of inhibin. From these results it was concluded that both Sertoli cells isolated from normal immature rat testes and those from testes without spermatogenic cells can secrete inhibin-like activity in culture. A number of discrepancies with in-vivo observations was observed. Therefore, it is likely that the in-vivo situation is too complicated for direct study of the regulation of inhibin production, because of mutual interactions between the testicular compartments. J. Endocr. (1986) 109, 411–418
Testicle
Cite
Citations (49)
ABSTRACT The ontogeny of testicular inhibin/activin in ducks was investigated. Testicular localization of three inhibin/activin subunits (α, β A and β B ) was determined in embryonic and newly hatched ducks from 12 days of incubation to 1 day of age, in immature ducks and in adult ducks. In the duck embryonic testis, positive α‐subunit immunostaining was first detected in the Leydig cells and Sertoli cells on day 15 of incubation, whereas β A ‐subunit and β B ‐subunit immunostaining were found in Sertoli cells and primary germ cells on day 18 of incubation. In 1 month old ducks, intense staining of α‐subunit was present in the seminiferous epithelium consistent with localization in Sertoli cells and primary germ cells, and the immunostaining of the β A ‐ and β B ‐subunit was also present in Sertoli cells and primary germ cells. Specific immunostaining with inhibin/activin α‐, β A ‐ and β B ‐subunits antisera occurred in Sertoli cells in the adult duck testes. In conclusion, it was shown that, in the duck testis, the majority of α‐, β A ‐ and β B ‐subunits are colocalized in Sertoli cells with a certain degree of staining in germ cells and the α‐subunit is present in Leydig cells of embryonic testes before day 18 of incubation. These results indicate that Sertoli cells and possibly germ cells in the embryonic testes of late stage of incubation and newly hatched ducks, immature ducks and mature ducks may produce bioactive inhibin dimers, inhibin A and inhibin B, as a possible regulator of follicle‐stimulating hormone secretion. Free inhibin/activin subunits and their dimers may also play an autocrine/paracrine role in the development of the testis and spermatogenesis. Furthermore, early onset of the α‐subunit in duck testes indicates that it may have an autocrine/paracrine effect on steroid hormones, which is important for sex differentiation.
Immunostaining
Testicle
Cite
Citations (1)