The role of activin, follistatin and inhibin in testicular physiology.

The role of the inhibins, activins and follistatins in testicular function are being more clearly defined following studies describing the cellular localisation of these proteins to the testis and the availability of specific assay systems enabling measurement of these proteins. Taken together with the results of targetted gene inactivation experiments, several concepts emerge. Inhibin B is predominantly produced by the Sertoli cell in many adult male mammals whereas there is a perinatal peak of inhibin A in the rat. In contrast, activin A has its highest concentrations in the immediate post-natal period during which it is involved in the developmental regulation of both germ cells and Sertoli cells being modulated by follistatin. Activin A levels are considerably lower in the adult testis but Sertoli cell production is stimulated by interleukin-1 and inhibited by FSH. Little is known about the production of activin B due to the absence of a suitable assay but the beta(B) subunit mRNA is expressed in germ cells and Sertoli cells and is stage-dependent. This pattern of expression suggest that it may be involved in autocrine or paracrine actions within the seminiferous epithelium.

Marked extension of proliferation of rat Sertoli cells in culture using recombinant human FSH.

Previous studies indicate that proliferation of rat Sertoli cells in culture can only be maintained until the equivalent of days 10-12 after birth, irrespective of the age of the donor animal. This report describes methods for the isolation and culture of Sertoli cells from day 6 rat testes, which can proliferate in culture for 20-24 days (that is, until the equivalent of days 26-30 after birth). Cells were isolated by enzymatic digestion of seminiferous cords followed by selective depletion of contaminating peritubular cells by adhesion to a polystyrene surface. The purity of the Sertoli cells was assessed using a combination of markers to be > 99.5%. Proliferation was assayed using tritiated thymidine incorporation and further verified by bromodeoxyuridine histochemistry and flow cytometry. Sertoli cells proliferated at basal levels in Dulbecco's modified Eagle's medium (DMEM)-F12 media alone, and proliferation was stimulated further by addition of recombinant human FSH to the culture media. After 20-24 days in culture, proliferation rapidly ceased, and cells assumed abnormal morphology and detached from the culture vessel; these events are consistent with the cells undergoing classic rodent cell senescence. The method described provides a useful tool for investigating the control of Sertoli cell division. Furthermore, these findings indicate that the timely differentiation of Sertoli cells is not dependent solely on an intrinsic timing mechanism, as has been suggested previously.

Developmental expression of thyroid hormone receptors in the rat testis.

Sertoli cell proliferation in the rat is completed by Days 15-20 postnatally. Thyroid hormones appear to regulate the duration of Sertoli cell proliferation, affecting adult Sertoli cell number and hence the capacity of the testis to produce sperm. In the present study, a combination of immunohistochemistry, immunoblot analysis, and reverse transcription-polymerase chain reaction was used to demonstrate the expression pattern of thyroid hormone receptors (TR) in the juvenile and adult rat testis. The results indicated that TRalpha1 was expressed in proliferating Sertoli cell nuclei, its expression decreasing coincident with the cessation of proliferation. TRalpha2, TRalpha3, and TRbeta1 mRNAs were expressed at low levels during development; however, the corresponding protein was not detected by immunoblot analysis. In addition, TRalpha1 was found to be expressed in germ cells from intermediate spermatogonia to mid-cycle pachytene spermatocytes. Immunohistochemistry also demonstrated TR expression in a subset of interstitial cells. The demonstration of TR expression in germ cells undergoing spermatogenic differentiation suggests a possible role for thyroid hormones in the adult testis.