Leslie L. Heckert

The molecular biology of the FSH receptor

The actions of follicle stimulating hormone (FSH) mediated through its receptor are necessary for the proper functioning of mammalian gonads. The FSH receptor is localized on granulosa cells of the ovary and Sertoli cells of the testis. The expression of the FSH receptor (FSHR) in Sertoli cells varies in vivo as a function of the stage of the cycle of the seminiferous epithelium and in culture as a result of the addition of exogenous hormones. The gene for the FSH receptor is large and has been shown to be related in structure to the genes for luteinizing hormone (LH) receptor and thyroid stimulating hormone (TSH) receptor. The promoter region of the gene for FSHR does not contain a TATA box and has multiple transcriptional start sites. Less than 280 bp of the promoter are sufficient in transient transfection assays to direct expression of the chloramphenicol acetyl transferase gene (CAT) in a number of different cell types including non-gonadal cells. However, the promoter does direct the expression of a marker gene only into testis and ovary of transgenic mice.

Expression of the FSH receptor in the testis.

FSH has multiple and changing roles in the regulation of spermatogenesis. The first function of FSH is to increase the number of Sertoli cells by stimulation of their mitotic activity. During the prepubertal phase of development, FSH is important for the maturation of the Sertoli cells. Hormonal stimulation of tight junction formation and specific protein secretion are essential. In the adult rat, some of the functions carried out by FSH in prepubertal animals are assumed by testosterone. However, there is evidence that even in the adult rat, FSH is important for quantitatively normal spermatogenesis. The gene for the FSH receptor is large (greater than 85 kb) and complex (10 introns) and is structurally similar to the genes for the LH and TSH receptor. The promoter region of the FSHR gene has been identified and is active in the expression of transgenes in transfected Sertoli cells. We have shown that the FSH receptor mRNA is present in the testes of the adult rat and that the levels of this mRNA are changing during the cycle of the seminiferous epithelium. The presence of relatively high levels of FSHR mRNA in stages XIV-II of the cycle and the relatively low levels in stages VII-VIII suggest that the FSH receptor is carefully regulated in adult rats and presumably has an important function in spermatogenesis. The levels of FSHR mRNA in cultured Sertoli cells are immediately reduced in the presence of FSH or phorbol esters, but the levels soon return to normal.

Expression of Receptors during the Cycle of the Seminiferous Epitheliuma

The data presented in this manuscript are all based on some inferences drawn from past experimental observations. The first is that the synchronized testis model is representative of the normal testis. Support for this premise comes from the studies on SGP-2, SGP-1 and transferrin where results using in situ hybridization and Northern blots are similar for normal and for synchronized testes. The second inference is that normalizing all of the data to the relative levels of SGP-1 mRNA adjusts for the possible differential loading of mRNA samples. The logic of this practice is based on the observation obtained using in situ hybridization that SGP-1 mRNA levels did not change across the cycle. The third assumption was that total mRNA levels do not change greatly across the cycle. Wholesale changes in testicular mRNA such as the doubling of all of the mRNA transcripts per testis would not be accounted for by these studies. We feel that this is an unlikely complication because of the strong correlation between much of the data and the known biology. In addition, there is a strong correlation between our data on the FSHR mRNA and the binding data for FSH obtained by another laboratory and different techniques. The available data in the literature reveals that most of the Sertoli cell products which change in relative concentrations during the cycle of the seminiferous epithelium appear to have a maximum in either stages VII or IX or in stages XIII to III. Thus, the Sertoli cells in the cycle could be described has having two different functional modes. In mode A maximal levels of mRNA for a specific Sertoli cell product are roughly found in stages VII-IX and in mode B the maximal levels are found in stages XIII to III (Fig. 5). The distribution of the receptor mRNA and ABP mRNA can also be described in terms of these two modes of Sertoli cell function. Transferrin receptors, retinoic acid receptors, and androgen-binding protein appear to fall into mode A while FSH and androgen receptors fall into mode B (Table 1). Products which have antithetical functions such as FSH and inhibin or cystatin and cathepsin L are found in different modes. We propose that most of the actions of the Sertoli cell during the cycle can be specified by the dual modes described above rather than by an infinite number of operational modes.(ABSTRACT TRUNCATED AT 400 WORDS)

The Changing Functions of Follicle Stimulating Hormone in the Testes of Prenatal, Newborn, Immature, and Adult Rats

The target cells for the action of follicle stimulating hormone (FSH) in the testes of mammals are the Sertoli cells (Sc). The function of the Sc includes the physical and biochemical support for germ cell development into spermatozoa. Sc create an environment where germ cells are provided with metabolites, nutrients, and regulatory components. Thus, by exerting influence on the Sc and their functions, FSH indirectly influences spermatogenesis (1, 2). It is evident that in the rat, FSH is necessary to initiate the first wave of spermatogenesis, but its function in the adult rat testis is not well defined (3). The purpose of this manuscript is to review the probable actions of FSH during the development and maturation of the rat testes and describe recent experiments on the ontogeny and expression of the FSH receptor (FSH-R).

Two Regions Within the Proximal Steroidogenic Factor 1 Promoter Drive Somatic Cell-Specific Activity in Developing Gonads of the Female Mouse

Targets of steroidogenic factor 1 (SF1; also known as NR5A1 and AD4BP) have been identified within cells at every level of the hypothalamic-pituitary-gonadal and -adrenal axes, revealing SF1 to be a master regulator of major endocrine systems. Mouse embryos express SF1 in the genital ridge until Embryonic Day 13.5 (E13.5). Thereafter, expression persists in the male and is substantially lower in the female gonad until birth. We hypothesize that the sexually dimorphic expression of Sf1 during gonadogenesis is mediated by sex-specific regulation of its promoter. To investigate dimorphic regulation within the fetal gonad, we developed an experimental strategy using transient transfection of E13.5 gonad explant cultures and evaluated various Sf1 promoter constructs for sexually dimorphic DNA elements. The proximal Sf1 promoter correctly targeted reporter activity to SF1-expressing cells in both XY and XX gonads. Stepwise deletion of sequences from the Sf1 promoter revealed two regions that affected regulation within female gonads. Mutation of both sequences together did not cause further disruption of reporter activity, suggesting the two sites might work in concert to promote activity in female somatic cells. Results from gel mobility shift assays and fetal gonad-chromatin immunoprecipitation showed that TCFAP2 binds to one of the two female-specific sites within the proximal promoter of Sf1. Together, we show that transient transfection experiments performed within developing testes and ovaries are a powerful tool to uncover elements within the Sf1 promoter that contribute to sex-specific expression.

Expression of the FSH Receptor in the Sertoli Cells

The regulation of cell-specific genes could result from the interaction of unique transcription factors with specific promoter sequences. In the testes of mammals, follicle stimulating hormone receptors (FSH-Rs) are apparently located only on the Sertoli cells. Thus, the gene for the FSH-R and the gene for the mullerian inhibiting substance (MIS) are the only gene products known that are unique to the Sertoli cells. Our interest in gene expression in the Sertoli cells has stimulated our studies of the expression of the FSH-R gene in an effort to dissect important cell-specific regulatory elements.

Regulation of Expression of the FSH Receptor

Follicle stimulating hormone (follitropin [FSH]) is a pituitary glycoprotein hormone that is essential for normal reproduction in both male and female mammals. Sertoli cells and granulosa cells are the target cells for the action of FSH in the testes and ovaries, respectively. The function of the Sertoli cells includes the physical and biochemical support for germ cell development into spermatozoa. Sertoli cells create an environment where germ cells are provided with metabolites, nutrients, and physical support. Granulosa cells have similar overall functions in the ovary; that is, they play a role in the overall support of the ovum and the development of the follicle (reviewed in 1–3). According to this scenario, FSH indirectly influences spermatogenesis and oogenesis by exerting influences on the corresponding somatic cells (1, 2, 4–7). The primary action of FSH is mediated by increased concentrations of intracellular cAMP (8, 9). There is also evidence that FSH can alter intracellular calcium levels through mechanisms that are independent of both the protein kinase C pathway and the adenylate cyclase activity (10, 11).