8 The molecular biology of the ovary and testis

8 The molecular biology of the ovary and testis R. N. CLAYTON

Application of biochemical techniques to study of the physiology of ovarian and testicular function has been in progress for many years. Early work investigated the steroidogenic enzyme cascade using classical biochemical techniques. This was followed by detailed analysis of the mechanism of gonadotrophin action in the respective target cells, including characterization of receptors for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and subsequently the signal transduction mechanisms linking receptors and cellular responses (Catt et ~al, '1980). More recently research has focused on the modulation of gonadotrophin action by intragonadal peptides, including growth factors (Adashi et al, 1985; Hsueh et al, 1985), insulin, inhibin and activin. The whole field of paracrine/autocrine action of these factors has identified some of the ways by which the different cellular compartments within the gonads communicate with each other, although at the same time this has highlighted the complexity of such heterogeneous tissues. With the widespread availability of recombinant D N A methods, these have been adopted by reproductive biologists as further tools to explore gonadal physiology. As such, much use is made of in vivo animal models and m vitro cell culture systems since these are readily accessible. However, the results must necessarily be treated with caution when extrapolated to humans. Despite much progress in this area we have still not reached the stage where the molecular pathogenesis of common clinical gonadal problems, such as polycystic ovarian disease and oligoasthenospermia, can be explained. As a consequence there have been few major breakthroughs in the treatment of these conditions. For example, ovulation induction still relies on the rather empirical treatment with antioestrogens and, if that fails, exogenous gonadotrophins in one form or another. The recent characterization of the inhibin-activin family of peptides using recombinant D N A techniques is an example where much initial promise remains to be translated into tangible clinical benefit. In writing this chapter, therefore, I am conscious that the clinical relevance of some of the basic research may currently seem remote but I am sure it is only a matter of time before benefits emerge. Consequently, I have selected areas in which significant recent advances have been made in our understanding of the physiology of the gonads by the application of recombinant D N A technology. Bailli&re's ClinicalEndocrinology and Metabolism--Vol. 2, No. 4, November 1988

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GONADAL DIFFERENTIATION AND SEX DETERMINATION

For 30 years it has been known that gender in humans is determined by the presence of a Y chromosome and if this is absent the phenotypic sex is female (Jacobs and Strong, 1959). The minor histocompatibility antigen, H-Y, was considered for some years to be the testis determining gene (Watchel et al, 1975). However, although H-Y and the recently defined testis determining factor (TDF) (Page et al, 1987) are both located on the Y chromosome, they map to different parts (McLaren et al, 1984; Simpson et al, 1987). Thus, it is now firmly established that, although male specific, H-Y is not TDF. Occasionally, examples of infertile men have been identified who are XX males and have been shown to carry a small part of the Y chromosome (Page et al, 1985). Conversely, occasional females are XY and carry small deletions of the Y chromosome (Disteche et al, 1986). These quirks of nature, identified by classical cytogenetic methods, opened the way to the cloning of the testis determining factor which is both necessary and sufficient for male sex determination in humans. This task was made possible because two individuals were particularly informative, enabling the responsible area of the Y to be narrowed down to 300 kilobase pairs (kb) of DNA. These are represented schematically in Figure 1, and imply that region 1A2 is the critical region of the short arm of the Y chromosome. About 200 kb from this region has been cloned, within which is an open reading frame encoding 404 amino acids. The intriguing thing about this sequence is its composition of 13 repeats of 28-32 residues, each containing two cysteine and two histidine residues characteristic of zinc-finger D N A binding proteins. (Page et al, 1987). This leads to the hypothesis that TDF is a transcriptional regulatory Pseudoautosomal

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protein, although its target genes are as yet unknown. Clearly of crucial importance to further understanding the process of testis differentiation, on which all future male phenotypic development is dependent, will be the identification and functional characterization of the genes and their proteins whose expression is dependent on TDF. Because of the zinc-finger motif, TDF has been redesignated ZFY (ZF for Zinc finger) and its homologue on the X chromosome (see below) is called ZFX. What other evidence is there that this region of the Y chromosome carries gene(s) primarily responsible for testis determination? Is this the only region of the genome with this function, or are there other areas involved? As yet there is no direct experimental proof for this, although several pieces of circumstantial evidence corroborate the story. Firstly, within the cloned 1A region there are several sequences that are highly conserved amongst D N A of various mammals (Page et al, 1987). Evolutionary conservation, though not synonymous with, is very suggestive of functional significance. Secondly, a Y sequence with homology to one of the conserved sequences of the human Y I A region has been detected in D N A from Sxr (sex-reversed) XX male mice (Singh and Jones, 1982). Moreover, it is of considerable interest to find that a gene similar to TDF is present on the X chromosome of humans and all other mammals examined (Page et al, 1987). This is perhaps not so surprising since it is thought that the human Y chromosome evolved from the X chromosome. Because of the high degree of conservation, the X homologue is unlikely to be a pseudogene. Moreover, because of X inactivation in females it is likely that only one copy is functional in females whereas in males the two genes, one from the X and one from the Y, are functional. Is the product from the X chromosome gene (ZFX) required for functional activity of the ZFY (TDF) or vice versa? Is testis differentiation dependent on gene dosage? This would presuppose that both the ZFY and ZFX gene products have identical functions. Some further insight on the roles of ZFY and ZFX has been obtained by what, at first sight, appeared contradictory evidence. Sinclair et al (1988) have cast some doubt that the testis determining function of ZFY is the universal primary event in all species. In marsupials ZFY-like sequences were not found on the Y chromosome, although in these species sex determination is known to depend on this chromosome, as in mammals. Instead the ZFY probe hybridized to autosomes and, furthermore, the marsupial X chromosome does not apparently contain ZFX. One possibility is that ZFY shows cross-hybridization with the marsupial homologue of mammalian ZFX on autosomes (designated ZFA). That this is likely is supported by the observation that probe for the Duchenne muscular dystrophy gene (DMD), which is close to ZFX in humans, hybridizes close to the ZFY probe on wallaby chromosome 5 (Sinclair et al, 1988). How can the paradox of absent ZFY-like sequences be reconciled with the Y chromosome requirement for testis differentiation in wallabies, and the strong circumstantial evidence implicating ZFY as the primary switch in mammals? One possibility has been suggested by Hodgkin (1988) and assumes that the primary determinant of testis differentiation is ZFX (or ZFA in marsupials), not ZFY. However, for ZFX to be active, ZFY in mammals, and as yet unidentified

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genes on the Y chromosome of marsupials, are essential components of the system. Thus, any number of copies of ZFX or ZFA, without ZFY or another Y gene, cannot result in male sex. Activation of a positive switch to turn on either ZFX or an autosomal homologue, which are the putative ancestral primary sex determining genes, could account for environmental sex determination in reptiles. In these species male sex determination is autosomally mediated and only occurs once a critical environmental temperature is reached. Bull et al (1988) have shown that ZFA (in turtles) is transcribed once the critical temperature for sex determination is reached. Further evidence that ZFX might be the primary determinant of maleness comes from XY females who have defects in Xp21 (Bernstein et al, 1980), which is precisely where ZFX is located. While it is clear that in humans a single Y chromosome is essential for testis differentiation, a single X chromosome is insufficient for normal ovarian function. In XO human females fetal ovaries do develop but contain a reduced complement of oogonia and degenerate to leave 'streak' gonads without follicles by the time of birth. It is unclear why two X chromosomes are required for normal human ovarian development, given that one of these is transcriptionally inactive in every cell. In certain rodent species XO females have fertile ovaries, as do XY female wood lemmings (Fredga et al, 1976), suggesting that at least in a few species a single X chromosome is sufficient for normal ovarian function. Why this should be is unclear. ANDROGEN RECEPTOR GENE AND MALE SEXUAL DIFFERENTIATION

Whilst a prerequisite for male sexual differentiation is the presence of a testis, the normal male phenotype also requires appropriate production and action of testosterone, dihydrotestosterone, and mullerian inhibiting hormone (MIH). The principles of male sexual differentiation have been reviewed elsewhere (Griffin and Wilson, 1980) and will not be further described. However, recent developments have laid the foundations for the elucidation of the molecular basis of phenotypic abnormalities ranging from oligozoospermia, hypospadias and ambiguous genitalia, to complete testicular feminization in genetic and gonadal males due to abnormalities in the androgen receptor (AR) (Berkovitz et al, 1983; Hughes and Evans, 1988). Absent binding of synthetic androgens to cytosol preparations from cultured genital skin fibroblasts has been demonstrated in the majority of patients with the complete androgen insensitivity syndrome (CAIS/classical testicular feminization). However, a few patients with CAIS and most of those with partial androgen insensitivity (PAIS) show normal androgen binding to their receptor (Hughes and Evans, 1988) but clearly this is functionally defective. This implies a qualitative abnormality in the androgen receptor of which several types are described, including reduced binding affinity, increased dissociation rate of ligand from receptor, increased affinity for progesterone, decreased nuclear binding and thermolability of binding (Berkovitz et al, 1983; Hughes and Evans, 1988). These

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abnormalities suggest there are a number of subtle defects in the androgen receptor protein outwith the hormone binding domain (see below). Some of these may be single amino acid substitutions generated by single base changes in critical parts of the protein, analogous to sickle cell anaemia. Family studies suggest that CAIS is inherited in an X-linked recessive manner. Moreover, fibroblasts from mothers of CAIS patients consist of two populations of cells, one with absent androgen binding and the other with normal androgen binding, consistent with random inactivation of the X chromosome. Furthermore, in the analogous Tfm mouse model androgen insensitivity is linked to blood group antigens residing on the X chromosome. Definitive localization of the androgen receptor locus to the human X chromosome was provided by Migeon et al (1981) by somatic cell hybridization methods. Linkage analysis of families with androgen resistance shows close linkage of AR with the DXSI locus on the proximal long arm of the X chromosome (Wieacker et al 1987). Despite the chromosomal localization of the AR gene in 1981, it is only recently that the AR cDNA was cloned (Chang et al, 1988; Lubahn et al, 1988a). The cloning of human and rat AR cDNAs was achieved without prior knowledge of any amino acid sequence data. The cloning strategy assumed that the AR DNA binding domain would show a high degree of sequence homology with the same region of the steroid hormone superfamily of receptors (Evans, 1988, for review). Thus, initial screening with a synthetic oligonucleotide to the DNA binding region identified many positive clones and this was followed by secondary screening with oligonucleotides specific for non-AR steroid receptors to eliminate glucocorticoid, mineralocorticoid, oestrogen and progesterone receptor cDNAs. Although the initial cloned cDNA was not full length it directs synthesis of a protein (72kDa) with similar size characteristics to partially purified androgen receptor. The synthetic protein binds radiolabelled androgen with high affinity and specificity and is immunologically similar to the natural AR. The putative DNA binding domain shows 79% homology with glucocorticoid (GR), mineralocorticoid (MR), and progesterone (PR) receptor DNA binding domains (Figure 2), though is less homologous with similar regions of the oestrogen, vitamin D and triiodothyronine receptors. The carboxyterminal steroid binding domain has 50% homology with GR, MR and PR. Original cDNAs lacked a portion of the aminoterminal coding region. This domain (AB) is thought to confer maximal transactivation function to the steroid hormone receptors by virtue of its high negative charge. It will be of interest to learn whether the AR protein encoded by these cDNAs is functionally active in vitro. Full-length cDNAs from human and rat have now been characterized (Lubahn et al, 1988b; Tan et al, 1988) and encode proteins of 919 and 902 amino acids, respectively. The aminoterminal domain is long, relatively hydrophilic, and negatively charged, like the GR, PR and MR, and considerable differences between species occur in this region. The human AR cDNA detects mRNA transcripts of 9.4 and 7 kb in human prostate and 9.4 kb only in foreskin fibroblasts. Both these transcripts are much larger than the cDNA (3.6 kb), implying longer 3'-untranslated regions in 'normal'

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Maximum activity DNA I

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Figure 2. Schematic representation of homologybetweenthe human glucocorticoidreceptor protein (GR), mineralocorticoid (MR), progesterone (PR), androgen (AR) and oestrogen (ER) receptors. The extent of homologywithin the variousdomainsis indicatedin the boxesas a percentage. DNA, DNA binding domain; Hormone, hormone binding domain.

mRNA. In the rat prostate the 10 kb mRNA is negatively regulated by testosterone (Tan et al, 1988). Antibodies generated to a synthetic peptide from the predicted amino acid sequence react predominantly with the nuclei of human prostatic epithelial cells and may prove useful in the determination of androgen receptor status of human prostatic carcinoma specimens. The partial A R cDNA was hybridized to clones from an X chromosome library (Chang et al, 1988; Lubahn et al, 1988a). With availability of these cDNAs it will be possible to define the precise A R abnormality in any patient and family with androgen insensitivity syndrome. As with other single gene disorders, different mutations affecting different domains of the protein will doubtless arise. Moreover, once the A R gene abnormality is determined, accurate antenatal diagnosis of affected male fetuses at an early stage is a realistic possibility in families with one already affected member. Thus, it can be predicted that individuals with CAIS who are androgen receptor positive will have a normal C-terminal hormone binding domain, but are lacking a functional D N A binding domain. Receptor negative patients might have gene deletions, mutations in the hormone binding domain and possibly in the D N A binding domain as well. Individuals with androgen binding and partial insensitivity will be interesting. They might have changes to the N-terminal domain of the protein which is not an absolute requirement for receptor activity but is necessary for maximum activation of transcription of steroid responsive genes (Evans, 1988). Thus in PAlS it could be envisaged that during development certain tissues, such as the urogenital sinus, have a requirement for maximal A R activity, whilst others, e.g. breast, brain and skeleton, may acquire a male phenotype with lesser androgen receptor activity. These hypotheses are now amenable to testing.

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It has been suggested that oligoasthenospermia could be due to subtle abnormalities in the androgen receptor (Aiman and Griffin, 1982; Morrow et al, 1987). It is possible that some cases of postpubertal gynaecomastia might also reflect mild androgen insensitivity (Migeon et al, 1984). With the recent advances it should soon be possible to answer these questions. INHIBINS, ACTIVINS AND CONTROL OF FSH SECRETION AND GONADAL FUNCTION Inhibins

In 1932 McCullagh coined the term 'inhibin' to refer to a non-steroidal factor from the gonads which specifically reduced FSH secretion. Since then, numerous reports from animal and human studies have confirmed that FSH secretion is under negative feedback control from a gonadal product(s) other than sex steroids, and numerous attempts have been made to purify the water-soluble product(s) (reviewed in de Jong, 1979; de Jong and Robertson, 1985). Two recent reviews on the characterization, structure and biological activities of inhibin have been published (McLachlan et al, 1988; Ying, 1988). Reports abound of follicular fluid and testicular extracts with 'inhibin-like' activity with molecular sizes ranging from 10000--100 000 kDa. This variability arose because of the variety and specificity of bioassays and sources of unpurified inhibin, as well as the inability, until recently, of classical protein purification processes to isolate 'pure' material. For this reason, there was considerable scepticism about the very existence of specific FSH inhibiting proteins from follicular and rete testes fluid, a view reinforced by the reports from Sheth et al (1984) and Seidah et al (1984) of a 14 kDa protein from human seminal plasma with FSH lowering activity. This material was subsequently shown to be unrelated to gonadal inhibins and originates from the prostate. However, in 1985 highly homogeneous preparations of inhibin from ovarian follicular fluid were purified from several species by four groups (Ling et al, 1985; Miyamoto et al, 1985; Rivier et al, 1985; Robertson et al, 1985). The key to the success was the use of pituitary cell cultures in which reproducible and sensitive suppression of basal or GnRH-stimulated FSH release was specific and not complicated by non-specific effects or cellular toxicity. These assays showed no inhibition of LH secretion or of other anterior pituitary hormones. Enrichment of inhibin activity of 5000-10 000fold was achieved and all groups reported the presence of a single molecular protein species of molecular weight 30-32 kDa. This was shown to be a glycoprotein heterodimer composed of two disulphide linked subunits termed a and [3 which have molecular sizes of 18 and 14 kDa, respectively (Figure 3). It is now known that two [3 subunits (~A, ~B) exist in ovarian follicular fluid. N-terminal amino acid sequence from the three subunits enabled the design of oligonucleotide probes with which to screen cDNA libraries from porcine (Mason et al, 1985), human (Mason et al, 1986), rat (Esch et al, 1987a,b) and

994

R . N . CLAYTON Inhibin A

FSH inhibitors

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Figure 3. Combinations of the inhibin subunits to form the dimers with FSH stimulatory and inhibitory activity. These have all been isolated from follicular fluid with the exception of PB dimer. Molecular size (kDa) are indicated beside and inside the dimers and monomers, respectively.

bovine (Forage et al, 1986) ovarian cDNA libraries. These cDNAs predicted amino acid sequences for larger precursor peptides for each subunit from which the C-terminal portion is cleaved to form the mature subunit for combination. Several dibasic amino acid cleavage sites are present within the precursor peptides, yielding a number of potential sizes for heterodimers ranging from 120-32 kDa (see Ying, 1988). This presumably accounts for the variability in molecular sizes observed during the early days of attempts at purification of inhibin from follicular fluid. However, the predominant molecular size protein in ovarian follicular fluid is 32 kDa inhibin A, or B, with the larger forms being processing intermediates. There is one glycosylation site in the mature oLchain and none in the t3 chain. There is > 8 0 % homology between the mature a subunit of human, porcine, bovine and rodent inhibin, the mature [3A subunits are identical, and only 1-3 amino acids differ between the [3Bsubunits. There is also a high degree of conservation in the precursor region which, together with the positions of the 7-9 cysteine residues, suggests that both [3subunits and the subunit genes arose from a single common ancestor. A remarkable amino acid homology between the inhibin p subunits and transforming growth factor-[3 (TGF-p) is observed. TGF-[3 is a 25kDa homo dimer of two 12.5 kDa subunits and has a number of biological actions, including inhibition and stimulation of mitogenesis, interaction with other growth factors, augmentation of FSH action in granulosa cells (Knecht et al, 1986; Ying et al, 1986) and stimulation of FSH release by cultured pituitary cells (cf. activin). These similarities suggest that inhibin may also have growth regulatory actions within the ovary (see below). Inhibin also shares homology with the C-terminal portion of mullerian inhibiting hormone, a

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140 kDa glycoprotein hormone produced by the developing testis to prevent development of the fallopian tubes and uterus in male embryos (Cate et al, 1986). Another protein showing C-terminal homology with inhibins, TGF-[3, and MIH is the decapentaplegic protein which is involved in embryogenesis of the fruit fly Drosophila (see McLachlan et al, 1988, for schematic representation of conserved residues). With the availability of cDNA probes for the specific inhibin subunits, studies on the sites of synthesis and regulation of gene expression have been performed. These show a single ~ subunit mRNA transcript in preparations from the ovary, which increases with FSH treatment. Moreover, in situ hybridization histochemistry localizes expression of the gene predominantly to granulosa cells, though some expression is seen in luteal cells (Woodruff et al, 1987; Meunier et al, 1988). Testicular RNA shows a single band on Northern blots, when probed with an ovarian inhibin cDNA probe (Esch et al, 1987b; Toebosch et al, 1988), suggesting that inhibin mRNA in the male is the same as in females. The Sertoli cell is the probable source of testicular inhibin since it has been localized there immunologically. Intriguingly, inhibin a subunit mRNA, but not [3, was found in the three zones of the sheep adrenal cortex and was increased by ACTH,(Crawford et al, 1987). Inhibin a mRNA has also been found in human placenta (Mayo et al, 1986) and is the source of circulating inhibin during early human pregnancy, when its rise parallels that of human chorionic gonadotrophin (hCG), oestradiol and progesterone (McLachlan et al, 1987a). The stimulation of inhibin production by hCG from cultured human trophoblasts and the increase in hCG by incubation of the cells with an inhibin antibody suggests a paracrine role for inhibin in placental hCG production (Petraglia et al, 1987). In respect of suppression of gonadotrophin secretion in vitro, 32kDa inhibin has a profound effect on both basal and gonadotrophin-releasing hormone (GnRH) stimulated FSH secretion and synthesis, but at higher concentrations inhibits release of LH (Farnworth et al, 1988). The decreased sensitivity to GnRH in the presence of inhibin may relate to reduction in GnRH receptors on gonadotrophs (Wang et al, 1988). There are indications that inhibin can synergize with oestradiol to suppress FSH secretion in vitro, as well as in vivo. Although inhibin receptors and the intracellular mediators of inhibin action in gonadotrophs have not yet been identified, the peptide has been shown to reduce specifically FSH-[3 subunit mRNA levels in vivo (Mercer et at, 1987). With the availability of purified bovine follicular fluid 32 kDa inhibin, McLachlan et al (1986b, 1987a,b) developed radioimmunoassays with the sensitivity and specificity required to measure the peptide in human serum. Undetectable levels in postmenopausal or castrate serum attest to the gonadal origin of the circulating material. Subsequently, synthetic Nterminal peptides from the o~ subunit were used to generate antibodies capable of measuring heterodimeric inhibin in rat serum and conditioned media from granulosa or Sertoli cells, thus enabling detailed study of inhibin physiology. Complementary studies have been performed using a highly sensitive bioassay based on suppression of FSH release from cultured ovine pituitary cells (Tsonis et al, 1986, 1988).

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Follicular fluid inhibin concentration correlates positively with oestradiol, testosterone and androstenedione levels, and serum inhibin correlates with serum oestradiol during ovulation induction (McLachlan et al, 1986a) as well as with the number of developing follicles seen by ultrasound. Moreover, during spontaneous menstrual cycles serum inhibin increases in the late follicular phase in parallel with oestradiol and at a time when FSH is at a nadir. Thus, the developing follicle is the major source of circulating inhibin in women and, synergistically with oestradiol, suppresses serum FSH. However, this inhibition is clearly overridden at the time of the preovulatory LH and FSH surges although, interestingly, the surge of FSH is always less than that of LH, perhaps implying a selective inhibitory action of inhibin, even at this time in the cycle. Inhibin is also produced by the corpus luteum, with highest levels observed in the mid-luteal phase when they correlate with serum progesterone (McLachlan et al, 1987a), suggesting that inhibin production is also regulated by LH. In vivo studies in other animals indicate a similar relationship between inhibin, oestradiol production and FSH, where FSH is the major stimulant. This is confirmed by in vitro studies showing that cultured rat granulosa cells secrete inhibin in response to FSH in a dose and cAMP-dependent fashion (Bicsak et al, 1986). Insulin-like growth factor I (IGF-I) also stimulates inhibin production and this may play a part in mediating the stimulation of follicular growth observed with growth hormone (Homburg et al, 1988). In contrast, agents such as GnRH and epidermal growth factor, which inhibit FSH dependent functions in granulosa cells, also reduce inhibin production. Studies of the inhibin-FSH axis in males have been less extensive, though early studies showing monotrophic elevation in serum FSH in proportion to damage to the seminiferous epithelium implied that the Sertoli cell was the source of this material. Radioimmunoassay of inhibin from Sertoli cell cultures confirmed this suggestion and showed that, as in the ovary, inhibin production was FSH dependent (Bicsak et a11987). Serum inhibin levels rise during puberty in boys, as they do in girls, indicating dependence of circulating inhibin on germinal epithelial development (Burger et al, 1988). Whether disordered inhibin physiology is in any way related to male infertility remains to be seen.

Activins

The discovery of activins as homo- or heterodimers (Figure 3) of the 13 subunits of inhibin was made when side-fractions from high-performance liquid chromatography (HPLC) purification of inhibin were found with FSH-releasing activity in the bioassay. NH2-terminal sequencing of purified follicular fluid FSH-releasing proteins confirmed their identify with the NH2 terminal portion of inhibin 13 subunits (Ling et al, 1986; Vale et al, 1986). Although there is striking similarity between activins and TGF-13, the activins do not have TGF-13 bioactivity, although TGF-13 has activin-like action in releasing FSH from cultured pituitary cells. Both the 13A13Ahomodimer and 13n13Bactivin are equipotent at releasing

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FSH without affecting LH, and this activity is antagonized by inhibin. Activin is more potent at releasing FSH than GnRH and its action is delayed, being maximal after 24-48 h of incubation, in contrast to GnRH, whose action is rapid (< lh). Activin-induced FSH release is not inhibited by GnRH receptor antagonists, and maximal doses of GnRH and activin are synergistic. These features indicate separate receptors and mechanisms of action of these FSH-releasing molecules. Activin has not yet been identified in peripheral serum so its physiological role is unknown. Intriguingly, the [~A[~A activin homodimer has been isolated from a human leukaemia cell line (Etol et al, 1987) where it acts as a differentiation factor. Moreover, Yu et al (1987) have shown that activin A induces differentiation and haemoglobin production in a human erythroid cell line as well as potentiating erythropoietin-induced growth of bone marrow erythroid progenitor cells. Thus, the activins may have physiological actions not only on the pituitary and gonads but also on haemopoiesis. In view of their general stimulatory actions in several tissues perhaps the activins will be found elsewhere and might best be classified along with the transforming growth factor family of peptides. Follistatins

A single-chain glycosylated peptide, bearing no structural homology to the inhibins or activins, with FSH-inhibiting activity, has been isolated from follicular fluid and named follistatin, although its physiological function is unknown (Ueno et al, 1987). There are two forms of follistatin with molecular size similar to that of the inhibins, at 35 and 32 kDa. The proteins are rich in cysteine residues (36 in all), contain 315 amino acids and an acidic carboxyterminal domain with 6 residues identical to the tyrosine kinase domain of the EGF receptor. Human and porcine follistatin are highly conserved and contain 3 identical repeating domains, having about 50% homology with human pancreatic secretory trypsin inhibitor protein (see Ying, 1988). Like inhibin, follistatin inhibition of FSH secretion is not immediate and the two peptides exhibit additive activities in this respect. Follistatin also inhibits FSH stimulated oestrogen production from cultured rat granulosa cells (Ying, 1988). As with activins, the physiological role of follistatins awaits elucidation. INTRAGONADAL PARACRINE ACTIONS OF INHIBINS AND ACTIVINS

Many peptide growth factors, including IGF-I, insulin, TGF-[3 and epidermal growth factor (EGF) have been shown to influence FSH-induced aromatase activity in cultured rodent granulosa cells (see review by Adashi et al, 1985). Inhibins and activins may now be added to this list of presumed paracrine intragonadal modulators of steroidogenesis. As with in vivo studies, the ovary rather than the testis has received most attention with respect to paracrine modulation by growth factors. Thus, Ying et al (1986) showed that purified

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follicular fluid inhibin attenuated FSH-induced oestrogen production by granulosa cells, an action reversed by TGF-~ and activin. Moreover, LHstimulated androstenedione synthesis by theca interna is amplified by inhibin and reduced by activin (Hsueh et al, 1987). Thus, within the rat ovarian follicle inhibin appears to exert opposing effects on the two steroidogenic compartments. Purified rete testis fluid inhibin, presumably originating from Sertoli cells, markedly enhanced LH-stimulated testosterone production from neonatal rat Leydig cells, while activin attenuated this action of LH (Hsueh et al, 1987). However, it is not clear whether similar results are obtained in adult males. Neither inhibin, activin nor TGF-[3 have any effect on steroidogenesis in ovary or testis in the absence of gonadotrophins. On the basis of this information it is feasible that these peptides provide one means of cross-communication between Sertoli/Leydig cells, and granulosa/theca cells. As a consequence of the rapidly expanding knowledge afforded by the purification and molecular cloning of proteins from follicular fluid, it is necessary to redraw the classical feedback axis between the gonads and pituitary gland (Figures 4 and 5). More surprises may be in store when pure inhibin and activin preparations, and large quantities of their antibodies, become widely available for whole animal studies.

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Figure 4. Inter-relationships between ovarian peptides of the inhibin/activinfamily, both within the ovary (paracrine regulation) and at a distance on the pituitary and the hypothalamus. There is no evidence, to date, that inhibins/activins influence gonadotrophin-releasing hormone (GnRH) secretion. LH, luteinizing hormone; FSH, follicle-stimulating hormone; G.C., granulosa cell; +, stimulation; - , inhibition; - / + , inhibition and stimulation.

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®

5~'_

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Figure 5. Inter-relationships between testicular inhibin/activin peptides within the testis, and on the pituitary. GnRH, gonadotrophin-releasing hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; +, stimulation; - , inhibition.

REFERENCES Adashi EY, Resnick CE, d'Ercole AJ, Svoboda ME & van Wyk JJ (1985) Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function. Endocrine Reviews 6: 400-420. Aiman J & Griffin JE (1982) The frequency of androgen receptor deficiency in infertile men. Journal of Clinical Endocrinology and Metabolism 54: 725-732. Berkovitz CD, Brown TR & Migeon CJ (1983) Androgen receptors. In Clayton RN (ed.) Clinics in Endocrinology and Metabolism, vol. 12, pp 155-173. Philadelphia: WB Saunders. Bernstein R, Koo GC & Wachtel SS (1980) Abnormality of the X chromosome in human 46 XY female siblings with dysgenetic ovaries. Science 207: 768-769. Bicsak TA, Tucker EM, Cappel Set al (1986) Hormonal regulation of granulosa cell inhibin. Endocrinology 119: 2711-2719. Bicsak TA, Vale W, Vaughan J, Tucker EM, Cappel S & Hsueh AJW (1987) Hormonal regulation of inhibin production by cultured Sertoli cells. Molecular and Cellular Endocrinology 49: 211-217. Bull, J J, Hillis DM & O'Steen S (1988) Mammalian ZFY sequences exist in reptiles regardless of sex-determining mechanism. Science 242: 567-569. Burger HG, McLachlan RI, Bangah M, et al (1983) Serum inhibin concentrations rise throughout normal male and female puberty. Journal of Clinical Endocrinology and Metabolism 67: 689-694. Cate RL, Mattaliano RJ, Hession C et al (1986) Isolation of the bovine and human genes for mullerian inhibiting substance and expression of the human gene in animal cells. Cell 45: 685-698. Catt KJ, Harwood JP, Clayton RN et al (1980) Regulation of peptide hormone receptors and gonadal steroidogenesis. Recent Progress in Hormone Research 36: 557-622. Chang C, Kokoutis J & Liao S (1988). Molecular cloning of human and rat complementary DNA encoding androgen receptors. Science 240: 324--326.

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