Abstract

Androgens are required for the development of male internal and external genitalia. Androgen action is mediated by an intracellular receptor which acts as a transcription factor following activation by ligand binding. The aim of the present study was to define the time of appearance of androgen receptor (AR) in the male fetal rat gonad using immunohistochemistry. Intact fetuses (days 13·5–16·5) or testicular tissue (days 16·5–20·5 and days 3–7 postnatal) were fixed in Bouins' solution and processed into paraffin wax. On day 16·5 nuclear AR were present in mesenchymal cells surrounding the Wolffian duct but those around the Mullerian duct were receptor negative. During the following day (17–18) the abundance of nuclear staining increased, becoming detectable in the epithelial cells of the Wolffian ducts. Within the testis some nuclear staining was apparent at day 17 but was confined to interstitial cells surrounding the seminiferous cords. As development of the testis proceeded the abundance of nuclear AR in peritubular and elongated mesenchymal cells increased. AR were not detected in fetal Leydig cells expressing 3β-hydroxysteroid dehydrogenase nor in the ovaries or associated ducts of female fetuses at the same ages.

In conclusion, in the rat we have found AR expression detectable by immunohistochemistry in mesonephric mesenchyme to be confined to that underlying the Wolffian ducts and to be absent from the area around the degenerating Mullerian duct. On and after day 17 of gestation AR is present in Wolffian duct epithelial cell nuclei and within the testis it is confined to peritubular and interstitial cells which may have migrated from the mesonephros. Fetal Leydig cells were receptor negative. Within the seminiferous cords AR in Sertoli cells remained low until after birth and some perinuclear staining was detected in cells thought to be gonocytes. We believe this to be the first report of immunolocalisation of AR to fetal testicular interstitial cells.

Journal of Endocrinology (1995) 147, 285–293

Abstract

Anti-Mullerian hormone (AMH) and androgenic steroids are key factors regulating the masculinisation of the internal and external genitalia during fetal development. AMH is produced in Sertoli cells and causes regression of the Mullerian ducts in the male. 3β-Hydroxysteroid dehydrogenase (3β-HSD) is one of the key steroidogenic enzymes regulating testosterone production in Leydig cells. The objective of this experiment was to elucidate the development of the ovine fetal testes by identifying the spatio-temporal expression of AMH, 3β-HSD and androgen receptor expression within them. Fetuses from days 30 and 40 of gestation were fixed intact, while the gonads were dissected from the fetuses on days 70, 100 and 130 of gestation. Tissue was fixed in Bouin's fixative for 6 h, processed into paraffin wax and sections immunostained using rabbit anti-human AMH, 3β-HSD or androgen receptor antibodies.

While seminiferous cords were absent on day 30 of gestation, pre-cord organisation was apparent and the gonad could be clearly distinguished from surrounding tissue by the presence of AMH and 3β-HSD immunopositive cells. Androgen receptor expression was not apparent at this stage. By day 40 of gestation the testis was organised into distinct seminiferous cords and intense immunostaining for AMH and 3β-HSD was present in Sertoli cells within the cords and Leydig cells in the interstitium respectively. Androgen receptor immunopositive cells were present in the interstitium but cells destined to develop into rete testis were immunonegative. By day 70 of gestation, the rete testis was organised in the centre of the testis and was strongly androgen receptor immunopositive. AMH and 3β-HSD expression was present in Sertoli and Leydig cells respectively. The expression of AMH, 3β-HSD and androgen receptor in the 100 and 130 day gestation fetuses was similar to that identified in the 70 day fetuses. In conclusion, Sertoli and Leydig precursor cells are present in the gonad prior to seminiferous cord formation and contain AMH and 3β-HSD at all stages of gestation examined. While androgen receptor immunoexpression was present in nuclei of interstitial cells from day 40 of gestation and in the rete testis from day 70 of gestation, Sertoli cells were immunonegative for androgen receptor at all of the stages examined.

Journal of Endocrinology (1997) 153, 27–32

Abstract

The aim of this study was to explore whether pituitary adenylate cyclase activating polypeptide (PACAP) could regulate protein synthesis by enriched preparations of spermatocytes and spermatids from the adult rat testis. Spermatocytes and spermatids were incubated for 8 h or 24 h in the absence (control) or presence of PACAP-27, PACAP-38, vasoactive intestinal peptide (VIP) or dibutyryl adenosine-3′,5′-cyclic monophosphate (db-cAMP). Total synthesis of intracellular and secreted proteins, during the incubation periods, was assessed and selected samples were analysed by 2-D SDS-PAGE. PACAP-38 (200 nm), VIP (200 nm) and db-cAMP (1 mm) significantly increased the synthesis of spermatocyte-secreted and intracellular proteins by 8 h and 24 h. Synthesis of both intracellular and secreted proteins by spermatids was significantly inhibited at 8 h and 24 h with PACAP, VIP and db-cAMP. The abundance of four germ cell-secreted proteins (GSP1, GSP2, GSP3 and phosphatidyethanolamine-binding protein (PEBP)), which can be identified in both spermatocyte and spermatid culture medium, and β-actin, which can only be identified in spermatid culture medium, was analysed. PACAP-38 and db-cAMP significantly increased the incorporation of label into GSP1, GSP2, GSP3 and PEBP, derived from spermatocyte culture medium, at 8 h and 24 h. In contrast PACAP-38 inhibited the incorporation of label into GSP1 and β-actin, derived from spermatid culture medium, at 24 h. The results show that PACAP can regulate synthesis of both secreted and intracellular proteins by spermatids and spermatocytes in vitro. This effect is mimicked with high doses of db-cAMP (>1 mm), suggesting that PACAP may act via a pathway that involves changes in cyclic AMP concentration in the germ cells.

Journal of Endocrinology (1995) 144, 215–223

Abstract

The sites of action and the physiological role of oestrogens in the male reproductive tract are poorly understood. We have undertaken a systematic study of the immunoexpression of oestrogen receptor-α (ERα) in the male rat from late fetal life through to adulthood and compared the findings with results obtained in the marmoset monkey (Callithrix jacchus) from neonatal to adult life. The testes, rete testis, efferent ducts and epididymis were examined from normal male rats (aged 4, 8, 10, 15, 20, 25, 38, 48 and 90 days) and from male rat fetuses on days 17·5 and 18·5 of gestation; comparable tissues were examined from neonatal, infantile, peripubertal and adult marmosets aged 8, 18–24, 54–62 and 92–112 weeks respectively. Immunolocalisation of ERa used antigen retrieval and a monoclonal antibody directed to the N-terminus, which had proved superior to six other antisera tested. ERa was immunoexpressed in interstitial cells, including the fetal/neonatal generation of Leydig cells, in both the rat and marmoset. In the rat, the adult generation of Leydig cells were also immunopositive for ERa whereas the comparable cells in the marmoset were only weakly immunopositive. ERa was not expressed in Sertoli cells, peritubular myoid cells, blood vessels or germ cells at any time in either species. In late fetal life in the rat, ERa was immunoexpressed in cells surrounding the mesonephric tubules, whereas postnatally it was expressed in the epithelium of the rete testis and efferent ducts at all ages from 4 to 90 days; this immunoexpression was most pronounced in the efferent ducts. In the marmoset, the efferent ducts, but not the rete testis, also showed intense immunoexpression of ERa. Apart from sporadic immunostaining for ERa in the epididymal duct of the rat in the neonatal period, the caput, corpus and cauda epididymis were negative for immunoexpression of ERa at all ages in both species. These findings suggest that the main actions of oestrogens in the male reproductive tract, mediated by ERa, are related to the development and function of the efferent ducts and the Leydig cells. In consideration of data from this and previous studies of oestrogen binding, we predict possible sites of expression of other oestrogen receptors (e.g. ERβ) in Sertoli cells and the epididymis. Interactive effects, related to the relative levels of androgens and oestrogens, could be physiologically important in the excurrent ducts of the adult testis.

Journal of Endocrinology (1997) 153, 485–495

ABSTRACT

In-vitro data from experiments on rats implicate granulosa cells as primary sites of hormone-dependent ovarian inhibin biosynthesis, but no equivalent data exist for primates. We have used the common marmoset (Callithrix jacchus) to investigate inhibin biosynthesis in primate granulosa cells in vitro and to determine its relationship to preovulatory follicular development. To relate the production of immunoactive inhibin to follicular maturity, we studied primary granulosa cell cultures from follicles at progressive stages of preovulatory development. Granulosa cells from 'large' (≥2·0 mm diameter) follicles expressed high rates of inhibin production and steroidogenesis (progesterone), and were positively regulated by human (h)LH in vitro. Less mature granulosa cells from 'medium' (1·1–1·9 mm) and 'small' (≤ 1·0 mm) follicles expressed proportionately lower rates of inhibin production and steroidogenesis, but each parameter was stimulated in a dose- and time-dependent manner by hFSH in vitro. The stimulatory action of hFSH on immunoactive inhibin was augmented by the presence of testosterone or oestradiol; testosterone (but not oestradiol) also augmented the steroidogenic response to hFSH. Marmoset luteal tissue also produced inhibin in vitro and expressed an ∼1·5 kb inhibin α-subunit mRNA, confirming the corpus luteum as a source of ovarian inhibin in primates.

These results provide direct experimental evidence that primate granulosa cells produce inhibin. They suggest that production of inhibin by immature granulosa cells is initially induced by FSH and subject to modulation by follicular steroids. During advanced preovulatory development, granulosa cell inhibin production becomes directly responsive to LH, thereby indicating a role for LH in the control of peri- and postovulatory inhibin secretion by the primate ovary.

Journal of Endocrinology (1989) 123, 65–73