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Age-related morphologic development of human adrenal zona reticularis (ZR) has not been well examined. Therefore, in this study, 44 human young adrenal autopsy specimens retrieved from large archival files (n=252) were examined for immunohistochemical and morphometric analyses. Results demonstrated that ZR became discernible around 4 years of age, and both thickness and ratio per total cortex of ZR increased in an age-dependent fashion thereafter, although there was no significant increment in total thickness of developing adrenal cortex. We further evaluated immunoreactivity of both KI67 and BCL2 in order to clarify the equilibrium between cell proliferation and apoptosis in the homeostasis of developing human adrenals. Results demonstrated that proliferative adrenocortical cells were predominantly detected in the zona glomerulosa and partly in outer zona fasciculata (ZF) before 4 years of age and in ZR after 4 years of age, but the number of these cells markedly decreased around 20 years of age. The number of BCL2-positive cells increased in ZR and decreased in ZF during development. Adrenal androgen synthesizing type 5 17beta-hydroxysteroid dehydrogenase (HSD17B5 or AKR1C3 as listed in the Hugo Database) was almost confined to ZR of human adrenals throughout development. HSD17B5 immunoreactivity in ZR became discernible and increased from around 9 years of age. Results of our present study support the theory of age-dependent adrenocortical cell migration and also indicated that ZR development is not only associated with adrenarche, but may play important roles in an initiation of puberty.

Unlabelled: Luteinising hormone (LH) appears to be important for the establishment of the adult-type Leydig cell population. The role of LH in the initial steps of stem Leydig cell/precursor cell differentiation is less clear. The aim of the present study was to elucidate the role of LH in the differentiation of spindle-shaped mesenchymal-like cells into Leydig cell progenitors. Interstitial cells were isolated from rat testes at three different ages reflecting different phases in development, and cultured in the presence of increasing concentrations of LH (ranging from 0.01 to 10 ng/ml). Cells were isolated from 10-day-old rats when stem Leydig cells/precursor cells are abundant; 13-day-old rats when the first 3beta-hydroxysteroid dehydrogenase (3beta-HSD)-positive Leydig cell progenitors have developed in the strain of rats used in this study; and 18-day-old rats just prior to the wave of progenitor proliferation. Immunohistochemistry revealed that before stem Leydig cells differentiate into progenitor cells, they acquire functional LH receptors and become precursor cells. This was confirmed by an in vivo immunohistochemical double-labelling experiment. Addition of LH to the cultures increased the percentage of LH/3beta-HSD-labelled Leydig cell progenitors, while the percentage of cells solely expressing the LH receptor decreased. Cell proliferation was negligible, suggesting that the increase in 3beta-HSD-positive cells is the result of precursor cell differentiation. When interstitial cells were isolated from 13-day-old rats and to a lesser extent from 10-day-old rats, a small proportion of the precursors could develop into progenitor cells independent of the presence of LH., In Conclusion: before Leydig stem cells differentiate into 3beta-HSD-positive progenitor cells, they acquire LH receptors and become precursor cells. LH appears to be essential, even at very low doses for the differentiation of these precursor cells into 3beta-HSD-positive progenitors, although a subpopulation of precursor cells can develop into progenitors independently of LH.

Although the human and the nonhuman primate fetal adrenal glands undergo a highly unique pattern of cortical zone-specific intrauterine growth and development, studies of the regulatory components of the cell cycle responsible for this growth have not been conducted. Therefore, the present study determined expression of the cell cycle regulators, cyclin D1 and cyclin E, and their cyclin-dependent kinases, Cdk2, Cdk4, and Cdk6, and Ki67 a marker of cell proliferation within the baboon fetal adrenal cortex during advancing stages of gestation. Fetal adrenal glands were obtained on days 60 (early), 100 (mid), and 160-170 (late) of gestation (term = 184 days). Mean (+/- s.e.) cyclin D1 mRNA levels, determined by RT-PCR and expressed relative to 18S rRNA, were similar at early (0.85 +/- 0.09) and mid (1.04 +/- 0.08) gestation, then decreased (P < 0.001, ANOVA) approximately 50% by late gestation (0.57 +/- 0.04). Cyclin E mRNA levels were also similar at early (2.03 +/- 0.07) and mid (1.63 +/- 0.31) gestation, and decreased by 70% (P < 0.001) in late gestation (0.53 +/- 0.09). Coinciding with the decrease in cyclin D1 and cyclin E, the percentage of Ki67 positive cells in the definitive zone decreased twofold (P < 0.01) between mid (28.2 +/- 3.6) and late (13.8 +/- 1.7) gestation. The cyclin D1 and cyclin E proteins, determined by immunocytochemistry, were expressed at high levels in the definitive zone of baboon fetal adrenal gland, where they decreased between mid- and late gestation. In contrast, immunocytochemical expression of the functionally important steroidogenic enzyme Delta(5)-3beta-hydroxysteroid dehydrogenase (3beta-HSD) became abundant in the definitive and transitional zones with advancing pregnancy. However, fetal adrenal Cdk2, Cdk4, and Cdk6 mRNA levels and protein immunoexpression were similar in the baboon fetal adrenal at early-, mid-, and late gestation. In summary, expression of cyclin D1, cyclin E, and Ki67 decreased, while 3beta-HSD expression increased, in the fetal adrenal cortex, particularly in the definitive zone, between mid- and late-baboon gestation. We propose that a developmental decline in cellular proliferation permits functional differentiation of fetal adrenal cortical cells, leading to increased production of steroid hormones important for placental estrogen synthesis and maturation of organ systems within the developing fetus.

The hepatocyte growth factor (HGF) is a pleiotropic cytokine able to regulate different cellular functions. HGF action is mediated by its receptor, c-met, a glycoprotein with tyrosine kinase activity. We previously demonstrated that c-met is expressed in the newly formed seminiferous cords of the mice embryonic testes and that HGF acts as a morphogenetic factor. In this paper, we report that at 15.5 days post-coitum (dpc) c-met is expressed in the testicular cords, whereas at 18.5 dpc c-met expression is almost exclusively localized in the interstitial tissue of the testis in particular in the fetal Leydig cells. In addition, we demonstrate that HGF gene is expressed during the fetal period of testis development, heavily detectable in the interstitial compartment of 18.5 dpc testes. Interestingly, HGF is not expressed in the Leydig cells that, as above reported, express the HGF receptor. Looking for the functional role of HGF on Leydig cells, we evaluated the amount of testosterone secreted by testes isolated from 18.5 dpc embryos and cultured in the presence of HGF. The results of the in vitro organ culture show that, at this age, HGF increases the amount of testosterone secreted in the culture medium. On the contrary, HGF does not modulate the amount of testosterone secreted by testes isolated from 15.5 dpc embryos. In conclusion, we report that HGF is produced in the interstitial compartment of the developing testis but not by the Leydig cells. Conversely, the HGF receptor c-met is expressed in the Leydig cells and HGF modulates Leydig cell function during the late period of prenatal development.

The biosynthesis of steroid hormones in endocrine steroid-secreting glands results from a series of successive steps involving both cytochrome P450 enzymes, which are mixed-function oxidases, and steroid dehydrogenases. So far, the subcellular distribution of steroidogenic enzymes has been mostly studied following subcellular fractionation, performed in placenta and adrenal cortex. In order to determine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural localization of the three key enzymes: P450 side chain cleavage (scc) which converts cholesterol to pregnenolone; 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) which catalyzes the conversion of 3 beta-hydroxy-5-ene steroids to 3-oxo-4-ene steroids (progesterone and androstenedione); and P450(c17) which is responsible for the transformation of C(21) into C(19) steroids (dehydroepiandrosterone and androstenedione). Immunogold labeling was used to localize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P450(scc) and 3 beta-HSD immunoreactivities were detected in the reticular, fascicular and glomerular zones. P450(scc) was exclusively found in large mitochondria. In contrast, 3 beta-HSD antigenic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P450(c17) could not be detected in adrenocortical cells. In the testis, the three enzymes were only found in Leydig cells. Immunolabeling for P450(scc) and 3 beta-HSD was restricted to mitochondria, while P450(c17) immunoreactivity was exclusively observed in ER. In the ovary, P450(scc) and 3 beta-HSD immunoreactivities were found in granulosa, theca interna and corpus luteum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P450(c17) could also be detected in theca interna cells of large developing and mature follicles. As observed in Leydig cells, P450(c17) immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P450(scc), 3 beta-HSD and P450(c17) have the same association with cytoplasmic organelles (with the exception of 3 beta-HSD in Leydig cells), suggesting similar intracellular pathways for biosynthesis of steroid hormones.

Gap junctions are intercellular protein channels which provide a pathway for the exchange of ions and small molecules. This exchange of materials allows metabolic coupling of cells. Gap junction channels are made up of connexins, integral membrane proteins encoded by a multigene family. Rat testes contain mRNAs for at least five different connexins: Cx26, Cx32, Cx33, Cx37 and Cx43. Immunocytochemical studies have shown that Cx43 assembles gap junctions between Leydig cells. The present study investigated the expression and regulation of the Cx43 gene in rat Leydig cells. Purified Leydig cells were obtained from 40- to 80-day-old Sprague-Dawley rats using a combination of arterial perfusion, collagenase digestion, centrifugal elutriation and Percoll gradient centrifugation. Leydig cells from 20- and 30-day-old rats were isolated without arterial perfusion or centrifugal elutriation. Cx43 mRNA was present in 20-day-old rat Leydig cells, reached a plateau at day 40, and remained at high levels in 65- and 80-day-old rat Leydig cells. To evaluate the regulation of Cx43 gene expression, Leydig cells were cultured overnight and then treated with human chorionic gonadotropin (hCG) for variable periods of time. Addition of hCG (10 ng/ml) increased cytochrome P450 side-chain cleavage and steroidogenic acute regulatory protein mRNA levels and testosterone formation. However, Cx43 mRNA levels were inhibited by hCG in a time- and dose-dependent manner. Cx43 mRNA levels decreased 27% as early as 2 h after the addition of hCG and decreased 60% by 24 h. Treatment of Leydig cells with 8-bromo-cAMP (0.1 mM) for 6 and 24 h also reduced Cx43 mRNA levels by 36 and 56% respectively. Primary cultured Leydig cells stained strongly positive with anti-Cx43 monoclonal antibody. Treatment with hCG for 24 h reduced Cx43 signals and caused Cx43 to redistribute to the periphery of the cells. To evaluate the regulation of Cx43 in vivo, rats were treated with hCG (300 ng i.p.) and testes were removed 24 h later. Frozen section of testes revealed that these interstitial cells stained positive for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) by histochemical staining and were positive for Cx43 by immunofluorescence staining. The adjacent seminiferous tubules stained only weakly positive for Cx43. Twenty-four hours after hCG treatment, 3beta-HSD activity increased while Cx43 immunostaining of Leydig cells was reduced. In conclusion, gap junction channels of Leydig cells are regulated by hCG both in vivo and in vitro. hCG increased Leydig cell steroidogenesis and steroidogenic enzyme mRNA levels but caused a redistribution of Cx43.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) play a key role in the synthesis of sex steroids. The hallmark of this family of enzymes is the interconversion, through their oxydoreductive reactivity at position C17, of 17-keto- and 17beta-hydroxy-steroids. Because this reaction essentially transforms steroids having low binding activity for the steroid receptor to their more potent 17beta-hydroxysteroids isoforms, it is crucial to the control of the physiological activities of both estrogens and androgens. The human placenta produces large amounts of progesterone and estrogens throughout pregnancy. The placental type 1 17beta-HSD enzyme (E17beta-HSD) catalyzes the reduction of the low activity estrogen, estrone, into the potent estrogen, estradiol. We studied the cell-specific expression of type 1 17beta-HSD in human term placental villous tissue by combining in situ hybridization to localize type 1 17beta-HSD mRNA with immunohistochemistry using an antibody against human placental lactogen, a trophoblast marker. Immunolocalization of E17beta-HSD was also performed. To ascertain whether other steroidogenic enzymes are present in the same cell type, cytochrome P450 cholesterol side-chain cleavage (P450scc), P450 aromatase, and type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were also localized by immunostaining. Our results showed that the syncytium is the major steroidogenic unit of the fetal term villi. In fact, type 1 17beta-HSD mRNA and protein, as well as P450scc, P450 aromatase, and 3beta-HSD immunoreactivities were found in these cells. In addition, our results revealed undoubtedly that extravillous cytotrophoblasts (CTBs), e.g. those from which cell columns of anchoring villous originate, also express the type 1 17beta-HSD gene. However, CTBs lying beneath the syncytial layer, e.g. those from which syncytiotrophoblasts develop, contained barely detectable amounts of type 1 17beta-HSD mRNA as determined by in situ hybridization. These findings, along with those from other laboratories confirm the primordial role of the syncytium in the synthesis of steroids during pregnancy. In addition, our results indicate for the first time that CTBs differentiating along the invasive pathway contain type 1 17beta-HSD mRNA.

Hypoxemia represents a major stress for the fetus, and is associated with alterations and adaptations in cardiovascular, metabolic and endocrine responses, which in turn may affect tissue growth and differentiation. To determine the effects of hypoxemia on fetal adrenal activity and growth, we subjected sheep fetuses at days 126-130 and 134-136 (term 145 days) to reduced PaO2 by reducing the maternal fraction of oxygen for 48 h (mean reduction of 6.8 mmHg), without change in arterial pH or PaCO2. This stimulus resulted in similar increases in the plasma immunoreactiveACTH response at both ages. Among adrenal steroids, plasma cortisol (C21Delta4) rose in both groups of animals, but plasma androstenedione (C19Delta4) declined marginally, resulting in a pronounced increase in the cortisol:androstenedione ratio in the plasma that was greater and more sustained in the older fetuses. In the younger fetuses, after 48 h of hypoxemia, there were no significant changes in mRNAs encoding steroidogenic enzymes in the fetal adrenal gland. However, in the older fetuses, hypoxemia resulted in significantly increased levels of mRNAs encoding P450scc, P450C21 and 3beta-hydroxysteroid dehydrogenase, but not for P450C17, in the fetal adrenal gland. Levels of IGF-II mRNA in the fetal adrenal gland fell in both groups of fetuses, and this response was greater at the later gestational age. We conclude that sustained hypoxemia is a potent stimulus which activates adrenal steroidogenesis in the late gestation fetal sheep. The resultant increase in cortisol synthesis is associated with decreased expression of adrenal IGF-II mRNA. We speculate that this relationship might influence patterns of fetal organ growth and differentiative function in response to fetal stress such as hypoxemia.

The ontogeny of inhibin secretion in the testis of rats was investigated. Testicular localization, content of immunoactive and bioactive inhibin and its molecular size in fetal and neonatal rats (from 16 days of gestation to 5 days of age) were determined. Strong immunostaining with an antiserum against a polypeptide of porcine inhibin alpha-subunit was noted in testicular interstitial cells from 16 days of gestation. Co-localization of inhibin alpha-subunit and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was observed in the interstitial cells until 2 days of age. Immunoreactive inhibin alpha-subunit in the interstitial tissue had disappeared by 5 days of age, although 3 beta HSD-positive cells were still detected. Weak immunostaining for the inhibin alpha-subunit was detected in the seminiferous tubules, probably in the cytoplasm of Sertoli cells, from 20 days of gestation onward. No inhibin alpha-subunit immunostaining was observed in germ cells throughout the experimental period. Testicular inhibin was detected at 16 days of gestation (49.5 +/- 6.7 pg per testis) by RIA. Testicular immunoreactive inhibin showed a tendency to increase during fetal life and levels were maintained at a similar value after birth (697.0 +/- 46.9 pg per testis at 5 days of age). Inhibin bioactivity and its molecular size in testicular homogenate was examined at 17 days of gestation and 0 and 5 days of age. Although no bioactivity was detected at 17 days of gestation, bioactivity was noted at 0 and 5 days of age (177.7 and 1303.9 pg per testis respectively). Immunoblot analysis with antiserum against inhibin alpha-subunit revealed only approximately 40 kDa molecular masses in the testis at 17 days of gestation, probably inhibin-related proteins, but not inhibin. At 0 and 5 days of age, a protein of 30 kDa molecular mass, possibly inhibin, was detected as well as material of approximately 40 kDa molecular mass. FSH in the plasma was first detected at 19 days of gestation (1197.0 ng/l), increased towards birth, and thereafter decreased (4588.5 +/- 572.3 ng/l at 21 days of gestation and 2400.0 +/- 179.6 ng/l at 5 days of age). These results indicate that Leydig cells in fetal and neonatal rats produce inhibin-related substances with no inhibin bioactivity, whereas Sertoli cells begin to produce inhibin during the perinatal period as a possible regulator of FSH secretion.

We have investigated the expression of cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type 1 genes during human trophoblast differentiation in culture and the modulation of their steady-state mRNA levels by steroids. During the first 24 to 48 h after plating, mononucleated cells aggregated, forming colonies. After 60 h in culture, cell diameters were increased and nuclei appeared centrally distributed within large cells, consistent with syncytiotrophoblast formation. During these striking morphological changes in culture the expression and activity levels of 3 beta-HSD type 1 and P450scc increased significantly as isolated cytotrophoblasts progressed to a differentiated state, with P450scc and 3 beta-HSD type 1 mRNAs activities being more abundant in cells cultured for 48 to 72 h. In the same culture, however, the amount of 3 beta-HSD protein decreased during the first 12 to 24 h by 50% compared with freshly isolated trophoblasts but remained at these levels throughout the culture period. The specific activity of the 3 beta-HSD as determined with pregnenolone or dehydroepiandrosterone was similar but increased with time as syncytiotrophoblast was formed in vitro. These observations provide additional evidence that the expression of these two progesterone-synthesizing enzymes is coincident and that they reach their maximum steady-state mRNA levels at a time when syncytium formation occurs in vitro. Incubation of trophoblast cells with progesterone or estradiol increased the abundance of P450scc and 3 beta-HSD type 1 mRNAs but had no significant effect on the amount of 3 beta-HSD protein. These observations of the regulation of 3 beta-HSD type 1 mRNA levels by steroids suggest a complex relationship of the mechanisms regulating transcription/mRNA processing and transduction of the 3 beta-HSD type 1 gene.

The presence of non-specific esterase activity is correlated with different Leydig cell characteristics: 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), human chorionic gonadotrophin binding and LH-stimulated steroid production. This indicates that esterase can be used as a marker enzyme for Leydig cells. Esterase, however, has also been used as a marker enzyme for macrophages. We have compared, using biochemical and histochemical techniques, the esterase activity of Leydig cell preparations from mature and immature rats and of preparations enriched in testicular or peritoneal macrophages. Leydig cells were identified by staining for 3 beta-HSD, and macrophages by phagocytosis of fluorescent beads. Leydig cell preparations from mature rats showed an approximately 400-fold higher esterase activity than peritoneal macrophage preparations and an approximately 50-fold higher activity than testicular macrophage preparations. Leydig cell preparations from mature rats showed a 60-fold higher esterase activity than Leydig cell preparations from immature rats. Differences in esterase activity were also demonstrated histochemically. Leydig cells from mature rats showed positive esterase staining after 30 s at room temperature. Testicular macrophages showed esterase activity after staining for 3 min. Only approximately 25% of the 3 beta-HSD-positive cells from immature rats showed esterase activity after staining for 6 min. Esterase is therefore a useful marker enzyme for Leydig cells from mature rats and can be of help in studies concerning the development of these cells.

Detailed histochemical localization of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 17 beta-HSD was made in the goat testis using both NAD and NADP coenzymes. The substrates used for 3 beta-HSD were dehydroepiandrosterone (DHA) and pregnenolone whereas 17 beta-HSD was localized with testosterone and oestradiol. In general, the activity of the enzymes varied with the cell type, substrate and coenzyme. In seminiferous tubules, DHA and NAD were the preferred substrate and coenzyme respectively for 3 beta-HSD. In addition, in interstitial tissue, NAD was the preferred coenzyme with DHA whereas no such preference existed with pregnenolone. 17 beta-Hydroxysteroid dehydrogenase showed a similar pattern in the two main compartments of the testis, as testosterone and oestradiol were equally utilized and NAD was the preferred coenzyme in both these compartments. The activities of the enzymes increased during the process of spermiogenesis and were higher in seminiferous tubules than in interstitial tissue, especially in elongated spermatids and spermatozoa.

The capacity for steroid production in the corpora lutea (CL) of bank voles was examined by enzyme histochemistry for 3 beta-hydroxysteroid dehydrogenase. The results revealed similar activity in the CL resulting from sterile matings and the CL of pregnancy 2 days after mating. Six days after mating, the CL of pregnancy showed a strong reaction, while there was almost no reaction in the CL from sterile matings, which were clearly regressed at this time. It is suggested that the CL from sterile matings produce progesterone before regression and that this progesterone may prime the luteotrophic complex, increasing fertility of subsequent matings.