mRNA Levels Of Steroidogenic Enzymes Research Articles

Alpha-Lipoic Acid Ameliorates Impaired Steroidogenesis in Human Granulosa Cells Induced by Advanced Glycation End-Products.

Ovarian granulosa cells (GCs) are essential for follicular development. Ovarian advanced glycation end-products (AGEs) accumulation is related to GCs dysfunction. Alpha-lipoic acid (ALA) illustrates therapeutic capabilities for infertility-related disorders. Therefore, this study assessed the effects of ALA on AGEs-induced GCs hormonal dysfunction. The study was conducted from October 2021 to September 2022 at the Department of Medical Genetics, Shiraz University of Medical Sciences. Isolated GCs (n=50) were divided into control, human glycated albumin (HGA), HGA+ALA, and ALA treatments. Steroidogenic enzymes and AGE receptor (RAGE) genes were assessed by qRT-PCR. Steroid hormones and RAGE protein were evaluated using ELISA and Western blotting. Data were analyzed using GraphPad Prism software (ver. 9), and P<0.05 was considered significant. Our findings showed that HGA treatment significantly (P=0.0001) increased RAGE (by 140.66%), STAR (by 117.65%), 3β-HSD (by 165.68%), and 17β-HSD (by 122.15%) expression, while it decreased CYP19A1 (by 68.37%) expression. RAGE protein level (by 267.10%) was also increased in HGA-treated GCs. A significant decrease in estradiol (by 59.66%) and a slight and sharp elevation in progesterone (by 30.40%) and total testosterone (by 158.24%) levels was also observed. ALA treatment ameliorated the HGA-induced changes in steroidogenic enzyme mRNA levels (P=0.001) and steroid hormone secretion (P=0.010). This work shows that ALA therapy likely corrects hormonal dysfunctions caused by AGEs in luteinized GCs. This effect is probably achieved by decreased RAGE expression. Clinical research is needed to understand how AGEs and ALA interact in the ovary, which might lead to a more targeted ovarian dysfunction therapy.

SAT267 BMP4, GDF11 And TGFβ2 Repress Adrenal Cell Cortisol Production By Blocking CYP17A1 And CYP11B1 Expression

Abstract Disclosure: E.C. Fischbach: None. A. Lopez: None. M. Ullenbruch: None. J. Rege: None. W.E. Rainey: None. Introduction: The adrenal cortex is responsible for the biosynthesis of multiple steroid hormones that regulate electrolyte and carbohydrate balance. Cortisol, the principal glucocorticoid in humans, is primarily produced in the middle zone of the cortex known as the zona fasciculata (ZF). The enzymes responsible for cortisol biosynthesis include cytochrome P450c17 (17α-hydroxylase), encoded by CYP17A1 and cytochrome P450c11 (11β-hydroxylase), encoded by CYP11B1. Several paracrine/autocrine regulators, including members of the transforming growth factor beta (TGFβ) superfamily, have been implicated in controlling adrenocortical cell homeostasis and steroid hormone production. Previously, we reported a bone morphogenetic protein 4 (BMP4) expression gradient in the adrenal cortex with higher levels in the zona glomerulosa (ZG) and outer ZF compared to the zona reticularis (ZR). In this study, we sought to compare adrenal expression of members of the TGFβ family including BMP4, growth differentiation factor 11 (GDF11), and transforming growth factor beta 2 (TGFβ2), and determine their effect on adrenal cell cortisol biosynthesis. Methods: Growth factor mRNA levels were studied using microarray analysis of laser captured ZG, ZF and ZR from human adrenals. The adrenal cell line, HAC15 was used to determine the effects of the TGFβ family members alone and in combination. Output measures included cell cortisol production and mRNA levels of steroidogenic enzymes using ELISA and quantitative RT-PCR, respectively. Results: Microarray analysis indicated higher mRNA levels of BMP4, GDF11 and TGFβ2 in the ZG compared with the ZF and ZR regions of the adrenal. Treatment of HAC15 cells with either BMP4 (50 ng/mL), GDF11 (50 ng/mL) or TGFβ2 (10 ng/mL) significantly inhibited cortisol production, as well as CYP11B1 and CYP17A1 mRNA levels. The inhibition was observed under basal and forskolin-stimulated conditions. Furthermore, the inhibitory effects on cortisol production and enzyme expression were additive when factors were added together. Summary: Overall, our findings suggest that the human adrenal has a gradient of multiple members of the TGFβ family. The additive action of these growth factors on adrenal cell differentiation may explain, in part, the inability of the ZG to produce cortisol and its low expression of CYP11B1 and CYP17A1. Presentation: Saturday, June 17, 2023

Interaction of Orexin and Bone Morphogenetic Proteins in Steroidogenesis by Human Adrenocortical Cells.

Orexins are neuropeptides that play important roles in sleep-wake regulation and food intake in the central nervous system, but their receptors are also expressed in peripheral tissues, including the endocrine system. In the present study, we investigated the functions of orexin in adrenal steroidogenesis using human adrenocortical H295R cells by focusing on its interaction with adrenocortical bone morphogenetic proteins (BMPs) that induce adrenocortical steroidogenesis. Treatment with orexin A increased the mRNA levels of steroidogenic enzymes including StAR, CYP11B2, CYP17, and HSD3B1, and these effects of orexin A were further enhanced in the presence of forskolin. Interestingly, orexin A treatment suppressed the BMP-receptor signaling detected by Smad1/5/9 phosphorylation and Id-1 expression through upregulation of inhibitory Smad7. Orexin A also suppressed endogenous BMP-6 expression but increased the expression of the type-II receptor of ActRII in H295R cells. Moreover, treatment with BMP-6 downregulated the mRNA level of OX1R, but not that of OX2R, expressed in H295R cells. In conclusion, the results indicate that both orexin and BMP-6 accelerate adrenocortical steroidogenesis in human adrenocortical cells; both pathways mutually inhibit each other, thereby leading to a fine-tuning of adrenocortical steroidogenesis.

Different cell compositions and a novel somatic KCNJ5 variant found in a patient with bilateral adrenocortical adenomas secreting aldosterone and cortisol.

This study aimed to explore the possible pathogenesis of a rare case of co-existing Cushing's syndrome (CS) and primary aldosteronism (PA) caused by bilateral adrenocortical adenomas secreting aldosterone and cortisol, respectively. A 41-year-old Chinese woman with severe hypertension and hypokalemia for 5 and 2 years, respectively, was referred to our hospital. She had a Cushingoid appearance. Preoperative endocrinological examinations revealed autonomous cortisol and aldosterone secretion. Computed tomography revealed bilateral adrenal adenomas. Subsequently, adrenal vein sampling and sequential left and right partial adrenalectomy indicated the presence of a left aldosterone-producing tumor and a right cortisol-producing tumor. Pathological examination included immunohistochemical analysis of the resected specimens. Secretions of aldosterone and cortisol were observed both in vivo and in vitro. Further, whole-exome sequencing was performed for DNA that was extracted from peripheral blood leukocytes and bilateral adrenal adenomas in order to determine whether the patient had relevant variants associated with PA and CS. Immunohistochemical staining revealed that the left adenoma primarily comprised clear cells expressing CYP11B2, whereas the right adenoma comprised both eosinophilic compact and clear cells expressing CYP11B1. The mRNA levels of steroidogenic enzymes (including CYP11B1 and CYP17A1) were high in the right adenoma, whereas CYP11B2 was highly expressed in the left adenoma. A novel somatic heterozygous missense variant-KCNJ5 c.503T > G (p.L168R)-was detected in the left adrenal adenoma, but no other causative variants associated with PA and CS were detected in the peripheral blood or right adrenocortical adenoma. In the primary cell culture of the resected hyperplastic adrenal adenomas, verapamil and nifedipine, which are two calcium channel blockers, markedly inhibited the secretion of both aldosterone and cortisol. We present an extremely rare case of bilateral adrenocortical adenomas with distinct secretion of aldosterone and cortisol. The heterogeneity of the tumor cell compositions of aldosterone- and cortisol-producing adenoma (A/CPA) and somatic mutation of KCNJ5 may have led to different hormone secretions in the bilateral adrenal adenomas.

Derailed peripheral circadian genes in polycystic ovary syndrome patients alters peripheral conversion of androgens synthesis.

Do circadian genes exhibit an altered profile in peripheral blood mononuclear cells (PBMCs) of polycystic ovary syndrome (PCOS) patients and do they have a potential role in androgen excess? Our findings revealed that an impaired circadian clock could hamper the regulation of peripheral steroid metabolism in PCOS women. PCOS patients exhibit features of metabolic syndrome. Circadian rhythm disruption is involved in the development of metabolic diseases and subfertility. An association between shift work and the incidence of PCOS in females was recently reported. This is a retrospective case-referent study in which peripheral blood samples were obtained from 101 control and 101 PCOS subjects. PCOS diagnoses were based on Rotterdam Consensus criteria. This study comprised 101 women with PCOS and 101 control volunteers, as well as Swiss albino mice treated with dehydroepiandrosterone (DHEA) to induce PCOS development. Gene expression analyses of circadian and steroidogenesis genes in human PBMC and mice ovaries and blood were executed by quantitative real-time PCR. We observed aberrant expression of peripheral circadian clock genes in PCOS, with a significant reduction in the core clock genes, circadian locomotor output cycles kaput (CLOCK) (P ≤ 0.00001), brain and muscle ARNT-like 1 (BMAL1) (P ≤ 0.00001) and NPAS2 (P ≤ 0.001), and upregulation of their negative feedback loop genes, CRY1 (P ≤ 0.00003), CRY2 (P ≤ 0.00006), PER1 (P ≤ 0.003), PER2 (P ≤ 0.002), DEC1 (P ≤ 0.0001) and DEC2 (P ≤ 0.00005). Transcript levels of an additional feedback loop regulating BMAL1 showed varied expression, with reduced RORA (P ≤ 0.008) and increased NR1D1 (P ≤ 0.02) in PCOS patients in comparison with the control group. We also demonstrated the expression pattern of clock genes in PBMCs of PCOS women at three different time points. PCOS patients also exhibited increased mRNA levels of steroidogenic enzymes like StAR (P ≤ 0.0005), CYP17A1 (P ≤ 0.005), SRD5A1 (P ≤ 0.00006) and SRD5A2 (P ≤ 0.009). Knockdown of CLOCK/BMAL1 in PBMCs resulted in a significant reduction in estradiol production, by reducing CYP19A1 and a significant increase in dihydrotestosterone production, by upregulating SRD5A1 and SRD5A2 in PBMCs. Our data also showed that CYP17A1 as a direct CLOCK-BMAL1 target in PBMCs. Phenotypic classification of PCOS subgroups showed a higher variation in expression of clock genes and steroidogenesis genes with phenotype A of PCOS. In alignment with the above results, altered expression of ovarian core clock genes (Clock, Bmal1 and Per2) was found in DHEA-treated PCOS mice. The expression of peripheral blood core clock genes in DHEA-induced PCOS mice was less robust and showed a loss of periodicity in comparison with that of control mice. N/A. We could not evaluate the circadian oscillation of clock genes and clock-controlled genes over a 24-h period in the peripheral blood of control versus PCOS subjects. Additionally, circadian genes in the ovaries of PCOS women could not be evaluated due to limitations in sample availability, hence we employed the androgen excess mouse model of PCOS for ovarian circadian assessment. Clock genes were assessed in the whole ovary of the androgen excess mouse model of PCOS rather than in granulosa cells, which is another limitation of the present work. Our observations suggest that the biological clock is one of the contributing factors in androgen excess in PCOS, owing to its potential role in modulating peripheral androgen metabolism. Considering the increasing prevalence of PCOS and the rising frequency of delayed circadian rhythms and insufficient sleep among women, our study emphasizes the potential in modulating circadian rhythm as an important strategy in PCOS management, and further research on this aspect is highly warranted. This work was supported by the RGCB-DBT Core Funds and a grant (#BT/PR29996/MED/97/472/2020) from the Department of Biotechnology (DBT), India, to M.L. B.S.J. was supported by a DST/INSPIRE Fellowship/2015/IF150361 and M.B.K. was supported by the Research Fellowship from Council of Scientific & Industrial Research (CSIR) (10.2(5)/2007(ii).E.U.II). The authors declare no competing interests. N/A.

A Novel Antigonadotropic Role of Thyroid Stimulating Hormone on Leydig Cell-Derived Mouse Leydig Tumor Cells-1 Line.

Subclinical hypothyroid men characterized by a rise in only thyroid stimulating hormone (TSH) levels and normal thyroid hormone levels showed a fall in their serum progesterone and testosterone levels. This suggested a role of TSH in regulating Leydig cell steroidogenesis. Therefore, we investigated the direct role of TSH on steroid production and secretion using a mouse Leydig tumour cell line, MLTC-1. MLTC-1 cells were treated with different doses of TSH isolated from porcine pituitary as well as recombinant TSH. Steroid secretion was measured by radioimmunoassay. The mRNA levels of steroidogenic enzymes were quantitated by real time PCR whereas the corresponding protein levels were determined by Western blot. In MLTC-1 cells, pituitary TSH as well as recombinant TSH inhibited progesterone and testosterone secretion in a dose dependent manner. The inhibitory action of TSH on steroid secretion was unique and not mimicked by other anterior pituitary hormones including FSH and ACTH. Recombinant TSH showed no effect on StAR and CYP11A1, the enzymes catalysing the non-steroidogenic and steroidogenic rate-limiting steps of steroid synthesis respectively. Recombinant TSH was shown to inhibit steroidogenesis in MLTC-1 cells by inhibiting the 3β hydroxy steroid dehydrogenase mRNA and protein levels, the enzyme that catalyses the conversion of pregnenolone to progesterone. This inhibitory effect of TSH is probably direct as both mRNA and protein of the TSH receptor were shown to be present in the MLTC-1 cells.

Changes in Sex Steroids and Ovarian Steroidogenic Enzyme mRNA Levels in Artificially Maturing Japanese Eel (Anguilla japonica) and Naturally Maturing New Zealand Longfin Eel (Anguilla dieffenbachii) during Vitellogenesis

Repeated hormone injections are routinely used to induce advanced stages of oogenesis in freshwater eels, but this approach may result in aberrant germ cell development. To investigate the underlying causes, levels of sex steroids (testosterone, T; estradiol-17β, E2) and ovarian steroidogenic enzyme mRNAs were compared between artificially maturing Japanese eels and wild-caught, spontaneously maturing New Zealand longfin eels. The latter were employed as reference, as wild Japanese eels in advanced stages of oogenesis are near-impossible to catch. Serum T levels in artificially maturing Japanese eel changed with stage in a pattern that was comparable to that in longfin eels. Likewise, ovarian mRNA levels of most steroidogenic enzyme genes were not qualitatively dissimilar between both eel species when taking developmental stage into account. However, aromatase (cyp19a) mRNA levels, together with serum E2 levels, rapidly increased in artificially maturing Japanese eels in mid-late stages of oogenesis (gonadosomatic index, GSI = 13.8%), whereas no such increase was evident in longfin eels (GSI ~ 6.9%). In addition, sex steroid and target gene mRNA levels fluctuated drastically with each hormone injection. We contend that expression of most target genes, possibly even that of cyp19a, during induced oogenesis could be “normal”, with the drastic fluctuations due most likely to hormone delivery through repeated injections. The effects of these fluctuations on gamete quality remain unknown and resolving this issue may prove fruitful in the future to further artificial propagation of anguillid eels.

Transcriptional networks associated with 5-alpha-dihydrotestosterone in the fathead minnow (Pimephales promelas) ovary

Androgens play a significant role in regulating oogenesis in teleost fishes. The androgen dihydrotestosterone (DHT) is a potent non-aromatizable androgen involved in sexual differentiation in mammals; however, its actions are not well understood in teleost fish. To better characterize the physiological role of DHT in the fathead minnow (FHM) ovary on a temporal scale, in vitro assays for 17β-estradiol (E2) production were conducted in parallel with microarray analysis. Ovarian explants were incubated at different concentrations of DHT (10−6, 10−7, and 10−8M DHT) in three separate experiments conducted at 6, 9, and 12h. DHT treatment resulted in a rapid and consistent increase in E2 production from the ovary at all three time points. Therefore, DHT may act to shift the balance of metabolites in the steroidogenic pathway within the ovary. Major biological themes affected by DHT in the ovary in one or more of the time points included those related to blood (e.g. vasodilation, blood vessel contraction, clotting), lipids (e.g. lipid storage, cholesterol metabolism, lipid degradation) and reproduction (e.g. hormone and steroid metabolism). Gene networks related to immune responses and calcium signaling were also affected by DHT, suggesting that this androgen may play a role in regulating these processes in the ovary. This study detected no change in mRNA levels of steroidogenic enzymes (cyp19a1, star, 11βhsd, 17βhsd, srd5a isoforms), suggesting that the observed increase in E2 production is likely more dependent on the pre-existing gene or protein complement in the ovary rather than the de novo expression of transcripts. This study increases knowledge regarding the roles of DHT and androgens in general in the teleost ovary and identifies molecular signaling pathways that may be associated with increased E2 production.

Sex-specific differences in the effects of local androgen metabolism in the heart as an indicator for the risk of myocardial infarction

Aim Testosterone influences cardiovascular risk and disease in a sex-specific manner. The more potent androgen 5α-dihydrotestosterone (DHT) can be formed through conversion of testosterone by the enzyme 5α-reductase. We hypothesized that, because of the presence of DHT in coronary and myocardial tissues, a sexually dimorphic effect can be observed if differences exist in genetics or mRNA expression in androgen-metabolizing enzymes. Materials and methods mRNA levels of steroidogenic enzymes and the androgen receptor (AR) were investigated in human myocardial tissue samples. Subsequently, all participants in the baseline cohort of the Rotterdam Study (RSI) with successful genotyping and without prevalent myocardial infarction (MI, N=5199) were recruited to study the association between single nucleotide polymorphisms (SNPs) within SRD5A1, SRD5A2, and AKR1C3 and incident MI using Cox regression models. Significant results were replicated within the Atherosclerosis Risk in Communities cohort and the second cohort of the RSII. Results The expression of SRD5A1, AKR1C3, and AR was found in all myocardial samples, whereas HSD17B3 and SRD5A2 expression levels were low and undetectable, respectively. Myocardial SRD5A1 expression was higher in women than in men. Within SRD5A1, SNP rs248805G>A was significantly associated with incident MI in western European women (hazard ratio 1.49; 95% confidence interval 1.19-1.87). This SNP is tightly linked to the HinfI polymorphism in SRD5A1 (rs248793G>C), of which the minor allele has been associated with a higher DHT/T ratio. Conclusion Genetic variation in SRD5A1 is associated with an increased risk of MI in western European women, possibly because of the sex-specific potential of local androgen conversion and effect.

Steroidogenic enzymes, their related transcription factors and nuclear receptors in human sebaceous glands under normal and pathological conditions

The sebaceous gland is a major site of steroid synthesis in human skin, but details of the status of steroidogenic enzymes and their regulation in human sebaceous glands under normal and pathological conditions have rarely been reported. Therefore, in this study, we examined the status of steroidogenic enzymes, sex steroid receptors and transcription factors in human sebaceous glands under normal and pathological conditions to explore their possible roles in in situ steroid production in human skin. Immunohistochemical analysis was performed in a total of 59 human skin specimens, including 22 normal human sebaceous glands, 12 with sebaceous nevus, 12 with sebaceous gland hyperplasia, 3 with sebaceoma and 10 with sebaceous carcinoma. Immortalised human SZ95 sebocytes were treated with forskolin or vehicle for 3h, 6h, 12h or 24h, and the mRNA levels of steroidogenic enzymes were evaluated at each time point using quantitative RT-PCR (qPCR). The results of immunohistochemistry demonstrated the immunoreactivity of 3β-HSD1, CYP11A1, StAR, 17β-HSD5, CYP17A1, 5α-red1, PRB, AR and NGFI-B in normal human sebaceous gland, with lower levels of expression in pathological sebaceous glands. The results of the in vitro study also indicated that the expression levels of 3β-HSD1, CYP11A1, StAR, 5α-red1 and NGFI-B were elevated by forskolin. 3β-HSD1 and other steroidogenic enzymes were expressed in sebaceous glands resulting in in situ androgen and progesterone synthesis and their functions.

Comparison of cell types in the rat Leydig cell lineage after ethane dimethanesulfonate treatment

The objective of this study was to purify cells in the Leydig cell lineage following regeneration after ethane dimethanesulfonate (EDS) treatment and compare their steroidogenic capacity. Regenerated progenitor (RPLCs), immature (RILCs), and adult Leydig cells (RALCs) were isolated from testes 21, 28 and 56 days after EDS treatment respectively. Production rates for androgens including androsterone and 5α-androstane-17β, 3α-diol (DIOL), testosterone and androstenedione were measured in RPLCs, RILCs and RALCs in media after 3-h in vitro culture with 100 ng/ml LH. Steady-state mRNA levels of steroidogenic enzymes and their activities were measured in freshly isolated cells. Compared to adult Leydig cells (ALCs) isolated from normal 90-day-old rat testes, which primarily produce testosterone (69.73%), RPLCs and RILCs primarily produced androsterone (70.21%) and DIOL (69.79%) respectively. Leydig cells isolated from testes 56 days post-EDS showed equivalent capacity of steroidogenesis to ALCs and primarily produced testosterone (72.90%). RPLCs had cholesterol side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1 and 17α-hydroxylase but had almost no detectable 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities, while RILCs had increased 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities. Because RPLCs and RILCs had higher 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase activities they produced mainly 5α-reduced androgens. Real-time PCR confirmed the similar trends for the expressions of these steroidogenic enzymes. In conclusion, the purified RPLCs, RILCs and RALCs are similar to those of their counterparts during rat pubertal development.

Between-female variation in house sparrow yolk testosterone concentration is negatively associated with CYP19A1 (aromatase) mRNA expression in ovarian follicles

Maternally-derived yolk androgens influence the development and long-term phenotype of offspring in oviparous species. Between-female variation in the amounts of these yolk androgens has been associated with a number of social and environmental factors, suggesting that the variation is adaptive, but the mechanisms behind it are unknown. Using two different approaches, we tested the hypothesis that variation in yolk androgen levels across individuals is associated with variation in their capacity to synthesize androgens. First, we injected female house sparrows with exogenous gonadotropin-releasing hormone (GnRH) to maximally stimulate ovarian steroidogenesis. Second, we collected pre-ovulatory follicle tissue and quantified the mRNA expression of four key enzymes of the steroid synthesis pathway: steroidogenic acute regulatory protein (StAR), cytochrome P450-side chain cleavage enzyme (CYP11A1), 17β-hydroxysteroid dehydrogenase (HSD17B1), and aromatase (CYP19A1). Thirty minutes after GnRH injection, androgen concentrations in both the plasma and in the yolks of pre-ovulatory follicles were significantly elevated compared to controls. However, this measure of steroidogenic capacity did not explain variation in yolk testosterone levels, although physiological differences between house sparrows and more widely studied poultry models were revealed by this approach. Steroidogenic enzyme mRNA levels were detectable in all samples and were significantly lower in the most mature pre-ovulatory follicles. Of the four measured genes, CYP19A1 expression exhibited a significant negative relationship with yolk testosterone concentrations in laid eggs, revealing a key mechanism for between-female variation in yolk testosterone. Furthermore, this suggests that any factors which alter the expression of CYP19A1 within an individual female could have dramatic effects on offspring phenotype.

KCNJ5 mutations in aldosterone- and cortisol-co-secreting adrenal adenomas [Rapid Communication

Adrenal aldosterone-producing adenomas (APA) are rarely associated with the clear co-secretion of cortisol. Somatic mutations of the potassium channel KCNJ5 gene, with the hotspots G151R and L168R, have been recently identified in patients with APA. However, whether APAs that secrete cortisol have these mutations remains unclear. We examined three patients with APAs showing clear autonomous secretion of cortisol who possessed a 1 mg dexamethasone suppression test (DST) with a failure of the serum cortisol level to drop below 3.0 μg/dL, a morning plasma ACTH level of less than 10 pg/mL, and suppressed accumulation in the intact adrenal on (131)I- adosterol scintigraphy, or postoperative adrenal insufficiency. Laparoscopic adrenectomy revealed all tumors to be golden yellow, and histological examination confirmed them to be adrenocortical adenomas. All these patients required replacement therapy with hydrocortisone after surgery. Sequencing demonstrated that 2 of three cases showed a mutation of the KCNJ5 gene, one with c.451G>A, p.G151R and one with c.503T>G, p.L168R. Furthermore, the mRNA levels of steroidogenic enzymes including CYP11B1, CYP11B2, HSD3B2, CYP17A1, CYP11A1 and KCNJ5 in the 3 cases did not differ from those in 8 pure APAs not showing any of the above conditions for autonomous cortisol secretion. In addition, all 8 pure APAs harbored mutations of the KCNJ5 gene. These findings suggested that at least some aldosterone- and cortisol-co-secreting adrenal tumors have mutations of the KCNJ5 gene, suggesting the origin to be APA, and pure APAs may show a high incidence of KCNJ5 mutations.

Metastin Stimulates Aldosterone Synthesis in Human Adrenal Cells

Kisspeptins, including metastin, are encoded by the KiSS-1 gene and play an important role in regulating the hypothalamic gonadotropin-releasing hormone (GnRH) system via G protein-coupled receptor 54 (GPR54, also called KiSS-1R). Normally, metastin (also called Kp-54) levels are quite low, except during pregnancy, when levels increase 1000-fold over those found in men and nonpregnant women. However, the potential hormonal role of metastin in the fetal and maternal circulation is unknown. In this study, the authors examine the levels of GPR54 mRNA expression in human adult and fetal adrenals using quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). In addition, they examine the effects of metastin on steroidogenesis and steroidogenic enzyme mRNA levels in fetal adrenal cells and in the H295R adrenocortical cell line using enzyme immunoassay and RT-PCR techniques. The authors demonstrate that GPR54 mRNA is significantly higher (50-fold) in human fetal adrenals than in adult adrenals. Immunohistochemical studies have demonstrated that the GPR54 protein is predominantly expressed in the neocortex of human fetal adrenals in the third trimester. Metastin increases aldosterone production (approximately 2-fold) in both fetal neocortex adrenal cells and H295R adrenal cells, with a maximal increase seen at 100 nM. In addition, metastin increased angiotensin II (Ang II)-stimulated aldosterone production by approximately 1.5-fold. Metastin also increased the ability of the H295R cells to metabolize exogenously added pregnenolone to aldosterone but had no effect on the expression of aldosterone synthase (CYP11B2). These results suggest that the high fetal/maternal levels of metastin seen during pregnancy may affect adrenal production of aldosterone.

Testicular Expression of Steroidogenic Enzyme Genes Is Related to a Transient Increase in Serum 19-nortestosterone during Neonatal Development in Pigs

Cytochrome P450 aromatase is responsible for the biosynthesis of estrogen. It is also responsible for the endogenous production of 19-nortestosterone (nandrolone), an anabolic androgen unique to pigs. Plasma concentrations of 19-nortestosterone are highest between two and four weeks after birth in male pigs. In the present study, the physiology of 19-nortestosterone was investigated by measuring the mRNA levels of steroidogenic enzymes, estrogen receptors and androgen receptor in the tissues of growing pigs. The expression of aromatase, 17α-hydroxylase and 3β-hydroxysteroid dehydrogenase in the testes of male piglets increased between birth and two weeks of age, and then decreased progressively. Similar developmental expressional patterns were observed for 17α- hydroxylase and 3β-hydroxysteroid dehydrogenase in the ovaries of female piglets, but without significant aromatase expression. The major form of aromatase expressed in the testes of piglets was identified as type I. Expression of estrogen receptor-α and -β and androgen receptor genes was also detected in both testes and ovaries. A transient elevation of androgen receptor mRNA in male piglets at two weeks of age was also observed in testes. Significant expression of the androgen receptor gene, but not of estrogen receptor-α and -β genes, was also demonstrated in adipose tissue and muscle. We conclude that the observed increase in the testicular expression of aromatase in male pigs could account for the production of large amounts of 19-nortestosterone at between two and four weeks of age in males. Androgen receptor and 19-nortestosterone appeared to be important for testicular development and might contribute to sexual dimorphism in body composition and muscle development in juvenile pigs. (Key Words : Cytochrome P450 Aromatase, Pig Testis, 19- Nortestosterone)

Adenovirus-delivered DKK3/WNT4 and Steroidogenesis in Primary Cultures of Adrenocortical Cells

The Wnt family molecules Dickkopf-3 (DKK3) and WNT4 are present at higher concentrations in the zona glomerulosa than in the rest of the adrenal cortex. In order to study direct effects of these proteins on adrenocortical cell function, we created adenoviruses encoding human DKK3 and WNT4. When added to cultured human adrenocortical cells, DKK3 inhibited aldosterone and cortisol biosynthesis, either alone or together with cyclic AMP. WNT4 increased steroidogenesis when added alone but decreased it in the presence of cyclic AMP. A control adenovirus encoding GFP had no effect. RNA was prepared from cultured cells and was assayed by real-time PCR. CYP11A1 (cholesterol side-chain cleavage enzyme), HSD3B2 (3beta-hydroxysteroid dehydrogenase type II), CYP17 (17 alpha-hydroxylase), CYP21 (21-hydroxylase) and CYP11B1 (11 beta-hydroxylase) mRNAs were all increased by cyclic AMP, whereas CYP11B2 (aldosterone synthase) was unaffected. DKK3 decreased cyclic AMP-stimulated CYP17. WNT4 increased both CYP17 and CYP21 in the absence of cyclic AMP. Both DKK3 and WNT4 increased the level of CYP11B2. These data show that these Wnt signaling molecules have multiple actions on steroidogenesis in adrenocortical cells, including effects on overall steroidogenesis (aldosterone and cortisol biosynthesis) and distinct effects on steroidogenic enzyme mRNA levels. The co-localization of DKK3 and WNT4 in the glomerulosa and their stimulation of CYP11B2 imply an action on glomerulosa-specific function.

Thyroid hormones: their role in testicular steroidogenesis.

Thyroid hormones are important for growth and development of many tissues. Altered thyroid hormone status causes testicular abnormalities. For instance, juvenile hypothyroidism/neonatal transient hypothyroidism induces macroorchidism, increases testicular cell number (Sertoli, Leydig, and germ cells) and daily sperm production. Triiodothyronine (T3) receptors have been identified in sperm, developing germ cells, Sertoli, Leydig, and peritubular cells. T3 stimulates Sertoli cell lactate secretion as well as mRNA expression of inhibin-alpha, androgen receptor, IGF-I, and IGFBP-4. It also inhibits Sertoli cell mRNA expression of Müllerian inhibiting substance (MIS), aromatase, estradiol receptor, and androgen binding protein (ABP) and ABP secretion. T3 directly increases Leydig cell LH receptor numbers and mRNA levels of steroidogenic enzymes and steroidogenic acute regulatory protein. It stimulates basal and LH-induced secretion of progesterone, testosterone, and estradiol by Leydig cells. Steroidogenic factor-1 acts as a mediator for T3-induced Leydig cell steroidogenesis. Although the role of T3 on sperm, germ, and peritubular cells has not yet been completely studied, it is clear that T3 directly regulates Sertoli and Leydig cell functions. Further studies are required to elucidate the direct effect of T3 on sperm, germ, and peritubular cells.

Administration of estradiol-3-benzoate down-regulates the expression of testicular steroidogenic enzyme genes for testosterone production in the adult rat.

ABSTRACT. To study the role of estrogen in the testes, testosterone and testicular steroidogenic enzyme mRNA levels were investigated in male Sprague-Dawley rats 24 hr after intramuscular administration of a single dose of estradiol-3-benzoate (EB). EB administration resulted in a greater decrease in intra-testicular and serum testosterone in 10-week-old rats than in 3- or 5-week-old rats. A dose of 2 microg EB/kg had the lowest observed effect. The level of serum luteinizing hormone (LH) was unchanged at any dose. Semiquantitative RT-PCR analysis revealed that, of the four major testicular steroidogenic enzymes, mRNA levels of cytochrome P450 side-chain cleavage and 17beta-hydroxysteroid dehydrogenase type-III were significantly reduced, and mRNA levels of cytochrome P450 17alpha-hydroxylase/ C17-20 lyase (P450c17) were reduced severely and significantly, by EB administration. However, the level of 3beta-hydroxysteroid dehydrogenase type-I mRNA was not changed. In addition, the P450c17 mRNA level in EB-treated rats was much lower than that in the testes of hypophysectomized rats, with the level in the latter being equal to that in control rats. LH is secreted into blood periodically, the effects of estrogen on the LH secretion pattern of the pituitary gland, for example, in frequency and amplitude of LH pulse, were difficult to detect with the methods of the present study. The results indicated, at least, that EB administration down-regulates P450c17 gene expression predominantly, resulting in the inhibition of testosterone production. From the differences in the steroidogenic enzyme expressions between hypophysectomized and EB-treated rats, it was suggested that EB acts on the testis directly or indirectly though not via alteration of LH secretion and induces reduction of P450c17 mRNA level.

Expression and regulation of connexin43 in rat Leydig cells.

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.

Differential effects of placental restriction on IGF-II, ACTH receptor and steroidogenic enzyme mRNA levels in the foetal sheep adrenal.

We have investigated the effects of restriction of placental growth on foetal adrenal growth and adrenal expression of mRNAs for Insulin-like Growth Factor II (IGF-II), the IGF binding protein IGFBP-2, Steroidogenic Factor 1 (SF-1) and adrenocorticotrophic hormone (ACTH) receptor (ACTH-R) and the steroidogenic cytochrome P-450 enzymes: cholesterol side chain cleavage (CYP11A1), 17alpha-hydroxylase (CYP17) and 21-hydroxylase (CYP21A1); and 3beta-hydroxysteroid dehydrogenase/Delta5Delta4 isomerase (3betaHSD). Endometrial caruncles were removed from non-pregnant ewes before mating (placental restriction group; PR). The total adrenal: foetal weight ratio was higher in PR (n=6 foetuses) than in control foetuses (n=6 foetuses). There was no difference in plasma ACTH concentrations between the PR and control foetuses between 130 and 140 days gestation. Adrenal IGF-II mRNA levels were lower (P<0.05) in the PR group, however, adrenal IGFBP-2 mRNA levels were not different between the PR and control groups. Adrenal ACTH-R mRNA levels were also lower whilst CYP11A1 mRNA levels were increased (P<0.005) in the PR group. We conclude that foetal adrenal growth and steroidogenesis are stimulated as a consequence of foetal growth restriction and that factors other than ACTH are important in foetal adrenal activation during chronic, sustained hypoxaemia.