Differences in testosterone, androstenone, and skatole levels in plasma and fat between pubertal purebred Duroc and Landrace boars in response to human chorionic gonadotrophin stimulation

The concentrations of the boar taint compounds androstenone and skatole in plasma and fat, together with those of testosterone in plasma, were investigated in pubertal purebred Duroc and Landrace boars following stimulation with human chorionic gonadotrophin (hCG). Higher initial levels of androstenone and testosterone were found in Duroc than Landrace boars. Duroc boars, which were approximately ten days older than the Landrace boars, also showed a more advanced stage of spermatogenesis than Landrace boars. While Landrace boars had the highest skatole levels. Following stimulation with hCG the relative increases in testosterone, androstenone, and skatole concentrations were highest in Landrace boars. The level of androstenone in fat three days after hCG stimulation exceeded 1 μg/g fat in all stimulated boars. The decreases in plasma levels of androstenone and testosterone on Days 2 and 3 after hCG stimulation were more pronounced in Landrace than Duroc boars. However, unlike the plasma androstenone and testosterone levels, the plasma concentrations of skatole did not decrease on Days 2 and 3 following stimulation, but remained elevated on Day 3. These results indicate that the lower levels of testicular steroids in Landrace boars compared with Duroc boars was not due to a lower production capacity, but more likely to a faster dissapearance of steroids in Landrace boars. In the present study, age, live weight, and testicular development did not significantly contribute to the variation in fat androstenone. The present data and previous reports on candidate genes related to androstenone biosynthesis and metabolism suggests that future selection against factors associated with boar taint remains a possible solution for the problem of boar taint in the swine industry.     

Assaying progesterone,estradiol and cortisol concentrations in hair of Père David deer hinds: an alternative way to reflect seasonality of steroid secretion

The use of a hair hormone concentration assay is increasingly recognized as a useful and noninvasive technique for monitoring the endocrinological status of animals. However, few studies have focused on reproductive and stress hormones together. We used a chemiluminescent immunoassay to determine whether the progesterone, estradiol, and cortisol concentrations could be measured from hair and whether these hormone concentrations varied in different hair segments of captive Père David deer hinds. We found that progesterone, estradiol, and cortisol could be measured in the hair samples and that the progesterone concentration varied but the estradiol and cortisol concentrations did not among different hair segments. Contrary to the segmental decline in hair cortisol found in many studies, we found that progesterone concentration was higher near the tip than at the base of hair in Père David deer. This suggests that the variation in segmental hair steroid hormone concentration in seasonal molting animals may be mainly due to internal reproductive cycles and that hair steroid hormones may reflect long-term physiological changes and can thus be used for the conservation and management of wildlife.     

A Cell-Autonomous Molecular Cascade Initiated by AMP-Activated Protein Kinase Represses Steroidogenesis

Steroid hormones regulate essential physiological processes, and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by luteinizing hormone (LH) via its receptor leading to increased cyclic AMP (cAMP) production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Steroidogenesis then passively decreases with the degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP-to-AMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutively steroidogenic R2C cells. We have also determined that maximum AMPK activation following stimulation of steroidogenesis in MA-10 Leydig cells occurs when steroid hormone production has reached a plateau. Our data identify AMPK as a molecular rheostat that actively represses steroid hormone biosynthesis to preserve cellular energy homeostasis and prevent excess steroid production.     

Leydig cells, thyroid hormones and steroidogenesis

Leydig cells are the primary source of androgens in the mammalian testis. It is established that the luteinizing hormone (LH) produced by the anterior pituitary is required to maintain the structure and function of the Leydig cells in the postnatal testis. Until recent years, a role by the thyroid hormones on Leydig cells was not documented. It is evident now that thyroid hormones perform many functions in Leydig cells. For the process of postnatal Leydig cell differentiation, thyroid hormones are crucial. Thyroid hormones acutely stimulate Leydig cell steroidogenesis. Thyroid hormones cause proliferation of the cytoplasmic organelle peroxisome and stimulate the production of steroidogenic acute regulatory protein (StAR) and StAR mRNA in Leydig cells; both peroxisomes and StAR are linked with the transport of cholesterol, the obligatory intermediate in steroid hormone biosynthesis, into mitochondria. The presence of thyroid hormone receptors in Leydig cells and other cell types of the Leydig lineage is an issue that needs to be fully addressed in future studies. As thyroid hormones regulate many functions of Sertoli cells and the Sertoli cells regulate certain functions of Leydig cells, effects of thyroid hormones on Leydig cells mediated via the Sertoli cells are also reviewed in this paper. Additionally, out of all cell types in the testis, the thyrotropin releasing hormone (TRH), TRH mRNA and TRH receptor are present exclusively in Leydig cells. However, whether Leydig cells have a regulatory role on the hypothalamo-pituitary-thyroid axis is currently unknown.     

LH action in the Leydig cell: modulation by angiotensin II and corticotropin releasing hormone, and regulation of P450(17) alpha mRNA

Luteinizing hormone is the major regulator of Leydig cell differentiation and steroidogenic function. A number of hormones produced by the Leydig cell (e.g. estrogen, angiotensin, CRF, vasopressin) and the tubular compartment (inhibin, TGF beta), can influence both acute and long-term actions of LH. Conversely, hormones produced in the Leydig cells modulate tubular function (e.g. androgen, beta-endorphin, oxytocin). The LH stimulatory event can be negatively influenced by the action of angiotensin II through the guanyl nucleotide inhibitory unit of adenylate cyclase. We have recently discovered an action of corticotrophin releasing hormone through specific high-affinity low-capacity receptors in the Leydig cells which involves a pertussis toxin insensitive guanyl nucleotide regulatory unit with interaction between signalling pathways and resulting inhibition of LH induced cAMP generation and consequently of steroidogenesis. In contrast to other tissues the CRF receptor in the Leydig cells did not couple to Gs. CRF action is exerted through direct or indirect action of protein kinase C, at the level of the catalytic subunit of adenylate cyclase. Physiological increases in endogenous LH cause positive regulation of membrane receptors and steroidogenesis, while major elevations in circulating gonadotropin can induce down-regulation of LH receptors and desensitization of steroid responses in the adult cell. Gonadotropin-induced desensitization in adult rat tests include an estrogen mediated steroidogenic lesion of the microsomal enzymes 17 alpha-hydroxylase/17,20-desmolase. For further understanding of the regulation of this key enzyme of the androgen pathway the rat P450(17) alpha cDNA was cloned and sequenced. This cDNA expressed in COS-1 cells 17 alpha-hydroxylase/17,20-desmolase activities. From the deduced amino acid sequence, two transmembrane regions were identified, a signal peptide for insertion in the ER, and a 2nd transmembrane region separated from the first by 122 amino acids. The carboxy terminal non-transmembrane region possesses 4 hydrophobic clefts, of which cleft II would contain the putative steroid binding site for both hydroxylase and lyase activities. The rat cDNA was employed to evaluate the hormonal regulation of mRNA levels in adult and fetal Leydig cells. Low dose hCG treatment caused an early increase in mRNA levels followed by a return to control values at later times, while with higher desensitizing doses the initial increase in mRNA was followed by a marked reduction in mRNA at 24 h and a small recovery at 48 h. Fetal rat Leydig cells treated with E2 showed a 70% decrease in P450 mRNA levels, and testosterone production closely followed the changes in mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

恒河猴睾丸间质细胞对支持细胞分泌雌二醇的影响

采用无血清培养的方法,分析了促肾上腺激素皮质激素(adrenocorticotropic hormone,ACTH)、黄体生成素(luteinizing hormone,LH)、cAMP、内啡肽(endorphin)和纳络酮(naloxone)对原代共培养的恒河猴(Macaca mulatta)睾丸间质细胞与支持细胞雌二醇分泌水平的影响。结果显示:ACTH、LH、cAMP和纳络酮对原代共培养恒河猴睾丸间质细胞与支持细胞的雌二醇分泌水平具有促进作用,并且这种影响与共培养的间质细胞数量呈线性关系,即共培养的间质细胞数量增加,雌二醇分泌水平亦明显上升;而内啡肽对原代共培养恒河猴睾丸间质细胞与支持细胞的雌二醇分泌水平有明显的抑制作用。研究表明,恒河猴睾丸的间质细胞对支持细胞分泌雌二醇具有调节作用。     

Cortisol and hypothalamic-pituitary-gonadal axis hormones in follicular-phase women with fibromyalgia and chronic fatigue syndrome and effect of depressive symptoms on these hormones

We investigated abnormalities of the hypothalamic-pituitary-gonadal axis and cortisol concentrations in women with fibromyalgia and chronic fatigue syndrome (CFS) who were in the follicular phase of their menstrual cycle, and whether their scores for depressive symptoms were related to levels of these hormones. A total of 176 subjects participated - 46 healthy volunteers, 68 patients with fibromyalgia, and 62 patients with CFS. We examined concentrations of follicle-stimulating hormone, luteinizing hormone (LH), estradiol, progesterone, prolactin, and cortisol. Depressive symptoms were assessed using the Beck Depression Inventory (BDI). Cortisol levels were significantly lower in patients with fibromyalgia or CFS than in healthy controls (P < 0.05); there were no significant differences in other hormone levels between the three groups. Fibromyalgia patients with high BDI scores had significantly lower cortisol levels than controls (P < 0.05), and so did CFS patients, regardless of their BDI scores (P < 0.05). Among patients without depressive symptoms, cortisol levels were lower in CFS than in fibromyalgia (P < 0.05). Our study suggests that in spite of low morning cortisol concentrations, the only abnormalities in hypothalamic-pituitary-gonadal axis hormones among follicular-phase women with fibromyalgia or CFS are those of LH levels in fibromyalgia patients with a low BDI score. Depression may lower cortisol and LH levels, or, alternatively, low morning cortisol may be a biological factor that contributes to depressive symptoms in fibromyalgia. These parameters therefore must be taken into account in future investigations.     

Hormonal regulation of testicular steroid and cholesterol homeostasis

The male sex steroid, testosterone (T), is synthesized from cholesterol in the testicular Leydig cell under control of the pituitary gonadotropin LH. Unlike most cells that use cholesterol primarily for membrane synthesis, steroidogenic cells have additional requirements for cholesterol, because it is the essential precursor for all steroid hormones. Little is known about how Leydig cells satisfy their specialized cholesterol requirements for steroid synthesis. We show that in mice with a unique hypomorphic androgen mutation, which disrupts the feedback loop governing T synthesis, that genes involved in cholesterol biosynthesis/uptake and steroid biosynthesis are up-regulated. We identify LH as the central regulatory molecule that controls both steroidogenesis and Leydig cell cholesterol homeostasis in vivo. In addition to the primary defect caused by high levels of LH, absence of T signaling exacerbates the lipid homeostasis defect in Leydig cells by eliminating a short feedback loop. We show that T signaling can affect the synthesis of steroids and modulates the expression of genes involved in de novo cholesterol synthesis. Surprisingly, accumulation of active sterol response element-binding protein 2 is not required for up-regulation of genes involved in cholesterol biosynthesis and uptake in Leydig cells.     

Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation

The concentrations of six steroids and of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in follicular fluid from preovulatory and large atretic follicles of normal Holstein heifers and from preovulatory follicles of heifers treated with a hormonal regimen that induces superovulation. Follicular fluid from preovulatory follicles of normal animals obtained prior to the LH surge contained extremely high concentrations of estradiol (1.1 +/- 0.06 micrograms/ml), with estrone concentrations about 20-fold less. Androstenedione was the predominant aromatizable androgen (278 +/- 44 ng/ml; testosterone = 150 +/- 39 ng/ml). Pregnenolone (40 +/- 3 ng/ml) was consistently higher than progesterone (25 +/- 3 ng/ml). In fluid obtained at 15 and 24 h after the onset of estrus, estradiol concentrations had declined 6- and 12-fold, respectively; androgen concentrations had decreased 10- to 20-fold; and progesterone concentrations were increased, whereas pregnenolone concentrations had declined. Concentrations of LH and FSH in these follicles were similar to plasma levels of these hormones before and after the gonadotropin surges. The most striking difference between mean steroid levels in large atretic follicles (greater than 1 cm in diameter) and preovulatory follicles obtained before the LH surge was that estradiol concentrations were about 150 times lower in atretic follicles. Atretic follicles also had much lower concentrations of LH and slightly lower concentrations of FSH than preovulatory follicles. Hormone concentrations in follicles obtained at 12 h after the onset of estrus from heifers primed for superovulation were similar to those observed in normal preovulatory follicles at estrus + 15 h, except that estrogen concentrations were about 6-40 times lower and there was more variability among animals for both steroid and gonadotropin concentrations. Variability in the concentrations of reproductive hormones in fluid from heifers primed for superovulation suggests that the variations in numbers of normal embryos obtained with this treatment may be due, at least in part, to abnormal follicular steroidogenesis.     

Effects of transdermal application of 7-oxo-DHEA on the levels of steroid hormones, gonadotropins and lipids in healthy men

The aim of this study was to investigate the effect of 7-oxo-DHEA (dehydroepiandrosterone) on the serum levels of steroid sexual hormones, gonadotropins, lipids and lipoproteins in men. 7-oxo-DHEA was applied onto the skin as a gel to 10 volunteers aged 27 to 72 years for 5 consecutive days. The single dose contained 25 mg 7-oxo-DHEA. Serum concentrations of testosterone, estradiol, cortisol, androstenedione, luteinizing hormone (LH), follicle-stimulating hormone (FSH), sex hormone binding globulin (SHBG), total cholesterol, HDL- and LDL-cholesterol, triglycerides, apolipoprotein A-I and B and lipoprotein(a) were measured before the beginning and shortly after the end of the steroid application. After the treatment, we noted the following significant changes: a decline of testosterone and estradiol levels, increase of LH, HDL-cholesterol and apolipoprotein A-I levels. The decrease of total cholesterol levels was of the borderline significance. A slight but significant increase was found in apolipoprotein B and lipoprotein(a). The most expressive was the fall of the atherogenic index. We suggest that the gel containing 7-oxo-DHEA might be a suitable drug for improving the composition of the steroid and lipid parameters in elderly men.     

Cortisol and hypothalamic–pituitary–gonadal axis hormones in follicular-phase women with fibromyalgia and chronic fatigue syndrome and effect of depressive symptoms on these hormones

We investigated abnormalities of the hypothalamic–pituitary–gonadal axis and cortisol concentrations in women with fibromyalgia and chronic fatigue syndrome (CFS) who were in the follicular phase of their menstrual cycle, and whether their scores for depressive symptoms were related to levels of these hormones. A total of 176 subjects participated – 46 healthy volunteers, 68 patients with fibromyalgia, and 62 patients with CFS. We examined concentrations of follicle-stimulating hormone, luteinizing hormone (LH), estradiol, progesterone, prolactin, and cortisol. Depressive symptoms were assessed using the Beck Depression Inventory (BDI). Cortisol levels were significantly lower in patients with fibromyalgia or CFS than in healthy controls (P < 0.05); there were no significant differences in other hormone levels between the three groups.     

Modulatory actions of estradiol and progesterone on phorbol ester-stimulated LH secretion from cultured rat pituitary cells.

We compared the ability of estradiol and progesterone to modulate gonadotropin-releasing hormone (GnRH) and protein kinase C (PKC)-mediated luteinizing hormone (LH) secretion. Long-term (48 h) treatment of rat pituitary cells with 1 nM estradiol enhanced GnRH and phorbol ester (TPA)-stimulated LH secretion. This positive effect was facilitated by additional short-term (4 h) treatment with progesterone (100 nM). However, long-term progesterone treatment, which inhibited GnRH-stimulated LH secretion, did not influence TPA-stimulated gonadotropin release. These steroid actions occurred without an effect on the total amount of LH in the cell cultures (total LH = LH secreted + LH remaining in the cell) and neither the secretagogues nor the steroids altered total LH. Since GnRH or TPA-induced LH secretion depends on Ca2+ influx into the gonadotroph, we also analyzed the effects of estradiol and progesterone under physiological extracellular Ca2+ concentrations and in the absence of extracellular Ca2+. The steroids were able to influence GnRH or TPA-induced LH secretion under both conditions. However, when TPA was used as stimulus in Ca(2+)-deficient medium the relative changes induced by estradiol and progesterone were more pronounced, possibly indicating that the extracellular Ca(2+)-independent component of PKC-mediated LH secretion is more important for the regulation of the steroid effects. It is concluded that estradiol and progesterone might mediate their modulatory actions on GnRH-stimulated LH secretion via an influence on PKC. This effect can occur independently from de novo synthesis of LH and Ca2+ influx into gonadotrophs.     

Progesterone and estradiol in cat placenta-Biosynthesis and tissue concentration

Ovarian and placental steroids are essential for the maintenance of pregnancy. In some mammals it is evident that the placenta is responsible for the production of steroids. However, in the domestic cat, steroid secretion from the placenta has not yet been elucidated. Our study aimed to find out whether feline placentae are able to produce steroids. Placentae from different pregnancy stages were analyzed for mRNA expression of five steroidogenic enzymes (HSD3B1, CYP11A1, CYP17A1, HSD17B1 and CYP19A1) and for tissue concentrations of progesterone and estradiol. Steroidogenic enzymes responsible for the final steps of estradiol (CYP19A1) and progesterone synthesis (HSD3B) were expressed at very high levels and followed almost the same pattern over pregnancy as the intraplacental hormones themselves. By contrast, the other enzymes were found in very low quantities suggesting that biosynthesis occurs via extra-placental steroid precursors. The plasma steroid profiles measured by other groups differ from the placental hormone courses determined by us; therefore we conclude that the feline placenta can produce progesterone and estradiol.     

Evidence for a Sertoli cell, FSH-suppressible inhibiting factor(s) of testicular steroidogenic activity

By using a model of immature porcine Leydig and Sertoli cells cultured in serum free defined medium, we evidenced a paracrine control of Leydig cell steroidogenic activity by Sertoli cells via a secreted inhibiting protein(s). This protein(s), partially purified using gel filtration (M.W. 20,000-30,000) suppresses the steroidogenic responsiveness to LH/hCG by decreasing the specific LH/hCG binding (52% decrease) and hormone steroid biosynthesis (73% decrease) at a level(s) located between cAMP production and pregnenolone formation. The suppression of this inhibitor(s) by FSH, in a dose dependent manner, is one mechanism by which FSH "sensitizes" Leydig cell response to LH/hCG stimulation.     

Profiles of Steroid Hormones in Canine X-Linked Muscular Dystrophy via Stable Isotope Dilution LC-MS/MS

Golden retriever muscular dystrophy (GRMD) provides the best animal model for characterizing the disease progress of the human disorder, Duchenne muscular dystrophy (DMD). The purpose of this study was to determine steroid hormone concentration profiles in healthy golden retriever dogs (control group - CtGR) versus GRMD-gene carrier (CaGR) and affected female dogs (AfCR). Therefore, a sensitive and specific analytical method was developed and validated to determine the estradiol, progesterone, cortisol, and testosterone levels in the canine serum by isotope dilution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). To more accurately understand the dynamic nature of the serum steroid profile, the fluctuating levels of these four steroid hormones over the estrous cycle were compared across the three experimental groups using a multivariate statistical analysis. The concentration profiles of estradiol, cortisol, progesterone, and testosterone revealed a characteristic pattern for each studied group at each specific estrous phase. Additionally, several important changes in the serum concentrations of cortisol and estradiol in the CaGR and AfCR groups seem to be correlated with the status and progression of the muscular dystrophy. A comprehensive and quantitative monitoring of steroid profiles throughout the estrous cycle of normal and GRMD dogs were achieved. Significant differences in these profiles were observed between GRMD and healthy animals, most notably for estradiol. These findings contribute to a better understanding of both dog reproduction and the muscular dystrophy pathology. Our data open new venues for hormonal behavior studies in dystrophinopathies and that may affect the quality of life of DMD patients.     

Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta

Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.     

Validation of the mechanisms proposed for the stimulatory and inhibitory effects of progesterone on gonadotropin secretion in the estrogen-primed rat: a possible role for adrenal steroids.

The stimulatory and inhibitory effects of progesterone on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion were found to be dependent on the length of estrogen exposure in ovariectomized estrogen-primed rats. Progesterone suppressed LH and FSH secretion when administered 16 hours after a single injection of estradiol to ovariectomized rats. If the estradiol treatment was extended over 40 hours by two injections of estradiol 24 hours apart, progesterone administration led to a highly significant elevation of both serum LH and FSH levels 6 hours later. In addition to the direct stimulatory effect on LH and FSH release, progesterone, when injected 1 hour before, was able to antagonize the suppressive effect of a third injection of estradiol on LH and FSH release. In the immature ovariectomized estrogen-primed rat, 10 IU of ACTH brought about a release of progesterone and corticosterone 15 minutes later and LH and FSH 6 hours later. Progesterone, but not corticosterone, appeared to be responsible for the effect of ACTH on gonadotropin release. The synthetic corticosteroid triamcinolone acetonide brought about LH and FSH release in the afternoon, while cortisol, similar to corticosterone, was unable to do so. Nevertheless, triamcinolone acetonide and cortisol brought about increased secretion of FSH the following morning.     

The ultrastructure of the cells of Leydig in the white-crowned sparrow (Zonotrichia leucophrys gambelii) in relation to plasma levels of luteinizing hormone and testosterone

In male White-crowned Sparrows subjected to 20 h daily photoperiods there is an approximately 3-fold increase in the plasma concentration of luteinizing hormone (LH) on the first long day after which a quasi-stable level is maintained for at least 42 days. This increase is followed by an increase in numbers of cells of Leydig and an enhancement of their steroidogenic features, a decrease in transitional interstitial cells, and an increase in plasma level of testosterone. With the decline in plasma LH, as photorefractoriness develops, the steroidogenic features of the cells of Leydig undergo disorganization. For as yet unexplainable reasons the plasma levels of testosterone decline before the decrease in plasma LH and before the degeneration of the steroidogenic features of the cells of Leydig.