Pituitary Androgen Receptor Signalling Regulates Prolactin but Not Gonadotrophins in the Male Mouse

Production of the androgen testosterone is controlled by a negative feedback loop within the hypothalamic-pituitary-gonadal (HPG) axis. Stimulation of testicular Leydig cells by pituitary luteinising hormone (LH) is under the control of hypothalamic gonadotrophin releasing hormone (GnRH), while suppression of LH secretion by the pituitary is controlled by circulating testosterone. Exactly how androgens exert their feedback control of gonadotrophin secretion (and whether this is at the level of the pituitary), as well as the role of AR in other pituitary cell types remains unclear. To investigate these questions, we exploited a transgenic mouse line (Foxg1^Cre/+; AR^fl/y) which lacks androgen receptor in the pituitary gland. Both circulating testosterone and gonadotrophins are unchanged in adulthood, demonstrating that AR signalling is dispensable in the male mouse pituitary for testosterone-dependent regulation of LH secretion. In contrast, Foxg1^Cre/+; AR^fl/y males have a significant increase in circulating prolactin, suggesting that, rather than controlling gonadotrophins, AR-signalling in the pituitary acts to suppress aberrant prolactin production in males.     

Inhibition of polyamine synthesis blocks urinary secretion of beta-glucuronidase from mouse kidney

The effect of inhibition of polyamine synthesis on castrated male mouse kidney beta-glucuronidase induction and secretion by testosterone was studied. Inhibition of the activities of polyamine synthesis key-enzymes, L-ornithine and S-adenosyl-L-methionine decarboxylases, was performed with the combined treatment of 2-difluoromethylornithine and methylglyoxal' bis(guanylhydrazone). Blockage of polyamine synthesis did not affect testosterone-induced increase in renal beta-glucuronidase but blocked its secretion into the urine. After withdrawal of inhibitor-treatment beta-glucuronidase secretion normalized, and repeated testosterone administration produced undisturbed beta-glucuronidase secretion peak in urine suggesting that blockage of beta-glucuronidase secretion was not due to the tissue damage produced by inhibitors. These results indicate that the stimulation of renal polyamine synthesis by testosterone is not necessary for the induction of beta-glucuronidase but is required for the urinary secretion of this protein.     

Comparison of androgen biosynthesis in isolated leydig cells from rat and mouse testis: incubation and superfusion studies

Production of testosterone by highly purified Leydig cells prepared from rat and mouse testes is compared. Testosterone formation is improved to a higher degree in rat (2.7-fold) than in mouse (1.7-fold) cells by collagenase treatment of the testis compared with mechanical isolation. Mouse Leydig cells respond to exogenous stimuli (choriogonadotropin, dibutyryl cyclic AMP) with 2.4-fold higher testosterone secretion than rat cells. A 1.7-fold increased conversion of androgen precursors to testosterone by mouse compared with rat Leydig cells is demonstrated in static incubations as well as in steady-state superfusion experiments and can be derived from enhanced androstenedione reduction and a less inhibitory effect of progesterone on this process in mouse Leydig cells.     

The dynamics of the testosterone response of perifused mouse Leydig cells to hCG and arginine vasopressin

The effect of hCG and Arginine-Vasopressin (AVP) on testosterone production by purified mouse Leydig cells was examined under dynamic conditions in a perifusion system. A rapid and dose-dependent increase in testosterone release was induced by a 5 min exposure of the cells to increasing concentrations of hCG (0.01 to 1 ng/ml). The testosterone response to hCG was Gaussian in distribution with a peak value by 100 min. A 12 h pretreatment of Leydig cells with 10(-5) M AVP enhanced testosterone accumulation in the perfusate under basal conditions, but markedly reduced the hCG-stimulated testosterone production. The basal and hCG-stimulated testosterone secretion profiles by freshly isolated Leydig cells were, however, unaffected by the continuous presence of the same dose of AVP. These results support the finding that AVP acts directly on Leydig cells. They support the hypothesis of a possible role of neurohypophysial peptides on reproductive functions in the mouse by modulating steroidogenesis at the testicular level.     

c-myb对人绒毛膜促性腺激素诱导的大鼠睾丸间质细胞睾酮分泌的影响

采用离体细胞体外孵育法,观察反义c-myb寡脱氧核苷酸(oligodeoxynucletides,ODN)对人绒毛膜促性隙激素(humanchorionic-gonadotropin hormone,hCG)诱导的人鼠间质细胞睾酮分泌的影响,并进一步探讨了外源性二丁酰cAMP(dbcAMP)、Ca^2 以及蛋白质抑制剂放线菌酮(cycloheximide,CYX)对间质细胞中c-Myb蛋白表达和睾酮分泌的作用。结果表明,反义c-myb ODN呈剂量依赖性地抑制hCG诱导的离体间质细胞的睾酮分泌,同时使间质细胞中c-Myb蛋白免疫组化染色下降：而无义tat ODN没有相应的作用。100μmol／L的dbc AMP可进一步促使hCG秀导的间质细胞分泌睾酮,并且使间质细胞中c-Myb蛋白免疫组化染色IOD值升高,与hCG组相比,具有统计学意义。钙离子通道阻断剂维拉帕米(10μmol／L)和蛋白质抑制剂放线菌酮(50μg／ml)可使hCG诱导的大鼠间质细胞的睾酮分泌下降,并使间质细胞的c-Myb蛋白免疫组化染色降低。该结果说明c-myb参与hCG诱导的大鼠间质细胞睾酮分泌作用。     

Insulin-like peptide 3 stimulates testosterone secretion in mouse Leydig cells via cAMP pathway

Testicular Leydig cells secrete insulin-like peptide 3 (INSL3) and express its receptor, RXFP2. However, the effects of INSL3 on endocrine function of Leydig cells are unknown. The present study examines the effects of INSL3 on mouse Leydig cells taking testosterone and cAMP secretions as endpoints. Leydig cells were isolated from testicular interstitial cells obtained from 8-week-old male mice. Cells were then plated in the presence or absence of mouse, human, canine or bovine INSL3 (0-100ng/ml) for 18h in multiwell-plates (96 wells) in different cell densities (2500, 5000, 10,000 or 20,000 cells per well). The effects of bovine INSL3 (100ng/ml) on testosterone secretion by Leydig cells were examined in the presence or absence of, an adenylate cyclase inhibitor, SQ 22536 (1μM) or INSL3 antagonist (bovine and human; 100ng/ml). Testosterone and cAMP in spent medium were measured by enzyme immunoassay. All INSL3 species tested significantly stimulated the testosterone secretion in Leydig cells, and the maximum stimulation was observed with 100ng/ml bovine INSL3 at the lowest Leydig cell density (2500 cells per well). Moreover, bovine INSL3 (100ng/ml) significantly stimulated the cAMP production from Leydig cells maximally at 1h, and remained significantly elevated even at 18h. SQ 22536 and INSL3 antagonists (bovine and human) significantly reduced INSL3-stimulated testosterone secretion from Leydig cells. Taken together, stimulatory effects of INSL3 on testosterone secretion in Leydig cells are exerted via the activation of cAMP, suggesting a new autocrine function of INSL3 in males.     

The interaction of testosterone and gonadotropins in stimulating estradiol and progesterone secretion by cultures of corpus luteum cells isolated from pigs in early and midluteal phase.

The first objective of this research was to define the capacity of corpora lutea of pig to secrete estradiol in the presence of an androgen substrate which was testosterone. The second objective was to define the synergism between gonadotropic hormones such as LH, FSH, and PRL and testosterone as measured by estradiol and progesterone secretion by two types of porcine luteal cells. Luteal cells were collected from newly forming corpora lutea (0-3 days after ovulation) and from mature corpora lutea (8-10 days after ovulation). After dispersion, luteal cells were suspended in medium M199 supplemented with 10% of calf serum and grown as monolayers at 37 degrees C. Control cultures were grown in medium alone while other cultures were supplemented with either testosterone alone at a concentration of 1 x 10(-7) M or with 10, 100, 500 ng LH plus testosterone, 10, 100, 500 ng FSH plus testosterone or 10, 100, 500 ng PRL plus testosterone. After 2 days of cultivation all cultures were terminated and media were frozen at 20 degrees C for further steroid analysis. Testosterone added to the culture medium in the absence of gonadotropins was without effect on estradiol and progesterone secretion by luteal cells collected in the corpora lutea of the early luteal phase. On the other hand testosterone added to the medium significantly increased progesterone and estradiol secretion by cultured luteal cells collected in the midluteal phase of the cycle. No additive stimulatory action of gonadotropins and testosterone on progesterone secretion was observed in cultures of luteal cells from the early luteal phase but this was not the case in cultures of luteal cells from the midluteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seasonal changes in testosterone and corticosterone levels in four social classes of a desert dwelling sociable rodent

Animals have to adjust their physiology to seasonal changes, in response to variation in food availability, social tactics and reproduction. I compared basal corticosterone and testosterone levels in free ranging striped mouse from a desert habitat, comparing between the sexes, breeding and philopatric non-breeding individuals, and between the breeding and the non-breeding season. I expected differences between breeders and non-breeders and between seasons with high and low food availability. Basal serum corticosterone was measured from 132 different individuals and serum testosterone from 176 different individuals of free living striped mice. Corticosterone and testosterone levels were independent of age, body weight and not influenced by carrying a transmitter. The levels of corticosterone and testosterone declined by approximately 50% from the breeding to the non-breeding season in breeding females as well as non-breeding males and females. In contrast, breeding males showed much lower corticosterone levels during the breeding season than all other classes, and were the only class that showed an increase of corticosterone from the breeding to the non-breeding season. As a result, breeding males had similar corticosterone levels as other social classes during the non-breeding season. During the breeding season, breeding males had much higher testosterone levels than other classes, which decreased significantly from the breeding to the non-breeding season. My results support the prediction that corticosterone decreases during periods of low food abundance. Variation in the pattern of hormonal secretion in striped mice might assist them to cope with seasonal changes in energy demand in a desert habitat.     

Action of gonadotropins on androgen synthesis by embryonic testes of mouse and rat]

Testes from mouse and rat embryos were cultured in vitro in the presence of 14C-sodium acetate and luteinizing hormone or chorionic gonadotrophin. There is an increase in testosterone and androstenedione synthesis from sodium acetate under both treatments. Sensitivity to gonadotrophins exists already at a relatively early stage, when the testis, through testosterone secretion, is building up the genital tract. But the role of the hypophysis in normal androgen secretion will have to be proven.     

Nitric oxide control of steroidogenesis: endocrine effects of NG-nitro-L-arginine and comparisons to alcohol.

Recent studies suggest that nitric oxide (NO) may regulate hormone biosynthesis and secretion. This was tested by treating male rats with NG-nitro-L-arginine methyl ester (NAME), a NO synthase inhibitor, and measuring serum and testicular interstitial fluid testosterone and serum corticosterone, luteinizing hormone (LH), and prolactin (PRL). The effect of NG-nitro-L-arginine (NA), a less-soluble form of the same NO synthase inhibitor, on the reproductive suppressant actions of alcohol was also examined. NAME increased testosterone and corticosterone secretion dose-dependently without affecting LH and PRL secretion. The alcohol-induced suppression of testosterone or LH secretion was not altered by treatment with NA. Although effects of NAME and NA on other systems may be involved, these results indicate that testicular and adrenal steroidogenesis are negatively regulated by endogenous NO and that NO does not regulate LH and PRL secretion or inhibit the testicular steroidogenic pathway in the same way as alcohol.     

Determination of fructose in sperm: application in the monitoring of a male hormonal contraceptive

This work describes a simple and reproducible method of determining the fructose content of sperm in order to assess the impregnation of the genital tract with testosterone in men in whom spermatogenesis is blocked by a combination of progesterone and androgens. The colorimetric method of Karvonen and Malm was used with certain modification. The fructose content of sperm is colorimetrically determined by the indol in a concentrated chlorhydric milieu, after deproteinization by cadmium hydroxide. Instructions are given for preparation of the aqueous solution of indol, the sulfuric solution of cadmium sulfate and the fructose solution, as well as for the procedure itself and calculation of the fructose concentration. The method was designed to monitor men taking 20 mg/daily of medroxyprogesterone acetate and applying testosterone daily to the torso. The progesterone inhibits spermatogenesis in inhibiting follicle stimulating hormone and luteinizing hormone and also blocks testosterone secretion by the testicles. The addition of testosterone is designed to maintain a normal level of testosterone to avoid feminization.     

In vitro steroid secretion by intact bovine ovarian follicles in a superfusion system

A superfusion system for single intact follicles is described. Bovine follicles were superfused for 5 to 10 hours. Progesterone and testosterone secretion was stable after an adaptation period of 150 min. A 5-minute stimulation with hCG resulted in a 2-fold increase in progesterone secretion. Superfusion for 450 minutes of 3 small follicles of similar size and in the same ovary next to each other showed different secretion patterns of progesterone and testosterone. Superfusion for 150 minutes of follicles obtained from 2 pairs of ovaries showed that follicular size is not a sufficient criterion to predict the progesterone and testosterone secretion pattern. Closeness of follicles (big or small) to a corpus luteum did not impair their ability to secrete progesterone and testosterone. It is concluded that a superfusion system as has been described in this report is an effective and desirable method to study the physiology and pathophysiology of single intact follicles.     

Assessment of reproductive status in male echidnas

This study reports the development and application of techniques to assess the reproductive status of male echidnas. The pattern of testosterone secretion over a 24-h period in five echidnas was documented. Testosterone secretion after injection i.m. of either 1000 IU hCG (n=4) or 4 microg GnRH agonist (n=6) was determined to establish whether this could be used as a practical index of the prevailing steroidogenic capacity of the testes. hCG (1000 IU) was also used to assess seasonal changes in testosterone secretion in six echidnas over a 13-month period. Seasonal changes in testicular volume were examined by transabdominal ultrasonography. Electroejaculation was attempted to monitor seasonal changes in sperm production, which was also determined by spermatorrhea. There was no apparent diurnal pattern of testosterone secretion in echidnas and circulating concentrations of testosterone remained relatively low (maximum 1.2 ng/mL) and stable over 24h. Injection of hCG resulted in an increase (P<0.01; n=4) in testosterone concentration with a peak (2.9+/-0.3 ng/mL) approximately 4h after injection. GnRH also induced an increase (P<0.01; n=6) in circulating testosterone that was apparent after 1h (2.6+/-0.3 ng/mL) and concentrations remained elevated (3.4+/-0.3 ng/mL) for up to 8h after injection. Seasonal changes in testosterone secretion determined after injection of hCG, increased (P=0.03; n=6) from late-autumn, peaked in late-winter, and decreased by early-spring. Testicular volume followed a similar seasonal pattern (P<0.01; n=6) with an increase from late-autumn, peak in winter and a decline in mid-spring. There was no seasonal change in live weight. Electroejaculation was attempted throughout two breeding seasons but no semen was obtained. Spermatorrhoea in the echidna was described for the first time and was subsequently used to assess seasonal sperm production. Spermatozoa were found in the urine from June to September. This study has demonstrated that exogenous hormones can be used to obtain an index of the prevailing steroidogenic capacity of the testes in echidnas, which is not apparent with repetitive non-stimulated samples over 24 h. The assessment of testosterone secretion after injection of trophic hormones provides a valuable and practical procedure for the assessment of reproductive status. Testicular ultrasonography and spermatorrhea are useful in assessing reproductive status and in this study were successfully used to determine seasonal reproduction in captive echidnas.     

Cortisol affects testicular development in male common carp, Cyprinus carpio L., but not via an effect on LH secretion

Previous work showed that prolonged elevated cortisol levels, implicated in the stress adaptation, inhibits testicular pubertal development in male common carp, as well as an impairment of the synthesis of the 11-oxygenated androgens. This may be a direct effect of cortisol on the testis or via the gonadotropin secretion by the pituitary. The aim of the present study was to investigate whether cortisol has an effect on pituitary LH secretion. Juvenile common carp were fed with cortisol containing food pellets. Elevated cortisol levels blocked the increase in testosterone levels and pituitary LH content, but induced higher plasma LH levels at the end of puberty. The in vitro LH release capacity was correlated to the pituitary LH content. At the final stage of pubertal development, when a significant difference in pituitary LH content was observed, sGnRHa-induced LH release was also decreased. Testosterone has been shown to induce development of pituitary gonadotrophs, leading to an increase in LH content and GnRH-inducible LH release, but a decrease in plasma LH levels. We observed decreased plasma testosterone levels as a consequence of prolonged cortisol treatment. It is hypothesised that cortisol inhibits the testicular testosterone secretion and thereby, prevents LH storage. In vitro, this leads to a reduced GnRH-inducible LH release, but in vivo to increased LH plasma levels. It is very unlikely that the impaired testicular development is due to an effect of cortisol on LH secretion.     

Brain and plasma levels of testosterone, dihydrotestosterone and estradiol in the one-day-old rat.

Current evidence indicates that the secretion of testosterone during perinatal life is essential in organizing the male brain which subsequently directs the male pattern of gonadotrophin (GTH) secretion and adult male sexual behavior in the rat. It has been hypothesized that testosterone is converted into estradiol enzymatically in the brain prior to its action. In the absence of testosterone and with the resultant low levels of estradiol, female patterns of gonadotrophin secretion and behavior result. In order to investigate this hypothesis further, the endogenous levels of gonadal steroids in the plasmas and brains of 24–48 hr old male and female rats were determined. Pooled samples were analyzed for testosterone, dihydrotestosterone and estradiol by radioimmunoassay. Testosterone levels in male brain and plasma samples were significantly (10-fold) higher than those in the female brain and plasma samples. Brain levels of estradiol were significantly higher in the male than in the female neonate, while plasma levels were identical. Whether the higher level of estradiol in the male brain is due to enzymatic conversion from testosterone within the brain differences in permeability or some other mechanism cannot be stated at this point. The significantly higher brain levels of both testosterone and estradiol in male neonates do fit the pattern predicted by the present concept of sexual differentiation. Dihydrotestosterone levels in brain and plasma of male rats were about 25% of those of testosterone. However in females the brain levels of dihydrotestosterone were significantly higher than testosterone even though the plasma levels of these hormones were identical. This may reflect a protective mechanism through which permeability of testosterone is lowered in the neonatal female brain during the critical period or simply a functional conversion of testosterone to dihydrotestosterone in the female during this period.     

Testosterone increases osteoprotegerin mRNA expression in mouse osteoblast cells.

The role that androgens play in the regulation of bone metabolism has been substantiated in animals and humans. We previously demonstrated that testosterone inhibits osteoclast differentiation stimulated by parathyroid hormone through the androgen receptor in mouse bone-cell cultures. However, the details of this mechanism are still unknown. The present study was aimed at examining whether testosterone would affect the mRNA levels of osteoprotegerin (OPG) and receptor activator of Nf kappa B ligand (RANKL) in mouse bone-cell cultures as well as mouse osteoblastic cell-line, MC3T3-E1 cells by employing semi-quantitative RT-PCR. Testosterone increased OPG mRNA expression in both mouse bone-cell cultures and MC3T3-E1 cells. 10-8 M PTH-(1-34) as well as 10-8M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibited OPG mRNA expression in mouse bone cells. 10-8 M testosterone antagonized OPG mRNA expression inhibited by 10-8 M PTH-(1-34), but failed to affect OPG mRNA expression inhibited by 10-8 M 1,25(OH)2D3. 10-8 M alpha-dehydrotestosterone, a non-aromatizable androgen, increased OPG mRNA expression. On the other hand, testosterone did not affect RANKL mRNA expression in MC3T3-E1 or mouse bone cells. In conclusion, the present study demonstrated that testosterone increased OPG mRNA expression in mouse bone-cell cultures and the osteoblastic cell line. These effects are likely to take place through the androgen receptor.     

Preparation of monoclonal antibodies able to discriminate between testosterone and 5α-dihydrotestosterone

A procedure for production of monoclonal antibodies to testosterone is described. The method involves immunization of rats with a bovine serum albumin conjugate of testosterone 3-(0-carboxymethyl)oxime followed by polyethylene glycol induced hybridization of the immune lymphocytes with mouse myeloma cells. The resulting hybridomas were cloned and the antibodies produced by each clone were characterized. All the antibodies obtained showed high affinity for testosterone, (K_a = 10¹⁰ l/mol), but clones differed widely in the degree of cross-reaction of the antibodies with other steroids, such as 5α-dihydrotestosterone (range 2–100%) and androstenedione (< 0.1–4%). Large quantities of the selected specific antibodies can be obtained by mass growth of the hybridoma line in culture or as tumors in irradiated or nude mice. Monoclonal antibody preparations may improve standardization of immunoassay methods.     

Ketanserin, an antagonist of 5-HT2A receptor of serotonin, inhibits testosterone secretion by rat Leydig cells in vitro.

The effect of ketanserin, an antagonist of 5-HT2A receptor of serotonin, added to the culture medium, on basal and LH-stimulated testosterone secretion was studied in primary cultures of adolescent rat Leydig cells. Ketanserin decreased the basal secretion of testosterone but showed an insignificant influence on the LH-stimulated process. It can be concluded that ketanserin may affect the testosterone-secreting cells by an indirect action at the vascular level as well as directly at the level of Leydig cells, at least in adolescent rats, leading to down-regulation of the basal testosterone secretion.     

Effect of uni- and bilateral cryptorchidism on testicular inhibin and testosterone secretion in rats

The effect of uni- and bilateral cryptorchidism on testicular inhibin and testosterone secretion and their relationships to gonadotropins were studied in rats. Mature Wistar male rats weighing approximately 300 g were made either uni- or bilaterally cryptorchid. Testicular inhibin and testosterone content and plasma levels of LH and FSH were examined 2 weeks later. A similar remarkable decrease in testicular inhibin content was found in uni- and bilaterally cryptorchid testes. On the other hand, the testicular testosterone content was significantly decreased only in unilaterally cryptorchid testis with an inverse increase in the contralateral testis. Plasma testosterone levels were normal and plasma LH and FSH increased significantly in both of the cryptorchid groups. These results showed that cryptorchidism impairs both Sertoli and Leydig cell functions. While testosterone production was compensated by increased LH for 2 weeks, neither inhibin secretion nor storage changed in cryptorchid or contralateral testes during the same period.     

The influence of dehydroepiandrosterone on basal and gonadotrophin-stimulated testosterone secretion by Mongolian gerbil interstitial cells incubated or superfused in vitro

1. Testosterone secretion by Mongolian gerbil interstitial cells incubated in the absence of HCG linearly increased with cell concentration (1 x 10(5) cells: 0.6 ng/4 hr, 10 x 10(5) cells: 8.0 ng/4 hr). Addition of 100 mIU HCG resulted in a drastic increase of testosterone secretion which was linear between concentrations of 1 x 10(5) and 4 x 10(5) cells. 2. Compared to HCG-stimulated testosterone release, secretion was significantly higher by cells incubated with 60-100 ng DHEA. 3. During the 4-hr incubation period, 53-69% of added progesterone and 72-88% of added dehydroepiandrosterone (DHEA) were converted to testosterone by cells freshly prepared or stored for 1-3 days at 4 degrees C. On the other hand, prolonged storage at 4 degrees C resulted in a marked decrease of HCG-stimulated testosterone secretion. 4. Testosterone secretion by interstitial cells superfused in vitro increased with the length of HCG (100 mIU/ml) application from 0.08 to 0.22 ng/10(6) cells/min (10 and 60 min, respectively). A much faster and pronounced elevation was found when cells were stimulated with DHEA (200 ng/ml: 0.06-0.80 ng/10(6) cells/min, 0 and 20 min, respectively). 5. After interstitial cells have been stimulated with a DHEA (200 ng/ml) pulse for 30 min and then superfused with medium only for an additional 30 min, testosterone secretion remained significantly elevated and could not be further stimulated by superfusing medium which contained as much as 100 mIU/ml HCG.