Sexual differentiation of the steroid feedback mechanism regulating follicle-stimulating hormone secretion in the Syrian hamster

The ability of gonadal steroid hormones to influence tonic follicle-stimulating hormone (FSH) secretion was investigated in Syrian hamsters. In Experiment 1, males were castrated as adults, and administered testosterone in 20-, 30-, 40-, and 50-mm silastic capsules (s.c.) at 67, 74, 81, and 88 days, respectively. Circulating FSH was reduced by testosterone in a dose-dependent manner. A similar FSH response to testosterone in adulthood was evident in neonatally androgenized hamsters given testosterone proprionate (TP) on Days 0 and 1 of life. By contrast, the absence of gonadal androgens during the neonatal period (females ovariectomized at 60 days of age and males orchidectomized at birth) resulted in only a partial suppression of circulating FSH by even the highest dose of testosterone during adulthood. Treatment with estradiol benzoate at birth failed to produce a masculine response to androgen in adulthood. In Experiment 2, using a similar protocol, the nonaromatizable androgen, dihydrotestosterone, produced a dose-dependent suppression in serum FSH in males castrated in adulthood (30-, 60-, 90-mm capsules). However, dihydrotestosterone failed to alter the hypersecretion of FSH produced by orchidectomy at birth in males or in females ovariectomized at 60 days of age and treated neonatally with either vehicle or TP. In Experiment 3, treatment with estradiol (10-, 20-, 30-mm capsules) decreased serum FSH in gonadectomized hamsters in a dose-dependent manner; males and females treated neonatally with TP were more responsive to estradiol as adults compared to neonatally orchidectomized males or females treated with vehicle at birth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aromatizable and 5alpha-reduced androgens: differentiation between central and peripheral effects on male rat sexual behavior.

Sexual behavior was observed in the 5 wk after castration in four separate experiments. In each, 50 male rats were allocated equally to the following treatment groups; intact controls; castrated controls; castrated + testosterone propionate, 75 μg/day; castrated + test steroid 1, 150 μg/day; castrated + test steroid 2, 150 μg/day. The following propionated compounds were used; dihydrotestosterone, androsterone, 3α- and 3β-androstanediols, androstenedione, androstanedione, 19-hydroxyandrostenedione, estradiol. With the exception of androstanedione, only aromatizable androgens or estrogen prevented post-castration increases in refractory period durations. However, aromatizable and 5α-reduced androgens stimulated penile spine growth. In each experiment there was a significant positive correlation between spine number and copulatory efficiency i.e., the ratio of intromission to mount frequencies.     

Physiological peripubertal activation of the ovary is not reproduced by pregnant mare serum gonadotropin (PMSG) administration

During the days preceding the first ovulation the ovary of the rat exhibits a remarkable increase in estradiol (E2) and progesterone (P) release in response to gonadotropins. No such increase is observed in the case of androgens (A, testosterone + dihydrotestosterone). The present experiments were undertaken to examine the possibility of reproducing these developmental events by stimulating the ovary with a gonadotropin that has substantial FSH-like activity. In vivo administration of pregnant mare serum gonadotropin (PMSG) to juvenile 29-day-old rats greatly increased the in vitro E2 and A response to human chorionic gonadotropin (hCG) measured 2 days later in the morning. The magnitude of the A response was significantly larger than that of ovaries from juvenile animals or rats in first proestrus. The E2 response was much greater than that of juvenile ovaries but similar to that of ovaries from late proestrous rats. In contrast, the P response to hCG was not enhanced by PMSG. In fact the response was similar to that of juvenile ovaries and markedly less than that of first proestrous rats. This decreased P response was not due to a greater conversion of P to its less active metabolite 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P). The results suggest that PMSG enhances the E2 and A response of immature ovaries to hCG at the expense of that of P. Treatment of immature rats with PMSG may represent a useful model to study E2 release from preovulatory ovaries, but it cannot be used to reproduce in its entirety the developmental changes in steroidal response to gonadotropins associated with normal puberty.     

Imprinting of male sex tissues by neonatal endogenous androgens in mice. Molecular alterations following exposure to cyproterone acetate

This study was conducted to evaluate the growth and biochemical responsiveness of the epididymis, vas deferens and seminal vesicles of adult mice exposed to cyproterone acetate during the first 10 days of life. Results indicate that the weight and protein content of sex accessory organs were significantly depressed, testosterone and dihydrotestosterone concentrations were unaffected or increased, the number of cytosolic androgen-binding sites was slightly or significantly reduced. The efficiency of exogenous testosterone in promoting growth and protein synthesis in target organs of castrated adult males was significantly lowered by neonatal cyproterone acetate treatment. It is concluded that a deficient androgenic stimulation during neonatal life induces a limited response of sex target organs to endogenous or exogenous androgens in adulthood.     

Interference with the preovulatory luteinizing hormone surge and blockade of ovulation in immature pregnant mare's serum gonadotropin-primed rats with the anti-androgenic drug, hydroxyflutamide

The involvement of androgens in the control of ovulation has been assessed by administration of the androgen antagonist, hydroxyflutamide, to prepubertal rats treated with pregnant mare's serum gonadotropin (PMSG) to induce first estrus and ovulation. Without human chorionic gonadotropin (hCG) injection, only 46% of rats that received six 5-mg, s.c. injections of hydroxyflutamide at 12-h intervals, beginning an hour before s.c. injection of 4 IU PMSG on Day-2 (Day 0 = the day of proestrus), had ovulated a mean of 1.3 +/- 0.4 oocytes per rat when killed on the morning of Day 1, whereas 92% of sesame oil-treated controls had ovulated a mean of 6.9 +/- 0.6 oocytes. After i.p. injection of hCG at 1600 h on Day 0, 92% of hydroxyflutamide-treated rats ovulated a mean of 8.3 +/- 1.2 oocytes compared to 100% of controls, which ovulated 7.3 +/- 0.4 oocytes per rat: these groups were not significantly different from each other, nor from control rats that received no hCG. Thus, exogenous hCG completely overcame the inhibitory effect of hydroxyflutamide on ovulation. Rats treated with PMSG and hydroxyflutamide without hCG were killed either on the morning of Day 0 to determine serum and ovarian steroid levels or on the afternoon of Day 0 to determine serum LH levels. Serum levels of estradiol-17 beta and testosterone in hydroxyflutamide-treated rats were significantly higher (178% and 75%, respectively; p less than 0.01) than levels observed in controls on the morning of Day 0. Ovarian concentrations of the steroids were also elevated in hydroxyflutamide-treated rats (p less than 0.01 for testosterone only).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of neonatal gonadal steroids on adult CA3 pyramidal neuron dendritic morphology and spatial memory in rats

The hippocampus is implicated in spatial cognition, which is sexually dimorphic and developmentally sensitive to gonadal steroids. Previously we have shown a sex difference in CA3 pyramidal cell layer volume and neuronal soma size that was reversible with neonatal castration in males or prenatal treatment of females with either testosterone propionate (TP) or a nonaromatizable androgen, dihydrotestosterone propionate, but not estradiol benzoate, all of which correlated with adult water maze navigation. The present study further investigates developmental androgen sensitivity of CA3 pyramidal neurons by measuring dendritic morphology and its relation to adult spatial ability. Female rats were injected with TP on postnatal day (P) 3 and P5 or ovariectomized (OVX) on P2, and male rats were castrated on P2, with or without testosterone replacement (Cas+T). Sham surgery controls were also included. Animals were tested on a water maze in adulthood, sacrificed, and CA3 pyramidal neurons were Golgi-stained and reconstructed in three dimensions using a computer-interfaced morphometry system. High-androgen groups (control males, Cas+T, TP females) performed better in spatial navigation and exhibited CA3 neurons with longer dendrites, a larger number of dendritic branches, and volumes of influence compared to low-androgen groups (control females, castrated males, OVX). Collectively, these findings indicate that the critical time period for organizational effects of androgens on the CA3 pyramidal neurons includes both prenatal and postnatal life, during which time androgens regulate developmental events such as somal growth and neuronal differentiation, all of which significantly contribute to establishing the sex difference in adult spatial navigation.     

Mounting behavior and lordosis behavior in castrated male rats treated with testosterone propionate, or with estradiol benzoate or dihydrotestosterone in combination with testosterone propinonate.

Treatment of prepuberally castrated male rats with testosterone propionate (TP, 50, 200, 500, or 1000 μg for 30 days) in adulthood stimulated the display of both mounting behavior and lordosis behavior. No correlation between mounting and lordosis behavior could be detected at any TP dose level. Treatment of prepuberally castrated male rats with either 1 μg estradiol benzoate (EB) or 500 μg dihydrotestosterone (DHT) for 60 days stimulated the display of mounting behavior in three of eight and four of eight rats, respectively. Treatment with 200 μg TP for the last 30 days of rats receiving either EB or DHT for 60 days resulted in an abrupt onset on mounting behavior as compared to rats treated with oil for 60 days. These results show additive effects of EB or DHT and TP upon mounting behavior by male rats and are interpreted as a support for the suggestion that testosterone to estrogen as well as testosterone to DHT conversion may be involved in the mechanism whereby testosterone activates the mounting behavior of castrated rats.     

Effects of aromatizable and nonaromatizable androgen treatments on luteinizing hormone receptors and ovulation induction in immature rats

The effects of androgen pretreatment on follicle-stimulating hormone (FSH)-stimulated luteinizing hormone (LH) receptor induction in ovarian granulosa cells was examined. Immature female rats were treated with various doses (0.1-5 mg/rat) of testosterone (T), 5 alpha-dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol), or 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). Subsequent follicular development was stimulated by treatment with ovine FSH. LH receptor induction in granulosa cells and ovulatory responses to 10 IU human chorionic gonadotropin (hCG) were examined. Since LH receptor induction requires the synergistic action of both FSH and estradiol, the effects of the androgen pretreatment on FSH-stimulated estradiol production were also examined. Dihydrotestosterone treatment at doses greater than 1 mg inhibited LH receptor induction by approximately 70%, which resulted in absent ovulatory responses. Treatment with 1 mg or more of T or 3 alpha-diol had no effect on LH receptor induction, yet the hCG-stimulated ovulation rate was reduced to 40% of that seen in vehicle-treated controls. 3 beta-Diol, at a dose of 1 mg/rat, did not affect LH receptor induction but did reduce hCG-stimulated ovulation responses. No significant effects of androgen treatment on ovarian or uterine weight or FSH-stimulated estradiol production were observed. These results suggest that androgens can act at multiple sites to inhibit ovarian follicular development and function. In addition these studies demonstrate that, although LH receptor induction is necessary, it may not be a sufficient condition to ensure ovulation of ovarian follicles.     

Unilateral sectioning of the superior ovarian nerve of rats with polycystic ovarian syndrome restores ovulation in the innervated ovary

The present study tested the hypothesis that if polycystic ovary syndrome (PCOS) results from activating the noradrenergic outflow to the ovary, unilaterally sectioning the superior ovarian nerve (SON) will result in ovulation by the denervated ovary, and the restoration of progesterone (P4), testosterone (T) and estradiol (E2) normal serum level. A single 2 mg dose of estradiol valerate (EV) to adult rats results in the development of a syndrome similar to the human PCOS. Ten-day old rats were injected with EV or vehicle solution (Vh) and were submitted to sham surgery, unilateral or bilateral sectioning of the SON at 24-days of age. The animals were sacrificed at 90 to 92 days of age, when they presented vaginal estrus preceded by a pro-estrus smear. In EV-treated animals, unilateral sectioning of the SON restored ovulation by the innervated ovary and unilateral or bilateral sectioning of the SON normalized testosterone and estradiol levels. These results suggest that aside from an increase in ovarian noradrenergic tone in the ovaries, in the pathogenesis of the PCOS participate other neural influences arriving to the ovaries via the SON, regulating spontaneous ovulation. Changes in P4, T and E2 serum levels induced by EV treatment seem to be controlled by neural signals arising from the abdominal wall and other signals arriving to the ovaries through the SON, and presents asymmetry.     

The role of aromatization in androgen stimulation of gerbil scent marking

The androgen dependent scent marking behavior of male Mongolian gerbils (Meriones unguiculatus) can be stimulated after castration by either testosterone or estrogen, but not by dihydrotestosterone (DHT). To determine if DHT fails to evoke scent marking because it cannot be aromatized to form an estrogen, two other nonaromatizable androgens, 1α-methyltestosterone and 6α-fluorotestosterone, were studied. 6α-Fluorotestosterone and its propionate ester stimulated scent marking in castrated male gerbils as effectively as testosterone and its ester did. Hence, an androgen's aromatizability does not determine its ability to influence gerbil scent marking behavior.     

Effects of androgens on serum concentrations of gonadotropins and ovarian steroids in gilts

To examine how androgens affect endocrine events associated with increased ovulation rate, gilts were injected with androgen receptor agonists, an antagonist, or a combination of both. Blood samples were collected hourly from Day 13 to estrus (Day 0 = onset of estrus) coincident with gilts (n = 6 per treatment) receiving daily treatments of vehicle (corn oil), 10 mg of testosterone, 10 mg of 5 alpha-dihydrotestosterone (dihydrotestosterone), 1.5 g of flutamide (an androgen receptor antagonist), testosterone plus flutamide, or dihydrotestosterone plus flutamide. Treatment of gilts with testosterone or dihydrotestosterone alone increased (P < 0.05) concentrations of FSH in serum, and these effects were blocked by cotreatment with flutamide. Estradiol-17beta and androstenedione concentrations in serum were increased (P < 0.05) at 2 h after injection of testosterone or testosterone plus flutamide but not after dihydrotestosterone treatment, probably because of the role of testosterone as a substrate for estradiol-17beta and androstenedione synthesis. There were no effects of the six treatments on serum concentrations of progesterone during luteolysis, but treating gilts with testosterone shortened (P < 0.05) the proestrous period. Total embryonic loss by Day 11 in gilts treated with dihydrotestosterone was reversed when gilts were cotreated with dihydrotestosterone plus flutamide. Results of this experiment indicated that androgen actions both increased FSH secretion and reduced embryonic survival by a mechanism(s) dependent on the androgen receptor.     

Increase in testosterone sensitivity induced by constant light in relation to melatonin injections in rats.

In this experiment we investigated whether the lack of the nocturnal melatonin peak under constant light would cause an increase in testosterone sensitivity. Castrated rats were kept under periodic or constant light for one week. They received a daily injection of vehicle, testosterone propionate (125 micrograms), melatonin (50 micrograms) or testosterone plus melatonin (125 micrograms + 50 micrograms). Serum and pituitary gonadotrophins and pineal melatonin were measured at the end of the experiment. Under constant light, testosterone injections reduced the serum luteinizing hormone concentration in castrated rats to that in intact rats, but, under periodic light, the decrease was smaller. Melatonin did not reverse the stronger effect of testosterone under constant light. The serum melatonin peak produced by the exogenous melatonin injection had a higher amplitude, shorter duration and earlier appearance than the physiological melatonin peak. Exogenous melatonin did not modify the physiological melatonin secretion, measured either as serum melatonin concentration or pineal melatonin content on the consecutive day. We conclude that the increase in testosterone negative feedback sensitivity of castrated rats under constant light was not due to the absence of the nocturnal melatonin pulse.     

Changes in gonadotrophin concentrations are not necessarily involved in ovarian compensation after unilateral ovariectomy in sheep

Ewes were unilaterally ovariectomized and/or hypophysectomized and treated with PMSG and hCG. For a given gonadotrophin treatment the ovulation rate per ewe was maintained, i.e. the ovulation rate of the remaining ovary was significantly increased (P less than 0.05), after the removal of one ovary in hypophysectomized and in pituitary-intact ewes. It is concluded that compensation of ovulation rate in the remaining ovary after unilateral ovariectomy in the sheep may be independent of feedback from the ovary and the release of gonadotrophins from the pituitary gland.     

Neonatal dihydrotestosterone and estrogen stimulation: Effects on sexual behavior of male rats

Male rats castrated on the second day after birth (Day 2) were, for the next 10 days, given daily injections of one of five steroids or steroid combinations: 200 μg of testosterone propionate (TP); 200 μg of dihydrotestosterone propionate (DHTP); 5 μg of estradiol benzoate (EB); 5 μg of estradiol benzoate plus 200 μg of dihydrotestosterone propionate; oil vehicle (VH). Control male rats castrated on Day 90 received a sham castration and oil vehicle in the neonatal period. All animals were given TP in adulthood and tested for male sexual behavior. There was no difference in mounting activity among the subjects. Day 2 DHTP subjects displayed intromissions but were incapable of ejaculating. The more frequent display of intromissions by Day 2 DHTP animals in comparison to Day 2 VH animals could be solely due to their larger and more highly developed penes. On the other hand, the ejaculatory failure of the Day 2 DHTP subjects was attributed to some deficiency in central neural processes controlling ejaculatory mechanisms rather than inadequate penile development. Equivocal results were obtained with the Day 2 EB and Day 2 EB-DHTP animals in that only a few animals in both groups showed an ejaculatory pattern.     

Changes in serum and ovarian steroids during reproductive development in the female guinea pig

This study was designed to measure ovarian hormones prior to and during the first estrous cycle in guinea pigs. Blood was obtained from 12 animals throughout the first estrous cycle. Ovaries and peripheral serum were obtained from 25 additional animals at various stages of development prior to and after first ovulation. Estradiol, progesterone, androstenedione, and testosterone were measured in all sera and half of the ovaries. The remaining ovaries were fixed for histology. Serum estradiol was nondetectable until a few days before first ovulation, but was present in the ovary throughout development. Serum progesterone was nondetectable until the day of ovulation, but the luteal phase pattern was similar to that observed in adults. Serum androgens were detectable throughout development, with androstenedione higher than testosterone. The immature ovary contained more testosterone than androstenedione, but this pattern was reversed after ovulation. These results indicate that the immature ovary in the guinea pig contains minimal amounts of estradiol and progesterone, the first estrous cycle is similar to that in adults, and that the pattern of ovarian androgen content changes during the peripubertal period.     

Sexual behavior of male golden hamsters receiving diverse androgen treatments

Four androgens were compared for their effectiveness in maintaining the sexual behavior of castrated male golden hamsters. Sexually experienced males were divided into 4 experimental treatment groups which received 500 μg daily of testosterone, androstenedione, dihydrotestosterone or androsterone. Control castrates were given oil. All animals were tested for sexual behavior every 2 wk for 10 wk following the onset of experimental treatment. Testosterone and androstenedione were the only androgens that maintained intromissions above the oil control level. However, testosterone, androstenedione and androsterone, but not dihydrotestosterone were effective in maintaining mounting behavior above the oil control level. No differences were detected between these 4 androgens in their maintenance of penile papillae.     

Exposure to androgens during in vitro maturation does not affect the developmental potential of porcine oocytes

Administration of exogenous androgens to pigs during the period of follicular development has been shown to positively affect ovulation rate and embryonic survival. The mechanisms of these actions are not known, but may include direct effects of androgens on the cumulus-oocyte complex (COC). The objective of this experiment was to assess the effects on embryonic development in vitro of exposure of COC to 0.26 and 2.6 microM testosterone (T) or dihydrotestosterone (DHT) during IVM. For IVM, COC were cultured for 44-46 h in protein-free tissue culture medium (TCM) 199 containing 10 IU/ml hCG and eCG and 10 ng/ml EGF. Oocytes were then stripped of cumulus cells, coincubated with 1 x 10(5) sperm/ml in modified TALP for 6 h, and cultured for 8 days in NCSU23. The proportions of oocytes that cleaved (Day 2) or developed to the morula (Day 6) or blastocyst (Day 6-8) stage were not different (P > 0.20) between oocytes exposed to androgens and oocytes not exposed to androgens. These results indicate that exposure to androgens during IVM does not affect the ability of oocytes to cleave or develop up to the blastocyst stage in vitro.     

Does androgen influence prolactin secretion?

In both intact and castrated male and female rats, administration of the A-ring reduced androgen, dihydrotestosterone (DHT), consistently failed to stimulate prolactin (PRL) secretion although it inhibited LH release and, in males, stimulated ventral prostate growth. In intact females, but not in the other types of rat, DHT actually suppressed PRL release. These findings do not support generalizations, based entirely on findings with testosterone, that both "androgens" and estrogens exert stimulatory actions on PRL secretion. The distinct stimulatory effects of testosterone and its esters on PRL secretion seem attributable, not to their androgenic actions per se, but to the ability of testosterone to form estrogenic metabolites. This ability does not appear to be shared by the "pure" androgen, DHT.     

Modulation of LH/hCG receptors and physical state of ovarian membranes in rat pseudopregnancy

Previous investigations have demonstrated that increased ovarian function during pseudopregnancy in the rat may be associated with alterations of the physical state of membranes. Changes in rigidity of membrane lipids were observed during the formation as well as regression of corpora lutea. The effects of cyclooxygenase inhibitors (indomethacin and acetylsalicylic acid (ASA)) and of selected steroids (estradiol, testosterone and dihydrotestosterone) on the functional state of luteinized ovaries were studied. The compounds were administered to the animals in silastic capsules on different days after hCG injection. ASA and indomethacin administration on days 10 and 11 after hCG injection resulted in an increase in the LH/hCG receptor binding activity and rigidity of ovarian membrane lipids, as determined by fluorescence polarization of 1,6-diphenyl-1,3,5 hexatriene (DPH) probe. This effect was apparent within 7 days after indomethacin and ASA treatment. Both estradiol and testosterone significantly increased the ovarian LH/hCG binding activity, however estradiol did not affect the membrane lipid rigidity. Unlike testosterone, the administration of dihydrotestosterone induced a decrease in membrane lipid rigidity and reduced the accessibility of the LH/hCG receptor. Inhibitors of prostaglandin F2alpha (PGF2alpha) synthesis, as the endogenous mediator of luteolysis, were shown to delay the regression of the corpora lutea and to prolong the luteal activity in pseudopregnant rats.