Effects of castration of immature rats on serum FSH and LH, and of various steroid treatments after castration

The intricate relationship between the gonads and pituitary gonadotropin secretion has been studied in the immature, 26-day-old rat. In male rats or chidectomized at this age, serum LH and FSH rose to significantly higher levels at 8 hours postcastration. A much later response was seen in ovariectomized females: at 24 hours and 48 hours for FSH and LH respectively. When groups of rats castrated at 26 days of age were treated with pharmacologic dosages of various steroids for 6 and 15 days postoperative, it was found that testosterone, 5alpha-dihydrotestosterone, and estradiol prevented the rise of both FSH and LH, in both sexes. A steroid-derived drug, 17alpha-ethinyl-testosterone-2, 3-isoxazol, was also effective, while progesterone alone was unable to suppress gonadotropins in either sex. Results reaffirm that the gonadal-hypophyseal relationhsip is sensitive before puberty. The marked sex difference in the response to castration is undoubtedly due to different gonadal hormones (androgen or estrogen) present at the time of castration, and their contributions to this feedback process. However it appears that hormones of either type can suppress both gonadotropins in both sexes. Results with 17alpha-ethinyl-testosterone-2, 3-isoxazol were particularly encouraging with respect to its clinical usefulness as a gonadotropin inhibitor with little or no biologic activity as a sex steroid.     

Effect of neonatal castration on the content of hypothalamic LHRH, pituitary LH and plasma LH in developing male rats.

The content of hypothalamic LHRH and concentration of LH in pituitary and plasma were measured on day 5, 7, 10, 14, 17, 22, 25, 30, 45, 52 and 60 in male rats which were bilaterally castrated on day 2. The levels of plasma LH were significantly higher in all the groups of castrated rats than in normal male rats of corresponding ages. The concentration of plasma LH did not rise progressively but showed day to day fluctuation apparently due to alteration of sexual differentiation of the hypothalamus. The concentration of pituitary LH was significantly lower in neonatally castrated rats compared to normal male rats except on days 17, 25 and 30. The content of hypothalamic LHRH declined initially following castration, but from day 17 onwards significantly higher levels of hypothalamic LHRH were maintained in neonatally castrated rats than in intact control. Initial decline in the content of hypothalamic LHRH may be because of stimulation of release of LHRH which exceeds maximal rate of synthesis and subsequent increase in the content of hypothalamic LHRH may be due to enhanced LHRH synthesis as a result of castration.     

Correlation of hypothalamic LHRH, pituitary LH and plasma LH in developing rats.

Hypothalamic LHRH, pituitary LH and plasma LH levels were measured in rats of both sexes from day 5-60 after birth. The content of hypothalamic LHRH was very high in one-week-old male and female rats. It declined gradually till day 17 in the female rat and sharply on day 10 in the male rat. Subsequently the content of hypothalamic LHRH increased and showed peak values on day 25 in the female rat and on day 45 in the male rat. It decreased markedly at respective times of puberty in both sexes (day 37 in the female rat and day 52-60 in the male rat). Results of the study suggest that maturation of hypothalamo-hypophyseal-axis proceeds in three distinct stages. Observations on days 17, 25 and 37 in the female rat and on days 5, 7, 10 and 22 in the male rat clearly show an inverse relationship between hypothalamic LHRH and plasma LH and a parallel relationship between pituitary and plasma LH. Marked decline in the content of hypothalamic LHRH at respective times of puberty in both sexes indicates that the release of threshold levels of LHRH from the hypothalamus may apparently be the event initiating the pubertal changes in rat.     

Effect of hypophysectomy and growth hormone replacement on hypothalamic GHRH

Long-term (7 and 14 days) hypophysectomy resulted in a striking decrease in growth hormone releasing hormone-like immunoreactivity (GHRH-LI) in the median eminence (ME) of adult male rats, evaluated by both radioimmunoassay and immunohistochemistry. Treatment with human GH (125 μg/rat, twice daily IP for 14 days) prevented, though partially, depletion of GHRH-LI from the ME, as assessed by both methods. These results demonstrate that circulating GH levels regulate the function of GHRH-producing structures, via a feedback mechanism.     

Effect of time after castration on secretion of LHRH and LH in the ram

Hypophysial portal blood and peripheral blood were obtained from conscious, unrestrained rams to measure simultaneously the secretion of LHRH and LH in entire rams and rams which had been castrated for 2-15 days (short-term castration) and for 1-6 months (long-term castration). The apparatus for portal blood collection was surgically implanted using a transnasal trans-sphenoidal approach and, 4-5 days later, portal blood and peripheral blood were collected simultaneously at 10-min intervals for 8-9 h from 15 sheep. LHRH was clearly secreted in pulses in all three physiological conditions, but there were marked differences in pulse frequencies, which averaged 1 pulse/2-4 h in entire rams, 1 pulse/70 min in short-term castrated rams and 1 pulse/36 min in long-term castrated rams. In entire and short-term castrated animals, LH profiles were also clearly pulsatile and each LHRH pulse in hypophysial portal blood was associated with an LH pulse in the peripheral blood. In long-term castrated animals, LH pulses were not as well defined, because of the high basal levels and small pulse amplitudes, and the temporal relationship between LHRH and LH pulses was not always clear. These results demonstrate the pulsatile nature of LHRH secretion under the three physiological conditions and suggest that the irregular LH profiles characteristic of long-term castrates are due to an inability of the pituitary gland to transduce accurately the hypothalamic signal. The very high frequency of the LHRH pulses may be one of the major reasons for this, and is probably also responsible for the high rate of LH secretion in the long-term castrated animal.     

Brain monoamines and sexual behavior in Japanese quail: Effects of castration and steroid replacement therapy

In the male Japanese quail, testosterone is required for the activation of sexual behavior. This steroid dependent process may rely heavily on mediation via monoaminergic neurons. These experiments were conducted to study the relationship between reproductive state (hormonal and behavioral components) and levels of monoamines in selected areas of the brain in Japanese quail. In Experiment 1, monoamine levels in a number of brain areas were compared in castrates, testosterone-implanted castrates, and intact males. Monoamine levels were comparable to those previously measured in Japanese quail, and there were no significant differences due to treatment. Plasma luteinizing hormone (LH) levels and recovery of cloacal gland area in implanted castrates confirmed the afficacy of treatments. In Experiment 2, the disappearance of dopamine (DA) and norepinephrine (NE) following administration of a-methyl-para-tyrosine (aMPT) was used as an indicator of turnover rate. Male and female quail were gonadectomized at 3 weeks of age. At the age of five weeks, some gonadectomized males and females were given implants containing testosterone. Only intact males and testosterone-implanted castrated males showed reproductive behavior. Plasma gonadotropin levels were elevated in gonadectomized birds and reduced in steroid-implanted gonadectomized birds. The aMPT treatment significantly reduced the levels of DA and NE in the telecephalon and the level of DA in the hypothalamus. After aMPT treatment, the disappearance of NE in the telecephalon and of DA in the hypothalamus were significantly different according to the sex or treatment of the birds or both. Significant interactions between these two factors were observed. Disappearance rate of NE in the telecephalon was decreased by castration of males and increased by ovariectomy of females. Both effects were counteracted by testosterone. Reverse effects were observed for DA disappearance in the hypothalamus (increase with castration in males and decrease with ovariectomy in females). These results give evidence for altered aminergic function in specific areas of the brain relative to altered reproductive state.     

Effects of sulpiride on levels of FSH, LH and steroid hormones

In order to study the effects of prolactin upon the gonadotrophins and steroid hormones, hyperprolactinaemia was induced by the administration of sulpiride. 12 men between the ages of 18 and 20 were given 3 capsules of 50 mg of sulpiride daily for a period of 15 days, and the following parameters being measured before and after the treatment: (prolactine, FSH, LH, testosterone, estradiol, ACTH and DHEA-S) by RIA, (cortisol) by fluorimetry and (etiocholanone, androsterone, pregnandiol, pregnantriol, pregnantriolone, 11-keto etiocholanone and 11-OH androsterone) by gas chromatography. Our results show that on termination of the treatment there was a significant rise in the prolactin and DHEA-S serum levels and a drop in the FSH serum levels but not of LH. In addition there was a marked increase in all the androgen levels studied, (etiocholanone, androsterone and 11-keto etiocholanone) with the exception of testosterone.     

Effects of pargyline on hypothalamic biogenic amines and serum prolactin. LH and TSH in male rats.

The time course effects of pargyline on hypothalamic biogenic amines and serum prolactin (PRL), LH and TSH were studied in adult male rats. The rats were killed at intervals of 1–6 hrs after pargyline injection. Hypothalamic dopamine (DA) rose 79% by 1 hr and was 41% above “0” time by 6 hrs. Norepinephrine (NE) increased 31% by 1 hr and remained at about this level through 6 hrs, whereas serotonin (5HT) increased from 42% by 1 hr and to 95% by 6 hrs. Serum PRL LH and TSH fell significantly during the first 2 hrs, but all had returned to pretreatment values by 4 hrs. Serum PRL was about 4-fold above pretreatment values by 6 hrs, but LH and TSH remained at pretreatment levels. Stimulation by pargyline of PRL release was potentiated by Lilly compound 110140, a serotonin reuptake inhibitor, and blocked by parachlorophenylalanine, a serotonin synthesis inhibitor. These results suggest that the inhibitory effects of pargyline on PRL, LH, and TSH release during the first 2 hrs were associated mainly with a rapid increase in DA, and subsequent elevation of PRL release was related to the increase in 5HT. Return of serum LH and TSH to pretreatment levels at 4 and 6 hrs appeared to be associated mainly with the decrease in DA and perhaps to elevated NE levels. These results suggest that changes in relative concentrations of hypothalamic amines are related to differential release of PRL, LH and TSH.     

Effects of morphine and naloxone on serum LH, FSH and prolactin levels and on hypothalamic content of LH-RF in proestrous rats.

Proestrus surges of serum LH, FSH and prolactin (PRL) were significantly reduced when morphine HCl (50 and 10 mg/kg) was administered to 4-day cycling rats just prior to the proestrous critical period. The inhibitory effect of morphine was reversed by naloxone, a morphine antagonist, at the dose which had no effect on the proestrus surges of serum LH, FSH or PRL. The hypothalamic LH-RF content of proestrous rats at 1800 hr (during the proestrus surge) was not significantly different from that at 1400 hr (before the surge) and was not affected by pretreatment with morphine or naloxone. Our results suggest that naloxone reverses the anti-ovulatory effect of morphine by antagonizing the inhibitory effect of morphine on preovulatory surges of gonadotropins or PRL.     

Effects of an LHRH agonist on endocrine function, LHRH receptor and LH/hCG receptor in the pituitary-gonadal axis of male rats

The effects of an LHRH agonist (LHRHa), [D-Ser (tBu)]6 des-Gly-NH210) ethylamide, on endocrine function and the LHRH and LH/hCG receptors in the pituitary-gonadal axis were examined. The LHRHa was injected at 100 ng/100 g body weight into male rats once a day for 4 weeks and its effects were observed until 2 weeks after the end of treatment. Due to LHRHa treatment, the plasma LH concentration began to increase on day 3, reached a peak on day 7, and then decreased, although it remained above the control level during the treatment. The pituitary LH content decreased on day 1, reached a minimum (about 40% of the control) between days 3 and 7, and then was maintained at 60% of the control level until week 4. In contrast, the pituitary LHRH receptor concentration increased only on day 3, and the association constant (Ka) remained unchanged during the observation period. The testis weight and plasma testosterone concentration began to decrease on day 3, reached the minimum on day 7 and remained at this level until week 4, and their levels were not completely restored to normal 2 weeks after cessation of treatment. The testicular LH/hCG receptor concentration was decreased on day 1, and markedly decreased to 10-15% of the control value between day 7 and week 4, but the Ka value was slightly increased during the treatment. However, these values had completely recovered 2 weeks after the cessation of treatment. The testicular LHRH receptor concentration increased between days 1 and 7, returned to the control level in weeks 2 and 4, and then decreased 2 weeks after cessation of treatment. Its Ka value was reduced in weeks 2 and 4. These data suggest that the inhibitory effect of LHRHa on the gonad in male rats is not due to reduced pituitary LH release, but to changes in the number and Ka values of gonadal receptors for LH/hCG and LHRH.     

New data on a second endogenous molecular form of mammalian hypothalamic LHRH, (hydroxyproline 9) LHRH]

An endogenous hydroxylated form of LHRH, (Hyp) LHRH, is able to displace LHRH bound to pituitary membrane preparations. In parallel, it stimulates release of both LH and FSH from pituitary cells in primary culture. The potency ratio of (Hyp)LHRH is approximately 1:20 and 1:5 with respect to the native decapeptide when peptidasic degradation is or is not inhibited. This correlates with a greater resistance of (Hyp) LHRH towards enzymatic degradation; in contrast to LHRH, the C-terminal (residues 6 to 10) end of (Hyp) LHRH is not degraded and generates C-terminal fragments which account for 64% of the LHRH immunoreactivity in extrahypothalamic areas as the hippocampus. Besides its weak gonadotropin releasing activity and its action or its localization in peripheral organs (placenta, gonads), a major role of the hydroxylated decapeptide may thus be to serve as a precursor of smaller active fragments on targets other than pituitary receptors.     

Effects and interactions of LH and LHRH agonist on testicular morphology and function in hypophysectomized rats

The effects of single or combined daily treatment with an LHRH agonist and low or high doses of LH upon the testes of adult hypophysectomized rats were studied for up to 2 weeks in which changes in testicular histology, particularly the interstitial tissue, were examined by morphometry and related to functional assessment of the Leydig cells in vivo and in vitro. Compared to saline-treated controls, LHRH agonist treatment did not alter testis volume or the composition of the seminiferous epithelium or any of the interstitial tissue components although serum testosterone and in-vitro testosterone production by isolated Leydig cells were significantly reduced. With 2 micrograms LH for treatment, testis volume was increased, spermatogenesis was qualitatively normal, total Leydig cell volume was increased, serum testosterone values were initially elevated but subsequently declined and in-vitro testosterone production was enhanced. Testis volume with 20 micrograms LH treatment was unchanged compared to saline treatment, the seminiferous epithelium exhibited severe disruption but total Leydig cell volume was greatly increased due to interstitial cell hyperplasia. This group showed elevated serum testosterone concentrations and major increases in testosterone production in vitro. Treatment with LHRH agonist with either dose of LH resulted in reduced testis volume, moderate to very severe focal spermatogenic disruption and increased total Leydig cell volume although serum testosterone values and in-vitro testosterone production were markedly reduced compared to control rats. It is concluded that, in the absence of the pituitary, LHRH agonist fails to disrupt spermatogenesis and the previously described antitesticular action of LHRH agonists in intact rats is therefore dependent upon the presence of LH, which alone or in combination with LHRH agonist, may focally disrupt spermatogenesis in hypophysectomized rats whereas the Leydig cells undergo hyperplasia. The findings show that impairment of spermatogenesis is accompanied by alterations of the interstitial tissue and suggest that communication between these two compartments is involved in the regulation of testicular function.     

Intracellular partitioning of androgen receptor immunoreactivity in the brain of the male syrian hamster: Effects of castration and steroid replacement

The effect of castration and steroid replacement on the intracellular partitioning of the androgen receptor in the brain of the male Syrian hamster was determined using immunocytochemistry. Androgen receptors were visualized using the PG-21 antibody (G. S. Prins) on 40-μm coronal brain sections from hamsters perfused with 4% paraformaldehyde with or without 0.4% glutaraldehyde. Control studies confirmed antibody specificity in gonad-intact and castrate males. In the normal adult male, androgen receptor immunocytochemistry reveals intense staining confined to the cell nucleus. Castration caused a gradual increase in cytoplasmic labelling within 2 weeks, accompanied by a reduction in nuclear staining intensity in androgen receptor-containing neurons throughout the brain. Cytoplasmic androgen receptor staining was eliminated after treatment of orchidectomized males for only 8 h with exogenous testosterone. Likewise, long-term exposure to testosterone and dihydrotestosterone, a nonaromatizable androgen, maintained nuclear androgen receptor immunoreactivity. However, exposure to low physiologic concentrations of estrogen was not effective in this regard. In addition, we determined that nuclear androgen receptor immunoreactivity decreases in response to inhibitory short-day photoperiod, but without an increase in cytoplasmic immunostaining. This appears to be due to the decrease in androgen production by the testis, rather than a direct photoperiodic effect, because testosterone supplementation to short-day males restored the intensity of nuclear androgen receptor immuno-reactivity to levels comparable to those in the intact male. These findings are compatible with a new model for the intracellular localization of androgen receptors, in which a subset of unoccupied receptors is located in the cell cytoplasm in the absence of ligand. They further demonstrate the repartitioning of such cytoplasmic receptors, thereby confirming and extending previous observations using biochemical techniques on the regulation of neuronal androgen receptors. © 1993 John Wiley & Sons, Inc.     

Effects of subchronic steroid therapy on vitamin E-deficient rats (38480).

Experiments were carried out to determine the effectiveness of steroid therapy in vitamin E-deficiency, as measured by autohemolysis of isolated RBC's body weight gain, serum creatine phosphokinase activity, and stabilization or labilization of isolated hepatic lysosomes. Results of such experiments would indicate whether triamcinolone acetonide could supplant vitamin E in vitamin E-deficiency states via its ability to stablize various membranes. Autohemolysis induced by vitamin E-deficiency could not be prevented by daily administration of triamcinolone. Daily dosages of 0.1 and 0.4 mg/kg (ip) triamcinolone given concomitantly with replacement vitamin E (at sufficient dosages to reverse the autohemolysis) resulted in an increased autohemolysis. No changes in lysosomal membrane fragility were noted when hepatic lysosomes were obtained from vitamin E-deficient rats with triamcinolone resulted in a greater attenuation of body-weight gain. Creatine phosphokinase levels were not augmented in vitamin E-deficient rats. Vitamin E-deficient rats supplemented with vitamin E and treated with triamcinolone, manifested an increase in creatine, phosphokinase. It was therefore concluded that although triamcinolone and vitamin E possess a common ability to stablize membranes and proteins, their mechanisms must be different since triamcinolone could not substitute for vitamin E in a deficiency state. Indeed, triamcinolone was found to be more toxic in the absence of vitamin E.