Effects of gonadal steroids on follicle-stimulating hormone and luteinizing hormone secretion by pituitary cells from castrated and intact male rats

The effectiveness of androgens in suppressing gonadotropin secretion declines with time following orchidectomy; however, the mechanism for this acquired resistance to androgen action is unknown. The role of the pituitary was studied by use of perifused rat pituitary cells and cells in monolayer culture. Pituitary cells from 7-wk-old intact male rats and rats that had been castrated 2 wk previously were treated with 10 nM testosterone (T) for 24 h; cells were then packed into perifusion chambers and stimulated with 2.5 nM GnRH for 2 min every hour for 8 h during which time T treatment was continued. T suppressed GnRH-stimulated LH secretion and LH pulse amplitude equally in both groups to approximately 60% of control values. Interpulse LH secretion was unchanged by T in either group. GnRH-stimulated FSH release was suppressed more (p less than 0.05) by T with cells from castrated rats than with cells from intact rats (76 +/- 4% vs. 90 +/- 2% of control; mean +/- SEM). By contrast, the action of T to increase interpulse basal FSH secretion was less (p less than 0.05) with cells from castrated rats (115 +/- 10% of control) than with cells from intact rats (146 +/- 6% of control). T treatment for 72 h also increased basal FSH secretion by pituitary cells in monolayer culture to a lesser extent with cells from castrated rats than with cells from intact rats (151 +/- 14% vs. 191 +/- 16% of control, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Variations in the number of pituitary LHRH receptors correlated with altered responsiveness to LHRH

Isolated pituitary cells from metestrous, ovariectomized (OVX), and ovariectomized-estradiol treated (OVX-EB) rats were employed to study the gonadotropin response to luteinizing hormone-releasing hormone (LHRH) challenge and to quantitate LHRH receptors, using a labeled LHRH analog. Ovariectomy (3–4 weeks post castration) resulted in a reduction of LHRH receptor concentration from 34.4 ± 2.1 in metestrous females to 14.3 ± 0.9 fmoles/10⁶ cells. Concomitantly, the luteinizing hormone (LH) response to a near-maximal dose of LHRH (5 ng/ml) decreased from a 3-fold stimulation in intact females to 1.13-fold stimulation in cells from OVX rats. Replacement therapy with EB (50 ug/rat for 2 days) to OVX rats restored LH response and LHRH binding sites (a 2.5-fold stimulation in LH secretion and 32.0 ± 2.1 fmoles/10⁶ cells, respectively). The LH response to LHRH stimulation was not altered after one day of EB treatment although the number of LHRH binding sites was increased. The changes in the number of LHRH binding sites were not accompanied by any alterations in the affinity of the LHRH analog ($\text{K}{}_{\mathrm{d}}\text{? 0.5 × 10}{}^{\mathrm{?9}}\text{M}$). It is concluded that variations in LHRH receptor number reflect the degree of pituitary sensitivity to LHRH and it may suggest that LHRH and estradiol modulation of gonadotropin release is mediated by these receptors.     

Androgen metabolism in the male hamster--1. Metabolism of testosterone in the pituitary gland and in the brain of animals exposed to different photoperiods

It is known that the metabolism of testosterone in the brain and in the anterior pituitary is different in mammalian and in photoperiodic avian species. In many mammalian species, testosterone is mainly metabolized to 5-alpha-reduced compounds (e.g. 17-beta-hydroxy-5-alpha-androstan- 3-one, 5 alpha-DHT and 3-alpha,17-beta-dihydroxy-5-alpha-androstane, 5-alpha,3-alpha-diol) and, to a smaller extent, to 4-androstene-3,17-dione (androstenedione), while in birds, androstenedione is the main testosterone metabolite and the conversion to the 5-alpha-reduced compounds is quantitatively negligible. In avian species, testosterone is also converted to 5-beta-reduced steroids (mainly 17-beta-hydroxy-5-beta-androstan-3-one, 5-beta-DHT and 3-alpha,17-beta-dihydroxy-5-beta-androstane, 5-beta,3-alpha-diol), and there is also evidence that in these species testosterone metabolism in the central structures may be influenced by the photoperiod. Since the hamster is a mammal whose reproductive cycle is controlled by day length, it has been analyzed whether: (a) the central structures of the hamster (cerebral cortex, hypothalamus and anterior pituitary) metabolize testosterone in vitro following a mammalian (5-alpha-reduced derivatives) or an avian (androstenedione and 5-beta-reduced compounds) pattern; and (b) the metabolism of testosterone in the same structures may be modified by the exposure to different photoperiods (LD 14:10 or LD 8:16). The present data indicate that no one of the hamster structures examined produces the 5-beta-reduced derivatives. Moreover, the formation of the 5 alpha-DHT is quantitatively low, and is not affected by the photoperiod. In contrast, androstenedione is formed in quite high yields and the exposure of the animals to 60 days of short photostimulation increases the formation of this steroid in the pituitary gland, but not in the brain structures. From these data, it appears that the central structures of the hamster metabolize testosterone with a pattern which is intermediate between that of birds and mammals.     

Evaluation of pituitary responsiveness to LHRH as a pregnancy test in ewes

Pregnant and nonpregnant ewes were injected with luteinizing hormone-releasing hormone (LHRH). Pituitary responsiveness, based on serum luteinizing hormone (LH), and follicle stimulating hormone (FSH) concentration, 2 hr after injection was then determined for each ewe, by radioimmunoassay (RIA) and correlated with the physiological reproductive state of each ewe. The serum LH release in pregnant ewes was significantly lower than that in nonpregnant ewes. Serum LH concentrations of pregnant ewes were further categorized according to whether the ewes were multiple (ML) or single lambing (SL). The responses by ML ewes were lower for LH than the SL responses. Follicle stimulating hormone responses were not different between pregnant or nonpregnant groups. Luteinizing hormone responses between pregnant ewes which were grouped according to 3 stages of pregnancy (1 to 5, 5 to 10 and 10 to 15 weeks pregnant) were not different from each other. Pregnancy diagnoses were made based on a fixed cut-off value, to which the LH response of each ewe to 5 mug LHRH was compared. Ewes whose response fell below this cut-off were diagnosed as pregnant. Accuracy of the diagnoses were determined by known lambing data. Diagnostic accuracy ranged from a low of 60% for nonpregnant, to a high of 95% for ML ewes. Accuracy for SL ewes (64%) was lower than for the overall pregnant group (79%), as well as that for ML ewes. Doses of LHRH, higher than 5 mug per ewe, generally produced LH release in pregnant ewes which was not significantly suppressed relative to responses of nonpregnant ewes. These results lead to the conclusion that gonadotropin response to exogenous LHRH injection is not an effective tool for pregnancy diagnosis.     

Enkephalins and pituitary hormone release: modification of responsiveness to LHRH.

An intraperitoneal injection of leucine-enkephalin into rats stimulates gonadotropin and prolactin release. To elucidate the mechanism of this releasing property of leucine-enkephalin, rat hemipituitaries were incubated with either enkephalin alone or enkephalin in combination with OHRH. Enkephalin alone had no effect on LH or prolactin release in vitro but potentiated the LH response to LHRH. Neither leucine-enkephalin nor LHRH alone had an effect on GH release; however, when combined, a GH response to LHRH occurred. These results suggest that leucine-enkephalin can modify the pituitary responsiveness to certain hypothalamic releasing hormones by a direct pituitary action.     

Modulation of pituitary responsiveness to LHRH by androgens in ovariectomized female rats.

The effect of androgens on pituitary response to luteinizing-hormore-releasing hormone (LHRH) and their ability to modify effects of 17beta-estradiol (E2) on pituitary responsiveness to LHRH were tested in ovariectomized rats maintained on a daily dose of 0.25 microgram estradiol benzoate per rat for 6 d before androgen administration. Testosterone propionate (TP) (4, 40, 400, or 4000 microgram per rat), administered 24 h before LHRH (500 ng per rat), had no significant effect on luteinizing hormone (LH) or follicle-stimulating hormone (FSH) response. Similar doses of dihydrotestosterone (DHT) did not significantly alter the LH response but significantly suppressed the FSH response. Even the lowest dose completely blocked the FSH response to LHRH. TP in combination with 4 or 400 microgram of E2 suppressed the stimulatory effect of E2 on both LH and FSH response to LHRH in a dose-related manner. DHT and E2 in combination affected LH response inconsistently, whereas their ratio determined FSH response; there was pronounced inhibition of FSH response in rats given high doses of DHT combined with low doses of E2; DHT inhibition of FSH response in animals receiving 4 microgram of DHT with 400 microgram E2 was partially overcome by the stimulatory effect of E2. Our results indicate that TP and DHT affect LH and FSH response to LHRH differently. The ratio of androgen to estrogen is important in determining the response to LHRH.     

Effects of season and testosterone treatment on gonadotrophin secretion and pituitary responsiveness to gonadotrophin-releasing hormone in castrated Romney and Poll Dorset rams.

In castrated rams (Romney and Poll Dorset, n = 8 for each breed), inhibition by testosterone treatment (administered via Silastic capsules) of luteinizing hormone (LH) pulse frequency, basal and mean LH concentrations, mean follicle-stimulating hormone (FSH) concentration, and the peak and total LH responses to exogenous gonadotrophin-releasing hormone (GnRH) were significantly (P less than 0.01) greater during the nonbreeding than during the breeding season. Poll Dorset rams were less sensitive to testosterone treatment than Romney rams. In rams not receiving testosterone treatment, LH pulse frequency was significantly (P less than 0.05) lower during the nonbreeding season than during the breeding season in the Romneys (15.8 +/- 0.9 versus 12.0 +/- 0.4 pulses in 8 h), but not in the Poll Dorsets (13.6 +/- 1.2 versus 12.8 +/- 0.8 pulses in 8 h). It is concluded that, in rams, season influences gonadotrophin secretion through a steroid-independent effect (directly on hypothalamic GnRH secretion) and a steroid-dependent effect (indirectly on the sensitivity of the hypothalamo-pituitary axis to the negative feedback of testosterone). The magnitude of these effects appears to be related to the seasonality of the breed.     

Release of LHRH in vitro and anterior pituitary responsiveness to LHRH in vivo during sexual maturation in pullets (Gallus domesticus)

An in-vitro superfusion technique was used to study basal and depolarization-induced (32 mmol K+/l) release of LHRH from the mediobasal hypothalamus (MBH) of pullets at 8-25 weeks of age. Plasma LH concentrations and the incremental change (delta LH) after an i.v. injection of 1 or 15 micrograms synthetic ovine LHRH/kg body weight were also determined. Between 8 and 25 weeks of age, significant (P less than 0.01) increases in basal and depolarization-induced release of LHRH (93 and 330%, respectively) were accompanied by a significant (P less than 0.01) rise in the residual LHRH content of MBH tissue (152%), observations which suggest that the ability of the hypothalamus to synthesize and secrete LHRH increases as sexual maturation proceeds. However, plasma LH, which reached a maximum concentration of 2.05 +/- 0.43 micrograms/l at 15 weeks, fell significantly (P less than 0.05) to 1.14 +/- 0.05 micrograms/l at 25 weeks. Since delta LH in response to exogenous LHRH showed a marked and progressive decline between 12 and 20 weeks of age, the low plasma concentration of LH typical of the mature hen is probably attributable to a direct negative-feedback action of ovarian steroids on the anterior pituitary gland rather than to an impaired secretion of LHRH from the median eminence. It is suggested that a dramatic increase in the responsiveness of LHRH nerve terminals in the MBH to depolarization by 32 mmol K+/l between 20 and 25 weeks of age (mean age at onset of lay 21.9 weeks; range 19-25 weeks) may reflect the development of hypothalamic responsiveness to the positive feedback action of progesterone.     

Gonadotropin secretion and testicular function in golden hamsters exposed to skeleton photoperiods with ultrashort light pulses

Both sexually mature and sexually regressed male golden hamsters were transferred to asymmetric skeleton photoperiods with night interruptions of varying duration, the short pulses occurring 14 h after "dawn." Testicular function and accompanying changes in follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone and spermatogenesis were observed. Sexually regressed animals exposed to a night-break of 6 seconds (sec) or longer exhibited maximal testicular development with a rapid rise in FSH secretion followed by a slower, more variable rise in LH. Full testicular size was achieved after 8 weeks. Night-breaks of 250 milliseconds (msec) or 1 sec induced testicular development and spermatogenesis but at a slower rate: levels of FSH and LH were still rising at the end of the experiment. Complete testicular maintenance was achieved by night-breaks of 1 sec or longer. Partial testicular regression was observed with a night-break of 250 msec. Night-breaks (60 sec) given less frequently than daily also stimulated testicular function and a night-break every 7 days increased FSH and LH secretion in sexually regressed hamsters, causing testicular development at a submaximal rate. Night-breaks given more frequently induced rapid testicular growth. Almost complete testicular maintenance of sexually mature hamsters was achieved with a 60-sec night-break at weekly intervals. Symmetric skeleton photoperiods also triggered testicular development in sexually regressed hamsters, with two 1-sec light pulses (14 h apart) being almost as effective as a normal long day. No difference in reproductive function was observed between animals on long days (14L:10D) and those exposed to maximally stimulatory skeleton photoperiods.     

Gonadotropin secretion and pituitary responsiveness to GnRH in mares with granulosa-theca cell tumor

Granulosa-theca cell tumors (GTCTs) are able to secrete variable amounts of sex steroids and immunoreactive inhibin (ir-INH). Although the pituitary appears to be affected by the presence of a GTCT, pituitary responsiveness to exogenous GnRH has not been examined. The aims of the present study were to: (i) assess the plasma hormone concentrations of ir-INH, gonadotropins and sex steroids in eight mares with GTCT and (ii) assess the responsiveness of pituitary gonadotroph cells to exogenous GnRH stimulus both before and after tumor removal. In seven mares, the contralateral ovary was firm, small and inactive. Histopathological observations of the tumors confirmed the presumptive diagnosis of a GTCT. Four mares, judged to be in vernal transition period (n=2) and in the breeding season (n=2), were used as controls. A single intravenous injection of 40 microg of GnRH agonist was given to each mare and blood samples were collected every 15 min from 2 h before to 4 h after injection. In four GTCT mares, this procedure was repeated 20 (n=2) and 90 (n=2) days after tumors removal. All plasma samples were analyzed for concentrations of ir-INH, LH, FSH, estradiol-17beta (E2), testosterone (T) by RIA and progesterone (P) by EIA. Results showed that E2 levels were significantly higher (P<0.001) in control animals compared to E2 levels in GTCT mares before and after surgery. P and T concentrations were not statistically different between the groups. Baseline levels of ir-INH were greater (P<0.05) in GTCT mares before surgery than in control mares, and decreased to undetectable levels after neoplasia ablation. Baseline FSH did not differ between control and GTCT animals either before or after the ovaries were removed. LH baseline values appeared to be higher for affected mares, but the difference was not statistically significant. Maximum release (MR) and area under the gonadotrophin release curve (AUC) after the GnRH challenge for both the gonadotrophins were similar between the groups.     

The development of aggressive and marking behavior in intact and castrated male hamsters.

In pairs consisting of two intact or two castrated male hamsters, the level of aggressive and flank-marking behavior increased with age. Dominance relationships were established early and remained stable. Castration prevented the appearance of the flank gland but did not alter the development of aggression, dominance, and marking. After the formation of intact-castrate pairs, the level of performance of the intacts was significantly higher than the castrates only for marking behavior. In a second experiment involving 49 intact-castrate pairs, there were no differences between intacts and castrates for any behavioral variables measured. Although testicular integrity may influence agonistic behavior in certain situations, these data indicate that it must be only one of several influences in determining the occurrence and outcome of an agonistic encounter.     

Specific activity of LHRH and TRH degrading enzymes in various tissues of normal and castrated male rats.

The chlorophyll composition and Hill activity of the leaf, developing seed parts and pod have been studied in three species of legumes, $\text{Lathyrus latifolius}$, $\text{Pisum sativum}$ and $\text{Vicia faba}$. The studies indicate that in all the species, the level of chlorophyll, on mg/g fresh weight basis, is maximum in the leaf. However, Hill activity studies show that the cotyledonary chloroplasts in all the cases have a higher Hill activity than the leaf chloroplasts. Thus, the Hill activities of the cotyledonary chloroplasts are 340% of the leaf chloroplasts in $\text{Lathyrus}$$\text{latifolius}$, 144% of the leaf chloroplasts in $\text{Pisum}$$\text{sativum}$ and 200% of the leaf chloroplasts in $\text{Vicia}$$\text{faba}$.