Dissociation of cyclic AMP accumulation from that of luteinizing hormone (LH) release in response to gonadotropin releasing hormone (GnRH) and cholera enterotoxin.

Anterior hemipituitaries from female rats were incubated $\text{in}\text{vitro}$ in Krebs Ringer bicarbonate buffer, pH 7.2 containing 2 mg/ml of glucose in the absence and in the presence of GnRH or cholera enterotoxin. Following this incubation, the pituitaries were separated from the medium and cAMP and LH were assayed in the tissue and the medium, respectively. Incubations with GnRH in the range of 25 ng/ml to 400 ng/ml resulted in increase in LH release into the medium. Cholera enterotoxin at a concentration of 1 μg/ml, by contrast, caused no release of LH into the medium, but caused a 5-fold increase in cAMP level and this effect was concentration dependent. Cholera enterotoxin did not interfere with the GnRH-mediated LH release. It is concluded from these experiments that the ability of GnRH to increase cAMP level may be independent of its ability to release LH.     

The induction of divergent gonadotropin secretion in vitro by variations in luteinizing hormone releasing hormone pulse regimen

The effects of luteinizing hormone releasing hormone (LHRH) pulse amplitude, duration, and frequency on divergent gonadotropin secretion were examined using superfused anterior pituitary cells from selected stages of the rat estrous cycle. Cells were stimulated with one of five LHRH regimens. With low-amplitude LHRH pulses (regimen 1) in the presence of potentially estrogenic phenol red, LH response in pituitary cells from proestrus 1900, estrus 0800, and diestrus 1,0800 were all significantly larger (P less than 0.05) than the other stages tested. In the absence of phenol red, responsiveness at proestrus 1900 was significantly larger than proestrus 0800, proestrus 1500, and estrus 0800 (P less than 0.01, 0.05, and 0.05, respectively); other cycle stages tested were smaller. No significant differences were observed between cycle stages for follicle-stimulating hormone (FSH) secretion in the presence or absence of phenol red. Because pituitary cells at proestrus 1900 were the most responsive to low-amplitude 4 ng LHRH pulses, they were also used to study the effects of LHRH pulses of increased amplitude or duration and decreased frequency. Increasing the amplitude (regimen 2) or the duration (regimens 3 to 5) increased FSH secretion; this effect was greatest with regimens 3 and 5. When regimens 3 and 5 were studied in pituitary cells obtained at proestrus 1500, FSH was significantly increased by both regimes, but most by regimen 5; furthermore, LH release was significantly reduced. When regimens 3 and 5 were studied in pituitary cells obtained at estrus 0800, FSH release was elevated most significantly by regimen 5. Thus, variations in LHRH pulse regimen were found to be capable of inducing significant divergence in FSH release from superfused anterior pituitary cells derived from specific stages of the estrous cycle.     

The participation of corticosterone in luteinizing hormone releasing hormone (LH-RH) action on luteinizing hormone (LH) release from anterior pituitary cells in vitro

Corticosterone alone was not able to stimulate release of luteinizing hormone (LH) from anterior pituitary cells $\text{in}$$\text{vitro}$, but corticosterone in combination with luteinizing hormone releasing hormone (LHRH) augmented the release of LH into the culture media. These results may indicate that corticosterone may have the capacity to activate membrane receptors for LHRH in the gonadotrophs.     

The effect of luteinizing hormone releasing hormone and anti-luteinizing hormone releasing hormone antibodies on chorionic gonadotropin and progesterone secretion by human placental villiin vitro

Gonadotropin releasing hormone has been located and found to be secreted by the human placenta in culture. Addition of the releasing hormone upto 1μg concentration in the placental cultures brings about stimulation of chorionic gonadotropin and progesterone secretion. Higher amounts of the decapeptide has an inhibitory influence on both the gonadotropin and the steroid production. The action of the releasing hormone on the placenta could be blocked by the anti-luteinizing hormone releasing hormone monoclonal antibodies indicating a possible site of action of the antibodies for control of fertility     

Hypothalamic luteinizing hormone-releasing hormone (LHRH) and LH secretion in aging female rats

Age-related changes in hypothalamic luteinizing hormone-releasing hormone (LHRH) and luteinizing hormone (LH) secretion were studied in young (6 months), middle-aged (12 months) and old (18 months) female rats. The LHRH levels in the mid-hypothalamic area were higher in intact middle-aged and old females than in young ones. Additionally, there was no age difference in the hypothalamic LHRH levels in male rats. In order to clarify the significance of this age-related increase in female rats, we examined the effects of progesterone treatment in estrogen-primed ovariectomized young and old rats on the LHRH levels in the median eminence (ME) and on plasma LH levels. We found phasic changes in ME-LHRH and plasma LH levels in estrogen-primed rats following progesterone treatment in rats of both ages, but the progesterone-induced change in ME-LHRH levels tended to be delayed in old rats compared with young females. This delay may correspond to the delayed onset, slow and low magnitude of plasma LH increase in old females. The ME-LHRH levels were generally higher in old rats than in young rats. Nevertheless, we found that the increase in plasma LH in response to progesterone treatment in estrogen-primed ovariectomized females was smaller in old rats than young rats. These results suggest that the LHRH secretory mechanism changes with age in female rats. Such alterations may result in the accumulation of LHRH in the mid-hypothalamic area and an increase in ME-LHRH.     

Changes in serum luteinizing hormone (LH) concentrations in response to luteinizing hormone releasing hormone (LHRH) in bull calves that attained puberty early or late

The objectives of this study were to determine if the response to luteinizing hormone releasing hormone (LHRH) could be used to select bull calves capable of early sexual maturation and to establish the optimum route and dose of LHRH. In Trial 1, at 4, 10 and 20 week of age, 20 calves were treated iv with 2 microg/kg body weight of LHRH 1 and 5h after commencing a 9-h period of blood sampling. Bulls were separated into early and late maturing (n=10), based on age at puberty (scrotal circumference (SC) of >or=28 cm). At 4 and 20 week of age, peak serum LH concentrations and area under the LH response curve in response to LHRH were lower (P<0.05) in early- versus late-maturing bulls. In Trial 2, calves at 20 week of age were given LHRH as follows: 2 microg/kg body weight iv (n=6), im (n=6) or sc (n=6); 5 microg/kg im (n=6), or ischio-rectally (ir, n=6) or sc (n=6); and 10 microg/kg im (n=6) or sc (n=6). Serum LH concentrations were at a plateau from 30 to 165 min after treatment with 5 microg/kg of LHRH (im or ir; P>0.05). We concluded that the LH responses to LHRH in calves at 4 and 20 week of age could facilitate the development of a simple test (one blood sample prior to treatment with LHRH and a second during the period of sustained response to LHRH) to select early-maturing bulls.     

Gonadotropin-releasing hormone (GnRH) inhibits ovulation induced with luteinizing hormone (LH) in proestrous hypophysectomized rats

In this paper we present evidence that a single low dose of the natural synthetic gonadotropin-releasing hormone (GnRH), inhibits ovulation induced by LH in proestrous-hypophysectomized rats. Rats hypophysectomized by the parapharyngeal route in the morning of proestrus received an intravenous injection of 100 or 300 ng GnRH at 1400 h immediately followed by 1.0 microgram LH per 100 g bw. In control groups, either one or both hormones were replaced with 0.9% NaCl. Ovulation was assessed the following morning by counting the ova present in oviductal flushings. All the rats treated with LH alone ovulated, and the addition of GnRH reduced significantly the number of ovulating rats and the number of ova per ovulating rat. In other groups of rats hypophysectomized in the morning of proestrus and treated in the same way, ovarian or adrenal secretory rates of estradiol and/or progesterone were measured after cannulation of the corresponding vein, in the afternoon of proestrus. In these animals, GnRH failed to inhibit either the ovarian progesterone surge observed 2 h after LH administration, or the adrenal progesterone secretion. All hypophysectomized rats showed lower ovarian secretory rate of estradiol than intact rats; this rate was not affected by treatment with LH or LH plus GnRH. The systemic estradiol levels in plasma of hypophysectomized rats were distributed within a range of 20 pg/ml to 50 pg/ml. The number of rats whose levels were above 21 pg/ml on estrus day was significantly higher in rats receiving 300 ng GnRH as compared to those receiving 100 ng GnRH, reaching values that surpassed the concentration found in intact, untreated animals at the same time of estrus. This effect did not depend on LH administration.     

Comparison of the effect of several gonadotropin releasing hormone antagonists on luteinizing hormone secretion, receptor binding and ovulation

The biological activity of three gonadotropin releasing hormone (GnRH) antagonists was evaluated in the following assays: suppression of GnRH-mediated luteinizing hormone (LH) secretion by cultured pituitary cells, suppression of the spontaneous LH release by ovariectomized rats, blockade of ovulation in regularly cycling females and inhibition of binding of a potent radiolabeled agonist to rat pituitary membrane homogenates. The peptides were: [Ac-delta 3Pro1,4FDPhe2, DTrp3,6]-GnRH (Antagonist 1); [Ac-delta 3Pro1,4FDPhe2,DNAL(2)3,6]-GnRH (Antagonist 2); and [Ac-DNAL(2)2,4FDPhe2,DTrp3,DArg6]-GnRH (Antagonist 3). All three antagonists exhibited similarly high potency in suppressing LH secretion in vitro, while Antagonist 1 was the most active peptide in the radioreceptor assay. When administered by gavage, Antagonist 3 exhibited the highest potency to inhibit LH secretion in gonadectomized rats and to block ovulation. Comparison of the oral versus the subcutaneous mode of administration of these analogs indicates that less than 1% is absorbed after gavage. However, these data demonstrate that the intragastric administration of GnRH antagonists can lower gonadotropin secretion and interfere with reproductive functions.     

Somatostatin affects morphology and secretion of pituitary luteinizing hormone (LH) cells in male rats

The somatostatin peptides (SRIH-14, SRIH-28) and their multiple receptors are generally associated with anti-proliferative and anti-secretory actions. This study compared, using standard morphometric measurements and terminal serum LH concentrations, effects of intracerebroventricular (icv) SRIH-14 and SRIH-28 in nanomolar amounts on immunohistochemically identified LH cells in pituitary glands of male rats. Rats received l microg/5 microl of SRIH-14 or SRIH-28 icv on days 1,3, and 5, whereas control rats received only icv saline. Animals were killed 5 days later for serum LH assays. Pituitarys were harvested for PAP immunohistochemistry and morphometry. Morphometric measurements were made by an observer blinded to the treatment group. Histochemically identified LH cells from both SRIH groups appeared smaller, often pycnotic and darkly stained compared to those from saline-treated rats. Both SRIH treatments reduced (p < 0.05) the quantitative morphometric measurements for cell volume, nuclear volume, and relative volume density. Both SRIH treatments also reduced serum LH concentration (p < 0.05), supporting the hypothesis that systemic physiology was altered. Collectively, the data support the opinion that nanomolar amounts of either SRIH peptide, acting on receptors reached from cerebrospinal fluid, exert an anti-secretory effect on LH cells of male rats. Modifications of central SRIH receptors may provide an approach for treatment of male sexual dysfunction and/or be of pathophysiologic significance in these disturbances.     

Conformation of gonadotropin releasing hormone.

The conformation of the gonadotropin releasing hormone (Gn-RH), whose primary sequence is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2, and of several of its structural analogues has been studied by circular dichroism, optical rotatory dispersion, and fluorescence spectroscopy. The effects of pH, guanidine, and temperature on fluorescence emission have also been examined. Titration data demonstrate that the histidine and tyrosine residues are free of any mutual interactions. The similarity of emission spectra in water and in guanidine hydrochloride solutions precludes significant interactions between the fluorescent groups and other residues. Neither the temperature nor the pH profiles of the emission intensities of either tyrosine or tryptophan reveal any fixed secondary structure in Gn-RH. Both the extent of alkaline quenching and the distance of 10-11 A calculated from F?rster energy transfer theory are in accord with a randomly coiled structure with only one residue between tyrosine and tryptophan. Furthermore, the circular dichroism spectrum and optical rotatory dispersion do not exhibit any contributions from peptide bonds in an ordered structure, although there is a perturbation of the peptide absorption region due to overlapping bands from side-chain chromophores. Gn-RH, therefore, appears to behave as a random coil polypeptide in water devoid of any intrachain residue interactions. This nonordered structure in Gn-RH and the lack of any significant differences in the physical-chemical properties of the hormone analogues indicate that a predetermined solution conformation is not required for biological activity. In contrast to its behavior in water, Gn-RH in trifluoroethanol exhibits a conformational transition, with the formation of a beta structure. Differences in conformational changes exhibited by several analogues in trifluoroethanol may be relevant to their relative biological activities at the receptor site.     

Independent actions of gonadotropin releasing hormone upon cyclic GMP production and luteinizing hormone release

Gonadotropin releasing hormone (GnRH) and its potent analog [D-Ser(tBu)6]des-Gly10-GnRH N-ethylamide elevate pituitary cyclic GMP levels while stimulating gonadotropin release in cultured pituitary cells. Addition of mycophenolic acid to pituitary cell cultures decreased basal and GnRH-induced cGMP production to undetectable levels, but did not reduce basal or GnRH-stimulated luteinizing hormone (LH) release. Elevation of endogenous cGMP levels by sodium nitroprusside, or addition of cGMP or its potent derivatives, was also without effect on basal or GnRH-stimulated LH release. These findings demonstrate that the elevation of intracellular cGMP during GnRH action does not mediate the release of LH by pituitary cells.