Effects of in vivo administration of human chorionic gonadotropin and ethanol on the processes of testicular receptor depletion and replenishment.

The effects of hCG (CR119) and ethanol (20% in saline, v/v) on the levels of gonadotropin receptor in mature rat testes are studied. The results indicate that 75 I.U. of hCG administered intraperitoneally is capable of depleting testicular tissue of all its gonadotropin binding sites within 24 hrs. after administration, and 11–14 days are required for these binding sites to be fully restored. The disappearance and reappearance patterns of testicular gonadotropin binding sites following $\text{in vivo}$ ethanol administration are very similar to those seen after gonadotropin administration, the only difference being that ethanol is much less immediate in its effect. The level of hLH in the serum of these rats is unaffected by the ethanol administered which seems to indicate that reduction of the number of gonadotropin binding sites in testes brought about by ethanol is through membrane-mediated mechanism. For rats receiving gonadotropin injections, no correlation sseems to exist between the concentration of gonadotropin in serum and the level of detectable gonadotropin binding sites in the testis.     

Effect of super-ovulatory doses of pregnant mare serum gonadotropin and human chorionic gonadotropin in blastocyst implantation in golden hamsters

The effect of super-ovulatory dose of pregnant mare serum gonadotropin and human chorionic gonadotropin on ovulation, advancement of ovulation, subsequent embryo development and implantation were studied in the hamster. Groups of hamsters received pregnant mare serum gonadotropin injection on day 1 of the estrous cycle followed by human chorionic gonadotropin injection either at 56 or 76 h later, pregnant mare serum gonadotropin alone on day 1 or human chorionic gonadotropin alone on day 3.The combination therapy (pregnant mare serum gonadotropin and human chorionic gonadotropin) resulted in super-ovulation (an average of 40 mature ova/animal) while human chorionic gonadotropin alone yielded an average of 10 mature ova/animal. Ovulation was advanced by 24 h by giving human chorionic gonadotropin at 56 h instead of 76 h after pregnant mare serum gonadotropin. Subsequent embryo development and implantation occurring under different hormonal regimens were studied. The ova obtained by giving human chorionic gonadotropin injection at 56 h were poorly fertilizablein vivo and hence the pregnancy rate was low (6 %). These ova however, were fertilizablein vitro, suggesting that the low fertilization rate and developmental failure may be due to inhibition of sperm capacitation/transport because of premature human chorionic gonadotropin administration. In the group receiving human chorionic gonadotropin alone on day 3 there was fertilization and cleavage, but no implantation occurred due to failure of functional corpora lutea. However, administration of progesterone and estrone from day 2 of gestation resulted in 80% implantation and sustenance of pregnancy. On the other hand, the pregnant mare serum gonadotropin and human chorionic gonadotropin combination therapy resulted in super-pregnancy. The number of fetuses present at term was higher in the group receiving pregnant mare serum gonadotropin alone than in the group receiving the combination therapy. Embryo resorption however was higher (37%) in the latter group compared with the former (9.5%). However, preimplantation embryos were found to be viable as evidenced by fluorescein diacetate staining.     

Physicochemical and biological characterizations of pregnant mare serum gonadotropin and its subunits.

Pregnant mare serum gonadotropin and its subunits have been further characterized. Ultracentrifugation of the gonadotropin at pH 1.3 and 11.5 showed little evidence of dissociation compared to pH 8.2. Highly purified subunits are obtained by urea dissociation and ion-exchange chromatography followed by gel-filtration. Circular dichroism spectra of the gonadotropin and its subunits are much like those of ovine lutropin and its subunits in that there is little evidence for secondary structure and one or more tyrosine residues are inaccessible in the intact gonadotropin compared to the subunits. The alpha-subunit possesses almost 3 times as much total carbohydrate as the beta-subunit; the individual sugar composition of each was determined as well as the amino acid composition. The alpha-subunit begins with the sequence NH2-Phe-Pro (Gly or Pro) ... and terminates with isoleucine. The beta-subunit has the sequence NH2-Ser-Pro-Gly ...; no C-terminal residue is detectable by either carboxypeptidase or hydrazinolysis. Biological studies show the gonadotropin to be active in assays specific for both lutropin and follitropin. Precipitin test in agar with rabbit antiserum against the gonadotropin show that the beta subunit cross-reacts whereas the alpha subunit does not.     

Different processing of LH/hCG receptors in cultured rat luteal cells and murine Leydig tumour cells (MLTC-1)

The metabolic fate of LH/hCG receptors after exposure to human chorionic gonadotropin (hCG) was examined in cultured rat luteal cells and murine Leydig tumour cells (MLTC-1). Kinetic studies performed after pulse-labelling of the cells with [125I]hCG indicated that the bound hormone was lost much more rapidly from the tumour cells than from the luteal cells (t1/2 = 4.5 and greater than 12 h, respectively). The tumour cells were also found to internalise and degrade the hormone more effectively than the luteal cells, as measured by disappearance of acid-releasable (i.e. surface-bound) radioactivity from the cells and by the appearance of trichloroacetic acid (TCA)-soluble label in the medium. In MLTC-1 cells, over 80% of the radioactivity released was TCA-soluble at all times examined, whereas in the luteal cells most (65-75%) was TCA-precipitable. Chemical cross-linking and analyses by SDS-PAGE of this material revealed that both cell types also released, in addition to intact hCG, two previously characterized receptor fragment-[125I]hCG complexes (Mr 96,000 and 74,000) (Kellokumpu & Rajaniemi, Endocrinology 116 (1985) 707) into the medium, although their amount was negligible in MLTC-1 cells. Possibly, due to rapid discharge of the ligand from its receptor, no similar complexes could be detected inside the MLTC-1 cells, suggesting that they were released directly from the cell surface. However, the Mr 74,000 complex was observed inside MLTC-1 cells if chloroquine, a lysosomotropic agent, was present during the incubations. This suggests that the internalised receptor also becomes degraded, at least when complexed to hCG. The results thus provide evidence that there exist two different mechanisms for proteolytic processing of LH/hCG receptors in these target cells. In tumour cells, the degradation seems to occur almost exclusively intracellularly, whereas in luteal cells a substantial portion of the receptors is also degraded at the cell surface.     

A regulator of G Protein signaling, RGS3, inhibits gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone (LH) secretion

Background  

Luteinizing hormone secreted by the anterior pituitary gland regulates gonadal function. Luteinizing hormone secretion is regulated both by alterations in gonadotrope responsiveness to hypothalamic gonadotropin releasing hormone and by alterations in gonadotropin releasing hormone secretion. The mechanisms that determine gonadotrope responsiveness are unknown but may involve regulators of G protein signaling (RGSs). These proteins act by antagonizing or abbreviating interaction of Gα proteins with effectors such as phospholipase Cβ. Previously, we reported that gonadotropin releasing hormone-stimulated second messenger inositol trisphosphate production was inhibited when RGS3 and gonadotropin releasing hormone receptor cDNAs were co-transfected into the COS cell line. Here, we present evidence for RGS3 inhibition of gonadotropin releasing hormone-induced luteinizing hormone secretion from cultured rat pituitary cells.     

Specific binding of 125I-labeled human chorionic gonadotropin to gonadal tissue: comparison of limited-point saturation analyses to Scatchard analyses for determining binding capacities and factors affecting estimates of binding capacity

Experiments were conducted to compare gonadotropin binding capacity calculated from limited-point saturation analyses to those obtained from Scatchard analyses, and to test the effects of membrane purity and source of gonadotropin receptors on determining the maximum percentage of radioiodinated hormone bound to receptors (maximum bindability). One- to four-point saturation analyses gave results comparable to results by Scatchard analyses when examining relative binding capacities of receptors. Crude testicular homogenates had lower estimates of maximum bindability of 125I-labeled human chorionic gonadotropin than more purified gonadotropin receptor preparations. Under similar preparation techniques, some gonadotropin receptor sources exhibited low maximum bindability.     

Recognition of the beta-subunit of human chorionic gonadotropin and sub-determinants by target tissue receptors.

The beta-subunit of human chorionic gonadotropin, purified immunochemically to eliminate undissociated human chorionic gonadotropin, induced testosterone production by mouse Leydig cells at concentrations 400-fold higher than human chorionic gonadotropin. Steroidogenesis was also stimulated by a synthetic fragment of the beta-subunit of human chorionic gonadotropin conforming to the peptide sequence residues 39--71, whereas peptide sequence residues 39--56 and three C-terminal fragments (residues 115--145, 111--145 and 101--145) failed to cause steroidogenesis. These studies suggest the presence in the beta-subunit of human chorionic gonadotropin of determinants recognized by the tissue receptors, a part of these determinants residing between amino acid residues 57--71.     

Stimulation of the formation of 13,14-dihydroprostaglandin F2 alpha by gonadotropin in rat ovary

Effects of pregnant mare serum gonadotropin and human chorionic gonadotropin on the formation of 13,14-dihydroprostaglandin F2 alpha, a biologically active compound, were investigated in rat ovarian homogenate. The mass number of the compound, which was formed prostaglandin F2 alpha via 13,14-dihydro-15-ketoprostaglandin F2 alpha in rat ovarian homogenate but was not produced in rat homogenate, accorded with that of the authentic 13,14-dihydroprostaglandin F2 alpha by negative ion chemical ionization mass spectrometry. In the present experiment, the radioactivity of [3H]prostaglandin F2 alpha added to ovarian homogenate was decreased linearly and immediately until the incubation time of 10 min. The formation of 13,14-dihydroprostaglandin F2 alpha was increased up to 60 min. The formation of 13,14-dihydroprostaglandin F2 alpha from prostaglandin F2 alpha was markedly increased by pregnant mare serum gonadotropin and human chorionic gonadotropin. However, there was no additive or synergistic effect of these hormones. The formation of 13,14-dihydroprostaglandin F2 alpha from 13,14-dihydro-15-ketoprostaglandin F2 alpha weas also greatly stimulated by pregnant mare serum gonadotropin and human chorionic gonadotropin. The formation of 13,14-dihydro-15-ketoprostaglandin F2 alpha steeply declined until 24 h after treatment with human chorionic gonadotropin in pregnant mare serum gonadotropin-primed rats. In contrast, the formation of 13,14-dihydroprostaglandin F2 alpha was markedly increased until 24 h after human chorionic gonadotropin treatment, and the level was about 2.5-fold higher than that at 0 h, 48 h after injection of pregnant mare serum gonadotropin.     

Observations on Immunochemical Assays of Human Chorionic Gonadotropin

Commercial immunochemical tests, “UCG” and “Prepuerin”, detected standardized human chorionic gonadotropin (HCG) in concentrations comparable to the rat ovarian hyperemia test. These in vitro systems also cross-reacted with pituitary luteinizing hormone (LH) but not with follicle-stimulating hormone (FSH) of animal origin. The latex agglutination slide tests required a higher concentration of HCG for a positive test than did the bioassay. In cases of abnormal pregnancies the immunochemical assays remained positive longer than the bioassay, which is apparently due to their detection of biologically inactive HCG. Several non-pregnant patients with ovarian cysts and cystic teratomas excreted in their urine some substances which reacted with the UCG test. After surgical removal of the tumour these substances disappeared from the urine.     

Modification of sialyl residues of gonadotropic hormones by reductive amination. Influence on biological activity and circulating half-life

The sialic acid residues of human chorionic gonadotropin, human lutropin and human follitropin were quantitatively modified by introduction of an amino compound. In radioreceptor assays, the modified chorionic gonadotropin, lutropin and follitropin saturated the receptors. However, in the low nanogram range, the gonadotropic binding was higher for the control compared to the modified sample.The hormonal activity of the chorionic gonadotropin was testedin vitro. The modified preparations were four- to thirteen-fold less stimulatory compared to the control but elicited the same maximal response. The biological activity of follitropin was determinedin vivo. In this case, the modified preparations were four- to five-fold less stimulatory than the control. Both the modified chorionic gonadotropin and follitropin preparations were found to act as agonists. Modification of the gonadotropin hormones did not significantly alter the immune recognition of these glycoproteins.The apparent circulating half-life in rats of the modified chorionic gonadotropin and follitropin was increased six- to nine-fold compared to that of native hormones; this might be a consequence of resistance of the modified sialyl residues to sialidases and the resultant slower exposure of terminal galactosyl residues; the plasma half-life of modified lutropin remained the same as that of the native hormone.Abbreviations hCG human chorionic gonadotropin - hLH human lutropin or luteinizing hormone - hFSF human follitropin or follicle stimulating hormone - mala methyl ester of alanine - hCG(ala, mala, etc.) human chorionic gonadotropin modified on sialicacid by reductive amination with alanine, methyl ester of alanine, etc. - IRP-HMG intact rat prostrate-human menopausal gonadotropin     

Regulation of Oxidative Activity of Neutrophils by Chorionic Gonadotropin: The Role of Female Steroid Sex Hormones

We studied the effect of the main sex hormone, chorionic gonadotropin, on the production of the active forms of oxygen and nitrogen by human neutrophils and the control of its effects by female steroid sex hormones. Gonadotropin proved to inhibit both total production of oxygen metabolites and NO synthesis by the cells. Gonadotropin-dependent regulation of oxidative activity of the neutrophils is controlled by female steroid sex hormones. At the same time, progesterone completely or partially inhibits the suppressive effects of gonadotropin, while estradiol has antagonistic, sensibilizing, or permissive effect on gonadotropin depending on the dose, incubation conditions, and activation status of the cells.     

Role of endogenous opiates in the modulation of gonadotropin secretion in infant monkeys (Macaca mulatta): effects of naltrexone

A potential inhibitory role of endogenous opioids in the gonadotropin decline from infancy to the prepubertal period in primates was assessed by examining the effect of the specific opioid antagonist naltrexone on gonadotropin levels in infant rhesus monkeys. Paradoxically, both chronic administration of naltrexone to neonatally castrate males as well as acute administration of graded doses to intact infant females resulted in gonadotropin suppression compared to appropriate vehicle-treated controls. Thus, naltrexone behaves as a gonadotropin secretory antagonist in infant monkeys and cannot be used to unmask a putative inhibitory mechanism involving endogenous opiates.     

Tissue specific compartmental analysis of gonadotropin stimulation of ovarian ornithine decarboxylase

Luteinizing hormone is known to stimulate the enzyme ornithine decarboxylase in the ovary. Highly purified human follicle stimulating hormone that is devoid of significant biologically active luteinizing hormone can also induce ornithine decarboxylase activity in intact immature rats with a time course of induction similar to that reported for luteinizing hormone. A maximum of 8–10-fold stimulation above controls was observed 4 h following intravenous administration of human follicle stimulating hormone. This stimulation followed a strict dose response relationship. Ovine luteinizing hormone and human chorionic gonadotropin always induced more ovarian ornithine decarboxylase activity than that achieved by maximally effective doses of follicle stimulating hormone. This could not be attributed solely to the ability of specific cell population to respond to the respective gonadotropins. Although granulosa cells contained little receptor for luteinizing hormone/human chorionic gonadotropin and the residual tissue contained little receptor for follicle stimulating hormone, each tissue responded to these gonadotropins in a manner suggestive of the mediation by one or more diffusable factors. A relationship between gonadotropin induced 3’5’-cyclic adenosine monophosphate (cyclic adenosine monophosphate) concentration and ornithine decarboxylase activity suggests that the mediation of gonadotropin stimulated ovarian ornithine decarboxylase is not solely through cyclic adenosine monophosphate, indicating the presence of other factors in the induction of gonadotropin increased ornithine decarboxylase activity.     

Dependence of secretory responses to gonadotropin-releasing hormone on diacylglycerol metabolism. Studies with a diacylglycerol lipase inhibitor, RHC 80267

The role of diacylglycerol (DG) as a source of arachidonic acid during gonadotropin-releasing hormone (GnRH) stimulation of gonadotropin secretion was analyzed in primary cultures of rat anterior pituitary cells. An inhibitor of DG lipase (RHC 80267, RHC) caused dose-dependent blockade of GnRH-stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. The DG lipase inhibitor did not alter gonadotropin responses to arachidonic acid, and addition of arachidonic acid reversed its inhibition of GnRH-stimulated LH and FSH release. In [3H]arachidonic acid-prelabeled cells, incubation with RHC increased the accumulation of [3H]DG. These results suggest that DG lipase participates in GnRH action and that arachidonic acid mobilization from DG is involved in the mechanism of gonadotropin release. Gonadotropin responses to tetradecanoyl phorbol acetate and dioctanoyl glycerol were not altered by RHC, and the addition of these activators of protein kinase C (Ca2+- and phospholipid-dependent enzyme) did not prevent the inhibition of GnRH-induced gonadotropin release by RHC. Activation of phospholipase A2 by melittin increased LH and FSH secretion, whereas blockade of this enzyme by quinacrine reduced GnRH-stimulated hormone release. However, RHC did not diminish the gonadotropin response to melittin. The inhibitory actions of RHC and quinacrine were additive and were reversed by concomitant treatment with arachidonic acid. Ionomycin also increased LH and FSH release, and the gonadotropin responses to the ionophore were unaltered by RHC but were reduced by quinacrine. Incubation of cells in Ca2+-depleted (+/- [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) medium reduced but did not abolish the LH and FSH releasing activity of GnRH. Treatment with RHC also reduced the gonadotropin responses to GnRH under Ca2+-depleted conditions. These observations indicate that RHC inhibition of GnRH action is not due to nonspecific actions on Ca2+ entry, protein kinase C activation and actions, nor phospholipase A2 enzyme activity. The results of this study provide further evidence for an extracellular Ca2+-independent mechanism of GnRH action, and suggest that GnRH causes mobilization of arachidonic acid by two distinct lipases, namely, phospholipase A2 and DG lipase, during stimulation of gonadotropin secretion.     

Regulation of gonadotropin secretion in the anterior pituitary

Studies on the regulation of gonadotropin secretion in dissociated pituitary cell cultures are described. Initial studies employing a ferritin-labelled analogue of gonadotropin hormone releasing hormone (GnRH) to localize its receptor sites on the gonadotropin cell surface that while these receptor sites initially have a random monodisperse distribution, binding of the ligand causes coarse aggregation and internalization of the GnRH receptor. These events are not due to the multivalency of the ligand and probably reflect redistributive events in vivo. By using an octapeptide analogue GnRH that binds to the GnRH receptor but lacks gonadotropin releasing activity in conjunction with sequence-specific antisera it is shown that antibodies that bind the octapeptide can induce the octapeptide to release gonadotropin. These data suggest that receptor aggregation is important in GnRH stimulation. Finally immunocytochemical studies are described in which golg-protein-A-antibody complexes are used to identify gonadotropins on ultrathin frozen sections of porcine pituitary cells. These studies indicate that in porcine gonadotropin cells the majority of the secretory granules contain both luteinizing hormone and follicle-stimulating hormone.     

Pike eel (Muraenesox cinereus) gonadotropin. Amino acid sequences of both alpha and beta subunits

The amino acid sequences of pike eel gonadotropin alpha and beta subunits have been determined by standard sequencing analytical methods. The alpha subunit is composed of 93 amino acid residues while the beta subunit comprises 113 amino acid residues. All the invariant half-cystine residues are in the same positions as those found in other gonadotropins. It is noteworthy that the first, putative glycosylation site (Asn56) found in the alpha subunit of other gonadotropins was replaced by Asp56 in the alpha subunit of pike eel gonadotropin. Similarity analyses indicate that both subunits are structurally more similar to other known fish gonadotropin subunits than to those of the mammalian gonadotropins.     

Effects of pinealectomy on pituitary gonadotrophs, pituitary gonadotropin potency and hypothalamic gonadotropin releasing activity in Notemigonus crysoleucas

The effects of pinealectomy on pituitary gonadotrophs, pituitary gonadotropin potency and hypothalamic gonadotropin releasing activity were examined in the cyprinid teleost, Notemigonus crysoleucas, exposed to various photoperiod-temperature regimes. In fish exposed to a long photoperiod-warm temperature regime, pinealectomy resulted in a decrease in gonadal activity, in hypothalamic gonadotropin releasing activity and an increase in pituitary gonadotropin potency. Fewer gonadotrophs were present in the pituitary of sham operated fish than in the pituitary of pinealectomized fish. Ovarian development was more rapid in sham operated than in pinealectomized fish exposed to a long photoperiod–low temperature regime. Pituitary gonadotropin activity was also greater in shams than in pinealectomized fish. A short photoperiod-warm temperature regime retarded ovarian development in N. crysoleucas. Pinealectomy reversed this trend. Gonadotrophs made up a greater area of the pituitary in pinealectomized fish than in shams under these conditions. Gonadotropin potency of the pituitary and hypothalamic gonadotropin releasing activity were also greater in pinealectomized fish than in shams. The area of the pituitary occupied by gonadotrophs was greater in pinealectomized than in sham operated animals maintained on a short photoperiod-low temperature regime. Pituitary gonadotropin activity was also greater in pinealectomized fish as compared to shams. Pituitary gonadotropin potency varies diurnally in animals maintained on both short and long photoperiods; the rhythm of variation differs depending on photoperiod. Pinealectomy alters the diurnal rhythm of pituitary gonadotropin potency in animals exposed to both long and short photoperiods. It is concluded that pinealectomy has a pronounced effect on reproductive activity in N. crysoleucas. The effects of pinealectomy on reproduction vary with photoperiod, but are mediated via the hypothalamus and pituitary. In fish exposed to long daylengths the pineal favours reproductive activity, but the epiphysis retards reproductive processes in animals maintained on short photoperiods.     

Evaluation of in vitro incubation systems for the study of gonadotropin release.

A detailed study was undertaken in order to determine if a pituitary-half incubation system were a suitable model for the study of anterior pituitary response to estradiol and LHRH. Considerable variation in the gonadotropin content of randomized pituitary halves was observed. Much less variation was found in matched halves. During the initial thirty minutes incubation of pituitary halves, a large spontaneous release of gonadotropins was observed. Time course secretion studies indicated that by four hours incubation, in the presence of 50 ng/ml LHRH, cumulative secretion of LH and FSH had far exceeded that of controls. Elevations in both cumulative secretion and rate of secretion were evident within 15-30 minutes of incubation. Regardless of LHRH dose, only 2-4% of either gonadotropin was secreted. Estradiol in the range of 10, 100, 500, 1,000 and 50,000 pg/ml had no significant effect on pituitary response to LHRH or on basal release, tissue levels or total gonadotropin. Based on these results, it was concluded that while the pituitary-half incubation system may be suitable for studying LHRH induced gonadotropin secretion, it is apparently of insufficient sensitivity to allow the collection of meaningful data concerning the effects of estradiol alone on gonadotropin secretion or estradiol modulation of LHRH induced gonadotropin secretion.