Effect of ethanol on follicle stimulating hormone-induced steroidogenic acute regulatory protein (StAR) in cultured rat granulosa cells.

Steroidogenic acute regulatory protein (StAR) plays a critical role in trophic hormone-stimulated steroid biosynthesis by facilitating the transfer of cholesterol across the mitochondrial membrane, where the cytochrome P450scc enzyme resides to initiate steroid hormone biosynthesis. Because follicle stimulating hormone (FSH) is a critically important regulator of estradiol (E2) synthesis in granulosa cells and because ethanol is known to suppress gonadotropin-stimulated ovarian steroidogenesis, we evaluated the effects of ethanol on FSH-stimulated StAR in ovarian granulosa cells. Granulosa cells from immature rats pretreated with pregnant mare serum gonadotropin were cultured for 24 h in serum-free medium, either alone (medium only) or with FSH (25 ng/ml) in the presence or absence of ethanol (50 mM). Real-time polymerase chain reaction (PCR) analysis showed increased (p < 0.01) expression of the StAR transcript in FSH-treated cells, when compared with cells that received medium only. The FSH stimulation of StAR transcript was blocked (p < 0.01) by the presence of ethanol. This effect coincided with a decrease in E2 secretion into the culture medium. We also examined whether ethanol could affect the production of cyclic AMP (cAMP), the main second messenger that mediates gonadotropin action within the ovary. FSH treatment of granulosa cells markedly increased (p < 0.001) cAMP levels, an effect that was not altered by ethanol. Importantly, FSH induced an increase (p < 0.01) in the release of prostaglandin E2 (PGE2), an effect that was blocked by ethanol. Real-time PCR analysis showed that ethanol had no effect on the expression of cyclooxygenase-1 (COX-1), but blocked (p < 0.01) FSH-stimulated expression of COX-2. These results demonstrate that ethanol is capable of inhibiting FSH-induced ovarian StAR and thus, contributing to suppressed E2 secretion, at least in part, through an inhibitory action on the COX-2-PGE2 pathway.

Effects of chronic systemic administration of opioid peptides, naloxone and N-acetyl beta-endorphin antiserum on gonadotropins and testicular functions in the rat.

Systemic administration of opioid peptides, methionine-enkephalin and beta-endorphin, chronically, lowered gonadotropin levels in plasma and had an inhibitory effect mainly on the testicular enzymes hyaluronidase, acid phosphatase and on incorporation of 3[H] thymidine in the tissue. When rats were similarly treated with opioid peptide antagonist naloxone and N-acetyl beta-endorphin antiserum, induced an opposite effect. This is either the direct effect of opioid peptides/antagonist on the gonads or it may be via the circulating levels of gonadotropin.

Studies on the effect of intratesticular administration of opioid peptides, naloxone or N-acetyl beta-endorphin antiserum on some testicular parameters in rats.

Opioid peptides have been localized in a variety of peripheral tissues like placenta, thyroid, pancreas, gastrointestinal tract, in the reproductive tract of male and female and in the testes of rats. Immunoassayable material was detected in extracts of gonads, reproductive tract and accessory reproductive organs. Studies with naloxone have suggested that beta-endorphin may have an important role in steroidogenesis and may have a role in regulating transport of luminal material. In our studies met-enkephalin, beta-endorphin, naloxone or N-acetyl beta-endorphin antiserum were microinjected intra testicularly once on alternate days for one week and autopsied 24 h after the last injection. Intratesticular administration of 25, 50 and 100 micrograms doses of naloxone induced significant decrease in in vitro secretion of testosterone per se, which was significantly greater with 50 micrograms dose than with those of the other two doses. A 25 micrograms dose had no effect on hyaluronidase or acid phosphatase activity while 50 micrograms dose significantly decreased the enzyme activity. One hundred micrograms dose also significantly decreased hyaluronidase activity. Intratesticular injection of 10 micrograms met-enkephalin or 1 microgram beta-endorphin significantly decreased hyaluronidase activity whereas 20 microliters N-acetyl beta-endorphin antiserum increased the specific activity of hyaluronidase. There was no change in the weight of the testes on treatment with the above agents.

Testicular lactate dehydrogenase and sorbitol dehydrogenase activity after intratesticular injection of dynorphin and morphine in male rats.

Testicular lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH) activity were measured at 1 and 4 hr following intratesticular injection of morphine and dynorphin. Twenty five and 50 micrograms doses of morphine sulfate significantly reduced LDH activity at 1 hr after injection. Five and 25 micrograms doses of dynorphin reduced LDH activity both at 1 and 4 hr after treatment. Testicular SDH activity was increased by morphine at 1 hr followed by a decrease at 4 hr. Both doses of dynorphin significantly reduced SDH activity at 1 and 4 hr after treatment. These results indicate paracrine regulatory role for opioids in testicular metabolism.

Physiological significance of neurotensin in pituitary glycoprotein hormone release as revealed by in vivo and in vitro studies with neurotensin antiserum.

The neuropeptide, neurotensin, is localized to neurons within the hypothalamus which project to the median eminence. It is released from the terminals in the median eminence into the hypophyseal portal vessels and is carried to the gland. The content in the pituitary gland cells may be partly related to the delivery of the peptide via the portal vessels, but it also appears to be produced directly in pituitary cells. The peptide has actions on the release of glycoprotein hormones from the pituitary and in the present experiments, we attempted to determine whether these actions of the peptide were physiologically significant by microinjecting purified antiserum directed against neurotensin into the third cerebral ventricle or intravenously into conscious freely moving rats. Blood samples were withdrawn from an indwelling intra-right atrial catheter. In ovariectomized rats with high levels of plasma gonadotropins because of removal of ovarian steroid negative feedback, the intraventricular injection of the higher (3 microliters) dose of neurotensin antiserum (NT-AS) induced a more than 2-fold increase in plasma LH within 2 h which was maintained until 3 h after the injection and returned to basal values in the 4th and 5th hour. The lower 1-microliter dose was ineffective and there was no response to the control normal rabbit serum (NRS) injections into the third ventricle in this and the other experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin suppression during pre and post-implantation periods on rat uterine glucosamine synthase activity.

Administration of bromocriptine (Bc), an ergot derivative having dopamine receptor agonist activity, to rats on day 1-5 of pregnancy prevented implantation of blastocysts and significantly suppressed uterine glucosamine 6-phosphate synthase activity. There was no effect on implantation or the enzyme activity when Bc was injected on day 7 or later of pregnancy. Injection of prolactin following Bc partially restored the enzyme activity and increased number of implantation sites. These results indicate that suppression of prolactin on day 1 to 5 of pregnancy causes failure of implantation. Bc on day 9 or later had no effect possibly due to the availability of placental LH/hCG to support the luteal cells.

Acute and short-term effects of intraventricular injection of somatostatin and LHRH on glutamate and GABA levels in rat brain.

Glutamate and GABA content in different regions of adult female rat brain were determined at 10 and 30 min following intraventricular injection of LHRH or somatostatin. Cerebral cortex, cerebellar and hypothalamic glutamate levels were significantly elevated at 30 min following injection of 1 micrograms somatostatin, whereas hypothalamic glutamate levels were elevated at 10 min following a 0.5 micrograms dose. LHRH at a dose of 1 micrograms elevated cerebellar and brain stem glutamate levels at 10 and 30 min, whereas a 0.5 micrograms dose significantly elevated cerebral cortex, cerebellar and hypothalamic glutamate levels at 30 min. Third ventricular injection of 1 micrograms somatostatin produced a significant decrease in hypothalamic GABA levels at 10 and 30 min, whereas a 0.5-microgram dose decreased brain stem GABA levels at 10 min. LHRH at a dose of 0.1 microgram significantly increased cerebral cortex and cerebellar GABA levels at 10 min and brain stem GABA levels at 10 and 30 min following injection. Intraventricular injection of LHRH at a dose of 0.5 microgram significantly elevated cerebral cortex, cerebellar and brain stem GABA levels at 30 min. Hypothalamic GABA levels were elevated at 10 and 30 min following 0.5 and 1 microgram intraventricular LHRH injection. The implications of these results are discussed in relation to probable interaction between these neuroactive amino acids and neuropeptides in the rat brain.

Anti-implantation role for substance P and neurotensin in rat.

Substance P (SP) and neurotensin (NT), two structurally related peptides with contrasting biological actions, have been shown to have some role in peripheral reproductive processes. Intrauterine microinjection of SP or NT on day 4 or 5 of pregnancy in the rat significantly reduced the number of viable fetuses, weight and glycogen content of the uterus. The number of viable fetuses, uterine weight or glycogen content were not modified when SP/NT was microinjected on day 8, 9, 10 or on day 14, 15 and 16. The results indicate that the peptides possibly exert a direct local alteration in uterine vascular permeability causing failure in implantation.

Control of anterior pituitary hormone secretion by neurotensin.

Neurotensin is localized in discrete populations of neuronal cell bodies with terminals in the hypothalamus and median eminence. High-affinity binding sites for neurotensin have been demonstrated not only in the hypothalamus but also in the pituitary gland. These studies suggest a role for neurotensin in control of hypothalamic-pituitary function. We initially demonstrated that neurotensin could block the release of prolactin in conscious, ovariectomized and male rats after its injection into the third ventricle, whereas intravenous injection of the peptide significantly elevated plasma prolactin and increased prolactin release by pituitaries incubated in vitro. These results suggested that neurotensin had opposite actions on prolactin release, an inhibitory effect at a hypothalamic site and an excitatory one at the pituitary. Further studies employing dopamine receptor blockers and inhibitors of catecholamine synthesis indicated that the action of the peptide to block prolactin release was probably mediated by release of dopamine, which then inhibited prolactin release by the pituitary gland directly. We have evaluated the physiological significance of the peptide in control of prolactin release by intraventricular injection of highly specific antiserum against neurotensin. The antiserum evoked dose-related elevations in plasma prolactin in intact males that were significant but smaller in magnitude than those seen in females, actions opposite to those of the peptide itself, which indicates that the inhibitory action of the peptide within the brain is physiologically significant. Intravenous injection of this antiserum produced a significant suppression of plasma prolactin in females but not males, which indicates that the previously demonstrated stimulatory effect of the peptide on prolactin release by the gland is also physiologically significant because immunoneutralization of the peptide resulted in a decline in plasma prolactin. Our earlier experiments revealed that neurotensin had a dose-related ability to inhibit LH release in ovariectomized and ovariectomized, estrogen progesterone-treated rats. Since it had no effect on the release of LH in vitro, we assigned a hypothalamic site for this action. It appears that this inhibitory effect of the peptide to suppress LH release is also physiologically significant since the intraventricular injection of the antiserum against the peptide produced a dose-related stimulation of LH release in ovariectomized and ovariectomized, estrogen progesterone-blocked rats. The mechanism by which endogenous neurotensin inhibits the release of LHRH has yet to be evaluated.(ABSTRACT TRUNCATED AT 400 WORDS)

Glutathione and gamma-glutamyl transpeptidase in the adult female rat brain after intraventricular injection of LHRH and somatostatin.

Glutathione content and glutamyl transpeptidase activity in different regions of adult female rat brain were determined at 10 and 30 min following intraventricular injection of LHRH and somatostatin. Hypothalamic glutathione levels were significantly elevated at 10 and 30 min after a single injection of a 0.1 micrograms dose of LHRH. On the contrary, glutathione levels significantly decreased in the hypothalamus, cerebral cortex and cerebellum at 10 and 30 min after 0.5 or 1 microgram dose. However, significant decrease in brain stem glutathione was evident at 30 min after 0.5 microgram and 10 min after the 1 microgram dose. Somatostatin at doses of 0.5 microgram and 1 microgram significantly decreased glutathione levels in all four brain regions both at 10 and 30 min following injection into the 3rd ventricle. Gamma-glutamyl transpeptidase activity in the hypothalamus and cerebral cortex was significantly elevated after intraventricular injection of LHRH. However, a significant increase in gamma-glutamyl transpeptidase activity in cerebellum and brain stem was seen only with 0.5 and 1 micrograms doses of LHRH. Somatostatin also significantly increased gamma-glutamyl transpeptidase activity in hypothalamus, cerebral cortex, brain stem and cerebellum. The decrease in glutathione levels with corresponding increase in gamma-glutamyl transpeptidase activity after intraventricular administration of LHRH and somatostatin suggests a possible interaction between glutathione and hypothalamic peptides.

Glutathione distribution in rat brain at different ages and the effect of intraventricular glutathione on gonadotropin levels in ovariectomized steroid-primed rats.

Glutathione levels were estimated in different regions of the brain of 21-, 30-, 40-, 42-, 45-day-old and adult female rats. Glutathione content in the cerebral cortex, cerebellum and the brain stem remained almost the same beginning from day 21 to sexually mature adult rats. There is a significant increase in hypothalamic glutathione content reaching a peak at puberty (42 days) and thereafter decreasing to the adult levels. Plasma gonadotropin levels were evaluated at 5 and 15 min after third ventricular injection of 15 and 30 microgram doses of glutathione in ovariectomized steroid-primed rats. Intraventricular injection of either 15 or 30 micrograms dose of glutathione significantly elevated plasma FSH levels. The 15 micrograms dose of glutathione significantly decreased plasma LH levels whereas 30 micrograms dose had no effect. Lower dose of glutathione inhibits LH release and stimulates FSH release whereas the higher dose of glutathione specifically elevates FSH levels without any change in LH levels suggesting a selective FSH releasing action of glutathione.

Use of antiserum to neurotensin reveals a physiological role for the peptide in rat prolactin release.

Previous studies have indicated that the brain peptide neurotensin can stimulate prolactin release by direct action on the pituitary gland, whereas its action within the hypothalamus is inhibitory. The inhibitory action is mediated by the release of dopamine into the hypophyseal portal veins, which deliver the neurotransmitter to the anterior pituitary gland to inhibit prolactin release. Our experiments were done to evaluate the physiologic significance of these neurotensin actions by injecting the globulin fraction of highly specific neurotensin antiserum either intravenously or intraventricularly. Injection into the third ventricle of either 1 or 3 microliter of neurotensin antiserum significantly increased plasma prolactin concentrations in (i) ovariectomized and (ii) ovariectomized estrogen- and progesterone-primed rats within 1 hr of injection. The response was more pronounced in the ovariectomized than in the ovariectomized estrogen- and progesterone-treated animals and was dose related. Intraventricular injection of these doses of neurotensin antiserum also evoked elevations in plasma prolactin in intact males, which were significant but smaller in magnitude than those seen in female rats. To evaluate the effect of the antiserum on the pituitary directly, the antiserum was injected intravenously at a dose of 40 microliter, which was sufficient to block the blood pressure-lowering effect of neurotensin. After the intravenous injection of antiserum, a highly significant suppression of plasma prolactin occurred, detectable when first measured at 1 hr after injection in both ovariectomized and ovariectomized estrogen- and progesterone-treated animals; however, the intravenous injection of antiserum had no significant effect on the prolactin release in males. These data indicate the physiological significance of the hypothalamic inhibitory actions of neurotensin on prolactin release, which are probably mediated by its stimulation of dopamine release that in turn, inhibits prolactin secretion by the lactotropes. The direct stimulatory effect of the peptide on prolactin release after its presumed release into portal vessels also appears to be physiologically significant in female but not in male rats.

A new non-hormonal antifertility drug DL-204: II. Effect on testicular hyaluronidase and gamma-glutamyl transpeptidase in male rats.

Effects of DL-204, 2-(3-ethoxyphenyl)-5,6-dihydro (5,1-a)-isoquinoline, a non-hormonal antifertility drug on testicular hyaluronidase and gamma-glutamyl transpeptidase levels, biochemical markers for testicular function, were evaluated in male rats. Treatment of 21-day-old rats with DL-204 for 7 or 15 days produced cryptorchid condition. Testicular hyaluronidase and gamma-glutamyl transpeptidase levels reveal that DL-204 acts on the testes, possibly in two ways; one, by reducing the gonadotropin levels thereby reducing the levels of androgens as reflected by reduced accessory reproductive organ weights and, secondly, by a direct action on the testes. Thus, we conclude that DL-204 is acting as an antispermatogenic agent, possibly acting in more than one way on the testes.

A new non-hormonal antifertility drug DL-204: I. Effects on testes and accessory glands of reproduction in male rats.

The effect of DL-204, 2-(3-ethoxyphenyl)-5,6-dihydro-5-triazole (5,1a)-isoquinoline, a non-hormonal post-implantational anti-fertility drug, on tissue weights, nucleic acids and total protein concentrations in the testes, ventral prostate and seminal vesicles were evaluated in immature and sexually mature rats, while changes in DNA synthesis were studied only in the immature rats. Treatment of 21-day-old rats at doses of 5mg and 10mg/kg bw once daily for 15 days had no effect on body weight but reduced the weights of testes and accessory glands of reproduction. The concentration of DNA increased while RNA and protein decreased significantly. Treatment of adult rats with DL-204 at a dose of 10mg/kg bw had no effect on body and testes weights but reduced ventral prostate and seminal vesicle weights. The concentration of RNA and protein decreased significantly, while DNA concentration was not altered. DL-204 treatment resulted in drastic decrease of RNA/DNA ratio, reflecting ribosomal loss and cytoplasmic shrinkage. The effects observed after DL-204 treatment are comparable to post-castration changes. DL-204 may be acting on testes and accessory reproductive organs by blocking androgen biosynthesis and/or by antagonizing the action of androgens. It may be acting directly on the normal function of the hypothalamo-pituitary-gonadal axis.

The effects of the cholecystokinin antagonist, proglumide, on prolactin secretion in the rat.

Since cholecystokinin produced important effects on prolactin secretion following its intraventricular injection in ovariectomized rats, we have evaluated the effects of the cholecystokinin antagonist, proglumide, to assess the physiologic significance of CCK in the control of prolactin release. Conscious rats of either sex were used following implantation of third ventricular and/or intravenous cannulae for the administration of proglumide. Blood samples were drawn from conscious animals at various times after injection of the compound. Intraventricular injection of 1 or 10 micrograms of proglumide produced a dramatic decline in plasma prolactin levels in either castrate or intact male rats. Similar results were found following the intravenous injection of 10 or 100 micrograms of the drug. These results contrasted sharply with the findings in ovariectomized females in which the intraventricular injection of the same two doses of proglumide used in males produced a dose-related elevation of prolactin which was opposite to the delayed lowering of prolactin following the intravenous injection of the same doses of the compound used in males. These results indicate that proglumide can lower prolactin in male rats and suggests a physiologically significant role of CCK in the control of prolactin secretion in the male. There appears to be a sex difference in the response since the results contrasted sharply in ovariectomized female rats. The results in the females are puzzling and it is apparent that further studies are needed to determine whether or not CCK has a physiologically significant role to play in prolactin secretion in the female. Since previous results have shown that CCK has no effect on the release of prolactin by the pituitary directly these interactions are presumably taking place in the hypothalamus.

Differential responses of hypothalamic cAMP and cGMP to substance P and neurotensin in ovariectomized rats.

Hypothalamic cAMP and cGMP levels in ovariectomized rats were evaluated after intravenous (IV) pulse injection and/or intraventricular (IVT) injection of substance P and/or neurotensin. Intravenous substance P lowered hypothalamic cAMP concentration whereas IVT injection of 2.5 micrograms substance P produced significant increase in cAMP levels. On the other hand, IV administration of neurotensin failed to alter hypothalamic cAMP levels while IVT injection induced significant decrease in cAMP. Intravenous pulse injection of substance P elevated hypothalamic cGMP levels while IVT injection decreased cGMP concentration. Hypothalamic cGMP concentration was not modified by IV administration of neurotensin. However, IVT injection of neurotensin significantly elevated cGMP levels. Since a number of neurotransmitters/neuropeptides exert their action through cyclic nucleotides the present results indicate differential responses of cAMP and cGMP to substance P and neurotensin and implicate a mediatory role for cAMP and cGMP in the neuroendocrine action of substance P and neurotensin.

The effects of the cholecystokinin antagonist, proglumide, on gonadotropin release in the rat.

Since cholecystokinin had manifest effects on anterior pituitary hormone secretion following its intraventricular injection in ovariectomized rats, we have evaluated the effects of the cholecystokinin antagonist, proglumide, to assess the physiologic significance of CCK in the control of gonadotropin secretion. Conscious rats of either sex were used following implantation of third ventricular and/or intravenous cannulae for the administration of proglumide. Blood samples were drawn from conscious animals at various times after the injection of the compound. In castrate female rats proglumide produced a small but significant increase in plasma LH whether administered by the intravenous or intraventricular route at the lower dose of 10 or 2 micrograms, respectively. The higher doses of 10 micrograms injected intraventricularly or 100 micrograms, injected intravenously failed to affect LH levels in these animals. In contrast there was a much larger increase in plasma LH in castrate males following intraventricular or intravenous injection of the lower doses of proglumide. Even after the higher doses, there was a slight increase in levels of LH by either route of injection. The results indicate that in the castrate animal proglumide can elevate LH levels by either route of injection but that the response is greater in castrate males than females. The reduction in response with the higher doses may reflect an agonist activity of proglumide. By contrast proglumide had no effect on plasma FSH except for a slight elevation observed following the intravenous or intraventricular injection of the lower doses of the compound in castrate males. The results favor a physiologically significant role of CCK in control of LH release in the rat.(ABSTRACT TRUNCATED AT 250 WORDS)

A new non-hormonal antifertility drug DL 111-IT: I. Effects on testes and accessory glands of reproduction in male rats.

Effect of DL-111, 3-(2-ethylphenyl)-5-(3-methoxyphenyl-1H-1,2,4-triazole, a non-hormonal postimplantational antifertility agent, on testicular and accessory reproductive organ weights and total protein, RNA and DNA concentrations were evaluated in immature and adult rats. Treatment of 21-day-old rats at doses of 2.5 mg and 5 mg/kg body weight decreased body weight, weights of testes and accessory glands of reproduction. RNA and protein concentrations decreased significantly with significant increase increase in DNA concentration in testes, epididymis, ventral prostate and seminal vesicles. DL-111 treatment of adult rats at doses of 5 mg and 10 mg/kg body weight had no effect on body weight, but significantly decreased weights of testes and accessory glands of reproduction. RNA and protein concentrations decreased significantly in all tissues studied while DNA concentration was not altered. RNA/DNA ratio decreased significantly, reflecting ribosomal loss and cytoplasmic shrinkage. These effects of DL-111 are comparable to post-castrational changes in accessory glands of reproduction. We presume that these changes are mediated by blocking the androgen biosynthesis and/or by interfering with normal function of hypothalamo-pituitary-gonadal axis.

A new non-hormonal antifertility drug DL 111-IT: II. Effects on testicular hyaluronidase activity in male rats.

Effects of DL-111, [3-(2-ethylphenyl)-5-(3-methoxyphenyl-1H-1,2,4-triazole] a non-hormonal antifertility agent, on testicular hyaluronidase activity, an accurate biochemical marker for testicular function, were evaluated in male rats. Treatment of 21-day-old rats with DL-111 sc for 7 or 15 days resulted in a significant fall in testicular weight and complete suppression of hyaluronidase activity. During the 30-day post-treatment the enzyme activity was restored to normal levels. Treatment of 40-day-old rats for 7 or 15 days also produced a significant decrease in testicular weight and hyaluronidase activity. Simultaneous administration of LH, PMSG or T with DL-111 to 21-day-old rats blocked the inhibitory activity of the drug as the enzyme activity was restored to untreated control levels. Administration of FSH along with DL-111 had no effect on suppressive action of the drug. These results suggest that in male rats DL-111 inhibits testicular activity by reducing LH levels, thereby reducing T levels as observed by reduced weights of testes and accessory glands of reproduction and hyaluronidase activity.