Role of gangliosides in gonadotropin and cholera enterotoxin stimulated steroidogenesis in isolated rat ovarian cells

Ovarian cells isolated from 26 day old rats responded to hCG (10 ng/ml) and cholera enterotoxin (100 ng/ml) $\text{in vitro}$ with a forty-five to fifty-fold increase in progesterone production. Both cholera enterotoxin and hCG-stimulated progesterone response was accompanied by a lag period. The duration of the lag period in the production of the progesterone depended on the concentration of gonadotropin or cholera enterotoxin, and with maximally stimulating dose it was 20–30 minutes. Addition of highly purified mixed gangliosides to the incubation medium abolished the stimulatory effect of cholera enterotoxin on progesterone response. In contrast, under identical experimental conditions, ganglioside addition produced no effect on progesterone response elicited by hCG or LH. Similarly mixed gangliosides did not prevent the specific binding of [¹²⁵I]hCG to the ovarian cells or to the membranes isolated from the ovary. In addition preincubation of [¹²⁵I]hCG with ganglioside did not alter the subsequent binding of the hormone to the ovarian cell surface receptor. These findings suggest that gangliosides are not involved in the hormone receptor interactions and subsequent receptor mediated physiological response.     

Effect on an antagonistic analog of gonadotropin releasing hormone upon ovarian granulosa cell function.

We have recently demonstrated that gonadotropin releasing hormone (GnRH) acts directly on ovarian granulosa cells to inhibit the follicle stimulating hormone (FSH)-induced increase in granulosa cell steroidogenesis $\text{in}$$\text{vitro}$. A GnRH antagonist, [D-pGlu¹, D-Phe², D-Trp^3,6] GnRH (A), which is known to antagonize GnRH-stimulated gonadotropin release by cultured pituitary cells, was tested in the granulosa cell system. GnRH (10^?8M) inhibited estrogen and progesterone production by FSH-treated granulosa cells $\text{in}$$\text{vitro}$, whereas the antagonist A (10^?6M) did not affect FSH stimulation of steroidogenesis. Antagonist A, when added together with GnRH and FSH, blocked the GnRH inhibition of FSH-induced steroidogenesis. Estrogen and progesterone production by granulosa cells was increased by 50% at a molar ratio (IDR₅₀) of $\text{20}\text{1}\text{and}\text{12}\text{1}$ ([antagonist]/[GnRH]), respectively. At 10^?6M, antagonist A completely prevented the GnRH (10^?8M) inhibition. A similar effect of antagonist A was seen in FSH-induced increase of luteinizing hormone (LH) receptor content. FSH treatment for 2 days $\text{in}$$\text{vitro}$ induced an 8-fold increase in LH receptor content in cultured granulosa cells; concomitant treatment with 10^?8M GnRH completely inhibited the FSH effect. Antagonist A (10^?6M), by itself, had no effect on the FSH action. However, when added together with FSH and GnRH, antagonist A completely abolished the inhibitory effect of GnRH. These results demonstrate that the direct inhibitory effect of GnRH on granulosa cell function can be prevented by a GnRH antagonist and that the GnRH action at the ovarian level may require stringent stereospecific interactions of these peptides with putative GnRH recognition sites.     

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.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the $\text{in vitro}$ release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all $\text{in vitro}$ experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

Insulin-like growth factor I enhances gonadotropin-releasing hormone-stimulated luteinizing hormone release from bovine anterior pituitary cells

The role of insulin-like growth factor I (IGF-I) in the release of luteinizing hormone (LH) is unclear in ruminants. In the present study, the effects of IGF-I on the release of LH stimulated by gonadotropin-releasing hormone (GnRH) were examined in primary cultures of bovine anterior pituitary (AP) cells, and the interaction between estradiol-17beta (E(2)) and IGF-I was characterized. GnRH(100nM)-stimulated LH release from the cultured cells was increased (P<0.05) 12, 24 and 36h after addition of IGF-I (250ng/ml), with a maximum at 12h (48.4ng/ml media versus 35.4ng/ml media in controls). IGF-I at concentrations of 25, 250 and 500ng/ml increased the release by 18.7, 24.2 and 28.9%, respectively (P<0.05), when compared with controls (37.2ng/ml media). E(2) (10nM), IGF-I (250ng/ml) and combined treatment of E(2) plus IGF-I also induced significant increases in LH release (P<0.05). The amounts of LH release after treatment with E(2) alone was 37.3% greater than with IGF-I alone (39.0ng/ml media versus 28.4ng/ml media) (P<0.05). When E(2) and IGF-I were added together (45.6ng/ml media), the release of LH was significantly greater than with either E(2) alone or IGF-I alone (P<0.05). E(2) (10nM) significantly (P<0.05) increased the amount of GnRH bound to the cells by 51.6% when compared with controls, however, IGF-I (250ng/ml) failed to increase GnRH binding. These results show that IGF-I enhances GnRH-stimulated LH release without changing the number of GnRH receptors in cattle, and IGF-I interacts with E(2) to increase the response to GnRH.     

Coincidence of down-regulation and desensitization in pituitary gonadotrophs stimulated by gonadotropin releasing hormone

The dynamics of gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) release was studied $\text{in}$$\text{vitro}$ by superfusion of cultured pituitary cells. Continuous exposure of the cells to GnRH resulted in desensitization of the gonadotroph responsiveness to further stimulation by the hormone. The refractory state was achieved within 4 hr of hormone introduction (10^?7 M) and was accompanied by down-regulation of GnRH receptors (50%) assayed by equilibration with [¹²⁵I]iodo-[D-Ala⁶]des-Gly¹⁰-GnRH N-ethylamide. The data indicate that GnRH can regulate the number of its own receptors, and that desensitization is accompanied by down-regulation.     

Postinhibitory overshoot of insulin release by gastric inhibitory polypeptide

Following intravenous infusion of somatostatin $\text{in vivo}$ occasionally there is a large rebound overshoot of insulin release. An $\text{in vitro}$ model to simulate this phenomenon was made by perfusing rat pancreas with gastric inhibitory polypeptide (GIP) during simultaneous perfusion with somatostatin. Adding GIP (100 ng/ml) to the perfusate for 2 minutes beginning either 3 or 9 minutes before terminating the somatostatin perfusion produced a large overshoot in insulin release. The magnitude of overshoot was greater when medium contained 300 mg/dl glucose that when it contained 150 mg/dl glucose. Perfusion with GIP for 2 minutes beginning 9 minutes before increasing the glucose concentration of the medium from 30 to 300 mg/dl elicited a large increase in both the acute and second-phase release of insulin. These suggest that post-inhibitory overshoot of insulin release after somatostatin may be produces $\text{in vitro}$ by the suppressed action of stimulatory hormones such as GIP. Prior infusion with GIP can also potentiate glucose-stimulated insulin increase.     

Progesterone and 20α-hydroxyprogesterone stimulate the in vitro release of GnRH by the isolated mediobasal hypothalamus

An $\text{in}$$\text{vitro}$ perifusion system was used to investigate the effect of progesterone (P₄) and 20α-hydroxyprogesterone (20α-OHP) on the release of GnRH from isolated hypothalamic tissue of the adult ovariectomized estradiol-17β primed rat. Pulse delivery of P₄ stimulated GnRH release not only from the isolated mediobasal hypothalamus-anterior hypothalamus-preoptic area (MBH-AHPOA) complex but also from the MBH alone. Release of GnRH from the MBH was also increased by 20α-OHP. These results suggest that the $\text{in}$$\text{vivo}$ increase of circulating P₄ or 20α-OHP associated with the initiation of the preovulatory LH surge may play a role in regulating the timing or amplitude of this surge by directly stimulating the release of GnRH from the MBH.     

Direct in vitro stimulation of pituitary LH release by alpha melanocyte stimulating hormone

The present $\text{in vitro}$ superfusion study demonstrates that synthetic α-MSH acts at the pituitary level, independent of the hypothalamus, to increase the release of LH in male but not in female rats.     

Restoration of responsiveness to gonadotropin releasing hormone (GnRH) in calcium-depleted rat pituitary cells.

GnRH-stimulated, but not basal, luteinizing hormone (LH) release from cultured pituitary cells requires extra-cellular calcium. The present studies were designed to show whether cells which had lost responsiveness to GnRH in the absence of extracellular calcium (“Ca²⁺-depleted cells”) could regain responsiveness by readdition of calcium to the media. The addition of calcium-containing medium to cells which were preincubated (75 min) in calcium-free medium resulted in elevated basal LH release. Addition of GnRH to the media in the presence of calcium did not cause additional stimulation of LH release above the elevated basal level. Incubation of Ca²⁺-depleted cells in calcium-containing media for 2 h before measuring responsiveness depressed the basal level to near that seen in control cells and GnRH was able to stimulate LH release, but not to as high a level as in control cells (which were preincubated in 1 mM Ca²⁺-containing media). After incubation of calcium depleted cells in calcium-containing media for 3 h or 5 h, the basal and stimulated levels of LH response were statistically indistinguishable from those seen in control cells.     

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.     

Effect of cortisol or adrenocorticotropic hormone on luteinizing hormone secretion by pig pituitary cells in vitro

The effect of cortisol or adrenocorticotropic hormone (ACTH) on basal and gonadotropin-releasing hormone (GnRH)-induced secretion of luteinizing hormone (LH) was studied in vitro using dispersed pig pituitary cells. Pig pituitary cells were dispersed with collagenase and DNAase and then grown in McCoy's 5a medium containing 10% dextran charcoal-pretreated horse serum and 2.5% fetal calf serum for 3 days. Cells were preincubated with cortisol or ACTH before GnRH was added. When pituitary cells were incubated with 400 micrograms cortisol/ml medium for 6 h or longer, increase basal secretion of LH was observed. However, GnRH-induced LH release was reduced by cortisol. The degree of this reduction was dependent on cortisol, and a concentration of cortisol higher than 100 micrograms/ml was needed. Cortisol also inhibited the 17 beta-estradiol-induced increase in GnRH response. ACTH-(1-24), ACTH-(1-39), or porcine ACTH had no influence on GnRH-induced LH secretion. Our results show that cortisol can act directly on pig pituitary to inhibit both normal and estradiol-sensitized LH responsiveness to GnRH.     

Effects of thyroidectomy and thyroxine replacement on the release of luteinizing hormone and gonadotropin-releasing hormone in vitro

The effects of thyroidectomy and thyroxine (T4) replacement on the release of luteinizing hormone (LH) and gonadotropin-releasing hormone (GnRH) in ovariectomized (Ovx) rats were studied. Immediately after ovariectomy, rats were thyroidectomized (Tx) or sham-Tx. The Ovx-Tx rats were injected subcutaneously with either saline or T4 (2 micrograms/100 g body weight) daily for 30 days before sacrifice. Sham-Tx rats were treated with saline only. Twenty hours after the last injection, the blood sample was obtained by decapitation. The excised anterior pituitary gland (AP) was bisected and incubated in vitro with or without 0.1, 0.5, 2.5, and 50 ng GnRH at 37 degrees C for 4 h. The mediobasal hypothalamus (MBH) was bisected and incubated with or without the AP of Ovx donor rat in vitro. Concentrations of LH and GnRH in the medium and that of LH in the serum were measured by radioimmunoassay. LH in the serum of Tx rats was higher than that in the serum of sham-Tx and Tx-T4 rats. Thyroidectomy resulted in an increase of LH release by Ovx rat AP, stimulated with or without 0.1 and 50 ng GnRH, as well as in an increase of immunoreactive GnRH release from MBH of Ovx rats in vitro. After a 4-hour incubation with donor APs, the LH in the medium containing MBH obtained from Tx rats was significantly higher than that obtained from sham-Tx and Tx-T4 rats. LH concentrations, in both sera and media, as well as GnRH concentration in the media of euthyroid and T4-replaced Tx groups were nonsignificantly different. These results suggest that T4 is inhibitory to the basal and GnRH-stimulated LH release as well as to the release of GnRH in the absence of ovarian hormones.     

Interrelationship between estrogen and thyroxine on the release of luteinizing hormone and gonadotropin-releasing hormone in vitro

The present experiments were designed to study the interaction between estradiol benzoate (EB) and thyroxine (T4) given in vivo on the responsiveness of pituitary luteinizing hormone (LH) to gonadotropin-releasing hormone (GnRH) and the release of GnRH in vitro. Ovariectomized-thyroidectomized (Ovx-Tx) rats were injected s.c. with saline or T4 (2 micrograms/100 g b.wt), and oil or EB (0.1 microgram) once daily for 40 days following a 2 x 2 factorial design. All animals were then decapitated and blood samples were collected. Anterior pituitaries (APs) were incubated in vitro with and without 0.1 ng GnRH at 37 degrees C for 4 h. Mediobasal hypothalami (MBHs) were excised and then incubated with and without APs from Ovx donor rats. Concentrations of LH and GnRH in the medium and that of LH in the serum were measured by radioimmunoassay. The LH level in media containing MBHs and donor APs was used as the index of bioactive GnRH release. In Ovx-Tx rats, T4 injections reduced the serum LH concentration, the pituitary LH response to GnRH, and the bioactive as well as the immunoreactive GnRH release. The serum LH levels and the spontaneous as well as the GnRH-stimulated release of LH in vitro were suppressed in Ovx-Tx rats following administration of EB. By contrast, the serum LH concentration, as well as pituitary LH response to GnRH and GnRH release in vitro, were higher in the group treated with both T4 and EB than in that treated with saline and EB. These results suggest that the differential changes in the LH secretion after thyroidectomy of Ovx versus non-Ovx rats are due to an antagonistic effect between T4 and estrogen on the response of pituitary LH to GnRH, and the release of GnRH.     

Chronic treatment with valium (diazepam) fails to affect the reproductive system of the male rat

Male rats treated chronically with high doses of Valium (50mg/ Kg/day; 10 days) failed to exhibit changes in their reproductive system. Testicular and prostate weights, serum testosterone (T) and LH were unaffected. Testes and pituitary tissue stimulated $\text{in}$$\text{vitro}$ with LH and GnRH, respectively, released normal amounts of T, LH and FSH. Brain benzodiazepine receptors were slightly but significantly elevated by Valium treatment as well as by castration. We conclude that the male reproductive system is resistant to chronic Valium treatment even though the brain levels of benzodiazepine receptors are not.     

Depression of amino acid transport in cultured rat hepatocytes by purified enterotoxin from Clostridiumperfringens

Rat liver parenchymal cells (hepatocytes) were isolated by a collagenase perfusion technique and maintained as monolayers in serum-free medium in collagen-coated culture dishes. Glucagon, in combination with dexamethasone, induced α-aminoisobutyric acid transport in these cells. Addition of purified $\text{Clostridium}\text{perfringens}$ enterotoxin to hepatocytes preinduced by glucagon and dexamethasone rapidly depressed (but did not abolish) α-aminoisobutyric acid transport. The toxin effect was dose dependent: 1000 or 300 ng/ml produced maximal depression whereas 100 or 40 ng/ml were without effect in 120 minutes. The effect was eliminated by pretreating the toxin with heat or specific antisera. The effect of enterotoxin on α-aminoisobutyric acid transport in two cultured rat hepatoma cell lines (H4-II-E-C3 and McA-RH 7777) was also investigated. Only the McA-RH 7777 cells were sensitive to the toxin suggesting that the enterotoxin may interact with specific membrane components of normal rat liver cells which are also present on some (but not all) cancerous rat liver cells.     

Release of progesterone from perifused, highly luteinized ovarian cells of rats: effects of luteinizing hormone and bovine serum albumin

Release of progesterone from enzymatically dispersed luteal cells of superovulated rats was studied using a multi-channeled perifusion system. Cells were perifused with protein-free medium for up to 5 h. Basal release of progesterone showed a steady decline during the first h of perifusion to a stable baseline where it remained throughout the experiment. A 30-min exposure of the luteal cells to increasing amounts of luteinizing hormone (LH) stimulated a dose-dependent increase in progesterone release. Similar results were observed when luteal cells were exposed to 0.2 or 1.0 mM dibutyryl (Bu)2 cAMP for 30 min. Exposure of the cells to 0, 1, 10, and 100 ng LH/ml protein-free medium for 230 min showed increased release of progesterone, although the dispersed cells perifused with 100 ng LH/ml protein-free medium were unable to maintain the maximal levels of progesterone release. The effect of bovine serum albumin (BSA) in the perifusion medium on the basal and LH-stimulated progesterone release was examined. Low concentrations of BSA (0.05%) had no effect, but 0.5% and 2.0% BSA significantly increased the basal release of progesterone. However, the addition of 0.05% BSA to the medium resulted in an increased progesterone release in response to 10 ng LH/ml medium. These results suggest that the in vitro perifusion system maintains physiologically viable cells which are responsive to either LH or (Bu)2 cAMP for at least 5 h. The effect of protein in the perifusion medium or progesterone release was demonstrated by the addition of BSA.     

Cyclo (His-Pro): a selective inhibitor of rat prolactin secretion in vitro

Cyclo (His-Pro) (10 ng/ml), inhibits KCl (59 mM) or thyrotropin-releasing hormone (10 ng/ml) stimulated, but not basal, release of prolactin from rat hemipituitaries $\text{in vitro}$. However, cyclo (His-Pro) has no effect on the basal or stimulated release of thyrotropin and growth hormone. Cyclo (His-Pro) does not inhibit the binding of thyrotropin-releasing hormone to pituitary membrane suggesting that cyclo (His-Pro) inhibition of prolactin release is not mediated via the pituitary TRH-receptor.     

抗孕53影响大鼠垂体前叶对GnRH的敏感性反应

本实验应用动情前期大鼠垂体前叶组织块离体培养方法,观察了 A 环失碳类甾体化合物—抗孕53对 LH 基础分泌和动员性分泌的影响。实验分为对照组、GnRH 组、抗孕53组、GnRH-抗孕53组。结果表明,GnRH 可显著促进 LH 分泌并产生自激作用。GnRH 的第一次作用后,LH 分泌增加量由对照组的0.7ng/ml 增加到4.3ng/ml,而当 GnRH 第二次作用后,这一效应显著增强,由对照组的0.5ng/ml 增加到6.8ng/ml,GnRH 的两次作用效应相比,差异极显著。抗孕53可部分抑制垂体对 GnRH 的敏感性反应。抗孕53作用后,LH 分泌增加量由 GnRH 第一次作用后的4.3减至2.5ng/ml,由 GnRH 第二次作用后的6.8降至4.1ng/ml。抗孕53不影响 LH 的基础分泌,与对照组相比,两组的 LH 分泌增加量无显著差异。抗孕53对垂体的这一作用可能是通过直接影响促性腺激素细胞的代谢及调节而实现。     

A teleost (Tilapia mossambica) gonadotropin that resembles luteinizing hormone.

A purified gonadotropin preparation was obtained from pituitaries of a teleost fish ($\text{Tilapia}$$\text{mossambica}$). This gonadotropin was found to resemble LH in that it behaved identically to mammalian and non-mammalian LHs in several chromatographic systems, and stimulated testerone production in isolated rat Leydig cells. In this assay, specific for LH, the $\text{Tilapia}$ gonadotropin was less potent than mammalian LHs but significantly more active than avian, reptilian or amphibian LHs. The $\text{Tilapia}$ gonadotropin was found to be a glycoprotein; preliminary amino acid composition data show resemblances to both mammalian and non-mammalian LHs.