Stimulatory action of prolactin on gonadotropin secretion in vitro

The action of prolactin (PRL) on the secretion of gonadotropin was investigated by means of a cell culture system of rat anterior pituitary gland. Anterior pituitary glands were removed from Wistar male rats, enzymatically digested and cultured. Luteinizing hormone (LH) release into medium was increased by adding PRL dose-dependently in the range between 10 ng/ml and 1 microgram/ml. This effect of PRL was further augmented by the presence of either gonadotropin-releasing hormone or estradiol. The intracellular LH concentration was also increased by PRL. PRL also caused an increase in follicle-stimulating hormone release into medium dose-dependently. In conclusion, PRL was shown to stimulate the secretion of gonadotropin at the pituitary level, thus suggesting a paracrine mode of PRL action in the anterior pituitary gland.     

In vitro secretion of progestins by rat luteal cells and their 20 alpha-hydroxysteroid dehydrogenase activity

Functionally active or regressing corpora lutea were harvested from pseudopregnant (psp) rats between days 5-8 of psp or day 15 of psp, respectively. They were enzymatically dispersed and cultured for 24 h to assess progestins in the medium and 20 alpha-hydroxysteroid dehydrogenase [20 alpha-HSD, catalyzing the conversion of progesterone to 20 alpha-dihydroprogesterone (20 alpha-OH-P)] activity in the cell. Though the active luteal cells retained low 20 alpha-HSD activity, they secreted 6-7 times more 20 alpha-OH-P than progesterone as the regressing luteal cells did. There was no significant difference between the total amounts of progestins in the 2 groups. When increasing doses of pregnenolone were added to the media, progesterone secretion from the active luteal cells was promoted and the progesterone to 20 alpha-OH-P ratio became comparable to the circulating progestins ratio during the mid-luteal phase. In contrast, from the regressing luteal cells only 20 alpha-OH-P secretion was promoted. These results indicate that an insufficient precursor supply results in the catabolism of a large part of synthesized progesterone before its release from luteal cells and suggest the presence of a high affinity but low capacity 20 alpha-HSD in active corpora lutea.     

Effects of leucine-enkephalin and methionine-enkephalin on prolactin and gonadotropin secretion and synthesis in vitro

In vivo, Enkephalins, stimulate PRL, inhibit LH and are inactive on FSH. However, in monolayer pituitary cell cultures, PRL, LH and FSH secretions and synthesis are not modified by Met-Enk. (5 microgram/ml) or Leu-Enk. (5 and 10 microgram/ml). But the simultaneous presence of LHRH and Enk. induces an increase in LH secretion and synthesis without modifying FSH and PRL. In conclusion 1) Enk do not act by themself at the pituitary level but 2) they are able to modify the responses induced by hypothalamic hormones.     

The effect of opioid peptides on ovine pituitary gonadotropin secretion in vitro

Although a hypothalamic site of action has been firmly established for opiate-mediated gonadotropin regulation, there have been several reports which indicate the possibility of a direct influence on the pituitary gland. The objective of this study was to further investigate this possibility in an in vitro pituitary perifusion system utilizing ovine tissue. Treatment with gamma-endorphin (GE) or human beta-endorphin (hBE) resulted in elevated basal LH release (p less than 0.05), followed by an inhibition in the response to a subsequent GnRH challenge (p less than 0.05). The stimulatory effect of hBE was found to be dose-responsive (p less than 0.01). PRL secretion was not similarly stimulated. Ovine beta-endorphin (oBE) had no effect on LH secretion, even though it differs from hBE by only 2 amino acids and contains the active GE sequence. Met-enkephalin also did not influence gonadotropin secretion. Naloxone pretreatment did not reverse the effects of hBE on gonadotropin release. It was found, however, that [D-pGlu1, D-Phe2, D-Trp3,6]-GnRH, a specific GnRH receptor antagonist, did reduce hBE-induced LH and FSH release (p less than 0.05). Naloxone pretreatment alone suppressed the response to GnRH (p less than 0.05). These data indicate that certain opioid peptides can influence ovine gonadotropin secretion in vitro by activating the GnRH receptor. Furthermore, a facilitory role is suggested for endogenous opiates in the local regulation of pituitary gonadotropin secretion.     

Steroidal modulation of in vitro gonadotropin secretion in prepubertal and adult male Siberian hamsters

The effects of exogenous gonadal steroids, testosterone (T), and 17beta-estradiol (E(2)) upon the hypothalamo-pituitary-gonadal axis were reported to be different between prepubertal and adult Siberian hamsters. Utilizing an in vitro static culture system, we investigated if age-related differences in steroid responsiveness occurs at the pituitary. Prepubertal (20 days old) or adult (140 days old) male Siberian hamsters were implanted with 1 mm silastic capsules containing undiluted T, E(2) or cholesterol (Ch, control). After 15 days, pituitaries were removed, incubated in vitro, and subjected to the following treatments: two baseline measurements, one challenge with 10ng/ml of D-Lys(6)-gonadotropin-releasing hormone (GnRH), and three post-challenge washes. Fractions were collected every 30 minutes and measured for follicle-stimulating hormone (FSH) and luteinizing hormone (LH). T and E(2 )reduced basal secretion of LH and FSH in juveniles but not adults. In juveniles, E(2) increased GnRH-induced FSH and LH secretion, while T augmented GnRH-induced FSH secretion but attenuated GnRH-induced LH secretion. Steroid treatment had no effect on GnRH-stimulated LH or FSH release in adults. The only effect of steroid hormones upon adult pituitaries was the more rapid return of gonadotropin secretion to baseline levels following a GnRH challenge. These data suggest both basal and GnRH-induced gonadotropin secretion are more sensitive to steroid treatment in juvenile hamsters than adults. Further, differential steroidal regulation of FSH and LH at the level of the pituitary in juveniles might be a mechanism for the change in sensitivity to the negative effects of steroid hormones that occurs during the pubertal transition.     

Anti-relaxin and relaxin on prolactin and gonadotropin secretion in vitro from porcine pituitary cells

This study examines the possibility of a feedback interaction between gonadal relaxin and the pituitary by investigating the impact of exogenous relaxin and ablation of endogenous with relaxin anti-relaxin serum on pituitary hormone secretion in vitro. Three wells were assigned to treatments: 0, 100 and 1000 ng ml⁻¹ of relaxin, 1:100, 1:1000 and 1:10000 titer of anti-relaxin. Relaxin significantly enhanced prolactin (PRL) secretion (P < 0.05) in long-term culture but had no effect on luteinizing hormone and follicle stimulating hormone secretion. Relaxin anti-serum stimulated a dose dependent increase (P < 0.05) in gonadotropin secretion at 48, 72 and 96 h. Luteinizing hormone and follicle stimulating hormone increased two-fold in 48 h cultures in response to 1:100 anti-relaxin serum in comparison with untreated controls. Anti-relaxin serum at 1:100 completely suppressed PRL secretion after either 48, 72, and 96 h of culture. At 48 h all levels of anti-relaxin serum completely suppressed PRL secretion. These results indicate that endogenous relaxin may be involved at the adenohypophysial level in modulating gonadotropin and PRL release in the pig.     

Mutations affecting gonadotropin secretion and action

A number of mutations are known to disturb the development and function of the hypothalamic-pituitary-gonadal axis. They affect hypothalamic-pituitary-gonadal function at multiple levels, from the migration of gonadotropin releasing hormone neurons to the hypothalamus right through to gonadotropin action in the ovary and testis. Most of the mutations are inactivating, causing various forms of hypogonadism. Exceptions are the activating mutations of the luteinizing hormone receptor, causing male-limited gonadotropin-independent precocious puberty. The human mutations and genetically modified animal models have clarified the molecular pathogenesis of hypogonadism and such disorders can now be diagnosed using molecular biological techniques, enabling selection of specific treatments and appropriate counselling of patients and their families.     

Letter: Prostaglandins and catecholamines in gonadotropin secretion

The hypothesized role of PGF in potentiating adrenergic transmission, known to be involved in gonadotropin secretion, was tested in McCormack-Mayer model involving use of 26-day old immature rats primed with 5 iu of PMS and given a single subcutaneous injection of progesterone 24 hours later. Aspirin was injected into the 3rd ventricle prior to the onset of the critical period on the day of the progesterone injection. Bilateral injections of aspirin into the anterior hypothalamic area (AHA) during onset of critical period were found to block the facilitatory effect of progesterone. Simultaneous injection of PGF2a or dopamine into AHA however fully restored ovulation blocked by aspirin. The results show that prostaglandins and catecholamines (CA) interact to induce adrenergic transmission normally required for gonadotropin secretion. The study also demonstrates the failure of aspirin in the dose used (60 to 100 mcg) to suppress prostaglandin synthesis in the relevant area of the hypothalamus, e.g., AHA, where CA fibers are known to most likely form synapses with LRF secreting neurons. This finding partly explains the failure of peripherally administered indomethacin to affect LH secretion in Patrono and Serra's experiment. Other studies cited suggest possible role of hypothalamic and/or pituitary prostaglandins in gonadotropin secretion. Subcutaneous administration of indomethacin may be more effective in inhibiting prostaglandin synthesis in the ovary than in the hypothalamus, possibly due to differential sensitivity of these tissues and/or poor penetration of the drug across the blood-brain barrier.     

Neuroendocrine control of gonadotropin secretion

Luteinizing hormone releasing hormone (LHRH), a hypothalmic peptide that is concentrated in granules of neurons, has the capacity to release gonadotropins (luteinizing hormone (LH) and follicle stimulating hormone) from the pituitary gland. LHRH has been found in hypophysial portal blood of rats, monkeys, and rabbits. Antibodies to LHRH depress plasma LH concentrations in castrated animals and evoke testicular atrophy, but passive immunization against LHRH does not block the LH surge induced by estrogen in monkeys. Estrogens, progestin, prolactin, and dopamine have marked effects on LH secretion, yet an association between these effects and altered hypophysial portal blood concentrations of LHRH is not established. In view of the paucity of evidence demonstrating such a cause and effect relationship, two alternative proposals have become tenable. One, hormones and neurotransmitters may not alter the levels of portal blood LHRH, but rather alter the frequency of pulsatile LHRH secretion. Two, hormones, such as estrogens, progesterone, and prolactin, may alter the responsiveness of the gonadotropin-secreting cells to LHRH by affecting the secretion of dopamine.     

Sex differences following gonadectomy in basal gonadotropin secretion rate of rat pituitary fragments in vitro

Sex differences in the acute response of circulating luteinizing hormone (LH) and follicle-stimulating hormone (FSH) to withdrawal from gonadal negative feedback in the rat are well established. To investigate postgonadectomy changes at the anterior pituitary level that may underlie dramatic in vivo sex differences, we used a computer-controlled pituitary perifusion system to measure in vitro basal secretion rates (BSRs) of LH and FSH following gonadectomy in the absence of exogenous gonadotropin-releasing hormone (GnRH). We compared BSRs of pituitaries removed from intact rats and from males and females 2 and 6 days post-gonadectomy. Glands were cut into quarters, placed into individual chambers, and perifused in Medium 199 at 10 ml/h for 4 h. In females (n = 12/gp), BSR of LH was not significantly elevated above intact levels by 2 days but had tripled by 6 days post-ovariectomy, while BSR of FSH had already doubled by 2 days and doubled again by 6 days. These changes in BSR in females paralleled changes in serum levels of both hormones. In males (n = 14/gp), although serum LH and FSH had increased 7-fold by 2 days post-orchidectomy, BSRs of LH and FSH had decreased to 75% and 64% of intact levels, respectively, by 6 days. These findings suggest important sex differences at the pituitary level in the responses to withdrawal from gonadal feedback that persist in culture in the absence of direct hypothalamic (GnRH) input.     

Steroid control of gonadotropin secretion

Current knowledge about the mechanism and site of action of estradiol (E2) and progesterone (P) during the menstrual cycle and the physiological role of androgens is reviewed. In normal women, the positive feedback effect of E2 at the pituitary level is the principal event of the follicular phase inducing the LH surge. P, by its negative feedback at the hypothalamic level and by its positive feedback at the pituitary level regulates GnRH and LH secretion during the luteal phase. Androgens do not directly play a role in gonadotropin regulation.     

Kisspeptins and the control of gonadotropin secretion in humans

The kisspeptin hormones are a family of peptides encoded by the KiSS-1 gene, which bind to the G-protein coupled receptor-54 (GPR54). Interactions between kisspeptin and GPR54 are thought to play a critical role in reproduction. In agreement with animal data, kisspeptin-54 administration acutely stimulates the release of gonadotrophins in both male and female healthy subjects, with no observed adverse effects. Furthermore, its potency is comparable to those of other gonadotrophin secretagogues studied. The kisspeptin-GPR54 system thus offers a novel means of therapeutically manipulating the hypothalamo-pituitary-gonadal (HPG) axis in humans. This article aims to provide a focused review of the experimental data which inform us how kisspeptin influences the HPG axis in humans.     

On feedback control of gonadotropin secretion

This note is an attempt to demonstrate that hypothalamic pulsatile GnRH secretion is not the result of a short-term, negative steroid hormone feedback. Clarification of this point is of importance for further modelling the control of gonads.     

The effect of exogenous progestins on endogenous progesterone secretion in pregnant mares

Twenty-four pregnant, light horse mares were used in a study to determine if an exogenous progestin or progesterone would alter serum concentrations of progesterone. On day 40 of gestation, mares were randomly assigned to one of three administration groups: 1) 250 mg of progesterone in oil every other day, 2) 22 mg of Altrenogest (Regumate, American Hoechst, Somerville, NJ 08876) orally every day, or 3) 10 ml of neobee oil (control) orally every day. The treatment period was from day 40 to 105. Pregnancy status was monitored on days 40, 60, 80, 100 and 105 and a single jugular blood sample was obtained daily from days 40 to 46, 69 to 75 and 99 to 105. Serum concentrations of progesterone were determined by radioimmunoassay. Concentrations of progesterone were similar (P>0.05) among groups at each sampling period. Overall concentrations of progesterone increased (P<0.001) from days 40 to 46. Injection of 250 mg of progesterone in oil failed (P>0.05) to maintain concentrations of progesterone in serum above baseline for 24 hr. Thus higher doses and/or more frequent injections would be needed in order to increase concentrations of progesterone above those seen in untreated controls. In summary, Altrenogest was found to be a nonstressful, convenient method of administering progestins to pregnant mares without altering their endogenous secretion of progesterone.     

Inappropriate gonadotropin secretion in the polycystic ovary syndrome

In this study was investigated the diagnostic significance of double stimulation test with (that is of 25 micrograms rapid injection intravenously twice at an interval of 120 minutes and the misure of maximal net increment of serum LH after the first GnRH injection expressed as delta 1 and after the second injection, expressed as delta 2) to discriminate patients with idiopatic hirsutism. This test was effectuated on 8 patients with PCO (presence of polycystic ovaries on Ecografya and/or Laparoscopy) and 8 patients with idiopatic hirsutism (presence of normal morphology ovaries). Basal LH, FSH, E1, E2 and delta 4 levels were also measured. The value of LH delta 2 were more elevated in patients with PCO (p less than 0,0002) than the patients with idiopatic hirsutism. Consequently it as been value of LH delta 2 to discriminate the two different groups of patients. In PCO patients were also found: -a positive linear correlation between LH delta 1 and basal concentration serum of E2 (p less than 0,001); -a significant increase of basal levels serum of delta 4 (p less than 0,02); while the values of basal LH and LH delta 1 were found superior only on 4 of the initial 8 patients, the basal values of E1 and E2 were at the superior found of the norm and basal FSH, FSH delta 1 and FSH delta 2 values were found normals.     

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.