Evaluation of pituitary responsiveness to LHRH as a pregnancy test in ewes

Pregnant and nonpregnant ewes were injected with luteinizing hormone-releasing hormone (LHRH). Pituitary responsiveness, based on serum luteinizing hormone (LH), and follicle stimulating hormone (FSH) concentration, 2 hr after injection was then determined for each ewe, by radioimmunoassay (RIA) and correlated with the physiological reproductive state of each ewe. The serum LH release in pregnant ewes was significantly lower than that in nonpregnant ewes. Serum LH concentrations of pregnant ewes were further categorized according to whether the ewes were multiple (ML) or single lambing (SL). The responses by ML ewes were lower for LH than the SL responses. Follicle stimulating hormone responses were not different between pregnant or nonpregnant groups. Luteinizing hormone responses between pregnant ewes which were grouped according to 3 stages of pregnancy (1 to 5, 5 to 10 and 10 to 15 weeks pregnant) were not different from each other. Pregnancy diagnoses were made based on a fixed cut-off value, to which the LH response of each ewe to 5 mug LHRH was compared. Ewes whose response fell below this cut-off were diagnosed as pregnant. Accuracy of the diagnoses were determined by known lambing data. Diagnostic accuracy ranged from a low of 60% for nonpregnant, to a high of 95% for ML ewes. Accuracy for SL ewes (64%) was lower than for the overall pregnant group (79%), as well as that for ML ewes. Doses of LHRH, higher than 5 mug per ewe, generally produced LH release in pregnant ewes which was not significantly suppressed relative to responses of nonpregnant ewes. These results lead to the conclusion that gonadotropin response to exogenous LHRH injection is not an effective tool for pregnancy diagnosis.     

Reduced Sertoli cell number and altered pituitary responsiveness in male lambs undernourished in utero

The nutritional status of females during pregnancy can have profound effects on the fetus reproductive system development that could affect the reproductive potential as later as in adulthood (fetal programming). The aim of the present study was to examine the effects of maternal nutrient restriction imposed during different periods of gestation on pituitary responsiveness at different ages postnatal and on Sertoli cell number in male offspring. Pregnant ewes were fed to 100% of Metabolizable Energy and Crude Protein requirements throughout pregnancy (Control) or to 50% from 0 to 30 (R1) or from 31 to 100 days of gestation (R2). Male lambs were selected and fed to appetite throughout the study. At 2, 5.5 and 10 months of age a GnRH challenge was conducted. At slaughter (10 months) testes were removed and examined histologically. Maternal undernutrition did not affect the time of the onset of puberty, defined as the first increase in plasma testosterone concentrations > or =1 ng/ml. The LH and FSH response to GnRH challenge did not differ between groups at 2 and 5.5 months but at 10 months of age a higher (P<0.05) FSH response was found in R2 group. Testes weight did not differ between groups at slaughter. Mean Sertoli cell number was significantly lower in animals of R2 group compared with Control (P<0.01). A smaller seminiferous tubules diameter was detected in R2 group (P<0.05), while the diameter of the lumen was similar in all groups. Collectively, these results provide clear evidence for a direct effect of nutrient restriction during pregnancy on Sertoli cell number in adulthood. The lower number of Sertoli cells is the most candidate factor for the higher pituitary activity through a reduction in the suppressive effect of inhibin.     

Modulation of pituitary responsiveness to LHRH by androgens in ovariectomized female rats.

The effect of androgens on pituitary response to luteinizing-hormore-releasing hormone (LHRH) and their ability to modify effects of 17beta-estradiol (E2) on pituitary responsiveness to LHRH were tested in ovariectomized rats maintained on a daily dose of 0.25 microgram estradiol benzoate per rat for 6 d before androgen administration. Testosterone propionate (TP) (4, 40, 400, or 4000 microgram per rat), administered 24 h before LHRH (500 ng per rat), had no significant effect on luteinizing hormone (LH) or follicle-stimulating hormone (FSH) response. Similar doses of dihydrotestosterone (DHT) did not significantly alter the LH response but significantly suppressed the FSH response. Even the lowest dose completely blocked the FSH response to LHRH. TP in combination with 4 or 400 microgram of E2 suppressed the stimulatory effect of E2 on both LH and FSH response to LHRH in a dose-related manner. DHT and E2 in combination affected LH response inconsistently, whereas their ratio determined FSH response; there was pronounced inhibition of FSH response in rats given high doses of DHT combined with low doses of E2; DHT inhibition of FSH response in animals receiving 4 microgram of DHT with 400 microgram E2 was partially overcome by the stimulatory effect of E2. Our results indicate that TP and DHT affect LH and FSH response to LHRH differently. The ratio of androgen to estrogen is important in determining the response to LHRH.     

Enkephalins and pituitary hormone release: modification of responsiveness to LHRH.

An intraperitoneal injection of leucine-enkephalin into rats stimulates gonadotropin and prolactin release. To elucidate the mechanism of this releasing property of leucine-enkephalin, rat hemipituitaries were incubated with either enkephalin alone or enkephalin in combination with OHRH. Enkephalin alone had no effect on LH or prolactin release in vitro but potentiated the LH response to LHRH. Neither leucine-enkephalin nor LHRH alone had an effect on GH release; however, when combined, a GH response to LHRH occurred. These results suggest that leucine-enkephalin can modify the pituitary responsiveness to certain hypothalamic releasing hormones by a direct pituitary action.     

Release of LHRH in vitro and anterior pituitary responsiveness to LHRH in vivo during sexual maturation in pullets (Gallus domesticus)

An in-vitro superfusion technique was used to study basal and depolarization-induced (32 mmol K+/l) release of LHRH from the mediobasal hypothalamus (MBH) of pullets at 8-25 weeks of age. Plasma LH concentrations and the incremental change (delta LH) after an i.v. injection of 1 or 15 micrograms synthetic ovine LHRH/kg body weight were also determined. Between 8 and 25 weeks of age, significant (P less than 0.01) increases in basal and depolarization-induced release of LHRH (93 and 330%, respectively) were accompanied by a significant (P less than 0.01) rise in the residual LHRH content of MBH tissue (152%), observations which suggest that the ability of the hypothalamus to synthesize and secrete LHRH increases as sexual maturation proceeds. However, plasma LH, which reached a maximum concentration of 2.05 +/- 0.43 micrograms/l at 15 weeks, fell significantly (P less than 0.05) to 1.14 +/- 0.05 micrograms/l at 25 weeks. Since delta LH in response to exogenous LHRH showed a marked and progressive decline between 12 and 20 weeks of age, the low plasma concentration of LH typical of the mature hen is probably attributable to a direct negative-feedback action of ovarian steroids on the anterior pituitary gland rather than to an impaired secretion of LHRH from the median eminence. It is suggested that a dramatic increase in the responsiveness of LHRH nerve terminals in the MBH to depolarization by 32 mmol K+/l between 20 and 25 weeks of age (mean age at onset of lay 21.9 weeks; range 19-25 weeks) may reflect the development of hypothalamic responsiveness to the positive feedback action of progesterone.     

Gonadotrophin secretion and pituitary responsiveness to LHRH in castrated and intact male rabbits exposed to different photoperiods

Adult male wild rabbits were exposed to at least 16 weeks of 16L:8 D before experiments began. Plasma LH and FSH concentrations increased significantly (P less than 0.001) when rabbits were castrated in 16L:8D but declined when rabbits were transferred to 8L:16D. Concentrations had returned to normal for castrated rabbits in 16L:8D by 74 days after the start of the 8L:16D treatment. Treatment of intact male rabbits with an injection of LHRH before and after transfer to short daylengths caused a transient increase in plasma LH which lasted 50-80 min and this produced a concomitant rise in plasma testosterone. The daylength change had no effect on this response even though testicular size declined after the transfer to short daylengths. Rabbits moulted in response to exposure to 8L:16D. This suggests that hypothalamic activity responds to photoperiod and that changes in pituitary responsiveness to LHRH and steroid negative feedback are unimportant.     

Relationship between pituitary responsiveness to Gn-RH and number of Gn-RH-binding sites in pituitary glands of beef cows

Changes in the ability of Gn-RH to induce gonadotrophin release with time after synchronization of oestrus was determined in 4 groups of 6 cows each. Cows were given Gn-RH at 40-min intervals for 6 h beginning at -24, 0, 18 or 36 h (time 0 = removal of progestagen implant). Changes in concentration (ng/ml) of serum LH after Gn-RH averaged 2.9, 6.2, 6.4 and 33.4, whereas serum FSH averaged 25.7, 35.8, 35.8 and 97.3. Thus the responsiveness of the pituitary to Gn-RH had increased by 36 h after implant removal. Other groups of cows subjected to the same synchronization scheme were slaughtered at 0 h, 24 h or at various times after onset of oestrous behaviour. Gn-RH binding to crude pituitary membrane preparations was assessed. There was no apparent change in the affinity constant of Gn-RH-binding sites with time after synchronization. The number of Gn-RH-binding sites remained unchanged until the period of oestrus when a significant decline with time was detected. We conclude that the increase in pituitary responsiveness to Gn-RH that occurs before the preovulatory gonadotrophin surge was not directly associated with changes in number or affinity of pituitary Gn-RH-binding sites in crude pituitary membrane preparations.     

Changes in pituitary and ovarian LHRH receptors after active immunization of female rats against LH or LHRH

The hypothalamic-pituitary-ovarian axis of female rats was disrupted at the site of LHRH stimulation by active immunization against LHRH or at the site of LH action by active immunization against LH. Active immunization against LH was associated with an increase in pituitary LHRH receptors to levels comparable to control values at pro-oestrus whereas immunization against LHRH led to a marked reduction in receptor numbers. Ovarian LHRH receptor concentrations were increased by both treatments. It is concluded, therefore, that (1) LHRH receptors in the pituitary and ovary are not concomitantly controlled, and (2) pituitary receptor numbers are primarily under positive autoregulatory control by LHRH and that ovarian LHRH receptor concentrations may be under long-term influence of LH.     

Somatostatin pretreatment increases the number of somatostatin receptors in GH4C1 pituitary cells and does not reduce cellular responsiveness to somatostatin

The GH4C1 pituitary cell line contains specific plasma membrane receptors for the inhibitory neuropeptide somatostatin (SRIF). Unlike other peptides which bind to cell surface receptors on these cells, SRIF is not rapidly internalized via receptor-mediated endocytosis. Here we examined the effects of chronic SRIF pretreatment on the subsequent ability of GH4C1 cells to bind and respond to this hormone. Treatment of cells with 100 nM SRIF increased [125I-Tyr1]SRIF binding to a maximum value of 220% of control after 20 h. Scatchard analysis demonstrated that the number, but not the affinity, of the receptors was altered. The effect of SRIF was dose-dependent (ED50 = 2.3 +/- 0.4 nM), was not mimicked by an inactive analog, and was specific for the SRIF receptor. Furthermore, pretreatment of cells with other agents, which mimic SRIF's action to decrease intracellular cAMP and free Ca2+ concentrations, did not mimic the SRIF-induced increase in receptor number. Thus, occupancy of the SRIF receptor was required for SRIF receptor up-regulation. Inhibition of protein synthesis with cycloheximide did not prevent the SRIF-induced increase in receptors, consistent with an effect of SRIF to either reduce receptor degradation or cause slow redistribution of preexisting receptors to the plasma membrane. In contrast to the effects on receptor binding, pretreating cells with SRIF did not alter either basal cAMP levels or the potency of SRIF to inhibit cAMP accumulation (ED50 = 0.5 +/- 0.2 nM). However, the maximum cAMP produced by stimulators of adenylyl cyclase was increased. The observation that chronic SRIF exposure did not cause homologous desensitization in GH4C1 cells and increased rather than decreased SRIF receptor number is consistent with the fact that this neuropeptide is not rapidly internalized by receptor-mediated endocytosis.     

White fat cell alpha-adrenergic receptors and responsiveness in altered thyroid status

[³H]-Dihydroergocryptine was used to identify α-adrenergic receptors in a crude adipocyte membrane fraction obtained from hyperthyroid, hypothyroid and euthyroid hamsters. Hyperthyroidism produced a 35–45 % decrease in the number of both the high and the low affinity [³H]-dihydroergocryptine binding sites but failed to affect the respective affinities of both these sites. On the other hand, binding of [³H]-dihydroergocryptine was unaltered in membranes of hypothyroid animals. Hamster adipocyte α-adrenergic responsiveness, reflected by the increment of epinephrine-stimulated cyclic AMP synthesis induced by phentolamine, was 50 % reduced by hyperthyroidism but unchanged by hypothyroidism. These results which demonstrate that thyroid hormones can regulate the density of adipocyte α-adrenergic receptors, suggest that in human fat cells where catecholamines produce opposite α- and β-adrenergic effects on lipolysis, the increased α-adrenergic responsiveness found in hyperthyroidism could be due, at least in part, to a reduction in the number of α-adrenergic receptors.     

The response of the anterior pituitary and testes to synthetic luteinizing hormone-releasing hormone (LHRH) and the effect of castration on pituitary responsiveness in the maturing chicken fed aflatoxin

The responsiveness of the anterior pituitary to exogenous luteinizing hormone-releasing hormone (LHRH; 20 micrograms/kg body weight) and the subsequent stimulation of testosterone secretion by the testes was studied after administration of dietary aflatoxin (10 ppm) to 9-wk-old male chickens. In both control and aflatoxin-treated males, there were significant (p less than 0.05) increases in plasma luteinizing hormone (LH) concentrations following LHRH administration, which peaked at 5 min post injection and declined thereafter. Plasma testosterone levels increased soon after the LHRH injection in control males, secondary to elevated LH levels in the peripheral circulation, and continued to increase throughout the experimental period. In contrast, this LH-induced elevation in plasma testosterone was delayed in aflatoxin-treated males, with no substantial increase until 20 min post-LHRH injection. In a subsequent experiment, castration of aflatoxin-fed males resulted in an altered response to exogenous LHRH, as compared to their intact counterparts. Based on these data, it appeared that while the LH-secretory capacity of the anterior pituitary was not diminished in birds receiving aflatoxin, the testicular response to exogenous LHRH was altered during aflatoxicosis. Additionally, the effect of castration on plasma LH profiles after LHRH administration provides preliminary evidence for extra-testicular effects of dietary aflatoxin on reproduction in the avian male.     

Superfused pituitary cell cultures: effects of culture conditions on apparent responsiveness to LHRH stimulation administered as short duration pulses

We wished to study estrous cycle related differences in LH and FSH responsiveness to pulsatile LHRH. Such studies are very difficult to perform in vivo under controlled conditions; therefore, an in vitro superfused anterior pituitary cell culture system was evaluated for its capacity to support differences in estrous stage associated LHRH responsiveness. Three vital culture system parameters were evaluated; these parameters were (1) culture medium composition, (2) duration allowed for cell attachment to microcarrier beads and (3) superfusion flow rate utilized during pulsatile LHRH stimulation. It was found that a culture system which utilized 10% Nu Serum in DMEM (final protein concentration of 1.8 mg/ml; final serum concentration of 2.5%), an attachment time of 48 hrs and a flow rate of 0.125 ml/min most successfully maximized LH responsiveness at the lowest serum concentration. These studies indicated that although one may be able to observe LHRH responsiveness under a wide range of culture conditions, responsiveness may nonetheless be maximized by judicious adjustment of culture conditions.     

Pulsatile GnRH administration stimulates the number of pituitary GnRH receptors in seasonally anoestrous ewes

Twenty seasonally anoestrous ewes were pretreated with progesterone for 4 days and divided into four equal groups. Ewes in Group 1 received no GnRH treatment and were slaughtered immediately after progesterone removal. Ewes in Groups 2, 3 and 4 received i.v. injections of 250 ng GnRH every 2 h for 36 h starting at the time of progesterone removal. Ewes in Group 2 were slaughtered immediately after the 36 h GnRH pulsing, while ewes in Groups 3 and 4 were given a bolus injection of 125 micrograms GnRH at this time and were slaughtered 2 and 10 h after the bolus injection, respectively. Blood samples were collected every 30 min from ewes in Group 4 only, from 4 h before the start of GnRH treatment until 10 h after the bolus injection. Pulsing with GnRH resulted in episodic release of LH, and the bolus injection of GnRH was immediately followed by a preovulatory type LH surge in those ewes in which an endogenous surge had no already begun. The pituitary GnRH receptor numbers were significantly higher for the ewes in Group 2 than for any of the other treatment groups, while there was no significant difference in the receptor numbers between Groups 1, 3 and 4. The results suggest an up-regulation of GnRH receptors resulting from pulsatile GnRH therapy.     

Ontogeny of pituitary responsiveness to corticotropin-releasing hormone in rat

The ontogeny of the pituitary's responsiveness to synthetic rat corticotropin-releasing hormone (CRH) in the late prenatal and early postnatal periods of rats was studied by a superfusion system using whole pituitaries. A significant increase of immunoreactive beta-endorphin (IR-beta-Ep) secretion in response to 10(-10) M CRH but not to 10(-11) M CRH was observed in pituitaries from the 15th day of gestation, the earliest day that we tested, whereas 10(-11) M CRH stimulated IR-beta-Ep release from the pituitaries of 17.5-day-old fetuses. Dose-related IR-beta-Ep secretions induced by 10(-12) M to 10(-10) M CRH were observed in pituitaries of 19.5- and 21.5-day-old fetuses, and 1-, 3- and 9-day-old newborn pups. CRH stimulated not only IR-beta-Ep and IR-adrenocorticotropic hormone (ACTH) but also IR-alpha-melanocyte-stimulating hormone (IR-alpha-MSH) secretions from fetal pituitaries. The content of IR-CRH in the hypothalamic extract from 15-day-old fetus was 6.6 +/- 3.6 pg/hypothalamus (mean +/- S.E.M.) and it gradually increased to reach 212.7 +/- 20.3 pg/hypothalamus on the 21.5th day of gestation. However, the content of IR-CRH in the hypothalamus dramatically decreased just after birth and then rapidly increased again from the 5th day after birth. These data indicate that the responsiveness of corticotrophs to CRH is already present on the 15th day of gestation, when the content of IR-CRH in the hypothalamus is extremely low and that the amount of hypothalamic IR-CRH dramatically dropped for several days just after birth in rats.     

Changes in pituitary prolactin responsiveness to TRH during pregnancy

Prolactin plasma concentration during pregnancy was determined in rats treated with thyrotropin-releasing hormone (TRH). Day 0 of pregnancy was defined as the day sperm were first found in the vagina. All blood samples were obtained in unanesthetized rats which had previously received a cannula in the right common carotid. On Day 8 of pregnancy, plasma prolactin concentrations reached a peak between 2400 and 0800 hr (lights on from 0600 to 1800 hr). Injection of TRH (1 microgram/kg body wt) via the carotid artery increased plasma prolactin levels within 5 min. The largest increase occurred when TRH was given during the prolactin surge, whereas much smaller effects were found when TRH was given at the beginning or after the end of the surge period. Thus, the sensitivity of the prolactin cell to TRH appears to be the greatest when the secretory activity of the cell is high. It was then determined whether there was any change in the sensitivity of the prolactin cell to TRH after the prolactin surges had disappeared at midpregnancy. Injection of TRH between 1100 and 1200 hr increased prolactin less on Day 12 than on Day 8 of pregnancy. Since placental lactogen (PL) levels in the plasma are high on Day 12 compared to Day 8, and are inhibitory to prolactin secretion, it was reasoned that PL may be the factor which caused the reduced sensitivity to TRH. However, hysterectomy on Day 11 failed to increase the pituitary responsiveness to TRH the next day. In summary, these data indicate that the pituitary responsiveness to factors that stimulate prolactin, such as TRH, varies with relation to the time of pregnancy or presence of the nocturnal surge. What cellular mechanism is responsible for these sensitivity changes is not known.