Effect of restricted feeding on the concentrations of growth hormone (GH), gonadotropins, and prolactin (PRL) in plasma, and on the amounts of messenger ribonucleic acid for GH, gonadotropin subunits, and PRL in the pituitary glands of adult ovariectomized ewes.

The effects of long term restricted feeding on the synthesis, storage, and release of GH, LH, FSH, and PRL were examined in adult ovariectomized ewes. Two groups of six ewes were fed a diet of either 1000 g/day (normal feeding) or 400-600 g/day (restricted feeding) hay for 20 weeks. Restricted feeding increased mean plasma GH concentrations and the amplitude of GH pulses, but did not affect GH pulse frequency. In contrast, mean plasma LH and FSH concentrations and LH pulse frequency were decreased by restricted feeding. Mean plasma PRL concentrations were unaffected by treatment. The levels of mRNA for GH in pituitary cytosol were increased by restricted feeding, but no changes were seen in mRNA levels of alpha-subunit, LH beta, FSH beta, or PRL. The pituitary contents of hormones measured did not change with the level of feeding. In conclusion, these data show that long term restricted feeding affects anterior pituitary function in adult ewes, presumably reflecting alterations in the secretion of hypothalamic releasing and inhibiting factors.

Cimetidine, a histamine H2 receptor antagonist, occupies androgen receptors.

The histamine H2 receptor antagonist cimetidine is in increasing usage in the medical management of peptic ulcer. In clinical trials, its most frequent side effect is gynecomastia. Such estrogenic/antiandrogenic manifestations are well known side effects of treatment with digitoxin or spirolactones. Both of these drugs share a common skeleton with the steroid hormones and have been shown to occupy estrogen and/or androgen receptors. Cimetidine has no measurable affinity for rat uterine estradiol receptors, but competes for tritiated dihydrotestosterone-binding sites in mouse kidney preparations with a displacement curve parallel to that for unlabeled dihydrotestosterone. Steroid receptor-mediated side effects, therefore, may not be confined to molecules with a common skeleton, such as steroids, spirolactones, and cardiac glycosides, but may extend to such apparently unrelated molecules as histamine antagonists and androgens.

Isolation and characterization of bovine follistatin cDNA.

Seven bovine follistatin (FS) cDNA clones were isolated from a bovine ovarian follicle cDNA library. The predicted amino acid sequences revealed that six of the cDNA clones represented an FS precursor of 344 amino acids which corresponded to a mature FS of 315 amino acids (FS 315), with one cDNA clone containing the entire coding sequence including 180 nucleotides of 5' untranslated sequence. The predicted amino acid sequence of the seventh cDNA clone, which differed in the 3' coding sequence, represented a precursor protein of 317 amino acids, corresponding to a mature FS of 288 amino acids (FS 288). This clone encoded an identical amino acid sequence to the other six cDNA clones except that the C terminal of FS 315 was truncated by 27 amino acids. The sequence of bovine FS was found to contain 36 cysteine residues and 2 potential N-linked glycosylation sites. The predicted amino acid sequence of bovine FS has overall sequence homologies of 98% with ovine FS and 97% with human FS.

Pro-opiomelanocortin mRNA levels fall in the fetal sheep pituitary before birth.

In order to clarify the corticotrophic capacity of the fetal sheep anterior pituitary in late gestation, we have measured the relative levels of messenger RNA for the ACTH precursor molecule pro-opiomelanocortin (POMC) in individual fetal sheep anterior pituitaries collected between 100 and 144 days of gestation. The mean relative POMC mRNA:poly(A)+ RNA ratio of the pituitary glands collected between 100 and 135 days (1.35 +/- 0.15) was significantly greater than the mean relative POMC mRNA:poly(A)+ RNA ratio of the pituitaries collected between 141 and 144 days (0.81 +/- 0.09). Northern blot analysis showed that a single band of RNA hybridized with the human POMC cDNA probe in adult and fetal sheep pituitaries. Our results do not contradict the hypothesis that an increase in basal ACTH concentrations after 140 days of gestation could reflect a change in the post-translational processing of POMC in the fetal sheep anterior pituitary.

Differential responses of post-natal rat ovarian cells to FSH and activin.

A role for activin in the acquisition of gonadotropin responsiveness by the post-natal rat ovary was investigated. The inhibin/activin subunits in terms of protein and mRNA, were localised in granulosa cells of the rat ovary at days 4, 8 and 12 after birth. A characteristic pattern of responses to FSH for inhibin and progesterone (P) production was established using a dispersed ovarian cell bioassay. P production by day 4, 8 and 12 cultures was stimulated by FSH, but only when iso-butyl-methyl-xanthine (MIX) was present. In contrast, a basal level of inhibin production was measured in day 4 cultures which was not responsive to FSH or MIX. In day 8 and 12 cultures, inhibin production was FSH-responsive, but only in the absence of MIX. The addition of activin to cultures of day 4, 8 and 12 ovarian cells induced FSH-responsive P production and stimulated both basal and FSH-stimulated inhibin production. These studies indicate a differential response of neonatal ovarian cells to FSH in terms of P and inhibin production. Activin may play a role in facilitating the effects of FSH on signal transduction pathways leading to inhibin and steroid production and therefore be part of the mechanism which determines responsiveness of granulosa cells to FSH.

Rapid and specific lowering of pituitary FSH beta mRNA levels by inhibin.

Hypothalamo-pituitary disconnected sheep were given gonadotropin releasing hormone pulses every 2 h for 1 week, and the effects of inhibin on mRNA for FSH beta, LH beta, alpha-subunit and prolactin examined. Levels of FSH beta mRNA were reduced to approximately 20% 6 h after administration, and to approximately 10% by 30 h; no change was seen in LH beta, alpha-subunit or prolactin mRNA. These data show that inhibin has a very rapid and specific effect on FSH beta mRNA levels directly at the level of the pituitary gland.

Regulation of anterior pituitary gonadotrophin subunit mRNA levels by gonadotrophin-releasing hormone in the ewe.

Abstract Levels of mRNA for the common a subunit and for the beta subunits of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in the pituitary glands of ovariectomized hypothalamo-pituitary disconnected ewes. A control group (n = 7) received 250 ng pulses of gonadotrophin-releasing hormone (GnRH) each hour for one week. To examine the effects of changing GnRH pulse amplitude four sheep were given 250 ng pulses of GnRH for one week and then 25 ng pulses for one week. Plasma LH and FSH concentrations were lowered by reducing the GnRH pulse amplitude but pituitary levels of mRNA for a subunit were increased. Levels of mRNA for FSHbeta and LHbeta were similar with 25 ng and 250 ng pulses of GnRH. To examine the importance of pulsatile versus continuous GnRH inputs, a group of sheep was given a constant infusion of 250 ng/h GnRH for one week. Compared to sheep given 250 ng pulses of GnRH the mRNA levels for LHbeta and FSHbeta were lower in sheep given a constant infusion of GnRH; levels of a subunit mRNA were similar in the two groups. To examine the short-term effects of removing GnRH inputs, ovariectomized, hypothalamo-pituitary disconnected ewes that had been receiving 250 ng pulses of GnRH each hour were deprived of GnRH for 6 h (n = 4) or 30 h prior to slaughter; levels of mRNA for the three subunits were similar to control values in both of these groups. These studies show that wide variation in GnRH pulse amplitude has little effect on mRNA levels for the gonadotrophin subunits but message levels are affected by the mode of GnRH input (constant versus pulsatile). The maintenance of gonadotrophin subunit mRNA levels for at least 30 h after GnRH deprivation suggests that these mRNA species have a long half-life or that transcription continues after GnRH withdrawal.

Short-term regulation of gonadotropin subunit mRNA levels by estrogen: studies in the hypothalamo-pituitary intact and hypothalamo-pituitary disconnected ewe.

In this study the levels of mRNA for the pituitary gonadotropin hormone subunits luteinizing hormone beta (LHbeta), follicle stimulating hormone beta (FSHbeta) and the common alpha-subunit were assessed during the acute feedback stages of estradiol benzoate (EB) action in ovariectomized (OVX) ewes with and without hypothalamo-pituitary disconnection (HPD). In OVX/HPD ewes maintained on hourly pulses of 250 micrograms of gonadotropin-releasing hormone (GnRH) a single i.m. injection of EB in oil caused a biphasic (decrease and then increase) change in plasma LH levels and a monophasic decrease in FSH levels. There was a decrease in pituitary alpha-subunit and FSHbeta mRNA levels during the acute negative (8 h post EB) and through the positive feedback (20 h post EB) stages of the response. No significant change was seen in LHbeta mRNA levels following treatment with EB. In hypothalamic-pituitary intact OVX ewes the same EB treatment as above caused a biphasic change in LH secretion with the positive feedback component being much greater than in GnRH-pulsed OVX-HPD ewes. The levels of mRNA for all three gonadotropin subunits were reduced by 8 h after EB injections and remained low throughout the positive feedback period. These data suggest that the LH surge in this experimental model does not require an increase in LHB mRNA levels. Furthermore, the fall in LHbeta subunit mRNA seen after estrogen injection of OVX ewes is most likely due to an effect of estrogen to decrease GnRH secretion, since pulsatile GnRH replacement prevents this effect. These data also show that estrogen feedback can effect rapid alterations in pituitary gonadotropin subunit mRNA levels. Short-term changes in FSHbeta mRNA are reflected in changes in FSH secretion; the same is not true for LH.

Development of large-scale fractionation methods. VI. An improved method for preparation of antihemophilic factor.

To increase purity and potency of the antihemophilic factor (AHF) concentrate prepared by the American Red Cross method, the following modifications were introduced: (1) A cold extraction step was incorporated to remove cold-soluble impurities. The cryoprecipitate (cryo) was extracted with 0.02 M Tris buffer, pH 7.0 (4 ml/g cryo) at 0 degrees C. Factor VIII loss in this step was negligible. (2) AHF was then recovered from the cold-insoluble portion of the cryo by extraction at 21 degrees C with the same buffer. To increase the AHF concentration, this second extraction step was carried out with a smaller buffer volume (2 ml instead of 3--4 ml/g cryo). The subsequent steps, deprothrombinization, filtration and lyophilization were essentially unchanged. To further increase factor VIII concentration, the dried AHF concentrate was reconstituted to 40 rather than 50% of the initial volume. AHF concentrate prepared on a large scale by this method was 20- to 30-fold concentrated and 40- to 50-fold purified over plasma at a recovery of about 250 factor VIII units per liter of plasma. The final product was readily soluble, clear and almost colorless upon reconstitution.

Wave-form analysis of intrauterine pressure curves with methods and models developed in cardiac research.

With the Hill model of muscle dynamics and a modified cardiac model, a series of computer programs have been generated which permit the description of certain aspects of uterine contractility and uterine function in human parturition. Patterns of labor relating to delivery outcome, dysfunctional uterine activity, and use of drugs are discussed with reference to the parameters measured in the study. Central tendencies among 70 spontaneous and induced labors (6,302 contractions) are presented and discussed. (Am. J. Obstet. Gynecol.

Luteinizing hormone-beta mRNA levels are regulated primarily by gonadotropin-releasing hormone and not by negative estrogen feedback on the pituitary.

Long-term effects of gonadotropin-releasing hormone (GnRH) and/or estrogen on pituitary mRNA levels for the beta-subunit of luteinizing hormone (LH-beta) were determined in anterior pituitary glands from ovariectomized (OVX) ewes. The relative roles of these two factors were assessed by studying hypothalamopituitary disconnected (HPD) ewes with appropriate hormonal treatments. Levels of LH-beta mRNA were increased by ovariectomy and substantially reduced by HPD. Treatment of OVX-HPD ewes with pulses of GnRH (250 ng each 2 h) for 1 week restored LH-beta mRNA levels to OVX levels, whereas treatment with estrogen alone did not alter the low levels found in OVX-HPD ewes. Combined GnRH and estrogen treatment for one week produced LH-beta mRNA levels that were similar to those found in OVX-HPD ewes given GnRH alone; plasma LH pulse amplitudes were also similar in these two groups. From these data we conclude that the long-term negative feedback effect of estrogen to reduce LH secretion is due to a primary inhibition of GnRH secretion and is not a pituitary effect of estrogen. Long-term regulation of LH-beta mRNA is thus primarily regulated by GnRH.

Regulation of follicle-stimulating hormone beta and common alpha-subunit messenger ribonucleic acid by gonadotropin-releasing hormone and estrogen in the sheep pituitary.

The effect of gonadotropin-releasing hormone (GnRH) and/or estradiol (E2) on pituitary messenger ribonucleic acid (mRNA) levels of luteinizing hormone beta (LH beta), follicle-stimulating hormone beta (FSH beta) and the common alpha-subunit were determined in anterior pituitary glands from ovariectomized (OVX) ewes. Hypothalamo-pituitary disconnected (HPD) ewes receiving appropriate hormonal treatment were used to assess the relative roles of GnRH and E2 in directly regulating FSH beta and alpha-subunit mRNA levels. Levels of LH beta mRNA were increased in OVX animals compared with intact controls, and E2 treatment of OVX animals significantly reduced mRNA levels of LH beta and FSH beta. HPD substantially reduced FSH beta and alpha-subunit mRNA levels. Treatment of OVX/HPD animals with pulses of GnRH (250 ng/2 h) for 1 week restored FSH beta and alpha-subunit mRNA to OVX levels. Combined GnRH and E2 treatment significantly lowered FSH beta mRNA levels, but resulted in a rise in alpha-subunit mRNA levels. Treatment of OVX/HPD ewes with E2 alone had no effect on FSH beta and alpha-subunit mRNA levels. These findings indicate that E2 acts directly on the pituitary to negatively regulate FSH beta mRNA levels, and to positively regulate alpha-subunit mRNA levels in the presence of GnRH.

Proopiomelanocortin messenger RNA levels are increased in the anterior pituitary of the sheep fetus after adrenalectomy in late gestation.

We have investigated the effect of bilateral adrenalectomy at 116-119 days' gestation on the levels of the messenger (m) RNA for proopiomelanocortin (POMC) in the anterior pituitary of the fetal sheep and in the ovine placentome during late gestation (134-136 days' gestation). After fetal adrenalectomy there was a significant (p less than 0.001) and sustained increase in circulating ACTH concentrations in the adrenalectomised group (1,838 +/- 155 ng/l at 130-136 days) when compared with the intact control group (131 +/- 25 ng/l at 130-136 days). The mean levels of POMCmRNA relative to 18S RNA were also significantly higher (p less than 0.001) in the adrenalectomised fetal sheep pituitaries (2.8 +/- 0.12; n = 4) than in the intact/control fetal sheep pituitaries (1.31 +/- 0.13; n = 4). In contrast to the findings in the anterior pituitary, POMCmRNA was not detected in RNA extracted from the placentomes of either the adrenalectomised or intact fetal sheep. There was also a significant arteriovenous difference in ACTH concentrations in the umbilical circulation in both adrenalectomised and intact fetal sheep at 134-136 days' gestation. This study demonstrates therefore that the fetal adrenals act to suppress POMCmRNA levels in late gestation and also that the increase in circulating ACTH after adrenalectomy originates from the pituitary and not the placentome.

Glucocorticoid regulation of proopiomelanocortin gene expression in the pituitary gland of hypothalamopituitary intact and hypothalamopituitary disconnected sheep.

Proopiomelanocortin (POMC) gene expression in the anterior pituitary (AP) gland has previously been shown to be positively regulated by CRF and AVP and negatively regulated by glucocorticoids. In the neurointermediate lobe (NIL) of the pituitary, however, POMC gene expression is under tonic inhibitory dopaminergic control. In the present study we have used hypothalamopituitary intact (HPI), ovariectomized (OVX), and OVX/hypothalamopituitary disconnected (OVX/HPD) ewes to examine direct (i.e. nonhypothalamic) effects of glucocorticoids on POMC gene expression in both the AP and the NIL. There was no difference between POMC mRNA levels in intact and OVX sheep. In intact animals treated with dexamethasone, AP POMC mRNA levels were half those of controls. POMC mRNA levels were increased 3-fold in OVX/HPD sheep, compared with OVX, and lowered by dexamethasone to half OVX/HPD levels. In the NIL, hypothalamopituitary disconnection resulted in slightly higher mean POMC mRNA levels than in intact animals but the large intragroup variation did not allow a significant change. Dexamethasone administration had no effect on NIL levels of POMC mRNA in intact or OVX/HPD sheep.