Thomas D. Landefeld

FOLLICLE-STIMULATING HORMONE BETA SUBUNIT MESSENGER RIBONUCLEIC ACID CONCENTRATIONS DURING THE RAT ESTROUS CYCLE

Serum follicle-stimulating hormone (FSH), pituitary FSH content and FSH beta subunit mRNA concentrations were measured at 1 to 3h intervals throughout the 4 day estrous cycle in rats. Serum FSH was stable (range 200-320 ng/ml) apart from the biphasic proestrus surge (5 fold elevation) which was present from 1800 h of proestrus through 0800 h on estrus. Basal FSH beta mRNA concentrations from late metestrus through the afternoon of proestrus were 0.10 +/- 0.04 f mol cDNA bound/100 micrograms pituitary DNA. The major increase in FSH beta mRNA began at 2000 h on proestrus, 2 h after the initial rise in serum FSH and peak mRNA concentrations (0.43 +/- 0.08 f mol cDNA bound) occurred at 0200 h on estrus. FSH beta subunit mRNA concentrations were again increased at 2300 h on estrus (peak 0.24 f mol cDNA bound) and remained elevated through 1700 h on metestrus. Pituitary FSH content was transiently increased during metestrus and diestrus, but was elevated at 1000 h through 1900 h on proestrus (peak 5-fold increase). FSH content fell rapidly at 2000 h and remained low until 1400 h on estrus when values again rose. These data show that FSH beta mRNA is increased 4-5 fold during the proestrus FSH surge, and a smaller increase occurs on metestrus in the absence of elevated FSH secretion. The increased concentrations of FSH beta mRNA occurred at different times to the previously reported changes in alpha and LH beta mRNAs. Therefore, the data suggest that different mechanisms are involved in the regulation of LH and FSH beta subunit gene expression during the 4-day estrous cycle in rats.

Regulation of LH beta subunit mRNA in the sheep pituitary gland during different feedback states of estradiol

The feedback effects of gonadal steroids on the amounts of in vitro translated luteinizing hormone (LH) beta subunit were examined using cell-free assays. These amounts were then correlated with serum and pituitary concentrations during various feedback states. RNA was prepared, translated and products identified by immunoprecipitation and gel electrophoresis. The amounts of beta subunit varied in a pattern similar to that observed for alpha subunit. In ovariectomized ewes, the amounts of beta were 2-3X those seen in negative feedback groups and slightly more than those seen in animals exhibiting an LH surge. The pituitary LH concentration in ovariectomized ewes was also higher than those seen in the other groups, however, the serum concentrations in the positive feedback group were the highest of all groups. These results provide evidence for: 1) a separate, but coordinate, control of gonadotropin subunit synthesis; and 2) a contribution of subunit synthesis to the effects of positive and negative steroid feedback on pituitary LH amounts.

Inhibin secretion during the rat estrous cycle: Relationships to FSH secretion and FSH beta subunit mRNA concentrations

Serum inhibin and FSH and FSH beta subunit mRNA levels were measured at 3h intervals throughout the 4 day estrous cycle in female rats and hourly between 1000 and 2400 h of proestrus. On proestrus, serum inhibin concentrations fell during the late morning-early afternoon, then increased transiently during the late afternoon gonadotropin surges. Inhibin levels decreased during the late evening of proestrus, coincident with the FSH surge-related rise in FSH beta mRNA levels. Serum inhibin remained relatively stable during estrus and early metestrus, but rose during the late evening of metestrus and remained elevated until early diestrus. FSH beta mRNA levels were elevated on late estrus and early metestrus and declined during the evening of metestrus as serum inhibin levels increased. These data show that concentrations of serum inhibin change during the estrous cycle and that a general inverse relationship exists between serum inhibin and FSH levels and FSH beta mRNA concentrations in the pituitary. This suggests that inhibin may inhibit FSH beta gene expression and FSH secretion during the 4 day cycle in female rats.

Hormonal measurement in rat anterior pituitary cell cultures: Loss of immunoreactive LH counteracted by fetal calf serum and bacitracin

Immunoassayable LH in media samples from rat anterior pituitary cell cultures declines during storage and only 20% of the LH remains after 4 weeks at -20 degrees C. The LH loss was not due to bacterial contamination or to damage to the hormone from repeated freezing and thawing. SDS-PAGE of 125IrLH in media samples showed greater recovery of 125IrLH when 1 mM bacitracin or 2% fetal calf serum were present in the medium. The ratio of intact: subunit 125IrLH was unchanged by the presence of bacitracin or fetal calf serum indicating that the loss of immunoreactive LH was not due to dissociation of intact hormone. LH appears to be irreversibly altered in stored culture media, a process which can be prevented by the addition of bacitracin or fetal calf serum to the media prior to storage. The use of either substance allows accurate and reproducible measurement of LH released from pituitary cells in culture.

Toward an Understanding of Interfaces Between Nutrition and Reproduction: The Growth-Restricted Lamb as a Model

For many basic and clinical scientists who focus on mechanisms underlying female puberty, the fundamental question has become, “How does the developing female determine when she has grown sufficiently to begin reproductive cycles?” Other investigators, interested in the reason for cessation of reproductive cycles after puberty in response to weight loss or high level exercise, are seeking an understanding about how the reproductive system senses these altered physiologic states.

The cell free synthesis of bovine lutropin β subunit

Summary RNA prepared from bovine pituitary glands directs the synthesis of trichloroacetic acid-insoluble proteins in the wheat germ cell-free system. Antisera specific to bovine lutropin (LH) beta subunit precipitated a product with a molecular weight of approximately 17,000, suggesting a precursor. Its specificity was demonstrated by the failure of antisera to other pituitary hormones, including LH alpha and thyrotropin (TSH) beta, to successfully precipitate it and also by its competition with unlabeled LH β for the beta antisera. The addition of microsomal membranes resulted in the partial processing of this 17,000 dalton protein to a product of higher molecular weight (Mr∼ 19,000), suggesting glycosylation.

Pit-1/ghf-1 transcription factor expression in rodent pituitaries

The Pit-1/GHF-1 (Pit-1) transcription factor is important for the development of anterior pituitary cells that produce GH and PRL. We examined the expression of Pit-1 mRNA in pituitary tissues from rats and mice. Analysis of pituitaries from normal and GHRH transgenic mice showed that Pit-1 transcripts were readily detected in normal, hyperplastic, and neoplastic pituitaries. A cell line (GHRH-CL1) established from a GhRH transgenic mouse pituitary tumor in our laboratory also expressed Pit-1 mRNA. Normal rat pituitaries and those with estrogen-induced PRL cell hyperplasia expressed Pit-1 mRNA. There was a decrease in Pit-1 mRNA in hyperplastic rat pituitaries concomitant with a decrease In GH mRNA amounts and an increase in PRL mRNA amounts after estrogen treatment. Similarly, analysis of GH3 cells in vitro showed that estrogen and bFGF modulated PRL but not Pit-1 mRNA levels. Pit-1 mRNA was localized by combined in situ hybridization and immunohistochemistry to predominantly GH and PRL cells, although some TSH and LH cells in the rat pituitary also expressed Pit-1 mRNA, indicating wide distribution of the mRNA for this transcription factor in various anterior pituitary cell types. Analysis of cell proliferation in normal rat pituitary and GH3 cells revealed that estrogen and bFGF stimulated cell proliferation in normal pituitaries but inhibited proliferation in GH3 cells, whereas Pit-1 transcripts remained unchanged in both groups of cultured cells. These results indicate that Pit-1 mRNA is readily detected in normal, hyperplastic, and neoplastic rodent pituitaries. Changes in Pit-1 mRNA amounts appear to correlate more closely with changes in GH than PRL mRNA levels in cultured pituitary cells.Endocr Pathol 4:146–154, 1993.

Prolactin messenger ribonucleic acid concentrations throughout the ovine estrous cycle: Assessment relative to prolactin serum and pituitary amounts

Prolactin (PRL) mRNA concentrations were assessed by nucleic acid hybridization assays in pituitaries of ewes representing the defined stages of the ovine estrous cycle. Concomitantly, pituitary and serum PRL concentrations were measured in these ewes using radioimmunoassays. It was observed that PRL serum, pituitary and mRNA concentrations tended to increase near the time of the gonadotropin preovulatory surge, particularly between 24 hrs before behavioral estrus to 5 hours after estrus. However, the changes in PRL mRNA, serum and pituitary concentrations were shown not to be statistically significant. These data suggest that PRL production during the sheep estrous cycle is maintained without dramatic changes in synthesis or secretion.

Development of Ovarian Responsiveness: Follicle Maturation and Luteinization

ABSTRACT Although granulosa cells may be isolated by simple expression from Graafian follicles, the viability and functional activities of the resulting cells have been markedly enhanced by incubating ovaries sequentially in EGTA and hypertonic sucrose prior to expression. It is believed that this treatment gently dissociates interconnecting gap junctions and thereby reduces the extent of cellular disruption that usually results from expression. By incubating the cells with [35S]-methionine at various times after injecting human chorionic gonadotropin (hCG), and then subjecting cellular homogenates to two dimensional electrophoresis, it was found that hCG-induced luteinization is accompanied by changes in synthesis of specific proteins, and that these changes occur prior to the onset of morphologic signs of luteinization. To obtain information on the fate of hCG bound to the granulosa cells, we labeled the non-identical subunits with different radioisotopes of iodine, and demonstrated that the dual labeled hormone was biologically active. The particulate fraction of granulosa cells showed a preferential retention of the beta subunit-associated radioactivity that was not seen in any other target or non-target control tissues. We conclude that a portion of the beta subunit of hCG is selectively retained by luteinizing granulosa cells. Whether or not this retention is causally related to the onset of luteinization remains to be determined.