James L. O'Conner

Evidence that progesterone modulates anterior pituitary neuropeptide Y levels during the progesterone-induced gonadotropin surge in the estrogen-primed intact immature female rat

In a previous study we reported that in vivo estrogen-priming alone, without subsequent progesterone-treatment, was sufficient to maximize NPY potentiation of gonadotropin hormone-releasing hormone responsiveness exhibited in vitro by the rat anterior pituitary. This observation suggests that the necessity, as reported by others, for both estrogen-priming and progesterone-treatment to maximize NPY potentiation of GnRH responsiveness in vivo may be due to progesterone acting primarily at the hypothalamus. Consequently, the current study was performed to determine whether progesterone facilitates gonadotropin secretion in vivo by acting to stimulate hypothalamic synthesis of NPY and the subsequent elevation of anterior pituitary tissue levels of NPY. Intact immature female rats were injected with estradiol at 1700 h on days 27 and 28. On day 29 at 0900 h, the animals received an injection of progesterone (2 mg/kg) or vehicle and were subsequently sacrificed at 1200, 1330 and 1500 h. Rats which received only estradiol injections were used as controls. Surge levels of serum LH and FSH were observed at 1330 and 1500 h. Hypothalamic levels of NPY mRNA at 1200 h on day 29 were higher (P < 0.01) in estradiol-primed rats which received progesterone; there was no accompanying statistically significant change in hypothalamic NPY content. NPY content in the anterior pituitary was significantly increased (P < 0.01) at 1200 h on day 29 in estradiol-primed rats which received progesterone; there was no accompanying significant change in anterior pituitary NPY mRNA levels. Hypothalamic GnRH mRNA content was significantly increased (P < 0.01) at 1330 h on day 29 concomitant with the peak of the gonadotropin surge in the estradiol-primed, progesterone-treated rat. The data indicate that progesterone modulates hypothalamic NPY mRNA and anterior pituitary NPY levels as well as GnRH mRNA levels and that modulation of NPY levels in the hypothalamic-pituitary axis occurs prior to modulation of GnRH gene expression. These studies support the hypothesis that in the estrogen-primed rat, progesterone facilitates the induction of the gonadotropin surge by maintaining hypothalamic synthesis of NPY as well as by modulating anterior pituitary NPY tissue levels.

Regulation of anterior pituitary gonadotropin subunit mRNA levels during the preovulatory gonadotropin surge: A physiological role of progesterone in regulating LH-β and FSH-β mRNA levels

In a previous study we demonstrated that in the ovariectomized estrogen-primed immature rat, progesterone induced a gonadotropin surge while the gonadotropin mRNA subunit levels were either suppressed or unaltered. This observation has now been confirmed using more frequent time points. Progesterone administered at 0900 h was found to suppress LH-beta mRNA levels at 1300, 1400, and 0800 h the next day, with no subsequent effects at 1000, 1200 or 1600 h. FSH-beta mRNA levels were unaffected by progesterone except for a slight elevation at 1400 h and a suppression at 0800 h. Progesterone was either suppressive or had no effect on alpha mRNA levels. Since elevations in LH-beta and FSH-beta mRNA levels were observed in the cycling rat, the observed differences in the ovariectomized estrogen-primed rat could be due to a higher basal synthesis occurring due to ovariectomy. This was indeed the case because LH-beta and FSH-beta mRNA levels were 3.7- and 42.7-fold higher in such animals as compared to intact estrogen-primed rats. In contrast to the ovariectomized estrogen-primed rats, in intact estrogen-primed rats LH-beta mRNA levels were increased at 1000 h and FSH-beta mRNA levels were increased at 1000, 1200 and 1300 h after the administration of progesterone. In pregnant mares serum gonadotropin-primed immature rats, LH-beta, FSH-beta and alpha-subunit mRNA levels were significantly elevated at 1800 and 2000 h, paralleling the serum LH and FSH surge. The progesterone antagonist RU486 (0.2 and 1.0 mg) significantly reduced serum LH and FSH levels at 2000 h. The lower dose reduced LH-beta and alpha-subunit mRNA levels at 2000 h and FSH-beta mRNA levels at 1800 h. The higher dose caused an increase in LH-beta mRNA levels at 1200 and 1800 h and a decrease in FSH-beta mRNA levels at 1800 and 2000 h. In conclusion, the present study provides evidence that preovulatory progesterone plays an important role in the increase in FSH-beta mRNA levels as well as the release of LH and FSH during the normal preovulatory gonadotropin surge. This relationship appears to be dependent on the ongoing rate of synthesis because this does not occur in the ovariectomized estrogen-primed rat in which synthesis is at a high basal level. Furthermore, the correlation with FSH appears to be tighter as compared to LH.

LH and FSH subunit mRNA concentrations during the progesterone-induced gonadotropin surge in ovariectomized estrogen-primed immature rats

Progesterone is able to bring about an LH and FSH surge in the estrogen-primed ovariectomized rat while dexamethasone brings about selective FSH release. The purpose of this study was to determine if progesterone and desamethasone-induced gonadotropin secretion is accompanied by changes in LHbeta and FSHbeta mRNA levels. Gonadotropin alpha-subunit, LHbeta-subunit, and FSHbeta-subunit mRNA levels in the pituitary of ovariectomized rats were suppressed by estrogen treatment and dexamethasone brought about a significant increase in FSHbeta mRNA within 1 h. Progesterone treatment (0900 h) led to a surge in serum LH levels, with peak values at 1400 h. LHbeta mRNA levels were slightly elevated by progesterone at 1400 h. However, an elevation of LHbeta at 1400 h was also observed in the dexamethasone group which did not show an increase in serum LH. Serum FSH levels were elevated at 1400 and 1600 h in the progesterone group and at 1600 h in the dexamethasone group after an initial fall at 1000 h. No correlation was observed between increases in serum FSH during these times with FSHbeta mRNA levels. In conclusion, the ability of progesterone to induce LH and FSH surges in the estrogen-primed ovariectomized rat was not associated with any clear correlative changes in the mRNAs for these hormones. On the other hand, dexamethasone did increase FSHbeta mRNA levels prior to elevating serum levels of FSH. Nevertheless, as a whole, steroid effects on the temporal secretory pattern of LH and/or FSH in the estrogenprimed ovariectomized rat were not mirrored by correlative changes in the mRNA levels for these hormones.

A possible role for progesterone in the preovulatory gonadotropin surge through modulation of LHRH degrading activity

The preovulatory surge of gonadotropins is triggered by estradiol and enhanced to its full magnitude by progesterone. Progesterone may exert this effect through several mechanisms. One of the mechanisms is through the ability of progesterone to induce an increase in the hypothalamic content and release of LHRH. The purpose of this study was to determine if progesterone might not act through yet another mechanism and facilitate LHRH release of the proestrous gonadotropin surge through modulation of luteinizing hormone releasing hormone (LHRH) degrading activity. Sixty-day-old Sprague-Dawley rats were ovariectomized; 14 days later, the estradiol-progesterone milieu of proestrous was mimicked in these animals through the use of estradiol containing silastic implants and subcutaneous progesterone injections. The LHRH degrading activity of the hypothalamus, pituitary and serum were monitored subsequently at preselected time points. In the hypothalamus, estradiol alone was capable of inducing significant increase in degrading activity; progesterone alone had no effect; however, progesterone subsequent to estradiol priming suppressed the increase induced by estradiol alone. In the pituitary, neither estradiol alone nor progesterone alone nor progesterone subsequent to estradiol priming had any significant effect on degrading activity. In the serum, estradiol induced a rapid and significant increase in activity; progesterone alone suppressed activity; progesterone subsequent to estradiol priming induced a similar but more rapid suppression. Therefore, the overall tendency was for estradiol to stimulate and progesterone to suppress LHRH degrading activity in the tissues studied. The results of this study indicate that progesterone has the capacity to suppress LHRH degrading activity and may be one of the mechanisms capable of increasing the availability of LHRH to the anterior pituitary gland thereby facilitating the preovulatory gonadotropin surges.

Luteinizing hormone releasing hormone of fixed pulse frequency and duration: a simplified system for studying the effect of varying pulse concentration on LH release from cytodex I attached anterior pituitary cells

The literature indicates agreement concerning basic differences in the behavior of the pituitary toward pulsatile and continuous luteinizing hormone releasing hormone (LHRH); however, conflicting results seem to exist concerning pituitary behavior toward pulsatile LHRH (Hopkins, 1977; Smith and Vale, 1981). Most superfusion studies have utilized pulses of 15-30 minutes during which the cells were exposed to pharmacological quantities of LHRH. Differences in results may have arisen because of the varying methodologies utilized to administer pulse frequency, pulse duration, and pulse concentration; therefore, the present studies utilized standardized methodology in which the LHRH pulse frequency and pulse duration were maintained constant while the pulse concentration was varied. Pulsatile LHRH of fixed concentration was associated with a relatively rapid loss of responsiveness, while small increases in each subsequent pulse served to prolong the period of responsiveness. The results indicated that seemingly small changes in the methological pattern of LHRH stimulation are capable of exerting an influence on the response to subsequent LHRH stimulation. Caution should therefore be exerted in comparing the results from different experiments utilizing different methodological designs for applying LHRH stimulation. In practical terms, these studies indicate that results must be interpreted carefully from experiments in which a fixed pool of pituitary cells has been repeatedly stimulated by LHRH. This is especially true with dose-response curves generated by this method and with experiments designed to study LHRH self-priming and desensitization.

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.

Superfused pituitary cell cultures: comparative responsiveness of cells derived from various stages of the estrous cycle to LHRH stimulation administered as short duration pulses

We have reinvestigated the question of maintenance of differential LHRH sensitivity in culture and further investigated the role of pulsatile LHRH in the in vitro release of pulsatile LH and FSH at different stages of the estrous cycle. Pituitaries were collected on each day of the 4 day cycle at 0800. In addition, pituitaries were also collected at 1500 and 1900 on proestrous. The cells were dispersed and exposed 48 hrs later to short duration 4 ng LHRH pulses; this dose was optimized for LH release and was applied at a frequency of 1 pulse/60 min. In terms of absolute magnitude of LH response, observed responsiveness was ranked in the following order: proestrous 1900 greater than estrous 0800 greater than diestrous 1 0800 greater than proestrous 1500 greater than diestrous 2 0800. Responsiveness was significantly greater at proestrous 1900 (p greater than 0.01), estrous 0800 (p greater than 0.05) and diestrous 1 0800 (p greater than 0.05) when compared to either of the other stages tested. The heightened LHRH sensitivity of proestrous was therefore maintained in cell culture indicating that the system should be valid for conducting studies on the control of gonadotropin secretion during this period. FSH did not respond in pulsatile manner to the LHRH levels employed further substantiating recent evidence that LHRH seems to function somehow less directly in FSH as compared to LH secretion.

LHRH inactivation by reconstituted horse and fetal bovine sera: assessment by reduction of immunoreactivity and biological activity in pituitary cell cultures

Lyophilized horse and fetal bovine sera are commonly incorporated into the growth media used for primary pituitary cell cultures. LHRH degrading activity has been assumed to exist in these preparations but has not actually been demonstrated. During our studies with pituitary cultures, it became necessary to ascertain if LHRH inactivating activity could be demonstrated in these sera. Luteinizing hormone releasing hormone (LHRH) was preincubated in either serum‐free medium or medium containing fetal bovine and horse serum. Whether LHRH was lost during these incubations was assessed by diminished immunoreactivity as indicated by radioimmunoassay (RIA) and by diminished biological activity as indicated by reduced release of LH from pituitary cell cultures. Both the RIA and bioassay results indicated LHRH inactivating activity; the loss of LHRH could be prevented by inclusion of bacitracin in the incubations.

The antiprogesterone steroid RU-486 does not impair gonadotropin-stimulated luteal adenylyl cyclase activity or gonadotropin release by pituitary cells.

Administration of the antiprogesterone synthetic steroid RU-486 (17 beta-hydroxy-11 beta-[4-dimethylaminophenyl-1]-17 alpha-[prop-l-ynyl]-estra-4,9-dien-3-one) in human and non-human primates induces menstruation and is promising as a new approach to fertility control. To explore the sites of action of RU-486, we investigated in this study the effects of RU-486 upon gonadotropin-stimulable adenylyl cyclase in membrane preparations obtained from human corpus luteum and upon LH and FSH release by a dispersed rat anterior pituitary cell culture. In the presence of a wide range of concentrations (10(-10) to 10(-6) M), RU-486 failed to alter basal or hCG-stimulated adenylyl cyclase activities under conditions allowing either maximal or submaximal hormonal activation. Additionally, enzyme stimulation by GMP-P(NH)P (100 microM), NaF (10 mM) or forskolin (100 microM) was not affected by a high concentration (10(-6) M) of RU-486. These data indicate that RU-486 does not affect gonadotropin receptor binding nor does it interfere with cAMP generation. It is unlikely, therefore, that the compound may modulate human luteal function through changes in plasma membrane lipid mobility or modifications of reactions occurring in plasma membranes as suggested for other steroids in several membrane systems. The present observations are compatible with previously published in vivo studies suggesting that RU-486 activity does not involve a direct antigonadotropic effect at the primate corpus luteum level. We also found that RU-486 (10(-12) to 10(-7) M) did not alter the basal release of gonadotropins by the pituitary cells, nor did the compound impair the response of these cells to maximally or submaximally effective concentrations of LHRH. Thus, these data suggest that the anti-reproductive actions of RU-486 involve no direct effect upon pituitary function. Taken together, these findings support the concept that RU-486 exerts its effects on the luteal phase exclusively by a local action upon the endometrium.

Localizing steroid dehydrogenase activity on acrylamide gels: The perils of “histochemistry”☆

Abstract Two methods were compared for the detection of solubilized human placental 3β-hydroxysteroid dehydrogenase activity in polyacrylamide gels following electrophoresis: (i) nitroblue tetrazolium-nicotinamide adenine dinucleotide (NBT-NAD) coupled histochemical incubations of entire gels and (2) direct radiochemical assay of 1.5-mm gel slices. Both procedures utilized pregnenolone as enzyme substrate. The histochemical method showed “nothing dehydrogenase” activity in that gels developed identical banding patterns in the presence and absence of substrate. The method also consistently failed to indicate enzyme activity at loci easily determined by radiochemical assay.