Progesterone and regulation of the follicle-stimulating hormone (FSH-beta) gene.

Progesterone (P) biphasically modulates follicle-stimulating hormone (FSH) secretion in the rat both in vivo and in vitro with the duration of estrogen priming determining the biphasic nature of the P action, probably through estrogen up-regulation of the anterior pituitary progesterone receptor (PR) levels. P has been also shown to regulate anterior pituitary levels of FSH-beta mRNA in the rat. Although the mechanism of this action has not been determined, steroids may regulate gene expression through the binding of liganded receptors to gene sequences known as hormone response elements (HRE); however, it is not known whether HRE's exist on the rat FSH-beta gene. We have localized a series of progesterone response elements (PRE)-like sequences on the rat FSH-beta gene and have begun testing the hypothesis that P modulates the expression of the rat FSH-beta gene through the direct binding of the P/PR complex to these PRE-like sequences. Electromobility shift assays indicate that these PRE-like sequences bind PR with high affinity and specificity. In addition, when a 361-base pair sequence, which contains the three PRE-like sequences localized in the upstream region of the gene, was cloned into a luciferase expression vector driven by a heterologous promoter and transiently transfected into anterior pituitary cell cultures, progestin stimulation elicited increased luciferase expression. These results indicated that the 361-base pair sequence conferred P-responsiveness to a heterologous promoter. The data further suggest that FSH synthesis in the rat is modulated by direct binding of PR to PRE-like sequences.

A 361 base pair region of the rat FSH-beta promoter contains multiple progesterone receptor-binding sequences and confers progesterone responsiveness.

The rat is frequently used as a model to study the role of progesterone (P) in regulating FSH secretion and synthesis. The ability of P to modulate rat FSH-beta mRNA levels suggests the presence of a functional hormone response element. We have found three PRE-like sequences upstream of the transcription start site in the rat FSH-beta gene. These sequences are herein referred to as PRE-like sequence #1, #2 and #3 with #1 being most distal from the start site. The current studies determined whether these PRE-like sequences bound P receptor (PR) and were functional in regulating the induction of expression by P. Electrophoretic mobility shift assays (EMSA) demonstrated that a single 289 base pair (bp) DNA fragment encompassing all three PRE-like sequences specifically bound PR. Further, PR bound with high affinity to double-stranded oligonucleotides representing individual PRE-like sequences #1, #2 and, with lower affinity to a double-stranded oligonucleotide representing PRE-like sequence, #3. We have cloned a 361 bp sequence from the promoter region of the rat FSH-beta gene encompassing all three PRE-like sequences into a luciferase reporter vector (pGL3-promoter) yielding pFSHbeta361-luc+ which when transiently transfected into primary rat pituitary cell cultures, conferred P-responsiveness to a heterologous promoter. P-responsiveness was dependent upon the presence of PR and was blocked by the PR antagonist RU-486. These data strongly suggest the presence of functional PRE's in the rat FSH-beta gene promoter.

Evidence that the posterior pituitary plays a role in neuropeptide Y and luteinizing hormone-releasing hormone-stimulated gonadotropin secretion in vitro.

Neuropeptide Y (NPY) has been shown to increase gonadotropin secretion directly at the level of the anterior pituitary (AP) in both the absence and the presence of luteinizing hormone-releasing hormone (LHRH). This is interesting because high-affinity 125I-NPY-binding sites have not been found in the AP. However, high-affinity 125I-NPY-binding sites have been localized in the posterior pituitary (PP), and it has been shown that removal of the PP alters luteinizing hormone (LH) secretion in vivo. The following studies were conducted to determine if gonadotropin responsiveness to either NPY alone or to NPY in combination with LHRH was significantly different in AP cells cultured in the presence compared with the absence of PP cells. The studies indicated that NPY-induced LH secretion was significantly greater in the presence of PP cells, while follicle-stimulating hormone (FSH) secretion was not significantly affected. LHRH-induced LH secretion was also significantly greater in the presence of PP cells; however, LHRH-induced FSH secretion was significantly decreased. NPY potentiated LHRH-induced LH secretion in AP cells cultured in both the presence and the absence of PP cells; however, the degree of potentiation was not significantly different whether PP cells were present or absent. These results indicate that the PP may play a role in the responsiveness of the AP to NPY and to LHRH but plays no apparent role in NPY potentiation of LHRH responsiveness.

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-beta and FSH-beta 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 mare's 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.

Direct anterior pituitary modulation of gonadotropin secretion by neuropeptide Y: role of gonadal steroids.

Neuropeptide Y (NPY) acts at the hypothalamus to stimulate LH secretion through increased LHRH secretion. NPY is also cosecreted into the portal system in a pulsatile manner with LHRH and may act at the anterior pituitary to increase LH secretion through potentiation of LHRH responsiveness and possibly through direct stimulation; however, controversy exists concerning this direct action of NPY at the pituitary. Utilizing dispersed anterior pituitary monolayer cell cultures, the first goal of this study was to determine the effects of vehicle, 10(-10) M LHRH, 10(-7) M NPY, 10(-6) M NPY, 10(-10) M LHRH + 10(-7) M NPY, and 10(-10) M LHRH + 10(-6) M NPY on LH and FSH responsiveness. Under these conditions, 10(-6) M NPY alone significantly stimulated LH secretion (630% above basal) and both 10(-7) M NPY and 10(-6) M NPY significantly potentiated LHRH-induced LH secretion (195 and 244% above LHRH alone, respectively). Neither 10(-7) M NPY nor 10(-6) M NPY alone stimulated FSH secretion; however, both 10(-7) M and 10(-6) M NPY significantly potentiated LHRH-induced FSH secretion (130 and 135% above LHRH alone, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Using a cationic carbocyanine dye to assess RNA loading in Northern gel analysis.

In Northern blotting, one must have a means of assessing the uniformity of RNA loaded into each lane of a gel. As an alternative to "common gene" controls and previously published nucleic acid dyes (ethidium bromide, acridine orange, methylene blue), we have utilized a cationic carbocyanine dye (Stains All) for the assessment of RNA gel loading uniformity over the range of 5-25 micrograms RNA/lane. The following protocol is suitable for messages of well-characterized mobility and utilizes xylene cyanol as a 4-kb marker; as such, it will migrate between 28S and 18S rRNA over a wide range of agarose concentrations. Optimally, it is best that the message(s) of interest should migrate either as a smaller species than 18S or as a larger species than 28S; this allows either the 28S or 18S ribosomal band to be separated from the message(s) of interest by severing the gel transversely at the xylene cyanol front. Severing the gel in such a manner makes it possible to simultaneously submit that portion of the gel containing either the 28S or 18S rRNA band to Stains All staining while immediately continuing with the transfer of that portion of the gel containing the mRNA of interest. We have found the dye to interact linearly with rRNA whether data were gathered by densitometrically scanning the gels themselves or photographs of the gels.

Determination of coexisting nuclear transcription rates and cytoplasmic mRNA levels for gonadotropin subunit genes in rat anterior pituitary.

Nuclear run-on transcription assays allow the study of those factors that regulate the expression of a given gene. Because previously published protocols may have been presented in abbreviated form or not developed for nuclei derived from specific tissues, we have established a comprehensive protocol for those who wish to measure transcription of the gonadotropin subunit genes (alpha, LH-beta, FSH-beta) in the anterior pituitary of the rat. The protocol also allows the determination of simultaneously existing cytoplasmic subunit mRNA levels within the same cells. An especially critical step in nuclear run-on transcription assays is the isolation of newly formed labeled mRNA transcripts from the DNA template. In previously published protocols such isolation has been accomplished through rather tedious, multistep methods. To simplify this isolation process, we have evaluated the use of the commercially available product RNAzol. Nascent mRNA transcripts are attached to chromatin and the DNA template; proteinase K and DNase I are typically utilized to disrupt this attachment, thereby facilitating subsequent extraction. We have found that strict adherence to proteinase K and DNase I digestion prior to RNA extraction with RNAzol, followed by two ethanol/NH4OAc precipitations, will enable one to successfully isolate nascent mRNA transcripts free of unincorporated nucleotides. In summary, we present a comprehensive protocol for determining transcription rates and cytoplasmic mRNA levels for the gonadotropin subunit genes in the anterior pituitary of the rat. RNAzol was found to simplify the extraction of newly formed transcripts and should prove applicable with transcription assays utilizing nuclei from a variety of tissue.

Estradiol and progesterone effects on relative luteinizing hormone and follicle stimulating hormone release induced from superfused anterior pituitary cell cultures by defined LHRH pulse regimens.

These studies examined the capacity of estradiol and progesterone to modulate relative luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion from superfused anterior pituitary cells when stimulated with luteinizing hormone releasing hormone (LHRH) pulse regimens of specific amplitude, duration and frequency. There was particular interest in whether such steroid and LHRH treatments induced evidence of divergent LH or FSH secretion. Pituitaries were recovered from adult, 2 week ovariectomized rats and cultured for 48 h with collagen coated Cytodex microcarrier beads. Cultures were preincubated either with or without estradiol (1 or 10 nM) for 48 h and were subsequently incubated for 3,6 or 12 h with 100 nM progesterone. All groups were then pulsed with 1 of 3 LHRH regimens; regimen 1 delivered 8 ng in a single 100 microliters bolus once/h; regimen 2 divided the 8 ng dose of regimen 1 into 3 equal doses administered at 4 min intervals thereby maintaining the 8 ng mass of regimen 1 while extending the duration of exposure; regimen 3 was the same as regimen 2 except that the 3 equal doses were administered at a pulse frequency of 1 per 2 h rather than 1 per h thereby not only maintaining the duration of exposure as in regimen 2 but also reducing the pulse frequency. 1 nM estrogen alone for 48 h had no effect on LHRH stimulated LH release regardless of regimen; however, FSH was increased when hourly pulses of increased duration were applied (regimen 2). When estrogen was increased to 10 nM, regardless of regimen, LH was predominantly inhibited while FSH was unaffected. When 1 nM estrogen was followed by progesterone, both LH and FSH were elevated at 6 h progesterone in response to regimen 2; with 10 nM estrogen however, a divergent response was observed in that LH release was elevated at 6 h while FSH was elevated at 3 h in response to regimens 2 and 3. These results first of all confirm that progesterone in combination with estrogen is capable of exerting both inhibitory and stimulatory effects on gonadotropin secretion; secondly, these studies show that, as a direct pituitary effect, the LHRH regimen and the gonadal steroid milieu are capable of interacting to significantly influence the relative secretion of LH and FSH. The data therefore suggest that the divergent gonadotropin secretion seen in various physiological states in vivo is due likely in part to a combination of estrogen and progesterone priming in combination with the hypothalamic LHRH secretory pattern.

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.

Effect of luteinizing hormone releasing hormone pulse characteristics on comparative luteinizing hormone and follicle stimulating hormone secretion from superfused rat anterior pituitary cell cultures.

We have shown that 4 ng luteinizing hormone releasing hormone (LHRH) pulses induced significantly greater luteinizing hormone (LH) release from proestrous rat superfused anterior pituitary cells with no cycle related differences in follicle stimulating hormone (FSH). Current studies gave 8 ng LHRH in various pulse regimens to study amplitude, duration and frequency effects on LH and FSH secretion from estrous 0800, proestrous 1500 and proestrous 1900 cells. Regimen 1 gave 8 ng LHRH as a single bolus once/h; regimen 2 divided the 8 ng into 3 equal 'minipulses' given at 4 min intervals to extend duration; regimen 3 gave the 3 'minipulses' at 10 min intervals, thereby further extending duration: regimen 4 was the same as regimen 2, except that the 3 'minipulses' were given at a pulse frequency of 2 h rather than 1 h. In experiment 1, all four regimens were employed at proestrus 1900. FSH was significantly elevated by all 8 ng regimens as compared to 4 ng pulses; further, 8 ng divided into 3 equal 'minipulses' separated by 4 min at 1 and 3 h frequencies (regimens 2 and 4) resulted in FSH secretion that was significantly greater than with either a single 8 ng bolus (regimen 1) or when the 'minipulses' were separated by 10 min (regimen 3). In experiment 2, at proestrus 1500, FSH response to the second pulse of regimen 4 was significantly greater than in regimen 2; LH release was significantly suppressed at pulse 2 compared to regimen 2 accentuating divergent FSH secretion. At estrus 0800, FSH response to the second pulse of regimen 4 was significantly stimulated FSH at proestrus 1900, 1500 and estrus 0800, FSH divergence was most marked at proestrus 1500. These data indicate a potential role for hypothalamic LHRH secretory pattern in inducing divergent gonadotropin secretion in the rat.

Control of buffer pH during agarose gel electrophoresis of glyoxylated RNA.

There is a shift in buffer pH routinely encountered during electrophoresis. If one is running glyoxylated RNA on agarose gels, this pH shift can have potentially detrimental effects on experimental objectives if the shift is allowed to proceed unabated; this is due to the fact that the RNA will deglyoxylate if the pH is allowed to rise above 8.0. In order to counteract the shift, the buffer may be continuously recirculated or totally replaced at predetermined intervals. Because constant recirculation may be technically bothersome to achieve and because repeated total buffer replacement may require large amounts of highly purified water, we have studied (a) the time course of the pH change encountered during the electrophoretic process and (b) whether or not simple manual mixing of the buffer system at specific intervals was sufficient to maintain pH within acceptable bounds. We found that, under the given conditions, the cathode pH had nearly reached 8.0 at 25 min and had soared to 10.5 at 40 min. We further found that manual mixing at 20-min intervals not only prevented the cathode pH from rising above 7.8, but also restored the buffer to its initial pH.

Cycle-related LHRH responsiveness of superfused pituitary cells in a Phenol red free medium.

Anterior pituitary cell cultures are frequently used in studying the control of gonadotropin secretion. Historically, many (if not most) of these studies have been performed in the presence of Phenol red as a pH indicator. Phenol red preparations, because of their potential estrogenic activity, may have influenced the results of previous studies defining the relative luteinizing hormone releasing hormone responsiveness of rat anterior pituitary-cells derived from various stages of the estrous cycle. We therefore felt it of interest to investigate this possibility by repeating our previous cycle-related superfusion studies [(1988) Life Sci. 42, 61-72] in the absence of these Phenol red preparations. Comparisons of data obtained in the presence or absence of Phenol red revealed cells derived from late proestrous (19.00) and cultured in the absence of Phenol red continued to evidence the highest LH responsiveness. However, diestrous 1 08.00 cells cultured in the absence of Phenol red were lower in responsiveness than previously observed in the presence of the substance and the responsiveness of proestrous 08.00 and 15.00 in the presence was lower in comparison to the same stages in the absence of Phenol red. The results suggest that Phenol red preparations are capable of modulating LHRH responsiveness in superfusion and that the effect is more pronounced at certain cycle stages than at others.

Ether as an anesthetic for decapitation in the rat: gonadotropin secretion by subsequently established anterior pituitary cell cultures.

Historically, for the establishment of dispersed anterior pituitary cell cultures, rodents have been killed by decapitation without anesthesia. Because decapitation fails to induce immediate unconsciousness, the American Veterinary Medical Association Panel on Euthanasia has recently recommended that rodents should not be decapitated without previous anesthesia or sedation. Investigators are therefore confronted with the dilemma of wishing to euthanize rodents humanely yet not wishing to potentially compromise experimental data through the use of anesthetics. We present our observations on the effects of diethyl ether anesthesia administered prior to decapitation on the gonadotropin secretory characteristics exhibited in vitro by cultured rat anterior pituitary cells. Neither light nor complete (surgical level) ether anesthesia had any statistically significant effect on either luteinizing hormone or follicle-stimulating hormone responsiveness or cell content of luteinizing hormone and follicle-stimulating hormone or DNA content. The data indicate that ether anesthesia would appear to be acceptable for those studies involving subsequent in vitro luteinizing hormone and follicle-stimulating hormone secretion.

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.

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.

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.

Selective modulation of FSH and LH secretion by steroids.

Significant divergence between the pattern of FSH and LH secretion has been observed in the ovulatory cycle, after ovariectomy and during puberty. The presence of an FSH-releasing factor, gonadal FSH inhibiting and releasing peptides and changes in the pulsatile pattern of LHRH secretion are among the postulates used to explain the divergent secretion of FSH and LH. Experiments in our laboratory have shown considerable evidence of differential regulation of FSH and LH secretion by steroids in the absence of gonadal regulatory peptides. Natural and synthetic estrogens show significant differences in the suppression of FSH and LH in the ovariectomized rat using a standard uterine response to the estrogen as the end point. In the immature ovariectomized rat treated with a low dose of estradiol that is sufficient for the synthesis of progesterone receptors to ensure progesterone sensitivity, but not large enough to induce estrogen triggered LH surges, progesterone administration resulted in a pattern of LH and FSH secretion similar to that observed on the day of proestrus in the cycling rat. Selective secretion of FSH was induced in the estrogen primed immature rat model by the administration of progesterone metabolite 5 alpha-dihydroporgesterone (5 alpha-DHP) while selective LH secretion was induced by 3 alpha, 5 alpha-tetrahydroprogesterone (3 alpha,5 alpha-THP). The selective secretion of FSH and LH induced by progesterone metabolites was confirmed in the immature female rat primed with PMSG and maintained in constant light. 5 alpha-DHP was also able to induce a greater release of FSH when administered to the adult cycling rat on proestrus. The priming of the pituitary gonadotrope in secreting a high baseline level of FSH or responding to LHRH in releasing a greater amount of FSH appeared to be an important factor in selective FSH release and such priming can be brought about by 5 alpha-DHP in the absence of gonadal regulatory peptides.

Hypothalamic and pituitary enzymatic degradation of luteinizing hormone-releasing hormone during the 4-day estrous cycle of the rat. Assessment by high-performance liquid chromatography.

Luteinizing hormone-releasing hormone (LHRH) degrading activity may be of physiological significance as a mechanism capable of partial regulation of hypothalamic LHRH release as well as LHRH levels at the gonadotroph. The possibility of cyclic fluctuations in LHRH-degrading activity was investigated in female rat hypothalami and pituitaries. These tissues were collected at selected time points during the 4-day estrous cycle, homogenized, and centrifuged at 100,000 g. Supernatants were incubated with synthetic LHRH, the reactions terminated, and the decapeptide and its products separated by high-performance liquid chromatography. Degradation of LHRH incubated with active cytosol was estimated by comparison of integrated LHRH peak area with that from incubations with heat-inactivated cytosol. Hypothalamic LHRH degradation was depressed during the latter hours of diestrus 2, a time during which the LHRH content in the hypothalamus has been reported to be increasing. From diestrus 24.00 h to proestrus 15.00 h, there was a significant increase in degrading activity. This was then followed by a decline from 15.00 to 18.00 h proestrus; at the time of the LH surge, the activity had not undergone significant increase in comparison to 18.00 h. Pituitary LHRH degradation was significantly increased during the 6-hour period preceding the surge, but was significantly depressed at the surge. The hypothalamic reduction in activity associated with diestrus 2 as well as the hypothalamic and pituitary reductions associated with proestrus may represent a permissive effect allowing increased LHRH accumulation in the hypothalamus and its prolonged action in the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

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.