Gonadotropin-releasing hormone neuron cell biology.

For those who do not work directly with this famed tissue on the underside of the brain, the hypothalamus can appear to be an intimidating network of neurons possessing a complex intercellular wiring diagram and offering a catalog of secretory products with autocrine, paracrine, and endocrine activities. For those who have been seduced into studying the multifunctional hypothalamus, especially its central role in reproductive biology, things recently have gotten a whole lot better.

Functional cross-talk between receptors for peptide and steroid hormones.

Communication between a cell surface peptide hormone receptor and an intracellular steroid hormone receptor can take various routes, as dictated by the physiology of a particular cell type. There is increasing evidence for a novel route which requires that a peptide hormone receptor pathway converge on a steroid hormone receptor, leading to its activation. One consequence of such a process can be signal amplification for the peptide hormone receptor agonist. This is exemplified by the self-potentiating action of GnRH, which is a critical component in events leading to a surge in LH secretion and ovulation. One signaling pathway stimulated by the GnRH receptor may entail a phosphorylation cascade resulting in progesterone-independent modulation of progesterone receptor activity.

Activation of the progesterone receptor by the gonadotropin-releasing hormone self-priming signaling pathway.

Signal amplification is fundamental to the normal operation of the preovulatory LH surge and is achieved through processes such as GnRH self-priming and augmentation of stimulated LH secretion by progesterone. We have proposed a model for GnRH self-priming that requires cross-communication between a GnRH receptor-activated protein kinase A pathway and the progesterone receptor (PR) to achieve amplification of the GnRH signal. We found that a pulse of GnRH administered to gonadotrope-enriched pituitary cells cultured in medium containing charcoal-treated serum plus estradiol (E2) potentiated the LH secretory response to subsequent GnRH pulses, and this potentiation could be blocked by a PR antagonist, RU486, in the absence of progesterone. Similarly, exposure of gonadotrope-enriched cultures to forskolin augmented the response to a pulse of GnRH, and the augmentation due to cAMP elevation could be reduced by RU486 in the absence of progesterone. To directly test whether stimulation with either GnRH or a cAMP analog results in transactivation of the endogenous PR, we used rat anterior pituitary cells cultured in the presence of E2 and transfected with reporter plasmids containing progesterone-responsive elements (PRE) and either a E1b or a thymidine kinase (tk) promoter linked to the chloramphenicol acetyltransferase (CAT) gene. For pituitary cells transfected with the PRE-E1b-CAT plasmid, exposure to either progesterone, GnRH, or 8-bromo-cAMP (8BrcAMP) for 6 h resulted in an induction of CAT activity which could be suppressed by coincubation with RU486. RU486 by itself had no effect on CAT activity. Similar results were obtained when a plasmid containing a different promoter (PRE-tk-CAT) was used. For cells transfected with a construct lacking a PRE (pSV2CAT), 8BrcAMP was without effect on CAT expression. When cells were made PR-deficient by omission of E2 from the incubation medium and transfected with PRE-E1b-CAT, neither progesterone, GnRH, nor 8BrcAMP was able to induce CAT activity. In summary we found that either GnRH or 8BrcAMP is able to stimulate transcription of reporter genes linked to two different PRE-containing promoters in anterior pituitary cells that contain endogenous PR; this occurred in the absence of progesterone and was suppressed by a PR antagonist. A simple interpretation of these data is that a GnRH-triggered signaling cascade can result in progesterone-independent transactivation of the PR. We propose that, in the normal operation of the preovulatory LH surge, the pathways for GnRH self-priming and progesterone augmentation converge at the PR and that the pathways serve as physiological redundancies to ensure the LH surge.

Steroid and pulsatile gonadotropin-releasing hormone (GnRH) regulation of luteinizing hormone and GnRH receptor in a novel gonadotrope cell line.

Properties of a pituitary gonadotrope include the capacity to regulate gonadotropin synthesis and secretion in response to a GnRH signal. Progress in identifying the steps involved in these processes has been impeded by the lack of a homogeneous in vitro model of gonadotropes. This paper presents functional characterization of a L beta T2 gonadotrope cell line generated by tumorigenesis in transgenic mice carrying the rat LH beta-subunit regulatory region linked to the SV40 T-antigen oncogene. This cell line expresses LH beta, alpha-subunit, and GnRH-receptor (GnRH-R) mRNAs (though not FSH beta), responds to glucocorticoid treatment with a reversible dampening of proliferation, and responds to pulsatile, concentration-dependent GnRH administration with LH secretion. L beta T2 cells presented with four GnRH pulses (10 nM, 90-min interpulse interval) on each of 4 days respond with incremental increases in LH secretion on successive days. This increase was greatest (15-fold) in the presence of estradiol and dexamethasone. Part of the enhanced responsiveness is apparently due to an increase in GnRH-R; pulsatile GnRH treatment alone as well as steroid treatment alone led to an increase in GnRH-R mRNA levels. When secretion was stimulated on day 4 with 54 mM [K+] pulses, bypassing the GnRH-R, the LH-secretory response indicated that the GnRH pulse history as well as estradiol and dexamethasone have actions on L beta T2-secretory capacity distinct from changes in the GnRH-R. This increase can be explained in part by the marked up-regulation of LH beta, but not alpha-subunit, mRNA observed in GnRH-pulsed cells. In summary, L beta T2 clonal gonadotropes exhibit functional characteristics consistent with those of normal pituitary gonadotropes such as LH secretion via a regulated pathway and changes in GnRH-R and LH beta gene expression in response to signaling by GnRH and steroid hormones and therefore should be a useful tool for dissecting the cellular and molecular events involved in these fundamental gonadotrope properties.

Rapid augmentation by progesterone of agonist-stimulated luteinizing hormone secretion by cultured pituitary cells.

Progesterone acts bimodally at the hypothalamus and at the pituitary gland, the sequelae in vivo being either stimulation or inhibition of gonadotropin secretion depending on a host of preconditions. Pituitary cells in culture were studied to characterize the acute action of progesterone on LH secretion. Preliminary studies established that anterior pituitary cells from adult female rats cultured for three days in 10% charcoal treated fetal bovine serum (c/t FBS) resulted in LH secretory responses to GnRH pulses which were half that for cells cultured in untreated FBS or c/t FBS + 0.2 nM 17 beta-estradiol (E2). Under standardized culture conditions (c/t FBS + E2), GnRH self-potentiation was evident. With this system, 90 min exposure to 200 nM progesterone resulted in a 3-fold augmentation of GnRH-stimulated LH secretion without affecting baseline LH. This action was manifested by 45 but not 15 min of progesterone exposure and was inhibited by simultaneous addition of cycloheximide. The augmentation of agonist-stimulated LH release could be elicited up to 4-5 h after progesterone addition. The estimated half-maximal effect was 10(-9) M, and this concentration of progesterone required E2-pretreatment of the cultured cells. In summary, addition of progesterone to cultured anterior pituitary cells pretreated with E2 leads to a concentration-, time-, and protein synthesis-dependent augmentation of pulsatile GnRH-stimulated LH secretion within 45 min of progesterone exposure. This rapid and unambiguous progesterone action in pituitary cells could function in vivo to define the final magnitude of the preovulatory LH surge.

Acute progesterone and 17 beta-estradiol modulation of luteinizing hormone secretion by pituitaries of cycling rats superfused in vitro.

During the rat estrous cycle there are marked changes in the concentrations of serum 17 beta-estradiol (E2) and progesterone. The objective of the present study was to examine the acute effects of E2 and progesterone on isolated pituitaries obtained at 1230 h at each stage of the 4-day cycle. At time zero, single quartered anterior pituitaries were placed in a chamber and superfused at 0.5 ml/min with medium 199 with or without steroids for 360 min; sequential 10-min effluent samples were collected. LHRH (10 ng/ml) was administered as two successive 10-min pulses at 120 and 240 min. The proestrous and the diestrous-II pituitaries in vitro exhibited the characteristic LHRH self-priming response: an increased LH secretory response to a second pulse of LHRH in both the absence and presence of steroids in the superfusion medium; however, the magnitude of the response to the initial pulse of LHRH was reduced significantly when E2 (50 pg/ml) was present. The presence of progesterone (50 ng/ml) resulted in a marked augmentation of the LH secretory response to the first pulse of LHRH; when both E2 and progesterone were included in the superfusion medium, the stimulatory effect of progesterone was reduced. The progesterone augmentation of LHRH-induced LH secretion was present after 60 min of steroid exposure in vitro but was not demonstrable after only 10 min of exposure. Cycloheximide inhibited the progesterone augmentation and the LHRH self-priming without affecting the LH secretory response to the initial LHRH pulse in the absence of steroid. Progesterone augmentation was not observed on estrus and diestrous-I. The presence of E2 in the medium, however, resulted in a significant decrease in the response to the first pulse of LHRH for pituitaries from either estrus or diestrous-I. These data 1) demonstrate that E2 by itself suppresses LHRH-induced LH secretion by the proestrous pituitary and 2) provide proof for a direct stimulatory action of progesterone, in the absence of E2, on the proestrous pituitary and suggest a positive feedback role for progesterone during the preovulatory LH surge. (Endocrinology 108: 413, 1981)

Adenosine 3',5'-monophosphate primes the secretion of follicle-stimulating hormone.

We investigated whether cAMP acts as a mediator for LHRH in either its immediate FSH release action or its self-priming action. Pituitary pieces from cyclic female rats were superfused in vitro in the presence of Bu2cAMP, 8-bromo-cAMP, or forskolin or used as controls. For pituitary pieces from proestrous rats, the first significant increase in the baseline FSH secretion rate occurred after approximately 90 min of exposure to elevated cAMP resulting from forskolin treatment. By comparison, in the same system LHRH caused a 3-fold increase in FSH secretion during a 10-min exposure to the peptide. In contrast to its ineffectiveness as a secretagogue, cAMP elevation resulted in a several-fold augmentation of both LHRH- and elevated K+-stimulated FSH secretion from pituitary pieces from proestrous, but not estrous, rats; for these experiments, superfusion with a cAMP analog or forskolin for varying times preceded a 10-min pulse of either 8 nM LHRH or 47 mM K+. Augmentation of K+-stimulated secretion was evident after 30 min of cAMP elevation. Priming of LHRH-stimulated FSH secretion required 30-90 min of pretreatment with cAMP; longer exposures to cAMP analogs or forskolin were coincident with greater potentiation. Cycloheximide prevented Bu2cAMP augmentation of LHRH-stimulated FSH secretion. These data show that cAMP does not mimic the FSH release action of LHRH, but does augment LHRH- or K+-stimulated FSH secretion with characteristics that lead us to suggest that cAMP mediates, at least in part, the self-priming function of LHRH.

The timing of progesterone-induced ribonucleic acid and protein synthesis for augmentation of luteinizing hormone secretion.

Progesterone addition to pituitary cells pretreated with estradiol leads within 45 min to an unambiguous augmentation of pulsatile GnRH-stimulated LH secretion. To investigate this rapid action, we established the kinetics of early events through manipulation of RNA synthesis, protein synthesis, and progesterone-receptor binding. Female rat pituitary cells cultured in medium containing charcoal-treated serum plus 0.2 nM estradiol were changed to 0.1% BSA-medium +/- 200 nM progesterone at time 0; at 90 and 150 min the cells were challenged with 1 nM GnRH 15-min pulses. The 3-fold augmentation of GnRH-stimulated LH secretion induced by progesterone was inhibited completely by simultaneous addition of 1 microM actinomycin D or emetine as was GnRH self-priming. In another series, the ability of cycloheximide to completely block progesterone augmentation was gradually diminished with delay of addition, but even 90 min after progesterone (30 min before GnRH pulse) cycloheximide resulted in 50% blockade of augmentation. In contrast, inhibition of RNA synthesis 60-90 min after progesterone introduction had little or no effect on progesterone augmentation. The temporal profile of inhibition by the progesterone antagonist RU486 was indistinguishable from that resulting from blockade of RNA synthesis and suggests that continual activation of the receptor is required for continued RNA synthesis. In summary: 1) both RNA and protein synthesis are required for GnRH self-priming; and 2) progesterone augmentation of GnRH-stimulated LH secretion requires RNA synthesis and synthesis of protein(s) which appear to be turning over rapidly, accumulating slowly, or both.

Tetrodotoxin augmentation of secretagogue-induced luteinizing hormone secretion.

Gonadotrophs are known to possess voltage-sensitive Na channels. We used two experimental systems, proestrous rat anterior pituitary tissue superfusion and 17 beta-estradiol-treated rat anterior pituitary cells in primary culture, to examine the effect of Na channel inhibition on LH secretion. We found that a blocker of voltage-sensitive Na channels, tetrodotoxin (TTX), significantly augments LHRH- and elevated extracellular [K+]-induced LH secretion 20-90%. The augmentation of LHRH-induced secretion was demonstrable for both experimental systems and was independent of the time of TTX exposure. These results differ from previous studies in which TTX was without effect or was found to inhibit LH secretion. These discrepancies may be explained, in part, by the demonstration that TTX augmentation requires relatively low TTX concentrations (10(-6)-10(-8) M) and is not demonstrable at higher concentrations, requires submaximal LHRH concentrations (10(-10)-10(-9) M), and requires exposure of cultured cells to 17 beta-estradiol. The site of TTX action could be either directly on gonadotroph voltage-sensitive Na channels or indirect via modulation of Na channels of a paracrine modulator of gonadotroph function. The mechanism by which TTX Na channel blockade augments secretagogue-induced LH secretion is unknown; however, the data are interpreted as favoring direct action of TTX on the gonadotroph, with Na channel blockade affecting a site or sites common to both LHRH and elevated extracellular [K+]. Whether the inhibition of Na channels is one of the several effects of LHRH-receptor interaction remains to be determined.

Modification of luteinizing hormone secretion by activators of Ca2+/phospholipid-dependent protein kinase.

We investigated the role of Ca2+/phospholipid-dependent protein kinase (protein kinase C) in LH secretion using rat anterior pituitary pieces obtained at known stages of the estrous cycle and superfused in vitro. Secretagogues were administered as 10-min (LHRH) or 30-min (all others) pulses. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) results 2 h later in an amplification of LHRH-induced LH secretion in a concentration (1 nM to 1 microM)-and protein synthesis-dependent manner in proestrous, but not estrous, pituitaries; the diacylglycerol analog 1-oleoyl-2-acetylglycerol (OAG) also augments subsequent LHRH-induced secretion. At 1 microM, PMA alone increases the LH secretory rate, but with a pattern different from that induced by LHRH; the characteristics of the PMA response are affected by prior exposure to LHRH, estrous cycle stage, and cycloheximide. Pretreatment with either 8-bromo-cAMP or forskolin results in augmentation of subsequent LHRH-induced secretion without affecting baseline secretion. If the cells are exposed simultaneously to forskolin and OAG, but not 8-bromo-cAMP and OAG, the augmentation is dampened. This preliminary result suggests a possible interaction between protein kinase C and cAMP-dependent protein kinase in LH secretion regulation. We conclude that, regarding initiation of LH release, protein kinase C appears to be but one of a complex of mediators required for the secretory response to LHRH. Regarding the amplification of LHRH-induced release, activation of protein kinase C may be a component of the LHRH self-priming response.

Progesterone regulation of the progesterone receptor in rat gonadotropes.

For rat pituitary cells, progesterone receptor (PR) protein localizes to gonadotropes and PR messenger RNA is induced by E2 and rapidly but transiently down-regulated by progesterone. Here we quantitatively establish the down-regulatory effect of progesterone on PR protein and evaluate possible mechanisms. Nuclear PR-immunoreactivity (PR-IR) in gonadotropes, identified by dual immunofluorescence, was analyzed by quantitative confocal microscopy. Pituitary cells from female rats were cultured +/- 0.2 nM E2 for 3 days. We confirmed the E2 requirement for PR induction in gonadotropes and determined that the increase in PR-IR required about 24 h. After removal of E2, PR-IR decreases were not found until 24-36 h. Addition of progesterone (40 nM) to E2-treated cells led to a dramatic loss in PR-IR by 9 h (26% of control); by 24 h, PR-IR was barely detectable. Reappearance of nuclear PR-IR required progesterone removal (8-fold increase by 12 h after progesterone removal) and protein synthesis (cycloheximide inhibited the reappearance of PR-IR). Although progesterone decreased PR-IR whether or not E2 was present concurrent with progesterone, the recovery of PR-IR required E2. RU486 completely blocked progesterone-induced PR down-regulation. Because the sustained progesterone-induced loss of PR protein did not correlate with previously reported temporal changes in PR messenger RNA levels, we examined a role for protein degradation. When cells were coincubated with progesterone and the proteasome inhibitor, MG132 (1 microM), the expected decrease in PR protein was abrogated. In summary, progesterone leads to a rapid and extensive reduction in nuclear PR protein in gonadotropes. The progesterone-dependent down-regulation of PR occurs, at least in part, by a proteasome-mediated pathway. Recovery of PR protein requires removal of progesterone, the presence of E2, and protein synthesis. These dynamic changes in nuclear PR levels coincide with the temporal extent of the preovulatory LH surge in rats and could provide a basis for progesterone's biphasic action on LH secretion.

Luteinizing hormone secretion from wild-type and progesterone receptor knockout mouse anterior pituitary cells.

The progesterone receptor (PR) has a central role in the hypothalamo-pituitary events culminating in the preovulatory LH surge, and mice with genetically ablated PR provide a model for dissecting cellular pathways subserving this role. The aims of this study were to determine 1) whether the GnRH self-priming response and acute progesterone augmentation of secretagogue-stimulated LH secretion are present in cultured wild-type (WT) mouse pituitary cells, and 2) whether the PR is essential for self-priming by comparing the responses in PR knockout (PRKO) cells. Pituitary cells from ovariectomized WT or PRKO mice cultured +/- 17beta-estradiol (E(2)) for 3 days were challenged with hourly pulses of 1 nM GnRH or 54 mM K(+). A background of E(2) had no effect on the initial LH secretory response for either WT or PRKO cells. However, for subsequent GnRH pulses, E(2) was permissive for the GnRH self-priming response in WT cells. PRKO cells exhibited a blunted GnRH self-priming response. Exposure to progesterone for 90 min before secretagogue stimulation resulted in a modest (1.5-fold) augmentation of the LH response to GnRH but not K(+) pulses in WT cells; progesterone had no effect in PRKO cells. Unlike in the rat, the PR antagonists RU486 or ZK98299 failed to prevent potentiation of LH secretory responses to multiple GnRH pulses in WT cells. Although RU486 blocked progesterone augmentation of the initial GnRH pulse, it was ineffective in blocking progesterone's action after multiple GnRH pulses. In WT cells, 8- bromo-cAMP (8-Br-cAMP) was able to substitute for the GnRH priming pulse; 8-Br-cAMP also augmented GnRH-stimulated secretion in PRKO cells but less effectively. 8-Br-cAMP augmented K(+)-stimulated LH secretion in WT and PRKO cells equally. These results suggest that, although mouse gonadotropes show GnRH self-priming, they have adapted strategies different than rat cells for amplifying the GnRH signal as shown by the residual self-priming in PRKO cells, the modest or absent augmentation by acute progesterone of GnRH- or K(+)-stimulated secretion in WT cells, and the reduced ability of PR antagonists to interfere with GnRH self-priming and progesterone augmentation. We speculate that the adaptations could involve, at least in part, differences in the ratio of PR isoforms.

A pathway for luteinizing hormone releasing-hormone self-potentiation: cross-talk with the progesterone receptor.

This study investigates the signaling pathways that lead to acute augmentation of secretagogue-induced LH secretion, the physiologically relevant manifestation of which is LHRH self-potentiation. The consequence of LHRH self-potentiation is an augmented LH secretory response to subsequent exposure to the peptide. Although the mechanism for LHRH self-potentiation remains obscure, the second messenger cAMP and the steroid hormone progesterone share common characteristics in their acute augmentation of secretagogue-induced pituitary LH secretion, suggesting that cross-talk between the peptide and steroid hormone pathways may occur. The progesterone receptor would represent a point of convergence of several effectors known to augment secretagogue-induced LH secretion. In rat anterior pituitary cells cultured in the absence of progesterone, it was found that the progesterone receptor antagonist RU486 (2 nM) inhibits LHRH self-potentiation induced by hourly pulses of 1 nM LHRH. In the absence of added progesterone, RU486 also suppresses the augmentation of LHRH-stimulated LH secretion which is a consequence of increasing [cAMP]i with either 8-bromo-cAMP (1 mM) or forskolin (1 microM) treatment. The extent of the suppression of the cAMP action in the presence of RU486 is similar to that found with the RNA synthesis inhibitor, actinomycin D. The data are consistent with the hypothesis that a LHRH-stimulated protein kinase A cascade acts, in part, through transcriptional activation of the progesterone receptor. It is concluded that the mechanism of LHRH self-potentiation requires cross-talk with the progesterone receptor.

The existence of a possible short-loop negative feedback action of LH in proestrous rats.

The responsiveness of the hypothalamus to electrochemical stimulation (ECS) and of the pituitary to lutenizing hormone-releasing hormone (LHRH) was examined following either endogenous LH release or exogenous LH infusion on the afternoon of proestrus in rats that had received Nembutal to block the LH surge. In all animals, sequential jugular blood samples were taken and LH was determined by radioimmunoassay. Unilateral ECS of the medial preoptic area (MPOA) resulted in a peak plasma LH of 121.1 +/- 8.6 ng/ml 120 min after stimulation; ECS of the contralateral MPOA of the same animals at 200 min resulted in a peak plasma LH of 66.9 +/- 5.6 ng/ml or half that obtained following the first ECS. To eliminate a pituitary role in this phenomenon, 250 ng LHRH was given iv at 200 min following unilateral MPOA-ECS. The peak in plasma LH following LHRH (510.7 +/- 60 ng/ml) was not depressed and, in fact, was significantly greater than that obtained in response to LHRH without prior ECS-induced LH release (143.9 +/- 19.6 ng/ml). To compare the responses obtained following exogenous LH, purified rat LH (4 mug) was infused iv over 60 min. When plasma LH had returned to pre-infusion levels, an iv pulse of 250 ng LHRH was administered; the peak in plasma LH was similar to that obtained in response to LHRH in animals without prior LH infusion. Finally, in another group of LH-infused rats (4 mug/60 min), MPOA-ECS was performed, resulting in a plasma LH peak half that obtained without prior exogenous LH exposure. These studies suggest decreased hypothalamic sensitivity to stimulation on proestrus due to a negative feedback of either LH or LH-induced steroids.

Androgen modulation of luteinizing hormone secretion by female rat gonadotropes.

In the female, androgens can have negative and positive actions in the regulation of LH, but it is not clear how they may function during the reproductive cycle. Toward resolving these potentially conflicting roles for androgen, we used an in vitro model of preovulatory gonadotropes to examine the effect of proestrous levels of testosterone (1.7 nM) or dihydrotestosterone (DHT; 0.7 nM) on LH secretion in response to pulsatile GnRH (1 nM) or elevated extracellular K+ (54 mM). For female rat pituitary cells cultured in 17beta-estradiol (E2)-containing medium, androgen treatment for 16 h, but not for 4 h, inhibited the LH secretory response to a pulse of either GnRH or K+ by about 60% and suppressed the acute augmentation action of 20 nM progesterone on GnRH- or K+-induced LH secretion. In the absence of E2, DHT also decreased LH secretion induced by a pulse of GnRH. DHT's suppressive effect on progesterone could be partially overcome with increased progesterone (200 nM) or by removal of DHT during progesterone exposure. For pituitary cells transfected with a reporter plasmid containing three progesterone response elements, DHT only partially suppressed progesterone-stimulated transcriptional activity. The positive action of androgen (16 h) on LH secretion was elicited by multiple GnRH pulses with a latency of about 2 h after the first pulse; this facilitatory action of androgen did not require an E2 background and, therefore, is distinct from GnRH self priming. In summary, these data demonstrate both facilitatory and inhibitory actions of androgen on LH secretion function in female gonadotropes in vitro in the absence or presence of E2; these actions occur with a time course suggestive of a role for androgen in shaping the preovulatory LH surge. Androgen also markedly suppresses progesterone augmentation of stimulated LH secretion, which could be due in part to interference with the trans-activation function of the progesterone receptor.

PR localization and anterior pituitary cell populations in vitro in ovariectomized wild-type and PR-knockout mice.

The PR is critical for normal female reproductive function in mammals, including primates, and the PR-knockout mouse is an important model for establishing PR targets. For example, LH secretion is significantly altered both in vivo and in vitro in female PR-knockout mice, but to establish specific mechanisms affected by the absence of the PR in the mouse requires characterization of wild-type mouse cell biology. As steps toward this, the aims were to establish whether altered LH secretion in PR-knockout mice reflects altered mouse gonadotrope cell lineage during development secondary to PR deletion and to test the assumption that PR in wild-type mouse pituitaries has the same exclusive gonadotrope localization and E2 and progesterone regulation as in rat and monkey pituitaries. As an in vitro model, dispersed pituitary cells from 2-wk ovariectomized wild-type or PR-knockout mice were cultured +/- E2 for 3 d. These cells were subjected to dual immunofluorescence staining for PR and LH, PRL, or GH. The proportion of LH-gonadotropes (8-9%) and somatotropes (26-29%) was not different for PR-knockout and wild-type cultures with or without E2. Lactotrope composition (41-42%) was the same in wild-type and PR-knockout, and E2 resulted in a similar and significant increase in the proportion (57-59%) for both mouse types. Nuclear PR immunoreactivity was absent in all PR-knockout pituitary cells. For wild-type, all LH gonadotropes showed nuclear PR immunoreactivity that was up-regulated by E2 (>10-fold increase). Progesterone exposure for 10 h but not 3 h led to a 40% decrease in PR immunoreactivity in LH-gonadotropes. Unexpectedly, PR immunoreactivity also localized to all lactotropes and was up-regulated by E2 and down-regulated by progesterone. In summary, the absence of PR has no effect on the proportion of LH gonadotropes, lactotropes, and somatotropes in ovariectomized PR-knockout mouse pituitary cultures. For ovariectomized wild-type mice, gonadotropes in the in vitro model contain PR that is up-regulated by E2, but the downregulation by progesterone is modest, compared with that previously reported for an in vitro rat model. In contrast to rats and monkeys, E2-dependent PR also is present in lactotropes of ovariectomized wild-type mice. These results underscore the risks in assuming identical cell biology between rats and mice.

The ovine pars tuberalis: a naturally occurring source of partially purified gonadotropes which secrete luteinizing hormone in vitro.

The pars tuberalis and pars distalis are constant subdivisions of the vertebrate adenohypophysis. Unlike the pars distalis, however, direct evidence of an endocrine function for the pars tuberalis is lacking. The present immunocytochemical study shows the ovine pars tuberalis to be unique in that 1) its only immunoreactive hormone-containing cell type is the gonadotrope; and 2) the vast majority of pars tuberalis cells contain no identifiable pituitary hormones. In vitro superfusion studies demonstrate that the ovine pars tuberalis is capable of baseline and GnRH-stimulated gonadotropin secretion which, on a per mg basis, is qualitatively and quantitatively similar to the secretory capacity of the pars distalis.

Membrane-specific association of annexin I and annexin II in anterior pituitary cells.

Calpactins are members of the annexin family of structurally related Ca2(+)-dependent membrane binding proteins. Recent studies suggest a role for calpactins in the membrane fusion event of exocytosis. We show in this work that two members of the annexin family which are immunologically related to calpactin I (p36, annexin II) and calpactin II (p35, annexin I) are present in anterior pituitary cells. When sheep adenohypophyseal cells are disrupted in the absence of a Ca2+ chelator, immunoreactive calpactins associate with the crude vesicle fraction. Further purification of this subcellular fraction by sucrose density gradient centrifugation revealed a differential distribution: calpactin I was associated with secretory granule membranes and with plasma membranes, whereas calpactin II was found primarily with the plasma membrane fraction. Consistent with the Ca2+ and phospholipid binding properties of the calpactins, extraction of these proteins from the pituitary membranous fractions required sequential treatment with a detergent, octylglucoside, in the presence of 1 mM Ca2+ followed by solubilization with EGTA. Calpactins contain sites for phosphorylation by protein kinase C, and in this study we found phosphoprotein substrates for protein kinase C associated with secretory granule and plasma membranes which could be immunoprecipitated with calpactin antisera. In summary, the characteristics in anterior pituitary secretory cells of these two members of the annexin family lend support to the hypothesis that calpactins, potentially regulated by Ca2+ and by phosphorylation, may have a role in exocytosis.

Steroid regulation of progesterone receptor expression in cultured rat gonadotropes.

During the preovulatory period, the pituitary action of progesterone is biphasic, moving from a severalfold augmentation of the gonadotropin release action of GnRH to a suppression of GnRH efficacy, which occurs in rats over a period of about 12 h, but the extent to which these biphasic effects are dependent on alterations in progesterone receptor (PR) expression is not known. To address this, as well as the localization of PR in cultured rat pituitary cells, we used cells from ovariectomized rats cultured +/- 0.2 nM E2 with acute progesterone treatment on day 3. Northern blot of poly(A+) RNA extracts showed multiple PR messenger RNA (mRNA) transcripts between 4.8-10.2 kb; E2 treatment led to a 5- to 6-fold increase in the predominant PR mRNA transcripts (5.1 and 10.1 kb). In the presence of E2, 200 nM progesterone resulted in a decrease in steady-state PR mRNA levels by 3 h of exposure, with the greatest decrease around 6 h (50% of E2 control) and recovery by 12 h. Similarly treated pituitary cultures were subjected to dual immunofluorescence staining for LH and PR. In the absence of E2, PR was undetectable. In the presence of E2, essentially all LH-positive cells were positive for PR and only 1-2% of PR-immunopositive cells were negative for LH, possibly reflecting FSH-exclusive gonadotropes. PR staining was predominantly nuclear, but 20 nM progesterone led to a gradual increase in cytoplasmic staining, with the nuclear-to-cytoplasmic ratio decreasing to near unity by 9-12 h of exposure. In summary, we show for the first time, that PR colocalizes with LH in cultured female rat pituitary cells and that E2 induces expression of PR mRNA, as well as PR protein, in rat gonadotropes. In the presence of E2, progesterone causes a rapid but transient down-regulation of PR message; recovery of PR mRNA is accompanied by an increase in cytoplasmic PR, suggestive of an increase in synthesis. These dynamic changes implicate the gonadotrope PR as having a significant role within the temporal context of the rat preovulatory period.

Characteristics of the adenohypophyseal Ca2+-phospholipid-dependent protein kinase.

The Ca2+-phospholipid-dependent protein kinase, initially described by Takai et al. (J. Biol. Chem. 254, 3692-3695, 1979), has been identified in the anterior pituitary gland of the rat and sheep. The enzyme is essentially undetectable in initial cell extracts but marked activity is manifest following DEAE chromatography, suggesting the potential presence of an endogenous inhibitor of this enzyme. Two forms of this protein kinase exist in both sheep and rat anterior pituitary gland, both of which are similarly dependent upon Ca2+, phosphatidyl serine and diacylglycerol. Several endogenous substrates for this protein kinase have been observed in both the pars distalis and pars tuberalis of the sheep adenohypophysis.