Pituitary Glands Of Female Rats Research Articles

Changes in the expressions of annexin A1, annexin A5, inhibin/activin subunits, and vitamin D receptor mRNAs in pituitary glands of female rats during the estrous cycle: correlation analyses among these factors.

Pituitary gonadotropin secretion is regulated by several pituitary factors as well as GnRH and ovarian hormones. To elucidate the regulatory mechanisms of pituitary gonadotropin secretions, we observed changes in the mRNA levels of pituitary factors, namely annexin A1 (Anxa1) and Anxa5, inhibin/activin subunits, follistatin (Fst), and vitamin D receptor (Vdr), in female rat pituitary glands during the estrous cycle. Additionally, levels of LHβ subunit (Lhb), FSHβ subunit (Fshb), and GnRH receptor (Gnrh-r) mRNA were examined. During proestrus, Anxa1, Anxa5, Vdr, and inhibin α-subunit (Inha) exhibited the lowest levels, while during estrus, activin βB-subunit (Actbb), Lhb, and Gnrh-r were the lowest. Moreover, Fshb exhibited the highest value during metestrus, whereas Fst did not differ significantly. Correlation analyses revealed 16 statistically significant gene combinations. In particular, four combinations, namely Anxa5 and Inha, Anxa5 and Actbb, Inha and Vdr, and Inha and Actbb, were highly significant (P<0.0001), while four combinations, Anxa1 and Anxa5, Anxa1 and Vdr, Anxa5 and Vdr, and Lhb and Gnrh-r, were moderately significant (P<0.001). The remaining eight combinations that exhibited statistical significance were Anxa1 and Inha, Anxa1 and Actbb, Vdr and Actbb, Anxa1 and Fshb, Inha and Lhb, Actbb and Fshb, Actbb and Lhb, and Fst and Fshb (P<0.05). These results highlight strong correlations among Anxa1, Anxa5, Vdr, Inha, and Actbb, thereby suggesting that an interaction among ANXA1, ANXA5, and VDR may lead to further communications with inhibin and/or activin in the pituitary gland.

Sequential preovulatory expression of a gonadotropin-releasing hormone-inducible gene, Nr4a3, and its suppressor Anxa5 in the pituitary gland of female rats.

Functional relationship between nuclear receptor subfamily 4 group A member 3 (Nr4a3) and annexin A5 (Anxa5), which are two gonadotropin-releasing hormone (GnRH)-inducible genes, has been established while evaluating pituitary gonadotropes in relation to follicle-stimulating hormone beta (Fshb) expression. However, the physiological variations that arise due to the differential expression of these genes in the pituitary gland during rat estrous cycle remain unknown. This study aimed to evaluate the Nr4a3 and Anxa5 mRNA expression during the estrous cycle in rats in comparison with the expression of the gonadotropin subunit genes, luteinizing hormone beta (Lhb) and Fshb. Nr4a3 mRNA expression showed a single peak at 1400 h of proestrus during the 4-d estrous cycle. Anxa5 mRNA level was elevated along with increased Fshb mRNA expression after the decline of Nr4a3 mRNA until 2300 h. Lhb mRNA expression levels were not significantly changed during the estrous cycle. Notably, addition of a GnRH antagonist at 1100 h completely eradicated luteinizing hormone secretion at 1400 h and 1700 h of proestrus, and significantly reduced the Nr4a3 mRNA expression level at both the time points. These results suggest that GnRH is, at least partly, responsible for the increase in pituitary Nr4a3, and that the interaction between NR4A3 and ANXA5 is required to regulate Fshb expression during the preovulatory gonadotropin surge.

Gonadotropin-releasing hormone and kisseptin-10 regulate nuclear receptor subfamily 5 group a member 1/catenin beta 1/ nuclear receptor subfamily 0 group B member 1 activity in female rat anterior pituitary gland.

In this study, we tested the hypothesis that modulation of endogenous gonadotropin-releasing hormone (Gnrh) neuronal network activity alters the mRNA expression of nuclear receptor subfamily 5 group A member 1 (Nr5a1), through one of the component of Wnt pathway signaling - catenin beta 1 (Ctnnb1) (its co-activator), and its co-repressor nuclear receptor subfamily 0, group B member 1 (Nr0b1) in the female rat pituitary gland in vivo. Adult ovariectomized rats were given a serial infusion of Gnrh, kisspeptin-10, Gnrh + Gnrh antagonist (Antide), or kisspeptin-10 + kisspeptin antagonist (kisspeptin-234) into the third ventricle of the brain. The anterior pituitary and blood was used to mRNA and protein expression analysis. We demonstrated that Gnrh up-regulates Nr5a1 mRNA expression in the anterior pituitary and induces NR5A1 depletion in gonadotropes. Gnrh administration increased both Ctnnb1 mRNA expression and protein synthesis, and induced activation of cellular Ctnnb1 via translocation from the gonadotropes cytoplasm to nucleus. After kisspeptin-10 treatment, up-regulation of Nr0b1 mRNA and protein expression in the anterior pituitary was observed. These data indicate that Gnrh-neuron-mediated network activity alters Nr5a1 gene transcription and translation in gonadotrope cells and this effect may result from the changes induced in the Ctnnb1 and Nr0b1 gene/protein expression balance.

In vivo oestrogenic modulation of Egr1 and Pitx1 gene expression in female rat pituitary gland.

EGR1 and PITX1 are transcription factors required for gonadotroph cell Lhb promoter activation. To determine changes in Egr1 and Pitx1 mRNA levels in central and peripheral pituitary stimulations, an in vivo model based on i.c.v. pulsatile (1 pulse/0.5 h over 2 h) GnRH agonist (1.5 nM buserelin) or antagonist (2 nM antide) microinjections was used. The microinjections were given to ovariectomised and 17β-oestradiol (E2) (3×20 μg), ERA (ESR1) agonist propyl pyrazole triol (PPT) (3×0.5 mg), ERB (ESR2) agonist diarylpropionitrile (DPN) (3×0.5 mg) s.c. pre-treated rats 30 min after last pulse anterior pituitaries were excised. Relative mRNA expression was determined by quantitative RT-PCR (qRT-PCR). Results revealed a gene-specific response for GnRH and/or oestrogenic stimulations in vivo. Buserelin pulses enhanced Egr1 expression by 66% in ovariectomised rats, whereas the oestradiol-supplemented+i.c.v. NaCl-microinjected group showed a 50% increase in Egr1 mRNA expression. The oestrogenic signal was transmitted via ERA (ESR1) and ERB (ESR2) activation as administration of PPT and DPN resulted in 97 and 62%, respectively, elevation in Egr1 mRNA expression. A synergistic action of GnRH agonist and 17β-oestradiol (E2) stimulation of the Egr1 gene transcription in vivo were found. GnRHR activity did not affect Pitx1 mRNA expression; regardless of NaCl, buserelin or antide i.c.v. pulses, s.c. oestrogenic supplementation (with E2, PPT or DPN) consistently decreased (by -46, -48 and -41% respectively) the Pitx1 mRNA in the anterior pituitary gland. Orchestrated Egr1 and Pitx1 activities depending on specific central and peripheral regulatory inputs could be responsible for physiologically variable Lhb gene promoter activation in vivo.

Immunohistomorphometric Features of ACTH Cells in Juvenile Rats after Treatment with Estradiol or Human Chorionic Gonadotropin

Immunohistomorphometric Features of ACTH Cells in Juvenile Rats after Treatment with Estradiol or Human Chorionic GonadotropinEstradiol and human chorionic gonadotropin (hCG) are very important in controlling the secretory activity of hormone producing cells in the female rat pituitary glands. The aim of the present study was to examine the morphometric parameters of immunohistochemically labeled ACTH cells in juvenile (16th day) female rat pituitaries after treatment with five doses of estradiol dipropionate (EDP) and two doses of hCG during the neonatal period of life. The controls were treated on the same schedule with an equivalent volume of vehicle. All animals were sacrificed 24 h after the last treatment. ACTH-producing cells were studied using the peroxidase-antiperoxidase immunohistochemical procedure. The absolute and relative pituitary weights were increased (p&lt;0.05) only in the EDP treated group by 120.0% and by 121.1% respectively, in comparison with the controls. In this group, the volume of ACTH cells, volume of their nuclei and volume density were significantly decreased (p&lt;0.05) by 6.4%, 33.3% and 46.2% respectively, compared to the corresponding controls. After treatment with hCG, there were no significant (p&gt;0.05) changes neither in the volume of ACTH cells nor in the volume of their nuclei, in comparison with the controls. On the basis of the results obtained in our study, it can be concluded that EDP, injected into female rats during the neonatal period of life, has an inhibitory effect on the immunohistomorphometric parameters of ACTH cells, but such an effect is not clearly expressed after treatment with hCG.

Estrogen Suppresses Retinaldehyde Dehydrogenase 1 Expression in the Anterior Pituitary Glands of Female Rats

Retinoic acid (RA) plays a critical role in cell growth and tissue development. RA is also a regulating factor of pituitary function. RA is synthesized from retinoids through oxidation processes. The oxidation of retinal to RA is catalyzed by the retinaldehyde dehydrogenases (RALDHs), including RALDH1, RALDH2 and RALDH3. Recently, we demonstrated that RALDH1 is expressed in the anterior pituitary glands of adult male rats. However, the expression of RALDH1 in the female pituitary gland and the regulation of RALDH1 expression have not been determined. Therefore, we examined the expression of RALDH1 mRNA in the pituitary glands of adult female rats. By in situ hybridization with digoxigenin-labeled cRNA probes and quantitative real-time PCR analysis, we found that the expression level of RALDH1 was significantly lower in estrus as compared to proestrus, metestrus and diestrus. RALDH1 mRNA levels increased after ovariectomy and decreased remarkably after a 1-week treatment with 17beta-estradiol implants. Estradiol (0.01-100 microg per rat) dose-dependently decreased the expression of RALDH1 in the pituitary glands after 24 hours of subcutaneous administration. These results clearly show that RALDH1 mRNA expression is suppressed by estrogen. We speculate that the generation of RA is regulated by estrogen and that RA plays a role in the estrus cycle through paracrine and/or autocrine mechanisms in the anterior pituitary gland of female rats.

Estradiol suppresses NMU mRNA expression during sexual maturation in the female rat pituitary

Neuromedin U (NMU) suppresses food intake and gonadotropin secretion. However, the developmental transition of NMU expression in the pituitary gland and the regulation of NMU expression are unclear. The objective of this study was to examine the transition of the expression of NMU mRNA in the pituitary glands of female rats from the juvenile period to the mature period of development. Furthermore, factors such as estradiol, insulin, leptin, and inhibin A, whose expressions change throughout puberty and which affect gonadotropin secretion in pituitary cell culture, were examined. In the pituitary gland, the expression of NMU mRNA was significantly lower in 8-week-old rats than in 4- and 6-week-old rats. In the pituitary cell culture, the expressions of NMU mRNA in the estradiol- and insulin-treated groups were significantly lower than in the control group. These results suggest that the expression of NMU mRNA in the female rat pituitary is reduced as the rats develop from the pubertal to the mature period. Additionally, the reduction of NMU expression in the pituitary may be related to the increases in serum estradiol and insulin levels that occur during the pubertal period, which may negate NMU suppression of gonadotropin secretion.

Region‐Specific Expression and Hormonal Regulation of the First Exon Variants of Rat Prolactin Receptor mRNA in Rat Brain and Anterior Pituitary Gland

Recent studies have revealed the occurrence of five first exon variants of the rat prolactin receptor mRNA, suggesting that multiple promoters direct prolactin receptor transcription in response to different regulatory factors. In the present study, regional expression of these first exon variants, as well as two prolactin receptor subtypes generated by alternative splicing, was examined in the brains and anterior pituitary glands of female rats. Expression of the long-form was detected in the choroid plexus, hypothalamus, hippocampus, cerebral cortex and anterior pituitary gland, whereas the short form was detected only in the choroid plexus. E1-3 mRNA, a first exon variant, was detected in the choroid plexus, hypothalamus, and anterior pituitary gland, whereas E1-4 was detected only in the choroid plexus. Other variants were not detectable by the polymerase chain reaction protocol employed in this study. Ovariectomy increased the short form in the choroid plexus and the E1-3 expression in the choroid plexus and pituitary gland, but changes in the long-form and E1-4 expression were minimal. Replacement of oestrogens and prolactin suggest that oestrogens down-regulate E1-3 expression in the choroid plexus and pituitary gland, and that the negative effect of oestrogen is mediated by prolactin in the pituitary gland. The present results revealed the region-specific promoter usage in prolactin receptor mRNA transcription, as well as the involvement of oestrogens in the regulation of E1-3 mRNA expression in the brain and pituitary gland.

Alteration in Prolactin Secretion in Female Ovariectomized Rats by some Endocrine Disrupting Chemicals

There is growing concern that many environmentally important chemicals may be capable of causing a variety of reproductive and endocrine disorders. Recent data seem to suggest that various xenobiotics have the potential to physiologically mimic important estrogen-regulated processes. It has also been shown that certain endocrine disrupting chemicals may have tumorpromoting potential via their ability to alter hormonal control. The purpose of this study is to examine the effects of several chemicals that have been implicated in endocrine disruption on the estrogen-mediated release of prolactin from the adult female rat pituitary gland. Ovariectomized female Sprague Dawley rats animals received DDT, methoxychlor, toxaphene, dieldrin, endosulfan, 17β-estradiol (E-17), and 30 μg/kg or the corn oil vehicle daily for seven days. Immediately following the exposures, blood samples were collected and plasma prolactin levels were measured by radioimmunoassay using [125I] rat prolactin as a tracer. Brain (TIDA) dopamine and DOPAC were also determined.

Postnatal differentiation of the immunohistochemical expression of aromatase P450 in the rat pituitary gland.

At our laboratory, we have recently demonstrated the immunohistochemical expression of aromatase P450 in the pituitary glands of adult rats; this expression was seen to be sex-dependent. In order to determine whether the changes in the expression of the enzyme are related to changes in the gonadal sphere and whether the expression of the enzyme is related to the postnatal differentiation of hypophyseal cytology, in the present work we performed an immunohistochemical study in the rat pituitary gland from birth to old age. The immunohistochemical reaction to aromatase was evident and very generalized at 7 days after birth, with no large differences between the male and female animals. At 14 days the immunohistochemical reaction was decreased in the females, with no changes in the males. At 17 days, aromatase immunoreactivity in the pituitary glands of female rats was very weak whereas the males showed large numbers of reactive cells. These observations were further pronounced at 21 days and 2 months of life. At 24 months, the immunoreactivity found in the pituitary glands of the male rats had almost completely disappeared. Our results show that a postnatal differentiation in the immunohistochemical expression of aromatase occurs; this is tightly linked to sexual activity and is lost in old age. This suggests that hypophyseal aromatase would be related to the mechanisms of action of gonadal steroids on hypophyseal differentiation and secretion.

Oestrogenic regulation of an egr-1 transgene in rat anterior pituitary.

The C(2)H(2) zinc-finger transcription factor Egr-1 has previously been shown to play an essential role within the endocrine system as a molecular determinant of LH beta-subunit synthesis in anterior pituitary gonadotrophs. The extent to which Egr-1 may be a dynamic mediator of changes in gonadotroph function during the oestrous cycle is currently unclear. We have recently produced a novel rat transgenic model of egr-1 gene function in which proximal regions of the rat egr-1 gene drive expression of a reporter gene. In the present study, we have investigated the expression and physiological regulation of our egr-1/d4 enhanced green fluorescent protein (EGFP) transgene in the female rat pituitary gland. In situ hybridization analysis has revealed anterior pituitary-specific expression that is limited to a sub-population of cells that includes immunohistochemically defined gonadotrophs. Expression of the transgene is up-regulated 5-fold following ovariectomy. The transgene also exhibits regulated expression during the oestrous cycle, mRNA levels being significantly raised on pro-oestrus. Using an explant culture system, we have also demonstrated a direct stimulatory effect of 17beta-oestradiol on anterior pituitary transgene and egr-1 expression. The acute response of egr-1 to an oestrogenic stimulus is attenuated by the MEK (MAPK kinase) inhibitor U0126, and is accompanied by increased levels of phospho-p44/42 MAPK protein, suggesting regulation of egr-1 through a MAPK-linked pathway in the pituitary. These findings provide further evidence of cyclical endocrine regulation of egr-1 in the rat, demonstrate that proximal sequences of the egr-1 gene mediate endocrine-regulated expression, and indicate a novel pathway through which pituitary transcriptional responses to oestrogen may be mediated.

Calcitonin Expression in Rat Anterior Pituitary Gland Is Regulated by Ovarian Steroid Hormones

Gonadotroph-derived calcitonin-like peptide (pit-CT) is a potent inhibitor of lactotroph function. We investigated the effect of ovarian hormones on pit-CT mRNA expression in the anterior pituitary (AP) gland of cycling female rats. Levels of mRNAs for pit-CT, CT receptor, prolactin (PRL), and beta-LH during 4-d estrous cycle were determined. In a second study, the effects of estrogens and progesterone on pit-CT and PRL mRNA levels were investigated. In a third group, the effect of estrogen or progesterone depletion on pit-CT mRNA expression was studied. In a fourth group, the effect of passive pit-CT immunization on PRL and LH mRNA expression was examined. Pit-CT mRNA levels varied during estrous cycle. They were highest in diestrus, but lowest in the evening of proestrus. CT-receptor mRNA levels displayed smaller fluctuations. Estrogen repletion caused a decline in pit-CT mRNA expression in ovariectomized rats, but progesterone produced a marked increase. ICI 182,780 prevented the decline of pit-CT mRNA levels during late proestrus-estrus, but RU 486 attenuated pit-CT mRNA levels. Passive CT immunization in diestrus altered PRL and LH mRNA expression, and advanced the estrus cycle. These results suggest that pit-CT mRNA expression is regulated by ovarian hormones, and depletion of pit-CT advances their estrous cycle.

The hormonal status modulates the effect of neurokinin A on prolactin secretion in female rats.

We have previously reported that neurokinin A (NKA), a tachykinin closely related to substance P, increases the release of prolactin (PRL) from the anterior pituitary gland of male rats, but not from pituitaries of ovariectomized (OVX) female rats. In this study, we evaluated the influence of estrogens in the action of NKA on PRL secretion in female rats. NKA stimulated the in vitro release of PRL from pituitary glands of OVX-chronically estrogenized rats, and of proestrus and estrus rats, but had no effect in anterior pituitaries of diestrus rats. In addition, we observed that cultured anterior pituitary cells of OVX rats responded to NKA only when they were incubated for 3 days in the presence of estradiol 10(-9) M. This effect was blocked by L-659,877, an NK-2 receptor antagonist. We also studied the action of NKA on PRL release during lactation. The response of anterior pituitary cells to NKA was variable over this period. The maximal sensitivity to NKA was observed at day 10 of lactation. Furthermore, the blockade of endogenous NKA by the administration of an anti-NKA serum to lactating rats reduced the PRL surge induced by the suckling stimulus. These results show that the responsiveness of the anterior pituitary gland of female rats to NKA is modulated by the endocrine environment, and suggest that NKA may participate in the control of PRL secretion during the estrus cycle and lactation.

Intracellular antagonistic interaction between GnRH and gonadotrophin surge-inhibiting/attenuating factor bioactivity downstream of second messengers involved in the self-priming process.

The antagonistic control of LH concentrations by GnRH and the putative ovarian factor gonadotrophin surge-inhibiting or -attenuating factor (GnSIF/AF) was studied by perifusion of female rat pituitary glands in vitro. LH release and GnRH self-priming were measured in response to five (0.32-10 nmol l-1) or three (10 nmol l-1) 10 min pulses of GnRH. In the latter case, pulses were preceded by five 10 min pulses of either 1 mmol 8-bromo-cAMP l-1 plus 10 mmol theophylline l-1 or 1 mumol phorbol 12-myristate 13-acetate l-1. The stimulations were carried out in the presence or absence of steroid-free bovine follicular fluid, which possesses GnSIF/AF bioactivity, to study the effect of GnSIF/AF on the self-priming process during successive stimulations by GnRH. First, the effect of follicular fluid was studied on GnRH-induced LH release from pituitary glands collected during the ovarian cycle. Only when a clear self-priming effect was evident, as on day 2 of dioestrus and the day of pro-oestrus, follicular fluid antagonized the self-priming effect of GnRH. Second, when glands on day 2 of dioestrus were used, self-priming was displayed by various combinations of GnRH and follicular fluid. The pulses with the lowest concentrations of GnRH together with a high concentration of follicular fluid, however, led to stable low amplitude LH pulses. Finally, priming of the pituitary LH response to GnRH with 8-bromo-cAMP plus theophylline or phorbol 12-myristate 13-acetate was inhibited by follicular fluid. These results confirm the control of LH release by GnRH and GnSIF/AF bioactivity. The effect of GnSIF/AF in follicular fluid is most pronounced on the days before the LH surge, showing evidence of its important role in maintaining low LH concentrations during this period. GnSIF/AF neutralizes downstream actions of second messengers involved in GnRH self-priming.

Age-related changes in the numbers of mammotrophs, somatotrophs and mammosomatotrophs in the anterior pituitary gland of female rats: A flow cytometric study

Age-related changes in the numbers of mammotrophs, somatotrophs, mammosomatotrophs, and the cells of other types in the anterior pituitary of female Wistar rat were measured by flow cytometry. The mammotrophs increased with age, and the somatotrophs decreased with senescence. The mammosomatotrophs increased remarkably in senescent rats, and these cells of the rats older than 21 months were about 10 times more than those of 3, and 12–13 months old rats. This result indicates that the stability of gene expression in cell differentiation is reduced in the aging process of the anterior pituitary.

Further studies on the regulation, localization and function of the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in the rat anterior pituitary gland.

Recent evidence shows that thyrotrophin-releasing hormone (TRH) immunoreactivity in the rat anterior pituitary gland is accounted for by the TRH-like tripeptide pyroglutamyl-glutamyl-prolineamide (pGlu-Glu-ProNH2, < EEP-NH2). The present study was undertaken to investigate further the regulation, localization and possible intrapituitary function of < EEP-NH2. Anterior pituitary levels of < EEP-NH2 were determined between days 5 and 35 of life, during the oestrous cycle and after treatment with the luteinizing hormone-releasing hormone (LHRH) antagonist Org 30276. Treatment of adult males with the LHRH antagonist either for 1 day (500 micrograms/100 g body weight) or for 5 days (50 micrograms/100 g body weight) reduced anterior pituitary < EEP-NH2 levels by 25-30% (P < 0.05 versus saline-treated controls). Anterior pituitary < EEP-NH2 increased between days 5 and 35 of life. In females, these levels were 2- to 3-fold higher (P < 0.05) than in males between days 15 and 25 after birth; these changes corresponded with the higher plasma follicle-stimulating hormone (FSH) levels in the female rats. After day 25, < EEP-NH2 levels in female rats decreased in parallel with a decrease in plasma FSH. Injections with the LHRH antagonist (500 micrograms/100 g body weight), starting on day 22 of life, led to reduced contents of < EEP-NH2 in the anterior pituitary gland of female rats on days 26 and 30 (55 and 35% decrease respectively). Levels of < EEP-NH2 in the anterior pituitary gland did not change significantly during the oestrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of correlation of distribution of prolactin (PRL) charge isoforms with induction of PRL storage.

GH4C1 cells, rat pituitary tumor cells, produce PRL, but store little relative to normal lactotrophs; treating cells with estradiol, insulin, and epidermal growth factor increases both PRL storage and accumulation of dense core granules. Normal lactotrophs contain several differently charged forms of PRL. We investigated whether inducing PRL storage in GH4C1 cells altered the production of these forms. Four forms of PRL that differed by charge were found by immunoblots of two-dimensional gels of extracts of female rat pituitary glands and of secretory granules isolated from the glands. Four forms were also secreted by GH4C1 cells. The relative abundance of the four forms in the medium of GH4C1 cells, determined by [35S]amino acid incorporation for 24h, was 1.2 +/- 0.58%, 91.3 +/- 1.09%, 6.3 +/- 0.72%, and 1.2 +/- 0.39% of total PRL (mean +/- SEM), from the most basic to the most acidic, respectively. Treatment with 1 nM estradiol, 300 nM insulin, and 10 nM epidermal growth factor did not significantly change the relative abundance of the forms. All four forms also were found in GH4C1 cells after 2 h of incubation with [35S]amino acids, although no incorporation of 32PO4 was detectable over the same incubation time. We conclude that GH4C1 cells produce four forms of PRL that differ by charge, as normal lactotrophs do. The increase in storage of PRL caused by insulin, estrogen, and epidermal growth factor does not result in or is caused by increased secretion of a specific form.

The physicochemical, immunological and biological properties of rat pituitary and plasma LH

A comparison was made between the properties of LH derived from female rat pituitary glands and plasma. Samples were collected from adult intact rats 5 h before or at the time of the pro-oestrous preovulatory LH surge; 27-day-old rats untreated or given 5 IU pregnant mares' serum gonadotrophin (PMSG) s.c. on day 25, which induced LH release 54 h later and adult ovariectomized rats untreated or primed with either 5 micrograms oestradiol benzoate s.c. or 5 micrograms oestradiol benzoate followed 48 h later by 0.5 mg progesterone s.c., which induced LH release 4-6 h later. All pituitary LH samples were totally bound to an anionic ion-exchange resin (DE52), while only a small proportion of the plasma LH was bound. Only 0-10% plasma LH obtained from intact, ovariectomized (with and without steroids) and untreated immature rats was bound, while a greater proportion of bound LH (36%) was noted in rats treated with PMSG. Gel filtration indicated only slight differences between pituitary and plasma LH, the former eluting marginally earlier than whole plasma and the unbound and bound plasma forms derived after separation by DE52 resin. Affinity chromatography (Concanavalin A and Glycine maximus) showed that LH from both sources possesses high mannose oligosaccharides and that plasma LH does not bear terminal N-acetyl galactosamine residues, although 20% of the pituitary form does. Plasma obtained from pro-oestrous rats had greater bioactivity than had pituitary LH in stimulating testosterone from Leydig cells and progesterone from granulosa cells in vitro, and inducing ovulation in immature rats in vivo. Leydig cell bioassays for LH in fractions obtained from ion-exchange separation indicate that steroidogenic activity of unbound plasma LH is greater than bound pituitary LH when they were collected at times of enhanced release. When release was inhibited (oestrogen-primed ovariectomized rats or immature rats), the steroidogenic activity of plasma and pituitary LH were similar and an acidic steroidogenic component was present in the plasma that was not recognized immunogenically as LH. In summary, pituitary LH undergoes a conversion on release into the plasma that involves a change in binding characteristics on an ion-exchange resin. In conditions when LH release is enhanced there is an increase in bioactivity of plasma LH owing to modification either by steroids or some other plasma factor(s) that perhaps influence the structure of LH directly or by steroids acting indirectly to alter GnRH release, which then modifies LH structure. These structural changes are minor and probably involve alterations in the glycosyl attachments.

In Situ Hybridization Study of Gonadotropin-Releasing Hormone (GnRH) Receptor mRNA in Female Rat Pituitary Gland during Estrous Cycle and after Ovariectomy.

Expression of rat GnRH receptor mRNA in female rat pituitary was examined by in situ hybridization using 35S-labeled antisense RNA probe. Specific signals were detected in the pars distalis. For detailed analysis of the cellular expression of GnRH receptor mRNA, the pituitaries were double-stained by in situ hybridization of GnRH receptor mRNA and antisera against some pituitary hormones (LH, FSH, TSH and prolactin). The expression of GnRH receptor mRNA was detected in all the immunoreactive gonadotropes, but was not detectable in TSH or prolactin cells. Cellular expression of GnRH receptor mRNA in immunoreactive gonadotropes was compared during the estrous cycle and after ovariectomy. In the pituitary of normal cycling rats, the mean value of the cellular expression of the mRNA in estrus was significantly smaller than those of other phases of the cycle. In ovariectomized rats, the cellular expression was significantly in-creased 4 days after the operation as compared with normal cycling rats, and the elevated level of cellular expression was maintained until 21 days after ovariectomy. These results suggest that GnRH receptor expression in the pituitary is preferentially localized in gonadotropes, and the extent of cellular expression varies during the estrous cycle and after ovariectomy.

Dopamine D 2 receptor mRNA in the pituitary during the oestrous cycle, pregnancy and lactation in the rat

We have used the technique of quantitative in situ hybridization in order to study the changes in the levels of expression of D 2 receptor mRNA in the anterior pituitary gland of female rats at different stages of the reproductive cycle. Plasma prolactin levels in the same animals were determined by radioimmunoassay. Rats in the prooestrous, oestrous, dioestrous 1, dioestrous 2 phases of the oestrous cycle and in pregnant, lactating, ovariectomised and ovariectomised animals treated with diethylstilbestrol (DES) have been examined. Our results show that expression of D 2 receptor mRNA in the anterior pituitary gland varies during the oestrous cycle, with the lowest expression measured during oestrus. Expression levels increased during dioestrus 1, reaching the highest values in dioestrus 2 and declining again in prooestrus. Expression of D 2 receptor mRNA was reduced during pregnancy when compared to lactating animals. In ovariectomised animals, the level of D 2 receptor mRNA was similar to that observed in intact animals during oestrus. Ovariectomised animals treated with DES showed dramatically increased prolactin levels, while D 2 receptor mRNA remained low. Prolactin secretion might be controlled not only by variations in the release and plasma concentrations of dopamine itself, but also by modulation of D 2 receptor expression in pituitary cells. Our results suggest that the variations in D 2 receptor density in the anterior pituitary cells at different physiological states are, at least to some extent, regulated at the level of gene expression.