Female Pituitary Research Articles

Sexual dimorphism in the age changes of the pituitary lactotrophs in rats

It is known that aging is associated with alterations in hypothalamic and pituitary functions. In the present study, we have undertaken a quantitative immunohistochemical assessment of the lactotroph cell population as well as prolactin (PRL) secretion, in male and female rats of different ages. Pituitaries from young (3 months), old (20 months) and senescent (29 months) male and female Sprague-Dawley rats were processed for the immunohistochemical detection of lactotrophs. Serum PRL was measured by a homologous RIA. Additionally, the in vitro PRL secretory activity was estimated by perifusion of pituitary cells from senescent animals. Analysis of morphometric parameters revealed age-related changes of PRL cell population in animals of both sexes. The cell density (CD), surface density (SD) and volume density (VD) decreased with age in both male and female rats. However, CD as well as SD appeared to have increased in females when compared to males, either in young or old animals, while VD was higher only in old females. The pituitaries of senescent females displayed chromophobic microadenomas on a background of diffuse PRL cell hyperplasia. Prolactin serum levels showed a marked increase with age in females, but only a modest elevation in males. In senescent females, PRL production per cell was reduced. We conclude that in rats, there exists a clear sexual dimorphism in the age-related changes of pituitary PRL cells.

Breed Differences in Expression of Inhibin/Activin Subunits in Porcine Anterior Pituitary Glands

Chinese Meishan (MS) boars have greater plasma FSH concentrations than European White Composite boars, but this difference does not occur in females of these breeds. To understand this disparity, we studied expression of the follistatin gene and of genes for the inhibin/activinα -, βA-, and βB-subunits in porcine anterior pituitary glands using quantitative reverse transcription-PCR and ribonuclease protection techniques. We found that 1) the inhibin/activin βA- and βB-subunits and follistatin were expressed in porcine pituitary; 2) the α-subunit was not detected in the porcine pituitary, but was highly expressed in porcine follicles; and 3) the βB-subunit gene is more abundantly expressed (2-fold greater) in MS boar pituitaries than in pituitaries of White Composite boars. We conclude that this is not due to a breed difference, because the expression levels of this gene were similar in pituitaries of females of these breeds. No breed differences were detected for other genes screened in this study. From these observations, we propose that activin B, a dimer ofβ B-subunits and a stimulator of FSH secretion, may be partially responsible for the elevated plasma FSH concentrations in MS boars, and intrapituitary inhibin plays no or a very minimal role.

The follicle-stimulating hormone beta-subunit gene of the common brushtail possum (Trichosurus vulpecula): analysis of cDNA sequence and expression.

Reverse transcription-PCR has been used to obtain a cDNA sequence from the follicle-stimulating hormone (FSH) beta-subunit gene of the Australian brushtail possum (Trichosurus vulpecula). Comparisons of the possum FSHbeta-mRNA coding region nucleotide sequence with that of six eutherian mammal homologues reveals a mean percent identity of 77.3% and 76.8% at the nucleotide and predicted amino acid-sequence levels respectively. Furthermore, the predicted amino acid sequence of the possum FSHbeta mature protein shows evolutionary conservation of twelve cysteine residues and two potential N-linked glycosylation sites. The protein lacks the CAGY motif present in most reported glycoprotein beta-subunit sequences. The translation termination codon and consensus polyadenylation sequence overlap, a feature observed in other mammalian FSHbeta genes. Northern hybridization of total RNA from adult female possum pituitary revealed three hybridizing transcripts of approximately 2.8, 1.2 and 0.5 kb which may arise from utilizing alternative polyadenylation signals. In situ hybridization localized the FSHbeta transcripts to a sub-population of anterior pituitary cells interpreted as being gonadotropes. In summary the results indicate considerable evolutionary conservation of the structure of the FSH beta-subunit gene between the marsupial and eutherian mammalian lineages.

The proximal 350 bp of 5′-flanking sequence of the human α-subunit glycoprotein hormone gene functions in the pituitary gland, but not the placenta, in transgenic mice

To understand better the minimal DNA sequence requirements for regulated expression of the human α-subunit glycoprotein hormone gene (Hα), two lines of transgenic mice were constructed that contained a fusion gene (Hα-350CAT) consisting of only 350 bp of 5'-flanking sequence of Hα linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). CAT activity was detectable in pituitary, but not in brain, heart, kidney, liver, lung, pancreas, or spleen in transgenic mice. Gonadectomy increased (p<0.05) CAT activity in the pituitaries of males (6.5±1.4% conversion/μg protein; mean ± SEM) and females (14.5±4.2) compared to intact males (1.2±0.3) and females (6.7±1.0). In addition, administration of a GnRH antagonist (antide; 60 μg/injection; one injection every other day) for 10 d to gonadectomized animals decreased (p<0.05) CAT activity in males (3.5±0.8) and females (2.9±0.5) compared to gonadectomized animals that received saline. Antide also reduced (p<0.05) serum concentrations of luteinizing hormone in gonadectomized males and females compared to gonadectomized animals that received saline. Surprisingly, CAT activity in the placenta of Hα-350CAT transgenic mice was not detectable (<3 SD above the mean of CAT activity in placenta from nontransgenic mice;n=77). Thus, expression of the human α-subunit promoter in the placenta of transgenic mice appears to require DNA sequences upstream of the proximal 350 bp of 5'-flanking sequence, whereas the proximal 350 bp of the human α-subunit gene contains sufficient DNA sequence to target pituitary-specific expression and confer responsiveness to gonadal hormones and GnRH.

Gonadal steroids regulate rat anterior pituitary levels of TSH-releasing hormone- and pyroglutamyl-glutamyl-proline amide-like immunoreactivity.

The novel peptide, pyroglutamylglutamylprolineamide (pGlu-Glu-ProNH2, EEP), which has structural and immunological similarities to TRH (pGlu-His-ProNH2) has recently been shown to contribute to total TRH-like immunoreactivity (t-TRH-LI) detected in the rabbit prostate and rat and porcine anterior pituitary. This study was undertaken to determine the effects of gonadal steroids on t-TRH-LI and its components in the rat hypothalamus and pituitary. EEP-like immunoreactivity (EEP-LI) was separated from TRH-LI by ion exchange chromatography and detected by TRH RIA. Although male and female posterior pituitary and hypothalamic t-TRH-LI levels were similar, the mean t-TRH-LI in female anterior pituitaries was significantly lower than that in males, 10.3 +/- 2.9 pmol/g vs. 24.4 +/- 2.5 pmol/g (P < 0.01). Anion exchange analysis of control anterior pituitary samples distinguished two peaks of t-TRH-LI, corresponding to [125I]-TRH marker and [3H]-EEP markers. In control female anterior pituitaries EEP-LI accounted for 26.0 +/- 2.4% of t-TRH-LI, whereas in males it accounted for 43.3 +/- 5.3% of the total. Hypothalamic and posterior pituitary samples only contained t-TRH-LI corresponding to [125I]-TRH markers. There was no significant change in hypothalamic and posterior pituitary levels of t-TRH-LI after ovariectomy or orchidectomy. Anterior pituitary levels of t-TRH-LI, however, were increased by an estimated 6-fold after ovariectomy and 2-fold after orchidectomy. After ovariectomy, the proportions of t-TRH-LI accounted for by TRH-LI and EEP-LI were reversed in the female. EEP-LI now accounted for the majority of t-TRH-LI, constituting an increase of approximately 21-fold in pituitary EEP-LI levels. The changes in the levels of pituitary TRH-LI and EEP-LI induced by ovariectomy were reversed by 17-beta-estradiol. As in the ovariectomized samples EEP-LI was increased (2-fold) by orchidectomy. Both TRH-LI, which increased 1.6-fold, and EEP-LI were restored to control values after testosterone replacement. These findings confirm the hypothesis that pituitary TRH-LI and EEP-LI are regulated by gonadal status. The fact that these changes were not observed in the hypothalamus and posterior pituitary suggests that TRH-LI and EEP-LI have specific functional significance in the pituitary gland.

Acute sex differences in serum LH levels in gonadectomized rats: investigation of pituitary response to GnRH pulse frequency and prolactin secretion as etiological agents.

Gonadectomy results in a striking sex difference in plasma LH levels in adult rats; by 2 days postgonadectomy, serum LH is 5- to 10-fold higher in males than in females. Despite 25 years of work, the site of this sex difference has yet to be determined. Since gonadectomy elevates GnRH pulse frequency, we asked whether a sex difference in pituitary LH secretory response to GnRH pulse frequency underlies the sex difference in serum LH postgonadectomy. Pituitaries from intact and 2-day postgonadectomized male and female rats were perifused for 8 h, receiving either no GnRH (basal) or GnRH pulses (50 ng/ml) at 1 pulse/1.5 h, 1/h or 2/h. Five-minute perifusate fractions were analyzed for LH, FSH, and PRL by RIA. At 1 GnRH pulse/1.5 h, but not other frequencies, pituitaries from gonadectomized males released 1.4-2.8 times more LH per pulse than female pituitaries. Such in vitro sex differences in LH release were not due to higher in vitro prolactin release by female than male pituitaries as suppression of pituitary prolactin secretion by perifusion with the dopamine agonist bromocriptine failed to erase sex differences. Since GnRH pulse frequency in vivo exceeds 1/h following castration, it is unlikely that weak sex differences at frequencies slower than 1/h are relevant to differences in serum LH.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine control of gametogenesis and spawning induction in the carp

The gonadotropic regulation of spermatogenesis and spermiogenesis is apparently mediated by 11-ketotestosterone (11-KT) secreted by Leydig cells. However, the acquisition of sperm motility requires the stimulation of the sperm duct by gonadotropin, or by 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P) which is produced by spermatozoa from 17α-hydroxyprogesterone (17-OH-P). The theca of the ovarian follicle performs most of the steroidogenic steps culminating in the formation of testosterone which diffuses into the granulosa layer to be aromatized to estradiol. A parallel two-cell type mechanism operates towards final oocyte maturation: theca cells produce 17-OH-P which diffuses into the granulosa cells to be converted to 17,20-P. Nevertheless, this model does not apply to all teleost fish. The synthesis of vitellogenin (VTG) by the liver is regulated by estradiol in synergism with growth hormone. The uptake of VTG, through low-affinity/high-capacity binding sites on the plasma membrane of the oocytes is stimulated by GTH-I. The proteins forming the chorion are produced by the liver and carried in the circulation to be incorporated at the periphery of the oocyte. The cascade of hormones involved in final oocyte maturation includes GTH-II which stimulates the formation of 17,20-P, or other maturation-inducing steroids, which bind to receptors on the oocyte membrane and activate the maturation-promoting factor (MPF). This factor is composed of cyclin B and a kinase of the CDC2 type. Ovulation involves the formation of prostaglandins and synthesis of proteases which degrade part of the follicular wall to form a rupture site through which the egg can emerge. Contractility of the follicular wall may take part in the process of ovulation. There is ample evidence for gonadotropin duality in salmonids. The two GTHs share a common α-subunit but differ considerably in their β-subunits. Also in the carp two GTHs have been isolated from pituitaries of adult females, and although shown to be chemically distinct, their function is quite similar. However, gonadotropin duality in cyprinid fish has not been confirmed by other laboratories and further research is required to elucidate this point. The release of GTH from the pituitary is regulated by various brain hormones, the most active being gonadotropin-releasing hormone (GnRH), which stimulates the release, and dopamine, which is inhibitory. Spawning induction in cyprinid fish is currently carried out by the hypophyseal approach (hypophysation or administration of chorionic gonadotropin) or by the hypothalamic approach (GnRH superactive analogues in combination with dopamine antagonists — the Linpe method). Comparison between the two approaches under the conditions of Israeli hatcheries has shown that both are suitable for spawning induction in carp, provided that the GTH content of the pituitary extract is calibrated. However, the Linpe method is advantageous because of the availability and lower cost of the compounds required for hypothalamic manipulations. In addition, the hazard of spreading pathogens along with pituitary material is a point to be considered. Adapting the Linpe method to Israeli cyprinid culture has required research int selecting the most suitable GnRH analogue and the dopamine antagonist, and the most suitable mode of their administration. In addition, the research examined the proper time of injection, the response latency and its temperature dependence. The results of the study indicate that the response to spawning induction by either approach cannot be assumed to be the same in every country due to differences in the environment, hatchery procedures and carp lines. In order to establish an optimal procedure for spawning induction at a given site, a study under the specific local conditions should be carried out using previous results only as guidelines.

All prepro-VIP-derived peptides, except , are expressed in the female rat anterior pituitary and increased by estrogen

The expression of VIP precursor products: prepro-VIP(22-79), peptide histidine isoleucine (PHI), peptide histidine valine (PHV), prepro-VIP(111-122), VIP, prepro-VIP(156-170), and prepro-VIP mRNA in the anterior pituitary of estrogen-treated, ovariectomized rats, of ovariectomized controls, and of sham-operated controls was examined. Using radioimmunoassays based on antisera against each of the prepro-VIP sequences, we found that all sequences were expressed and markedly induced by estrogen, except PHI and PHV, which both were undetectable. By immunohistochemistry, it appeared that the number of cells immunoreactive for each of these sequences was increased in the estrogen-treated animals. However, PHI/PHV-immunoreactive cells could not be detected, despite the use of four different PHI antisera with different specificities. Estrogen treatment increased the prepro-VIP mRNA as judged by Northern blotting. In situ hybridization signals for both VIP mRNA and PHI mRNA were observed in few pituitary cells from control animals whereas strong positive signals were observed in a larger number of cells after estrogen treatment. The findings show that estrogen causes activation of the VIP gene expression in anterior pituitary cells, and that the absence of PHI and PHV probably is due to translational or posttranslational events.

Thyrotropin-releasing hormone downregulates pyroglutamyl peptidase II activity in adenohypophyseal cells.

Pyroglutamyl peptidase II (PPII) is a thyrotropin-releasing hormone (TRH) hydrolyzing ectoenzyme with a narrow specificity. In the adenohypophysis, it is present on lactotropes. This study was undertaken in order to determine whether TRH itself regulates PPII activity in the adenohypophysis. After 5 days in culture, dispersed cells from female pituitaries expressed detectable levels of PPII activity when 10(-8) M 3,3',5'-triiodo-L-thyronine was present throughout the culture. 10(-6) M TRH decreased PPII activity with a maximal effect (down to 46% of initial values) at 16 h and an ED50 of 10(-9) M. [3Me-His2]TRH, a potent agonist of the TRH receptor was effective at lower concentrations (ED50: 1.6 x 10(-10) M). Phorbol-12-myristate-13-acetate (PMA; 10(-6) M), a protein kinase C (PKC) activator, diminished PPII activity to 61% or initial values with an ED50 of 2.2 x 10(-8) M. Maximal effects of PMA and TRH were not additive. Neither PMA nor TRH effects were reversed by inhibitors of protein kinases (1-(5-isoquinolinesulfonyl)-2-methyl-piperazine or sphingosine or staurosporine); TRH-induced downregulation of the enzyme was not modified by PMA pretreatment. TRH had no effect on two other ectopeptidases, endopeptidase 24.11 and dipeptidyl aminopeptidase IV. These data demonstrate that TRH specifically downregulates PPII activity in adenohypophyseal cells through TRH receptor activation and suggest that the activation of a presumably calcium-independent PKC mimics the TRH effect. TRH regulation of PPII activity may contribute to adjust lactotrope responsiveness to TRH.

Intracellular mediation of GnRH action on GTH release in tilapia

The objective of the present study was to confirm previous results on the mediation of GnRH signal in tilapia by providing evidence from experiments in cultured pituitary cells and from perifusion experiments using a GnRH-antagonist. After 4 days in culture under identical conditions, cells taken from pituitaries of fish maintained at 26°C were more sensitive to GnRHa ([D-Ala(6), Pro(9)-NEt]-LHRH) than those taken from fish maintained at 19°C. Cells from female pituitaries were more responsive than those from males. taGTH release in culture was augmented by Ca(2+) ionophore (A23187; 1-100 μM) or ionomycin (0.02-10 μM). The response of perifused pituitary to GnRH was reduced by nimodipine (1-10 μM) indicating that Ca(2+) influx via voltage-sensitive Ca(2+) channels is involved in the stimulation of GTH release. Activation of protein kinase C by OAG (1-oleyl-2-acetyl glycerol; 0.16-160 μM) or TPA (1-O-tetra-decanoyl phorbol-13-acetate; 1.25-125 nM) resulted in a dose-dependent stimulation of taGTH release from cultured cells. Arachidonic acid (0.33-330 μM) also augmented the release of taGTH from the culture. Four sequential pulses of sGnRH (100 nM) at 2h intervals resulted in surges of taGTH release from perifused pituitary fragments; the surges were similar in magnitude with no signs of desensitization. Sequential stimulation with graded doses of sGnRH (0.1 nM to 1 μM) in the presence of GnRH-antagonist ([Pro(2,6), Trp(3)]-GnRH) resulted in an attenuation of taGTH release. However, the GnRH-antagonist did not alter the pattern of forskolin-stimulated GTH release, indicating that forskolin stimulation is exerted at the level of the adenohypophyseal cells. It is concluded that, as in other vertebrates, the transduction of GnRH stimulation of GTH release involves Ca(2+) influx through voltage-sensitive Ca(2+) channels, mobilization of the ion from intracellular sources, arachidonic acid and activation of PKC. Adenylate cyclase-cAMP system us also involved in the mediation but its relationship with other transduction cascades requires further investigations.

Development of Gonadotrophs and Thyrotrophs in the Female Foetal Sheep Pituitary: Immunocytochemical Localization Studies

In order to investigate the ontogenesis of cell types in the pituitary gland, anterior pituitaries were collected from female foetal sheep at days 70, 100 and 130 of gestation (term = 145 days). Cells containing the common alpha-subunit and the specific beta-subunits of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) were immunolocalized using the avidin-biotin immunoperoxidase technique. LH beta-containing cells were first detected in the foetal pituitary by day 70 of gestation. The number and intensity of staining of these LH beta cells increased by day 100 but had declined again by day 130. Immunopositive alpha-subunit and FSH beta-cells appeared by day 100 of gestation and had further increased in number and staining intensity by day 130. Cells containing TSH beta were present at day 70 and progressively increased in abundance and intensity through gestation. These data indicate that the development of LH- and FSH-containing cells in the female foetal sheep pituitary is differentially regulated during foetal life, and that in the sheep free alpha-subunit is not produced in significant amounts before the specific beta-subunits.

The 22K variant of rat prolactin: evidence for identity to prolactin-(1-173), storage in secretory granules, and regulated release.

Western blot analyses of the rat pituitary have detected a 22K PRL variant distinct from intact PRL (25K). We recently reported that glandular kallikrein (GK), an estrogen-induced lactotroph protease, can process PRL in vitro from a 25K form to a 22K form in a thiol-dependent cleavage at Arg174-Arg175 to remove 23 amino acids. We also detected an estrogen- and thiol-induced 22K PRL variant in the rat pituitary comigrating with a PRL product generated by in vitro processing with GK and carboxypeptidase-B. This study addressed whether the in vivo 22K PRL variant originates through a GK-like cleavage and is a regulated secretory product of the rat pituitary. A polyclonal antipeptide antiserum was raised against a synthetic peptide [PRL-(163-173)] representing the new C-terminus after GK and carboxypeptidase-B processing. In slot and Western blots, this antiserum (CT-antiserum) specifically recognized PRL processed in vitro by GK and carboxypeptidase-B and did not recognize intact PRL or PRL cleaved by GK alone. Western blot analysis of rat pituitary extracts with CT-antiserum specifically detected an estrogen- and thiol-induced 22K band that comigrated with a PRL product generated by in vitro processing with GK and carboxypeptidase-B. This 22K band was concentrated in subcellular fractions of the pituitary enriched in secretory granules. During short term incubations in medium 199, pituitaries from normal adult female rats released substantial amounts of 22K PRL; in contrast, male pituitaries did not release detectable 22K PRL. The release of 22K PRL from female pituitaries was powerfully blocked by bromocriptine, a dopaminergic agonist. GK was also released from pituitaries of female, but not male, rats, and GK release was inhibited by bromocriptine. The results identify the 22K PRL variant as PRL-(1-173), which is consistent with GK-like processing at Arg174-Arg175, followed by carboxypeptidase-B-like processing. The results also show that 22K PRL is a natural female-specific secretory product of the rat pituitary under inhibitory dopaminergic control.

Inhibition of In Vitro Pituitary Gonadotropin Secretion by 17β-Estradiol in the Frog Rana pipiens

We tested the effects of low concentrations of 17β-estradiol (E 2) on the in vitro pituitary responsiveness to gonadotropin-releasing hormone (GnRH) in frogs ( Rana pipiens). We first showed that the pH indicator phenosulfonpthalein (phenol red. PR) at levels normally used in the culture medium did not significantly affect pituitary basal or GnRH-stimulated gonadotropin secretion in male glands, as may occur in mammals. Incubation of male glands with E 2 at 10 pg/ml for 24 hr in medium lacking PR did not alter basal follicle-stimulating hormone (FSH) or luteinizing hormone (LH) secretion, but significantly attenuated responsiveness of both gonadotropins to GnRH (0.2, 1, and 5 ng/ml); E 2 did not affect responses at the highest dose of GnRH (25 ng/ml). GnRH responsiveness of female pituitaries was also inhibited by E 2 at all doses of GnRH; 1 ng/ml E 2 had greater effects than did 10 pg/ml. The present results demonstrate that E 2 at concentrations commonly found in the circulation of both sexes have a consistent direct inhibitory effect on FSH and LH secretion at the level of the pituitary.

B-HT 920 activates dopamine D2 receptors coupled to inhibition of adenylate cyclase activity.

In homogenates of female rat anterior pituitary, the azepine derivative B-HT 920 inhibited the forskolin-stimulated adenylate cyclase activity with an EC50 value of 0.35 microM. In male rat anterior pituitary, B-HT 920 curtailed the stimulation of adenylate cyclase activity by vasoactive intestinal peptide with an EC50 of 0.20 microM. In synaptic plasma membranes of rat striatum, B-HT 920 significantly reduced basal adenylate cyclase activity with an EC50 of 0.68 microM. Both in pituitary and striatum, the B-HT 920 inhibition was counteracted by the dopamine (DA) D2 receptor antagonist 1-sulpiride, but not by the alpha 2-adrenergic antagonist yohimbine. These results indicate that B-HT 920 is capable of activating DA D2 receptors negatively coupled to adenylate cyclase activity.

Carcinogenicity study of γ-oryzanol in F344 rats

The carcinogenic potential of γ-oryzanol, a drug mainly used for the treatment of hyperlipidaemia, was studied in F344 rats. Groups of 50 males and 50 females were fed a diet containing 0 (control), 200, 600 or 2000 mg γ-oryzanol/kg body weight/day for 2 yr. Although females in the highest dose group (2000 mg/kg body weight) showed a slight decrease in body weight at 104 wk, there were no treatment-related changes in general condition, food consumption, mortality, organ weight or haematology. Histopathological examination showed various tumours in all groups, including the control group. In the control and 2000-mg/kg groups, high tumour incidences were observed in the testes, pituitary and thyroid of males, and in the pituitary, uterus and mammary gland of females; however, there was no significant increase in the incidence of any tumours between the control and the 2000-mg/kg groups. The findings indicate that under the experimental conditions described γ-oryzanol was not carcinogenic in F344 rats.

Immunoelectron Microscopic Studies of Gonadotrophs in the Male and Female Rat Anterior Pituitaries, with Special Reference to their Changes with Aging.

Gonadotrophs immunocytochemically identified with an antibody to LH beta were studied in both male and female rats of various ages, viz., 2, 14 and 23 months after birth. The rat gonadotrophs are classified into two cell types, i.e., Type I containing large (300-700 nm) and small (150-200 nm) secretory granules, and Type II containing only small (100-200 nm) secretory granules. In normal young adults, the male rat pituitary contains a large number of Type I cells (more than 90% of all gonadotrophs), whereas the female pituitary contains much more Type II cells (more than 70%) than Type I cells. With age, Type I gonadotrophs in the male rat pituitary decrease remarkably to less than 25%, while Type II cells constitute more than 70% at the age of 23 months; the ratio of Type I to Type II comes to be the same as in the female. The main cause of this change is the decrease in the number of large secretory granules as the animal ages. FSH and LH levels in both the pituitary and serum were determined by radioimmunoassay. Pituitary FSH content decreased gradually in the male with age, while that in the female showed no change. LH content in the male decreased slowly, but LH in the female increased with aging. The large secretory granules indicating the presence of FSH markedly decreased with aging. The Type II cells contain only small secretory granules which probably contain LH. Thus the results of morphological counting of each type of gonadotroph and those of radioimmunoassay of gonadotropins during aging were essentially parallel. Some signs of degeneration were observed in the gonadotrophs in the aged rat pituitaries.

Estrous cycle dependent regulation of peptidylarginine deiminase transcripts in female rat pituitary

Northern blot hybridization demonstrated 4.5-5.0 kilobase peptidylarginine deiminase mRNA in poly (A)+ RNA-enriched fractions of both male and female pituitaries. Dot blot analysis of total RNA fractions showed more than 50-fold sex difference in the mRNA content. The female pituitary mRNA content showed at least a 50-fold variation during the estrous cycle characterized by elevation at diestrus and proestrus followed by a rapid decline at estrus. The data were discussed in comparison with the sex difference and estrous cycle dependence of the pituitary enzyme content reported previously.

Galanin and vasoactive intestinal polypeptide are colocalised with classical pituitary hormones and show plasticity of expression

The identity of galanin- and vasoactive intestinal polypeptide-(VIP) immunoreactive (IR) cells in the rat anterior pituitary was investigated using immunocytochemistry and, since levels of both peptides are stimulated by oestrogen, the effect of oestrogen treatment and gonadectomy on the expression of both peptides was examined. In normal male rats, few galanin-IR and very few VIP-IR cells were found. Colocalisation studies performed on 2-microns serial paraffin sections revealed that in these animals galanin IR was present in somatotrophs and thyrotrophs. In normal females in dioestrus many lactotrophs, in addition to somatotrophs and thyrotrophs, expressed galanin, but very few VIP-IR cells were seen. In cryostat sections of normal rat pituitaries, slightly more VIP-IR cells were present. Oestrogen treatment in females produced an increase in frequency of galanin-IR cells, the vast majority of which were lactotrophs, and more VIP-IR cells, identified as lactotrophs, also appeared. VIP was present in a subset of galanin-IR lactotrophs after oestrogen treatment. After ovariectomy female pituitaries resembled those of normal males, with few galanin positive cells none of which were lactotrophs, and hardly any VIP-IR cells. Thus these two peptides are present in specific endocrine cell types of rat anterior pituitary and display plasticity of expression in different cell types under the influence of oestrogen. Their roles in control of pituitary hormone secretion are supported by these findings, and it is possible that both peptides act in a paracrine fashion within the pituitary.

Estrone sulfatase activity in rat brain and pituitary: effects of gonadectomy and the estrous cycle

Estrone sulfatase activity was characterized in microsomal preparations from rat brain and anterior pituitary. No differences in apparent K m were found in hypothalamic-preoptic area between male (7.5 μM) and female (7.4 μM) rats. Apparent K m 's of anterior pituitaries from males (14.5 μM) and females (22.5 μM) were higher than those found in brain. Estrone sulfatase activity was equally inhibited by estradiol-17β-3-sulfate, dehydroepiandrosterone-3-sulfate and estrone-3-sulfate indicating a broad range of substrate specificity for this enzyme. Sulfatase activity in female anterior pituitary was found to be twice that of male. Sulfatase activity was distributed similarly in brain tissues between sexes with cerebellum ⩾ medial basal hypothalamus > preoptic area = cortex. Following gonadectomy, sulfatase activity in anterior pituitary of males was significantly greater than activity found in intact animals ( P < 0.05). This increase in activity, however, was unaffected by treatment with testosterone, dihydrotestosterone or estradiol-17β. Gonadectomy did not change sulfatase activity in brains of males or females or in pituitaries of females. However, sulfatase activity in pituitary glands of females changed significantly ( P < 0.05) with stages of the estrous cycle (metestrus < diestrus < proestrus < estrus). These data indicate sulfatase activity in rat anterior pituitary gland may be controlled by gonadal factors while sulfatase activity in brain is regulated differently.

Immunocytochemical study of peptidylarginine deiminase: localization of its immunoreactivity in prolactin cells of female rat pituitaries.

We have performed an immunocytochemical study of peptidylarginine deiminase (EC 3.5.3.15) in rat pituitaries. Male and female rat pituitaries were fixed by brief transcardial perfusion of Bouin Hollande Sublimate solution. Immunoperoxidase staining of cryosections obtained from female pituitaries with an antiserum to rat skeletal muscle peptidylarginine deiminase yielded specific staining of numerous cells in the pars distalis. In contrast, few immunopositive cells were noted in the pars distalis of male pituitaries. At least some of the immunopositive cells in female pars distalis were also stained with an antiserum to rat PRL. No such coincidence of immunoreactivities was noted when antisera to other anterior pituitary hormones were used. Biochemical and immunocytochemical examination of peptidylarginine deiminase during the pregnant period revealed its marked depletion on day 7 and its substantial restoration on day 14, showing apparently negative correlation with the known changes in the secretory activities of lactotrophs. This and the female-dominant occurrence of peptidylarginine deiminase in lactotrophs suggest significant involvement of the enzyme in the function of lactotrophs.