Prolactin Is a Strong Candidate for the Regulation of Luteal Steroidogenesis in Vizcachas (Lagostomus maximus).

Prolactin (PRL) is essential for the maintenance of the corpora lutea and the production of progesterone (P4) during gestation of mice and rats, which makes it a key factor for their successful reproduction. Unlike these rodents and the vast majority of mammals, female vizcachas (Lagostomus maximus) have a peculiar reproductive biology characterized by an ovulatory event during pregnancy that generates secondary corpora lutea with a consequent increment of the circulating P4. We found that, although the expression of pituitary PRL increased steadily during pregnancy, its ovarian receptor (PRLR) reached its maximum in midpregnancy and drastically decreased at term pregnancy. The luteinizing hormone receptor (LHR) exhibited a similar profile than PRLR. Maximum P4 and LH blood levels were recorded at midpregnancy as well. Remarkably, the P4-sinthesizing enzyme 3ß-HSD accompanied the expression pattern of PRLR/LHR throughout gestation. Instead, the luteolytic enzyme 20α-HSD showed low expression at early and midpregnancy, but reached its maximum at the end of gestation, when PRLR/LHR/3ß-HSD expressions and circulating P4 were minimal. In conclusion, both the PRLR and LHR expressions in the ovary would define the success of gestation in vizcachas by modulating the levels of 20α-HSD and 3ß-HSD, which ultimately determine the level of serum P4 throughout gestation.

Orexin A and B in vitro modify orexins receptors expression and gonadotropins secretion of anterior pituitary cells of proestrous rats.

AIM: Orexin A and orexin B (hypocretins) are neuropeptides synthesized mainly by neurons located in the lateral hypothalamus and projections throughout the brain. They are agonists at both the orexin 1 and orexin 2G protein-coupled receptors. They have been related to arousal, sleep and feeding, autonomic and neuroendocrine functions. Their role in the brain control of gonadotropins secretion was postulated in rodents and humans. Previously, we demonstrated the participation of the orexinergic system in attaining successful reproduction in in vivo studies. METHODS: We studied in vitro the effects of both neuropeptides, in the presence or absence of selective antagonists, on the mRNA expression of orexin 1 and orexin 2 receptors in anterior pituitary cells of proestrous rats, as well as the direct effects on FSH and LH secretion. RESULTS: Both orexin A and orexin B increased FSH and LH secretion; these effects were suppressed by the orexin 1 receptor blocking agent SB-334867 and the orexin 2 receptor antagonists JNJ-10397049. Orexin A and orexin B decreased OX1 receptor mRNA expression and this effect was modified only when both blocking agents were present. Neither orexin A nor the blocking drugs by themselves modified OX2 receptor mRNA expression. Orexin B treatment increased the mRNA expression of OX2 receptor. The effect was abolished only by the OX2 receptor antagonist. CONCLUSION: In an in vitro model, we demonstrated a direct effect of orexins on gonadotropins release and orexins receptors expression, underlining the hypothesis that orexins participate in the brain control of pituitary functions.

Sex differences in insulin resistance in GABAB1 knockout mice.

AIMS: We have previously demonstrated that the absence of functional GABA B receptors (GABABRs) disturbs glucose homeostasis in GABAB1KO mice. The aim of this work was to extend our studies of these alterations in GABAB1KO mice and investigate the sexual differences therein. MAIN METHODS: Male and female, GABAB1KO and WT mice were used. Glucose and insulin tolerance tests (GTT and ITT), and insulin and glucagon secretion tests (IST and GST) were performed. Blood glucose, serum insulin and hyperglycemic hormones were determined, and HOMA-IR calculated. Skeletal muscle insulin receptor ß subunit (IRß), insulin receptor substrates 1/2 (IRS1, IRS2) and hexokinase-II levels were determined by Western blot. Skeletal muscle insulin sensitivity was assessed by in vivo insulin-induced Akt phosphorylation (Western blot). Food intake and hypothalamic NPY mRNA expression (by qPCR) were also evaluated. KEY FINDINGS: Fasted insulin and HOMA-IR were augmented in GABAB1KO males, with no alterations in females. Areas under the curve (AUC) for GTT and ITT were increased in GABAB1KO mice of both genders, indicating compromised insulin sensitivity. No genotype differences were observed in IST, GST or in IRß, IRS1, IRS2 and hexokinase-II expression. Akt activation was severely impaired in GABAB1KO males while no alterations were observed in females. GABAB1KO mice showed increased food intake and NPY expression. SIGNIFICANCE: Glucose metabolism and energy balance disruptions were more pronounced in GABAB1KO males, which develop peripheral insulin resistance probably due to augmented insulin secretion. Metabolic alterations in females were milder and possibly due to previously described reproductive disorders, such as persistent estrus.

Oligodeoxynucleotide IMT504: lack of effect on immune parameters during islet regeneration in single dose streptozotocin-induced diabetes.

BACKGROUND: We have shown that oligodeoxynucleotide IMT504 improved blood glucose and islet beta-cell content in streptozotocin (STZ)-induced diabetic rats, inducing early expression of progenitor markers. Here we determined the effect of IMT504 on islet infiltration and on immunomodulatory proteins indoleamine 2,3-dioxygenase (IDO) and TNF-α-stimulated gene/protein 6 (TSG-6) in islets of STZ-diabetic rats, at the time of progenitor markers expression. METHODS: Male rats were i.p. injected with STZ [60 mg/kg body weight (BW)] or citrate buffer (control) (day 1). Starting on day 4, STZ animals were daily treated with saline (STZ-saline) or IMT504 (20 mg/kg BW/day s.c., STZ-IMT504) and killed after two consecutive decreases in blood glucose. Islet area and insulin expression, CD3 (T lymphocytes), CD68 (macrophages), IDO and TSG-6 immunostainings were determined. Islet infiltration was also evaluated by haematoxylin staining. RESULTS: STZ-induced diabetes in rats, with an important decrease in islet area was reversed by IMT504. Diabetes development did not involve islet infiltration, determined by haematoxylin and by the absence of significant T lymphocyte and macrophage presence. IMT504 did not induce changes in these parameters. IDO was not expressed in controls; the percentages of IDO-positive islets were very low and similar in STZ-saline and STZ-IMT504. Scarce TSG-6 was expressed in all groups, without significant differences. CONCLUSIONS: IMT504 improved insulin content but did not alter IDO or TSG-6 staining in islets of STZ-diabetic rats, suggesting that they do not participate in the IMT504-induced repair process. IMT504 did not per se modify leukocyte presence in islets of diabetic rats.

Oligodeoxynucleotide IMT504 induces a marked recovery in a streptozotocin-induced model of diabetes in rats: correlation with an early increase in the expression of nestin and neurogenin 3 progenitor cell markers.

AIMS/HYPOTHESIS: IMT504 is an oligonucleotide that promotes tissue repair in bone injury and neuropathic pain models by stimulating progenitor cells. Here we evaluated the effect of IMT504 on the recovery of islet function in a streptozotocin (STZ)-induced model of diabetes in the rat. METHODS: Male Sprague-Dawley rats were injected with STZ (60 mg/kg, i.p., day 1) or citrate buffer (Control). Animals with glycaemia between 11 and 20 mmol/l on day 4 were injected with IMT504 (4 mg/animal in saline, s.c., STZ-IMT504) or with saline (STZ-Saline) for 10 days. Glycaemia and water and food intake were recorded for 33 days. Intraperitoneal glucose tolerance tests (IPGTTs) were performed on day 30. On day 35, overnight-fasted animals were killed and blood samples and pancreases collected for hormonal and histological studies. A second group of STZ-IMT504 rats was killed, together with Control and STZ-Saline rats, after two consecutive days of blood glucose decreases after the beginning of IMT504 treatment. Pancreases were collected and proliferating cell nuclear antigen (PCNA), nestin and neurogenin 3 (NGN3) detected by immunohistochemistry. RESULTS: IMT504 greatly improved blood glucose and food and water intakes in STZ-IMT504 rats by day 8, as well as IPGTTs on day 30. Significant increases in islet number and beta cell content were observed in STZ-IMT504 rats (day 33). Furthermore, after two to five IMT504 injections, blood glucose decreased, and an increase in pancreatic nestin (mainly in endothelial cells), PCNA and NGN3 production (in islets) was observed in STZ-IMT504 rats. CONCLUSIONS/INTERPRETATION: IMT504 induced a marked recovery of STZ-induced diabetes that correlated with early production of progenitor cell markers, such as nestin and NGN3.

Role of orexins in the hypothalamic-pituitary-ovarian relationships.

Appropriate nutritional and vigilance states are needed for reproduction. In previous works, we described the influence of the hormonal milieu of proestrus on the orexinergic system and we found that orexin receptor 1 expression in the hypothalamus, but not other neural areas, and the adenohypophysis was under the influence of oestradiol and the time of the day. Information from the sexual hormonal milieu of proestrous afternoon impacts on various components of the orexinergic system and alertness on this particular night of proestrus would be of importance for successful reproduction. In this review, we summarize the available experimental data supporting the participation of orexins in the hypothalamic-pituitary-ovarian relationships. All together, these results suggest a role of the orexinergic system as an integrative link among vital functions such as reproduction, food intake, alertness and the inner biological clock.

GABAB receptors and glucose homeostasis: evaluation in GABAB receptor knockout mice.

GABA has been proposed to inhibit insulin secretion through GABAB receptors (GABABRs) in pancreatic beta-cells. We investigated whether GABABRs participated in the regulation of glucose homeostasis in vivo. The animals used in this study were adult male and female BALB/C mice, mice deficient in the GABAB1 subunit of the GABABR (GABAB(-/-)), and wild types (WT). Blood glucose was measured under fasting/fed conditions and in glucose tolerance tests (GTTs) with a Lifescan Glucose meter, and serum insulin was measured by ELISA. Pancreatic insulin content and islet insulin were released by RIA. Western blots for the GABAB1 subunit in islet membranes and immunohistochemistry for insulin and GABAB1 were performed in both genotypes. BALB/C mice preinjected with Baclofen (GABABR agonist, 7.5 mg/kg ip) presented impaired GTTs and decreased insulin secretion compared with saline-preinjected controls. GABAB(-/-) mice showed fasting and fed glucose levels similar to WT. GABAB(-/-) mice showed improved GTTs at moderate glucose overloads (2 g/kg). Baclofen pretreatment did not modify GTTs in GABAB(-/-) mice, whereas it impaired normal glycemia reinstatement in WT. Baclofen inhibited glucose-stimulated insulin secretion in WT isolated islets but was without effect in GABAB(-/-) islets. In GABAB(-/-) males, pancreatic insulin content was increased, basal and glucose-stimulated insulin secretion were augmented, and impaired insulin tolerance test and increased homeostatic model assessment of insulin resistance index were determined. Immunohistochemistry for insulin demonstrated an increase of very large islets in GABAB(-/-) males. Results demonstrate that GABABRs are involved in the regulation of glucose homeostasis in vivo and that the constitutive absence of GABABRs induces alterations in pancreatic histology, physiology, and insulin resistance.

Experimental data supporting the expression of the highly conserved GnRH-II in the brain and pituitary gland of rats.

The second GnRH form, originally identified in chickens (cGnRH-II or GnRH-II), is the most ubiquitous peptide of the GnRH neuropeptide family, being present from jawed fish to human beings. However, the presence of GnRH-II in such an important experimental model as the rat is still an object of discussion. Here we present chromatographic, immunologic and biologic activity evidence supporting the expression of GnRH-II in the rat. Olfactory bulb, hypothalamus, remnant brain and anterior pituitary from a pool of 50 female adult rats were extracted and subjected to RP-HPLC on a C-18 column. The fractions were collected and evaluated by using two different RIA systems, specific for GnRH-I and GnRH-II respectively. Under these conditions the GnRH-I standard eluted in fraction 21 (f21) was only detected with the GnRH-I RIA system, whereas the GnRH-II standard was only detected in the fraction 27 (f27) by using a GnRH-II RIA system. In the olfactory bulbs extract, the fractions analyzed by the GnRH-I RIA systems showed a single peak in f21, whereas by using the GnRH-II RIA system a single peak at f27 was observed. In the hypothalamus GnRH-I was detected in f21 meanwhile GnRH-II could not be detected. When the remnant brain and pituitary gland extracts were analyzed, both GnRH forms were detected. To the best of our knowledge, this is the first report concerning GnRH-II detection in a mammalian pituitary. Serial dilutions of f27 and GnRH-II presented similar displacement of radioiodinated-GnRH-II, demonstrating that both molecules share immunological properties. Moreover, after 60 min stimulation, both f27 and GnRH-II had similar LH and FSH releasing activity in 12 day-old rat pituitary primary cell cultures. However, we failed to characterize the GnRH-II gene in this model. These results provide strong evidence for the expression of GnRH-II in the rat brain and pituitary gland.

Differential gonadotropin releasing hormone (GnRH) expression, autoregulation and effects in two models of rat luteinized ovarian cells.

GnRH has been suggested to participate in corpus luteum function. Here we studied the expression of GnRH mRNA and peptide in two models of rat luteinized tissues: ovarian cells from PMSG-hCG treated prepubertal rats (SPO) and from intrasplenic ovarian tumors (Luteoma). A GnRH autoregulatory effect was evaluated as well as its action on cell proliferation and apoptosis. GnRH mRNA was present in SPO, isolated corpora lutea from SPO and Luteoma from 1 week to 7 months of development. In vitro cultures of Luteoma cells expressed 2-fold higher GnRH mRNA and 10-fold higher GnRH peptide than SPO cells. Buserelin (GnRH analog) increased GnRH mRNA and peptide expression in SPO but not in Luteoma cells. While basal proliferation was very low in Luteoma cells, SPO cells showed a significant increase in cell number by both the thymidine and the MTS methods after 72 h in culture. Buserelin induced a decrease in cell number in both cell types to a similar degree. Although basal apoptosis levels were higher in SPO than in Luteoma cells, Buserelin-induced apoptosis was only detected in Luteoma cells after 48 h treatment. These results show that the two types of rat, luteinized tissues, Luteoma and SPO, markedly differed in some intrinsic properties and in their local GnRH systems. Luteoma cells proliferate very weakly, express and secrete high amounts of GnRH, do not show an autoregulatory effect and respond to the decapeptide with apoptosis stimulation. In contrast SPO cells proliferate significantly, secrete low levels of GnRH but possess a positive, autoregulatory mechanism and respond to GnRH stimulation with impairment of proliferation.

Oestradiol restores cell proliferation in dentate gyrus and subventricular zone of streptozotocin-diabetic mice.

Type 1 diabetes mellitus correlates with several brain disturbances, including hypersensitivity to stress, cognitive impairment, increased risk of stroke and dementia. Within the central nervous system, the hippocampus is considered a special target for alterations associated with diabetes. Neurogenesis is a plastic event restricted to few adult brain areas: the subgranular zone of the dentate gyrus and the subventricular zone (SVZ). First, we studied the ability for neurogenesis in the dentate gyrus and SVZ of chronic diabetic mice induced by streptozotocin (STZ). Using bromodeoxyuridine (BrdU) labelling of cells in the S-phase, we observed a strong reduction in cell proliferation rate in both brain regions of diabetic mice killed 20 days after STZ administration. Second, because oestrogens are active neuroprotective agents, we investigated whether 17beta-oestradiol (200 micro g pellet implant in cholesterol during 10 days) restored brain cell proliferation in the diabetic mouse brain. Our results demonstrated a complete reversibility of dentate gyrus cell proliferation in oestrogen-treated diabetic mice. This plasticity change was not exclusive to the hippocampus because oestrogen treatment restored BrdU incorporation into newborn cells of the SVZ region of diabetic animals. Oestrogen treatment did not alter the hyperglycemic status of STZ-diabetic mice. Moreover, oestrogen did not modify BrdU incorporation in control animals. These data show that oestrogen treatment strongly stimulates brain neurogenesis of diabetic mice and open up new venues for understanding the potential neuroprotective role of steroid hormones in diabetic encephalopathy.

Gonadotropins and inhibins along the development of a luteinized rat ovarian tumor.

Luteinized intrasplenic ovarian tumors develop in response to high circulating gonadotropins. The relationship between tumor development, gonadotropins and inhibins was studied. Tumor-bearing animals were sacrificed weekly along the first 6 weeks of development. Inhibins were measured by enzyme-linked immunosorbent assay (ELISA), serum gonadotropins, GH and IGF-1 by RIA. Inhibin subunit mRNAs were determined by Northern blot. Tumor histology was examined. Ovarian grafts grew significantly along development. LH increased ten-fold on week 1; a further significant increment was observed on week 3. FSH peaked on weeks 1 and 2 and fell significantly thereafter. Serum inhibins markedly increased on weeks 3-5. Tumor inhibin A content and mRNA levels for alpha and beta A subunits also increased on week 3. Inverse correlations between inhibins and FSH and direct correlations between inhibins and LH were observed. Tumor inhibin A and IGF-1 contents correlated significantly. Increasing levels of luteinization were observed along tumor development. These luteinized tumors develop mainly in response to LH, since growth continues under FSH inhibition. The active inhibin secretion and the positive correlation between inhibins and LH suggests that LH may be the main driving force behind this production, while growth factors produced by the gonads may also participate in their regulation.

Development of an experimental ovarian tumor: immunocytochemical analysis.

OBJECTIVE: The aim of the present work was to study whether immunocytochemical parameters present in the normal ovary were altered after tumor development under high gonadotropin levels. METHODS: Ovarian tumors (luteoma): castrated female rats had an ovary grafted into the spleen; tumors were left to develop for 1, 2, 3 or 7 months. The presence of apoptotic cells (TUNEL method) and the expression of proliferating cell nuclear antigen (PCNA), gap junction protein (Cx43), steroidogenic acute regulatory protein (StAR), aromatase and synaptosome-associated protein of 25 kDa (SNAP-25) were determined by immunocytochemistry. Some of these findings were confirmed by RT-PCR (Cx43, StAR, SNAP-25). Inhibin subunit mRNAs were investigated by Northern blot. RESULTS: PCNA staining of tumors was mainly found in granulosa cells of transforming follicles and was absent from luteinized follicles. A nearly complete absence of apoptosis was observed. Cx43 was mainly found in follicles, while it was very weakly expressed or absent in luteinized follicles. StAR protein expression, indicating active steroidogenesis, was demonstrated only in luteinized follicles and in thecal cells, but was absent from granulosa cells. Aromatase immunoreactivity was very intense in granulosa and also present in luteal cells. Membrane-associated and cytoplasmic SNAP-25 immunostaining was determined in patches of endocrine cells in the follicles, as well as in the luteinized follicles. The expression of mRNAs for Cx43, StAR and SNAP-25 (RT-PCR) and inhibin subunits (Northern blots) were confirmed in 1-, 3- and 7-month-old tumors. CONCLUSIONS: These results indicated that luteoma most likely develop from unruptured follicles by hypertrophy and proliferation of follicular cells. Circulating gonadotropins seem to play a fundamental role in maintaining the expression of proteins typically expressed in normal ovary, while avoiding apoptosis in this tissue.

Actions of immunosuppressor drugs on the development of an experimental ovarian tumor.

Immunosuppression has been related to the incidence of tumor apparition, including endocrine tumors. The intrasplenic ovarian tumor (luteoma) is a typical benign endocrine tumor that develops under high gonadotropin stimulation and, from the immunological perspective, is located in a critical organ involved in immune response. To establish if immunosuppression could alter the development of this experimental tumor, the effects of cyclosporin A (CsA) and dexamethasone (Dex) were evaluated. After surgery, tumor-bearing and sham animals were kept without treatment for 4 weeks; thereafter, they were distributed into CsA (25 mg/kg), Dex (0.1 mg/kg), or vehicle (75:25 castor oil:ethanol) groups and were injected on alternate days for 50 days. Body weight was evaluated weekly. Animals were sacrificed after a jugular vein blood sample was obtained. Thymi were weighed. Tumors were measured and placed in formaline for histological studies. Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), and estradiol were measured by radioimmunoassay. Hematological parameters were determined. CsA induced a significant decrease in survival rates both in tumor-bearing and sham animals (P < 0.01). Dex significantly impaired weight increase in both groups of animals. CsA induced a significant weight loss in sham animals, not observed in tumor-bearing animals. Dex induced thymus weight loss in both groups, whereas CsA induced thymus weight loss only in sham animals. Only Dex induced a decrease in lymphocyte number in both groups. CsA induced an increase in monocyte number only in sham animals. Treatments did not alter LH, FSH, or estradiol, whereas PRL was increased by CsA only in sham rats. Neither Dex nor CsA induced any significant variations in tumor volume, nor did they alter tumor histology. In addition, no visible metastases or alterations in other organs were observed. We conclude that, though immunological parameters were altered by the treatments, immunosuppressor drugs did not condition tumor development. In addition, tumors secrete one or more factor/s that counteract CsA effect.

Structure and biological activity of gonadotropin-releasing hormone isoforms isolated from rat and hamster brains.

Rat and hamster brain tissues were used to investigate the possible existence of a follicle stimulating hormone (FSH)-releasing factor with similar characteristics to the lamprey gonadotropin-releasing hormone III (lGnRH-III) form proposed in previous reports. The present studies involved isolation and purification of the molecule by high-performance liquid chromatography (HPLC), identification by radioimmunoassay, sequence analysis by automated Edman degradation, mass spectrometry and examination of biological activity. Hypothalamic extracts from both species contained an HPLC fraction that was immunoreactive to GnRH and coeluted with lGnRH-III and 9-hydroxyproline mGnRH ([Hyp(9)]GnRH). Determination of primary structure from purified total brain material demonstrated that the isolated molecule was [Hyp(9)]GnRH. This is the first report showing the presence of the posttranslationally modified form already known as [Hyp(9)]GnRH by primary sequence analysis. The biological activity of distinct GnRH peptides was also tested in vitro for gonadotropin release using rat pituitary primary cell cultures. The results showed that [Hyp(9)]GnRH stimulated both luteinizing hormone and FSH release, as already reported, whereas lGnRH-III had no action on the secretion of either gonadotropin.

GABA(B) receptors in anterior pituitary cells. Mechanism of action coupled to endocrine effects.

The activation of pituitary GABA(B) receptors by the specific agonist baclofen inhibits pituitary hormone secretion in vitro. Here we studied the mechanism of action of GABA(B) receptors in rat adenohypophysis. Anterior pituitary cells were obtained by trypsinization and were either plated for hormonal studies and cAMP determination or incubated in FURA 2AM for calcium measurements. Baclofen (BACL: 1 x 10(-5) M) significantly inhibited basal and thyrotropic releasing hormone (TRH)-stimulated (1 x 10(-7) M) PRL secretion in anterior pituitary cells from proestrous rats. In the presence of pertussis toxin (PTX: 150 ng/ml, 20 h), which leads to the uncoupling of the G(i/o)-protein from the receptor, both effects of BACL were abolished while the effect of dopamine (DA: 1 x 10(-8) M), used as an inhibitory control, was reduced from 70 to 25%. PTX also reversed BACL-induced inhibition of gonadotropin-releasing hormone (GnRH)-elicited luteinizing hormone (LH) secretion in anterior pituitary cells from 15-day-old female rats. In addition, though working in a pituitary mixed cell population, in which only some cell types possess GABA(B) receptors, BACL (1 x 10(-5) M) attenuated the forskolin-induced (0.5 microM) increase in cAMP. This effect was prevented by co-incubation with the antagonist 2 hydroxysaclofen and by preincubation with PTX. BACL (5 x 10(-5) M) and DA (5 x 10(-7) M) inhibited basal intracellular calcium concentrations ([Ca(2+)](i)) in pituitary cells and the effect of the latter was significantly stronger. The effect of BACL on [Ca(2+)](i) was abolished after preincubation with PTX. In the presence of the potassium channel blocking agents barium (200 microM and 1 mM) and tetraethylammonium (10 mM), BACL was still able to inhibit [Ca(2+)](i). Blockade of voltage-sensitive calcium channels (VSCC) with either verapamil (5 x 10(-6) M) or nifedipine (1 x 10(-6) M) completely abolished the effect of BACL on [Ca(2+)](i). In the presence of 12.5 mM potassium concentration baclofen significantly inhibited [Ca(2+)](i). In conclusion, our results describe the negative coupling of adenohypophyseal GABA(B) receptors to VSCC through PTX-sensitive G-proteins. These characteristics suggest a resemblance of these receptors to the typical presynaptic GABA(B) sites described in the central nervous system.

Progestin regulation of galanin and prolactin gene expression in oestrogen-induced pituitary tumours.

Galanin is a peptide widely distributed in the hypothalamic-pituitary axis. In the female rat pituitary, galanin is mainly present in lactotrophs, where it regulates their secretion and proliferation. Galanin expression is increased in oestrogen-induced prolactinomas, and it has been proposed that oestrogen effects on lactotroph function and proliferation could be mediated by galanin. Previous studies from our laboratory demonstrated that the synthetic progestin levonorgestrel antagonizes pituitary tumorigenesis of rats given oestrogen, reducing the number of proliferating cells and increasing cell death by nonapoptotic mechanism(s). To elucidate the role of galanin in levonorgestrel effects on the tumours, we examined galanin and prolactin mRNA and peptide expression in prolactinomas of rats receiving the progestin. Levonorgestrel reduced the pituitary weight and serum prolactin concentrations in oestrogen-treated rats. Galanin mRNA expression (determined by in situ hybridization), and the number of galanin expressing cells (determined by immunocytochemistry) were also reduced by the progestin in tumour-bearing rats. However, neither prolactin mRNA content, nor the number of prolactin-expressing cells, were modified by levonorgestrel treatment of oestrogen-receiving rats. The present study suggests that levonorgestrel controls pituitary growth by diminishing galanin expression. In contrast, changes in serum prolactin concentration seem to be more related to the reduction in tumour size, since the reduction in galanin expression was not large enough to regulate prolactin mRNA expression or the percentage of lactotrophs.

Ontogenic expression of anterior pituitary GABA(B) receptor subunits.

gamma-Aminobutyric acid (GABA) is involved in the neuroendocrine control of hypophyseal secretion, acting both in the central nervous system and directly at the pituitary. We have characterized the properties of anterior pituitary GABA(B) receptors. In this work the ontogeny of rat anterior pituitary GABA(B) receptors and the pattern of subunit expression in rats of both sexes were determined. Western blot analysis showed a temporal decrease in GABA(B) subunits GABA(B(1a)) and GABA(B(1b)) expression in female anterior pituitary membranes from day 4 to adulthood, with GABA(B(1a)) being significantly more abundant than GABA(B(1b)) at early stages of development; the GABA(B(2)) subunit was barely detectable. In the male, GABA(B(1a)) followed a similar pattern and appeared to be significantly less abundant than in 4- and 12-day-old females; GABA(B(1b)) and GABA(B(2)) expression in the male was barely detectable. Scatchard plot analysis showed a temporal decrease in binding sites in female anterior pituitary membranes, in agreement with the western blot results. The number of binding sites was significantly higher in female than in male 4-day-old membranes. Dissociation constant values were similar for both sexes at all ages studied. This study reports for the first time the ontogeny of anterior pituitary GABA(B) receptors, showing a particular developmental pattern of subunit expression and a clear sexual dimorphism.

Sexual dimorphism in diethylstilbestrol-induced prolactin pituitary tumors in F344 rats.

Female F344 rats treated chronically with diethylstilbestrol (DES) develop prolactin (PRL)-producing pituitary tumors. These tumors are larger in female than in male rats. To investigate gender differences in DES-induced pituitary tumor formation, we employed female and male rats and neonatally androgenized females, which received 100 microg of testosterone propionate (TP) after birth. At 3 months of age, all rats were deprived of their gonads and divided into control and DES-treated groups. Forty days after beginning treatment, control pituitary weight and serum PRL were similar in gonadectomized males (GDX), ovariectomized females (OVX) and androgenized-ovariectomized females (OVX + TP), but weight of DES-induced tumors was 2.5-fold higher and serum PRL 5.6-fold higher in OVX + DES than in GDX + DES or OXV + TP + DES (p<0.001). At the pituitary level, nuclear estrogen receptors (NE(2)R) amounted to >100 fmol/mg DNA in all rats receiving DES. However, NE(2)R were lower in OVX + DES (101.3+/-9.0 fmol/mg DNA) than in GDX + DES (174.6 +/-16.8; p<0.05) and in OXV + DES + TP (150.3+/-27.7; p<0.05). A similar profile was found for cytosolic progestin receptors. Using electron microscopy (EM), hyperplasia/hypertrophy of lactotropes was found in all DES-stimulated pituitaries. However, tumors of OVX + DES rats were enriched in hyperstimulated typical lactotropes, i.e., cells with high rate of hormonal synthesis, processing and secretion. Instead, tumors from GDX + DES and OVX + TP + DES rats were a mixture of typical and atypical lactotropes, i.e. a cell subpopulation with refractory secretory response and a few gonadotropes. In agreement with these data, immunoreactive pituitary PRL was lower in OVX + DES than in OVX + TP + DES and GDX + DES groups. Thus, differences in the sensitivity to DES, serum and tumor PRL, NE(2)R and progestin receptors between estrogenized female rats on one side and male and TP-androgenized females on the other, may by due in part to heterogeneity of cell populations. Our data further suggest that neonatal hypothalamic exposure to androgens, as in normal males or androgenized females with masculinization of hypothalamic centers, may condition the response to DES stimulation later in life.

Reproductive effects of hexachlorobenzene in female rats.

Hexachlorobenzene (HCB) is a polyhalogenated aromatic hydrocarbon widely distributed in the environment. In animal testing, HCB has been shown to be a reproductive toxin. Previous investigations of the effects of HCB on ovarian function have yielded equivocal results. Thus, the effects of chronic administration of HCB (1 g kg(-1) body wt.) on the ovary and pituitary hormone levels, hepatic and uterine oestradiol receptors, ovarian histopathological changes and oestrus cycle characteristics were investigated in spontaneously cycling rats. Our data demonstrate that HCB treatment, under the conditions of the present study, reduced circulating levels of oestradiol and prolactin without differences in serum concentrations of progesterone. Follicle-stimulating hormone serum levels were elevated. Hexachlorobenzene treatment resulted in irregularity of cycles, characterized mainly as prolonged periods of oestrus with a reduced number of ova recovered. In addition, HCB administration resulted in significantly decreased uterine nuclear oestrogen receptor levels. Histopathological examination revealed degenerative changes of the ovarian follicles and germinal epithelium and increased numbers of atresic follicles.

Calcium influx and intracellular stores in angiotensin II stimulation of normal and hyperplastic pituitary cells.

In rat pituitary cells from estrogen-induced hyperplasia, angiotensin II (ANG II) does not evoke a clear spike elevation of intracellular Ca2+ concentration ([Ca2+]i) but induces a plateau increase. The present work was undertaken to establish whether this difference was related to a differential participation of intracellular and/or plasma membrane Ca2+ channels. We first tested the effect of 10 nM ANG II on [Ca2+]i in the absence of extracellular Ca2+ in cells depolarized with 25 mM K+ or in the presence of blockers of L-type voltage-sensitive Ca2+ channels (VSCC). These treatments did not alter spike elevation in [Ca2+]i in controls but reduced plateau levels in hyperplastic cells. Intracellular Ca2+ stores were similar in both groups, as assessed by thapsigargin treatment, but this drug abolished spike increase in controls and scarcely modified plateau levels in hyperplastic cells. Finally, inositol trisphosphate (InsP3) production in response to ANG II was significantly higher in control cells. We conclude that the observed plateau rise in hyperplastic cells results mainly from Ca2+ influx through VSCC. In contrast, in control cells, the ANG II-induced spike increase in [Ca2+]i results from mobilization of Ca2+ from thapsigargin-sensitive internal channels, activated by higher inositol 1,4,5-trisphosphate generation.