Differential and interactive effects of ligand-bound progesterone receptor A and B isoforms on tyrosine hydroxylase promoter activity.

The classical progesterone receptors (PRs) are expressed in some hypothalamic dopaminergic and brainstem noradrenergic neurones. Progesterone influences prolactin and luteinising hormone release from the anterior pituitary gland, in part by regulating the activity of these catecholaminergic neurones. The present study aimed to determine the effects of PRs on tyrosine hydroxylase (TH) promoter activity. When CAD, SK-N-SH and CV-1 cells were transfected with TH promoter constructs and PR-A or PR-B expression vectors, progesterone treatment caused three- to six-fold increases in TH-9.0 kb promoter activity in PR-B expressing cells, although only a modest increase or no change in PR-A expressing cells. Using CAD cells, deletional analysis mapped the site of PR action to the -1403 to -1304 bp region of the TH promoter. Mutational analysis of putative regulatory sequences in this region indicated that multiple DNA elements are required for complete PR-B transactivation. Electrophoretic mobility shift assays were unable to demonstrate direct PR-B binding to TH promoter DNA sequences. However, chromatin immunoprecipitation analysis indicated PR-B was recruited to the TH promoter. Two different PR-B DNA binding domain mutants had opposing effects on PR-B-mediated TH promoter activation. A GS to AA mutation located in the p-box of the first zinc finger of PR-B inhibited progesterone transactivation of the TH promoter, whereas a C to A mutation in the zinc finger increased transactivation. PR-A was able to inhibit PR-B transactivation in a dose-dependent manner, although the degree of PR-A inhibition was dependent on the TH promoter deletion construct. These data indicate that ligand-bound PR-B is recruited to DNA elements in the TH promoter and acts as a transcriptional activator of the TH gene, and also that changes in the ratio of PR-A to PR-B may affect the ability of progesterone to increase TH expression.

Mu and kappa opioid receptor expression in the mediobasal hypothalamus and effectiveness of selective antagonists on prolactin release during lactation.

Endogenous opioid peptides are involved in prolactin release during lactation, in part by decreasing tuberoinfundibular dopaminergic (TIDA) neuronal activity. Both mu (mu) and kappa (kappa) opioid receptors have a role in the suckling-induced prolactin rise after 4-5 h up deprivation. The aim of this study was to investigate effects of mu opioid receptor antagonist, beta-funaltrexamine (beta-FNA), and kappa opioid receptor antagonist, nor-binaltorphimine (nor-BNI), on prolactin secretion and TIDA neuronal activity in lactating rats after 18 h pup deprivation. After 4 h separation from pups, the suckling-induced prolactin rise was abolished by 16 microg nor-BNI and 5 microg beta-FNA, coincident with increased dihydroxyphenylacetic acid (DOPAC):dopamine ratio in the stalk-median eminence (SME). However, after 18 h pups separation, these same doses of nor-BNI and beta-FNA did not alter the prolactin surge or DOPAC:dopamine ratios in the SME. Higher doses of nor-BNI (32 microg) and beta-FNA (10 microg) were required to inhibit suckling-induced prolactin secretion. beta-FNA (10 microg) increased the DOPAC:dopamine ratio in the SME, whereas nor-BNI (32 microg) treatment had no effect. The mu and kappa opioid receptor mRNA levels in the mediobasal hypothalamus were similar to suckled control rats after 4 h pup deprivation, but increased 1.4-fold after 18 h pup deprivation. These data support involvement of endogenous opioidergic systems in the suckling-induced prolactin rise after a prolonged (18 h) period of pup deprivation, as well as the shorter (4 h) pup deprivation period previously reported. Suppression of TIDA neuronal activity likely played a part in mu opioid receptor input to the suckling-induced prolactin rise after both 4 h and 18 h separation, whereas non-dopaminergic input was implicated with kappa opioid receptors after 18 h pup deprivation. Increased mu and kappa opioid receptors gene expression in the mediobasal hypothalamus may contribute to reduced effectiveness of opioid receptor antagonists to block suckling-induced prolactin release after 18 h pup deprivation.

Gene expression profiles of intracellular and membrane progesterone receptor isoforms in the mediobasal hypothalamus during pro-oestrus.

Progesterone action is mediated by its binding to specific receptors. Two progesterone receptor (PR) isoforms (PRA and PRB), three membrane progesterone receptor (mPR) subtypes (mPRalpha, mPRbeta and mPRgamma) and at least one progesterone membrane-binding protein [PR membrane component 1 (PRmc1)] have been identified in reproductive tissues and brain of various species. In the present study, we examined gene expression patterns for PR isoforms, mPR subtypes and PRmc1 in the rat mediobasal hypothalamus (MBH) during pro-oestrus. The mRNA level for each receptor subtype was quantified by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) at the time points: 13.00 h on dioestrous day 2; 09.00, 13.00, 17.00 and 22.00 h on pro-oestrus; and 13.00 h on oestrus. For PR, one primer set amplified PRA+PRB, whereas a second primer set amplified PRB. As expected, PRA+PRB mRNA expression was greater than PRB in MBH tissue. PRB mRNA levels increased throughout the day on pro-oestrus, with the highest levels being observed at 17.00 h. PRB mRNA levels in the MBH were increased by 2.4- and 3.0-fold at 13.00 and 17.00 h, respectively, on pro-oestrus compared to 13.00 h on dioestrous day 2. There were differential mRNA expression levels for mPRs and PRmc1 in the MBH, with the highest expression for PRmc1 and the lowest for mPRgamma. The mPRalpha mRNA contents at 13.00 and 17.00 h on pro-oestrus were increased by 1.5-fold compared to that at 13.00 h on dioestrous day 2. The mPRbeta mRNA levels at 13.00 and 17.00 h on pro-oestrus were 2.5- and 2.4-fold higher compared to that at 13.00 h on dioestrous day 2, respectively. PRA+PRB, mPRgamma and PRmc1 mRNA levels did not vary on pro-oestrus. These findings suggest that the higher expression of PRB, mPRalpha and mPRbeta in the MBH on pro-oestrous afternoon may influence both genomic and nongenomic mechanisms of progesterone action during the critical pre-ovulatory period.

Analysis of partner of inscuteable (mPins) expression in the developing mouse eye.

PURPOSE: Asymmetric cell division (ACD) is the fundamental mechanism underlying the generation of cellular diversity in invertebrates and vertebrates. During Drosophila neuroblast division, this process involves stabilization of the apical complex and interaction between the Inscuteable (Insc) and Partner of inscuteable (Pins) proteins. Both cell-intrinsic factors and cell-cell interactions seem to contribute to cell fate decisions in the retina. The Pins protein is known to play a major role in the asymmetric segregation of cell fate determinants during development of the central nervous system in general, but its role in asymmetric cell divisions and retinoblast cell fate has never been explored. The primary aim of this study was to determine the spatial distribution and time course of mouse homolog of Drosophila Partner of Inscuteable (mPins) expression in the developing and adult mouse eye. METHODS: The expression pattern of mPins was studied in the mouse eye from embryonic (E) stage E11.5 until adulthood, by semiquantitative RT-PCR, in situ hybridization, and immunohistochemistry. In addition, variations in mRNA and protein levels for mPins were analyzed in the developing postnatal and adult lens, by semiquantitative RT-PCR, western blot analysis, in situ hybridization, and immunohistochemistry. RESULTS: We detected mPins mRNA at early stages of mouse embryonic eye development, particularly in the neuroblastic layer. In early postnatal development, mPins mRNA was still detected in the neuroblastic layer, but also began to be detectable in the ganglion cell layer. Thereafter, mPins mRNA was found throughout the retina. This pattern was maintained in differentiated adult retina. Immunohistochemical studies showed that mPins protein was present in the neuroblastic layer and the ganglion cell layer during the early stages of postnatal retinal development. At these stages, mPins protein was colocalized with Numb protein, a marker of the ACD. At later postnatal stages, mPins protein was present in all retinal nuclear layers and in the inner plexiform layer. It continued to be detected in these layers in the differentiated retina; the outer plexiform layer and the photoreceptor inner segments also began to display positive immunostaining for mPins. In the adult retina, mPins was also detected in the retinal pigment epithelium and choroidal melanocytes. Throughout development, mPins protein was detected in nonretinal tissues, including the cornea, ciliary body, and lens. We focused our attention on lens development and showed that mPins protein was first detected at E14.5. The most striking results obtained concerned the lens, in which mPins protein distribution switched from the anterior to the posterior region of the lens during embryonic development. Interestingly, in the postnatal and adult lens, mPins protein was detected in all lens cells and fibers. CONCLUSIONS: We provide the first demonstration that mPins protein is expressed from embryonic stages until adulthood in the mouse eye. These results suggest that mPins plays important roles in eye development. This work provides preliminary evidence strongly supporting a role for mPins in the asymmetric division of retinoblasts, and in the structure and functions of adult mouse retina. However, the link between the presence of mPins in different ocular compartments and the possible occurrence of asymmetric cell divisions in these compartments remains to be clarified. Further studies are required to elucidate the in vitro and in vivo functions of mPins in the developing and adult human eye.

The RNA-binding protein Musashi-1 is produced in the developing and adult mouse eye.

PURPOSE: Musashi-1 (Msi1) is an RNA-binding protein produced in various types of stem cells including neural stem/progenitor cells and astroglial progenitor cells in the vertebrate central nervous system. Other RNA-binding proteins such as Pumilio-1, Pumilio-2, Staufen-1, and Staufen-2 have been characterized as potential markers of several types of stem or progenitor cells. We investigated the involvement of Msi1 in mouse eye development and adult mouse eye functions by analyzing the profile of Msi1 production in all ocular structures during development and adulthood. METHODS: We studied Msi1 production by in situ hybridization and immunohistochemistry of ocular tissue sections and by semi-quantitative RT-PCR and western blot analysis from the embryonic stage of 12.5 days post coitum (E12.5 dpc) when the first retinal ganglion cells (RGCs) begin to appear to the adult stage when all retinal cell types are present. RESULTS: Msi1 mRNA was present at all studied stages of eye development. Msi1 protein was detected in the primitive neuroblastic layer (NbL), the ganglion cell layer (GCL), and in all major differentiated neurons of postnatal developing and adult retinae. During postnatal developing stages, faint diffuse Msi1 protein staining is converted to a more specific distribution once mouse retina is fully differentiated. The most striking result of our study concerns the large amounts of Msi1 protein and mRNA in several unexpected sites of adult mouse eyes including the corneal epithelium and endothelium, stromal keratocytes, progenitor cells of the limbus, equatorial lens stem cells, differentiated lens epithelial cells, and differentiating lens fibers. Msi1 was also found in the pigmented and nonpigmented cells of the ciliary processes, the melanocytes of the ciliary body, the retinal pigment epithelium, differentiated retinal neurons, and most probably in the retinal glial cells such as Müller glial cells, astrocytes, and the oligodendocytes surrounding the axons of the optic nerve. Msi1 expression was detected in the outer plexiform layer, the inner plexiform layer, and the nerve fiber layer of fully differentiated adult retina. CONCLUSIONS: We provide here the first demonstration that the RNA-binding protein, Msi1, is produced in mouse eyes from embryonic stages until adulthood. The relationship between the presence of Msi1 in developing ocular compartments and the possible stem/progenitor cell characteristics of these compartments remains unclear. Finally, the expression of Msi1 in several different cell types in the adult eye is extremely intriguing and should lead to further attempts to unravel the role of Msi1 in cellular and subcellular RNA metabolism and in the control of translational processes in adult eye cells particularly in adult neuronal dendrites, axons, and synapses.

Gamma-D crystallin gene (CRYGD) mutation causes autosomal dominant congenital cerulean cataracts.

Congenital cataracts are a major cause of bilateral visual impairment in childhood. We mapped the gene responsible for autosomal congenital cerulean cataracts to chromosome 2q33-35 in a four generation family of Moroccan descent. The maximum lod score (7.19 at recombination fraction theta=0) was obtained for marker D2S2208 near the gamma-crystallin gene (CRYG) cluster. Sequencing of the coding regions of the CRYGA, B, C, and D genes showed the presence of a heterozygous C>A transversion in exon 2 of CRYGD that is associated with cataracts in this family. This mutation resulted in a proline to threonine substitution at amino acid 23 of the protein in the first of the four Greek key motifs that characterise this protein. We show that although the x ray crystallography modelling does not indicate any change of the backbone conformation, the mutation affects a region of the Greek key motif that is important for determining the topology of this protein fold. Our data suggest strongly that the proline to threonine substitution may alter the protein folding or decrease the thermodynamic stability or solubility of the protein. Furthermore, this is the first report of a mutation in this gene resulting in autosomal dominant congenital cerulean cataracts.

Regulation of prolactin secretion during pregnancy and lactation.

Prolactin plays major roles in maintaining the corpora lutea of pregnancy and in the synthesis of milk during lactation. The hypothalamic mechanisms involved in these functions have been investigated. Mating leads to a surge of prolactin and programs daily surges during early pregnancy. The expression of Fos-immunoreactivity shows that mating activates several hypothalamic nuclei, particularly the arcuate nucleus and medial preoptic area. In the arcuate nucleus, mating is associated with Fos expression in beta-endorphin neurons, and infusion of naloxone blocks both mating-induced and diurnal prolactin surges. Tyrosine hydroxylase-immunoreactive dopamine neurons appear not to participate in surge generation. However, after day 10 of gestation the secretion of placental lactogens suppresses prolactin secretion via activation of dopamine neurons without involvement of beta-endorphin neurons. Intracerebroventricular implantation of placental lactogen-secreting cells will block pregnancy prolactin surges, increase Fos expression in dopamine neurons, and increase tyrosine hydroxylase activity. During lactation the mechanisms regulating dopamine and beta-endorphin neurons are further modified. In early lactation a prolactin-induced increase in tyrosine hydroxylase activity leads to negative feedback, but this effect is lost by mid-lactation. Overriding this negative feedback is the inhibitory effect that suckling has on dopaminergic activity. This may involve beta-endorphin-mediated inhibition of dopamine neurons, as naloxone causes a marked increase in tyrosine hydroxylase activity and suppression of circulating prolactin. However, removal of tonic dopamine inhibition is not sufficient to account for the high levels of prolactin attained during lactation, and additional releasing factors are probably involved. In situ hybrization histochemistry for the most recent candidate, prolactin-releasing peptide, suggests that this may involve brain stem neurons that co-localize noradrenaline. Thus, prolactin secretion during pregnancy and lactation involve complex interactions of regulatory factors and plasticity of neuronal responsiveness.

Calmodulin and a cyclic nucleotide-dependent protein kinase facilitate the prolactin-induced increase in tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons.

Many aspects of tuberoinfundibular dopaminergic neuronal function are increased by elevated prolactin (PRL) levels, including the activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine. This study evaluated the roles of calmodulin, cyclic nucleotide-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II in the PRL-induced increase in tyrosine hydroxylase activity. Ovariectomized rats were treated with haloperidol or ovine PRL (oPRL) for 20-30 h before the experiment, respectively. Treatment with haloperidol increased circulating PRL levels 8-fold and tyrosine hydroxylase activity in the stalk-median eminence 1.8-fold. Treatment with oPRL increased tyrosine hydroxylase activity 1.9-fold. W-7, a calmodulin antagonist, reversed both the haloperidol- and oPRL-induced increase in tyrosine hydroxylase activity to control levels. H-8, a cyclic nucleotide-dependent protein kinase inhibitor, also reversed the haloperidol induced increase in tyrosine hydroxylase activity. KN62, a selective calcium/calmodulin-dependent protein kinase II inhibitor, attenuated the haloperidol-induced increase in tyrosine hydroxylase activity, but KNO4, a structurally related control compound, had no effect. By contrast, the oPRL- and haloperidol-induced increases in tyrosine hydroxylase activity were not altered by KN93, a selective calcium/calmodulin-dependent protein kinase II inhibitor. These data indicate that calmodulin and a cyclic nucleotide-dependent protein kinase contribute to the PRL-induced increase in tyrosine hydroxylase activity, but the role of calcium/calmodulin-dependent protein kinase II is still unclear.

Calcitonin inhibition of prolactin secretion in lactating rats: mechanism of action.

The effects of intracerebroventricular (10 ng/rat) or intravenous (10 or 40 microg/15 min/rat) administration of salmon calcitonin (sCT) on the prolactin (PRL) response to suckling and the activity of tyrosine hydroxylase (TH) were examined in lactating rats. Plasma concentration of PRL increased dramatically in control rats after the onset of the suckling stimulus, while administration of sCT resulted in inhibition of PRL response to suckling. The action of sCT was much more effective with intracerebroventricular administration, which totally blocked PRL release, compared to intravenous administration. The intracerebroventricular administration of sCT increased TH activity of tuberoinfundibular dopamine neuron (TIDA) in the stalk-median eminence, as measured by DOPA accumulation, while completely suppressing the PRL response to suckling. Injection of alpha-methyl-p-tyrosine (alpha-MT; 50 mg/kg), an inhibitor of TH and thus dopamine synthesis, increased PRL levels, and suckling caused a further increase in plasma concentrations of PRL. Injection of sCT (intracerebroventricularly) did not inhibit the PRL response to suckling in the presence of a depletion of dopamine. These results suggest that sCT inhibition of PRL secretion in lactating rats is mediated mainly by TIDA neurons without involvement of other neuroendocrine mechanisms.

Estradiol attenuates the forskolin-induced increase in hypothalamic tyrosine hydroxylase activity.

The purpose of this study was to evaluate interactions between estradiol and the 3',5' cyclic adenosine monophosphate (cAMP) signaling pathway to regulate tyrosine hydroxylase (TH) activity in hypothalamic dopaminergic neurons. The first experiment examined the ability of forskolin to activate TH in the tuberoinfundibular dopaminergic neurons of adult ovariectomized rats with or without estradiol treatment. Estradiol treatment reduced both basal and forskolin-stimulated TH activity in the median eminence. The second group of experiments examined the effect of estradiol on the forskolin-induced activation of TH in fetal hypothalamic cells cultures. Estradiol decreased basal TH activity in the hypothalamic cell cultures to 80% of control levels. Forskolin treatment for 1 h increased TH activity in a concentration-dependent manner in control and estradiol-treated cells, but estradiol attenuated the stimulatory response to 0.01-10 microM forskolin. The suppressive effect of estradiol on cAMP-dependent activation of TH was evident with 1-12 h of forskolin treatment. The responses to other activators of the cAMP- protein kinase A pathway, including dibutyryl cAMP and 8-bromo-cAMP, and to a depolarizing stimulus were blunted in estradiol-treated cultures. Forskolin treatment for 1 h increased radiolabeled phosphate incorporation into TH protein in control but not estradiol-treated cells, suggesting that estradiol interferes with the ability of the cAMP pathway to phosphorylate TH. Forskolin caused a time-dependent increase in TH mRNA signal levels in control cultures. The magnitude of the forskolin-induced increase in TH mRNA levels was less in the estradiol-treated cells after 6 h of forskolin treatment, indicating that estradiol hinders cAMP-regulated TH gene expression. These data indicate that estradiol attenuates the ability of hypothalamic dopaminergic neurons to respond to cAMP-dependent stimulation by interfering with phosphorylation mechanisms in the short term and control of TH mRNA levels in the long term.

Pituitary hormone gene expression and secretion in human growth hormone-releasing hormone transgenic mice: focus on lactotroph function.

The human GH-releasing hormone (hGHRH) transgenic mouse has a hyperplastic anterior pituitary gland that eventually develops into an adenoma. We showed previously that the number of lactotrophs in the male hGHRH transgenic mouse is increased 2-fold, yet there is no concomitant increase in plasma levels of PRL. To further elucidate underlying changes in lactotroph function in the hGHRH transgenic mouse, the objectives of this study were to 1) examine the relative differences in PRL gene expression in transgenic mice and their siblings, 2) quantify PRL secretion at the level of the individual cell, 3) determine whether tyrosine hydroxylase gene expression and/or activity are altered in the hypothalamus of transgenic mice, and 4) assess dopamine receptor gene expression and functional sensitivity in lactotrophs of transgenic mice. Total PRL messenger RNA (mRNA) levels were increased nearly 5-fold in the hGHRH transgenic mouse, whereas the concentrations of PRL mRNA (PRL mRNA per microg total RNA) were unchanged. In contrast, total PRL contents were unchanged, whereas the concentrations of PRL (micrograms of PRL per mg total protein) were decreased 3-fold. Hypothalamic tyrosine hydroxylase steady state mRNA levels were not altered in the hGHRH transgenic mice, but hypothalamic tyrosine hydroxylase activity was increased 2-fold in transgenic mice. Dopamine D2 receptor mRNA concentrations in the anterior pituitary were increased 2.5-fold in hGHRH transgenic mice, and total pituitary D2 receptor mRNA levels were increased nearly 10-fold. Furthermore, the basal secretory capacity of lactotrophs from transgenic mice was increased significantly at the level of the single cell, and dopamine inhibited the secretion of PRL to a greater extent in hGHRH transgenic mice. Thus, although the total number of lactotrophs is increased 2-fold in hGHRH transgenic mice, the present data are consistent with the hypothesis that increased hypothalamic dopamine synthesis and release coupled with an increase in D2 dopamine receptor gene expression and functional sensitivity in the pituitary result in normal plasma levels of PRL.

3,5 cyclic adenosine monophosphate mediates the salmon calcitonin-induced increase in hypothalamic tyrosine hydroxylase activity.

This study examined the effect of salmon calcitonin (sCT) on hypothalamic tyrosine hydroxylase (TH) activity and evaluated the cellular signaling mechanisms involved in the response. Fetal hypothalamic cells were cultured in a defined medium and treated with sCT and/or specific protein kinase inhibitors on day 14 in vitro. sCT (0.1-10 nM) increased both TH activity and cellular cAMP content in a concentration-dependent manner. sCT (10 nM) increased TH activity to 150-175% of control values and resulted in a 10-fold increase in cellular cAMP content. Both the C1a and C1b CT receptor isoforms were present in the cultures, as assessed by RT-PCR. Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), a cAMP antagonist, and H-8, a cyclic nucleotide kinase inhibitor, blocked the sCT-induced increase in TH activity, with complete abolition of the response observed at concentrations of 1 mM and 5 microM, respectively. sCT (10 nM) increased radiolabeled phosphate incorporation into TH protein to 169% of control values and 1 mM Rp-cAMPS completely blocked this effect. In contrast, neither Calphostin C, a protein kinase C inhibitor, nor U-73122, a phospholipase C inhibitor, significantly altered the ability of sCT to increase TH activity. Likewise, the sCT-induced increase in TH activity was observed after pretreating the cells with either BAPTA/AM, an intracellular calcium chelator, or thapsigargin, an inhibitor of the endoplasmic reticulum calcium pump. These data indicate that sCT has a profound stimulatory effect on TH activity in fetal hypothalamic cells and that enhanced phosphorylation of TH coincides with the sCT-induced increase in enzyme activity. Moreover, CT receptors, which are linked to cAMP production, are expressed in the hypothalamic cells and a cAMP-dependent mechanism mediates the sCT-induced activation and phosphorylation of TH.

Elevated level of follicular fluid vascular endothelial growth factor is a marker of diminished pregnancy potential.

OBJECTIVE: To evaluate whether differences in follicular fluid vascular endothelial growth factor (FF VEGF) concentrations are observed between women achieving a clinical pregnancy and those failing to conceive. DESIGN: Retrospective chart review and analysis of FF VEGF concentrations. SETTING: University teaching center. PATIENT(S): Fifty-seven women < or =42 years of age undergoing follicular aspiration in preparation for IVF or GIFT. INTERVENTION(S): Analysis of FF VEGF concentrations and chart review of a single IVF or GIFT cycle. MAIN OUTCOME MEASURE(S): Follicular fluid VEGF concentrations, clinical pregnancy rate, age, ampules of gonadotropins used, oocytes retrieved, peak estradiol serum concentrations, day 3 FSH levels, and fertilization rate. RESULT(S): Women who did not conceive had higher FF VEGF concentrations than women achieving a clinical pregnancy (4.409 + 2,387 versus 2.793 +/- 1,180 pg/mL: P < .001). A negative correlation was observed between FF VEGF concentrations and peak estradiol levels and number of oocytes retrieved. A positive correlation was found for FF VEGF and patient's age and ampules of gonadotropins used. CONCLUSION(S): Elevated FF VEGF concentrations are associated with poor conception rates after IVF or GIFT.

Dimeric inhibin: a direct marker of ovarian aging.

OBJECTIVE: To investigate whether luteal secretion of inhibin-a is altered in the perimenopausal transition and to evaluate whether luteal inhibin secretion is correlated with other markers of ovarian reserve such as FSH and inhibin-b. DESIGN: Prospective study. SETTING: Reproductive Endocrinology Laboratories at The Ohio State University. PATIENT(S): Twenty-five women 39-52 years of age with regular menstrual cycles. INTERVENTION(S): Daily urine samples were monitored (LH predictor kit) to identify the day of ovulation. Blood samples obtained on days 6 and 8 after the LH surge and on day 3 of the subsequent follicular phase were assayed for FSH, E2, progesterone. inhibin-a, and inhibin-b. MAIN OUTCOME MEASURE(S): Serum levels of inhibin-a, inhibin-b, FSH, E2, and progesterone. RESULT(S): Luteal phase inhibin-a and follicular phase inhibin-b were correlated inversely with age in perimenopausal women. In addition, luteal phase inhibin-a and follicular phase inhibin-b levels were correlated inversely with follicular phase FSH levels. CONCLUSION(S): Both luteal phase inhibin-a and follicular phase inhibin-b levels are correlated inversely with age during the fifth decade of life. These findings suggest that corpus luteum function is altered during the perimenopausal transition. Moreover, these direct measures of ovarian function may be more sensitive indicators of "ovarian reserve" than indirect indicators such as pituitary FSH secretion.

Endogenous opioid peptides contribute to suckling-induced prolactin release by suppressing tyrosine hydroxylase activity and messenger ribonucleic acid levels in tuberoinfundibular dopaminergic neurons.

The endogenous opioid peptides have been implicated in the control of the suckling-induced PRL rise during lactation. This study examined the role of the endogenous opioid peptides in suppressing tuberoinfundibular dopaminergic neuronal activity during lactation. In the first experiment, lactating rats were constantly exposed to pups. Naloxone (NAL; 60 mg/kg x h; i.v.), an opioid receptor antagonist, or saline was infused for 12 h. Blood was collected before and at 2-h intervals during the infusion. NAL suppressed circulating PRL levels to less than 36% of control values at 4, 6, 8, and 12 h after the onset of the infusion. Tyrosine hydroxylase (TH) activity in the stalk-median eminence and TH messenger RNA signal levels in the arcuate nucleus were determined at the end of the NAL infusion. TH activity and TH messenger RNA signal levels were increased 2.5- and 2.7-fold, respectively, after the 12-h NAL infusion. Even though the time spent with their pups was similar between the two groups, the pups in the NAL-treated group failed to gain weight during the 12-h NAL infusion period, whereas the control litters (8 pups) gained 5 g. In a second experiment, pups were removed from the dams before the 12-h NAL infusion and were returned after 11 h. Blood was collected before the infusion, at 3-h intervals during the pup separation period, and at 15-min intervals after reunion with the pups. Plasma PRL in control and NAL-treated rats was low (1-15 ng/ml) and similar during the separation period. The suckling-induced PRL surge in NAL-treated rats was markedly attenuated to 9-25% of control levels (350-650 ng/ml). After a 1-h suckling episode, TH activity in the stalk-median eminence of NAL-treated rats was 4.5-fold greater than controls. Litter weight gains were significantly less in NAL-treated rats during the 1-h suckling episode. These data indicate that the endogenous opioid peptides are an integral component for increasing PRL release in response to suckling and they act to decrease tuberoinfundibular dopaminergic neuronal activity during lactation, in part, by suppressing TH gene expression.

Effects of chronic hyperprolactinemia on tuberoinfundibular dopaminergic neurons.

Prolactin (PRL) secretion is under the inhibitory regulation of the tuberoinfundibular dopaminergic (TIDA) system. Short-term elevation in PRL levels has been shown to increase the activity of TIDA neurons, however, the responsiveness of TIDA neurons to chronically elevated serum PRL levels is controversial. The purpose of this study was to investigate the effects of prolonged elevations of serum PRL on TIDA neuronal activity. Female Sprague-Dawley rats (2-3 months old) were ovariectomized and implanted (s.c.) with haloperidol (HAL), a dopamine receptor antagonist for 6 or 9 months to produce hyperprolactinemia. Ovariectomized, sham-implanted rats were used as controls. Other groups of intact rats were implanted with HAL or sham-implanted for 9 months and then were implanted with PRL-producing MMQ cells for 6 weeks to further increase circulating PRL levels. TIDA neuronal activity was measured in terms of tyrosine hydroxylase (TH) activity in the stalk-median eminence and was correlated with changes in serum PRL levels. After 6 months of treatment, TH activity in HAL-treated rats was 130% higher than that in the control rats. After 9 months of treatment, TH activity in HAL-treated rats was 81% higher than that in control rats. This increase was significantly less than the increase that occurred after 6 months of treatment. Nine months of HAL-induced hyperprolactinemia followed by implantation of PRL-producing MMQ cells, which resulted in very high levels of PRL, did not increase TH activity in the stalk-median eminence. These results demonstrate that hyperprolactinemia over a prolonged period reduces the responsiveness of TIDA neurons, and these effects vary depending on the duration and intensity of hyperprolactinemia.

Semicircadian rhythms of c-Fos expression in several hypothalamic areas during pregnancy in the rat: relationship to prolactin secretion.

Prolactin (PRL) serves an important luteotrophic function in the rat during early pregnancy, expressed as a nocturnal surge in the early morning and a diurnal surge in the late afternoon. Several areas of the hypothalamus, including the preoptic area (POA), the suprachiasmatic nucleus (SCN) and the ventromedial and dorsomedial nuclei (VM-DM) have been implicated in PRL surges. We investigated the temporal relationship between neuronal activity as measured by c-Fos immunocytochemistry in these areas and PRL secretion during early and late pregnancy. Brains were collected at nine time points (24:00, 02:00, 04:00, 06:00, 10:00, 14:00, 16:00, 18:00 and 20:00 h) on days 6-7 and three time points (02:00, 14:00 and 18:00 h) on days 14-15 of pregnancy. Plasma PRL levels determined by radioimmunoassay revealed two surges with peaks at 02:00 and 18:00 h and a trough at 14:00 h on days 6-7, which were absent on days 14-15 of pregnancy. The number of neurons expressing c-Fos in the anterior medial preoptic nucleus, the medial preoptic area and the medial preoptic nucleus, but not the anteroventral preoptic nucleus of the POA, and the VM-DM, showed a semicircadian rhythm which was maximal at 02:00 h or/and 04:00 and 18:00 h and reached the lowest value at 14:00 h, in parallel with the PRL surges in early pregnancy. However, the temporal pattern of c-Fos in these areas was reversed during late pregnancy, with a peak at 14:00 h and low levels at 02:00 and 18:00 h. PRL surges were absent and levels were uniformly low during these times. Neuronal activity in the SCN did not show any correlation with PRL surges. The dorsomedial subdivision of the SCN showed high neuronal activity during the daytime in both stages of pregnancy. Neuronal activity in the ventrolateral subdivision of the SCN was high during the nighttime in early pregnancy, however it exhibited high levels during the daytime in late pregnancy. These results suggest that the two daily surges of PRL secretion during the first half of pregnancy might be related to the temporal rhythm of neuronal activity in the POA and the VM-DM, and a major change in the pattern of neuronal activity in these hypothalamic areas might result in termination of the PRL surges at midpregnancy.

Follicular fluid vascular endothelial growth factor concentrations are elevated in women of advanced reproductive age undergoing ovulation induction.

OBJECTIVE(S): To determine whether follicular fluid (FF) from women of advanced reproductive age had a relative deficiency of the angiogenic cytokine vascular endothelial growth factor/vascular permeability factor. Furthermore, we sought to determine whether luteinized granulosa cells secrete vascular endothelial growth factor/vascular permeability factor in response to hypoxia. DESIGN: Retrospective cohort study. SETTING: University teaching hospital. PATIENTS: Women undergoing follicular aspiration after superovulation in preparation for IVF-ET. Women of advanced reproductive age consisted of 21 women > or = 38 years old (range, 38 to 46 years); 15 subjects < or = 30 years served as the control population. INTERVENTION(S): Granulosa cells and FF were collected by transvaginal aspiration 35 hours after hCG. Granulosa cells from two women were cultured for 24 and 48 hours in M199 + 10% fetal bovine serum in 1% O2-5% CO2-94% N2 (hypoxic) or 95% air-5% CO2 (normoxic) without or with 0.1 mol/L cobalt chloride. MAIN OUTCOME MEASURE(S): Pooled FF vascular endothelial growth factor/vascular permeability factor concentrations and media vascular endothelial growth factor/vascular permeability factor accumulation at 24 and 48 hours were determined. RESULT(S): Follicular fluid vascular endothelial growth factor/vascular permeability factor concentrations were higher in advanced reproductive age women compared with younger women (3,735 +/- 2,155 versus 2,205 +/- 952 pg/mL, mean +/- SD). Accumulation of vascular endothelial growth factor/vascular permeability factor at 24 and 48 hours was 391 +/- 54 and 744 +/- 2 pg/mL in media maintained in 5% CO2 and air. Cobalt chloride induced a marked increase in vascular endothelial growth factor/vascular permeability factor (2,008 +/- 52 pg/mL at 24 hours and 3,630 +/- 519 pg/mL at 48 hours). An intermediate but significant increase in vascular endothelial growth factor/vascular permeability factor (733 +/- 35 pg/mL at 24 hours and 2,675 +/- 864 pg/mL at 48 hours) was observed with 1% O2 compared with normoxic controls. CONCLUSION(S): After hMG and hCG administration the FF from women of advanced reproductive age showed increased vascular endothelial growth factor/vascular permeability factor concentrations compared with younger women. Increased vascular endothelial growth factor/vascular permeability factor concentrations could be consistent with a hypoxic environment within follicles of older women.

Prolactin (PRL) receptors are colocalized in dopaminergic neurons in fetal hypothalamic cell cultures: effect of PRL on tyrosine hydroxylase activity.

This study examined the responsiveness of dopaminergic neurons to PRL and the expression of PRL receptors in fetal hypothalamic cells. Hypothalamic cells were cultured in medium containing 5 or 25 mM potassium (K+) with or without 5% FBS. Rat PRL (rPRL) treatment (10-1000 ng/ml) for 10 days increased tyrosine hydroxylase (TH) activity 1.6- to 1.8-fold in dopaminergic neurons cultured in serum-containing medium with 25 mM K+, but not in defined medium or any medium with 5 mM K+. The rPRL-induced increase in TH activity was observed at 10-1000 ng/ml after both 1 and 10 days of rPRL treatment, whereas 1 ng/ml was not effective. TH activity was not altered after 1-12 h of rPRL treatment (100 ng/ml), but was increased 1.4-fold after 1-3 days and 1.8-fold after 5-10 days. The colocalization of PRL receptors and TH was evaluated by double labeled immunocytochemistry. PRL receptor immunostaining was observed in most TH-immunoreactive cells cultured in either defined or serum-containing medium with or without 10 days of rPRL treatment (100 ng/ml). As assessed by reverse transcriptase-PCR, the long form, but not the short form, of the PRL receptor was expressed in the hypothalamic cells regardless of medium composition, similar to the expression pattern in adult mediobasal hypothalamus from ovariectomized rats. These data indicate that a factor present in FBS imparts PRL responsiveness to hypothalamic dopaminergic neurons in vitro. The effective PRL concentrations and the time course for PRL's action in vitro are within the physiological range in vivo. The colocalization of PRL receptor in dopaminergic neurons provides anatomical evidence for a direct effect of PRL, with the long form of the PRL receptor being the predominant form in the hypothalamic cells.

Identification of gonadotrophin surge-inhibiting factor (GnSIF)/attenuin in human follicular fluid.

Evidence from several laboratories suggests that the ovaries of many species produce a non-steroidal factor called gonadotrophin surge-inhibiting or attenuating factor (GnSIF) which may regulate the response of the pituitary to gonadotrophin-releasing hormone (GnRH) and as such, may modulate the timing and/or amplitude of the luteinizing hormone (LH) surge. We have recently isolated a candidate GnSIF from porcine follicular fluid (PFF). Porcine GnSiF is a 69 kDa protein which has undetectable inhibin and follistatin immunological and biological activity. The present study was designed to purify and identify GnSIF from human follicular fluid. GnSIF activity was measured as suppression of GnRH-stimulated LH secretion from rat pituitary cells in primary culture. Human follicular fluid (approximately 500 ml) was recovered from patients undergoing in-vitro fertilization (IVF). GnSIF was purified by heparin-sepharose, Q-sepharose, S-sepharose, and hydrophobic interaction chromatography followed by isoelectric focusing. Gel electrophoresis and Western blot were used to identify human GnSIF and compare it with porcine GnSIF. Using these steps, we obtained a highly-purified preparation of GnSIF that manifests in-vitro bioactivity and chromatography characteristics similar to those observed for porcine GnSIF and that hybridizes with a porcine GnSIF antibody. Following treatment with human chorionic gonadotrophin/human menopausal gonadotrophin (HMG/HCG), human follicular fluid contained roughly 25% of the GnSIF (per mg protein) present in porcine follicular fluid. We conclude that GnSIF is present in human follicular fluid and may participate in the regulation of gonadotrophin secretion in this species.