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Sphingolipids are involved in the regulation of cell proliferation. It has been reported that diacylglycerol and sphingosine-1-phosphate generation, during the synthesis of phospho-sphingolipids, is necessary for both, G1-S transition of cell cycle during the sustained activation of protein kinase C in various cell models (MDCK, Saccharomyces and Entamoeba ) and AKT pathway activation. During the estrous cycle of the rat, AKT signaling is the main pathway involved in the regulation of uterine cell proliferation. The aim of the present study was to investigate the role of sphingolipid synthesis during proliferation of uterine cells in the estrous cycle of the rat. On metestrus day, when both luminal and glandular uterine epithelia present the maximal BrdU-labeled cells (S phase cells), there was an increase in the relative abundance of total sphingomyelins, as compared to estrus day. Myriocin, a sphingolipid synthesis inhibitor administered on estrus day, before the new cell cycle of epithelial cells is initiated, decreased the abundance of sphingomyelin, accompanied by proliferation arrest in uterine epithelial cells on metestrus day. In order to study the sphingolipid signaling pathway affected by myriocin, we evaluated the activation of the PKC-AKT-GSK3b-Cyclin D3 pathway. We observed that total and phosphorylated protein kinase C diminished in uterine epithelial cells of myriocin treated animals. Interestingly, cyclin D3 nuclear localization was blocked by myriocin, concomitantly with a decrease in nuclear pRb expression. In conclusion, we demonstrate that sphingolipid synthesis and signaling are involved in uterine epithelial cell proliferation during the estrous cycle of the rat., (© 2018 Society for Reproduction and Fertility.)

Tanycytes are special ependymal cells located in the ventrolateral wall and floor of the third ventricle having processes extending nuclei that regulate reproductive functions and around of vessels in median eminance. The aquaporins (AQPs) are a family of transmembrane proteins that transport water and glycerol. AQP-7 and -9 are permeable to other small molecules as glycerol and therefore called aquaglyceroporins. In this study, we aimed to show localization of AQP-7 and -9 in epithelial cells of choroid plexus and tanycytes during female mouse estrus cycle. AQP-7 and -9 proteins were detected in α2 and β1 tanycytes in prœstrus stage. Interestingly, there is no staining in estrus stage in any type of tanycytes. We observed weak immunoreactivity in α1, α2 and β1 tanycyte cells in metestrus stage for AQP-7 and α1 for AQP-9 protein. AQP-7 and -9 showed intense immunoreactivity in α2, β1 and β2 tanycyte cells during diestrus stage. Consequently, AQP-7 and -9 showed differential staining pattern in different stages of mouse estrus cycle. In the light of our findings and other recent publications, we suggest that AQP-7 and -9-mediated glycerol transport in tanycyte cells might be under hormonal control to use glycerol as a potential energy substrate during mouse estrus cycle., (Copyright © 2016. Published by Elsevier Masson SAS.)

The neuropeptide kisspeptin (Kiss1) is integral to the advent of puberty and the generation of cyclical LH surges. Although many complex actions of Kiss1 are known, the mechanisms governing the processing/regulation of this peptide have not been unveiled. The metallo enzyme, endopeptidase 24.15 (thimet oligopeptidase), has been demonstrated to play a key role in the processing and thus the duration of action of the reproductive neuropeptide, GnRH, which signals downstream of Kiss1. Initial in silico modeling implied that Kiss1 could also be a putative substrate for EP24.15. Coincubation of Kiss1 and EP24.15 demonstrated multiple cleavages of the peptide predominantly between Arg29-Gly30 and Ser47-Phe48 (corresponding to Ser5-Phe6 in Kiss-10; Kiss-10 as a substrate had an additional cleavage between Phe6-Gly7) as determined by mass spectrometry. Vmax for the reaction was 2.37±0.09 pmol/min · ng with a Km of 19.68 ± 2.53μM, which is comparable with other known substrates of EP24.15. EP24.15 immunoreactivity, as previously demonstrated, is distributed in cell bodies, nuclei, and processes throughout the hypothalamus. Kiss1 immunoreactivity is localized primarily to cell bodies and fibers within the mediobasal and anteroventral-periventricular hypothalamus. Double-label immunohistochemistry indicated coexpression of EP24.15 and Kiss1, implicating that the regulation of Kiss1 by EP24.15 could occur in vivo. Further studies will be directed at determining the precise temporal sequence of EP24.15 effects on Kiss1 as it relates to the control of reproductive hormone secretion and treatment of fertility issues.

Background: Progesterone (P4) may modulate oviductal functions to promote early embryo development in cattle. In addition to its nuclear receptor (PR), P4 may mediate its actions through P4 receptor membrane component 1 (PGRMC1) and its relative, PGRMC2. Two successive experiments were undertaken to characterise the expression of PR, PGRMC1 and PGRMC2 in the bovine oviduct during the post-ovulation period, and to relate their expression to the presence of an embryo, the proximity of the CL and to the region of the oviduct., Methods: In the first experiment (Exp. I), whole oviduct sections were collected from Holstein cows at Day 1.5, Day 4 and Day 5 post-ovulation (n = 2 cows per stage). The expression of PR, PGRMC1 and PGRMC2 was studied in the ampulla and isthmus by RT-PCR, western-blot and immunohistochemistry. In Exp. II, oviduct epithelial cells were collected from cyclic and pregnant Charolais cows (n = 4 cows per status) at Day 3.5 post-ovulation and mRNA expression of PR, PGRMC1 and PGRMC2 was examined in the ampulla and isthmus by real-time quantitative PCR., Results: In Exp. I, PR, PGRMC1 and PGRMC2 were expressed in all oviduct samples. PGRMC1 was mainly localised in the luminal epithelium whereas PR and PGRMC2 were localised in the epithelium as well as in the muscle and stroma layers of the oviduct. The expression was primarily nuclear for PR, primarily cytoplasmic for PGRMC1 and both nuclear and cytoplasmic for PGRMC2. In Exp. II, mRNA levels for PR, PGRMC1 and PGRMC2 were not affected by either the pregnancy status or the side relative to the CL. However, the expression of PR and PGRMC2 varied significantly with the region of the oviduct: PR was more highly expressed in the isthmus whereas PGRMC2 was more highly expressed in the ampulla., Conclusions: This is the first evidence of PGRMC2 expression in the bovine oviduct. Our findings suggest that P4 regulates the functions of the bovine oviduct in a region-specific manner and through both classical and non classical pathways during the post-ovulation period.

This is the first study reporting the locations of neurons expressing CART peptide (cocaine-amphetamine-regulated transcript) throughout all the nuclear and paleocortical formations of the amygdaloid body (AB) and demonstrating the effects of sex steroids on immunoreactivity. The immunocytochemical reaction was performed on frontal brain sections from adult rats (seven females in estrus, seven females in metestrus, and seven males). The proportions of immunoreactive neurons to the total number of neurons in adjacent sections stained by the Nissl method were assessed in estrus and metestrus. In the dorsomedial and posterior cortical nuclei and the lateral capsular subnucleus of the central field, the relative numbers of immunoreactive neurons at estrus were significantly greater than at metestrus. These results provide evidence of the involvement of the olfactory and integrative centers of the AB in the pathogenesis of drug dependence and show that new and efficient methods of gene therapy might be developed using the intranasal route for drug administration.

The location of CART peptide in the paleoamygdala neurons was studied by immunocytochemical reaction. Significant differences in the number of immunoreactive cells and optical density of CART-positive neurons detected over the course of the estrous cycle indicate modulating effects of sex steroids on the expression of CART peptide.

During the study of all nuclear and paleocortical structures of amygdala, the CART-peptide (cocaine-amphetamine-regulated transcript) expressing neurons were for the first time demonstrated in this region, and their immunoreactivity was shown to be influenced by sex steroids. Immunocytochemical reaction was performed on frontal slices of adult rat brain (7 female rats in estrus stage, 7 female rats in metestrus stage and 7 male rats). The portion of immunopositive neurons in animals in estrus and metestrus was counted in relation to their numbers in adjoining slices (stained using Nissl's method). The relative number of immunoreactive neurons in dorsomedial, posterior cortical nuclei and latero-capsular subnucleus of the central nucleus was found to be significantly greater in estrus than in metestrus. The data obtained show that olfactory and integrative centers of amygdala may be involved in the pathogenesis of drug addiction and indicate the possibility of development of new effective methods of gene therapy with the application of an intranasal route of drug delivery.

Rat luteinizing hormone beta (Lhb) gene transcription is stimulated by hypothalamic gonadotropin-releasing hormone 1 (GnRH1), and this response may be modulated by other signaling pathways such as cAMP. Here we characterize the ability of cAMP, alone or with GnRH1, to stimulate Lhb gene transcription in mouse pituitary and clonal gonadotroph cells. Both cAMP and pituitary adenylyl cyclase-activating peptide increase GnRH1 stimulation of luciferase activity in pituitaries of mice expressing the rat Lhb-luciferase transgene, suggesting cAMP and GnRH1 pathways interact in vivo. cAMP stimulation of the Lhb-luciferase transgene was similar between females in metestrus and proestrus, but GnRH1 stimulation was greater at proestrus. Additive effects with combined treatments were observed at metestrus and proestrus. Elevated intracellular cAMP stimulated Lhb promoter activity in LbetaT2 clonal gonadotroph cells, alone and with GnRH1. In LbetaT2 cells, cAMP stimulation of the Lhb promoter was eliminated by inhibition of protein kinase A (PKA); GnRH1 stimulation was partially suppressed by either PKA or protein kinase C inhibitors. Only the proximal GnRH1-responsive region of the promoter was required for cAMP stimulation, and mutation of the 3' NR5A1 site diminished the response. Regulation of primary mRNA transcripts from the endogenous Lhb gene by cAMP and GnRH1 correlated with results from the Lhb-luciferase transgene or transfected promoter. Occupancy of the endogenous promoter by EGR1 was increased by GnRH1 with or without forskolin, but forskolin alone had little effect. Thus, cAMP stimulation of Lhb promoter activity, and enhancement of GnRH1 stimulation, occurs in multiple physiological states independent of steroid status, via a PKA-dependent mechanism.

The hypothalamic arcuate nucleus integrates different hormonal and neural signals to control neuroendocrine events, feeding, energy balance and reproduction. Previous studies have shown that in adult female rats the arcuate nucleus undergoes a cyclic fluctuation in the number of axo-somatic synapses during the estrous cycle, in parallel to the variation of ovarian hormone levels in plasma. In the present study we have used an unbiased stereological analysis in conjunction with postembedding immunocytochemistry to assess whether the synaptic remodeling during the estrous cycle in rats is specific for certain types of synapses. Our findings indicate that there is a significant decrease in the number of GABAergic axo-somatic synapses on proestrus afternoon and estrus day compared with other days of the estrous cycle. This decrease in GABAergic synapses is accompanied by an increase in the number of dendritic spine synapses. The synaptic density appears to cycle back to proestrus morning values on metestrus day. In contrast, the number of synapses on dendritic shafts does not change during the cycle. These results indicate that a rapid and selective synaptic turnover of arcuate synapses occurs in physiological circumstances.

Certain female reproductive tissues are known to express the non-neuronal cholinergic system. Using different experimental approaches, we tested the hypothesis that acetylcholine (ACh) in the porcine oviduct may also be derived from non-neuronal structures. Immunohistochemistry was performed to detect acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) in different segments of the oviduct of cyclic and pregnant sows. Immunohistochemical experiments revealed strong immunoexpression of ChAT in the entire oviductal epithelium at metoestrus. Thereby, a particular pronounced staining was found in the supranuclear region of almost all epithelial cells. Immunostaining of ChAT decreased markedly during dioestrus and prooestrus stages, respectively. At prooestrus, ChAT immunoreactivity was confined to ciliated cells. Furthermore, we found elevated level of staining intensity of ChAT in the pregnant oviduct at day 13. Using the same ChAT antibody for Western blot analyses, we detected immunoreactive bands of MW 69,000 and 46,000 mainly in ampulla, while MW 58,000 and 30,000 forms were present mainly in infundibulum and isthmus. Furthermore ACh was detected by HPLC and fluorimetric methods in oviductal epithelium. In conclusion, we show expression of ChAT in oviductal epithelial cells at different stages of the oestrus cycle and pregnancy, indicating that these cells can synthesize ACh in a cycle-dependent manner. These results suggest as yet unexplored roles of epithelial ACh in the oviduct.

Since splenic immune functions are depressed in metestrus females following trauma-hemorrhage, we hypothesized that administration of the androgen receptor antagonist flutamide at the onset of resuscitation will maintain the immune function of the spleen following trauma-hemorrhage. Female C57BL6/J mice (metestrus state, 8-12 weeks old), underwent laparotomy and hemorrhagic shock (35.0+/-5.0 mm Hg for 90 min) and received 17beta-estradiol (50 microg/25 g), flutamide (625 microg/25 g) or 17beta-estradiol+flutamide. Four hours after resuscitation, the in vitro productive capacity of different cytokines (TNF-alpha, IL-6, IL-10, and IFN-gamma) by splenic MPhi and splenocytes were determined by flow cytometry. A significantly decreased cytokine production by both splenocytes and splenic MPhi was observed following trauma-hemorrhage compared to shams. Administration of 17beta-estradiol, flutamide and 17beta-estradiol+flutamide following trauma-hemorrhage resulted in a significant increase in the in vitro IL-6 release by splenic MPhi. The TNF-alpha productive capacity, however, was only restored by 17beta-estradiol and 17beta-estradiol+flutamide administration following trauma-hemorrhage. No significant effect of either treatment was observed with regard to the suppressed splenic MPhi IL-10 release. Anti-CD3 stimulation, administration of 17beta-estradiol and 17beta-estradiol+flutamide, but not the administration of flutamide alone resulted in a significant increased release of TNF-alpha, IL-6 and IFN-gamma compared to vehicle-treated animals. No significant effect of either treatment was found on IL-10 productive capacity. These results collectively suggest that flutamide administration following trauma-hemorrhage in females has beneficial effects on splenic immune function. However, flutamide administration in combination with estrogen does not provide any significant, additional effects over 17beta-estradiol administration alone.

Objective: This study was designed to elucidate whether there were differences in the hormonal responses of the parameters involving triacylglycerol (TG) deposition and mobilization in adipose tissue among the stages of the estrous cycle in female rats., Measurements: Adipose tissue was obtained from the parametrial region in female rats at each stage of the estrous cycle. Lipoprotein lipase (LPL) activity in the extracts of acetone/ether powders of the tissues was measured as a parameter for TG deposition. Norepinephrine-stimulated lipolysis in isolated fat cells was measured as a parameter for TG mobilization., Results: LPL activity changed periodically during the estrous cycle; the activity level was highest at diestrus, began to decrease at proestrus, reached a minimum at estrus, began to increase again at metestrus-1, and increased further at metestrus-2. At diestrus and proestrus, LPL activity was increased with an increase in plasma insulin levels, suggesting that plasma insulin was the predominant up-regulator of LPL. But at estrus, metestrus-1 and metestrus-2, LPL activity remained low even when plasma insulin levels were high, indicating that it was not up-regulated by plasma insulin. Norepinephrine-stimulated lipolysis in fat cells was high at estrus and metestrus-1 and low at diestrus., Conclusion: The hormonal responses of LPL activity and lipolysis in adipose tissue differed depending on the stage of the estrous cycle.

The aim of this immunohistochemical study was to describe the cellular distribution of the estrogen receptor-alpha (ERalpha), progesterone receptor (PR) and androgen receptor (AR) in canine uterine tubes. Samples of uterine tubes were taken from dogs in different stages of the estrous cycle, and dogs that were pregnant or had just delivered. Nuclear staining for sex steroid hormone receptors was observed in the surface epithelium, stromal cells and smooth muscle cells of the muscular layer. Only slight differences in staining pattern were observed between the ampulla and fimbriae. The staining for ERalpha and PR showed changes throughout the estrous cycle. Some of these changes were related to changing concentrations of sex steroid hormones. High staining scores for ERalpha and PR were found during proestrus and low scores during early metestrus. The staining for AR showed only minor cyclic changes. However, during proestrus and estrus, cytoplasmic staining for AR was observed in differentiated secretory epithelial cells, when nuclear staining in these cells was nearly absent. For the three hormone receptors, stromal cells generally stained with a higher intensity than epithelial cells. It is likely that many steroid hormone actions on the epithelium are mediated through stromal cells. During pregnancy, rather high staining scores were found for ERalpha and AR in the uterine tube. This is in contrast to observations in the canine pregnant uterus.

The importance of androgens for the female reproductive system has been investigated for decades and a number of androgen sensitive processes has now been identified in female reproductive organs. For carnivore species no data were available so far about uterine androgen sensitivity and its regulation. The present study therefore aimed to investigate whether androgen receptors (AR) are present in the dog uterus, whether they are regulated throughout the ovarian cycle and whether pyometra affects their expression rate. Uterine tissue samples were collected from 28 bitches of different ages and various breeds. The samples were grouped according to the stage of estrous cycle (metestrus ME or anestrus AE) and the pathological status of the uterus (i.e. suffering from pyometra or not). Androgen receptor mRNA (AR mRNA) was quantified from 500 ng of total RNA isolated from the tissue samples using an internally standardized reverse transcription polymerase chain reaction (RT-PCR) described previously. The amount of total RNA extractable per g tissue was elevated during pyometra. The successful amplification of the expected 172 bp fragment from canine uterine RNA together with the confirmation of the identity of this fragment by sequence analysis, demonstrates that AR is expressed in this particular tissue. Comparing the expression rates in uteri from bitches during ME or AE being healthy (H) or suffering from pyometra (P), the only significant (p < 0.01) difference was found between H and P uteri during ME with 3.5-fold lower expression rates in P. Although the same seems true for AE bitches, a significant difference could not be demonstrated due to the low number (n = 2) of diseased animals in the AE group. There was no evident effect of the stage of ovarian cycle on uterine AR mRNA levels.

Epidermal growth factor (EGF) stimulates gonadotropin secretion, suggesting that it may regulate gonadotrope functions. These responses may be modulated by changes in expression of EGF receptors (EGFR), especially during the estrous cycle. To test this hypothesis, EGFR and pituitary hormones were detected by dual immunocytochemistry. Pituitary cells from metestrous rats contained 41 +/- 4% cells labeled for EGFR. This peak was followed by a decline to 17.6 +/- 2% of cells from proestrous rats. The percentages of metestrous pituitary cells with EGFR and each hormone were: PRL, 11.8 +/- 1; ACTH, 9.9 +/- 1.8%; GH, 8.2 +/- 0.6%; TSH, 6.3 +/- 0.8%; FSH, 4 +/- 0.6%; and LH, 2.6 +/- 0.6%. The relatively low percentages of gonadotropes may have reflected the low expression of LH or FSH antigens during metestrus. Dual labeling for EGFR and LHbeta or FSHbeta messenger RNAs (mRNAs) showed a significant increase in the percentages of pituitary cells with LHbeta mRNA and EGFR (to 5.7% of pituitary cells), but there were no increases in the EGF target cells bearing FSHbeta mRNA. When gonadotropin antigens were detected in EGF target cells during other stages of the cycle, there was an increase to reach a peak of 6.6-7% of pituitary cells by the morning of proestrus (or 40-50% of gonadotropes). To summarize, EGFR are seen in few gonadotropes during the metestrous peak, although more LH cells (but not FSH cells) can be identified by their content of LHbeta mRNA. This suggests that EGFR is expressed initially in monohormonal LH gonadotropes. The peak expression of EGFR by gonadotropes during diestrus and proestrus suggests that EGF may be involved in the development of the gonadotropes as they approach surge secretory activity. It also may help stimulate the transcription of new gonadotropin beta-subunit mRNA seen late in proestrus, early in estrus.

Rat mixed anterior pituitary cells from cycling females were microscopically demonstrated to produce epidermal growth factor (EGF), in culture, using a reverse hemolytic plaque assay. Anterior pituitary cells of proestrous female rats secrete more EGF than those of metestrous rats, as evaluated by the mean area and the percentage of EGF plaque-forming cells. Estradiol-17 beta (10 nM) treatment of metestrous culture for 24 h increased significantly the size of EGF plaque-forming cells (from 1290 +/- 58 to 2566 +/- 57 microns 2, P < 0.01) and the percentage of EGF plaque-forming cells (from 20.57 to 28.19%; P < 0.01) to a level as high as observed in proestrous cultures (basal mean area 2171 +/- 157 microns 2, and basal percentage of EGF plaque-forming cells 22.71%). These results suggest that the hormonal status of female rats influences EGF secretion in the anterior pituitary gland and that estradiol is implicated in this phenomenon.

The physiological role of lactoferrin (LF), the major estrogen-inducible protein in the murine uterus, is unclear; however, LF may be a useful marker for the study of estrogen action in the uterus. Thus, the expression of LF mRNA and the localization of the protein in genital tract tissues and secretions of female mice (6-8 wk old) at different stages of the estrous cycle were investigated. Uterine luminal fluid (ULF) was analyzed for LF by means of gel electrophoresis and Western blot techniques; LF mRNA and protein were identified in reproductive tract tissues through in situ hybridization and immunocytochemistry. At diestrus, the level of LF mRNA was low, and staining for the protein was very light in uterine epithelial cells; LF was undetectable in ULF. At proestrus, LF mRNA and protein increased in the uterine epithelium and LF was readily detectable in ULF. LF mRNA and protein reached the highest levels at estrus. At early metestrus as compared to estrus, LF mRNA and protein were detected in decreasing amounts in uterine epithelial cells; the protein was undetected in ULF. By late metestrus and diestrus, LF mRNA and protein returned to a low level, and the protein was undetectable in ULF. LF protein was also demonstrated by immunocytochemistry in the epithelium of the oviduct, cervix, and vagina. LF protein fluctuation similar to that observed in the uterus was seen in these tissues; however, the uterus demonstrated the most dramatic changes in the number of epithelial cells involved in LF production during the estrous cycle. In summary, LF mRNA and its expression in uterine epithelial cells of the mouse varied with the stage of the estrous cycle. These results, combined with previously reported findings that LF is a major constituent of mouse ULF under the influence of estrogen, suggest that LF may play an important role in normal reproductive processes.

Peripheral and central atrial natriuretic factor (ANF) concentrations were measured across the rat's estrous cycle. Vaginal smears were obtained from adult Sprague-Dawley rats maintained under controlled illumination (L/D: 14/10, onset 05.00 h). ANF concentrations in plasma, cardiac atria, pituitary and nine microdissected brain regions of females (n = 5-13) were determined by radioimmunoassay during either early proestrus (09.00-11.00 h), late proestrus (17.00-19.00 h), estrus (09.00-11.00 h), early metestrus (09.00-11.00 h) and late metestrus (17.00-19.00 h). Patterns of cyclic ANF immunoreactivity in plasma and atria were inversely related to each other, with plasma levels being significantly elevated during early metestrus when atrial levels were significantly decreased. Statistically significant central fluctuations in ANF levels during the estrous cycle were only found in the hypothalamic periventricular region (hPVA) and in the dorsal raphe (DR). ANF levels declined in both regions after late proestrus. Results indicate a relationship between ANF activity and cyclic patterns of fluid volume regulation and with phasic reproductive hormonal events.

Alpha and LH beta subunit mRNAs were measured in pituitaries of 4-day cycling rats during the estrous cycle. A two-fold increase in alpha mRNA occurred between 0800-2000 h on diestrus, but alpha mRNA concentrations were stable during other days of the cycle. LH beta mRNA concentrations were low during estrus and metestrus (11-16 pg cDNA bound/100 micrograms pituitary DNA), but were elevated (27-30 pg) between 0800-2000 h on diestrus. A second increase in LH beta mRNA was observed on the afternoon of proestrus, prior to the onset of the LH surge with maximum values (45 pg) coincident with peak LH secretion. LH beta mRNA concentrations declined rapidly and had fallen to basal values by midnight on proestrus. These data show that alpha and LH beta mRNAs change in a similar manner during metestrus, diestrus and estrus, suggesting coordinate regulation of alpha and LH beta gene expression at these times. During the LH surge, however, LH beta mRNA alone is increased, suggesting that the LH beta gene can be differentially expressed at times when maximum LH secretion is occurring.

LH and FSH basal secretion rates (BSR) in vitro of pituitary fragments from female rats at metestrus or following ovariectomy parallel changes seen in serum gonadotropins. These findings led us to suspect that in vitro BSR of fragments depended on prior exposure to GnRH or lingering GnRH in the culture. We compared pituitaries removed from rats in two cycle stages, proestrus (PRO) and metestrus (MET). Rats were given a single injection of a potent GnRH antagonist, Antag-WY 45760-[Ac-beta(2)-D-Nal1,4-F-D-Phe2,D-Trp3,D-Arg6]-LHRH, or oil prior to sacrifice at 09.00 h on PRO or MET. Pituitary fragments were perifused for 8 h. To test the effects of Antag in vitro some pituitaries from oil-injected rats were perifused with medium containing 1 microM Antag for the first 4 h. Starting at 5 h all chambers received hourly 10-min pulses of 1 microM GnRH. In PRO rats Antag injection in vivo lowered LH BSR to 50% and FSH to 70% of oil-treated controls. Surprisingly, Antag administered in vitro suppressed LH BSR to 30% and FSH to 55% of controls. In pituitaries from MET rats, BSR or LH and FSH were 20 and 70% of control PRO values and were unaffected by Antag treatment in vivo or in vitro. In PRO pituitaries LH and FSH responses to the first GnRH pulse were blunted by Antag in vivo but responses to subsequent pulses were unaffected; MET responses were not different from controls. In vitro Antag suppressed GnRH-stimulated release to 10-20% of controls at both cycle stages.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous work has shown that the immature rat uterus contains epidermal growth factor (EGF) receptors and that tissue levels of this receptor are increased by the administration of exogenous estrogens. This study was undertaken to determine if estrogen administration also elevated EGF receptor levels in the mature animal and if the growth factor receptor levels varied in concert with endogenous estrogens throughout the estrous cycle. In the mature, castrate rat administration of estradiol, but not non-estrogenic steroids, causes a 2-3-fold elevation of uterine EGF receptors as judged by ligand binding. This increase is maximum in 18 h and is due to an increase in the number of binding sites. In cycling animals EGF receptor levels are low at metestrus, rise at diestrus, reach a maximum (approximately twice metestrus values) at proestrus, and then return at estrus to metestrus levels. These changes in EGF receptor levels parallel changes in plasma estrogens and occupied nuclear estrogen receptor reported by other workers. These results indicate that uterine EGF receptors are increased by exogenous estrogens in both mature and immature animals, and support a physiological role for estrogens in the regulation of this growth factor receptor.

Serum follicle-stimulating hormone (FSH), pituitary FSH content and FSH beta subunit mRNA concentrations were measured at 1 to 3h intervals throughout the 4 day estrous cycle in rats. Serum FSH was stable (range 200-320 ng/ml) apart from the biphasic proestrus surge (5 fold elevation) which was present from 1800h of proestrus through 0800 h on estrus. Basal FSH beta mRNA concentrations from late metestrus through the afternoon of proestrus were 0.10 +/- 0.04 f mol cDNA bound/100 micrograms pituitary DNA. The major increase in FSH beta mRNA began at 2000 h on proestrus, 2 h after the initial rise in serum FSH and peak mRNA concentrations (0.43 +/- 0.08 f mol cDNA bound) occurred at 0200 h on estrus. FSH beta subunit mRNA concentrations were again increased at 2300 h on estrus (peak 0.24 f mol cDNA bound) and remained elevated through 1700 h on metestrus. Pituitary FSH content was transiently increased during metestrus and diestrus, but was elevated at 1000 h through 1900 h on proestrus (peak 5-fold increase). FSH content fell rapidly at 2000h and remained low until 1400 h on estrus when values again rose. These data show that FSH beta mRNA is increased 4-5 fold during the proestrus FSH surge, and a smaller increase occurs on metestrus in the absence of elevated FSH secretion. The increased concentrations of FSH beta mRNA occurred at different times to the previously reported changes in alpha and LH beta mRNAs. Therefore, the data suggest that different mechanisms are involved in the regulation of LH and FSH beta subunit gene expression during the 4-day estrous cycle in rats.

3H-dihydroalprenolol (DHA) -- receptor binding was studied in membrane preparations from metestrous uterine tissue, both in presence and absence of exogenous prostaglandin (PG) F2 alpha at 10(-9) M. In addition, the uptake of 3H-noradrenaline (NA) by uterine segments from estrous and metestrous rats and the influences of PGF2 alpha (10(-9) M), cocaine (10(-5) M) corticosterone (5.10(-5) M), normetanephrine (10(-6) M) or acetylsalicylic acid (ASA: 10(-4) M), were explored. The Scatchard analysis of experimental data with 3H-DHA with or without added PGF2 alpha indicates the existence of a single class of high affinity receptors and no differences were found, in presence of PGF2 alpha, regarding the control dissociation constant or the control maximal sites of specific binding. On the other hand, the uptake of 3H-NA by uterine segments at metestrus was significantly greater than at estrus. In metestrous uteri PGF2 alpha (10(-9) M) reduced significantly NA uptake. ASA enhanced NA uptake by uteri from estrous rats, an influence prevented by PGF2 mu. In uterine segments isolated at estrus, cocaine, corticosterone and normetanephrine failed to alter 3H-NA uptake, whereas in preparations isolated at metestrus, corticosterone and normetanephrine reduced the uptake, but cocaine did not evoke any influence. Results are discussed in terms of previous findings documenting an amplification of the negative inotropic influence of NA mediated by the activation of beta-adrenoceptors, both in estrous or in metestrous preparations incubated with PGF2 alpha. Such previous findings cannot be explained by changes in the number of NA receptors or by a greater affinity of tissue receptors for the agonist, but rather by differences in NA uptake controlling its effective concentration at the biophase, near receptor sites. Interrelationships along sex hormones (estradiol), prostaglandins (PGF2 alpha) and catecholamines (NA) in uteri, are also discussed.