Oxytocin receptor expression in the midbrain dorsal raphe is dynamic across female reproduction in rats.

Central oxytocin receptor (OTR) expression is extremely sensitive to circulating steroid hormones and OTRs influence many of the neurobehavioural adaptations associated with female reproduction (e.g., postpartum caregiving, aggression, cognition, affective responses). Changes in central OTR expression across female reproduction have often been studied, but almost all of such research has focused on the forebrain, ignoring hormone-sensitive midbrain sites such as the serotonergic dorsal raphe (DR) that are also critical for postpartum behaviours. To investigate the effects of female reproductive state on OTRs in the DR, we first used autoradiography to examine OTR binding across four female reproductive states in laboratory rats: dioestrous virgin, pregnancy day 10, the day of parturition and postpartum day 7. OTR binding in the rostral DR (but not other DR subregions) was approximately 250% higher in parturient rats compared to dioestrous virgins and dropped back down to virgin levels by postpartum day 7. Given the chemical heterogeneity of the DR, we then examined OTR expression in the three most abundant neuronal phenotypes of the DR (i.e., serotonin, GABA and dopamine) in dioestrous virgins and recently parturient females. Using dual-label immunohistochemistry and in situ hybridisation, we found that twice as many dopaminergic cells in the parturient rostral DR contained OTR immunoreactivity compared to that found in virgins. On the other hand, mothers had fewer rostral DR GABAergic cells expressing OTRs than did virgins. OTR expression in serotonin cells did not differ between the two groups. Overall, these results suggest that the rostral subregion of the midbrain DR is uniquely sensitive to oxytocin around the time of parturition, with subpopulations of cells that become more sensitive (i.e., dopamine), less sensitive (i.e., GABA) and show no change (i.e., serotonin) to this neuropeptide. This dynamic OTR signalling in the female DR may help drive the numerous behavioural changes across female reproduction that are necessary for successful motherhood.

Cysteine-Cysteine Motif Chemokine Receptor 5 Expression in Letrozole-Induced Polycystic Ovary Syndrome Mice.

Polycystic ovary syndrome (PCOS), which affects 5-10% of women of reproductive age, is associated with reproductive and metabolic disorders, such as chronic anovulation, infertility, insulin resistance, and type 2 diabetes. However, the mechanism of PCOS is still unknown. Therefore, this study used a letrozole-exposed mouse model in which mice were orally fed letrozole for 20 weeks to investigate the effects of letrozole on the severity of reproductive and metabolic consequences and the expression of cysteine-cysteine motif chemokine receptor 5 (CCR5) in letrozole-induced PCOS mice. The letrozole-treated mice showed a disrupted estrous cycle and were arrested in the diestrus phase. Letrozole treatment also increased plasma testosterone levels, decreased estradiol levels, and caused multicystic follicle formation. Furthermore, histological analysis of the perigonadal white adipose tissue (pgWAT) showed no significant difference in the size and number of adipocytes between the letrozole-treated mice and the control group. Further, the letrozole-treated mice demonstrated glucose intolerance and insulin resistance during oral glucose and insulin tolerance testing. Additionally, the expression of CCR5 and cysteine-cysteine motif ligand 5 (CCL5) were significantly higher in the pgWAT of the letrozole-treated mice compared with the control group. CCR5 and CCL5 were also significantly correlated with the homeostasis model assessment of insulin resistance (HOMA-IR). Finally, the mechanisms of insulin resistance in PCOS may be caused by an increase in serine phosphorylation and a decrease in Akt phosphorylation.

Qualitative analysis and functional classification of the uterine proteome of mares in oestrus and dioestrus.

Quantitative analysis of the uterine flush fluid proteome of mares in oestrus and dioestrus has been previously reported. The objectives of this study were to: a) evaluate qualitative differences in the uterine flush fluid proteome between mares in oestrus and mares in dioestrus and b) perform a functional classification of proteins either unique to each stage or common between the two stages. Uterine flush fluid samples were collected from 8 light breed mares in either oestrus (n = 5) or dioestrus (n = 3). Proteomic analysis of the samples was conducted using liquid chromatography-tandem mass spectrometry. Proteins exclusively detected in oestrus or dioestrus and those common to both stages were identified using the Scaffold software (version 4.4.8, Proteome Software Inc., Portland, OR). The identified proteins were classified into gene ontology (GO) categories (cellular component [CC], molecular function [MF] and biological process [BP]) using the PANTHER (www.pantherdb.org) classification system version 14.0. Of 172 proteins identified, 51 and 28 were exclusively detected in mares in oestrus and dioestrus, respectively, and 93 proteins were common to both stages. The most represented terms in various GO categories were similar among the three subsets of proteins. The most represented CC terms were extracellular region and cell, the most represented MF terms were catalytic activity and binding, and the most represented BP terms were metabolic process and cellular process. In conclusion, proteomic analysis of the uterine flush fluid enabled the identification of subsets of proteins unique to oestrus or dioestrus, or common to both stages. The results of this study can serve as a baseline for future research focused on finding stage-specific protein markers or evaluating differences in the uterine flush fluid proteome between normal mares and those with uterine disease.

Chronic exposure to low dose of bisphenol A causes follicular atresia by inhibiting kisspeptin neurons in anteroventral periventricular nucleus in female mice.

Bisphenol-A (BPA) is an estrogenic chemical extensively used in industrial and household applications. The present study was conducted to investigate the effect of chronic exposure to BPA on the adult female neuroendocrine system. Herein, we found that expose of adult female mice to BPA (50 µg/kg) by oral gavage for 60 days (BPA mice) prolonged diestrus and decreased serum 17ß-estradiol (E2) concentration by reducing the number of antral follicles and corpora luteum. In comparison with controls, the levels of serum luteinizing hormone (LH), follicle stimulating hormone (FSH), hypothalamic gonadotrophin releasing hormone (GnRH) and the expression of kisspeptin in anteroventral periventricular nucleus (AVPV) decreased in BPA mice, which could be reversed by injecting kisspeptin-10 (i.c.v.). Treatment with BPA or estrogen receptor α (ERα) antagonist MPP, but not ERß antagonist PHTPP inhibited E2-induced AVPV-kisspeptin expression in ovariectomized mice. Use of ERα agonist PPT rather than ERß agonist DPN enhanced AVPV-kisspepetin expression, which decreased after treatment with BPA. The amplitude of the proestrus LH surge decreased in mice exposed to BPA, but was recovered by administering kisspeptin-10. The present study provides in vivo evidence that chronic exposure to a low dose of BPA suppressed ERα-induced activation of AVPV-kisspeptin neurons, leading to prolonged diestrus and reduced ovulation in adult female mice.

"Rapid actions of the neurosteroid allopregnanolone on ovarian and hypothalamic steroidogenesis: Central and peripheral modulation".

The present study aimed to determine whether an i.c.v. administration of allopregnanolone (ALLO) rapidly modifies the hypothalamic and ovarian 3ß-hydroxysteroid dehydrogenase (3ß-HSD) enzymatic activity and gene expression in in vivo and ex vivo systems in pro-oestrus (PE) and dioestrus I (DI) rats. Animals were injected with vehicle, ALLO, bicuculline or bicuculline plus ALLO and were then killed. In the in vivo experiment, the hypothalamus, ovaries and serum were extracted and analysed. In the ex vivo experiment, the superior mesenteric ganglion - ovarian nerve plexus - ovary system was extracted and incubated during 120 minutes at 37 ºC. The serum and ovarian compartment fluids were used to determine progesterone by radioimmunoanalysis. In the in vivo experiments, ALLO caused a decrease in hypothalamic and ovarian 3ß-HSD enzymatic activity during PE. During DI, ALLO increased hypothalamic and ovarian 3ß-HSD activity and gene expression. The ovarian 3ß-HSD activity increased in both stages in the ex vivo system; gene expression increased only during DI. ALLO induced an increase in serum progesterone only in D1 and in the ovarian incubation liquids in both stages. All findings were reversed by an injection of bicuculline before ALLO. Ovarian steroidogenic changes could be attributed to signals coming from ganglion neurones, which are affected by the acute central neurosteroid stimulation. The i.c.v. administration of ALLO via the GABAergic system altered 3ß-HSD activity and gene expression, modulating the neuroendocrine axis. The present study reveals the action that ALLO exerts on the GABAA receptor in both the central and peripheral nervous system and its relationship with hormonal variations. ALLO is involved in the "fine tuning" of neurosecretory functions as a potent modulator of reproductive processes in female rats.

Analysis of the uterine flush fluid proteome of healthy mares and mares with endometritis or fibrotic endometrial degeneration.

The objective of this study was to evaluate the differences in the uterine flush fluid proteome between healthy mares and mares with endometritis or fibrotic endometrial degeneration (FED). Uterine flush fluid samples were collected from healthy mares (n=8; oestrus n=5 and dioestrus n=3) and mares with endometritis (n=23; oestrus n=14 and dioestrus n=9) or FED (n=7; oestrus n=6 and dioestrus n=1). Proteomic analysis was performed using label-free liquid chromatography-tandem mass spectrometry. Of 216 proteins identified during oestrus, 127 were common to all three groups, one protein was exclusively detected in healthy mares, 47 proteins were exclusively detected in mares with endometritis and four proteins were exclusively detected in mares with FED. Of 188 proteins identified during dioestrus, 113 proteins were common between healthy mares and mares with endometritis, eight proteins were exclusively detected in healthy mares and 67 proteins were exclusively detected in mares with endometritis. Quantitative analysis revealed a subset of proteins differing in abundance between the three groups during oestrus and between healthy mares and mares with endometritis during dioestrus. These results provide a springboard for evaluation of specific proteins as biomarkers of uterine health and disease and for investigation of their roles in the establishment and maintenance of pregnancy.

Estrogen and progesterone dependent expression of visfatin/NAMPT regulates proliferation and apoptosis in mice uterus during estrous cycle.

Visfatin is an adipokine which has an endocrine effect on reproductive functions and regulates ovarian steroidogenesis. There is scant information about the expression, regulation, and functions of visfatin in the mammalian uterus. The present study examined expression and localization of visfatin in the mouse uterus at various stages of the natural estrous cycle, effects of estrogen and progesterone on localization and expression of visfatin in the ovariectomised mouse uterus and effect of visfatin inhibition by a specific inhibitor, FK866 on proliferation and apoptosis in the uterus. Western blot analysis of visfatin showed high expression in proestrus and metestrus while it declined in estrus and diestrus. Immulocalization study also showed strong immunostaining in the cells of endometrium, myometrium, luminal and glandular epithelium during proestrus and metestrus that estrus and diestrus. The uterine visfatin expression closely related to the increased estrogen levels in proestrus and suppressed when progesterone rose to a high level in diestrus. The treatment with estrogen to ovariectomised mice up-regulates visfatin, PCNA, and active caspase3 whereas progesterone up-regulates PCNA and down-regulates visfatin and active caspase3 expression in mouse uterus. The co-treatment with estrogen and progesterone up-regulates visfatin and down-regulates PCNA and active caspase3. In vitro study showed endogenous visfatin inhibition by FK866 increased expression of PCNA and BCL2 increased catalase activity while FK866 treatment decreased expression of active caspase3 and BAX with decreased SOD and GPx activity. BrdU labeling showed that inhibition of visfatin modulates the uterine proliferation. This study showed that expression of visfatin protein is steroid dependent in mouse uterus which is involved in the regulation of proliferation and apoptosis via modulating antioxidant system in the uterus of mice during the reproductive cycle.

In Vitro Mimicking of Estrous Cycle Stages: Dissecting the Impact of Estradiol and Progesterone on Oviduct Epithelium.

We have previously mimicked the morphological and functional changes occurring in the oviduct epithelium during the estrous cycle in vitro by using an air-liquid interface (ALI) culture system and basolateral application of 17ß-estradiol (E2) and progesterone (P4). In the current study we aimed to explore the transcriptomic changes elicited by E2 and P4 together during estrous cycle simulation and to dissect the individual effects of E2 and P4 on oviduct epithelium physiology. Primary porcine oviduct epithelial cells (POECs) (N = 6 animals) were cultured at the ALI. After differentiation for 11 days, we sequentially simulated diestrus (10 days) and estrus (2.5 days) by adding serum levels of E2 and P4 to the basolateral compartment either in combination (mix trial) or separately (P4 trial and E2 trial, respectively). Cell response was evaluated by microarray analysis (mix and P4 trials), quantitative RT-PCR, and histomorphometry (all trials). When we compared simulated diestrus with estrus stage in the mix trial, there were 169 (142 upregulated and 27 downregulated) differentially expressed genes (DEGs; fold change ≥1.5). In the P4 trial, 108 DEGs (83 upregulated and 25 downregulated) were detected. Gene enrichment analysis revealed that immune-related pathways were exclusively affected in the mix trial. In both mix and P4 trials, POECs exhibited in vivo-like morphological changes regarding epithelium height and portion of ciliated cells. However, E2 alone did not trigger morphological changes. We deduce that P4 mainly drives structural variations, and E2 is imperative for regulating immune function of the oviduct epithelium during estrous cycle.

Influence of the estrus cycle of the mouse on the disposition of SHetA2 after vaginal administration.

SHetA2 is a novel compound with the potential to treat cervical dysplasia, but has poor water solubility. A vaginal suppository formulation was able to achieve therapeutic concentrations in the cervix of mice, but these concentrations were variable. Histological analysis indicated that mice in the same group were in different stages of their estrous cycle, which is known to induce anatomical changes in their gynecological tissues. We investigated the effects of these changes on the pharmacokinetics and pharmacodynamics of SHetA2 when administered vaginally. Mice were synchronized to be either in estrous or diestrus stage for administration of the SHetA2 suppository. Pharmacokinetic parameters were calculated from the SHetA2 concentrations vs. time data. The reduction in the expression of cyclin D1 protein in the cervix was used as pharmacodynamic endpoint. Mice dosed during diestrus had a larger AUCcervix (335 µg mL h-1), higher Cmax (121.8 ±â€¯38.7 µg/g) and longer t1/2-cervix (30.3 h) compared to mice dosed during estrus (120 µg mL h-1, 44.6 ±â€¯29.5 µg/g and 3.6 h respectively). Therapeutic concentrations of SHetA2 were maintained for 48 h in the cervix of mice dosed during diestrus and for only 12 h in the estrus group. The treatment reduced the expression of cyclin D1 protein in the cervix of mice in the estrus to a larger extent. These results indicate that the estrous cycle of mice influences significantly the disposition of SHetA2 after vaginal administration and may also influence its efficacy.

Differential expression of the receptors for thyroid hormone, thyroid stimulating hormone, vitamin D and retinoic acid and extracellular signal-regulated kinase in uterus of rats under influence of sex-steroids.

Sex-steroids play important role in modulating uterine functions. We hypothesized that these hormones affect expression of proteins in the uterus related to thyroid hormone action. Therefore, changes in expression levels of receptors for thyroid hormone (TRα-1 and TRß-1), thyroid stimulating hormone (TSHR), vitamin D (VDR) and retinoid acid (RAR) as well as extracellular signal-regulated kinase (ERK1/2) in uterus were investigated under sex-steroid influence. METHODS: Two rat models were used: (i) ovariectomised, sex-steroid replaced and (ii) intact, at different phases of oestrous cycle. A day after completion of sex-steroid treatment or following identification of oestrous cycle phases, rats were sacrificed and expression and distribution of these proteins in uterus were identified by Western blotting and immunohistochemistry, respectively. RESULTS: Expression of TRα-1, TRß-1, TSHR, VDR, RAR and ERK1/2 in uterus was higher following estradiol (E2) treatment and at estrus phase of oestrous cycle when E2 levels were high. A relatively lower expression was observed following progesterone (P) treatment and at diestrus phases of oestrous cycle when P levels were high. Under E2 influence, TRα, TRß, TSHR, VDR, RAR and ERK1/2 were distributed in luminal and glandular epithelia while under P influence, TSHR, VDR abn RAR were distributed in the stroma. CONCLUSIONS: Differential expression and distribution of TRα-1, TRß-1, TSHR, VDR, RAR and ERK1/2 in different uterine compartments could explain differential action of thyroid hormone, TSH, vitamin D, and retinoic acid in uterus under different sex-steroid conditions.

Evaluation of cell proliferation and endometrial thickness of bitches in different periods of diestrus

ABSTRACT This study aimed to determine the histological features of the endometrium of bitches, as well as the cell proliferation at specific moments of diestrus, 10, 20, 30, 40, 50 and 60 days post ovulation, correlating the endometrial thickness with the uterine cell proliferation and the metabolic state (weight, blood glucose and plasma cholesterol) of the animals. Therefore, the right and left uterine horns of 26 clinically healthy bitches submitted to ovariohysterectomy were histologically analyzed 10, 20, 30, 40, 50 and 60 days post ovulation. The hematoxylin-eosin and AgNOR staining techniques were performed. All parameters were evaluated by ANOVA and post-hoc Tukey test (p<0.05). The correlation between endometrial thickness and uterine cell proliferation, weight, blood glucose and plasma cholesterol of animals was observed using the Pearson method (p<0.05). In the present study, it is concluded that endometrial thickness does not differ at any of the moments analyzed in diestrus. The endometrial thickness is not influenced by hormones, weight, blood glucose or serum cholesterol of bitches in this phase of the estrous cycle. However, there is greater cell proliferation in the endometrium at day 40 compared to day 60 post ovulation under the influence of the endocrine profile.

Evaluation of cell proliferation and endometrial thickness of bitches in different periods of diestrus.

This study aimed to determine the histological features of the endometrium of bitches, as well as the cell proliferation at specific moments of diestrus, 10, 20, 30, 40, 50 and 60 days post ovulation, correlating the endometrial thickness with the uterine cell proliferation and the metabolic state (weight, blood glucose and plasma cholesterol) of the animals. Therefore, the right and left uterine horns of 26 clinically healthy bitches submitted to ovariohysterectomy were histologically analyzed 10, 20, 30, 40, 50 and 60 days post ovulation. The hematoxylin-eosin and AgNOR staining techniques were performed. All parameters were evaluated by ANOVA and post-hoc Tukey test (p<0.05). The correlation between endometrial thickness and uterine cell proliferation, weight, blood glucose and plasma cholesterol of animals was observed using the Pearson method (p<0.05). In the present study, it is concluded that endometrial thickness does not differ at any of the moments analyzed in diestrus. The endometrial thickness is not influenced by hormones, weight, blood glucose or serum cholesterol of bitches in this phase of the estrous cycle. However, there is greater cell proliferation in the endometrium at day 40 compared to day 60 post ovulation under the influence of the endocrine profile.

Neuromodulatory effect of oestradiol in the metabolism of ovarian progesterone and oestradiol during dioestrus II: participation of the superior mesenteric ganglion.

The aims of the present study were to determine: (1) whether oestradiol (E2) in the superior mesenteric ganglion (SMG) modifies the release of ovarian progesterone (P4), androstenedione (A2) and E2, the activity and gene expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 20α-HSD and the expression of P450 aromatase (Cyp19a1) and (2) whether any such modifications are related to changes in ovarian nitric oxide (NO) and noradrenaline (NA) levels during dioestrus II. Using an ex vivo SMG-ovarian nervous plexus-ovary system, ovarian P4 release was measured following the addition E2 plus tamoxifen (Txf) (10-6M) to the ganglion, whereas A2, E2, NA and NO were measured following the addition of E2 alone. Steroids were measured by radioimmunoassay, NA concentrations were determined by HPLC and gene expression was evaluated using reverse transcription-polymerase chain reaction. Oestradiol in the ganglion decreased ovarian P4, E2 and NA release, as well as 3ß-HSD activity, but increased the release of A2 and nitrites, as well as the 20α-HSD expression and its activity. No changes were observed in Cyp19a1 gene expression. The addition of E2 plus Txf to the ganglion reversed the effects of E2 alone. The action of oestradiol in SMG favours the beginning of functional luteolysis, due to an increase in NO release and a decrease in NA in the ovary. These results may help elucidate the role of E2 in hormone-dependent pathologies in women.

Peri-ovulatory endocrine regulation of the prostanoid pathways in the bovine uterus at early dioestrus.

We hypothesised that different endocrine profiles associated with pre-ovulatory follicle (POF) size would impact on uterine prostanoid pathways and thereby modulate the histotroph composition. Beef cows (n=15 per group) were hormonally manipulated to have small (SF-SCL group) or large (LF-LCL group) pre-ovulatory follicles (POF) and corpora lutea (CL). Seven days after induction of ovulation, animals were slaughtered and uterine tissues and flushings were collected for quantification of prostanoids. The POF and CL size and the circulating progesterone concentrations at Day 7 were greater (P<0.05) in the LF-LCL cows than in the SF-SCL group, as expected. The abundance of 5 out of 19 genes involved in prostanoid regulation was different between groups. Transcript abundance of prostaglandin F2α, E2 and I2 synthases was upregulated (P<0.05) and phospholipase A2 was downregulated (P<0.05) in endometrium of the LF-LCL group. No difference (P>0.1) in prostanoid concentrations in the endometrium or in uterine flushings was detected between groups. However, prostaglandin F2α and E2 concentrations in the uterine flushings were positively correlated with the abundance of transcripts for prostaglandin endoperoxide synthase 2 (0.779 and 0.865, respectively; P<0.002). We conclude that endometrial gene expression related to prostanoid synthesis is modulated by the peri-ovulatory endocrine profile associated with POF size, but at early dioestrus differences in transcript abundance were not reflected in changes in prostanoid concentrations in the uterine tissue and fluid.

Analysis of LH receptor in canine ovarian follicles throughout the estrous cycle.

The aim of this study was to determine the mRNA LHR and LHR protein expression pattern in the canine ovarian follicles at different stage of development throughout the estrous cycle. Dog ovaries were obtained from 1-6y bitches at proestrus/estrus, anestrus and diestrus stages following ovariohysterectomy. Follicular cells were mechanically recovered from follicles distributed into four types (preantral, small antral, medium antral and large antral). Total RNA extraction was performed and the evaluation of gene expression levels was achieved by relative quantification q-PCR analysis. Intrafollicular amounts of LHR were assessed by western blot method. All results were evaluated by ANOVA. The expression levels of mRNA LHR in follicular cells were observed in every stage of development, however this gene expression varied over the estrous cycle. LHR transcripts increased (P < 0.05) from preantral to antral stage. There were not differences in LHR gene expression among follicles at preantral stages; however, at antral stages the lowest (P < 0.05) LHR mRNA expression was found at anestrus and the highest (P < 0.05) at proestrus/estrus. The LHR protein was also detected in dog follicles in all reproductive phases with patterns varying with stage of follicular development over the reproductive cycle. The antibody against human LHR revealed two bands at ∼90 and ∼67 kDa, probably representing the matured protein and its precursor respectively. Both bands LHR appeared already at preantral follicles increasing (P < 0.05) with growth. A high proportion of LHR was presented as immature forms in all follicles stages during different phases of the estrous cycle. In conclusion, the gene and protein of LHR are differentially expressed in dog follicles over the estrous cycle, increasing with growth and the precursor protein is the most predominant LHR form present in canine follicles.

PR-independent neurosteroid regulation of α2-GABA-A receptors in the hippocampus subfields.

Progesterone (P) binding to the intracellular progesterone receptors (PRs) plays a key role in epilepsy via modulation of GABA-A receptor plasticity in the brain. This is thought to occur via conversion of P to neurosteroids such as allopregnanolone, an allosteric modulator of GABA-A receptors. In the female brain, the composition of GABA-A receptors is not static and undergoes dynamic spatial changes in response to fluctuations in P and neurosteroid levels. Synaptic α2-containing GABA-A receptors contribute to phasic neuronal excitability and seizure susceptibility. However, the mechanisms underlying α2-subunit plasticity remain unclear. Here, we utilized the neurosteroid synthesis inhibitor finasteride and PR knockout mice to investigate the role of PRs in α2-subunit in the hippocampus. α2-Subunit expression was significantly upregulated during the high-P state of diestrous stage and with P treatment in wildtype and PR knockout mice. In contrast, there was no change in α2-subunit expression when metabolism of P into neurosteroids was blocked by finasteride in both genotypes. These findings suggest that ovarian cycle-related P and neurosteroids regulate α2-GABA-A receptor expression in the hippocampus via a non-PR pathway, which may be relevant to menstrual-cycle related brain conditions.

Hexokinase 2 drives glycogen accumulation in equine endometrium at day 12 of diestrus and pregnancy.

BACKGROUND: Secretion of histotroph during the prolonged pre-implantation phase in mares is crucial to pregnancy maintenance, manifested as increased embryonic loss in mares with age-related endometrial degeneration. Glycogen content of uterine histotroph is higher during the progesterone-dominated phase of the estrous cycle in mares, but regulatory mechanisms are not well understood. METHODS: mRNA expression of glycogen-metabolizing enzymes (HK1, HK2, GSK3B, GYS1, PEPCK, PKM, PYGM) in endometrial samples were compared among mares in anestrus, estrus, and at Day 12 of diestrus and pregnancy. In addition, hexokinase 2 (HK2) activity was assessed using a colorimetric assay. RESULTS: HK2 was the key regulator of glycogen accumulation during diestrus and pregnancy; hexokinase transcript abundance and enzyme activity were significantly higher during diestrus and pregnancy than estrus and anestrus. In addition, despite similar relative transcript abundance, hexokinase activity was significantly greater in the pregnant versus diestrous endometrium. Therefore, we inferred there was regulation of hexokinase activity through phosphorylation, in addition to its regulation at the transcriptional level during early pregnancy. Based on immunohistochemistry, HK2 was localized primarily in luminal and glandular epithelial cells, with weaker staining in stromal cells. CONCLUSION: Among glycogen metabolizing enzymes identified, expression of HK2 was significantly greater during the progesterone-dominated phase of the cycle.

High-fat diet with stress impaired islets' insulin secretion by reducing plasma estradiol and pancreatic GLUT2 protein levels in rats' proestrus phase.

This study was conducted to determine whether two estrus phases (proestrus and diestrus) in female rats may influence the metabolic response to a high-fat diet and/or stress, focusing on pancreatic insulin secretion and content. Animals were divided into high-fat and normal diet groups, then each group was subdivided into stress and non-stress groups, and finally, each one of these was divided into proestrus and diestrus subgroups. At the end of high-fat diet treatment, foot-shock stress was applied to the animals. Then, blood samples were taken to measure plasma factors. Finally, the pancreas was removed for determination of glucose transporter 2 (GLUT2) protein levels and assessment of insulin content and secretion of the isolated islets. In the normal and high-fat diet groups, stress increased plasma corticosterone concentration in both phases. In both study phases, high-fat diet consumption decreased estradiol and increased leptin plasma levels. In the high-fat diet group in response to high glucose concentration, a reduction in insulin secretion was observed in the proestrus phase compared with the same phase in the normal diet group in the presence and absence of stress. Also, high-fat diet decreased the insulin content of islets in the proestrus phase compared with the normal diet. High-fat diet and/or stress caused a reduction in islet GLUT2 protein levels in both phases. In conclusion, it seems possible that high-fat diet alone or combined with foot-shock, predispose female rats to impaired insulin secretion, at least in part, by interfering with estradiol levels in the proestrus phase and decreasing pancreatic GLUT2 protein levels.

The participation of the muscarinic receptors in the preoptic-anterior hypothalamic areas in the regulation of ovulation depends on the ovary.

BACKGROUND: Muscarinic receptors (mAChRs) of the preoptic and anterior hypothalamus areas (POA-AHA) regulate ovulation in an asymmetric manner during the estrous cycle. The aims of the present study were to analyze the effects of a temporal blockade of mAChRs on either side of the POA-AHA performed in diestrus-2 rats on ovulation, the levels of estradiol, follicle stimulating hormone (FSH) and luteinizing hormone (LH) and the mechanisms involved in changes in ovulation. METHODS: Cyclic rats on diestrus-2 day were anesthetized and randomly assigned to the following groups: 1) microinjection of 1 µl of saline or atropine solution (62.5 ng) in the left or right POA-AHA; 2) removal (unilateral ovariectomty, ULO) of the left (L-ULO) or right (R-ULO) ovary, and 3) rats microinjected with atropine into the left or right POA-AHA plus L-ULO or R-ULO. The ovulation rate and the number of ova shed were measured during the predicted estrus, as well as the levels of estradiol, FSH and LH during the predicted proestrus and the effects of injecting synthetic LH-releasing hormone (LHRH) or estradiol benzoate (EB). RESULTS: Atropine in the left POA-AHA decreased both the ovulation rate and estradiol and LH levels on the afternoon of proestrus, also LHRH or EB injection restored ovulation. L- or R-ULO resulted in a lower ovulation rate and smaller number of ova shed, and only injection of LHRH restored ovulation. EB injection at diestrus-2 restored ovulation in animals with L-ULO only. The levels of estradiol, FSH and LH in rats with L-ULO were higher than in animals with unilateral laparotomy. In the group microinjected with atropine in the left POA-AHA, ovulation was similar to that in ULO rats. In contrast, atropine in the right POA-AHA of ULO rats blocked ovulation, an action that was restored by either LHRH or EB injection. CONCLUSIONS: These results indicated that the removal of a single ovary at noon on diestrus-2 day perturbed the neuronal pathways regulating LH secretion, which was mediated by the muscarinic system connecting the right POA-AHA and the ovaries.

Phospholipid Profile and Distribution in the Receptive Oviduct and Uterus During Early Diestrus in Cattle.

Phospholipid metabolism and signaling influences on early pregnancy events in cattle are unknown. This study aimed to characterize global phospholipid composition of oviduct and uterus during early diestrus in a model of contrasting embryo receptivity. Beef cows were treated to ovulate a larger (LF-LCL group, associated with greater receptivity) or smaller (SF-SCL group) follicle and, consequently, to present greater or smaller plasma concentrations of estradiol during proestrus-estrus, as well as progesterone during early diestrus. Oviduct and uterus (4 days after gonadotropin-releasing hormone-induced ovulation; D4) as well as the uterus (D7) were collected, and lipid profiles were monitored by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This technique allowed the identification and tissue localization of sphingomyelins (SM), phosphatidylcholines (PC), ceramides (Cer), and phosphatidylethanolamines (PE). Multivariate statistics were used to separate samples into groups with distinctly different phospholipid profiles in the uterus at D4 and D7. Different abundance of ions corresponding to specific lipids were detected on D4 (Cer [42:1], PC [31:0], PC [32:1], PC [34:4], and PC [36:4] greater for LF-LCL group; and PC [38:7], PC [38:5], PC [38:4], PC [40:7], and PC [40:6] greater for SF-SCL group) and D7 (SM [34:2], SM [34:1], PC [32:1], and PC [35:2] greater for LF-LCL group). The MALDI-MS imaging showed the spatial distributions of major phospholipids. In conclusion, distinct phospholipid profiles were associated with animals treated to show contrasting receptivity to the embryo. Functional roles of the identified phospholipids on uterine function and preimplantation embryo development deserve further studies.