Estrus Cycle In Mice Research Articles

Temporal Variations in Density and Distribution of Neutrophils with Associated Morphological Changes in Uterus at All Stages of Estrus Cycle in Mice

Temporal Variations in Density and Distribution of Neutrophils with Associated Morphological Changes in Uterus at All Stages of Estrus Cycle in Mice

Active glucose transport varies by small intestinal region and oestrous cycle stage in mice.

What is the central question of this study? Body mass and food intake change during the female ovarian cycle: does glucose transport by the small intestine also vary? What is the main finding and its importance? We have optimised Ussing chamber methodology to measure region-specific active glucose transport in the small intestine of adult C57BL/6 mice. Our study provides the first evidence that jejunal active glucose transport changes during the oestrous cycle in mice, and is higher at pro-oestrus than oestrus. These results demonstrate adaptation in active glucose uptake, concurrent with previously reported changes in food intake. Food intake changes across the ovarian cycle in rodents and humans, with a nadir during the pre-ovulatory phase and a peak during the luteal phase. However, it is unknown whether the rate of intestinal glucose absorption also changes. We therefore mounted small intestinal sections from C57BL/6 female mice (8-9weeks old) in Ussing chambers and measured active ex vivo glucose transport via the change in short-circuit current (∆Isc ) induced by glucose. Tissue viability was confirmed by a positive ∆Isc response to 100µM carbachol following each experiment. Active glucose transport, assessed after addition of 5, 10, 25 or 45mM d-glucose to the mucosal chamber, was highest at 45mM glucose in the distal jejunum compared to duodenum and ileum (P<0.01). Incubation with the sodium-glucose cotransporter 1 (SGLT1) inhibitor phlorizin reduced active glucose transport in a dose-dependent manner in all regions (P<0.01). Active glucose uptake induced by addition of 45mM glucose to the mucosal chamber in the absence or presence of phlorizin was assessed in jejunum at each oestrous cycle stage (n=9-10 mice per stage). Overall, active glucose uptake was lower at oestrus compared to pro-oestrus (P=0.025). This study establishes an ex vivo method to measure region-specific glucose transport in the mouse small intestine. Our results provide the first direct evidence that SGLT1-mediated glucose transport in the jejunum changes across the ovarian cycle. The mechanisms underlying these adaptations in nutrient absorption remain to be elucidated.

In vivo arterial stiffness, but not isolated artery endothelial function, varies with the mouse estrus cycle

Due to the increasing inclusion of female mice in research, it is important to understand the influence of the estrus cycle on vascular function. In humans, endothelial function and arterial stiffness vary throughout the menstrual cycle; however, these functional changes have yet to be investigated for the mouse estrus cycle. During the estrus cycle, circulating estrogen is highest during the proestrus and estrus phases and lowest during diestrus, while progesterone peaks during estrus. Estrogens bind to estrogen receptor α (ERα), estrogen receptor β (ERβ), and estrogen G‐protein coupled receptor (GPR30) in vascular cells. Activation of ERα and GPR30 increases nitric oxide (NO) bioavailability. The purpose of this study was to determine the effect of the estrus cycle phase on arterial stiffness, vascular endothelial function, and estrogen receptor expression.In young female C57Bl/6J mice (n=23; 5.96 ± 0.04 months), we determined the estrus cycle via vaginal cytology and plasma hormone concentrations. We assessed large artery stiffness via aortic pulse wave velocity (PWV). Endothelial function was assessed by dose responses to acetylcholine (ACh) and insulin in pressurized mesenteric arteries (MAs) and posterior cerebral arteries (PCAs). NO‐mediated dilation was determined by the difference in vasodilation to ACh in the presence vs. absence of the NO synthase inhibitor L‐NAME. Endothelium independent dilation was assessed by the dose responses to sodium nitroprusside. We quantified gene expression of ERα (Esr1), ERβ (Esr2), and GPR30 (Gper1) in MAs and PCAs.Plasma progesterone concentrations were higher for mice in estrus (5.7 ± 1.1 ng/mL) compared to proestrus (2.5 ± 0.6 ng/mL; p=0.04) and tended to be higher in diestrus (3.9 ± 1.1 ng/mL, p=0.14), verifying the cytology classifications. Plasma 17β‐estradiol and estrone were below detection in most mice. There was a higher plasma testosterone concentration during diestrus (0.08 ± 0.02 ng/mL) compared to other stages (estrus 0.02 ± 0.00 ng/mL, p=0.006; proestrus 0.01 ± 0.00 ng/mL, p=0.001).Aortic PWV was lower for mice in estrus compared with mice in diestrus (3.0 ± 0.1 vs. 3.4 ± 0.1 m/s, p=0.005). Aortic PWV for mice in proestrus did not differ from the other stages (3.2 ± 0.1 m/s, p&gt;0.05). The MA and PCA responses to ACh, insulin and sodium nitroprusside, as well as NO‐mediated dilation, were not different between estrus cycle phases (p&gt;0.05). PCA Esr2 gene expression for mice in estrus was 57% lower than diestrus (p=0.008) and 54% lower than proestrus (p=0.04). Gene expression did not differ between estrus cycle phases for Esr1 and Gper1 in either the MA or PCA (p&gt;0.05), and MA Esr2 was not detectable.In summary, we found that the estrus phase in mice is associated with lower in vivo large artery stiffness. In contrast, there were no differences between estrus cycle phase in ex vivo resistance artery endothelial function. Changes to estrogen receptor gene expression during the estrus cycle are limited to ERβ in the cerebral arteries. These results suggest that estrus phase should be considered when measuring in vivo arterial stiffness in young female mice.

Impact of the mouse estrus cycle on cannabinoid receptor agonist-induced molecular and behavioral outcomes.

Sexual dimorphisms are observed in cannabinoid pharmacology. It is widely reported that female animals are more sensitive to the cataleptic, hypothermic, antinociceptive, and anti‐locomotive effects of cannabinoid receptor agonists such as CP55,940. Despite awareness of these sex differences, there is little consideration for the pharmacodynamic differences within females. The mouse estrus cycle spans 4–5 days and consists of four sex hormone‐mediated phases: proestrus, estrus, metestrus, and diestrus. The endocannabinoid system interacts with female sex hormones including β‐estradiol, which may influence receptor expression throughout the estrus cycle. In the current study, sexually mature female C57BL/6 mice in either proestrus or metestrus were administered either 1 mg/kg i.p. of the cannabinoid receptor agonist CP55,940 or vehicle. Mice then underwent the tetrad battery of behavioral assays measuring catalepsy, internal body temperature, thermal nociception, and locomotion. Compared with female mice in metestrus, those in proestrus were more sensitive to the anti‐nociceptive effects of CP55,940. A similar trend was observed in CP55,940‐induced catalepsy; however, this difference was not significant. As for cannabinoid receptor expression in brain regions underlying antinociception, the spine tissue of proestrus mice that received CP55,940 exhibited increased expression of cannabinoid receptor type 1 relative to treatment‐matched mice in metestrus. These results affirm the importance of testing cannabinoid effects in the context of the female estrus cycle.

Pulsed administration for physiological estrogen replacement in mice.

Estrogens are important regulators of body physiology and have major effects on metabolism, bone, the immune- and central nervous systems. The specific mechanisms underlying the effects of estrogens on various cells, tissues and organs are unclear and mouse models constitute a powerful experimental tool to define the physiological and pathological properties of estrogens. Menopause can be mimicked in animal models by surgical removal of the ovaries and replacement therapy with 17β-estradiol in ovariectomized (OVX) mice is a common technique used to determine specific effects of the hormone. However, these studies are complicated by the non-monotonic dose-response of estradiol, when given as therapy. Increased knowledge of how to distribute estradiol in terms of solvent, dose, and administration frequency, is required in order to accurately mimic physiological conditions in studies where estradiol treatment is performed. In this study, mice were OVX and treated with physiological doses of 17β-estradiol-3-benzoate (E2) dissolved in miglyol or PBS. Subcutaneous injections were performed every 4 days to resemble the estrus cycle in mice. Results show that OVX induces an osteoporotic phenotype, fat accumulation and impairment of the locomotor ability, as expected. Pulsed administration of physiological doses of E2 dissolved in miglyol rescues the phenotypes induced by OVX. However, when E2 is dissolved in PBS the effects are less pronounced, possibly due to rapid wash out of the steroid.

Resveratrol ameliorates malathion-induced estrus cycle disorder through attenuating the ovarian tissue oxidative stress, autophagy and apoptosis

Malathion is a high-efficiency organic phosphorus broad-spectrum insecticide which is commonly used in agricultural production, sanitation and epidemic prevention. Although the toxic effects of malathion on animal reproduction have been partially evaluated, its function, regulatory mechanism and antidote in estrus cycle and reproductive damage remain generally unclear. Here, the results showed that malathion disrupted the normal estrus cycle in mice, reduced the secretion of ovarian hormones, increased the amount of reactive oxygen species (ROS), and promoted autophagy and apoptosis in the ovary. Interestingly, we found that an antioxidant resveratrol could inhibit the disorders of estrus cycle and steroid hormone synthesis, reduced the abnormality of ROS accumulation, autophagy and apoptosis in malathion-exposed ovarian tissue. Furthermore, compared with those of the control group, malathion induced autophagy and apoptosis in the granular cells, whereas resveratrol attenuated these effects of malathion. Therefore, disadvantages of malathion exposure on estrus cycle disorder could partly reverse by resveratrol supplement. Overall, resveratrol may be a potential drug to prevent malathion-induced ovarian damages and estrus cycle disorder. Our findings provide new insights into ovarian response to malathion and resveratrol exposure.

Amoxicillin-induced gut dysbiosis influences estrous cycle in mice and cytokine expression in the ovary and the caecum.

Gut dysbiosis is caused by several factors, including the use of antibiotics. Since intestinal dysbiosis is associated with a wide range of immunopathological and reproductive conditions, the main goal of this study was to evaluate amoxicillin-induced gut dysbiosis and its influence on the oestrous cycle in mice. Mice were treated with amoxicillin or PBS, and faecal microbiota was evaluated by 16S rDNA metagenomic sequencing. The oestrous cycle was evaluated by vaginal cytology, vaginal opening and flow cytometry. After the induction of gut dysbiosis, the ovaries and the caecum were analysed to differential expression of IL-1β and IL-10 genes and histological analysis. Amoxicillin-treated mice presented differing bacterial groups in the faecal microbiota when compared to the PBS-treated group indicating that amoxicillin treatment-induced gut dysbiosis and they gained weight. The vaginal cytology analysis showed that amoxicillin-induced gut dysbiosis decreased the number of cells but increased the relative number of leucocytes and altered the oestrous cycle. IL-1β was shown to be upregulated in the caecum and in the ovary of the dysbiotic mice. On the other hand, IL-10 expression was shown to be diminished in both organs of the dysbiotic mice. The oocyte area from dysbiotic group presented lower than non-dysbiotic mice with increasing thickness of the pellucid zone. The follicular teak from dysbiotic mice showed lower thickness than non-dysbiotic mice. The results indicate that amoxicillin induces gut dysbiosis and influences the oestrous cycle and the inflammatory status of the ovary and the caecum.

Foxp3 heterozygosity does not overtly affect mammary gland development during puberty or the oestrous cycle in mice.

Female mice heterozygous for a genetic mutation in transcription factor forkhead box p3 (Foxp3) spontaneously develop mammary cancers; however, the underlying mechanism is not well understood. We hypothesised that increased cancer susceptibility is associated with an underlying perturbation in mammary gland development. The role of Foxp3 in mammary ductal morphogenesis was investigated in heterozygous Foxp3Sf/+ and wildtype Foxp3+/+ mice during puberty and at specific stages of the oestrous cycle. No differences in mammary ductal branching morphogenesis, terminal end bud formation or ductal elongation were observed in pubertal Foxp3Sf/+ mice compared with Foxp3+/+ mice. During adulthood, all mice underwent normal regular oestrous cycles. No differences in epithelial branching morphology were detected in mammary glands from mice at the oestrus, metoestrus, dioestrus and pro-oestrus stages of the cycle. Furthermore, abundance of Foxp3 mRNA and protein in the mammary gland and lymph nodes was not altered in Foxp3Sf/+ mice compared with Foxp3+/+ mice. These studies suggest that Foxp3 heterozygosity does not overtly affect mammary gland development during puberty or the oestrous cycle. Further studies are required to dissect the underlying mechanisms of increased mammary cancer susceptibility in Foxp3Sf/+ heterozygous mice and the function of this transcription factor in normal mammary gland development.

Expression of aquaporin-7 and aquaporin-9 in tanycyte cells and choroid plexus during mouse estrus cycle

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.

A sexually dimorphic effect of cholera toxin: rapid changes in colonic motility mediated via a 5-HT3 receptor-dependent pathway in female C57Bl/6 mice.

Cholera causes more than 100,000 deaths each year as a result of severe diarrhoea, vomiting and dehydration due to the actions of cholera toxin; more females than males are affected. Cholera toxin induces hypersecretion via release of mucosal serotonin and over-activation of enteric neurons, but its effects on gastrointestinal motility are not well characterized. We found that cholera toxin rapidly and reversibly reduces colonic motility in female mice in oestrus, but not in males or females in prooestrus, an effect mediated by 5-HT in the colonic mucosa and by 5-HT3 receptors. We show that the number of mucosal enterochromaffin cells containing 5-HT changes with the oestrous cycle in mice. These findings indicate that cholera toxin's effects on motility are rapid and depend on the oestrous cycle and therefore can help us better understand differences in responses in males and female patients. Extensive studies of the mechanisms responsible for the hypersecretion produced by cholera toxin (CT) have shown that this toxin produces a massive over-activation of enteric neural secretomotor circuits. The effects of CT on gastrointestinal motility, however, have not been adequately characterized. We investigated effects of luminal CT on neurally mediated motor activity in ex vivo male and female mouse full length colon preparations. We used video recording and spatiotemporal maps of contractile activity to quantify colonic migrating motor complexes (CMMCs) and resting colonic diameter. We compared effects of CT in female colon from wild-type and mice lacking tryptophan hydroxylase (TPH1KO). We also compared CMMCs in colons of female mice in oestrus with those in prooestrus. In female (but not male) colon, CT rapidly, reversibly and concentration-dependently inhibits CMMC frequency and induces a tonic constriction. These effects were blocked by granisetron (5-HT3 antagonist) and were absent from TPH1KO females. CT effects were prominent at oestrus but absent at prooestrus. The number of EC cells containing immunohistochemically demonstrable serotonin (5-HT) was 30% greater in female mice during oestrus than during prooestrus or in males. We conclude that CT inhibits CMMCs via release of mucosal 5-HT, which activates an inhibitory pathway involving 5-HT3 receptors. This effect is sex- and oestrous cycle-dependent and is probably due to an oestrous cycle-dependent change in the number of 5-HT-containing EC cells in the colonic mucosa.

151PD - Arid1A Role in Cell Cycle Regulation and Proliferation in Mouse and Human Gynaecological Tissues Reveals Potential Therapeutic Targets

ABSTRACT Aim: Ovarian clear cell carcinoma (OCCC) arises from endometriosis and can be considered an ectopic uterine cancer. ARID1A mutations occur as an early event in 50% of OCCC and 30% of high-grade endometrial cancers. We aimed to characterise the function of ARID1A in the normal uterus and ovary using an Arid1a mouse model and immortalised human ovarian surface epithelium cells. Methods: Arid1a expression during the mouse estrus cycle and its relationship to estrogen (ER), progesterone receptor and proliferation were assayed in a cohort of 28 mice. Illumina Beadchip arrays and MetaCore pathway analysis were used to assay ARID1A-driven transcriptional programmes in mouse uterus and, after siRNA-mediated knockdown, in mouse embryonic fibroblasts (MEF), immortalised human ovarian surface epithelium (IOSE) and JHOC-5 OCCC cells. SRB proliferation assays were performed in MEFs, IOSE and OCCC cell lines. Proliferation in the uterus was assessed 28 days after tamoxifen-induced knockout in Arid1afl/fl ROSA26Cre-ERT2 or wild type (WT) mice. Results: Arid1a expression was not hormonally regulated in the mouse uterus at either the mRNA or protein level, but Arid1a expression was correlated with stromal ER expression (r = 0.6, p = 0.0003). ARID1A-regulated genes in mouse uterus, MEFs and IOSE cells overlapped significantly (p = 1.7x10-13). Pathways involved in the G2/M checkpoint were significantly enriched in all 3 systems. The conserved core transcriptional program included AURKA, PLK1, PLK4, CCNB1 and CCND1. Conversely, genes involved in inflammation and cell adhesion pathways were ARID1A-regulated in JHOC-5 cells. ARID1A knockdown increased proliferation in MEFs, IOSE cells in an HNF1B-dependent manner and in 4 of 6 ARID1A-expressing OCCC cell lines. In vivo knockout in the uterine epithelium resulted in a dramatic increase in Ki67 staining in Arid1afl/fl compared to WT mice. Conclusions: A core ARID1A-driven transcriptional programme, conserved accross normal tissues and species appears to exist, centred around regulation of genes involved in the G2/M checkpoint. ARID1A loss promotes proliferation in normal tissues, providing important clues as to the mechanisms of ARID1A-driven carcinogenesis. Mitotic kinases emerge as potential therapeutic targets in ARID1A mutant tumours, an observation that is not evident from studies in cancer cell lines. Disclosure: All authors have declared no conflicts of interest.

Transplantation of Human Menstrual Blood Stem Cells to Treat Premature Ovarian Failure in Mouse Model

The incidence of premature ovarian failure (POF), also known as ovarian insufficiency, has been increasing in recent years. Although some treatments are currently available, improved treatment strategies are urgently required. Many researchers have reported that human endometrial stem cells (HuMenSCs), which exhibit stem/progenitor cell properties in vitro repaired damaged cells in vivo. Thus, we aimed to determine whether HuMenSCs can serve as cell therapy tools and be used for the treatment of POF. After treating with cyclophosphamide, on the first estrus period (we predicted mouse estrus cycle was generally 5 days), HuMenSCs were injected into a cyclophosphamide-induced mouse model of POF. The results revealed that the HuMenSCs could survive within POF mouse ovaries for at least 14 days in vivo; further, ovaries of the HuMenSCs-transplanted group expressed higher levels of ovarian markers [AMH, inhibin α/β, and follicle-stimulating hormone receptor (FSHR)], and the proliferative marker Ki67. In addition, the ovarian weight, plasma E2 level, and the number of normal follicles increased over time in the HuMenSC group compared with the control group. Further, microarray analysis of cDNA expression patterns revealed that, after HuMenSC transplantation, the gene mRNA expression patterns in the ovarian cells following stimulation of the host ovarian niche became increasingly similar to those observed in human ovarian tissue compared with the pretransplantation mRNA expression pattern in HuMenSCs. Hence, we can safely conclude that the mesenchymal stem cell properties and in vivo survival of HuMenSCs make them ideal seed cells for stem cell transplantation in the treatment of POF.

The Role of LEF1 in Endometrial Gland Formation and Carcinogenesis

Endometrial carcinoma is the most common gynecologic cancer, yet the mechanisms underlying this disease process are poorly understood. We hypothesized that Lef1 is required for endometrial gland formation within the uterus and is overexpressed in endometrial cancer. Using Lef1 knockout (KO) mice, we compared uterine gland development to wild-type (WT) controls, with respect to both morphology and expression of the Lef1 targets, cyclin D1 and MMP7. We characterized the dynamics of Lef1 protein expression during gland development and the mouse estrus cycle, by immunostaining and Western blot. Finally, we investigated the roles of cyclin D1 and MMP7 in gland and cancer formation in the mouse, and assessed the relevance of Lef1 to human cancer by comparing expression levels in cancerous and normal endometrial tissues. Lef1 upregulation in mouse endometrium correlates with the proliferative stages of the estrus cycle and gland development during the neonatal period. WT mice endometrial glands began to develop by day 5 and were easily identified by day 9, whereas Lef1 KO mice endometrial glands had not developed by day 9 although the endometrial lining was intact. We found that during gland development cyclin D1 is elevated and localized to the gland buds, and that this requires the presence of Lef1. We also noted that Lef1 protein was expressed at higher levels in endometrial cancers within mice and humans when compared to normal endometrium. Our loss-of-function data indicate that Lef1 is required for the formation of endometrial glands in the mouse uterus. Lef1 protein elevation corresponds to gland formation during development, and varies cyclically with the mouse estrus cycle, in parallel with gland regeneration. Finally, Lef1 is overexpressed in human and mouse endometrial tumors, consistent with it playing a role in gland proliferation.

Measuring the estrus cycle and its effect on superovulation in mice

This study compares superovulation efficiency during different stages of the estrus cycle in mice by investigating the pudendum appearance, vaginal epithelial cell type, and the follicular development dynamics, in relation to the estrus cycle. We determined the stages of the estrus cycle by observing the pudendum and making vaginal smears of the Kunming mouse, and ascertaining the follicular development by making paraffin sections. Based on the observation, mice in stages of proestrus, oestrus, metoestrus and dioestrus were superovulated respectively, and their follicular developments and embryo collection results were tracked to ascertain the appropriate estrus cycle stage for superovulation. Our data showed that pudendum appearance and vaginal smears could effectively distinguish different stages of the estrus cycle and that ovarian tissue observation showed that follicular development, corpus luteum formation and luteolysis were regular during the estrus cycle. Likewise, during the estrus cycle of the Kunming mouse, pudendum appearance, vaginal epithelial cell type and follicular development were relational to one another. Superovulating mice in different stages of the estrus cycle showed marked differences both in pregnancy rate and average number of embryos collected. Proestrus was significantly better than metoestrus and dioestrus (P<0.05), and better than oestrus, but the difference between them was not significant (P>0.05). We suggest that pudendum appearance and vaginal smears can be used to distinguish estrus cycle stages and that proestrus is the optimal stage of the estrus cycle for superovulation.

The c-Abl expression in uterine epithelium during the mouse estrus cycle

The epithelial cells of the mouse endometrium comprises the recurring physiologic changes such as proliferation and apoptosis induced by the reproductive hormones throughout the estrus cycle. The c-Abl is a protein tyrosine kinase, localized in the different cellular compartments such as nucleus, cytoplasm, mitochondria, and endoplasmic reticulum, interacts with different cellular proteins, including signaling adaptors, kinases, phosphatases, cell-cycle regulators, transcription factors and cytoskeletal proteins. Our hypothesis is that the c-Abl expression in the mouse uterine epithelium shows cyclic changes during the estrus cycle that is involved in regulation of the endometrial epithelial cells. The regulation of c-Abl gene and protein expression in the uterus of intact animals in the different cycle phases was investigated using immunohistochemistry, western blot and semi-quantitative RT-PCR. The immunohistochemistry results revealed that the luminal and the glandular epithelium (LE) and (GE), respectively, showed gradually increase in the expression of c-Abl from the proestrus (P) to the metestrus and followed by a decrease in the diestrus (D). c-Abl immunoreactivity was detected in the both LE and GE cells, especially the LE showed diminished the c-Abl protein expression on the D phase and the minimal value was detected on the P day. The c-Abl protein level in the LE was increased during the M, presenting a high correlation with the hormonal level of this cell type. The result of c-Abl RT-PCR analysis was compatible with pattern of c-Abl protein expression. In conclusion, the stage-specific protein pattern of the mammalian c-Abl tyrosine kinase presented a good correlation with the mouse estrus cycle and may have a regulative function during the uterine remodeling.

Changes in the Mouse Estrus Cycle in Response to Brca1 Inactivation Suggest a Potential Link between Risk Factors for Familial and Sporadic Ovarian Cancer

Menstrual cycle activity is the most important risk factor for sporadic serous ovarian carcinoma, whereas a germ-line mutation in BRCA1 is the most important risk factor for the familial form. Given the rarity of BRCA1 mutations in sporadic ovarian cancers, we hypothesized that BRCA1 influences the menstrual cycle in a way that mimics the factors underlying sporadic ovarian cancer predisposition, making BRCA1 mutations redundant in such cancers. We compared the length of each phase of the estrus cycle (equivalent to the human menstrual cycle) and of circulating levels of estradiol in control mice and in mice carrying a Brca1 mutation in their ovarian granulosa cells, two thirds of which develop ovarian or uterine epithelial tumors. We also compared the length of the different phases of the cycle in mutants that subsequently developed tumors with those in mutants that remained tumor-free. Mutant mice as well as oophorectomized wild-type mice harboring mutant ovarian grafts showed a relative increase in the average length of the proestrus phase of the estrus cycle, which corresponds to the estrogen-dominated follicular phase of the human menstrual cycle. Total circulating levels of estradiol were also increased in mutant mice injected with pregnant mare serum gonadotropins. The relative increase in proestrus length was highest in mutant mice that subsequently developed reproductive epithelial tumors. We conclude that loss of a functional Brca1 increases murine ovarian epithelial tumor predisposition by increasing estrogen stimulation in the absence of progesterone, recapitulating conditions associated with sporadic ovarian cancer predisposition in humans.

Expression of Frizzled 2 in the mouse ovary during oestrous cycle

The Wnt/beta-catenin pathway is a conserved signalling pathway that regulates gene expression and controls diverse developmental processes such as cell fate specification, cell proliferation and cell differentiation. To our knowledge, the potential role of this pathway in the adult ovary has been poorly addressed. To this end, we investigated the expression pattern of Frizzled 2 in the mouse ovary during oestrous cycle by real-time Q-PCR, in situ hybridization, Western blotting and immunohistochemistry. In this study, we used uterine wet weight and H3.2 mRNA level as two markers for classification of mouse oestrous cycle. We found that both uterine wet weight and H3.2 mRNA are the highest in the proestrus and decrease from oestrus to diestrus. During oestrous cycle, Frizzled 2 mRNA and protein exhibited the highest level in the proestrus stage and rapidly reduced from oestrus to diestrus. In situ hybridization results showed that the positive signals for Frizzled 2 are highly detected in the oocyte and stroma in proestrus stage, while moderate or weak Frizzled 2 mRNAs are localized in the oocyte, granulosa cells, stroma and corpus luteum from oestrus to diestrus. The localization pattern of Frizzled 2 protein is mostly consistent with its mRNA, but stronger Frizzled 2 proteins are present in the granulosa cells and membrane of oocyte in oestrous cycle. Our data suggest that Frizzled 2 may be involved in regulating follicle growth, oocyte maturation and luteinization during oestrous cycle.

Influence of Nutritionally Induced Anaemia on the Oestrous Cycle of Mice

Investigations were carried out to evaluate the probable effect of anaemia due to food restriction on the oestrous cycle of mice. Female mice were divided into four groups and maintained on normal and restricted diet for 21 days as follows: Group I, maintained on ad libitum food; Group II, 25% diet restriction (2.7 g/day/animal); Group III, 50% diet restriction (1.8 g/day/animal; Group IV, 75% diet restriction (0.9 g/day/animal). Following 21 days diest restriction, the starved mice exhibited weight loss. Haemoglobin concentration and plasma protein levels were found to be reduced in direct proportion to the degree of diet restriction. The red blood cell (RBC) count did not vary much. These observations suggest that diet restriction may interfere with haemoglobin synthesis. Underfed mice exhibited a significant reduction in the average number of oestrous cycles compared to that of normally fed mice. Thus a probable influence of anaemia on mouse oestrous cycle is evident.

Primary hypoxic tolerance and chemical preconditioning during estrus cycle in mice.

Exogenous application of estrogens or progesterone ameliorates hypoxic/ischemic cell damage. This study investigates whether values of primary and induced hypoxic tolerance vary endogenously during the estrus cycle in female mice. Population spike amplitude (PSA) and NADH were measured during hypoxic hypoxia and recovery in hippocampal slices from untreated control animals (C slices) and slices prepared from animals pretreated in vivo with a single intraperitoneal injection of 3-nitropropionate (3NP) (3NP slices) or acetylsalicylate (ASA) (ASA slices). Posthypoxic recovery of PSA was dose dependent in 3NP slices from males, with maximal recovery on pretreatment attained with 20 mg/kg 3NP (82+/-32% [mean+/-SD]; C slices, 38+/-29%; P<0.01). PSA recovered to 17+/-12% in C slices during proestrus, 43+/-23% during estrus, and 63+/-44% during diestrus. In 3NP slices, recovery of PSA increased to 57+/-36% (P<0. 05) during proestrus. Hypoxic tolerance was not increased in other stages of the estrus cycle. Hypoxic NADH increase during proestrus declined from 212+/-76% in C slices to 133+/-11% in 3NP slices (P<0. 05). Recovery of PSA in ASA slices was 75+/-36% (P<0.01 versus control) in males and 48+/-34% during proestrus (P<0.05 versus ASA slices from males). Primary and induced hypoxic tolerance are endogenously modulated during the estrus cycle. Differences in hypoxic oxidative energy metabolism mediate part of the differential tolerance. Experimental and clinical therapeutic strategies against cerebral ischemia/hypoxia need to consider sex-related dependence.

Brain neurosteroids during the mouse oestrous cycle

Concentrations of the neuroactive steroid 3 α,5 α-tetrahydroprogesterone (TH PROG or allopregnanolone) and its precursors progesterone (PROG) and 5 α-dihydroprogesterone (DH PROG) have been measured in mouse brain throughout the oestrous cycle. Plasma PROG concentrations were also measured for comparison. At each stage, circadian fluctuations were found in the concentrations of brain PROG and its metabolites. Such fluctuations were greater than those attributable to any particular stage of the oestrous cycle. Over the entire cycle, a significant correlation was found between brain TH PROG (or DH PROG) and PROG concentrations but not between brain TH PROG (or DH PROG) and plasma PROG concentrations. There was also no correlation between endogenous TH PROG (or DH PROG) and activity of the 5 α-reductase converting 3H-PROG to 3H-DH PROG in whole brain homogenates. Concentrations of another neuroactive steroid, pregnenolone sulphate (PREG S), in the brain during the oestrous cycle were in phase with plasma PROG but not brain PROG concentrations. Our results indicate that circadian and ovarian influences on the concentrations of PROG and its metabolite TH PROG in female whole mouse brain are caused predominantly by changes in the supply of PROG from within the tissue, whatever the contribution of peripheral sources.