Estrous Mice Research Articles

Modulation of pulsatile GnRH dynamics across the ovarian cycle via changes in the network excitability and basal activity of the arcuate kisspeptin network.

Pulsatile GnRH release is essential for normal reproductive function. Kisspeptin secreting neurons found in the arcuate nucleus, known as KNDy neurons for co-expressing neurokinin B, and dynorphin, drive pulsatile GnRH release. Furthermore, gonadal steroids regulate GnRH pulsatile dynamics across the ovarian cycle by altering KNDy neurons' signalling properties. However, the precise mechanism of regulation remains mostly unknown. To better understand these mechanisms, we start by perturbing the KNDy system at different stages of the estrous cycle using optogenetics. We find that optogenetic stimulation of KNDy neurons stimulates pulsatile GnRH/LH secretion in estrous mice but inhibits it in diestrous mice. These in vivo results in combination with mathematical modelling suggest that the transition between estrus and diestrus is underpinned by well-orchestrated changes in neuropeptide signalling and in the excitability of the KNDy population controlled via glutamate signalling. Guided by model predictions, we show that blocking glutamate signalling in diestrous animals inhibits LH pulses, and that optic stimulation of the KNDy population mitigates this inhibition. In estrous mice, disruption of glutamate signalling inhibits pulses generated via sustained low-frequency optic stimulation of the KNDy population, supporting the idea that the level of network excitability is critical for pulse generation. Our results reconcile previous puzzling findings regarding the estradiol-dependent effect that several neuromodulators have on the GnRH pulse generator dynamics. Therefore, we anticipate our model to be a cornerstone for a more quantitative understanding of the pathways via which gonadal steroids regulate GnRH pulse generator dynamics. Finally, our results could inform useful repurposing of drugs targeting the glutamate system in reproductive therapy.

The Origin of GnRH Pulse Generation: An Integrative Mathematical-Experimental Approach.

Fertility critically depends on the gonadotropin-releasing hormone (GnRH) pulse generator, a neural construct comprised of hypothalamic neurons coexpressing kisspeptin, neurokoinin-B and dynorphin. Here, using mathematical modeling and in vivo optogenetics we reveal for the first time how this neural construct initiates and sustains the appropriate ultradian frequency essential for reproduction. Prompted by mathematical modeling, we show experimentally using female estrous mice that robust pulsatile release of luteinizing hormone, a proxy for GnRH, emerges abruptly as we increase the basal activity of the neuronal network using continuous low-frequency optogenetic stimulation. Further increase in basal activity markedly increases pulse frequency and eventually leads to pulse termination. Additional model predictions that pulsatile dynamics emerge from nonlinear positive and negative feedback interactions mediated through neurokinin-B and dynorphin signaling respectively are confirmed neuropharmacologically. Our results shed light on the long-elusive GnRH pulse generator offering new horizons for reproductive health and wellbeing.SIGNIFICANCE STATEMENT The gonadotropin-releasing hormone (GnRH) pulse generator controls the pulsatile secretion of the gonadotropic hormones LH and FSH and is critical for fertility. The hypothalamic arcuate kisspeptin neurons are thought to represent the GnRH pulse generator, since their oscillatory activity is coincident with LH pulses in the blood; a proxy for GnRH pulses. However, the mechanisms underlying GnRH pulse generation remain elusive. We developed a mathematical model of the kisspeptin neuronal network and confirmed its predictions experimentally, showing how LH secretion is frequency-modulated as we increase the basal activity of the arcuate kisspeptin neurons in vivo using continuous optogenetic stimulation. Our model provides a quantitative framework for understanding the reproductive neuroendocrine system and opens new horizons for fertility regulation.

Repeated ovarian stimulation does not affect the expression level of proteins involved in cell cycle control in mouse ovaries and fallopian tubes

To understand if repeated cycles (2-4 rounds) of gonadotropin stimulation could affect intracellular localization/content of proteins controlling cell cycle progression in mouse fallopian tubes (FT) and ovaries. FT and ovaries of estrous mice (control) and of stimulated mice were analyzed to detect Oct-3/4, Sox-2, p53, β-catenin, pAKT and cyclin D1 localization/content. Spindles and chromosome alignment were analyzed in ovulated oocytes. After round 4, FT and ovaries of control and stimulated groups showed no differences in Oct-3/4, Sox-2 and β-catenin localization nor in Oct-3/4, Sox-2, p53, β-catenin and pAKT contents. Cyclin D1 level increased significantly in FT of treated mice. Oocytes number decreased meanwhile frequency of abnormal meiotic spindles increased with treatments. Repetitive stimulations affected oocyte spindle morphology but did not induce changes in a set of proteins involved in cell cycle progression, usually altered in ovarian cancer. The significant increase of cyclin D1 in the FT requires further investigation.

Estrous cycle variations in GABAA receptor phosphorylation enable rapid modulation by anabolic androgenic steroids in the medial preoptic area

Anabolic androgenic steroids (AAS), synthetic testosterone derivatives that are used for ergogenic purposes, alter neurotransmission and behaviors mediated by GABAA receptors. Some of these effects may reflect direct and rapid action of these synthetic steroids at the receptor. The ability of other natural allosteric steroid modulators to alter GABAA receptor-mediated currents is dependent upon the phosphorylation state of the receptor complex. Here we show that phosphorylation of the GABAA receptor complex immunoprecipitated by β2/β3 subunit-specific antibodies from the medial preoptic area (mPOA) of the mouse varies across the estrous cycle; with levels being significantly lower in estrus. Acute exposure to the AAS, 17α-methyltestosterone (17α-MeT), had no effect on the amplitude or kinetics of inhibitory postsynaptic currents in the mPOA of estrous mice when phosphorylation was low, but increased the amplitude of these currents from mice in diestrus, when it was high. Inclusion of the protein kinase C (PKC) inhibitor, calphostin, in the recording pipette eliminated the ability of 17α-MeT to enhance currents from diestrous animals, suggesting that PKC-receptor phosphorylation is critical for the allosteric modulation elicited by AAS during this phase. In addition, a single injection of 17α-MeT was found to impair an mPOA-mediated behavior (nest building) in diestrus, but not in estrus. PKC is known to target specific serine residues in the β3 subunit of the GABAA receptor. Although phosphorylation of these β3 serine residues showed a similar profile across the cycle, as did phosphoserine in mPOA lysates immunoprecipitated with β2/β3 antibody (lower in estrus than in diestrus or proestrus), the differences were not significant. These data suggest that the phosphorylation state of the receptor complex regulates both the ability of AAS to modulate receptor function in the mPOA and the expression of a simple mPOA-dependent behavior through a PKC-dependent mechanism that involves the β3 subunit and other sites within the GABAA receptor complex.

A proline rich acidic protein PRAP identified from uterine luminal fluid of estrous mice is able to enhance the estrogen responsiveness of Ishikawa cells

Using mice as experimental animals, proteins in the uterine luminal fluid (ULF) from both adults and diethylstilbestrol dipropionate (DES)-treated immature animals were resolved by 2D gel electrophoresis. Two of the protein spots, (a) and (b) around the positions of 18-20 kDa, in the adult ULF were not found in the DES-treated ULF. Automated Edman degradation established the same N-terminal sequences of AHQVPVKTKGKHVFP for the two protein spots. Two trypsin digests of spot (a) were analyzed using CID MS/MS to establish the peptide sequences DNQLGPLLPEPK and RPDAMTWVETEDILSHLR. These partial sequences were confirmed in the cDNA-deduced mouse proline rich acidic protein (PRAP). Using human Ishikawa cell line as a surrogate endometrial model, we demonstrated rapid entrance of exogenous PRAP into the cells and its ability to enhance alkaline phosphatase activity of the E(2) -stimulated cells. Further, the transcripts of five estrogen-responsive genes, including ALPP (Placental alkaline phosphtase), ALPPL (placental alkaline phosphatase-like 2), TGF (transforming growth factor), PR (progesterone receptor), and Wnt7a, were measured after the cell incubation in modified Eagle medium containing 0.1 nM E(2) , or 0-25 µM PRAP, or both together at 37°C for 48 h. As compared with the control, E(2) alone increased the transcripts of ALPP, ALPPL, TGF-α, and PR, and reduced the transcript of Wnt7a, whereas PRAP alone had a slight impact on their expression. E(2) together with PRAP greatly increased the E(2) -stimulated transcriptions of ALPP, ALPPL, TGF-α, and PR, and markedly reduced the E(2) -suppressed transcription of Wnt7a.

Chronic exposure to anabolic androgenic steroids alters activity and synaptic function in neuroendocrine control regions of the female mouse

Disruption of reproductive function is a hallmark of abuse of anabolic androgenic steroids (AAS) in female subjects. To understand the central actions of AAS, patch clamp recordings were made in estrous, diestrous and AAS-treated mice from gonadotropin releasing hormone (GnRH) neurons, neurons in the medial preoptic area (mPOA) and neurons in the anteroventroperiventricular nucleus (AVPV); regions known to provide GABAergic and kisspeptin inputs to the GnRH cells. Action potential (AP) frequency was significantly higher in GnRH neurons of estrous mice than in AAS-treated or diestrous animals. No significant differences in AAS-treated, estrous or diestrous mice were evident in the amplitude or kinetics of spontaneous postsynaptic currents (sPSCs), miniature PSCs or tonic currents mediated by GABA A receptors or in GABA A receptor subunit expression in GnRH neurons. In contrast, the frequency of GABA A receptor-mediated sPSCs in GnRH neurons showed an inverse correlation with AP frequency across the three hormonal states. Surprisingly, AP activity in the medial preoptic area (mPOA), a likely source of GABAergic afferents to GnRH cells, did not vary in concert with the sPSCs in the GnRH neurons. Furthermore, pharmacological blockade of GABA A receptors did not alter the pattern in which there was lower AP frequency in GnRH neurons of AAS-treated and diestrous versus estrous mice. These data suggest that AAS do not impose their effects either directly on GnRH neurons or on putative GABAergic afferents in the mPOA. AP activity recorded from neurons in kisspeptin-rich regions of the AVPV and the expression of kisspeptin mRNA and peptide did vary coordinately with AP activity in GnRH neurons. Our data demonstrate that AAS treatment imposes a “diestrous-like” pattern of activity in GnRH neurons and suggest that this effect may arise from suppression of presynaptic kisspeptin-mediated excitatory drive arising from the AVPV. The actions of AAS on neuroendocrine regulatory circuits may contribute the disruption of reproductive function observed in steroid abuse.

LPS Introduced at Mating Regulates KC Production in the Murine Uterus During Early Pregnancy.

An inflammatory cascade is elicited in the female reproductive tract following mating in mice. The resulting expression of cytokines and recruited leukocytes have roles in facilitating implantation through activating immunological tolerance and endometrial tissue remodelling. We have previously shown that seminal plasma acts to induce synthesis of GMCSF and IL6 in the female reproductive tract in response to TGFβ present in seminal fluid. Recently we have shown that chemokines, specifically the neutrophil chemoattractant KC (mouse homologue of human IL8), is dramatically upregulated after mating. The purpose of this study was to identify the active constituent of semen responsible for KC induction. Female mice were mated with either intact, seminal vesicle deficient or vasectomized males and uterine lavages were collected approximately 8 hours later when KC content was measured by KC specific ELISA. KC production was increased 13-fold, 6-fold and 10-fold respectively, indicating that neither the seminal plasma nor the sperm fraction of semen is independently necessary for induction. To investigate more precisely the identity of the KC inducing factor, an in vitro primary uterine epithelial cell culture system was employed. Uterine epithelial cells were harvested from estrous female mice and exposed to a range of doses of seminal vesicle fluid, recombinant TGFβ1 or lipopolysaccharide (LPS). Following addition of seminal vesicle fluid or TGFβ1, KC production was decreased by 100% and 58% respectively, however synthesis was increased ≈2-fold in a dose-responsive manner to LPS. In addition, instillation of LPS into the uterine luminal cavity of estrous mice acted to induce a 2-fold increase in KC production and to elicit neutrophil recruitment into the endometrial tissue. To further evaluate the contribution of LPS signalling via its receptor, toll-like receptor 4 (TLR4), to the post insemination induction of uterine KC, uterine fluid lavages were collected 8 hours after mating from female LPS hyporesponsive (C3H/HeJ) mice. Uterine KC production was increased 6-fold, similar to that observed in wild type mice, indicating that the increase in uterine KC content in response to mating is independent of LPS signalling via TLR4. Neutrophil recruitment into the uterine tissue following mating in C3H/HeJ mice was similar to that observed in wild type mice. Together these data indicate that while LPS derived from the male or lower female reproductive tract accessing the uterus after insemination can contribute to KC induction, factors other than LPS may contribute to this response, and/or LPS signalling may be mediated by a signalling pathway that is independent of the conventional LPS receptor, TLR4. These findings highlight the potential importance of microflora in the male and female reproductive tracts in influencing the immune environment during early pregnancy. This work was supported by Program Grant 453556 from the NHMRC of Australia.

Expression of Interleukin-18 Receptor mRNA in the Mouse Endometrium

Interleukin-18 (IL-18) is a proinflammatory cytokine involved in chronic inflammation, autoimmune diseases, and a variety of cancers, and is expressed in mouse uteri. Our previous study suggested that IL-18 acts as a paracrine factor, regulating endometrial function. To elucidate the physiological roles of IL-18 in the mouse endometrium, the expression of the IL-18 receptor (IL-18R) alpha subunit was analyzed. IL-18Ralpha mRNA was expressed in several mouse organs in addition to the endometrium. In situ hybridization analysis using a biotin-labeled mouse IL-18Ralpha riboprobe demonstrated that IL-18Ralpha mRNA expression was detected in glandular epithelial cells, stromal cells around uterine glands, and myometrial cells in the mouse uterus, suggesting that these cells are targets for IL-18. The uterine IL-18Ralpha mRNA expression level changed with the estrous cycle. The uterine IL-18Ralpha mRNA levels of estrous mice were higher than those of diestrous mice. In addition, the IL-18Ralpha mRNA levels in uteri at 3 and 14 days after ovariectomy were higher than those at diestrus and decreased following treatment with estradiol-17beta or progesterone. These findings suggest that IL-18Ralpha gene expression is regulated by estrogen and progesterone and that the uterine IL-18 system is involved in the regulation of uterine functions in a paracrine manner.

Uterine and serum cytokine arrays in the mouse during estrus

Cytokines govern uterine immunology and embryo receptivity and are increasingly recognized for their embryotrophic roles. While supplementing culture media with cytokines may improve embryo development/viability in vitro, little is known about their physiological profiles in vivo, and hence which are likely to be uterine immunoregulators and embryotrophins. Therefore, this study profiled 23 cytokines in uterine fluid and serum from individual naturally cycling estrous mice. Samples were analyzed by fluid-phase multiplex immunoassays for interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, eotaxin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon (IFN)-γ, keratinocyte-derived chemokine (KC), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α MIP)-1β regulated upon activation, normal T-cell expressed and secreted (RANTES) and tumor necrosis factor (TNF)-α. There was a marked divergence in cytokine concentrations between uterine fluid and serum. The former was dominated by G-CSF, eotaxin, KC and IL-1α, and had significantly higher levels of IL-1β, IL-2, IL-3, IL-4, IL-6, IL-9, GM-CSF, MIP-1α, MIP-1β and RANTES. Serum had significantly higher IL-12 (p40), IL-12 (p70), IL-17 and IFN-γ concentrations. No significant differences in IL-5, IL-10, IL-13, MCP-1 or TNF-α profiles were noted. These data indicated a strict compartmentalization of uterine cytokines, with G-CSF as a major cytokine at estrous. Results are discussed with respect to immune cell function, post-coital paternal antigen processing, estrous cyclicity, and endometrial angiogenesis, cell turnover and differentiation.

Estrogen Inhibits Interleukin-18 mRNA Expression in the Mouse Uterus

Interleukin-18 (IL-18) is a proinflammatory cytokine expressed in female reproductive organs in humans, rats and mice. The physiological roles of uterine IL-18 and the regulatory mechanisms of IL-18 gene expression are unclear. The present study aimed to clarify the effects of estradiol-17beta (E2) and progesterone (P4) on IL-18 mRNA expression in the mouse uterus. Distribution and expression levels of IL-18 mRNA were studied using an RNase protection assay. Expression of IL-18 mRNA was observed in all organs studied, including testes, ovaries and uteri. The uterine IL-18 mRNA level of estrous mice was higher than that of diestrous mice. E2 treatment (1, 5, 25 or 250 ng/mouse) decreased uterine IL-18 mRNA levels in ovariectomized mice dose-dependently. E2 treatment acutely decreased IL-18 mRNA levels 3 h after injection, but these levels returned to the initial level after 48 h. P4 treatment (1 mg/mouse) decreased uterine IL-18 mRNA levels after 12 h, but levels returned to the initial level after 48 h. Both uterine IL-18 and IL-18Ralpha mRNAs were detected in cultured endometrial epithelial and stromal cells. These results suggest that uterine IL-18 expression is reduced by sex steroid hormones and that IL-18 acts on endometrial cells in a paracrine or autocrine manner.

Diversity in Phenotype and Steroid Hormone Dependence in Dendritic Cells and Macrophages in the Mouse Uterus1

The dendritic cells and related antigen-presenting cells (APCs) that activate lymphocytes for acquired immunity in the female reproductive tract are not well characterized. The aim of the present study was to examine heterogeneity among uterine APCs in mice and, specifically, to determine whether phenotypically and functionally distinct subpopulations of dendritic cells and macrophages can be identified. Using immunohistochemistry, abundant cells expressing APC-restricted molecules major histocompatibility complex (MHC) class II, F4/80, class A scavenger receptor, macrosialin, and sialoadhesin were evident in estrous mice. Cells expressing the costimulatory molecule B7-2 were rarely observed. Flow cytometric analysis revealed three subpopulations of uterine APCs. Undifferentiated macrophages were F4/80-positive (+), MHC class II-negative (-) cells, of which 70-80% expressed CD11b, but few expressed class A scavenger receptor, macrosialin, or sialoadhesin. Mature macrophages were F4/80+/MHC class II+ cells, of which approximately 50% expressed CD11b, class A scavenger receptor, macrosialin, and sialoadhesin. Uterine dendritic cells were F4/ 80-/MHC class II+ cells, with stimulatory immunoaccessory function relative to uterine macrophages and heterogeneous expression of dendritic markers 33D1, DEC205, CD11c, and CD1. Experiments in ovariectomized mice showed that undifferentiated macrophages were steroid hormone dependent but that mature macrophages and dendritic cells persisted after depletion of ovarian steroid hormones, although with altered phenotypes. In summary, our findings identify three discrete populations of APCs inhabiting the murine uterus and suggest that both mature macrophages and dendritic cells differentiate from undifferentiated macrophage precursor cells. Plasticity in the ontogenetic and functional relationships between uterine dendritic cells and macrophages likely is critical in regulating immune responses conducive to reproductive success.

Evidence of altered neuropeptide Y content and neuropeptide Y1 receptor gene expression in the hypothalamus of pregnant transgenic mice.

Alterations in the density of neuropeptide Y (NPY)-immunoreactive fibers and of NPY1 receptor gene expression in the hypothalamus of Y1R/LacZ transgenic pregnant mice were investigated. In the paraventricular nucleus of mice on the 18th d of pregnancy NPY immunoreactivity was significantly decreased, and NPY1 receptor gene expression, as measured by histochemical staining of beta-galactosidase and in situ hybridization of NPY1 receptor mRNA, was significantly increased compared with those in estrous mice. Conversely, pregnant transgenic mice displayed a significant induction of NPY immunoreactivity and a reduction of NPY1 receptor gene expression in the ventromedial nucleus. A significant increase in Y1R/LacZ transgene expression and NPY1 receptor mRNA, but no changes in NPY immunoreactivity were observed in the arcuate nucleus of mice on the 18th d of pregnancy. These results suggest that the elevated expression of NPY in the ventromedial nucleus may contribute to the state of leptin resistance that occurs during pregnancy.

Seminal transforming growth factor beta1 stimulates granulocyte-macrophage colony-stimulating factor production and inflammatory cell recruitment in the murine uterus.

Mating in rodents evokes an inflammatory-like reaction within the uterine endometrium, characterized by extensive infiltration and activation of macrophages, dendritic cells, and granulocytes. This response is initiated when seminal vesicle gland-derived factors in the ejaculate stimulate uterine epithelial cells to release proinflammatory cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF). Experiments in which seminal vesicle secretions were fractionated by Sephacryl S-400 chromatography and assayed in vitro for GM-CSF-stimulating activity revealed that the seminal moiety coeluted with transforming growth factor beta1 (TGFbeta1) in the 150-440-kDa range and was neutralized by anti-TGFbeta1 antibodies. Comparable amounts of recombinant TGFbeta1 stimulated GM-CSF release in cultures of uterine epithelial cells from estrous mice and, when instilled into the uterine lumen, caused an increase in GM-CSF content and an infiltration of leukocytes into the endometrium similar to the postmating response. These results show that seminal vesicular fluid contains TGFbeta1 at levels sufficient to be the primary causative agent in the postmating inflammatory cascade through induction of GM-CSF synthesis by uterine epithelial cells. Seminal TGFbeta1 is thus implicated as a key factor in initiation of the remodeling events and immunological changes that occur in the uterus during the preimplantation period of pregnancy.

Tyrosine kinase inhibition decreases Muc-1 expression in mouse epithelial cells.

Mouse uterine epithelial cells (UEC) express high levels of both messenger RNA (mRNA) and protein encoding the polymorphic mucin glycoprotein, Muc-1, under most conditions in vivo and in vitro. Although steroid hormones modulate Muc-1 expression in vivo, it is not clear if these actions are mediated directly by steroid hormone receptors or indirectly by modulation of key intracellular signal transduction cascades. To address the latter issue, we examined the effects of a wide variety of modulators of signal transduction cascades on the expression of Muc-1 in primary cultures of polarized mouse UEC. Transient exposure of UEC to agents that inhibit tyrosine kinases by distinct mechanisms, i.e., tyrphostin, genistein, and staurosporine, consistently and significantly reduced Muc-1 expression. In contrast, a variety of agents that modulate protein kinase A- or C-dependent pathways had little or no effect on Muc-1. The effect of tyrphostin proved to be similar in magnitude at both the level of Muc-1 protein and mRNA expression. Transient transfection assays of mouse UEC and a murine mammary epithelial cell line, NMuMG, with mouse Muc-1 promoter-CAT reporter constructs demonstrated a similar (50-60%) degree of tyrphostin inhibition. These observations suggested an action at the level of Muc-1 gene expression. Levels of 100,000 g soluble tyrosine kinase activity in mouse UEC freshly isolated from estrous stage (high-level Muc-1 expression) and day 4 of pregnancy (low-level Muc-1 expression) correlated with Muc-1 expression. Furthermore, pretreatment of day 4 pregnant mice with the anti-progestin, RU486, an agent previously shown to restore or maintain high levels of Muc-1 expression, also restored soluble tyrosine kinase activity to levels similar to that observed in estrous stage mice. Collectively, these results indicate that tyrosine kinase activity is required to maintain high level Muc-1 expression in mice.

Rapid and high yield induction of endometrial adenocarcinomas in CD-1 mice by a single intrauterine administration of N-ethyl-N-nitrosourea combined with chronic 17β-estradiol treatment

Induction of uterine endometrial adenocarcinomas in mice by N-ethyl-N-nitrosourea (ENU) and 17 beta-estradiol (E2) was examined. Illumination-induced persistent estrous CD-1 mice were divided into three groups at 10 weeks of age. Group 1 was given a single intra-uterine administration of polyethylene glycol (PEG) 1 week later, while Groups 2 and 3 received ENU (12.5 mg/kg), dissolved in PEG, in the same manner. Group 3 mice were also implanted with E2 pellets s.c. 1 week previously, and thereafter the pellets were renewed every 8 weeks throughout the experiment. At the termination (week 15 after the ENU treatment), all surviving mice were killed and the development of uterine proliferative lesions was assessed. All groups demonstrated endometrial hyperplasias, the severity being greatest in the ENU plus E2-treated animals (Group 3). The incidence of adenocarcinomas in Group 3 (20/29, 69%) was significantly higher than in Group 1 (0/25, 0%) or 2 (0/29, 0%). At 10 weeks after the ENU-treatment, serum E2 and progesterone concentrations in Group 3 were significantly higher and lower, respectively, than those in Groups 1 and 2. Consequently, the E2/progesterone (E2:P) ratio in Group 3 was significantly increased. These results indicate that a continuing high level of serum E2 and low level of progesterone are important for endometrial adenocarcinoma development in mice, with an increased E2:P ratio acting as a promoter for development of the endometrial lesions initiated by ENU treatment.

Ovarian steroid hormones regulate granulocyte-macrophage colony-stimulating factor synthesis by uterine epithelial cells in the mouse.

Uterine epithelial cells have been shown by in vitro studies to be a potent source of the inflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), and the luminal and glandular epithelium has been confirmed as the predominant site of GM-CSF expression in the intact endometrium by in situ hybridization. To examine the role of ovarian steroid hormones in GM-CSF synthesis, GM-CSF bioactivity has been measured in the supernatants of short-term primary cultures of endometrial cells prepared from mice in which steroid levels were perturbed by ovariectomy and steroid replacement or by steroid antagonists. GM-CSF production was found to fluctuate in cells harvested at different times during the estrous cycle, peaking at estrus. Endometrial cells derived from ovariectomized mice produced 25-fold less GM-CSF than did cells from estrous mice, and production was increased if ovariectomized mice were pretreated with estrogen, but not progesterone, 3 h or more before harvest. This estrogen-induced increase was inhibited by coadministration of progesterone or by induction of a decidual response and was blocked by the estrogen antagonist ZK 119,010. By contrast, pretreatment of mice with the anti-progestin RU486 significantly elevated GM-CSF output in cells from ovariectomized mice given estrogen and progesterone in combination and antagonized the inhibition of GM-CSF release seen in cells harvested from mice treated with hCG. These studies demonstrate that GM-CSF synthesis and/or release by uterine epithelial cells is stimulated by estrogen, with progesterone having a moderate inhibitory effect. Analysis of GM-CSF mRNA expression in uterine epithelial cell cultures and in intact uteri from steroid hormone-treated ovariectomized mice by quantitative reverse transcription-polymerase chain reaction indicated that the effects of estrogen and progesterone on GM-CSF release are mediated at least in part at the transcriptional level. These findings implicate GM-CSF as a local mediator of steroid-driven remodeling events in the cycling and preimplantation endometrium, possibly acting through the recruitment and behavioral regulation of granulocytes and macrophages.

Reproductive organ blood flow measured using radioactive microspheres in diestrous and estrous mice

Blood flow is a primary mechanism controlling reproductive organ functions. In the present study, radioactive microsphere techniques were adapted to measure ovarian, uterine, and vaginal blood flow levels in C57BL mice. Anesthetized animals were tracheostomized and the left carotid artery was cannulated. The heart was exposed and 113Sn-labeled spheres (15 microns size, 2 microCi, 0.1 ml) were injected via the left ventricle. Reference sample was obtained by carotid artery blood "free flowing" into a tared microfuge tube for 1 min. The animal was killed, and selected tissues were excised for weighing and radioactivity measurement to determine flow. Absence of differences in flow levels (ml.min-1.g-1) to paired nonreproductive organs (adrenals and kidneys) validated the procedure. Blood flow levels were significantly higher in the ovaries, but not in the uterus and vagina of estrous mice vs. diestrous mice. Comparison of left vs. right ovaries suggested consistent blood flow distribution during diestrus. Ovarian blood flow level is enhanced during estrus and, in addition, is highly nonuniform regarding right-left flow distribution. Nonuniform ovarian blood flow distribution in estrous mice leads us to speculate that alternating right-left (i.e. nonuniform) ovulation predominates during each murine estrous cycle.

Estrous Mice and Vulnerability to Weasel Predation

L'aptitude d'un predateur (Mustela nivalis) a utiliser le comportement et les signaux de femelles (Peromyscus maniculatus bairdi) en rut pour detecter et capturer ces proies

Stress-induced responses and open-field behavior in estrous and nonestrous mice

Three experiments were performed to compare the reactions of estrous and nonestrous female CD-1 mice to stressful stimuli. These experiments revealed no effect of estrous state on (a) response latencies to thermal pain in the mouse hot-place test, (b) immediate, active responses to electric foot shock, or (c) open field ambulation under no stress condition or under stress conditions provided by foot shock or bright light. Estrous mice were found to defecate less in a small open field, and to ambulate more in the center of a large open field, than nonestrous mice, but neither of these effects interacted with an effect of stress condition. An incidental finding was a diurnal effect on response latencies in the mouse hot-place test. These experiments, combined with previous experiments with female CD-1 mice, support the conclusion that estrus results in decreased avoidance of potentially dangerous or stressful places, but does not affect the animal's reactivity to unavoidable stressful stimuli.

The neurons containing dense core vesicles in the mouse hypothalamic arcuate nucleus: distribution and influence of ovarian activity.

The hypothalamic arcuate nucleus (ARC) was electron microscopically studied in diestrous (D) and estrous (E) mice, and mice ovariectomized (OX) 3 weeks before. In the middle region of the ARC (mARC), the number of dense core vesicles (DCVs) appearing in a section of each neuronal perikaryon was examined, and the location of the neurons containing DCVs was mapped. About 10% of the neurons in the mARC contained many (5 or more) DVCs in the perikarya in a section. These neurons mainly localized in the ventromedial portion of the mARC, which shows that the cytoarchitecture of the mouse ARC is heterogeneous. The localization and the frequency of these neurons were not influenced by the variance of ovarian activity. A DCV was composed of an electron-dense core and a membrane halo, and was likely to be formed in the cisternae of the Golgi apparatus. The size of DCVs was estimated at 100 nm as the median diameter and was stable independent of the ovarian activity. However, the formation of DCVs was likely influenced by the ovarian activity, because DCVs in D mice were more intimately associated with the Golgi apparatus than in E or OX mice. In the neuronal perikarya of the ventromedial portion of the mARC, Nissl bodies developed in OX mice and nucleolus-like bodies developed in E mice. These results suggest that the neurons of the ventromedial portion of the mARC change their function according to the variance of ovarian activity and, therefore, are involved in the estrogen feedback mechanism.