Evaluation of the vaginal and urinary microbiota of healthy cycling bitches.

BACKGROUND: While the urogenital microbiota has recently been characterized in healthy male and female dogs, the influence of sex hormones on the urogenital microbiome of bitches is still unknown. A deeper understanding of the cyclic changes in urinary and vaginal microbiota would allow us to compare the bacterial populations in healthy dogs and assess the impact of the microbiome on various urogenital diseases. Therefore, the aim of this study was to characterize and compare the urogenital microbiota during different phases of the estrous cycle in healthy female dogs. DNA extraction, 16 S rDNA library preparation, sequencing and informatic analysis were performed to determine the vaginal and urinary microbiota in 10 healthy beagle dogs at each phase of the estrous cycle. RESULTS: There were no significant differences in alpha and beta diversity of the urinary microbiota across the different cycle phases. Similarly, alpha diversity, richness and evenness of vaginal bacterial populations were not significantly different across the cycle phases. However, there were significant differences in vaginal beta diversity between the different cycle phases, except for between anestrus and diestrus. CONCLUSION: This study strongly suggests that estrogen influences the abundance of the vaginal microbiota in healthy female dogs, but does not appear to affect the urinary microbiome. Furthermore, our data facilitate a deeper understanding of the native urinary and vaginal microbiota in healthy female dogs.

Expression and localization of anti-Müllerian hormone and its receptors in bovine corpus luteum.

Although anti-Müllerian hormone (AMH) is involved in the regulation of granulosa cell function in female animals, its role in tissues other than ovarian follicles remains poorly understood. It has also been suggested that cows with high circulating AMH concentrations have increased fertility; however, the mechanism has not been elucidated. This study was conducted to identify the presence of the AMH-signaling system and its target cells in the bovine corpus luteum formed from an ovulated follicle. Immunoblotting revealed that the proteolytically cleaved C-terminal region in AMH (AMHC), a biologically active peptide, was present in trace amounts in the early corpus luteum and significantly increased during the mid to regressed stages. AMHC and cleaved N-terminal region (AMHN) in AMH generate a noncovalent isoform that improves the activity of AMH signaling. An immunohistochemical analysis revealed that AMHC, AMHN, and type II AMH receptor (AMHR2) were localized to luteal cells during the entire estrous cycle. AMH in the corpus luteum seemed to be newly synthesized since AMH expression was detected. These findings suggest that AMH signaling is involved in the regulation of luteal cell function through an autocrine and post-translational processing mechanism. The level of AMHR2 and mRNA expression of AMHR2 and type I AMH receptors (activin-like kinase 2, 3, and 6) were highest in the mid stage. Thus, AMH signaling in the corpus luteum may also be regulated by changes in the receptor levels. Since the transforming growth factor-beta superfamily, to which AMH belongs, is a multifunctional polypeptide growth factor, further studies are needed to evaluate whether AMH signaling has a role in facilitating or inhibiting luteal cell functions.

Differential effects of deep brain stimulation on reinstatement of cocaine seeking in male and female rats.

There are currently no FDA-approved treatments for cocaine use disorder. Recent preclinical and clinical studies showed that deep brain stimulation (DBS) in limbic regions reduced drug seeking behavior. Our previous work indicated that DBS of the nucleus accumbens shell attenuated reinstatement of cocaine seeking, a model of relapse, in male rats. The current experiments were designed to evaluate the effect of electrical DBS on cocaine reinstatement in female rats across the estrous cycle. Rats were allowed to self-administer cocaine and lever responding was subsequently extinguished. Cocaine seeking was reinstated by an acute injection of experimenter-delivered cocaine. The effect of nucleus accumbens shell DBS vs. sham stimulation on cocaine-primed reinstatement was evaluated in female and male rats using a within-subjects counterbalanced design. Consistent with previous work, accumbens shell DBS suppressed cocaine seeking in male rats. In sharp contrast, accumbens shell DBS had no effect on cocaine reinstatement in female rats evaluated in either the estrus or non-estrus phases. These results suggest that changes across the estrous cycle are not responsible for the differences in the effect of DBS on cocaine reinstatement between female and male rats.

Proliferation and apoptosis dynamics of the normal canine mammary gland during the oestrous cycle evaluated by Ki-67 and Caspase-3 indexes.

Understanding the normal physiology of the canine mammary gland (CMG) is crucial, as it provides a foundational reference for understanding canine mammary neoplasms. The relation between the Proliferation Index (PI) indicated by Ki-67 expression, along with the Apoptotic Index (AI) determined through Caspase-3 expression during the oestrous cycle, is inadequately documented in existing literature. This study seeks to offer insights into the interplay between PI and AI in the CMG across oestrous cycle phases. An extensive investigation was conducted on a diverse case series of bitches (n = 18). Oestrous cycle stages were determined through vaginal cytology, histological examination of the reproductive tract and serum progesterone and oestradiol concentrations. The entire mammary chain was histologically examined, and proliferation and apoptosis were assessed via double immunohistochemistry employing anti-Ki-67 and Caspase-3 antibodies. PI and AI were evaluated through a systematic random sampling approach, counting a minimum of 200 cells for each cell type. There was a significantly higher PI during early dioestrus in all mammary gland components, with a greater proportion of positive cells observed in epithelial cells compared to stromal cells. The highest PI was detected in epithelial cells within the end buds. Significant differences were found in Ki-67 labelling across the cranial mammary glands. A positive and strong correlation was noted between progesterone concentration and PI in epithelial cells. The AI remained consistently low throughout the oestrous cycle, with few differences observed across histological components. Caspase-3 labelling displayed the highest positivity in caudal mammary pairs. A negative and moderate correlation was identified between progesterone concentration and AI in interlobular mesenchymal cells. This study highlights the influence of endocrine regulation on cell proliferation indices in mammary tissue, emphasizing the need to consider these hormonal variations in toxicopathological studies involving canine mammary gland.

Imaging the dynamics of murine uterine contractions in early pregnancy†.

Uterine muscle contractility is essential for reproductive processes including sperm and embryo transport, and during the uterine cycle to remove menstrual effluent. Even still, uterine contractions have primarily been studied in the context of preterm labor. This is partly due to a lack of methods for studying the uterine muscle contractility in the intact organ. Here, we describe an imaging-based method to evaluate mouse uterine contractility of both the longitudinal and circular muscles in the cycling stages and in early pregnancy. By transforming the image-based data into three-dimensional spatiotemporal contractility maps, we calculate waveform characteristics of muscle contractions, including amplitude, frequency, wavelength, and velocity. We report that the native organ is highly contractile during the progesterone-dominant diestrus stage of the cycle when compared to the estrogen-dominant proestrus and estrus stages. We also observed that during the first phase of uterine embryo movement when clustered embryos move toward the middle of the uterine horn, contractions are dynamic and non-uniform between different segments of the uterine horn. In the second phase of embryo movement, contractions are more uniform and rhythmic throughout the uterine horn. Finally, in Lpar3-/- uteri, which display faster embryo movement, we observe global and regional increases in contractility. Our method provides a means to understand the wave characteristics of uterine smooth muscle in response to modulators and in genetic mutants. Better understanding uterine contractility in the early pregnancy stages is critical for the advancement of artificial reproductive technologies and a possibility of modulating embryo movement during clinical embryo transfers.

The cycle of stress: A systematic review of the impact of chronic psychological stress models on the rodent estrous cycle.

Stress is known to impair reproduction through interactions between the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes. However, while it is well accepted that stress can alter estrous cycle regularity, a key indicator of female's HPG axis function, effects of different types of psychological stress have been inconsistent. This systematic review evaluated the impact of rodent models of psychological stress on estrous cyclicity, while reporting biological parameters pertaining to HPA or HPG axis function assessed within these studies. We performed a systematic database search and included articles that implemented a psychological stress model in rodents and reported estrous cyclicity for at least two cycles after initiation of stress. Of the 32 studies included, 62.5% reported post-stress alterations to estrous cyclicity, with Chronic Mild Stress (CMS) models showing the most conclusive effects. Twenty-five studies measured HPG or HPA axis markers, with cycle disruptions being commonly observed in parallel with altered estradiol and increased corticosterone levels. Our review highlights gaps in reporting estrous cyclicity assessments and makes recommendations to improve comparability between studies.

[Individual hormonal profiles of blood progesterone and estradiol-17ß during the course of a reproductive cycle in mares]. / Individueller Konzentrationsverlauf von Progesteron und Östradiol-17ß im Blut bei Stuten während des Zyklus.

OBJECT AND AIM: This study presents the individual course of estradiol-17ß and progesterone concentrations in blood during the reproductive cycle in mares in order to point out physiological differences between individual animals and to aid in the interpretation of hormone values. MATERIAL AND METHODS: Concentrations of estradiol-17ß and progesterone were determined in seven mares over the course of their cycle. One mare was excluded from the study due to a physiologically deviating cycle. In addition, the mares' ovaries were examined via ultrasound on a daily basis in order to match the hormone values to morphological changes of the ovaries. RESULTS: In some cases, the mares showed considerable individual differences in their hormone concentrations, which also differed from the published comparative values in the literature. For example, two mares showed progesterone levels above basal levels at the time of ovulation. The postovulatory progesterone concentrations of the mares are characterized by marked fluctuations, which makes it difficult to provide reference values in the different sections of the corpus luteum phase. The length of the plateau phases averaged 12.3±1.5 days. The mare with double ovulation showed the highest progesterone concentrations. CONCLUSION: The measurement of plasma progesterone levels in mares should be interpreted only in the context of other test results. The very wide variation in estradiol-17ß concentrations makes it questionable whether the determination of this hormone value is of diagnostic value. CLINICAL RELEVANCE: When interpreting steroid hormone values in the ingravid cycle of a mare, the individual concentration courses must be taken into consideration, as they may deviate significantly from the published reference values.

Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle.

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.

Sex- and cycle-dependent changes in spine density and size in hippocampal CA2 neurons.

Sex hormones affect structural and functional plasticity in the rodent hippocampus. However, hormone levels not only differ between males and females, but also fluctuate across the female estrous cycle. While sex- and cycle-dependent differences in dendritic spine density and morphology have been found in the rodent CA1 region, but not in the CA3 or the dentate gyrus, comparable structural data on CA2, i.e. the hippocampal region involved in social recognition memory, is so far lacking. In this study, we, therefore, used wildtype male and female mice in diestrus or proestrus to analyze spines on dendritic segments from identified CA2 neurons. In basal stratum oriens, we found no differences in spine density, but a significant shift towards larger spine head areas in male mice compared to females. Conversely, in apical stratum radiatum diestrus females had a significantly higher spine density, and females in either cycle stage had a significant shift towards larger spine head areas as compared to males, with diestrus females showing the larger shift. Our results provide further evidence for the sexual dimorphism of hippocampal area CA2, and underscore the importance of considering not only the sex, but also the stage of the estrous cycle when interpreting morphological data.

Generalization of a positive-feature interoceptive morphine occasion setter across the rat estrous cycle.

INTRODUCTION: Interoceptive stimuli elicited by drug administration acquire conditioned modulatory properties of the induction of conditioned appetitive behaviours by exteroceptive cues. This effect may be modeled using a drug discrimination task in which the drug stimulus is trained as a positive-feature (FP) occasion setter (OS) that disambiguates the relation between an exteroceptive light conditioned stimulus (CS) and a sucrose unconditioned stimulus (US). We previously reported that females are less sensitive to generalization of a FP morphine OS than males, so we investigated the role of endogenous ovarian hormones in this difference. METHODS: Male and female rats received intermixed injections of 3.2 mg/kg morphine or saline before each daily training session. Training consisted of 8 presentations of the CS, each followed by access to sucrose on morphine, but not saline sessions. Following acquisiton, rats were tested for generalization of the morphine stimulus to 0, 1.0, 3.2, and 5.4 mg/kg morphine. Female rats were monitored for estrous cyclicity using vaginal cytology throughout the study. RESULTS: Both sexes acquired stable drug discrimination. A gradient of generalization was measured across morphine doses and this behaviour did not differ by sex, nor did it differ across the estrous cycle in females. CONCLUSIONS: Morphine generalization is independent of fluctuations in levels of sex and endogenous gonadal hormones in females under these experimental conditions.

Consistent and effective method to define the mouse estrous cycle stage by a deep learning-based model.

The mouse estrous cycle is divided into four stages: proestrus (P), estrus (E), metestrus (M), and diestrus (D). The estrous cycle affects reproductive hormone levels in a wide variety of tissues. Therefore, to obtain reliable results from female mice, it is important to know the estrous cycle stage during sampling. The stage can be analyzed from a vaginal smear under a microscope. However, it is time-consuming, and the results vary between evaluators. Here, we present an accurate and reproducible method for staging the mouse estrous cycle in digital whole-slide images (WSIs) of vaginal smears. We developed a model using a deep convolutional neural network (CNN) in a cloud-based platform, Aiforia Create. The CNN was trained by supervised pixel-level multiclass semantic segmentation of image features from 171 hematoxylin-stained samples. The model was validated by comparing the results obtained by CNN with those of four independent researchers. The validation data included three separate studies comprising altogether 148 slides. The total agreement attested by the Fleiss kappa value between the validators and the CNN was excellent (0.75), and when D, E, and P were analyzed separately, the kappa values were 0.89, 0.79, and 0.74, respectively. The M stage is short and not well defined by the researchers. Thus, identification of the M stage by the CNN was challenging due to the lack of proper ground truth, and the kappa value was 0.26. We conclude that our model is reliable and effective for classifying the estrous cycle stages in female mice.

Successful equine in vitro embryo production by ICSI - effect of season, mares' age, breed, and phase of the estrous cycle on embryo production.

This retrospective study aimed at identifying factors that contribute to the success of equine in vitro embryo production by intracytoplasmic sperm injection (ICSI). A total of 7993 ovum pick-up (OPU) sessions were performed, totaling 2540 donor mares and semen from 396 stallions. Oocytes were aspirated at multiple sites in Brazil and were sent to the laboratory, within 6 h from OPU, in pre-maturation medium where they were in vitro matured (IVM) followed by ICSI and in vitro embryo culture for 7-8 days. The number of recovered oocytes, matured oocytes, cleaved embryos and blastocysts were used to explore the effect of age and breed of the donor mare, time of year in which the mare was aspirated and phase of the estrous cycle on the day of follicular aspiration. Mares between 6 and 15 years old were superior to other age groups in most parameters evaluated, including the average number of blastocysts per OPU. The impact of age was similar when evaluated within two breeds, American Quarter Horse (AQHA) and Warmblood mares. We observed that breed (AQHA, Warmblood, Crioulo, Lusitano and Mangalarga) had an important effect on most of the parameter evaluated, including number of oocytes recovered, blastocysts produced per OPU, and blastocyst rates. The overall impact of season was less pronounced than age and breed, with the only statistically significant difference being a higher rate of oocyte maturation during the summer season. Finally, most of the parameters evaluated were superior in follicular phase mares, with or without dominant follicle than luteal phase mares. In conclusion, this retrospective study revealed that breed, age, season and stage of estrous at the time of OPU are all important parameters for the success of equine embryo production by ICSI. This technology enables producing embryos all-year-round from mares of different breeds and ages from OPU-derived oocytes collected at multiple sites.

Effect of age on androgens pattern in cyclic mares.

Androgens are produced in both sexes. In females produced by the adrenal gland and the ovaries they play a crucial role in regulating ovarian function, estrogen synthesis and follicular growth. Age leads to a reduction in androgen concentrations, although, at present, these mechanisms are not elucidated in mares. The objective of this study was to evaluate the concentrations of testosterone (T), androstenedione (A4) and dehydroepiandrosterone (DHEA) in mares of different ages. Blood samples were drawn from seventy cyclic Spanish Purebred mares belonging to five age groups: 3-5 years, 6-9 years, 10-13 years, 14-16 years and > 16 years. The concentrations of T, A4 and DHEA were determined by EIA, validated specifically for horses. Mares aged 3-5, 6-9 and 10-13 years had higher T concentrations (P < 0.05) than mares aged >16 years, and mares aged 6-9 years had also higher concentrations than those 14-16 years old (P < 0.05). A4 concentrations were lower (P < 0.05) in mares >16 years old when compared with those of other age groups. DHEA concentrations were lower (P < 0.05) in mares 14-16 years and > 16 years old when compared with those of other age groups. DHEA was positively correlated with T (r = 0.61; P < 0.05) and A4 (r = 0.51; P < 0.05). Age induces reduction in androgens' synthesis in physiologically cyclic Spanish Purebred mares. These physiological variations must be duly considered for a correct and objective interpretation of the analytical data.

Sex- and estrous-related response patterns for alcohol depend critically on the level of compulsion-like challenge.

Alcohol use disorder is a substantial social and economic burden. During the last years, the number of women with drinking problems has been increasing, and one main concern is that they are particularly more vulnerable to negative consequences of alcohol. However, little is known about female-specific response patterns for alcohol, and potential underlying differences in brain mechanisms, including for compulsion-like alcohol drinking (when intake persists despite adverse consequences). We used lickometry to assess behavioral microstructure in adult Wistar male and female rats (n = 28-30) during alcohol-only drinking or moderate- or higher-challenge alcohol compulsion (10 or 60 mg/l quinine in alcohol, respectively). Estrous stages were determined and related to drinking levels and patterns of responding to alcohol, as was ovariectomy. Our findings showed that females (where we didn't determine estrus stage) had similar total licks in a session as males, but significantly longer licking bouts under alcohol-only and moderate-challenge, suggesting greater persistence. Further, greater intake under alcohol-only and moderate-challenge was related to faster licking in males, while female consumption was not related to licking speed. Thus, females could have increased persistence without greater vigor, unlike males. However, under higher-challenge, faster licking did predict higher intake in females, similar to males. To better understand female higher-challenge responding, we examined drinking in relation to phases of the estrous cycle. Higher-challenge had longer bouts only in late diestrus. In addition, ovariectomy led to longer bouts only under higher-challenge, suggesting that conditions with reduced hormone levels could increase female persistence for alcohol under higher-challenge. However, ovariectomy also reduced alcohol-only and moderate-challenge drinking but did not reduce bout length. Thus, intake level and response strategy could be regulated somewhat differently by ovarian hormones. Finally, moderate-challenge licking speed was less variable during early diestrus, and we previously showed more stereotyped responding specifically under moderate-challenge in males. By combining behavioral microstructure and sex- and estrus-related changes in drinking patterns, our results suggest that females have greater persistence for alcohol under lower-challenge drinking, while late diestrus and ovariectomy unmasked greater persistence under higher-challenge. Together, our novel insights could help develop more effective and personalized treatments for problematic alcohol use.

Role of steroid hormones in the maintenance of focal adhesions in bovine oviductal epithelial cells.

The oviduct, the organ of the female reproductive system where fertilization and early embryonic development occur, provides an optimal environment for the final maturation of oocytes, storage, and sperm capacitation and transport of gametes and embryos. During the estrous cycle, the oviduct is affected by ovarian sex hormones, resulting in changes aimed at maintaining an appropriate microenvironment. Normal cell migration is tightly regulated, its role being essential for the development and maintenance of organ and tissue functions as well as for regeneration following injury. Due to their involvement in focal contact formations, focal adhesion kinase (PTK2) and paxillin (PXN) are key proteins in the study of cell migration and adhesion. The objective of this work was to compare the expression of PTK2 and PXN in oviductal cells along the estrous cycle and to determine if their expression is regulated by the presence of 17-ß estradiol (E2) and/or progesterone (P4). No transcripts of PTK2 or of PXN were detected in cells corresponding to the luteal phase. Additionally, hormonal stimulation experiments on bovine oviductal cell cultures (BOECs) were carried out, where P4 inhibited the expression of both genes. Migration assays demonstrated that P4 reduced BOECs migration capacity. P4 treatment also reduced cell adhesion, while E2 increased the number of adhered cells. In conclusion, the presence of E2 and P4 regulates the expression of genes involved in the formation of focal contacts and modifies the migration and adhesion of BOECs. Understanding the effect of steroid hormones on BOECs is critical to grasp the impact of steroid control on oviductal function and its contribution to establishing successful pregnancies.

Characterizing the relationship between gonadotropin releasing hormone (GnRH), kisspeptin, and RFamide related peptide 3 (RFRP-3) neurons in the equine hypothalamus across the estrous cycle and in the anovulatory seasons.

To understand better the role that kisspeptin plays in regulating seasonal and estrous cycle changes in the mare, this study investigated the number, location and interactions between GnRH, kisspeptin and RFRP-3 neurons in the equine hypothalamus. Hypothalami were collected from mares during the non-breeding season, vernal transition and various stages of the breeding season. Fluorescent immunohistochemistry was used to label the neuropeptides of interest. GnRH cells were observed primarily in the arcuate nucleus (ARC), while very few labeled cells were identified in the pre-optic area (POA). Kisspeptin cells were identified primarily in the ARC, with a small number of cells observed dorsal to the ARC, surrounding the third ventricle (3V). The mean number of kisspeptin cells varied between animals and typically showed no pattern associated with season or stage of estrous cycle, but a seasonal difference was identified in the ARC population. Small numbers of RFRP-3 cells were observed in the ARC, ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH). The mean number of RFRP-3 cells appeared higher in pre-ovulatory animals compared to all other stages. The percentage of GnRH cell bodies with kisspeptin appositions did not change with season or stage of estrous cycle. The percentage of kisspeptin cells receiving inputs from RFRP-3 fibers did not vary with season or stage of estrous cycle. These interactions suggest the possibility of the presence of an ultra-short loop feedback system between these three peptides. The changes in RFRP-3 neurons suggest the possibility of a role in the regulation of reproduction in the horse, but it is unlikely to be as a gonadotropin inhibitory factor.

Changes in fecal microbiota during estrous cycle in healthy thoroughbred mares.

Gut microbiota plays a crucial role in various physiological processes, including the regulation of the reproductive system and steroid sex hormones. Throughout the normal estrous cycle of healthy mares, the levels of estradiol-17ß (E2) and progesterone (P4) in the blood exhibit periodic changes. To investigate the relationship between cyclic changes in steroid sex hormones and the gut microbiome of mares, we analyzed the fecal microbiota composition in healthy mares during the typical estrous cycle. Blood and fecal samples from five healthy mares were collected, E2 and P4 levels in serum were analyzed using radioimmunoassay (RIA), and the gut microbiome was analyzed by 16S rRNA sequencing. The overall richness and composition of the gut microbiota remained relatively stable during the normal estrous cycle in mares. The Linear Discriminant Analysis Effect Size analysis of the microbial composition during the follicular and luteal phases identified the Rhodococcus genus as differentially abundant. These findings indicate that the mare's gut microbiota's significant composition remains consistent throughout the estrous cycle. At the same time, specific low-abundance pathogenic bacteria exhibit changes that align with sexual hormonal fluctuations.

Role of chemokines in regulating luteal and uterine functions in pregnant cows.

Pregnancy is intricately regulated by the interactions between various bioactive substances secreted by the conceptus, uterus, and corpus luteum (CL). Interferon-τ, synthesized and secreted by the conceptus, plays a central role in the interaction mechanism of maternal recognition in cows. Chemokines, chemotaxis mediators that are primarily secreted by immune cells, regulate various reproductive responses in various species. Although there are scattered reports on the potential roles of chemokines in the bovine CL and the uterus during the estrous cycle, there is little information on chemokines in these organs during pregnancy. Therefore, in this review, we discuss the possible physiological roles of chemokines in the CL and uterus of pregnant cows, focusing on our recent findings on chemokines and changes in their receptor expression in the CL and endometrium of cows at some stages of pregnancy.

Postweaning Social Isolation Alters Puberty Onset by Suppressing Electrical Activity of Arcuate Kisspeptin Neurons.

INTRODUCTION: Postweaning social isolation (PWSI) in rodents is an advanced psychosocial stress model in early life. Some psychosocial stress, such as restrain and isolation, disrupts reproductive physiology in young and adult periods. Mechanisms of early-life stress effects on central regulation of reproduction need to be elucidated. We have investigated the effects of PWSI on function of arcuate kisspeptin (ARCKISS1) neurons by using electrophysiological techniques combining with monitoring of puberty onset and estrous cycle in male and female Kiss1-Cre mice. METHODS: Female mice were monitored for puberty onset with vaginal opening examination during social isolation. After isolation, the estrous cycle of female mice was monitored with vaginal cytology. Anxiety-like behavior of mice was determined by an elevated plus maze test. Effects of PWSI on electrophysiology of ARCKISS1 neurons were investigated by the patch clamp method after intracranial injection of AAV-GFP virus into arcuate nucleus of Kiss1-Cre mice after the isolation period. RESULTS: We found that both male and female isolated mice showed anxiety-like behavior. PWSI caused delay in vaginal opening and extension in estrous cycle length. Spontaneous-firing rates of ARCKISS1 neurons were significantly lower in the isolated male and female mice. The peak amplitude of inhibitory postsynaptic currents to ARCKISS1 neurons was higher in the isolated mice, while frequency of excitatory postsynaptic currents was higher in group-housed mice. CONCLUSION: These findings demonstrate that PWSI alters pre- and postpubertal reproductive physiology through metabolic and electrophysiological pathways.

Track-by-Day: A standardized approach to estrous cycle monitoring in biobehavioral research.

Despite known sex differences in brain function, female subjects are underrepresented in preclinical neuroscience research. This is driven in part by concerns about variability arising from estrous cycle-related hormone fluctuations, especially in fear- and anxiety-related research where there are conflicting reports as to whether and how the cycle influences behavior. The inconsistency may arise from a lack of common standards for tracking and reporting the cycle as opposed to inherent unpredictability in the cycle itself. The rat estrous cycle is conventionally tracked by assigning vaginal cytology smears to one of four qualitatively-defined stages. Although the cytology stages are of unequal length, the stage names are often, but not always, used to refer to the four cycle days. Subjective staging criteria and inconsistent use of terminology are not necessarily a problem in research on the cycle itself, but can lead to irreproducibility in neuroscience studies that treat the stages as independent grouping factors. We propose the explicit use of cycle days as independent variables, which we term Track-by-Day to differentiate it from traditional stage-based tracking, and that days be indexed to the only cytology feature that is a direct and rapid consequence of a hormonal event: a cornified cell layer formed in response to the pre-ovulatory 17ß-estradiol peak. Here we demonstrate that cycle length is robustly regular with this method, and that the method outperforms traditional staging in detecting estrous cycle effects on Pavlovian fear conditioning and on a separate proxy for hormonal changes, uterine histology.