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Adenosine is a ubiquitous neuromodulator that plays a role in sleep, vasodilation, and immune response and manipulating the adenosine system could be therapeutic for Parkinson's disease or ischemic stroke. Spontaneous transient adenosine release provides rapid neuromodulation; however, little is known about the effect of sex as a biological variable on adenosine signaling and this is vital information for designing therapeutics. Here, we investigate sex differences in spontaneous, transient adenosine release using fast-scan cyclic voltammetry to measure adenosine in vivo in the hippocampus CA1, basolateral amygdala, and prefrontal cortex. The frequency and concentration of transient adenosine release were compared by sex and brain region, and in females, the stage of estrous. Females had larger concentration transients in the hippocampus (0.161 ± 0.003 µM) and the amygdala (0.182 ± 0.006 µM) than males (hippocampus: 0.134 ± 0.003, amygdala: 0.115 ± 0.002 µM), but the males had a higher frequency of events. In the prefrontal cortex, the trends were reversed. Males had higher concentrations (0.189 ± 0.003 µM) than females (0.170 ± 0.002 µM), but females had higher frequencies. Examining stages of the estrous cycle, in the hippocampus, adenosine transients are higher concentration during proestrus and diestrus. In the cortex, adenosine transients were higher in concentration during proestrus, but were lower during all other stages. Thus, sex and estrous cycle differences in spontaneous adenosine are complex, and not completely consistent from region to region. Understanding these complex differences in spontaneous adenosine between the sexes and during different stages of estrous is important for designing effective treatments manipulating adenosine as a neuromodulator., (© 2020 International Society for Neurochemistry.)

Based on the previous studies, neurokinin B (NKB) participation in the modulation of prolactin secretion at the pituitary level can be assumed, but information concerning this topic is largely insufficient. Therefore, in the present study, we aimed: 1) to evaluate changes in the expression of NKB precursor (Tac3) and its receptor (Tacr3) genes as well as the content of NKB and TACR3 proteins in the porcine anterior pituitary throughout the estrous cycle (days 2 - 3, 9 - 10, 12 - 13, 15 - 16, 19 - 20); 2) to determine in vitro the influence of NKB on the expression of Prl, D2r and Trhr genes in the anterior pituitary cells (incubated for 4 h) as well as on prolactin secretion by these cells (incubated for 4 and 24 h) during chosen days of the estrous cycle (9 - 10, 15 - 16, 19 - 20). The experiments have shown alterations in the expression of Tacr3 mRNA and TACR3 protein content, but not in Tac3 mRNA and NKB protein. The treatment with NKB stimulated the expression of Prl (days 15 - 16), D2r (days 9 - 10) and Trhr (days 19 - 20) genes, but its potential to modulate prolactin secretion was observed only following 24-h incubation, specifically inhibition by NKB alone and stimulation by NKB with dopamine on days 19 - 10 of the cycle. These results indicate some implications of NKB in the modulation of prolactin secretion at the pituitary level in cyclic pigs, however further experiments are required to better clarify its role in this process.

The gonadotropin-releasing hormone-gonadotropin inhibitor (GnRH-GnIH) system in the hypothalamus of mammals is the key factor that controls the entire reproductive system. The aim of this study was to immunolocalize GnIH (RFRP-3) in the hypothalamus during the estrous cycle and to study the effect of putrescine on the expression of GnRH-I and GnIH through both in vivo and in vitro (GT1-7 cells) approach and the circulatory levels of GnRH-I, GnIH, and gonadotropins were also investigated. The study also aims in analyzing all the immunofluorescence images by measuring the relative pixel count of an image. This study showed the effect of putrescine on the morphology of ovary, uterus, and the expression of the steroidogenic acute regulatory protein in the ovary. This study showed GnIH expression was intense during the diestrus and moderate during proestrus and estrus, whereas mild staining during the metestrus. The study further showed that putrescine supplementation to adult female rats increased both GnRH-I expression in the hypothalamus as well as the GnRH-I levels in circulation. The study, for the first time, also showed that putrescine supplementation decreased the expression and release of GnIH. These effects of upregulating GnRH-I expression and downregulating GnIH expression were confirmed by in vitro experiments using GT1-7 cells. Putrescine supplementation also increased the gonadotropin levels in the serum. To summarize, putrescine can regulate the hypothalamic-pituitary-gonadal axis by increasing the GnRH-I, luteinizing hormone, and follicle-stimulating hormone levels and suppressing GnIH levels. This is the first report showing the simultaneous effects of putrescine on the regulation of both GnRH-I and GnIH in the hypothalamus., (© 2020 Wiley Periodicals, Inc.)

To achieve self-sustaining and genetically diverse populations ex situ, captive breeding programmes must ensure good overall rates of reproduction, and equal contribution across individuals. Previous research in the critically endangered eastern black rhinoceros (Diceros bicornis michaeli) revealed a high incidence of irregular oestrous cyclicity; in particular extended cycle duration among nulliparous females and acyclic periods in parous females that have not bred for several years. Irregular ovarian activity could play a role in reduced reproductive output; however, the mechanisms underlying these anomalies are poorly understood. The aim of this study was to measure faecal glucocorticoid metabolite (fGCM) concentrations and variability prior to and during periods of regular and irregular ovarian activity, and determine if adrenal activity influences the occurrence of different cycle types in this species. Faecal samples were collected every other day from parous (N = 6) and nulliparous (N = 12) females at eight European institutions for periods of 9-15 months. Concentration and variability in fGCM were compared between periods of regular and irregular cyclicity and between different cycle types (<20 days, 20-40 days, >40 days, acyclic) using generalized linear mixed models. Concentrations of fGCM were influenced by season and higher during the luteal than the follicular phase of the oestrous cycle. Taking this into account, fGCMs were increased during periods of irregular cyclicity (all types combined and during cycles >40 days in length) compared to 20-40 day cycles. This was predominantly driven by nulliparous females. The variation in fGCM concentration also differed between periods of regular and irregular cyclicity; higher standard deviation in fGCM preceded irregular cycles and >40 day cycles compared to 20-40 day cycles. These results suggest that although fGCM concentrations fluctuate across the oestrous cycle in this species, changes in adrenal activity at specific times could be one factor associated with irregular ovarian activity in the black rhinoceros., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The gonadal steroids estradiol and progesterone exert critical suppressive and stimulatory actions upon the brain to control gonadotropin-releasing hormone (GnRH) release that drives the estrous/menstrual cycle. A simple model for understanding these interactions is proposed in which the activity of the "GnRH pulse generator" is restrained by post-ovulation progesterone secretion to bring about the estrus/luteal phase slowing of pulsatile gonadotropin release, while the activity of the "GnRH surge generator" is primed by the rising follicular phase levels of estradiol to generate the pre-ovulatory surge. The physiological fluctuations in estradiol levels across the cycle are considered to clamp the GnRH pulse generator output at a constant level. Independent pulse and surge generator circuitries regulate the excitability of different compartments of the GnRH neuron. As such, GnRH secretion through the cycle is determined simply by the summed influence of the estradiol-clamped, progesterone-regulated pulse and estradiol-regulated surge generators on the GnRH neuron., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

This is the first study describing the relation between the phase of the oestrous cycle and the number of mast cells (MCs) in the canine ovaries and uterine horns. The total number of MCs was counted in cortex ovarii, medulla ovarii, endometrium and myometrium. The number of MCs was compared in all of these areas among the bitches in the early follicular phase, luteal phase and anoestrus. MCs were the most numerous in the early follicular phase and in the luteal phase; however, they were significantly less numerous during anoestrus. Based on the results, it was concluded that the number of MCs in ovaries and uterine horns fluctuates during the oestrous cycle. The results of this study may contribute to clarifying the role of MCs in the reproductive organs of bitches., (© 2020 Blackwell Verlag GmbH.)

In female mammals, cycles in reproductive function depend both on the biological clock synchronized to the light/dark cycle and on a balance between the negative and positive feedbacks of estradiol, whose concentration varies during oocyte maturation. In women, studies report that chronodisruptive environments such as shiftwork may impair fertility and gestational success. The objective of this study was to explore the effects of shifted light/dark cycles on both the robustness of the estrous cycles and the timing of the preovulatory luteinizing hormone (LH) surge in female mice. When mice were exposed to a single 10-hour phase advance or 10-hour phase delay, the occurrence and timing of the LH surge and estrous cyclicity were recovered at the third estrous cycle. By contrast, when mice were exposed to chronic shifts (successive rotations of 10-hoursour phase advances for 3 days followed by 10-hour phase delays for 4 days), they exhibited a severely impaired reproductive activity. Most mice had no preovulatory LH surge at the beginning of the chronic shifts. Furthermore, the gestational success of mice exposed to chronic shifts was reduced, because the number of pups was 2 times lower in shifted than in control mice. In conclusion, this study reports that exposure of female mice to a single phase shift has minor reproductive effects, whereas exposure to chronically disrupted light/dark cycles markedly impairs the occurrence of the preovulatory LH surge, leading to reduced fertility., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Androgen exposure of female fetuses could be an important factor in the development of polycystic ovary syndrome (PCOS) in subsequent generations. The present study aimed to investigate the transgenerational effects of PCOS on the growth, reproduction, and metabolism of the first- and second-generation offspring in rats. Female F0 rats received excessive dehydroepiandrosterone (DHEA) exposure to establish PCOS or the same amount of vehicle as controls. These F0 females were crossed with normal males to obtain control (C) and DHEA (D) F1 offspring, whereas F2 offspring were obtained by inter-crossing between F1 rats for 4 groups: (1) C♂-C♀; (2) D♂-C♀; (3) C♂-D♀ and (4) D♂-D♀. Compared with control groups, F1 and F2 offspring with ancestral DHEA exposure showed higher body weight with increasing age. In addition, female F1 and F2 offspring with ancestral DHEA exposure exhibited PCOS-like reproductive and metabolic phenotypes, including disrupted estrous cycles and polycystic ovaries, as well as increased serum levels of testosterone, impaired glucose tolerance and widespread metabolic abnormalities. Male offspring with ancestral DHEA exposure exhibited lower quality of sperms. These findings confirm the negative effects of excessive androgen exposure of female fetuses on subsequent generations., (Copyright © 2020 Zhang, Yi, Li, Li, Zhao and Qiao.)

Reproduction is a major component of an animal's life history strategy. Species with plasticity in their reproductive biology are likely to be successful as an invasive species, as they can adapt their reproductive effort during various phases of a biological invasion. Silver carp (Hypophthalmicthys molitrix), an invasive cyprinid in North America, display wide variation in reproductive strategies across both their native and introduced ranges, though the specifics of silver carp reproduction in the Illinois River have not been established. We assessed reproductive status using histological and endocrinological methods in silver carp between April and October 2018, with additional histological data from August to October 2017. Here, we show that female silver carp are batch spawners with asynchronous, indeterminate oocyte recruitment, while male silver carp utilize a determinate pattern of spermatogenesis which ceases in the early summer. High plasma testosterone levels in females could be responsible for regulating oocyte development. Our results suggest that silver carp have high spawning activity in the early summer (May-June), but outside of the peak spawning period, female silver carp can maintain spawning-capable status by adjusting rates of gametogenesis and atresia in response to environmental conditions, while males regress their gonads as early as July. The results of this study are compared to reports of silver carp reproduction in other North American rivers as well as in Asia., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Treatment options are limited for the approximately 40% of postmenopausal women worldwide who suffer from female sexual dysfunction (FSD). Neural stimulation has shown potential as a treatment for genital arousal FSD, however the mechanisms for its improvement are unknown. One potential cause of some cases of genital arousal FSD are changes to the composition of the vaginal microbiota, which is associated with vulvovaginal atrophy. The primary hypothesis of this study was that neural stimulation may induce healthy changes in the vaginal microbiome, thereby improving genital arousal FSD symptoms. In this study we used healthy rats, which are a common animal model for sexual function, however the rat vaginal microbiome is understudied. Thus this study also sought to examine the composition of the rat vaginal microbiota. Treatment rats (n = 5) received 30 minutes of cutaneous electrical stimulation targeting the genital branch of the pudendal nerve, and Control animals (n = 4) had 30-minute sessions without stimulation. Vaginal lavage samples were taken during a 14-day baseline period including multiple estrous periods and after twice-weekly 30-minute sessions across a six-week trial period. Analysis of 16S rRNA gene sequences was used to characterize the rat vaginal microbiota in baseline samples and determine the effect of stimulation. We found that the rat vaginal microbiota is dominated by Proteobacteria, Firmicutes, and Actinobacteria, which changed in relative abundance during the estrous cycle and in relationship to each other. While the overall stimulation effects were unclear in these healthy rats, some Treatment animals had less alteration in microbiota composition between sequential samples than Control animals, suggesting that stimulation may help stabilize the vaginal microbiome. Future studies may consider additional physiological parameters, in addition to the microbiome composition, to further examine vaginal health and the effects of stimulation., Competing Interests: No competing interests.

Actions of kisspeptins (KISSs) and RFamide-related peptide-3 (RFRP-3) at the hypothalamus modulate female reproduction. The action of KISS and RFRP-3 in the pituitary gland of pigs during the estrous cycle has not yet been delineated. The present study was conducted to assess the effect of KISS and RFRP-3 on relative abundance of αGSU and βLH mRNA transcript and LH secretion in vitro by pituitary cells of gilts during the estrous cycle. The cells were isolated from gilts on Days 2-3, 10-12, 15-16 and 19-20 of the estrous cycle and cultured in vitro without inclusion of GnRH (control) or with GnRH (100 ng/ml), KISS (10 -6 M, 10 -7 M) and RFRP-3 (10 -6 M, 10 -7 M) alone or in combination. The relative abundance of α-GSU and β-LH mRNAs was examined. Treatment with KISS increased the synthesis and/or secretion of LH by pituitary cells and RFRP-3 inhibited the synthesis and secretion of LH in the presence of GnRH on Days 10-12 and 15-16 of the estrous cycle. The synthesis and secretion of LH was greater when there was treatment with KISS and GnRH during the late follicular phase. Treatments with KISS and RFRP-3 affected the synthesis and/or secretion of LH during the luteal phase and luteolysis. In conclusion, KISS and RFRP-3 apparently affects the synthesis and secretion of LH by pituitary cells of estrous cyclic pigs. There appears to be a greater effect of KISS in modulation of LH secretion than RFRP-3 in pigs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aim of this study was to determine the change of Ras and its guanosine triphosphatases (GTPases) proteins in the bovine corpus luteum (CL) during estrous cycle and investigate protein-protein interaction between hormone receptors and Ras proteins via angiogenetic and apoptotic factors using bioinformatics database. The bovine CLs at proliferation phase (PP), secretion phase (SP), and regression phase (RP) were dissected from abattoir ovaries (n = 4/stage), whole of the tissue samples was used to analyze two-dimensional electrophoresis (2-DE), mRNA, and protein analysis. The protein-protein interaction between the Ras GTPases proteins and hormone receptors were analyzed using Search Tool for the Retrieval of Interacting Genes (STRING) database. The Ras protein activator like 3 (RASAL3), Ras GTPase activating protein 3 (RASA3), Ras guanine nucleotide exchange factors 1 beta (RasGEF1B) were discovered by the 2-DE and mass spectrometry in bovine CLs, and the protein spots of RASA3 and RASAL3 were significantly increased in the SPCL compared to the PPCL, whereas the RasGEF1B was reduced in the PPCL (P < 0.05). The mRNA and proteins expression of progesterone receptor, estrogen receptor alpha (ERα), vascular endothelial growth factor A (VEGFA), angiopoietin 1 (Ang1), VEGF receptor2 (VEGFR2), and Tie2 were significantly increased, but intrinsic and extrinsic apoptotic factors were decreased in PPCL and SPCL compared to RPCL (P < 0.05). Based on STRING database, we determined that RasGEF1B is activated by ERα via VEGFA and VEGFR2, then RasGEF1B activates H-Ras and R-Ras. In addition, the RasGAP protein was significantly increased, however, the RasGEF, H-Ras and R-Ras proteins were reduced in SPCL compared to PPCL and RPCL (P < 0.05). In summary, the RasGEF and Ras proteins were raised during the development, whereas the RasGAP was increased when development was completed, then the Ras and its GTPases dramatically decreased at the regression in bovine CL. In conclusion, these results suggest that Ras and Ras GTPases could be changed during development and regression, activated by the ERα via angiogenetic signaling during proliferation, and may be important to understanding of the Ras and its GTPases system for estrous cycle in bovine CL., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

All extant species in the Rhinocerotidae family are experiencing escalating threats in the wild, making self-sustaining captive populations essential genetic reservoirs for species survival. Assisted reproductive technologies (ARTs) will become increasingly important for achieving and maintaining ex situ population sustainability and genetic diversity. Previous reports have shown that a large proportion of captive southern white rhinoceros (SWR) females are irregularly cyclic or acyclic, and that cycling females display two different estrous cycle lengths of approximately 30 or 70 days. It has been suggested that the longer estrous cycle length is infertile or subfertile, as no term pregnancies have been observed following long cycles. Here we report the achievement of two pregnancies following long luteal phases, using ovulation induction and artificial insemination with either fresh or frozen-thawed semen. One female SWR conceived on the first insemination attempt and gave birth to a live offspring. A second female conceived twice in consecutive long cycles although the first embryo was resorbed by 33 days post-insemination. A pregnancy from this female's second insemination is ongoing with expected parturition in November 2019. Whether prolonged estrous cycles in SWR are subfertile or infertile in natural breeding situations remains unclear. However, our findings demonstrate that the application of ARTs following prolonged cycles can result the successful establishment of pregnancies in SWR. Therefore, with ARTs, female SWR otherwise considered nonreproductive due to long estrous cycles may still have the potential for representation and contribution to the ex situ population., (© 2019 The Authors. Zoo Biology Published by Wiley Periodicals, Inc.)

This research was conducted to evaluate a method for determining ovarian volume using ultrasonography; specifically, how the day of the estrous cycle affected ovarian volume, and the application of a formula for adjusted ovarian volume (ADJ VOL) and its relationship to actual ovarian volume. Cows (n = 22) were estrous synchronized and subjected to serial transrectal ultrasonographic evaluations, performed every 48 h from the day prior to ovulation until day 9 of the subsequent estrous cycle. Measurements obtained from recorded ultrasonographic images were used to determine actual ovarian volume cm 3 = [0.523 X (D1 X D2 X D3)] and adjusted ovarian volume (ADJ VOL) cm 3 = [0.523 X (D1 X D2 X D3)]- 43 (πrCL 2 ) - 43 (πrF 2 ) on days 1, 3, 5, 7, 9 of the estrous cycle. A third objective was to evaluate the concordance correlation coefficient (ρc) between post-mortem ovarian measurements and the water displacement test. Ovarian volume increased with each day of the estrous cycle from Day 1 to Day 7. Day of the estrous cycle affected ovarian volume (P <  0.001). When applying the formula for ADJ VOL, there was not an effect of day of the estrous cycle (P  = 0.509). Ovarian volume, results ex-vivo, were very consistent with the displacement volume results (ρ c = 0.942). If large ovarian structures are present, and the day of the estrous cycle is unknown, then the formula for ADJ VOL can be used to estimate ovarian volume on Day 1 of the estrous cycle., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Forkhead Box L2 (FOXL2) is a member of the FOXL class of transcription factors, which are essential for ovarian differentiation and function. In the endometrium, FOXL2 is also thought to be important in cattle; however, it is not clear how its expression is regulated. The maternal recognition of pregnancy signal in cattle, interferon-Tau, does not regulate FOXL2 expression. Therefore, in the present study, we examined whether the ovarian steroid hormones that orchestrate implantation regulate FOXL2 gene expression in ruminants. In sheep, we confirmed that FOXL2 mRNA and protein was expressed in the endometrium across the oestrous cycle (day 4 to day 15 post- oestrus ). Similar to the bovine endometrium, ovine FOXL2 endometrial expression was low during the luteal phase of the oestrous cycle (4 to 12 days post- oestrus ) and at implantation (15 days post- oestrus ) while mRNA and protein expression significantly increased during the luteolytic phase (day 15 post- oestrus in cycle). In pregnant ewes, inhibition of progesterone production by trilostane during the day 5 to 16 period prevented the rise in progesterone concentrations and led to a significant increase of FOXL2 expression in caruncles compared with the control group (1.4-fold, p < 0.05). Ovariectomized ewes or cows that were supplemented with exogenous progesterone for 12 days or 6 days, respectively, had lower endometrial FOXL2 expression compared with control ovariectomized females (sheep, mRNA, 1.8-fold; protein, 2.4-fold; cattle; mRNA, 2.2-fold; p < 0.05). Exogenous oestradiol treatments for 12 days in sheep or 2 days in cattle did not affect FOXL2 endometrial expression compared with control ovariectomized females, except at the protein level in both endometrial areas in the sheep. Moreover, treating bovine endometrial explants with exogenous progesterone for 48h reduced FOXL2 expression. Using in vitro assays with COS7 cells we also demonstrated that progesterone regulates the FOXL2 promoter activity through the progesterone receptor. Collectively, our findings imply that endometrial FOXL2 is, as a direct target of progesterone, involved in early pregnancy and implantation.

The steroid hormones are instrumental for the growth of mammary epithelial cells. Our previous study indicates that hormones regulate the expression of Rspondin-1 (Rspo1). Yet, the regulatory mechanism remains unknown. In the current study, we identify Amphiregulin (Areg) as a novel upstream regulator of Rspo1 expression mediating the hormonal influence. In response to hormonal signaling, Areg emanating from estrogen receptor (ER)-positive luminal cells, induce the expression of Rspo1 in ER-negative luminal cells. The paracrine action of Areg on Rspo1 expression is dependent on Egfr. Our data reveal a novel Estrogen-Areg-Rspo1 regulatory axis in the mammary gland, providing new evidence for the orchestrated action of systemic hormones and local growth factors., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Vaginal cytology evaluation is an economic, non-invasive technique for indirect monitoring of fluctuations in estrogen concentrations, and thus progression of the estrous cycle. This technique is widely used in domestic dogs for determining timing of artificial insemination. There, however, are only a few reports on the vaginal cytology of non-domestic felids, including lions. This study was conducted, therefore, to describe the vaginal epithelial changes throughout the reproductive cycle of African lions, and to investigate the efficacy of vaginal cytology assessments for predicting reproductive stages. During a 12-month period, reproductive behavioral data and vaginal swabs were collected daily from five lionesses. In total, 541 vaginal smears were evaluated for the proportion of mucosal epithelial cells, neutrophils, bacterial cells, and amount of mucous, cellular debris. One single swab with a large proportion of superficial cells, absence of neutrophils, large number of bacteria, without cellular debris was sufficient for detecting lionesses in estrus. Likewise, one cytology sample with a large proportion of parabasal and intermediate cells, few neutrophils, few bacteria, and large amount of mucous, cellular debris enabled detection of females in advanced diestrus or gestation. To distinguish lionesses in early diestrus from those in an inter-estrous period, at least two consecutive swabs were necessary for satisfactory classification. Overall, evaluation of vaginal cytology samples was an effective technique for differentiation among different stages of the reproductive cycle, confirmation of estrus, and pregnancy diagnosis in lionesses. This technique, therefore, has the potential for application in classifying different stages of the reproductive cycle in other feline species., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The effects of gonadotropin, serum and follicular fluid on the in vitro maturation of canine oocytes were examined. Additionally, spindle size and spindle migration in MI-stage oocytes derived by in vivo or in vitro maturation were evaluated for the first time. Mature oocytes collected from beagle dog ovaries were divided into two experiments. In experiment I, oocytes were cultured in basic TCM 199 medium supplemented with different levels of P4, E2 and FSH. In experiment II, oocytes in the estrus or anestrus stage were cultured in basic medium supplemented with 30% or 40% canine serum plus 20% or 10% follicular fluid. Our results showed that in experiment I, more oocytes reached MI-MII (18.57%) after supplementation with 1 IU/ml FSH+ 5 IU/ml P4 + 5 IU/ml E2 than after supplementation with other levels of reagents. However, there were no significant differences among the groups (three different concentration groups and a control group) with respect to the proportions of oocytes that resumed meiosis, completed meiosis or degenerated. In experiment II, the number of oocytes from the estrus stage that reached MI-MII in TCM 199 medium supplemented with 40% canine serum and 10% follicular fluid (46.72%) was significantly higher (p < 0.01) than the number of oocytes from the anestrus stage that reached MI-MII in medium supplemented with 30% canine serum and 20% follicular fluid (21.84%). In addition, the degeneration rate was significantly lower (p < 0.05) in the 40% canine serum/10% follicular fluid group from follicular stage than in the other three groups. The average spindle length of the MI-stage oocytes that matured in vivo was significantly (p < 0.01) longer than that of the MI-stage oocytes that matured in vitro (21.75 vs. 14.39 μm). These results suggest that supplementation of the culture medium with 40% estrus serum and 10% follicular fluid had a positive influence on the in vitro maturation of canine oocytes and greatly affected spindle size in MI-stage oocytes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The current study was conducted to determine the optimal timing of artificial insemination and vaginal mucous characteristics relative to the onset of estrus in goats. Does (n = 257) were estrous synchronized using hormonal treatments. Intracervical inseminations with fresh semen were performed at 0, 12, 24 and 36 h after the onset of estrus. Characteristics of vaginal mucus (i.e., color, consistency, and volume) were observed and graded at the time of AI to calculate cumulative mucous score. The vaginal electrical resistance (VER) was recorded (Draminski® detector) at the time of AI. Pregnancy rate was less (P < 0.05) when inseminations occurred at 0 h (28.6%) compared with 12 (58.3%) and 24 h (56.4%) after the onset of estrus; however, pregnancy rate at 36 h (54.5%) did not differ (P> 0.05) compared with inseminations at 0, 12 or 24 h after estrous onset. Relative odds for pregnancy rate at 12, 24 and 36 h were 5.24, 5.20 and 3.29 times greater compared with 0 h. Cumulative mucous score varied (P < 0.05) relative to the onset of estrus and correlated well (P < 0.05) with the color and consistency than volume of the mucus. The VER was less (P < 0.05) at 12 than 0 and 36 h after estrous onset. In conclusion, goats can be inseminated between 12-36 h after the onset of estrus; however, the chances of pregnancy are greater with inseminations at the 12 or 24 h time periods., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interests and have no financial or personal relationship(s) which may have inappropriately influenced the research and write-up of this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Orexin A (OXA) is primarily known for its involvement in the regulation of feeding behaviour, energy metabolism and sleep/wake cycle. Nevertheless, studies indicate its engagement in the regulation of the porcine reproductive system. Therefore, the aim of this study was to investigate OXA effect (1, 10, 100 nM), in the presence or absence of the selective orexin receptor type 1 antagonist (SB-3348667; 1 μM), on the gene expression of key steroidogenic enzymes: steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase (HSD3B1), as well as on progesterone (P 4 ) and androstenedione (A 4 ) secretion. Endometrial and myometrial tissue explants were collected from gilts on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy, and on days 10 to 11 of the oestrous cycle (n = 5 per studied period of pregnancy or mid-luteal phase of the oestrous cycle). Gene expression was evaluated by real-time PCR. The level of steroid hormones secreted into the culture medium was examined by radioimmunoassay (RIA). In the present study, in the endometrium, OXA significantly stimulated StAR expression on days 12 to 13, CYP11A1 expression on days 27 to 28 and HSD3B1 expression on days 15 to 16 of pregnancy. Further, in this tissue, OXA decreased StAR mRNA level on days 10 to 11, CYP11A1 mRNA level on days 15 to 16, as well as HSD3B1 mRNA level on days 10 to 11 and 12 to 13 of gestation. Regarding the myometrium, OXA stimulated CYP11A1 gene expression on days 15 to 16 of pregnancy. In this tissue, OXA decreased StAR transcript content on days 15 to 16 and CYP11A1 mRNA level on days 27 to 28. We also demonstrated that OXA alone enhanced P 4 secretion in the endometrium on days 10 to 11 and 12 to 13 of gestation. OXA alone has no significant effect on endometrial and myometrial A 4 secretion, whereas OXA in combination with OX1R antagonist increased this hormone secretion during all studied stages of pregnancy. Therefore, we can conclude that OXA may affect de novo synthesis and secretion of P 4 and A 4 in the porcine uterus via participating in the regulation of key steroidogenic enzymes gene expression, as well as modulating steroid hormones secretion during early pregnancy and mid-luteal phase of the oestrous cycle in pigs. However, further research is required to explain the exact role of OXA in the porcine uterus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Women are at greater risk than men for developing posttraumatic stress disorder (PTSD) after trauma exposure. Sleep, especially rapid-eye-movement sleep (REMS), has been considered a contributing factor to the development of PTSD symptoms through its effects on the processing of emotional memories. However, it remains unknown if sex and sex hormones play a role in the hypothesized impact of sleep on the development of PTSD. Animal models have methodological advantages over human studies in investigating this research question; however, animal models of sleep in PTSD have been tested only with males. C57BL/6 mice (7 males and 15 females) were exposed to 15 footshocks in a footshock chamber, and 5 min after the last footshock, were returned to their home cages for telemetric electroencephalographic sleep recording. Nine to thirteen days later, mice were returned to the footshock chamber for 10 min without footshocks. Fear recall rates were computed by comparing freezing behaviors in the footshock chamber immediately after the footshocks to those during fear context reexposure. Males had significantly lower recall rates compared to metestrous females (that received footshocks on metestrus). Overall, males slept more than both proestrous females (that received footshocks on proestrus) and metestrous females during the dark period. Regression analyses revealed that average REMS episode durations after footshocks were differentially associated with recall rates across groups, such that the association was positive in males, but negative in proestrous females. Results suggest that both sex and the estrous cycle modulate the associations between REMS continuity and fear memory consolidation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Parkinson disease (PD) is a progressive, neurological disease that affects millions of individuals worldwide. Although instability, rigidity, tremor, and bradykinesia are considered hallmark motor signs of the disease, these are not apparent until mid-to-late stage. In addition to limb motor impairment, individuals with PD also exhibit early-onset speech dysfunction and reduced vocal intelligibility as well as anhedonia and anxiety. Many of these clinical signs vary according to sex in humans with PD. In this study, a translational genetic rat model of early-onset PD (Pink1-/-) was used to address significant gaps in knowledge concerning sex-specific characteristics of limb sensorimotor deficits, vocal motor dysfunction, and changes in affective state. Traditional behavioral tests of limb function, ultrasonic vocalization, anxiety, and anhedonia in the Pink1-/- female rat and wildtype controls were used to test the hypothesis that behavioral performance would significantly differ between genotypes, and that these differences would increase with disease progression (age of the rat). Results demonstrate that Pink1-/- female rats do not exhibit limb sensorimotor deficits but do have significantly reduced intensity (loudness) of vocalizations, and present with anhedonia and anxiety by 8 months of age. Consistent with an early-disease model, Pink1-/- female rats do not exhibit significant decreases in nigrostriatal catecholamines/metabolites, as measured by HPLC. These results are significant in expanding knowledge of early-onset deficits in the female Pink1-/- genetic rat model of PD., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The timing of the first postpartum ovulation is an important factor affecting the timing of estrous resumption in dairy cows. The first postpartum ovulation is delayed in cows producing large amounts of milk with an intensive negative energy balance. The antral follicle count (AFC) and serum anti-Müllerian hormone concentrations are known to be indicators of the ovarian reserve, which is the number and quality of follicles left in a pair of ovaries and known as an indicator of female fertility. Cows with higher AFC have been proven to show higher pregnancy rate and shorter calving to conception intervals; however, the relationship between the timing of the first postpartum ovulation and ovarian reserve remains unclear. Therefore, this study examined the relationships between postpartum follicular dynamics, the ovarian cycle, nutritional status, and ovarian reserve., Methods: Transrectal ultrasonography was conducted from calving to 70-120 days in milk (DIM) in 26 cows to monitor AFC, follicular dynamics and the ovarian cycle. Body weight (BW) and milk yield were used as indicators of nutritional status., Results: The first postpartum ovulation was significantly later in cows with low AFC (< 25) than in those with high AFC (≥25), while changes in BW from calving to the nadir and milk production were similar in both groups. The present results also suggested that cows with low AFC and a delayed first postpartum ovulation had a shorter first ovarian cycle after the first postpartum ovulation. The mean DIM of the first postpartum artificial insemination (AI) and days open (days from calving to AI with which pregnancy was achieved) were similar in high and low AFC groups., Conclusions: The first postpartum ovulation was significantly earlier in cows with high AFC than in those with low AFC. The assumed reason for this result was higher sensitivity to luteinizing hormone and larger androstenedione and estradiol production in follicles in high AFC cows. Therefore, cows with high AFC may be more fertile than those with low AFC while their milk production increase and BW decrease; it means they are in negative energy balance. (340/350 words).

Uterine has a pivotal role in implantation and conceptus development. To prepare a conducive uterine condition for possibly new gestation during the estrous cycle, uterine endometrium undergoes dramatic remodeling. In addition, angiogenesis is an indispensable biological process of endometrium remodeling. Furthermore, essential protein expressions related to important biological processes of endometrium remodeling, which are vascular endothelial growth factor (VEGF), myoglobin (MYG), collagen type IV (COL4), fucosyltransferase IV (FUT4), and cysteine-rich protein 2 (CRP2), were detected in the endometrial tissue reported in many previous studies and recently discovered in histotroph substrates during the estrous cycle. Those proteins, which are liable for provoking new vessel development, cell proliferation, cell adhesion, and cell migration, were expressed higher in the histotroph during the luteal phase than follicular phase. Histotroph proteins considerably contribute to endometrium remodeling during the estrous cycle. To that end, the following review will discuss and highlight the relevant information and evidence of the uterine fluid proteins as endometrial-secreted factors that adequately indicate the potential role of the uterine secretions to be involved in the endometrial remodeling process., (© 2020 Japanese Society of Animal Science.)

For unknown reasons, reproductive success varies among zoos in managed red river hogs. In response to urine exposure from novel conspecifics, we hypothesized that males with low libido would exhibit increased concentrations of testosterone metabolites and that acyclic and/or non-breeding females would be induced to cycle or cycle more regularly. Estrous cycle length and progesterone metabolites in same-sex housed females were compared prior to and following exposure to novel red river hog male urine. Male testosterone metabolites and female progesterone metabolites as well as estrous cycle length were compared among: 1) proven-breeder females and males; 2) non-breeding females newly paired with novel males; 3) non-breeding females and males exposed to urine from novel females and males. Fecal samples were collected 3-5 times per week for eight to 12 months, lyophilized, extracted, and assayed for progesterone and testosterone metabolites with validated enzyme immunoassays. Introduction of female urine resulted in an increased number of estrous cycles per female per month, and decreased luteal and increased follicular progesterone metabolites in females. Introduction of male urine resulted in an increase in testosterone metabolites in males. Average progesterone metabolites for pregnant proven-breeder females were more than double that for pregnant females newly paired to novel males. An interaction between season and treatment group, as well as the acyclicity of females from July through November irrespective of treatment group, suggest that season may confound and warrant judicious interpretation of the results. Additionally, females housed with pregnant females were either acyclic or did not carry their pregnancies to term, indicating that reproductive suppression may occur in females. In conclusion, urine may be a cost-effective and efficient means to manipulate estrous cycling in managed red river hogs. Furthermore, careful consideration of the number of females in a managed herd is recommended to avoid reproductive suppression., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The application of behavioral economic demand theory in addiction science has proved useful for evaluating individual characteristics underlying abuse liability. Two factors that have received comparably little attention within this literature are sex and gonadal hormones. We determined cocaine and remifentanil demand in male and female rats using a within-session procedure. Cocaine and remifentanil demand were evaluated for 15 consecutive days using a balanced, crossover design that randomized drug order. This design allowed for the evaluation of temporal and exposure effects on two independent dimensions of demand, unconstrained demand (Q 0 ) and demand elasticity (α). Estrous cyclicity was tracked to determine the contribution of phase to demand. No overall sex differences were observed. Increased unconstrained demand for cocaine and remifentanil was observed in females during periods in which estrogen was high (eg, estrus phase). Unconstrained remifentanil demand escalated over the 15-day testing period, but escalation was not observed for cocaine or for demand elasticity. A significant exposure effect was also observed in which greater prior remifentanil intake increased unconstrained cocaine demand and reduced cocaine demand elasticity. These effects were directionally specific as no significant effects of prior cocaine exposure were observed on remifentanil demand measures. These data suggest that unconstrained demand and demand elasticity do not differ between male and female subjects; however, that unconstrained demand is associated with estrous cyclicity. These findings also suggest that opioid exposure enhances subsequent demand for psychomotor stimulants, which may be important when considering recent increases in nonmedical prescription opioid use in the United States., (© 2019 The Authors.Addiction Biology published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

Anti-Müllerian hormone (AMH) is produced by granulosa cells of early-antral follicles found on the ovary. After production, it enters circulation and can be detected from a blood sample with an ELISA. Multiple works have found that circulating AMH is a reliable marker of the antral follicle population (AFP) of an animal as well as directly correlated to an animal's response to a superovulation protocol. Research has also found high repeatability within an animal's oestrous cycle. Further use of AMH may be valuable as a reproductive management tool, based on previous research linking productive life with circulating AMH in heifers and success to various breeding protocols by AMH concentration. The aim of this review was to summarize previous works describing basic function of AMH as well as explore recent research examining AMH as a reproductive tool and measurement of fertility in dairy animals., (© 2019 Blackwell Verlag GmbH.)

For the timed re-insemination at the minimal interbreeding interval, cows were treated with a progesterone (P 4 )-releasing intravaginal device from Days 13-15 to 21 post-insemination (Day 0 = estrus), followed by plasma P 4 assay on Day 23 and then subjected to the Experiments 1 and 2. In Experiment 1, of 18 cows, 6 cows were determined as luteolysis with low (<1 ng/ml) plasma P 4 concentrations on Day 23 and ovulated on Days 24 (3 cows), 25 (1 cow), and 26 (1 cow) except a cow affected by ovarian quiescence. In Experiment 2, all cows were treated with GnRH on Day 23. Cows with low (<1 ng/ml) plasma P 4 concentrations on Day 23 were diagnosed as non-pregnant and subjected to the re-insemination in the morning of Day 24 irrespective of estrous signs. Of 36 cows, 15 cows were diagnosed as being non-pregnant on Day 23. Fourteen cows of the non-pregnant animals were re-inseminated in the morning of Day 24 irrespective of estrous signs and the pregnancy rate of re-insemination was 36%. The conception rates of initial and re-inseminations were 50% (18/36) and 36% (5/14), respectively. The overall pregnancy rate by adding the rates of initial and re-inseminations was 64% (23/36)., (© 2020 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

The uterus is dynamically regulated in response to various signaling triggered by hormones during the estrous cycle. The Hippo signaling pathway is known as an important signaling for regulating cellular processes during development by balancing between cell growth and apoptosis. Serine/threonine protein kinase 3/4 (STK3/4) is a key component of the Hippo signaling network. However, the regulation of STK3/4-Hippo signaling in the uterus is little known. In this study, we investigated the regulation and expression of STK3/4 in the uterine endometrium during the estrous cycle. STK3/4 expression was dynamically regulated in the uterus during the estrous cycle. STK3/4 protein expression was gradually increased from the diestrus stage and reached the highest in the estrus stage. STK3/4 was exclusively localized in the luminal and glandular epithelial cells of the uterus, and phosphorylated STK3/4 was also increased at the estrus stage. Moreover, the increase of STK3/4 expression in uteri was induced by administration of estradiol, but not by progesterone injection in ovariectomized mice. Pretreatment with an estrogen receptor antagonist ICI 182,780 reduced estrogen-induced STK3/4 expression and its phosphorylation. The estrogen-induced STK3/4 expression was related to the increase in phosphorylation of downstream targets including LATS1/2 and YAP. These findings suggest that STK3/4-Hippo signaling acts a novel signaling pathway in the uterine epithelium and STK3/4-Hippo is one of key molecules for connecting between the estrogen downstream signaling pathway and the Hippo signaling pathway leading to regulate dynamic uterine epithelium during the estrous cycle.

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., (Copyright © 2019 the authors.)

In Pavlovian renewal paradigms, intact female rats have previously failed to exhibit renewal of appetitive behavior after extinction. However, when treated with exogenous estradiol, female rats exhibit robust renewal behavior. The current study aims to investigate whether the estrous cycle can influence renewal of appetitive behaviors and activity in brain areas known to support the renewal effect. We further aimed to examine whether the estrous cycle would similarly affect renewal of two different types of appetitive behaviors. We first establish that rats in the proestrous stage of the estrous cycle during extinction exhibit elevated renewal behavior compared with rats in either metestrous/diestrous stages, and only rats in proestrus during extinction training (but not during the renewal test) exhibit elevated renewal behavior. Furthermore, we show that this estrous cycle dependent effect on renewal only applies to the conditioned approach behavior toward the food delivery site but not the conditioned approach behavior toward the light cue associated with food delivery. Finally, we examined FOS activity within the prelimbic and infralimbic areas of the medial prefrontal cortex, the dorsal and ventral hippocampal formation, the paraventricular nucleus of the thalamus, the nucleus accumbens, and areas of the amygdala. Particularly in the hippocampus and amygdala, FOS expression which corresponded to the behavioral differences between groups was observed. Results from this study suggest that context information processing may vary as a function of endogenous female hormones across the gonadal hormone cycle and that encoding and retrieval of this information is accomplished in a state-specific manner. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

The objective of this study was to examine the impact of a bovine respiratory disease complex (BRDC) vaccine with a temperature-sensitive modified live vaccine (MLV) infectious bovine rhinotracheitis (IBR) component on oestrous cycle parameters and the follicular pool. Twenty-four Holstein heifers (12.4 ± 0.5 months) previously calfhood vaccinated with an IBR MLV component were enrolled in two replicates (Spring; n = 10 and Fall; n = 14) and were blocked by pre-vaccination bovine viral diarrhoea (BVD) serum neutralizing (SN) titres. Upon enrolment, heifers were oestrous synchronized with sampling beginning at detected oestrus. At their second heat, heifers were vaccinated with a BRDC calfhood vaccine with a MLV (MLV; n = 12) or killed (K; n = 12) IBR component and sampled for two additional cycles. Serum samples for oestrogen (E2) and progesterone (P4) as well as ultrasound data of ovarian structures were collected every other day. Serum samples for anti-Müllerian hormone (AMH) were collected at oestrus and mid-cycle for each cycle, and serum for titres was collected prior to and following vaccination. Data were analysed with the PROC MIXED and GLM procedures of SAS. There was no difference in pre- or post-vaccination titres between MLV and K heifers (p > .5). Vaccination had no impact on P4 concentrations, P4 area under the curve, luteal tissue area, peak E2 production or oestrous cycle length (p > .05). Cycle number did impact AMH concentration (p < .05). In MLV heifers, AMH concentration was highest in cycle 1 (p < .05) while cycles 2 and 3 did not differ (p > .05). This was also true for the K heifers in the Fall replicate (p < .05). Within cycle 2, AMH concentrations were numerically lower between vaccine types (K = 308.22 ± 33.3 pg/ml, MLV = 181.13 ± 32.9 pg/ml; p > .05). Although no differences were seen in overall cycle parameters, differences in AMH concentrations may indicate a reduction of the follicular pool following vaccination and requires further investigation., (© 2019 Blackwell Verlag GmbH.)

The objectives of this study were to investigate the carryover effects of differing pre- and postweaning planes of nutrition on prepubertal reproductive tract development and postpubertal estrous cycle characteristics in Holstein heifers. Heifer calves (n = 36) were randomly assigned to receive either a low or a high (5 or 10 L of whole milk/d, respectively) preweaning diet from 1 to 7 wk of age and either a low or a high (70 or 85% of concentrate dry total mixed ration, respectively) postweaning diet from 11 to 25 wk of age. Starting at wk 26 of age, heifers were subjected to weekly transrectal ultrasonography until wk 33 or until first ovulation to assess endometrial thickness and ovarian follicular count and size in the prepubertal phase. In a subset of heifers (n = 28), ovarian ultrasonography continued weekly until at least the second ovulation was confirmed; thereafter, ovarian dynamics (through ultrasonography) and blood progesterone (P4) were assessed every 2 d throughout 1 complete estrous cycle in the postpubertal phase. In the prepubertal phase, endometrial thickness (12.0 ± 0.4 vs. 10.8 ± 0.3 mm) and largest follicle size (11.8 ± 0.3 vs. 10.9 ± 0.2 mm) were greater in heifers fed the high postweaning diet than in those fed the low postweaning diet. Furthermore, the number of class 2 (6-9 mm) follicles was greater in heifers fed the high preweaning diet than in those fed the low preweaning diet (1.6 ± 0.1 vs. 1.1 ± 0.1), whereas the number of class 3 (>9 mm) follicles was greater in heifers fed the high postweaning diet than in those fed the low postweaning diet (1.2 ± 0.1 vs. 1.0 ± 0.1). In the postpubertal phase, overall corpus luteum and P4 dynamics did not differ among pre- or postweaning treatments; however, P4 at 4 d preceding luteolysis was lesser in heifers fed the high postweaning diet than in those fed the low postweaning diet (6.1 ± 0.4 vs. 7.7 ± 0.4 ng/mL). In addition, compared with heifers fed the low postweaning diet, those fed the high postweaning diet had a greater number of antral follicles (31.4 ± 2.2 vs. 21.4 ± 2.3) and tended to have more class 3 follicles (3.6 ± 0.3 vs. 2.7 ± 0.3). Results indicate positive carryover effects of increasing the preweaning plane of nutrition from 5 to 10 L of whole milk/d on prepubertal follicular growth in Holstein heifers. Furthermore, an increased postweaning plane of nutrition (85 vs. 70% of concentrate dry total mixed ration) advanced reproductive development through greater endometrial thickness and follicular growth in the prepubertal phase and increased the population of antral follicles in the postpubertal estrous cycle., (Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. All rights reserved.)

This study was conducted to elucidate mare cervical dilation mechanisms by testing two hypotheses: (i) the proportion of collagen staining in histological samples of mare cervices and (ii) the abundance of hormone receptors in the equine cervix differ with stage of the oestrous cycle and site within the cervix. Tissues and jugular vein blood samples were collected from 15 mares. Collagen content was assessed using Masson's Trichome staining. Receptor abundance was assessed using RT-PCR, qRT-PCR and immunohistochemistry. In sub-epithelial stroma, there was less collagen during the follicular than luteal phase, in the caudal- (P =  0.029), mid- (P =  0.0000) and cranial (P =  0.001) cervical tissue. In the deep stroma, there was less collagen staining during the follicular stage in the mid- (P =  0.004) and cranial- (P =  0.041) cervical regions. There were PTGER2, PTGER3, PGR and ESR1 mRNA transcripts in the cervix. A greater proportion of cells were positive for ESR1 protein during the follicular phase in sub-epithelial (P =  0.019) and deep (P =  0.013) stroma. The abundance of ESR1 in the epithelium was negatively correlated with collagen staining in sub-epithelial (P =  0.007) and deep (P =  0.005) stroma. The results of the study provide new information about the cervical biology of mares by increasing the knowledge about collagen content and the relationship between collagen content and ESR1 protein abundance during the oestrous cycle which indicates the ESR1 receptor is a candidate for involvement in control of cervical dilation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The use of animal models for behavioral and pharmaceutical testing is employed in many different fields of research but often relies solely on male animals. When females are included, the existing literature frequently offers inconsistent results regarding the effects of sex and/or estrous cycle on anxiety-like behaviors. Our current study sought to establish baseline or normative behaviors in three commonly employed tests of anxiety-like behavior, and determine any sex or cycle differences. Anxiety-like behaviors in male and naturally-cycling female Sprague-Dawley rats were assessed using elevated plus maze, open field, and a social interaction/avoidance paradigm. Female rats were examined once daily to determine their stage of estrous. Results from the elevated plus maze but not the open field showed that female rats spent significantly more time in open areas than did male rats; however, there was no effect of estrous cycle stage. The social avoidance test revealed that female rats spent significantly more time in the interaction zone with an empty wire mesh cage (novel object), but there was no sex difference in time spent with an age- and sex- matched target rat. Females often exhibited greater locomotion as compared to males in social and non-social tests, but this was not related to primary anxiety measures in these tests. Overall, our findings indicate that outcomes differ in tests of anxiety-like behaviors, highlighting sex differences in the expression of anxiety-like behaviors that depend on the test employed. Importantly, the lack of estrous cycle effects suggest that for these anxiety-based tests, female Sprague-Dawley rats could be collapsed across the cycle phases to facilitate the inclusion of females in future behavioral experiments., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Naturally occurring alterations in estradiol influence food intake in females. However, how motivational responses to food cues are affected by the estrous cycle or ovarian hormones is unknown. In addition, while individual susceptibility to obesity is accompanied by enhanced incentive motivational responses to food cues and increased NAc intrinsic excitability in males, studies in females are absent. Therefore, we examined basal differences in intrinsic NAc excitability of obesity-prone vs. obesity-resistant females and determined how conditioned approach (a measure of cue-triggered motivation), food intake, and motivation for food vary with the cycle in naturally cycling female obesity-prone, obesity-resistant, and outbred Sprague-Dawley rats. Finally, we used ovariectomy followed by hormone treatment to determine the role of ovarian hormones in cue-triggered motivation in selectively-bred and outbred female rats. We found that intrinsic excitability of NAc MSNs and conditioned approach are enhanced in female obesity-prone vs. obesity-resistant rats. These effects were driven by greater MSN excitability and conditioned approach behavior during metestrus/diestrus vs. proestrus/estrus in obesity-prone but not obesity-resistant rats, despite similar regulation of food intake and food motivation by the cycle in these groups. Furthermore, estradiol and progesterone treatment reduced conditioned approach behavior in obesity-prone and outbred Sprague-Dawley females. To our knowledge, these data are the first to demonstrate cycle- and hormone-dependent effects on the motivational response to a food cue, and the only studies to date to determine how individual susceptibility to obesity influences NAc excitability, cue-triggered food-seeking, and differences in the regulation of these neurobehavioral responses by the estrous cycle., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Phthalates have a history of reproductive toxicity in animal models and associations with adverse reproductive outcomes in women. Human exposure to dibutyl phthalate (DBP) occurs via consumer products (7-10 μg/kg/day) and medications (1-233 μg/kg/day). Most DBP toxicity studies have focused on high supraphysiological exposure levels; thus, very little is known about exposures occurring at environmentally relevant levels. CD-1 female mice (80 days old) were treated with tocopherol-stripped corn oil (vehicle control) or DBP dissolved in oil at environmentally relevant (10 and 100 μg/kg/day) or higher (1000 μg/kg/day) levels for 30 days to evaluate effects on DNA damage response (DDR) pathway genes and folliculogenesis. DBP exposure caused dose-dependent effects on folliculogenesis and gene expression. Specifically, animals exposed to the high dose of DBP had more atretic follicles in their ovaries, while in those treated with environmentally relevant doses, follicle numbers were no different from vehicle-treated controls. DBP exposure significantly reduced the expression of DDR genes including those involved in homologous recombination (Atm, Brca1, Mre11a, Rad50), mismatch repair (Msh3, Msh6), and nucleotide excision repair (Xpc, Pcna) in a dose-specific manner. Interestingly, staining for the DNA damage marker, γH2AX, was similar between treatments. DBP exposure did not result in differential DNA methylation in the Brca1 promoter but significantly reduced transcript levels for the maintenance DNA methyltransferase, Dnmt1, in the ovary. Collectively, these findings show that oral exposure to environmentally relevant levels of DBP for 30 days does not significantly impact folliculogenesis in adult mice but leads to aberrant ovarian expression of DDR genes., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Addiction to psychostimulants is a major public health crisis that leads to significant morbidity and mortality, for which there are currently no FDA-approved pharmacotherapies. Female subjects have increased propensity to develop pathological substance use disorders after initial use, suggesting the possibility of different pathophysiological mechanisms between males and females. Recently, we identified the neuroactive cytokine granulocyte-colony stimulating factor (G-CSF) as a key mediator of neuronal and behavioral plasticity in response to cocaine in male mice. Here, we found that G-CSF potentiated the rewarding effects of cocaine in female mice as well; however, the dopaminergic mechanism linked to these effects was highly dependent on the ovarian hormone cycle. G-CSF treatment enhanced the ability of cocaine to inhibit dopamine clearance; however, this effect was observed specifically during pro/estrus, when circulating ovarian hormone levels were high. These findings demonstrate important sex differences in the synaptic effects of this translationally relevant neuroimmune modulator.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a therapeutic option for the treatment of inflammation. However, negative effects of non-selective NSAIDs for treatment of mares with endometritis have been described, including delayed uterine clearance and impairment of ovulations. Firocoxib is a specific cyclooxygenase-2 (COX-2) inhibitor and has the ability to act in the uterus of mares. We investigated the effects of firocoxib on ovulation rate, numbers of polymorphonuclear neutrophils (PMNs), and COX-2 protein levels in the endometrial tissue of susceptible mares after insemination. Two experiments were conducted. In experiment 1, twenty mares were evaluated in two consecutive estrous cycles broken into the following groups: Control - no pharmacological interference; Treatment - mares were treated with 0.2 mg/kg of firocoxib orally. The treatment began on the day of ovulation induction, and firocoxib was administered until one day after artificial insemination (AI). Ovulation was induced with 1 mg of deslorelin acetate and the mares were inseminated 24 h after the injection. Ovulation was confirmed 48 h after induction, and embryos were collected eight days after ovulation. Experiment 2: Nine mares susceptible to persistent mating-induced endometritis (PMIE) were artificially inseminated. The mares were examined with ultrasound and inseminated with fresh semen in two consecutive cycles, control and treated, in a cross-over study design. The amount of intrauterine fluid was measured, and endometrial samples were collected 24 h after AI. The number of PMNs was determined by endometrial cytology and biopsy, and COX-2 labeling in endometrial samples was evaluated by immunohistochemistry. Firocoxib treatment did not induce ovulatory failure or affect embryo recovery rate in Experiment 1. In Experiment 2, firocoxib treatment reduced inflammation after AI in mares as evidenced with results regarding PMN numbers/percentage and endometrial COX-2 staining. In conclusion, the proposed treatment with firocoxib reduced endometrial inflammation in mares susceptible to PMIE after breeding, with no adverse effects., (Copyright © 2019. Published by Elsevier Inc.)

The aim of the present study was to determine the differences in corpus luteum (CL) functionality between the first postpartum estrous cycle and the following cycle in lactating dairy cows. Luteal blood flow (LBF), luteal size and blood progesterone (P4) concentration were monitored during the first and second postpartum estrous cycle. During the first and second postpartum estrous cycle, the mean LBF value increased (p < .05) from early to late dioestrus, while it decreased rapidly in proestrus, resulting statistically lower (p < .05) than those registered in all previous phases. Statistically significant differences were not observed between overall LBF during first and second postpartum estrous cycle (p > .05). During the first postpartum estrous cycle, P4 blood concentrations showed a significant reduction (p < .05) from dioestrus to proestrus. A different trend of P4 concentrations was observed during the second postpartum estrous cycle, where mean P4 value registered in proestrus resulted statistically lower than those registered in the previous cycle phases (p < .05). The mean P4 concentration registered over the first postpartum estrous cycle resulted statistically lower (p < .05) than that registered during the second one. A significant correlation between P4 concentrations and LBF was registered only during the second postpartum estrous cycle. Results indicate that during the first postpartum estrous cycle, P4 concentration was independent of luteal blood flow and luteal size., (© 2019 Blackwell Verlag GmbH.)

The epidermal growth factor receptor (ErbB) family and its ligands are essential for the regulation of multiple cellular processes required for mammalian reproduction. The objectives of this study were to investigate the expression and localisation of ErbB subtypes (ErbB1-4) and selected ligands, namely epidermal growth factor (EGF), amphiregulin (AREG) and neuregulin (NRG), in the cervix and vagina of cycling cows and to determine possible steroid hormone-dependence of their expression using immunohistochemistry. All four ErbBs and EGF, AREG and NRG proteins were found to be localised in the nucleus and cytoplasm of different cells in the cervix and vagina, and their expression differed during the oestrous cycle. During the follicular phase, in both the cervix and vagina, ErbB1, ErbB2, ErbB3, ErbB4 and EGF expression was higher in the luminal epithelium (LE) than in stromal and smooth muscle (SM) cells (P<0.05). During the luteal phase, the expression of ErbB1, ErbB3 and EGF in the LE was significantly different from that in stromal and SM cells in the cervix, whereas the expression of EGF and AREG differed in the vagina compared to the cervix (P<0.05). Throughout the oestrous cycle, in both the cervix and vagina, although ErbB2/human epidermal growth factor receptor 2 expression in the LE and SM cells was significantly higher than in the stromal cells (P<0.05), NRG expression was similar in the LE, stromal and SM cells (P>0.05). Overall, these results suggest that all four ErbBs and the EGF, AREG and NRG proteins may collectively contribute to several cellular processes in the bovine cervix and vagina during the oestrous cycle.

A growing body of evidence demonstrates that estrogen and corticosterone (CORT) impact on cognition and emotion. On the one hand, ovarian hormones may have beneficial effects on several neurophysiological processes, including memory. On the other hand, chronic exposure to stressful conditions has negative effects on brain structures related to learning and memory. In the present study, we used the plus-maze discriminative avoidance task (PMDAT) to evaluate the influence of endogenous variations of sex hormones and exposure to different types of prolonged stressors on learning, memory, anxiety-like behavior and locomotion. Female Wistar rats were submitted to seven consecutive days of restraint stress (4 h/day), overcrowding (18 h/day) or social isolation (18 h/day) and tested in different phases of the estrous cycle. The main results showed that: (1) neither stress conditions nor estrous cycle modified PMDAT acquisition; (2) restraint stress and social isolation induced memory impairments; (3) this impairment was observed particularly in females in metestrus/diestrus; (4) stressed females in estrus displayed less risk assessment behavior, suggesting reduced anxiety-like behavior; (5) restraint stress and social isolation, but not overcrowding, elevated corticosterone levels. Taken together, our findings suggest that the phase of the estrous cycle is an important modulatory factor of the cognitive processing disrupted by stress in female rats. Negative effects were observed in metestrus/diestrus, indicating that the peak of sex hormones may protect females against stress-induced memory impairment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Consumption of unhealthy, energy-dense palatable food during early age leads to obesity in children and the onset of obesity during childhood has a profound effect on the reproductive health of women. In this study, the mechanism underlying diet-induced obesity on ovarian dysfunction was studied by exposing rats to cafeteria diet (CAFD) for two different durations. For that purpose, 21-day-old female Sprague Dawley rats were fed ad libitum with a standard diet (control group) and a cafeteria diet (CAFD group) for a period of 20 weeks (20 W) and 32 weeks (32 W). We observed obesity, hyperglycemia, hyperlipidemia, hyperleptinemia and hypoadiponectinemia in CAFD fed groups. Hyperinsulinemia, hypergonadotrophism, hypertestosteronemia and hyperprogesteronemia were observed in the 20 W-CAFD group. Conversely, in the 32 W-CAFD group hypersecretion declined to hyposecretion. The levels of estradiol remained low during both time periods. The duration of estrous cycle was extended in the CAFD fed rats. The ovary weight was higher in the 20 W-CAFD fed rats but it was drastically reduced over a longer duration cafeteria diet feeding. In the 20 W-CAFD fed rats, the protein levels of LHR, StAR, CYP11A1, 3β-HSD and 17β-HSD were increased but FSHR and CYP19A1 levels were decreased in the ovary. On the other hand, gonadotropin receptor and the protein levels of steroidogenic enzymes were decreased in the ovary of 32 W-CAFD fed rats. We conclude that the duration of energy-dense diet consumption has differential regulatory mechanism in altering the ovarian steroid production. In 20 W-CAFD fed rats, hypergonadotropic condition was observed whereas, 32 W-CAFD consumption induced hypogonadotropic hypogonadism., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Traumatized women are more likely than traumatized men to develop post-traumatic stress disorder (PTSD). Still, the inclusion of females in animal models of PTSD has largely been avoided, likely due to the variable hormone profile of female rodents. Because a valid animal model of PTSD that incorporates females is still needed, we examined the influence of estrous stage and ovarian hormones on the female rat response to a predator-based psychosocial stress model of PTSD. Female Sprague-Dawley rats were exposed to psychosocial stress or control conditions for 31 days. Stressed rats were given two cat exposures and daily social instability; control rats were handled daily. Beginning on Day 32, rats underwent physiological or behavioral testing. In Experiment 1, vaginal smears were collected on days of the first and second cat exposures and each day of behavioral testing to determine estrous stage. In Experiments 2 and 3, ovariectomized or sham control rats were exposed to stress or control conditions. Then, they were given behavioral testing (Exp 2), or their hearts were isolated and subjected to ischemia/reperfusion on a Langendorff isolated heart system (Exp 3). Chronic stress increased anxiety-like behavior, irrespective of estrous stage or ovariectomy condition. Ovariectomized females displayed greater startle responses and anxiety-like behavior than sham rats. Stress had no impact on myocardial sensitivity to ischemic injury; however, ovariectomized females exhibited greater ischemia-induced infarction than sham rats. These findings suggest that ovarian hormones may prevent anxiety-like behavior and be cardioprotective in non-stressed controls, but they do not interact with chronic stress to influence the development of PTSD-like sequelae in female rats., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: To study the effects of cold exposure on estrous cycle of female C57BL/6 mice., Methods: Twelve healthy female C57BL/6 mice were randomly divided into two groups: control group and cold exposure group, 6 in each group. Cold exposure group was exposed to 4℃, 4 h per day, while control group stayed in normal conditions. Vaginal smears were used to observe the estrous cycle. After 2 weeks, blood and uteri were collected from each mouse after anesthetized and weighted. Serum levels of estradiol(E2), follicle-stimulating hormone(FSH), luteinizing hormone(LH), prolactin(Prl) and progesterone(P) were determined by using mouse ELISA kits. The uterus and ovary pathological slices were prepared to observe the structural changes., Results: Compared with the control group, body weight gain showed no significant differences (P＞0.05). The cold group had significant lower coefficients of uterus and the diestrus phase was significantly increased after cold exposure (P＜0.01). Serum level of FSH in cold group was higher and Prl was lower significantly (P＜0.01). Pathological examination of uterus and ovary showed that uterine glands of cold group were expanded and the amount of follicles was decreased significantly., Conclusion: Cold exposure might increase mouse estrous cycle and affect their reproductive function.

This study examined the effects of bovine oviductal fluid (bOF) obtained during the follicular or luteal phase of the estrous cycle on ram sperm kinematics, capacitation status and plasma membrane (PM) integrity at various time points during the 24-h incubation period. Fresh ram spermatozoa were selected using the swim-up technique and then incubated separately with either follicular phase (FbOF) or luteal phase (LbOF) bovine oviductal fluid added to Fert-TALP medium (positive control - POSControl) or in Fert-TALP medium without capacitating agents (negative control - NEGControl) at 38 °C under 5% CO 2 . Incubation with FbOF or LbOF for 2 h and 4 h promoted an increase (P <  0.05) in most of the sperm motility parameters as compared with the NEGControl group, and bOF-induced changes in sperm kinematics were similar (P >  0.05) to those seen in the POSControl group. After 6 h of incubation, the stimulatory effect of FbOF or LbOF on ram sperm kinematics was no longer observed (P >  0.05). Sperm PM integrity was not affected (P >  0.05) by incubation in bOF-supplemented media or in absence of capacitating factors (NEGControl). Although neither FbOF nor LbOF had any effect on sperm capacitation rates, the proportion of acrosome-reacted spermatozoa was greater (P < 0.05) for bOF-containing media compared with the NEGControl group during the long incubation periods (18 h and 24 h). In conclusion, bOF from either follicular or luteal phase of the estrous cycle enhances ram sperm motility for up to 4 h and the rate of acrosome reaction after long (18-24 h) incubation periods without affecting sperm viability., (Copyright © 2019 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Adiponectin is an adipocyte derived cytokine implicated in energy homeostasis, insulin resistance and is involved in the regulation of reproduction both centrally and peripherally in animals. The present study was conducted to investigate adiponectin (ADIPOQ) and its receptors ADIPOR1 and ADIPOR2 abundance of mRNA transcript and protein in different stages of corpora lutea (CL) development during the estrous cycle of water buffalo and to determine the effect of adiponectin on cultured luteal cells of water buffalo (Bubalus bubalis). The results indicate adiponectin, ADIPOR1, and ADIPOR2 were present in buffalo corpora lutea (CL) throughout the estrous cycle. The abundance of adiponectin and its receptors was greater in the early and regressing and was less in mid- and late-stages of CL functionality. Adiponectin and its receptors were localized in the cytoplasm of small and large luteal cells. Furthermore, luteal cells were cultured in the in-vitro culture system and were treated with 1 and 10 μg/mL dose of adiponectin for 48 h. Adiponectin at both doses decreased (P < 0.05) progesterone (P 4 ) secretion from cultured luteal cells and also suppressed the abundance of factors involved in P 4 productionv [Steroidogenic Acute Regulatory Protein (STAR), cytochrome P45011A1 (CYP11A1) and 3β-hydroxysteroid dehydrogenase (HSD3B1) at the 10 μg/mL dose as compared to adiponectin non-supplemented cells]. In conclusion, results of the present study indicate adiponectin and its receptors are present in bubaline CL and adiponectin inhibits P 4 production in cultured luteal cells. The findings indicate adiponectin affects luteal dynamics and reproductive functions in water buffalo., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sperm capacitation, fertilization and embryo development take place in the oviduct during the periovulatory period of the estrous cycle. Phospholipids are crucial metabolites for sperm capacitation and early embryo development. The aim of this study was to monitor the abundance of phospholipids in the bovine oviductal fluid (OF) according to the stage of the estrous cycle and the side relative to ovulation. Pairs of bovine oviducts were collected in a slaughterhouse and classified into four stages of the estrous cycle: post-ovulatory (Post-ov), mid-luteal (Mid-lut), late-luteal (Late-lut) and pre-ovulatory (Pre-ov) phases (n = 17 cows/stage). Cell-free OF from oviducts ipsilateral and contralateral to the site of ovulation were analyzed using MALDI-TOF mass spectrometry. Lipid identification was achieved by high resolution mass spectrometry. A total of 274 lipid masses were detected in the mass range of 400-1000 Da, corresponding mostly to phosphatidylcholines (PC), lysoPC, phosphatidylethanolamine (PE), lysoPE and sphingomyelins (SM). Ipsilateral and contralateral OF did not differ in their lipid profiles at any stage of the cycle. However, 127 and 96 masses were differentially abundant between stages in ipsilateral and contralateral OF, respectively. Highest differences in lipid profiles were observed in the Pre-ov vs. Mid-lut and Pre-ov vs. Late-lut comparisons in both sides relative to ovulation. Differential abundance of specific molecules of PC, PE, SM and l-carnitine were observed at Pre-ov and Post-ov compared with the luteal phase. This work proposes new candidates potentially able to regulate sperm capacitation and early embryo development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Stress is a precipitating factor in depression and anxiety disorders. Patients with these disorders often show amygdala abnormalities. The basolateral amygdala (BLA) is integral in mood and emotion, and is sensitive to stress. While much is known about effects of stress on BLA neuron activity and morphology in males, less is known in females. We tested whether repeated stress exerts distinct effects on BLA in vivo neuronal activity and morphology of Golgi-stained BLA neurons [lateral (LAT) and basal (BA) nuclei] in adult female rats. Repeated restraint stress increased BLA neuronal firing and caused hypertrophy of BLA neurons in males, while it decreased LAT and BA neuronal firing and caused hypotrophy of neurons in the LAT of females. BLA neuronal activity and function, such as fear conditioning, shifts across the estrous cycle. Repeated stress disrupted this pattern of BLA activity and fear expression over the estrous cycle. The disruptive effects of stress on the pattern of BLA function across estrous may produce behavior that is non-optimal for a specific phase of the estrous cycle. The contrasting effects of stress may contribute to sex differences in the effects of stress on mood and psychiatric disorders.

The first follicular wave emerges soon after ovulation, and its dominant follicle (DF) develops during the first 8-10 days of the estrous cycle in cattle. And, the first-wave DF is a non-ovulatory follicle, because it develops during the first half of the estrous cycle simultaneously with the corpus luteum (CL), which produces and secretes progesterone. Regarding the characteristics of development and the mechanisms of deviation in the DF during the follicular wave, the first-wave DF has been well studied. However, the characteristics of the first-wave DF, such as growth, blood flow in the follicular wall, concentration of sex steroid hormones in the peripheral blood and follicular fluid, amounts of mRNA in granulosa cells, as well as the characteristics of the CL formed after the first-wave DF and the influence of the first-wave DF on fertility (conception rate), have not been well studied. Additionally, the first-wave DF synthesizes and secretes 17β-estradiol (E 2 ), and plasma E 2 concentration increases during the early stage of the estrous cycle. Consequently, there is a possibility that the first-wave DF might affect the fertility in cattle. In this review, to provide the new perspective on reproductive physiology in cattle, characteristics of the first-wave DF were examined in detail and its characteristics were compared with that of the second-wave DF. In addition, the locational effects of the first-wave DF and CL on conception rate are discussed.