Day Of Diestrus Research Articles

Are there sex differences in oxytocin and vasopressin V1a receptors ligand binding affinities?

There is substantial evidence for sex differences in the functioning of one of the most common receptor systems; G protein-coupled receptors (GPCRs). There are many points along the GPCR-mediated molecular signaling pathway at which males and females may differ, one of the first of which, chronologically, is in the stability of the interaction between the ligand and the receptor, or its binding affinity. Here we investigate the binding affinities of oxytocin (OT) and vasopressin (AVP) at the oxytocin receptor (OTR) and the vasopressin V1a receptor (V1aR), both of which are present in numerous in brain regions associated with social behavior. In order to investigate sex- and estrous cycle-dependent differences in ligand-receptor binding affinity, male (n = 6) Syrian hamsters (Mesocricetus auratus), females on the day of estrus (E females, n = 6), and females on the second day of diestrus (D2 females n = 6) were chosen for study. Brains from hamsters were mounted on slides and competition and saturation binding assays were conducted. We report a remarkable similarity in the binding affinities of OT and AVP in males and females. Small differences were detected, however, in receptor and ligand specificity in females depending on whether they were in the estrous or diestrous stage of their ovulatory cycle. These data suggest that sex differences in binding affinity are not a likely source of the many sex differences that have been observed in the effects of OT and AVP in hamsters and other species.

FRI279 Submaximal Loss Of KNDy Neurons Accelerates And Amplifies Pulsatile LH Secretion In Female Rats

Abstract Disclosure: A.C. Campideli-Santana: None. K.S. da Costa Silva: None. R. Araújo-Lopes: None. L.M. Antunes: None. R.E. Szawka: None. Kisspeptin is a powerful stimulator neuropeptide in the hypothalamus-pituitary-gonadal axis and is essential for fertility. Neurons in the arcuate nucleus (ARC) of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (KNDy) play a crucial role in the control of luteinizing hormone (LH) secretion, but the neuroendocrine mechanisms involved in this regulation are not fully understood. Our previous study demonstrated that the moderate loss of KNDy neurons amplifies the LH surge induced by estradiol in ovariectomized rats (1). The current study aimed to perform a longitudinal evaluation of female rats in which KNDy neurons were ablated to determine the integrated role of this neuronal population in the control of pulsatile LH secretion. Adult female rats underwent a neurochemical ablation of KNDy neurons via intra-ARC stereotaxic injections of neurokinin-3 receptor agonist conjugated with saporin (NK3-SAP), while control animals received blank-saporin (Control). Estrous cyclicity was monitored over 21 days before and after the stereotaxic surgery. Three weeks after surgery, blood samples (10 µL) were withdrawn from the tail tip at 6-minute intervals for 180 minutes on the day of diestrus. Rats were then ovariectomized and the ovaries were processed for histological analysis. Whole blood LH levels were measured by ELISA and brains were immunohistochemically labeled for neurokinin B in the ARC. NK3-SAP rats bearing a submaximal loss of KNDy neurons (between 55-95%; n = 8) displayed irregular estrous cycles. The ovarian weight and the number of corpora lutea and atretic follicles were unaltered, whereas the number of healthy early antral follicles decreased in NK3-SAP rats. The frequency of LH pulses was increased and both pulse amplitude and mean LH levels were higher in NK3-SAP compared with Control rats. Thus, the submaximal loss of KNDy neurons affects the regularity of estrous cyclicity but does not preclude ovulation, while seems to stimulate the maturation of early antral follicles. Notably, KNDy neuron loss accelerates and amplifies LH pulsatile secretion during the rat estrous cycle. These results suggest a new role for KNDy neurons in restraining the frequency and amplitude of LH pulses in ovary-intact animals, with possible implications in the rate of recruitment of early antral follicles. Reference: Campideli-Santana et al., J Neuroendocrinol. 2022 Nov;34(11):e13204. Support: FAPEMIG, CNPq, CAPES, and Pro-reitoria de Pesquisa-UFMG. Presentation: Friday, June 16, 2023

Ultrasonographic and Doppler characteristics of corpus luteum luteogenesis in Remonta Argentino jennies

The corpus luteum (CL) plays a crucial role in maintaining pregnancy and preparing the uterus for implantation of the fertilized egg. Transrectal Doppler ultrasonography of the ovary can be used as a diagnostic tool to assess the reproductive status and functionality of the CL. Our aim was to characterize size, echogenicity and degree of vascularization of the corpus luteum over five days after ovulation in Remonta Argentino jennies. Five healthy cycling jennies aged 9 to 14 years were monitored transrectally using the MyLabTMOne ultrasound (ESAOTE, InMed, Argentina) with a 5-10 MHz linear array transducer. After a preovulatory follicle was detected, jennies were monitored daily for ovulation (day 0) and ultrasonographic evaluation of the CL until day 5. Two ovulations and the respective CL formation were analyzed in each jenny (n=10 CL). Size and echogenicity were evaluated using B mode and CL vascularization using the Doppler-color function, with a pulse repetition frequency of 1.2 MHz. All ultrasound examinations were video recorded for analysis. Adobe Premiere Pro CS6® was used to collect three images from each video where the largest area of the CL and the highest Doppler signal were detected. Image J. version 1.52i software (National Institute of Health, USA) was used to analyze images and to determinethe area, number of gray pixels (echogenicity) and color area within the CL in Doppler mode. The average value from the three images was used to determine CL size (area in cm2), echogenicity (average gray pixel intensity) and the proportion of vascularized area (AV; area with blood flow/total CL area x 100). Spearman's test was used to correlate the variables and the changes recorded between days 1 and 5 of diestrus. The results obtained on each day were the following (values expressed as mean±SEM): Day 0: area 3.26±0.5; echogenicity 84.2±4.86; AV 5.54±1.75; Day 1: area 4.86±0.32; echogenicity 91.25±7.42; AV 1.84±0.26; Day 2: area 4.83±0.41; echogenicity 88.72±7.89; AV 3.51±0.81; Day 3: area 4.64±0.43; echogenicity 89.69±4.9; AV 4.54±0.67; Day 4: area 4.38±0.48; echogenicity 97.78±7.61; AV 5.8±1.06; Day 5: area 4.24±0.49; echogenicity 89.18±9.25; AV 8.66±1.15. No statistical correlation was observed either between variables or with the day of diestrus (p>0.05). Blood samples were collected daily between day 0 and day 5 of diestrus and progesterone determinations are ongoing. Our results contribute to a better understanding of CL formation in Remonta Argentino donkeys that could potentially be used to determine CL age and function.

Kisspeptin and Sex Steroid Hormone Receptors Are Upregulated at the Maternal‐Fetal Interface of the Preeclamptic‐like BPH/5 Mouse

The main features of preeclampsia (PE), a devastating syndrome that affects 5‐8% of pregnancies worldwide, are spontaneously recapitulated by the Blood Pressure High Subline 5 (BPH/5) mouse model. Comparable to women with early‐onset PE, BPH/5 females develop poor placentation with decreased trophoblast cell invasion, shallower placental discs and reduced placental endothelial remodelling than control C57 mice. Kisspeptins (KP), encoded by Kiss1, have been implicated in the etiology of PE: KP upregulation at the human maternal‐fetal interface is suggested to inhibit trophoblast cell invasion and angiogenesis. However, uterine KP expression pre‐pregnancy in women that later develop PE and the upstream signaling mechanisms leading to uterine/placental KP upregulation remain vastly unexplored. Interestingly, administration of estradiol‐17ß (E2) and/or progesterone (P4) to non‐pregnant ovariectomized CD‐1 mice resulted in uterine Kiss1upregulation. We have previously shown that BPH/5 females present abnormal circulating E2 and P4 throughout the estrous cycle and early pregnancy. Furthermore, KP and KP receptor (Kiss1r) are upregulated at the BPH/5 maternal‐fetal interface during the peak of decidualization, at embryonic day (e) 7.5. Therefore, the goal of this study was to investigate mRNA expression of Kiss1/Kiss1r,and the receptors for Progesterone (PR‐a/b/c)and Estrogen (ER‐αand GPER‐1)in BPH/5 non‐pregnant uterus during the first day of diestrus (NP‐D) and embryonic implantation sites (eIS) at e4.5. We hypothesized that the sex steroid hormone receptors would be upregulated in the uterus/eIS of BPH/5 during NP‐D and e4.5, respectively, potentially contributing with Kiss1/Kiss1rupregulation. Daily vaginal cytology samples were collected from adult BPH/5 and C57 females to determine the stage of the estrous cycle, and uterine samples were collected on the first day of cytologic diestrus (NP‐D, n = 3‐6/strain). Moreover, adult BPH/5 and C57 females were bred, and eIS were collected at e4.5 (n = 7‐9/strain). Gene expression was investigated using quantitative RT‐PCR and comparisons were performed using unpaired T‐tests. Welch’s corrections were performed for unequal variances and the significance level was set at α ≤ 0.05. Kiss1expression was higher in BPH/5 NP‐D uterus (p = 0.04) and e4.5 eIS (p = 0.01) when compared with C57. Contrarily, Kiss1rwas not different between BPH and C57 in NP‐D or e4.5 (p > 0.05). Interestingly, PR‐a/b/cand ER‐αwere also higher in the uterus/eIS of BPH/5 females both during NP‐D (p = 0.05 and 0.02, respectively) and e4.5 (p = 0.003 and 0.02, respectively). The trans‐membrane G‐protein‐coupled estrogen receptor 1 (GPER‐1), largely associated with rapid, non‐genomic cellular responses to estrogens, was also higher in BPH/5 eIS at e4.5 when compared with controls C57 (p < 0.001), but not different between groups in NP‐D (p > 0.05). In conclusion, uterine Kiss1upregulation precedes pregnancy in the BPH/5 mouse model. Furthermore, PR‐a/b/cand ER‐α expression in the NP‐D uterus and early maternal fetal interface parallels the expression levels of Kiss1in the PE‐like BPH/5 mouse.

Participation of the Cholinergic System in the Development of Polycystic Ovary Syndrome.

In rats with polycystic ovary syndrome (PCOS) induced by injection of estradiol valerate (EV), unilateral or bilateral section of the vagus nerve restores ovulatory function in 75% of animals, suggesting that the vagus nerve participates in the development of PCOS. Since the vagus nerve is a mixed nerve through which mainly cholinergic-type information passes, the objective of the present study was to analyze whether acetylcholine (ACh) is involved in the development of PCOS. Ten-day-old rats were injected with 2.0 mg EV, and at 60 days of age, they were microinjected on the day of diestrus in the bursa of the left or right ovary with 100 or 700 mg/kg of ovarian weight atropine, a blocker of muscarinic receptors, and sacrificed for histopathological examination after the surgery. Animals with PCOS microinjected with 100 mg of atropine showed a lack of ovulation, lower serum concentrations of progesterone and testosterone, and cysts. Histology of the ovaries of animals microinjected with 700 mg of atropine showed corpus luteum and follicles at different stages of development, which was accompanied by a lower concentration of progesterone and testosterone. These results allow us to suggest that in animals with PCOS, ACh, which passes through parasympathetic innervation, is an important component in the persistence and development of the pathophysiology.

Aglepristone Administration in Mid-Proestrus Reduces the LH Peak but Does Not Prevent Ovulation in the Bitch.

Simple SummaryThe role of preovulatory progesterone for LH release and ovulation in the bitch is not clear. The aim of the study was to evaluate the influence of administration of aglepristone in mid-proestrus on progesterone concentration, LH release, and occurrence of ovulation in the bitch. Experimental bitches (n = 7) were treated on days 4 and 5 of proestrus with aglepristone (Alizin®, Virbac) at the dose of 10 mg/kg body weight s.c. (i.e., the two treatments were 24 h apart). The progesterone concentration showed a similar pattern in both groups. The LH peak value and area under the curve for LH in bitches treated with aglepristone were significantly lower than those in control bitches. The ovulation occurred in all animals in both groups. The presented study showed that withdrawal of progesterone by administration of aglepristone in the mid-proestrus significantly reduced the preovulatory LH surge, but it had no effect on periovulatory progesterone concentration or the occurrence of ovulation.The aim of the study was to evaluate the influence of administration of aglepristone in mid-proestrus on progesterone concentration, LH release, and occurrence of ovulation in the bitch. Experimental bitches (n = 7) were treated on days 4 and 5 of proestrus with aglepristone at the dose of 10 mg/kg body weight s.c. (i.e., the two treatments were 24 h apart). Control animals (n = 7) received s.c. injections of saline. For progesterone determination, blood was collected daily until the first day of cytological diestrus. For LH determination, blood was collected daily and in the periovulatory phase every 8 h. The progesterone concentration showed a similar pattern in both groups. The LH peak value in bitches treated with aglepristone was significantly lower (p < 0.05) than that in control bitches (4.83 ± 1.20 vs. 13.66 ± 1.21 ng/mL). The area under the curve (AUC) for LH was significantly (p < 0.05) lower in treated than in control animals (6.85 ± 1.21 ng/mL/d vs. 12.25 ± 1.35 ng/mL/d). The ovulation occurred in all animals in both groups. The study showed that administration of aglepristone in the mid-proestrus significantly reduced the preovulatory LH surge, but it had no effect on progesterone concentration and the occurrence of ovulation.

Serum progesterone concentration, volume, and apoptosis of corpora lutea in early, middle and late diestrus in the bitch

The aim was to study number, volume, apoptosis of corpora lutea (CL), and serum P4 concentrations in early, middle, and late diestrus of dogs. Thirty-six bitches were ovari-hysterectomized (OVX): Early Diestrus (Group [G]1; OVX 20 days after end of estrus [DEH]); Mid-diestrus (GII; OVX between 21 and 40 days after DEH), and Late-diestrus (GIII; OVX between 41 and 60 days after DEH). Before OVX a blood sample was collected to quantify P4. After OVX, the number of CL (NCL) was recorded, CL measured using both ultrasonography (US) and caliper (CAL), and the volume (mm3) was calculated. Based on abundances of caspase-3, apoptotic luteal cells were detected. Bitches in early-diestrus had greater P4 concentrations than bitches in mid- and late-diestrus (23.52 ± 3.78 and 10.86 ± 3.88 ng/mL; P < 0.05). The NCL, cumulative USCLV, and CALCLV were similar among diestrus stages (P > 0.30). Bitches with CL (≥5) had twice the serum P4 concentrations as bitches with CL1−2 and CL3−4 (22.71 ± 3.70 and 10.97 ± 4.06 ng/mL; P < 0.05). There were correlations between P4 concentrations with USCLV, CALCLV, and NCL (r = 0.64, r = 0.67, r = 0.44; P < 0.0001). When serum P4 concentrations were adjusted for stages of diestrus, however, there were only correlations during early diestrus. The percentage of apoptotic cells was greater in GIII compared with GI and GII (13.75 ± 2.26 % compared with 4.5 ± 0.68 % and 4.6 ± 1.5 %, respectively; P < 0.05). As days of diestrus increased, number of apoptotic cells increased, and serum P4 concentrations decreased.

156 Oestrus response, corpus luteum function, and pregnancy rates following aromatase inhibitor treatment in beef heifers

In a previous study involving the use of the aromatase inhibitor letrozole to synchronise ovulation, incomplete luteolysis appeared to confound the synchronising effect in letrozole-treated heifers. Experiments were done to determine whether letrozole treatment interferes with luteolysis and affects the timing of oestrus (Experiment 1) and whether pregnancy rate is affected by the stage of the oestrous cycle during which letrozole treatment is initiated (Experiment 2). In Experiment 1, Hereford heifers were fitted with a HeatWatch sensor and given prostaglandin F2α (PGF2α). Ovulation (Day 0) was determined by daily ultrasonography, and on Day 3 heifers were given an intravaginal letrozole-releasing device or a sham device for 4 days (n=16 per group). Half of the heifers in each group were given PGF2α on Day 7 (PGF 1×) or on Day 7 and 7.5 (PGF 2×). Ultrasonography and blood sampling were done daily from device insertion to removal, every 12h for 72h following PGF2α and daily for 14 days following ovulation. In Experiment 2, heifers were randomly assigned to three groups (n=10-11 per group) in which a 4-day letrozole-releasing device treatment was initiated during metoestrus (Days 0-2), diestrus (Days 7-9), or pro-oestrus (Days 15-17). Prostaglandin F2α was given at device removal and again 12h later. Heifers were inseminated with frozen-thawed semen and given gonadotrophin-releasing hormone 66h after device removal. Ultrasonography and blood sampling were done twice daily from device removal until ovulation. End points examined included dominant follicle and corpus luteum (CL) diameters, onset of oestrus, and plasma concentrations of oestradiol determined by radioimmunoassay. Nominal data were compared using GENMOD, analysis of variance, or mixed model for repeated-measures and were expressed as means±s.e.m. Proportional data were analysed using chi-square. In Experiment 1, no main effects or interactions were detected between device or PGF treatment for onset of oestrus following PGF2α (overall 60.0±2.5h) or CL diameter profiles during luteolysis. Residual variation was reduced in the timing of the first PGF to ovulation in the PGF 2× group versus the PGF 1× group (5.6±2.2h vs. 15.2±3.2 h; P&amp;lt;0.01). The CL diameter following ovulation was greater in the PGF 2× group compared with the PGF 1× group (P=0.03). Circulating plasma oestradiol during treatment was greater (P=0.04) in both the PGF 1× and sham groups. In Experiment 2, a greater proportion of heifers in the pro-oestrus group ovulated ≤24h after device withdrawal compared with the metoestrus and diestrus groups combined (6/11 vs. 0/22, respectively; P&amp;lt;0.05). Pregnancy rate tended to be greater in the metoestrus group than in the diestrus group (10/11 vs. 6/10; P=0.08), and pregnancy rate in both groups was greater than that in the pro-oestrus group (0/11; P&amp;lt;0.05). In conclusion, letrozole treatment did not interfere with luteolysis, and PGF2α given 2× at a 12-h interval on Day 7 after ovulation decreased variance in the timing to ovulation. Treatment initiated during metoestrus and diestrus yielded greater pregnancy rates than that started during pro-oestrus and warrants efficacy testing in a large fixed-time AI field trial. This research was supported by Alberta Agriculture and Forestry and by Dechra.

Curtailing parturition observation and performing preparturient cesarean section in bitches

Properly planned elective cesarean section (CS) in bitches is considered safe and justified for some breeds. Therefore, planning a scheduled (fixed date and time) preparturient CS in bitches belonging to a subpopulation where a CS is unavoidable, seems justified. The first day of cytological diestrus (D0) was used to predict the date of parturition. The aims of this study were to (1) compare the mortality of puppies delivered by preparturient CS to those delivered by parturient CS, (2) determine the extent to which a preparturient CS (performed at 08:00 on D57, while the cervix is closed) would shorten gestation, (3) compare the level of progesterone at the time of preparturient CS to that at the time of parturient CS, (4) compare the change in hematocrit before and after CS for parturient- and preparturient CSs. Out of 99 gestations for which D0 was known and a preparturient CS planned, the CS was performed at the scheduled time in 61%, before the scheduled time because cervical dilatation had started in 32% and before the scheduled time in 7% because the bitches had started showing signs of impending parturition, although their cervices were still closed. This study showed high neonatal survival ratios and good Apgar scores after preparturient CS with 99% of puppies delivered by preparturient CS born alive and 98.8% surviving to 2 h. Performing preparturient CSs shortened gestation by four to 52 h. Progesterone level was significantly higher at the time of preparturient CSs than at the time of parturient CSs. Further studies are required to determine whether preparturient CSs on D57 are routinely safe in all breeds, does not affect long term survival of the puppies and to timeously identify bitches in which parturition starts prior to the date and time for the scheduled elective preparturient CS.

Conditioned disgust in rats (anticipatory nausea) to a context paired with the effects of the toxin LiCl: Influence of sex and the estrous cycle

The role of sex and estrous phase in the conditioning of toxin-induced disgust reactions (anticipatory nausea) to a novel context were examined in adult rats. Conditioned oral gaping responses have been shown to be a reliable index of nausea in rats. In Experiment 1 male and female rats were injected with LiCl (0, 64, 96, or 128 mg/kg) on each of 4 conditioning trials (72 h apart) and then placed in a novel context for 30 min. 72 h following the last conditioning trial each animal was re-exposed to this context in a toxin-free state for 10 min and disgust responses (gapes, forelimb flails, chin rubs, and paw treads) were scored from video records. A significant toxin dose-related monotonic increase in conditioned gaping showed a significantly greater increase in females, relative to males. In Experiment 2 female rats were conditioned, using the same paradigm, on either diestrus or proestrus days (trials 96 h apart) with LiCl (96 mg/kg) or saline control injections. Disgust responses were recorded on each of the 4 conditioning trials and a toxin-free test trial. Significant increases in conditioned disgust were obtained on proestrus relative to diestrus days on the toxin-free test trial. However no significant estrous cycle differences in disgust responding were obtained on the acquisition trials. The sex difference in conditioned gaping and the increased conditioned disgust responses in proestrus suggest that increased levels of estradiol likely enhance the strength of the association of the toxin-induced nausea with the novel context in rats.

Escherichia coli virulence influences the roles of sex hormone receptors in female dogs with simulated pyometra.

The present study aimed to investigate whether Escherichia coli virulence affects the roles of sex hormone receptors in female dogs with simulated pyometra. A total of 33 healthy, nulliparous, crossbred female dogs were divided into four groups, with 10 dogs in each of the three experimental groups and 3 dogs in the control group. Estradiol was administrated to female dogs in group 1 continuously at 0.6–4.8 mg/kg twice daily for 12 days (the dose doubled every three days), followed by intramuscular injection of 0.2–1.8 mg/kg progesterone. The progesterone was administrated with an initial dose of 0.2 µg/kg and increased 0.2 mg/kg every three days, twice daily until the maximum of 1.8 mg/kg for 24 days and maintained at 1.8 mg/kg for 19 days. Progesterone only was administrated at 1.8 mg/kg in group 2 (twice daily) for 55 continuous days and only estradiol was administered with an initial dose of 0.6 µg/kg (dose doubled every 3 days for 12 days) in group 3 twice daily and maintained at 4.8 mg/kg for the following 43 days. A strongly virulent E. coli strain, nau-b, and a weakly virulent strain, nau-i, were screened. On the 12th day of diestrus, 5 female dogs in each of the experimental groups were inoculated with E. coli nau-i strain, while the other five in each group were inoculated with nau-b strain. Histopathological changes of uterine tissues were microscopically observed 50 days after E. coli inoculation and hormone receptor expression levels were detected by quantitative polymerase chain reaction. Simulated pyometra was observed in dogs administrated with progesterone alone or progesterone combined with estradiol. The clinical symptoms and histopathological observation demonstrated that inoculation with strongly virulent E. coli strain, nau-b, caused earlier onset of pyometra symptoms and more severe pyometra symptoms compared with the weakly virulent E. coli strain, nau-i. Furthermore, estrogen and progesterone receptor levels in dogs with pyometra inoculated with E. coli strain nau-i was higher than those in dogs with pyometra inoculated with E. coli strain nau-b. These results indicated that E. coli affects the roles of sex hormone receptors in female dogs with simulated pyometra.

Prepubertal exposure to T-2 toxin advances pubertal onset and development in female rats via promoting the onset of hypothalamic-pituitary-gonadal axis function.

T-2 toxin, a naturally produced Type A trichothecene mycotoxin, has been shown to damage the reproductive and developmental functions in livestocks. However, whether T-2 toxin can disturb the pubertal onset and development following prepubertal exposure remains unclear. To clarify this point, infantile female Sprague-Dawley rats were given a daily intragastric administration of vehicle or T-2 toxin at a dose of 375 μg/kg body weight for 5 consecutive days from postnatal day (PND) 15-19 (PND15-PND19). The days of vaginal opening, first diestrus, and first estrus in regular estrous cycle were advanced following T-2 toxin treatment, indicating prepubertal exposure to T-2 toxin induced the advancement of puberty onset. The relative weights of uterus and ovaries and the incidence of corpora lutea were all increased in T-2 toxin-treated rats; serum hormone levels of luteinizing hormone and estradiol and the messenger RNA expressions of gonadotropin-releasing hormone (GnRH) and GnRH receptor also displayed marked increases following exposure to T-2 toxin, all of which were well consistent with the manifestations of the advanced puberty onset. In conclusion, the present study reveals that prepubertal exposure to a high level of T-2 toxin promotes puberty onset in infantile female rats by advancing the initiation of hypothalamic-pituitary-gonadal axis function in female rats.

155 COMPARISON OF CIRCULATING microRNAs BETWEEN PREGNANT AND NONPREGNANT MARES

MicroRNAs (miRNAs) are small, non-coding RNAs that are differentially expressed throughout the body, including the endometrium and chorion during gestation. Although they are produced by individual tissues, many of these miRNAs are excreted into circulation, either within exosomes or attached to protein carrier molecules. Circulating miRNAs are surprisingly stable, with an average half-life of 5 days. These characteristics suggest that circulating miRNAs have the potential to be powerful biomarkers following the characterisation of the normal population of miRNAs during pregnancy. Additionally, evaluation techniques must be considered, as some miRNAs are extremely sensitive to varying collection and analysis techniques. To evaluate the population of miRNAs in both pregnant and nonpregnant mares, whole blood was collected from mares at 10 months of gestation (n = 3), as well as at 9 days of diestrus (n = 3 for microarray; n = 4 for qPCR). Blood was collected using PAXgene blood miRNA kits (Qiagen, Valencia, CA, USA), and then stored at –20°C until use. RNA extraction was performed following the manufacturer’s instructions. Two different analysis platforms were compared. Quantitative RT-PCR (Lightcycler 480; Roche, Indianapolis, IN, USA) was used to evaluate 346 distinct miRNAs, whereas microarray (LC Sciences, Houston, TX, USA) was used to evaluate 275 miRNAs. In total, 222 of these miRNAs were analysed on both platforms for comparison purposes. Because of the large number of miRNAs evaluated, the robust response screening analysis method (JMP ver. 11.1; SAS Institute Inc., Cary, NC, USA) was used, and multiple comparisons were based upon a false discovery rate of P &lt; 0.1. Microarray counts were normalized by log10, whereas qPCR cycle threshold (Ct) counts were normalized by delta Ct (Ct – overall average Ct). Correlation was evaluated by the restricted maximum likelihood method (REML). Between pregnant and nonpregnant animals, qPCR analysis found 93 transcripts to be differentially expressed, whereas microarray analysis identified 15 transcripts. Of the transcripts evaluated on both platforms, 46% of transcripts identified as significant by microarray were also identified by qPCR analysis. The samples analysed by qPCR were highly correlated within themselves (r = 0.9655), as were those analysed by microarray (r = 0.8568). The microarray and qPCR platforms showed some degree of correlation between the platforms (r = –0.6980); this correlation was negative because of the different measurement methods between microarray and qPCR. These results indicate there is a correlation between the qPCR and microarray platforms; however, it may be unrealistic to expect consistently complementary results between the platforms. Overall, qRT-PCR provided more consistent results and appears to be a more powerful analysis tool. Additionally, these data suggest that there is a distinct population of circulating miRNAs of mares at 10 months gestation compared with nonpregnant individuals. These differences should be further explored to characterise the normal miRNA populations of healthy mares throughout gestation, as well as during various disease states.

Evidence for a Putative Circadian Kiss-Clock in the Hypothalamic AVPV in Female Mice.

The kisspeptin (Kp) neurons in the anteroventral periventricular nucleus (AVPV) are essential for the preovulatory LH surge, which is gated by circulating estradiol (E2) and the time of day. We investigated whether AVPV Kp neurons in intact female mice may be the site in which both E2 and daily signals are integrated and whether these neurons may host a circadian oscillator involved in the timed LH surge. In the afternoon of proestrous day, Kp immunoreactivity displayed a marked and transient decrease 2 hours before the LH surge. In contrast, Kp content was stable throughout the day of diestrus, when LH levels are constantly low. AVPV Kp neurons expressed the clock protein period 1 (PER1) with a daily rhythm that is phase delayed compared with the PER1 rhythm measured in the main clock of the suprachiasmatic nuclei (SCN). PER1 rhythm in the AVPV, but not in the SCN, exhibited a significant phase delay of 2.8 hours in diestrus as compared with proestrus. Isolated Kp-expressing AVPV explants from PER2::LUCIFERASE mice displayed sustained circadian oscillations of bioluminescence with a circadian period (23.2 h) significantly shorter than that of SCN explants (24.5 h). Furthermore, in AVPV explants incubated with E2 (10 nM to 1 μM), the circadian period was lengthened by 1 hour, whereas the SCN clock remained unaltered. In conclusion, these findings indicate that AVPV Kp neurons display an E2-dependent daily rhythm, which may possibly be driven by an intrinsic circadian clock acting in combination with the SCN timing signal.

Kisspeptin antagonist prevents RF9-induced reproductive changes in female rats.

The aim of this study was to determine the modulatory effects of peptide 234 (p234) (an antagonist of GPR54 receptors) on kisspeptin and RF9 (an RFamide-related peptide antagonist)-induced changes in reproductive functions and energy balance in female rats. Female Sprague-Dawley rats were weaned on postnatal day (pnd) 21. The animals were intracerebroventricularly cannulated under general anesthesia on pnd 23. Groups of female rats were injected with kisspeptin, RF9, p234, kisspeptin plus p234, or RF9 plus p234, daily. The experiments were ended on the day of first diestrus following pnd 60. Kisspeptin or RF9 alone advanced vaginal opening (VO), which was delayed by administration of kisspeptin antagonist alone. In the rats given kisspeptin plus p234 or RF9 plus p234, VO was not different from control rats. Kisspeptin and RF9 elicited significant elevations in circulating LH levels. Coadministrations of kisspeptin or RF9 with p234 decreased LH levels significantly. The use of p234 alone did not cause any significant change in LH secretion. Kisspeptin decreased both food intake and body weight while RF9 decreased only food intake without affecting body weight. The effects of kisspeptin on energy balance were also reversed by central administration of p234. In conclusion, kisspeptin antagonist, p234, modulates the effects of kisspeptin on reproductive functions and energy balance, whereas RF9 seems to exert only its effects on reproductive functions by means of GPR54 signaling in female rats.

C/EBPβ Isoforms Expression in the Rat Brain during the Estrous Cycle.

The CCAAT/enhancer-binding protein beta (C/EBPβ) is a transcription factor expressed in different areas of the brain that regulates the expression of several genes involved in cell differentiation and proliferation. This protein has three isoforms (LAP1, LAP2, and LIP) with different transcription activation potential. The role of female sex hormones in the expression pattern of C/EBPβ isoforms in the rat brain has not yet been described. In this study we demonstrate by western blot that the expression of the three C/EBPβ isoforms changes in different brain areas during the estrous cycle. In the cerebellum, LAP2 content diminished on diestrus and proestrus and LIP content diminished on proestrus and estrus days. In the prefrontal cortex, LIP content was higher on proestrus and estrus days. In the hippocampus, LAP isoforms presented a switch on diestrus day, since LAP1 content was the highest while that of LAP2 was the lowest. The LAP2 isoform was the most abundant one in all the three brain areas. The LAP/LIP ratio changed throughout the cycle and was tissue specific. These results suggest that C/EBPβ isoforms expression changes in a tissue-specific manner in the rat brain due to the changes in sex steroid hormone levels presented during the estrous cycle.

The effects of sensorial denervation on the ovarian function, by the local administration of capsaicin, depend on the day of the oestrous cycle when the treatment was performed.

There is evidence that sensory innervation plays a role in the regulation of puberty. The present study investigates the effects of functional sensorial desensitisation induced by capsaicin administration to adult female rats in the days of diestrus 1, diestrus 2, pro-oestrus or oestrus on ovulation and serum oestradiol and progesterone concentration. The animals were allotted at random to one of the following groups: (1) animals with capsaicin administration into the bursa ovarica (local administration) (2) animals with vehicle administration into the bursa ovarica and (3) untreated animals group. The animals treated were killed on the day of oestrus after three consecutive 4-day oestrous cycles. No differences were observed in oestrous cyclicity or the average number of ova shed between the sensorial desensitisation animals and the vehicle-treated groups. Capsaicin administration resulted in a significant increase in the intra-ovarian noradrenaline levels in the day of diestrus 2 and pro-oestrus. Serum oestradiol and progesterone concentrations were different, depending on the day of the oestrous cycle in which the treatment was performed. These results suggest that in adult normal female rats, ovarian sensorial innervations participate together with the sympathetic innervation in the ovarian function regulating the hormone secretion and this participation varies along the oestrous cycle.

Shortened Estrous Cycle Length, Increased FSH Levels, FSH Variance, Oocyte Spindle Aberrations, and Early Declining Fertility in Aging Senescence-Accelerated Mouse Prone-8 (SAMP8) Mice: Concomitant Characteristics of Human Midlife Female Reproductive Aging

Women experience a series of specific transitions in their reproductive function with age. Shortening of the menstrual cycle begins in the mid to late 30s and is regarded as the first sign of reproductive aging. Other early changes include elevation and increased variance of serum FSH levels, increased incidences of oocyte spindle aberrations and aneuploidy, and declining fertility. The goal of this study was to investigate whether the mouse strain senescence-accelerated mouse-prone-8 (SAMP8) is a suitable model for the study of these midlife reproductive aging characteristics. Midlife SAMP8 mice aged 6.5-7.85 months (midlife SAMP8) exhibited shortened estrous cycles compared with SAMP8 mice aged 2-3 months (young SAMP8, P = .0040). Midlife SAMP8 mice had high FSH levels compared with young SAMP8 mice, and mice with a single day of high FSH exhibited statistically elevated FSH throughout the cycle, ranging from 1.8- to 3.6-fold elevation on the days of proestrus, estrus, metestrus, and diestrus (P < .05). Midlife SAMP8 mice displayed more variance in FSH than young SAMP8 mice (P = .01). Midlife SAMP8 ovulated fewer oocytes (P = .0155). SAMP8 oocytes stained with fluorescently labeled antitubulin antibodies and scored in fluorescence microscopy exhibited increased incidence of meiotic spindle aberrations with age, from 2/126 (1.59%) in young SAMP8 to 38/139 (27.3%) in midlife SAMP8 (17.2-fold increase, P < .0001). Finally, SAMP8 exhibited declining fertility from 8.9 pups/litter in young SAMP8 to 3.5 pups/litter in midlife SAMP8 mice (P < .0001). The age at which these changes occur is younger than for most mouse strains, and their simultaneous occurrence within a single strain has not been described previously. We propose that SAMP8 mice are a model of midlife human female reproductive aging.

Dynamics of Vaginal pH in the Bitch during Proestrus and Estrus

The aim of this study was to investigate the dynamics of the pH of vaginal secretions during proestrus and estrus in bitches and its association with the optimal time for insemination and the values of steroid hormones. Sixteen bitches from different breeds and age were included in this investigation. Vaginal pH of each bitch was measured once daily, starting from the first day of vulval bleeding and continuing until the first day of cytological diestrus. In each bitch vaginal smears were taken to determine the onset of estrus and diestrus and blood samples were taken to determine serum progesterone (ng/ml) and estradiol-17β (pg/ml) for detection of the onset of ovulation and their influence on values and the dynamics of vaginal pH. Vaginal pH values in the bitches at the first day of proestrus were between 7.4 and 8.7. This parameter decreased with the progress of the proestrus and the entering into the estrus. Compared to the rest of the estrus, at the time of ovulation and on the next day, a consistent decrease of mean values (6.61±0.15 and 6.63±0.15 respectively) was observed (р<0,01). Individually at the time of ovulation vaginal pH ranged between 6.5 and 6.8. In the days following ovulation a gradual increase in mean values was detected. At the first day of diestrus vaginal pH values were measured significantly higher than those in estrus (р<0,001) and were between 7.4 and 8.3 (7.74±0.26). In our study we found that the vaginal pH values in the bitch are lowest at the time of ovulation, with individual variations and exceptions.

Changes in cyclic GMP level and phosphodiesterase activity during follicular development in the rat ovary.

Guanosine 3',5'-cyclic monophosphate (cGMP), as a second messenger, plays potential roles in ovarian functions. To elucidate the role of phosphodiesterase (PDE) in cGMP signaling during ovarian follicular development, the present study was conducted to investigate ovarian cGMP level and cGMP-PDE activity by radioimmunoassay (RIA) in postnatal rats, immature rats during gonadotropin-primed follicular development, ovulation and luteinization, adult rats during normal estrous cycle, and aged rats that spontaneously developed persistent estrus (PE). All four rat models were confirmed by histological examination of one ovary, and the other ovary was used for RIA. In postnatal rats, cGMP level was high at birth and decreased dramatically by Day 5, and then, it increased maximally at Day 10 and declined at Day 21. However, cGMP-PDE activity did not significantly change during Days 1 to 10, but increased significantly on Day 21. In immature female rats, cGMP level markedly decreased upon treatment with equine chorionic gonadotropin (eCG), while cGMP-PDE activity did not show any significant changes; however, ovarian cGMP level and cGMP-PDE activity increased after injection of an ovulatory dose of human chorionic gonadotropin (hCG) for induction of ovulation and luteinization. In adult rats during normal estrous cycle, cGMP level was high on proestrus and metestrus days, while cGMP-PDE activity was high on estrus day. In PE rats, ovarian cGMP level was similar to that in adult rats on estrus and diestrus days but lower than that on proestrus and metestrus days; ovarian cGMP-PDE activity was lower than that on estrus days but similar as the other estrous cycle days. In addition, there was a significant negative correlation between ovarian cGMP level and cGMP-PDE activity during normal estrous cycles in the adult rat (r = -0.7715, N = 16, P < 0.05), but not in the postnatal rat (r = -0.1055, N = 20, P > 0.05). Together, the results of our present study indicated that ovarian cGMP levels were not dependent on cGMP-PDE activity during early postnatal development, but highly dependent on cGMP-PDE activity in the adult rat. This implies that mechanisms of cGMP signaling involved in ovarian functions are stage-specific in the rat.