Irregular Estrus Research Articles

Phyllanthus muellerianus (Euphorbiaceae) Restores Ovarian Functions in Letrozole-Induced Polycystic Ovarian Syndrome in Rats.

Polycystic ovarian syndrome (PCOS) is one of the common causes or female infertility. Phyllanthus muellerianus (Euphorbiaceae) is a plant used to treat various ailments including frequent menstruation and anovulation. We investigated the effects of P. muellerianus extracts on estrus cyclicity, lipid profile, oxidative stress-related markers, sex hormones, and ovarian architecture in letrozole-induced PCOS in rats. After induction of PCOS using letrozole (1 mg/kg/day), normal (n=6), and PCOS (n=108; distributed into 18 groups of 6 animals/group) rats were treated orally for 7 or 14 days with distilled water (10 ml/kg/day), clomiphene citrate (2 mg/kg/day), metformin (500 mg/kg/day), and aqueous or methanolic extract of P. muellerianus (30, 60, and 120 mg/kg). Estrus cyclicity, body, and sexual organ (ovaries and uterus) weights, biochemical and histological parameters were measured. There were letrozole-induced PCOS characterized by irregular estrus cyclicity, elevated (p<0.05-0.01) glycaemia, ovarian weight, triglycerides, total cholesterol, LDL cholesterol, VLDL cholesterol, malondialdehyde, luteinizing hormone (LH), and testosterone concentrations, but there were low (p<0.05-0.001) HDL cholesterol, estradiol, progesterone, catalase, peroxidase, and superoxide dismutase levels, compared with control. PCOS rats had multiple cysts compared with control. These reproductive, biochemical, and structural alterations were alleviated by P. muellerianus extracts. For instance, P. muellerianus restored the estrus cyclicity with a remarkable effect after 14 days of treatment. Moreover, P. muellerianus significantly decreased (p<0.001) LH and testosterone (both extracts; 30, 60, and 120 mg/kg) levels, but increased (p<0.01) estradiol (aqueous extract; 60 mg/kg) concentration. Cystic follicles were also decreased after plant application. P. muellerianus alleviated reproductive, hormonal, and structural alterations in PCOS rats. This plant could be useful in the management/treatment of reproductive and metabolic disorders related to PCOS.

SUN-LB020 The Role of Estradiol and Diet on the Gut Microbiome

Extensive research has shown that diet-induced obesity induces a bidirectional relationship between the gut microbiome and physiological health. However, the majority of this research was conducted using only male subjects. This is particularly problematic because sex hormones strongly influence the gut microbiome. Women have a different composition of gut bacteria than men. Additionally, imbalances in the gut microbiome have also been linked to diseases that are both predominately found in women, such as: PCOS and endometriosis. Interestingly, these reproductive conditions are comorbid with obesity. Therefore, the objective of this study was to examine the impact of diet-induced obesity on the female gut microbiome before and after ovariectomy and hormone replacement. To induce obesity, female Sprague-Dawley rats were fed a HFHS (high fat, high sugar) diet for 10 weeks. After this initial diet exposure, pre-operative fecal samples were collected and fasting blood glucose and insulin levels were determined. Following these procedures, all animals underwent bilateral ovariectomies. Half of each diet group was given a constant release 17β-Estradiol pellet (OVX+E), while the other half were not (OVX). Ovaries were fixed in paraformaldehyde and prepared for histology. A second fecal sample was collected two weeks post operation. At termination, blood samples were collected and fat pads were removed and weighed. Fecal DNA was extracted and then analyzed using qPCR. Primers for the major bacterial phyla were utilized. We focused primarily on Firmicutes and Bacteriodetes because the ratio of these phyla (Firmicutes: Bacteriodetes) is an established measure of gut health. If the ratio shifts towards Firmicutes, it is thought that the organism displays a greater level of gut dysbiosis. After 10 weeks on the diet, the HFHS rats weighed significantly more than the control animals. HFHS rats also displayed higher fasting blood glucose levels, higher fasting insulin levels, and insulin resistance. Similarly, the HFHS rats showed significantly larger Firmicutes: Bacteriodetes ratios indicating gut microbiome imbalance. After removal of the ovaries and estradiol replacement, OVX+E rats, regardless of diet, lost weight compared their OVX counterparts. This estradiol-driven weight loss was also associated with healthier gut microbiome profiles in the OVX+E rats. Interestingly, HFHS animals had a greater number of irregular estrus cycles and showed elevated numbers of ovarian cysts compared to control-fed females. This study provides a novel insight into the complex relationship between sex hormones, obesity, and the gut microbiome. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Ablating astrocyte insulin receptors leads to delayed puberty and hypogonadism in mice.

Insulin resistance and obesity are associated with reduced gonadotropin-releasing hormone (GnRH) release and infertility. Mice that lack insulin receptors (IRs) throughout development in both neuronal and non-neuronal brain cells are known to exhibit subfertility due to hypogonadotropic hypogonadism. However, attempts to recapitulate this phenotype by targeting specific neurons have failed. To determine whether astrocytic insulin sensing plays a role in the regulation of fertility, we generated mice lacking IRs in astrocytes (astrocyte-specific insulin receptor deletion [IRKOGFAP] mice). IRKOGFAP males and females showed a delay in balanopreputial separation or vaginal opening and first estrous, respectively. In adulthood, IRKOGFAP female mice also exhibited longer, irregular estrus cycles, decreased pregnancy rates, and reduced litter sizes. IRKOGFAP mice show normal sexual behavior but hypothalamic-pituitary-gonadotropin (HPG) axis dysregulation, likely explaining their low fecundity. Histological examination of testes and ovaries showed impaired spermatogenesis and ovarian follicle maturation. Finally, reduced prostaglandin E synthase 2 (PGES2) levels were found in astrocytes isolated from these mice, suggesting a mechanism for low GnRH/luteinizing hormone (LH) secretion. These findings demonstrate that insulin sensing by astrocytes is indispensable for the function of the reproductive axis. Additional work is needed to elucidate the role of astrocytes in the maturation of hypothalamic reproductive circuits.

Effects of Azorubine Food Additive on Female Reproductive Organs and Hormones in Sprague Dawley Rat

The objective of the present study was to assess the effects of various doses of Azorubine which is a food additive on the female reproductive organs in Sprague Dawley rats. Twenty four female Sprgue Dawley rats were divided randomly into 4 equal groups. Group 1(control group), group, 2, 3 and 4 were received Azorubine (5, 10 and 20 mg/kg) orally, daily for 30 days respectively. Blood samples were taken for estimation of white blood cells, red blood cells, hemoglobin and platelets, in addition Luteinizing, follicular stimulation, estrogen and progesterone hormones from the sera. The reproductive hormones levels affected drastically under the effects of different doses of treatment like Luteinizing hormone (0.69±0.25, 0.60±0.75 and 0.55±0.63), Follicular Stimulation hormone (0.17±0.11, 0.13±0.33 and 0.3±0.45), Progesterone hormone (0.50±0.77, 0.14±0.56 and 0.10±0.85), and estrogen hormone (0.45+0.43, 0.30±0.29 and 0.14±0.27) hormones were decreased significantly (P˂0.05) in groups of rats treated with each 5, 10 and 20 mg/kg doses of Azorubine respectively. Histopathologically, the ovaries treated with 5 mg/kg doses of Azorubine showing follicles at the beginning stages of growth with no Graffian follicle while the ovaries with 10 mg/kg doses of Azorubine contain fully grown Graffian follicles with no follicles at various stages as well as those with 20 mg/kg doses of treatment displaying no mature Graffian follicle with many atretic and shrunk follicles. The hematological outcomes are significantly affected by this food additive. The results of this work is concluded that Azorubine can be considered as one of the most important causes of infertility, hormonal disturbances and irregular estrus cycle in the female rat.

Reproductive disorders in dairy cows of the southwest of Paraná

Reproductive efficiency of dairy cows is evaluated directly considering the calving interval, which is affected by nutrition, irregular estrus cycle, prolonged anestrus, and reproductive diseases. Reproductive diseases cause economic losses due to diagnosis and treatment costs, and reduction in milk production. Thus, the objective of this study was to report the occurrence of the main reproductive disorders in dairy cows of the Southwest of Paraná, South of Brazil. Data of ultrasound diagnoses were collected from 2274 cows of 70 rural properties in the municipalities of Realeza, Planalto, Capanema, Pérola do Oeste, and Santa Isabel do Oeste from January 2014 to May 2015. Reproductive disorders were found in 24.50% (557/2274) of the animals evaluated; ovarian cysts (follicular, and luteal) were the most significant disorders, found in 16.13% (367/2274) of the cases. Other disorders were found less frequently. The occurrence of follicular and luteal cysts was observed especially in primiparous animals in months of pasture transition.

IMPACT OF DIETARY RESTRICTION ON FOLLICULAR WAVES IN CATTLE WITH PARTICULAR REFERENCE TO ANOESTRUS

Reproductive failure (anoestrus) is a one of the major problem in cattle production of Bangladeshdue to under feeding and unavailability of balanced ration. This study was conducted for a period from July 2009 to June 2010 to determine the nutritional impact on the follicular growth using blood hormone assays and the rate of anoestrus in cows following dietary restriction and to develop a feasible feeding technique concerning the available feed ingredients in local market. A uniform group of 2-3 years old 15 heifers were selected and were grouped randomly into three named T1(60% feeding restriction), T2(40% feeding restriction) and T3 (standard feeding). Body condition score (BCS) and body weight were measured by standard method. Hormonal assays were analyzed to determine the impacts of restricted feeding on the ovarian follicular cycle using blood sera. The average BCS and body weight was decreased significantly (p&lt;0.01) in the animals of T1 and T2 compared to control T0. The average values of estrogen and progestrogen was decreased significantly (p&lt;0.01) in restricted feeding heifers (T1&amp; T2) than that of standard feeding heifers (T3). About 50% heifers showed irregular estrus and 50% showed anoestrus signs in the animals of T1 group. In addition, a total of 60% animals showed irregular estrus and 40% showed anoestrus condition in animals of T2 group.. On the other hand, the animals of control group showed regular estrous cycle with good hormonal assays. Standard feeding could improve the cattle production efficiency by overcoming the reproductive failure using available feed ingredients.

Factores predisponentes para la enfermedad quística ovárica bovina y su efecto en la eficiencia reproductiva

En el ganado lechero los quistes ováricos son anomalías habituales. Se caracterizan por presentar anestro, celos irregulares, aumento de intervalos reproductivos e infertilidad. Su origen es aún desconocido. Es importante estudiar su patogenia y posible vinculación a ciertos factores predisponentes en su presentación. El objetivo de este trabajo fue determinar ecográficamente la prevalencia y tipo de quistes ováricos. Se estudiaron 93 vacas que pertenecían a 23 ganaderías de la Hoya de Loja. Las formaciones quísticas se identificaron y clasificaron según el perfil ecográfico de 24 días, con 6 exploraciones cada 4 días. Las edades de los animales fluctuaron entre 2 y 12 años, con condición corporal &gt; 2. Para el análisis de datos se utilizó el PROC REG en el programa estadístico SPSS Statistics 21. Se determinó un incremento de 256,4 días abiertos en animales con quistes ováricos versus 114,8 días abiertos en las vacas sanas control. La prevalencia de quistes ováricos fue de 32 %, distribuidos en 21 % de quistes foliculares y 11 % de luteínicos. Además, se evidenció correlación entre presencia de quistes con condición corporal ≤ 3 (23 %), presencia de metritis (9 %) y utilización de productos hormonales (10 %). La manifestación clínica de los quistes ováricos se expresó con síntomas de anestro de vacas con quistes luteínicos (90 %), y la presencia de celos irregulares en vacas con quistes foliculares (40 %). En conclusión, los quistes ováricos son causa directa del incremento de los periodos parto-celo, parto-concepción y del intervalo entre partos en vacas lecheras en posparto.

Kisspeptin expression is decreased in the arcuate nucleus of hypothyroid female rats with irregular estrus cycles

Insufficiency of thyroid hormones inhibits gonadotropin release and results in dysregulation of reproductive function, although the precise mechanism of this disrupted gonadotropin secretion remains unclear. Kisspeptin is a neuropeptide that strongly stimulates gonadotropin secretion and plays an important role in reproductive function. To examine the involvement of kisspeptin in the dysregulation of gonadotropin secretion in hypothyroidism, we investigated Kiss1 mRNA expression and kisspeptin immunoreactivity in the hypothalamus of female rats treated with propylthiouracil (PTU). In the PTU-treated rats, serum thyroxine (T4) was significantly decreased, whereas thyroid stimulating hormone (TSH) levels were significantly increased. In addition, irregular estrus cycles were observed in PTU-treated rats. In situ hybridization and immunohistochemistry revealed significant reductions in the number of Kiss1 mRNA-expressing neurons and kisspeptin-immunoreactive neurons in the arcuate nucleus (ARC) but not in the anteroventral periventricular nucleus (AVPV) of the PTU-treated rats. Although the serum levels of luteinizing hormone (LH) and estradiol (E2) were unaffected, serum prolactin levels were significantly increased after PTU treatment. These data indicate that kisspeptin expression in the ARC is suppressed under thyroid hormone insufficiency, suggesting that the dysregulation of reproductive function in hypothyroidism is caused by inhibition of kisspeptin neurons in the ARC.

Neonatal isoflavone exposure interferes with the reproductive system of female Wistar rats

There is increasing concern about possible adverse effects of soy based infant formulas (SBIF) due to their high amount of isoflavones (ISO). The aim of the present study was to investigate effects of neonatal exposure to ISO on reproductive system of female Wistar rats.Animals were exposed to an ISO depleted diet or a diet enriched with an ISO extract (IRD; 508mg ISO/kg) during embryogenesis and adolescence. Pups of each group were fed daily by pipette with ISO-suspension (ISO+; 32mg ISO/kg bw) or placebo from postnatal day (PND) 1 until PND23 resulting in plasma concentrations similar to levels reported in infants fed SBIF.The visceral fat mass was reduced by long-term IRD. Vaginal epithelial height was increased at PND23 and vaginal opening was precocious in ISO+ groups. Later in life, more often irregular estrus cycles were observed in rats of ISO+ groups. In addition, FSH levels and uterine epithelial heights were increased at PND80 in ISO+ groups.In summary, the results indicate that neonatal ISO intake, resulting in plasma concentrations achievable through SBIF, has an estrogenic effect on prepubertal rats and influences female reproductive tract later in life.

Effects of maternal exposure to aflatoxin B1 during pregnancy on fertility output of dams and developmental, behavioral and reproductive consequences in female offspring using a rat model

A suboptimal in utero environment can have detrimental effects on the pregnancy and long-term adverse “programing” effects on the offspring. Aflatoxin B1 is one of the potent reproductive toxicants and currently detected in both milk and tissues. This article focuses on the effects of prenatal exposure to graded doses of aflatoxin B1 on the pregnancy outcomes of dams and postnatal developments of the female offspring, since these issues have ethological relevance in both animals and humans. Pregnant Wistar rats were injected intramuscularly with vehicle or aflatoxin B1 (10, 20, 50 or 100 μg/kg body weight/day) on days 12–19 of gestation. At parturition, newborns were observed for clinical signs of toxicity and survival. The female offspring were examined through a battery of tests in order to evaluate their developmental, behavioral and reproductive end points. All animals were born alive. The litter size of the aflatoxin B1 treated rats was comparable to the controls. However, the birth weight of the pups in the experimental group was significantly lower when compared to controls. Significant and persistent lags in cliff avoidance, negative geotaxis, surface rightening activity and ascending wire mesh, with a delay in elapsed time for vaginal opening were detected in the female progeny exposed to aflatoxin B1 during embryonic development. The locomotor activity and exploratory behavior in experimental females were significantly decreased than that of controls. Embryonic exposure to aflatoxin B1 also resulted in prolonged stress response, irregular estrus and suppressed fertility output in the progeny at their adulthood. These results indicate that in utero exposure to aflatoxin B1 severely compromised postnatal development of neonatal rats and caused irregular estrus that was accompanied by suppressed fertility output.

Mifepristone (RU486) Induces Polycystic Ovarian Syndrome in Female Wistar Rats with Features Analogous to Humans

Numerous inducers of polycystic ovarian syndrome (PCOS) at different doses have been proposed in several experimental animals but there is no consensus on an appropriate dose(s) that should ideally reproduce the key biochemical and clinical features of PCOS similar to those of humans. Therefore, this study was aimed at investigating an appropriate dose(s) for the induction of PCOS in female Wistar rats. Twenty-four female albino rats (190.00 ± 13.00 g) with 4-5 days of estrus cyclicity were completely randomized into 4 groups (A - D) of six animals each. Animals in group A (control) were subcutaneously administered 0.2 ml of pure olive oil, while those in groups B, C and D were subcutaneously administered same volume corresponding to 5.0, 7.5 and 10.0 mg of mifepristone in olive oil for 9 days starting from the day of estrus (Day 1). The estrus cycle, serum testosterone (T), estradiol (E), prolactin (Pr), follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P), insulin (Is), weight of the animals, fasting blood glucose (FBS) and ovarian morphology were monitored/evaluated/examined. The 5.0 mg of mifepristone extended the estrus stage for four days, increased (p<0.05) the levels of serum E, P, Pr, FSH, T, triacylglycerides (TAG), and total cholesterol (TC) as well as decreased the concentrations of LH and high density lipoprotein-cholesterol (HDL-C). There was no significant difference (p<0.05) in the Is concentration, animal body weights and FBS at day 10 in rats administered 5.0 mg of mifepristone. The 7.5 mg of mifepristone produced irregular estrus cycle, increased Pr, TAG, T, and TC concentrations and FBS whereas it decreased E, P, HDL-C, and LH. The Is, FSH and body weights of the animals were not significantly altered at 7.5 mg of mifepristone. The 10.0 mg of mifepristone produced irregular estrus cycle, increased the levels of E, TAG, Is, LH, T as well as decreased the levels of P and HDL-C. The levels of Pr, FSH, TC, body weights and FBS were not significantly altered at this dose. There was no ovarian follicular growth and atresia in the 5.0 and 7.5 mg mifepristone-treated rats whereas the 10 mg of mifepristone produced these histopathological features. Overall, the study concluded that subcutaneous administration of mifepristone (RU486) induces polycystic ovarian syndrome in rats through deprivation of progesterone with the 10 mg producing majority of the key biochemical and clinical features associated with PCOS in humans. The study, therefore, recommends the subcutaneous administration of mifepristone (RU486) on daily basis for 9 days as a good model for inducing PCOS in animals.

Liraglutide improves hypertension and metabolic perturbation in a rat model of polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder in women of reproductive age, with a prevalence of 5–8%. Type 2 diabetes and cardiovascular disease (CVD) are its long-term complications. Targeted therapies addressing both these complications together are lacking. Glucagon like peptide-1 (GLP-1) agonists that are used to treat type 2 diabetes mellitus have beneficial effects on the cardiovascular system. Hence we hypothesized that a GLP-1 agonist would improve both cardiovascular and metabolic outcomes in PCOS. To test this hypothesis, we used an established rat model of PCOS. Prepubertal female Sprague Dawley rats were sham-implanted or implanted s.c. with dihydrotestosterone (DHT) pellets (90 day release; 83μg/day). At 12 wks of age, sham implanted rats received saline injections and the DHT treated animals were administered either saline or liraglutide (0.2mg/kg s.c twice daily) for 4 weeks. Subgroups of rats were implanted with telemeters between 12-13 weeks of age to monitor blood pressure. DHT implanted rats had irregular estrus cycles and were significantly heavier than the control females at 12 weeks (mean± SEM 251.9±3.4 vs 216.8±3.4 respectively; p<0.05) and 4 weeks of treatment with liraglutide in DHT treated rats significantly decreased body weight (mean± SEM 294.75 ±3.2 in DHT+ saline vs 276.25±2.7 in DHT+ liraglutide group respectively; p<0.01). Liraglutide treatment in the DHT implanted rats significantly improved glucose excursion during oral glucose tolerance test (area under the curve: DHT+ saline 28674±310 vs 24990± 420 in DHT +liraglutide p <0.01). DHT rats were hypertensive and liraglutide treatment significantly improved mean arterial pressure. These results suggest that GLP-1 treatment could improve DHT–induced metabolic and blood pressure deficits associated with PCOS.

Estudo clínico, histopatológico e imunoistoquímico de neoplasias mamárias em cadelas

O objetivo foi realizar um estudo clínico e epidemiológico de neoplasias mamárias em cadelas, considerando-se histórico reprodutivo, exame físico, diagnóstico histopatológico e imunoistoquímico. Utilizaram-se 60 neoplasias mamárias, divididas em grupos (grupo 1 - benigno, e grupo 2 - maligno). Avaliaram-se dados do histórico reprodutivo, o exame físico e achados histopatológicos e imunoistoquímicos para fator de crescimento endotelial vascular. Ao estudo do histórico reprodutivo, encontraram-se 90% dos animais com irregularidade de cio, 86,63% das cadelas não foram medicadas com contraceptivos e 83,33% não eram castradas. Ao exame físico, não foi verificada diferença (p&gt;0,05) entre grupos ao se avaliar consistência das massas, regularidade da superfície tumoral e localização anatômica dos tumores. Quanto ao tamanho das massas, verificou-se diferença entre os grupos (p=0,0077), com 0,78±1,13cm para o grupo 1 e 1,81±2,29cm para o grupo 2. Diagnosticaram-se 40% de massas benignas e 60% de malignas, de acordo com os tipos de neoplasias. Para VEGF, verificaram-se valores médios de 2,22±0,89 para tumores malignos e 1,66±0,91 para benignos, com diferença entre grupos (p=0,0315). As neoplasias mamárias em cadelas não apresentam características de histórico reprodutivo e de exame clínico que auxiliem o diagnóstico diferencial, sendo a histopatologia o único método para conclusão do diagnóstico e a imunoistoquímica podendo ser utilizada para prognóstico da lesão.

477. Disappearance of Proestrus LH Surge Caused by the Decline in Activation of Metastin/Kisspeptin Neurons in Middle-Age Female Rats.

Reproductive function declines with aging and one of the earliest markers for this change in female rats is disappearance of the LH surge. The neuropeptide metastin/kisspeptin, encoded by the Kiss1 gene, plays an integral role as a potent activator of GnRH neurons in the reproductive function of mammals. We investigated the involvement of kisspeptin neurons in the hypothalamic anteroventral periventricular nucleus (AVPV) to disappearance of the LH surge in aged female rats. Young (Normal estrous cycle: YN) and middle-age (Normal estrous cycle: MN and Irregular estrous cycle: MI) rats were used. In the evening at proestrus or irregular estrus, LH surge and estradiol (YN: N=7, MN: N=38, MI: N=30) and Kiss1 mRNA and metastin/kisspeptin expression (N=7/each group) were determined in these females of 3 groups. In the result, although MI showed no LH surge with low estradiol level, Kiss1 mRNA and metastin/kisspeptin expression in MI was not different from YN and MN. To investigate the sensitivity of GnRH neurons to kisspeptin, rats were administered kisspeptin (10 and 100 nmol/kg, N=6/each group) subcutaneously. Administration of metastin/kisspeptin elevated plasma LH levels in all 3 groups. These data suggested that absent of metastin/kisspeptin in the AVPV was responsible for the disappearance of the LH surge in intact middle-age rats. To investigate the activation of metastin/kisspeptin neurons by estradiol in MI, rats were ovariectomised and treated with estradiol (OVX+E2), and blood samples taken for LH determination and brain samples for in situ hybridization (Kiss1 mRNA) and immunohistochemistry (cFos). OVX+E2 MI (N=5) showed extremely lower percentage of Kiss1 mRNA-positive cells with cFos immunopositivity than in OVX+E2 YN (N=4). Further, OVX+E2 MI (N=7) showed no elevation of LH levels while YN (N=7) showed clear elevation of LH level. These data of OVX+E2 rats suggested that the absence of elevation of LH levels was due to a decline in the activation of metastin/kisspeptin neurons by estradiol. In conclusion, the decline in metastin/kisspeptin neurons' activation by estrogenic positive-feedback might be responsible for the absence of metastin/kisspeptin secretion in the AVPV, and these neuroendocrinological changes resulted in disappearance of the LH surge in middle-age rat.

Follicular Stage-Specific Gene Expression Analysis in Rat Ovary.

It is well established that endocrine-disrupting chemical (EDC) exposures during early development can cause ovarian dysfunction by adulthood. Studies with the organochlorine pesticide methoxychlor (MXC), a model EDC with estrogenic and anti-estrogenic activities, have demonstrated that neonatal and fetal exposure [embryonic day 18 to postnatal day (PND) 7] lead to accelerated puberty, irregular estrus cycles, reduced litter sizes, reduced superovulatory response to gonadotropins, and premature reproductive aging. In PND 50-60 ovaries, there was a disruption of follicular progression with a large number of follicles found in the early antral follicle stage with a concomitant reduction in corpora lutea. Furthermore, immunohistochemical studies showed that MXC-treated ovaries had decreased levels of CYP11A1 and LHR proteins in mid-late antral follicle stages and reduced ESR2 expression in the preantral and early antral follicles. Interestingly, there was no significant effect on ESR2 expression at earlier follicular stages such as primary and secondary follicles. Other studies with granulosa cell (GC) cultures demonstrated an enhanced effect of HPTE (a major metabolite of MXC) only in cells stimulated with FSH suggesting that gonadotropin responsive stages of follicles/GCs were more likely to exhibit the MXC mediated detrimental effects. Therefore, to identify the specific stages and the molecular/epigenetic mechanisms involved in EDC-associated effects, follicular isolations were performed. The objectives were (1) to establish a robust follicle separation method by enzymatic digestion and (2) to compare the gene expression patterns at the various follicular stages. Enzymatic digestions were performed with Collagenase Type I and various size beads (Polysciences, Inc.) were utilized to categorize the follicles into three groups: (1) “small” (50-180 μm, up to and including early pre-antral), (2) “medium” (180-300 μm, late pre-antral and early antral), and (3) “large” (300-700, mid- and late-antral follicles. Granulosa cells were collected from the large follicles by puncturing them prior to the enzymatic digestion. In this manner, approximately 25-30 small, 10-20 medium follicles and ~6 x 10-5 GCs from large follicles, per rat (n = 3), were obtained and RNA was isolated. Gene expression analyses were conducted after whole transcriptome amplification and QPCR with Taqman assays. Small follicles had a 100- to 200-fold enrichment ( p < 0.001) of Gdf9 and Bmp15 transcripts while GC from the large follicles had an enrichment of 100- to 500-fold ( p < 0.001) in steroidogenic enzyme transcripts such as Cyp11a1, Cyp19a1, and Hsd17b1 . These data validated our isolation techniques. Currently these data are being compared to those obtained from laser capture microdissection technique. Together they provide a useful tool for future studies of EDC exposed ovaries wherein gonadotropin responsive follicular stage-dependent gene expression and epigenetic analyses can be performed. Supported by R56 ES017059 from NIEHS (MU) and TUBITAK– BIDEB 2219 International Postdoctoral Fellowship Program (KA).

Neonatal dexamethasone exposure down-regulates GnRH expression through the GnIH pathway in female mice

Synthetic glucocorticoid (dexamethasone; DEX) treatment during the neonatal stage is known to affect reproductive activity. However, it is still unknown whether neonatal stress activates gonadotropin-inhibitory hormone (GnIH) synthesizing cells in the dorsomedial hypothalamus (DMH), which could have pronounced suppressive action on gonadotropin-releasing hormone (GnRH) neurons, leading to delayed pubertal onset. This study was designed to determine the effect of neonatal DEX (1.0mg/kg) exposure on reproductive maturation. Therefore, GnRH, GnIH and GnIH receptors, G-protein coupled receptors (GPR) 147 and GPR74 mRNA levels were measured using quantitative real-time PCR in female mice at postnatal (P) days 21, 30 and in estrus stage mice, aged between P45–50. DEX-treated females of P45–50 had delayed vaginal opening, and irregular estrus cycles and lower GnRH expression in the preoptic area (POA) when compared with age-matched controls. The expression levels of GPR147 and GPR74 mRNA in the POA increased significantly in DEX-treated female mice of P21 and P45–50 compared to controls. In addition, GPR147 and GPR74 mRNA expression was observed in laser captured single GnRH neurons in the POA. Although there was no difference in GnIH mRNA expression in the DMH, immunostained GnIH cell numbers in the DMH increased in DEX-treated females of P45–50 compared to controls. Taken together, the results show that the delayed pubertal onset could be due to the inhibition of GnRH gene expression after neonatal DEX treatment, which may be accounted for in part by the inhibitory signals from the up-regulated GnIH–GnIH receptor pathway to the POA.

Infertility in WNIN Obese Mutant Rats—Causes?

We are maintaining two obese mutant rat strains (WNIN/Ob and WNIN/GR-Ob) in our animal facility since 1997. These rat colonies are perpetuated by crossing heterozygous littermates, since the obese phenotypes of both genders turned out be infertile. The present study revealed the reasons for this infertility. The male obese rats, though appeared normal in terms of sperm count, sperm motility and testis histology, however found wanting in terms of libido. This appeared to be due to low circulating testosterone levels seen in these animals, which should also account for low testis and accessory gland weights seen in them. The females exhibited delayed puberty, in terms of days taken for opening of vagina, irregular oestrus cycles and had small ovaries and short and stumpy uterine horns. The FSH peak observed in control lean animals during oestrus stage of the sexual cycle and also the E2 peak of normal oestrus cycle was conspicuously absent in these animals. They also showed elevated levels of progesterone throughout the sexual cycle. Thus the infertility seen in these mutants could be attributed to their abnormal gonadosteroid levels and the resulting anatomical and physiological defects.

Postnatal dexamethasone treatment delays the onset of puberty and attenuates the regulation of Kiss-1 mRNA expression by allopregnanolone in pubertal female mice

eurosteroid allopregnanolone (3 ,5 -tetrahydroprogesterone; THP), a otent modulator of GABAA receptors and kisspeptin have been shown to egulate theonset of puberty.Whether exposure topostnatal stress effects on HP regulated expression of kisspeptin and thereby delayed pubertal onset, emain unknown. Therefore, in this study, femalemicewere subcutaneously njected with synthetic glucocorticoid, dexamethasone (DEX) (1.0mg/kg) rom postnatal day (PN) 1–4 and intraperitoneally with THP (10mg/kg) for 3 aysduringpuberty (PN45). PostnatalDEX treatedmicehaddelayedonset of uberty by 1 week and irregular estrous cycle patterns compared to control ice of the same age. Normal cycling pubertal femalemice injectedwith THP howed a significant increase in Kiss1 mRNA expression in the anterovenral periventricular nucleus (AVPV) and no change in Kiss1mRNA expression evels in the arcuate nucleus (Arc). Postnatal DEX treatment inhibited the pregulation of Kiss1 mRNA expression by THP in the AVPV during puberty ut had no effect on Kiss1 mRNA expression levels in the Arc. These results emonstrate that exposure to DEX during early development disrupts the isspeptin–GABAA system in the AVPV resulting in irregular estrus cycle and elayed the onset of puberty.

The first ovulation of the breeding season in the mare: The effect of progesterone priming on pregnancy rate and breeding management (hCG response rate and number of services per cycle and mare)

The mare is a seasonally polyestrus breeder. In early spring, the mare enters a “transition period” between the anovulatory season and the first ovulation of the year. This period is characterized by irregular estrus cycles and high incidence of regressing dominant follicles. There is a belief that pregnancy rates resulting from the first ovulation of the season is lower than in subsequent ovulations, however this has never been studied critically. Progestagens are often used as an aid to manage the transition period. The objective of this study was to compare pregnancy rates of mares from the first ovulation of the year with: (a) mares on their second or subsequent ovulations and (b) mares with progesterone-primed first ovulations. A total of 136 thoroughbred mares were used in the study. The mares were classified into four groups: (1) mares mated at the first ovulation of the year ( n = 46); (2) mares mated in the first ovulation of the year after removal of a previously inserted progesterone device (CIDR) ( n = 29); (3) mares mated at the second or more ovulations of the year after prostaglandin-induced estrus ( n = 50); and (4) mares mated after spontaneous return to estrus ( n = 11). Pregnancy rates were not different in any of the groups studied: 65.2, 75.9, 76 and 72.7 for groups 1–4 respectively ( P > 0.05). Group 1 mares had the lowest response to hCG treatment which resulted in the highest number of services per cycle ( P < 0.05). In conclusion, although the use of progesterone priming did not affect pregnancy rates, it did improve the breeding management of transitional mares by enhancing the hCG response rate and consequently decreasing the number of services per cycle.

Ovarian status, uterine pathology and physical condition of anoestrus and culled buffaloes in Chitwan, Nepal

Buffaloes are the major production animals in most of the South Asian countries including Nepal. The buffalo, however, is a sluggish breeder and is beset with various constraints which adversely influence its fertility; such as, problems of silent heat coupled with poor expression of oestrus, irregular oestrus cycles, anoestrus, prolonged days open and low conception rates (Madan and Prakash, 2006). Among them, anoestrus is the most commonly encountered condition leading to reproductive failure and culling in buffaloes. However, the ovarian status of such buffaloes is poorly understood. The present study aims to investigate the ovarian status, uterine pathology and physical condition of anoestrus and culled buffaloes in Chitwan, Nepal. The buffaloes in Nepal are generally culled if they fail to conceive for long time or the production is seized.