In mares resistant to endometrial infection, periovulatory treatment with ecbolic drugs does not influence uterine clearance or luteal development.

We aimed to determine associations between experimentally impaired uterine clearance or treatment with ecbolic drugs on luteal development in estrous mares after insemination. In a crossover design, eight mares were treated with saline (CON), clenbuterol (CLEN), oxytocin (OXY) and carbetocin (CARB) from the day of first insemination until 2 days after ovulation. Between treatments, the mares rested for one cycle. Estrous mares were examined for the presence of free intrauterine fluid by transrectal ultrasound. Endometrial swabs for cytology and bacteriology were collected on days 1 and 14. Blood samples were collected daily before AI until day 14 after ovulation for determination of progesterone and PGF2α metabolites (PGFM). Differences between treatments were compared by a general linear model for repeated measures (SPSS 29). One mare was excluded because of a uterine infection in the control cycle. In all other mares, only minor amounts of free intrauterine fluid were present after insemination and decreased over time (P<0.05) with no treatment x time interaction. There was no effect of treatment on polymorphonucleated cells (PMN) in endometrial cytology after ovulation or PGFM secretion. Progesterone release from day 1-14 as well as pregnancy rate and conceptus size on day 14 was not influenced by treatment. In conclusion, treatment with clenbuterol does not impair uterine clearance in estrous mares resistant to endometritis. Repeated injection of the oxytocin analogue carbetocin during the early postovulatory period is not detrimental to corpus luteum function and can be recommended to enhance uterine clearance.

The effect of exogenous melatonin administration before superovulation on embryo yield in Assaf ewes.

The aim of this study was to determine the effect of exogenous melatonin administration on transferable embryos by increasing total antioxidant status before superovulation in Assaf ewes. Selected ewes were randomly divided into two equal groups: melatonin (n = 9) and control (n = 9). In the melatonin group, a melatonin implant (18 mg melatonin, Regulin®, Ceva, Turkey) was placed under the skin of the ear 7 days prior to insertion of the progesterone-containing sponge. In the control group, a physiological saline solution was injected under the skin of the ear on the same day. The same superovulation protocol was used in both groups. In addition, blood samples for determination of Glutathione peroxidase, superoxide dismutase, total antioxidant status and total oxidant status concentrations were collected on five different days, including the day of melatonin implant placement (Day-7), vaginal sponge insertion (Day 0), vaginal sponge removal (Day 11), mating (Day 12-13) and uterine flushing (Day 19). Embryos were collected by laparotomy on the 7th day after mating. Uterine flushing taken into petri dishes were scanned under a stereomicroscope, and the quality and developmental stages of the embryos were recorded. In the study, total corpus luteum count and total cell count were found to be higher in the control group than in the melatonin group (p < .05). When the results were evaluated in terms of oxidative stress index, a negative correlation was found between the total number of corpus luteum, number of cells obtained, count of transferable embryos and number of Grade 1 embryos on Day 0. There was also a positive correlation oxidative stress index and the number of unfertilized oocytes on Day-7. As a result, exogenous melatonin administration prior to superovulation during the breeding season is thought to have a negative effect on embryo yield and quality. Therefore, the use of exogenous melatonin in MOET studies during the breeding season is recommended to be investigated in new studies.

Effect of prostaglandin treatment on the estrus behaviour, follicular and luteal morphometry and serum hormone profile in sub-estrus buffaloes during non-breeding season.

Sub-estrus buffaloes do not exhibit estrus signs despite being cyclic contributing to extended service periods and inter-calving intervals causing significant economic loss. The present study described the effect of synthetic prostaglandin (PGF2α) on estrus behaviour, follicular and luteal morphometry, and serum estradiol (E2) and progesterone (P4) profile in sub-estrus buffaloes during the non-breeding season. The incidence of sub-estrus was 38.4% during the non-breeding season. The sub-estrus buffaloes (n = 33) were divided into two groups, viz., Control (n = 16) and PGF2α treatment (Inj. Cloprostenol 500 µg, i.m., n = 17). Estrus induction response was significantly greater in the treatment (100 vs. 18.75%, p < .001), and a relatively greater proportion of animals conceived in the treatment group (29.41 vs. 6.25%, p = .08). The time elapsed to induction of estrus and insemination following treatment was significantly lower in the treatment group than control. A significant increment in the follicle diameter (9.72 ± 0.45 vs. 13.00 ± 0.45 mm, P < .0001) and serum estradiol (E2) concentration (66.01 ± 11.92 vs. 104.9 ± 13.21 pg/mL, p = .003) observed at the post-treatment period in the PGF2α treatment group. At the same time, CL diameter was reduced significantly at a higher regression rate in the PGF2α treated buffaloes than those of control. Of the responded buffaloes, only 30% showed high-intensity estrus attributed to the expulsion of cervico-vaginal mucus (CVM), uterine tonicity, micturition, and mounting response by a teaser bull. From this study, it can be concluded that the administration of PGF2α could induce estrus in the sub-estrus buffaloes during the non-breeding season. Behavioural changes, along with sonographic observation of POF, regressing CL, and serum E2 and P4 concentration would be useful to determine the right time of insemination in sub-estrus buffaloes during non-breeding season.

In vitro effect of visfatin on endocrine functions of the porcine corpus luteum.

Previously, we demonstrated the expression of visfatin in porcine reproductive tissues and its effect on pituitary endocrinology. The objective of this study was to examine the visfatin effect on the secretion of steroid (P4, E2) and prostaglandin (PGE2, PGF2α), the mRNA and protein abundance of steroidogenic markers (STAR, CYP11A1, HSD3B, CYP19A1), prostaglandin receptors (PTGER2, PTGFR), insulin receptor (INSR), and activity of kinases (MAPK/ERK1/2, AKT, AMPK) in the porcine corpus luteum. We noted that the visfatin effect strongly depends on the phase of the estrous cycle: on days 2-3 and 14-16 it reduced P4, while on days 10-12 it stimulated P4. Visfatin increased secretion of E2 on days 2-3, PGE2 on days 2-3 and 10-12, reduced PGF2α release on days 14-16, as well as stimulated the expression of steroidogenic markers on days 10-12 of the estrous cycle. Moreover, visfatin elevated PTGER mRNA expression and decreased its protein level, while we noted the opposite changes for PTGFR. Additionally, visfatin activated ERK1/2, AKT, and AMPK, while reduced INSR phosphorylation. Interestingly, after inhibition of INSR and signalling pathways visfatin action was abolished. These findings suggest a regulatory role of visfatin in the porcine corpus luteum.

Effect of prostaglandin alone and in combination with trace minerals on the follicular and luteal dynamics, estrus response and pregnancy in sub-estrus buffaloes (Bubalus bubalis).

Sub-estrus is a condition when buffaloes do not display behavioural estrus signs, despite being in estrus and causes a delay in conception and increases the service period. The present study describes the effect of synthetic prostaglandin (PGF2α) alone and in combination with trace minerals on the follicular and corpus luteum (CL) dynamics, serum estradiol (E2) and progesterone (P4) concentration correlating estrus response and pregnancy outcome in sub-estrus buffaloes during the breeding season. A total of 50 sub-estrus buffaloes, identified through ultrasonography (USG) examination, were randomly allocated into three groups, viz. T1 (Synthetic PGF2α, Inj. Cloprostenol 500 µg, i.m, n = 17), T2 (Synthetic PGF2α + Trace mineral supplementation, Inj. Stimvet 1 mL/100 kg body weight, i.m., n = 17) and control (untreated; n = 16). Following treatment, 100% of sub-estrus buffaloes were induced estrus in the T1 and T2 groups, while only 18.75% were induced in the control. The CL diameter and serum P4 concentration were significantly lower at post-treatment, whereas the pre-ovulatory follicle (POF) size and serum E2 concentration were significantly higher in the T1 and T2 groups as compared to the control (p < .05). The buffaloes of the T2 group had a greater proportion of moderate intensities estrus than those of T1. Moreover, the proportion of buffaloes conceived in the T1 and T2 were 41.2% and 52.95%, respectively. The larger POF diameter and higher serum E2 concentration were associated with intense intensity estrus and higher conception rate (66.7%) in sub-estrus buffaloes. Similarly, CL regression rate, POF size and serum E2 concentration were relatively higher in the buffaloes conceived as compared to those not conceived. It is concluded that synthetic PGF2α in combination with trace minerals induces moderate to intense intensities estrus in a greater proportion of sub-estrus buffaloes and increases the conception rate during the breeding season. Moreover, behavioural estrus attributes correlating follicle and luteal morphometry, serum E2 and P4 concentration could be used to optimise the breeding time for augmenting the conception rate in sub-estrus buffaloes.

Expression dynamics of adipokines during induced ovulation in the bovine follicles and early corpus luteum.

The study aimed to assess the local gene expression of adipokine members, namely vaspin, adiponectin, visfatin, resistin and their associated receptors - heat shock 70 protein 5 (HSPA5), adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2) - in bovine follicles during the preovulatory period and early corpus luteum development. Follicles were collected before gonadotropin-releasing hormone (GnRH) treatment (0 h) and at 4, 10, 20, 25 and 60 h after GnRH application through transvaginal ovariectomy (n = 5 samples/group). Relative mRNA expression levels were quantified using real-time reverse transcription polymerase chain reaction (RT-qPCR). Vaspin exhibited high mRNA levels immediately 4 h after GnRH application, followed by a significant decrease. Adiponectin mRNA levels were elevated at 25 h after GnRH treatment. AdipoR2 exhibited late-stage upregulation, displaying increased expression at 20, 25 and 60 h following GnRH application. Visfatin showed upregulation at 20 h post-GnRH application. In conclusion, the observed changes in adipokine family members within preovulatory follicles, following experimentally induced ovulation, may constitute crucial components of the local mechanisms regulating final follicle growth and development.

Changes on corpus luteum structure and progesterone synthesis pathway after hCG or GnRH treatment during the early luteal phase in sheep.

This study investigated the effect of hCG or GnRH on structural changes of the corpora lutea (CL) and the regulation of the expression of steroidogenic enzymes involved in P4 secretion in post-ovulatory (po-CL) and accessory CL (acc-CL). Sixty-four ewes were assigned to three groups receiving: 300 IU of hCG (hCG) or 4 µg Buserelin (GnRH) or 1 mL of saline solution (Control) on Day (d) 4 post artificial insemination (FTAI). Laparoscopic ovarian were performed on d 4, 14 and, 21 post-FTAI to determine the numbers of CL. Blood samples were collected for serum LH and P4 analysis. On d 14 post-FTAI, both CL were removed from the ovary to determine large luteal cell (LLC) number and to evaluate the expression of steroidogenic enzymes (HSD3B1, STAR, CYP11A1). Only hCG and GnRH treated ewes generated acc-CL. The LLC in both po- and acc-CL were significantly greater in the hCG group compared to GnRH and Control groups (P<0.05). Overall, hCG group showed the greatest immunodetection of HSD3B1and STAR in both po- and acc-CL (P<0.05). rnRNA expression of HSD3B1, STAR and CYP11A1 in the acc-CL tended to be greater in hCG group than in GnRH group (P<0.1). The LH concentration was increased in GnRH group (P<0.05) and P4 concentration was greater in hCG group compared to the other groups (P<0.05). In conclusion, administration of hCG has a notably impact on acc-CL development and the expression of steroidogenic enzymes compared to GnRH treatment in ewes. This leads to elevated P4 concentration and improved luteal function.

Adropin may regulate ovarian functions by improving antioxidant potential in adult mouse.

The corpus luteum (CL) is a temporary endocrine gland that synthesizes progesterone. The luteal progesterone plays a central role in the regulation of the estrous cycle as well as the implantation and maintenance of pregnancy. Our previous study showed the expression of adropin and its receptor, GPR19, in the luteal cells and its significant role in luteinization. The aim of the present study was to investigate the in vitro effect of adropin on hCG-induced ovarian functions in adult mice. We also evaluated the effect of exogenous treatment with adropin on ovarian steroidogenesis and anti-oxidant parameters, with special emphasis on CL function. Our results demonstrated that adropin acts synergistically with hCG to promote ovarian steroidogenesis and survival by increasing the expression of StAR, 3ß-HSD, and aromatase proteins and decreasing the BAX/BCL2 ratio. Exogenous adropin treatment increased progesterone production by increasing the expression of GPR19, StAR and 3ß-HSD enzymes in the mouse ovary. Also, adropin inhibited the luteal oxidative stress by increasing nuclear translocation of NRF-2 in CL, which resulted in increased HO-1 expression and SOD, catalase activity. Decreased oxidative stress might inhibit the translocation of NF-κB into the nucleus of luteal cells, resulting into increased survival and decreased apoptosis, as evident by decreased lipid peroxidation, BAX/BCL2 ratio, caspase 3, active caspase 3 expression, and TUNEL-positive cells in adropin treated mice. Our findings suggest that adropin can be a promising candidate that can enhance the survivability of the CL.

Effects of repeated embryo flushing without PGF2α administration on luteal function, percentage of unwanted pregnancy and subsequent fertility in mares.

BACKGROUND: PGF2α is commonly given at the end of embryo flushing (EF) to shorten the interval to the next oestrus and ovulation. OBJECTIVES: To determine the effect of repeated EF on plasma progesterone concentration, percentage of mares with endometritis, unwanted pregnancy and subsequent fertility in mares flushed without the use of PGF2α. STUDY DESIGN: Controlled experiments. METHODS: Nine mares were inseminated in seven consecutive cycles (n = 63), to either perform an EF (n = 54) 7-9 days after ovulation or left pregnant (n = 9). PGF2α was not used to induce oestrus. Ultrasound examination and blood sampling were performed just before the EF and 72 h later to determine changes in progesterone concentration and signs of endometritis. RESULTS: The overall percentage of positive EF/pregnancy was 55.5% (30/54) and 66.7% (6/9), respectively. The likelihood of pregnancy/positive EF in the first three cycles was 55.5% (15/29). This was not different (p > 0.1) from the fertility of the last four cycles (69.4%, 25/36). In five EF cycles (9.3%), mares had signs of endometritis and early luteolysis (progesterone <2 ng/mL) 72 h after EF. The reduction in progesterone concentration by 72 h after EF was greater (p < 0.05) for Day 9 (-2.3 ± 0.7 ng/mL) than Day 7 (-1.0 ± 0.8 ng/mL) or Day 8 (-1.3 ± 1.1 ng/mL) cycles. The progesterone concentration in non-flushed mares did not vary significantly during the sampled period (Day 7-12). There were 5 cycles in which the donor mare remained pregnant after the EF, although four were from a single mare. MAIN LIMITATIONS: The mare population was limited to barren and maiden mares. The cycle order and operator allocation to each EF were not randomised. CONCLUSIONS: EF induces a subtle, but significant reduction in progesterone concentrations compared with non-EF cycles. However, the percentage of mares with EF-induced full luteolysis is low (9.3%). The fertility of mares after repeated EF without administration of PGF2α was unaffected; however, there is a considerable risk of unwanted pregnancy (5/27 = 18.5%) in donors from which an embryo was not recovered.

Role of thyroid stimulating hormone in the maintenance and functioning of the human corpus luteum.

PURPOSE: To evaluate the impact of high thyroid stimulating hormone (TSH) levels on human granulosa-luteal (hGL) cells. METHODS: hGL cells were isolated from follicular aspirates derived from patients undergoing IVF treatment without any thyroid disorder (serum TSH 0.5-2 mU/L). Cells were cultured at 37 °C in DMEM, supplemented with 5% FBS. The cells were treated with 1 nM LH and increasing concentrations of TSH. At the end of culture, conditioned medium and cells were collected to analyze progesterone production, cell viability, and mRNA levels of genes involved in the steroidogenesis process. Human ovarian tissues were analyzed for TSH receptor (TSHR) expression by IHC. RESULTS: The expression of TSHR was detected in human corpus luteum by IHC and in hGL by RT-PCR. In hGL cells, TSH treatment did not modulate progesterone production nor the expression of steroidogenic genes, such as p450scc and HSD3b 1/2. However, TSH induced a dose-dependent increase in cell death. Finally, TSH did not affect LH-induced p450scc and HSD3b1/2 expression while LH partially reverted TSH negative effect on cell death in hGL. CONCLUSIONS: Elevated TSH levels in hypothyroid women may be associated with impaired CL functioning and maintenance. These findings open a new line of research for the importance of the treatment of women with thyroid dysfunction that could contribute to the onset of infertility.

Insulin induces steroidogenesis in canine luteal cells via PI3K-MEK-MAPK.

Glucose uptake increases in canine luteal cells under insulin treatment. We hypothesize that insulin also increases luteal cell steroidogenesis. Dogs underwent elective ovariohysterectomy from days 10-60 post ovulation and their corpora lutea (CL) and blood samples were collected. Deep RNA sequencing determined differentially expressed genes in CL; those related to insulin signaling and steroidogenesis were validated in vivo by qPCR and their respective proteins by Western blotting and immunofluorescence. Next, luteal cell cultures were stimulated with insulin with or without inhibition of MAPK14, MAP2K1 and PI3K. Studied proteins except P450 aromatase showed the same expression pattern of coding genes in vivo. The expression of HSD3B and CYP19A1 was higher in insulin-treated cells (P < 0.005). Following respective pathway blockades, the culture medium had decreased concentrations of progesterone (P4) and 17b-estradiol (E2) (P < 0.01). Our results indicate that insulin increases HSD3B and CYP19A1 expression via MAPK and PI3K, and contributes to the regulation of P4 and E2 production in canine luteal cells.

Gestational exposure to acrylamide suppresses luteal endocrine function through dysregulation of ovarian angiogenesis, oxidative stress and apoptosis in mice.

The discovery of acrylamide in various carbohydrate-rich foods cooked at high temperatures has attracted public health concerns. This study aimed to elucidate the effects and mechanisms additional with acrylamide exposure on the luteal function in vivo during early- and mid-pregnancy. Mice were fed with different dosages of acrylamide (0, 10 and 50 mg/kg/day) by gavage from gestational days (GD) 3 to GD 8 or GD 13. The results indicated that acrylamide exposure significantly decreased levels of serum progesterone and estradiol, and the numbers and relative areas of ovarian corpora lutea. The expression levels of Hsd3b1, Cyp11a1 and Star mRNA markedly reduced in acrylamide-treated ovaries. Furthermore, acrylamide exposure obviously suppressed the activities of catalase and superoxide dismutase, but increased the levels of H2O2 and malondialdehyde. Additionally, acrylamide treatment significantly inhibited luteal angiogenesis and induced the apoptosis of ovarian cells by up-regulation of P53 and Bax protein and down-regulation of Bcl-2 protein. Thus, our results showed that gestational exposure to acrylamide significantly inhibited luteal endocrine function via dysregulation of ovarian angiogenesis, oxidative stress and apoptosis in vivo.

Ficus deltoidea ameliorates biochemical, hormonal, and histomorphometric changes in letrozole-induced polycystic ovarian syndrome rats.

BACKGROUND: Insulin resistance and hormonal imbalances are key features in the pathophysiology of polycystic ovarian syndrome (PCOS). We have previously shown that Ficus deltoidea var. deltoidea Jack (Moraceae) can improve insulin sensitivity and hormonal profile in PCOS female rats. However, biological characteristics underpinning the therapeutic effects of F. deltoidea for treating PCOS remain to be clarified. This study aims to investigate the biochemical, hormonal, and histomorphometric changes in letrozole (LTZ)-induced PCOS female rats following treatment with F. deltoidea. METHODS: PCOS was induced in rats except for normal control by administering LTZ at 1 mg/kg/day for 21 days. Methanolic extract of F. deltoidea leaf was then orally administered to the PCOS rats at the dose of 250, 500, or 1000 mg/kg/day, respectively for 15 consecutive days. Lipid profile was measured enzymatically in serum. The circulating concentrations of reproductive hormone and antioxidant enzymes were determined by ELISA assays. Ovarian and uterus histomorphometric changes were further observed by hematoxylin and eosin (H&E) staining. RESULTS: The results showed that treatment with F. deltoidea at the dose of 500 and 1000 mg/kg/day reduced insulin resistance, obesity indices, total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL), malondialdehyde (MDA), testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) to near-normal levels in PCOS rats. The levels of high-density lipoprotein cholesterol (HDL), estrogen, and superoxide dismutase (SOD) are also similar to those observed in normal control rats. Histomorphometric measurements confirmed that F. deltoidea increased the corpus luteum number and the endometrial thickness. CONCLUSIONS: F. deltoidea can reverse PCOS symptoms in female rats by improving insulin sensitivity, antioxidant activities, hormonal imbalance, and histological changes. These findings suggest the potential use of F. deltoidea as an adjuvant agent in the treatment program of PCOS.

Transcriptional profiling of equine endometrium before, during and after capsule disintegration during normal pregnancy and after oxytocin-induced luteostasis in non-pregnant mares.

The current study used RNA sequencing to determine transcriptional profiles of equine endometrium collected 14, 22, and 28 days after ovulation from pregnant mares. In addition, the transcriptomes of endometrial samples obtained 20 days after ovulation from pregnant mares, and from non-pregnant mares which displayed and failed to display extended luteal function following the administration of oxytocin, were determined and compared in order to delineate genes whose expressions depend on the presence of the conceptus as opposed to elevated progesterone alone. A mere fifty-five transcripts were differentially expressed between samples collected from mares at Day 22 and Day 28 of pregnancy. This likely reflects the longer-term exposure to a relatively constant, progesterone-dominated environment with little change in factors secreted by the conceptus that would affect endometrial gene expression. The complement system was amongst the canonical pathways significantly enriched in transcripts differentially expressed between Day 14 and Day 22/28 of pregnancy. The expression of complement components 7 and 8 was confirmed using in situ hybridization. The expression of SERPING1, an inhibitor of the complement system, was confirmed by immunohistochemistry. In line with the resumed capacity of the endometrium to produce prostaglandin, prostaglandin G/H synthase 1 was expressed at higher levels at Days 22 and 28 than at Day 14 of pregnancy. Our data suggest that this up-regulation is enhanced by the presence of the conceptus; samples obtained from mares at Day 20 of pregnancy had significantly higher levels of prostaglandin G/H synthase 1 transcript than mares with extended luteal function.

Varied effects of doxorubicin (DOX) on the corpus luteum of C57BL/6 mice during early pregnancy†.

Certain chemotherapeutic drugs are toxic to ovarian follicles. The corpus luteum (CL) is normally developed from an ovulated follicle for producing progesterone (P4) to support early pregnancy. To fill in the knowledge gap about effects of chemotherapy on the CL, we tested the hypothesis that chemotherapy may target endothelial cells and/or luteal cells in the CL to impair CL function in P4 steroidogenesis using doxorubicin (DOX) as a representative chemotherapeutic drug in mice. In both mixed background mice and C57BL/6 mice, a single intraperitoneal injection of DOX (10 mg/kg) on 0.5-day postcoitum (D0.5, postovulation) led to ~58% D3.5 mice with serum P4 levels lower than the serum P4 range in the phosphate buffer saline-treated control mice. Further studies in the C57BL/6 ovaries revealed that CLs from DOX-treated mice with low P4 levels had less defined luteal cords and disrupted collagen IV expression pattern, indicating disrupted capillary, accompanied with less differentiated luteal cells that had smaller cytoplasm and reduced StAR expression. DOX-treated ovaries had increased granulosa cell death in the growing follicles, reduced proliferating cell nuclear antigen-positive endothelial cells in the CLs, enlarged lipid droplets, and disrupted F-actin in the luteal cells. These novel data suggest that the proliferating endothelial cells in the developing CL may be the primary target of DOX to impair the vascular support for luteal cell differentiation and subsequently P4 steroidogenesis. This study fills in the knowledge gap about the toxic effects of chemotherapy on the CL and provides critical information for risk assessment of chemotherapy in premenopausal patients.

Effects of administration of exogenous estradiol benzoate on follicular, luteal, and uterine hemodynamics in beef cows.

Objectives of these two experiments were to determine if exogenous estradiol benzoate (EB) affects follicular, luteal, and uterine hemodynamics. In both experiments, 77 estrous-synchronized beef cows were assigned to one of two treatments: 1) Control (CON) or, 2) an injection of 1 mg EB the day before expected estrus (Day 0; Experiment 1) or on the day of estrus (Day 1; Experiment 2). There was transfer of an embryo (Day 7) into cows that expressed estrus. In Experiment 1, estradiol concentrations in circulation at Day 0 were greater in EB-treated cows (P = 0.003); however, concentrations of progesterone were only greater (P = 0.03) at Day 21 in cows of the EB-treated compared to those in the CON group. The follicular and luteal blood perfusion was similar, however, treatment with EB resulted in a greater uterine blood perfusion. In Experiment 2, treatment with EB did not affect size or blood perfusion of the corpus luteum (CL) on Day 7, 14, and 21. Only on Day 21, however, did pregnant cows have a larger CL than non-pregnant cows (P = 0.02). Blood perfusion to the CL was greater (P < 0.05) in all cows on Day 21 compared to 7 or 14 and those determined to be pregnant on Day 35 tended (P = 0.06) to have greater CL blood perfusion only on Day 21 compared to non-pregnant cows. In conclusion, EB treatment resulted in a greater blood perfusion of the uterus, and only affected the CL on Day 21 in Experiment 2.

Role of cAMP/PKA/CREB pathway and ß-arrestin 1 in LH induced luteolysis in pregnant rats.

Luteal dysfunction in pregnant women is associated with early pregnancy loss, making the study of structure and function of the corpus luteum (CL) critical. Luteinizing hormone (LH) plays a crucial role in the mammalian female reproduction majorly by regulating luteal development. In rats, the luteotropic roles of LH have been widely investigated but its role in the process of luteolysis has received little attention. In this study, we explored the luteolytic actions of LH during different stages of pregnancy in rats. Repeated administration of LH during the late and mid-stages of pregnancy led to functional luteolysis during both stages, while structural luteolysis was observed only during the late-stage. We analyzed the involvement of cAMP/PKA/CREB pathway, MAP kinases and ß-arrestins to elucidate the molecular mechanism of LH-mediated luteolysis. The results indicate that the repeated administration of LH causes LH/CGR desensitization along with an increase in ß-arrestin 1 expression, while luteal expression of MAP kinases remained unaffected. Further, siRNA-mediated depletion of ß-arrestin 1 in primary luteal-cell cultures prevents initiation of the luteolysis process to some extent during both the stages of pregnancy, underscoring its role in LH mediated-luteolysis. In conclusion, the luteolytic actions of LH appear to involve more than one signaling pathway and cAMP/PKA/CREB pathway appears to be the key regulator. This is the first report to show a positive correlation between ß-arrestin 1 and 20α-hsd expression. These findings have implications for our understanding of the molecular pathways that regulate luteolysis.

Neuromodulatory effect of GnRH from coeliac ganglion on luteal regression in the late pregnant rat.

The GnRH/GnRH receptor system has been found in several extrapituitary tissues, although its physiological significance has not yet been well established. Taking into account that the peripheral neural system can act as a modulator of pregnancy corpus luteum, the objective was to physiologically investigate the presence of the GnRH system in coeliac ganglion (CG) and to analyse its possible involvement in luteal regression through the superior ovarian nerve (SON) at the end of pregnancy in the rat. The integrated ex vivo CG-SON-Ovary system of rats on day 21 of pregnancy was used. Cetrorelix (CTX), a GnRH receptor antagonist, was added into the ganglionic compartment while the control systems were untreated. Ganglionic GnRH release was detected under basal conditions. Then, the CTX addition in CG increased it, which would indicate the blockade of the receptor. In turn, CTX in CG caused an increase in ovarian progesterone release. Furthermore, the luteal cells showed an increase in the expression of Hsd3b1 and a decrease in the expression of Akr1c3 (progesterone synthesis and degradation enzymes, respectively), reduced TUNEL staining according to an increase in the antioxidant defence system activity and low lipid peroxide levels. The ovarian and ganglionic nitric oxide (NO) release increased, while the luteal nitrotyrosine content, measured as nitrosative stress marker, decreased. CTX in CG decreased the ovarian noradrenaline release. The present study provides evidence that GnRH from CG may trigger neuronal signals that promote the luteal regression in late pregnancy by affecting the release of NO and noradrenaline in the ovary.

Effects of neonatal exposure to methoxychlor on corpus luteum in gilts: A transcriptomic analysis.

This study investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities, on luteal function in pigs. Piglets were injected subcutaneously with MXC (20 µg/kg body weight) or corn oil (control) between postnatal Days 1 and 10 (N = 5/group). Corpora lutea from sexually mature gilts were examined for luteal steroid and prostaglandin concentrations and processed for total RNA isolation and subsequent RNA sequencing. Intra-luteal concentrations of androstenedione and prostaglandin E2 were greater, while that of estrone was lower when compared to control. Fifty-three differentially expressed (DE) microRNAS (miRNAs) (p-adjusted <.05 and log2(fold change) ≥.5) and 359 DE genes (p-adjusted <.05 and log2(fold change) ≥1) were identified in luteal tissue in response to neonatal MXC treatment. MXC was found to affect the expression of genes related to lipogenesis, steroidogenesis, membrane transport, immune response, cell signaling and adhesion. These results suggest an earlier onset of structural luteolysis in pigs caused by MXC actions in neonates. Since negative correlation analysis showed the potential interactions of miRNAs with specific messenger RNAs, we propose that these miRNAs are potential mediators of the long-term MXC effect on the CL function in pigs.

Prenatal dexamethasone exposure and developmental programming of the ovary of the offspring: a structural study in the rat.

Overexposure to glucocorticoids during fetal development alters fetal organ growth and maturation patterns, which can result in adverse programming outcomes in adulthood. The aim of this study was to determine whether exposure to dexamethasone (Dx) during the fetal period programmed ovary development and function in infant (16-day-old) and peripubertal (38-day-old) female offspring. Pregnant Wistar rats were separated into control and Dx-treated (0.5mg kg-1) groups and were injected with Dx or an equivalent volume of vehicle on Days 16, 17 and 18 of gestation. Ovaries from 16- and 38-day-old female offspring were prepared for histological and stereological examination. The volume of the ovary and the number of primordial and primary follicles were significantly reduced in prenatally Dx-exposed infant and peripubertal female offspring compared with control offspring. The number of multilaminar follicles was decreased in infant female offspring. In peripubertal females, prenatal exposure to Dx increased the number of multilaminar and large follicles of all classes. Because vaginal opening did not occur up to Day 38 postpartum in the Dx-exposed offspring, the absence of ovulation and corpora lutea is confirmation that the onset of puberty had been delayed. We can conclude that overexposure to glucocorticoids early in life programs ovary development, which may affect fertility in adulthood.