Local Regulatory Changes of HSD11B1 and NR3C1 in the Follicular and Luteal Tissue During Experimentally Induced Ovulation in the Cow.

The objective of the study was to characterise the expression patterns of the two key components of cortisol action namely HSD11B1 (11-beta-hydroxysteroid dehydrogenase type 1) and NR3C1 (nuclear receptor subfamily 3, group C, member 1, also known as the glucocorticoid receptor) in superovulation induced bovine follicles during the periovulation and subsequent corpus luteum (CL) formation. Bovine ovaries containing preovulatory follicles or CL were timely defined during induced ovulation as follows: 0 h before GnRH (Gonadotropin-releasing hormone) application, and 4, 10, 20, 25 (follicles) and 60 h (early CL) after GnRH. The low mRNA expression of HSD11B1 and NR3C1 in the follicle group before the GnRH application increased significantly in the follicle group 20 h after GnRH and remained high afterward also in the early CL group. In contrast, the high NR3C1 mRNA decreased in follicles 25 h after GnRH (close to ovulation) and significantly increased again after ovulation (early CL). Our results indicated the involvement of HSD11B1 and NR3C1 as the two key components of cortisol action in the local mechanisms coordinating final follicle maturation, ovulation, follicular-luteal transition and CL development in the cow.

Identification of nonpregnant beef cows based on CL size vs. luteal blood perfusion at 21 days after timed artificial insemination.

The aim was to evaluate the efficiency of two different ultrasonographic systems, Doppler mode vs. Two-dimensional mode (B mode), to identify the pregnancy status of beef cows and heifers on day 21 (D21) after Timed Artificial Insemination (TAI). The experiment was performed on a commercial beef farm in central-west region of Brazil using 1895 Nelore heifers and cows. All females had ovulation synchronized for a TAI that was performed on D0. Twenty-one days after the TAI, all animals had their ovaries evaluated by ultrasound for pregnancy diagnosis based on the size of the corpus luteum (CL). Using B mode ultrasonography, females without a CL or with a CL ≤ 10 mm in diameter were considered nonpregnant, whereas females with a CL > 10 mm in diameter were considered potentially pregnant. After the B mode examination, the Doppler mode was turned on, and the CL was evaluated by the subjective percentage of blood perfusion in the total area of the CL. Using Doppler mode, females were considered nonpregnant if they had no CL or the CL had ≤25 % of the total area with detectable blood perfusion, whereas animals with >25 % blood perfusion in the CL were considered potentially pregnant. The results for each method (potentially pregnant or nonpregnant) were later compared with the gold standard technique, which was a pregnancy diagnosis on D33 after TAI using ultrasound with visualization of an embryonic heartbeat. The accuracy was determined using the 2 × 2 contingency table approach. The area under the curve using the receiver operating characteristic curve for Doppler mode and B mode were 0.929 and 0.902 (P < 0.01), respectively. There were almost no false negatives (designated non-pregnant but later pregnant at D33) with either technique (0.2 % vs. 0.3 %; P = 0.65 for Doppler mode vs. B mode, respectively). False positives (designated pregnant but non-pregnant on D33) were greater for B mode compared to Doppler (19.1 % vs. 14.0 %; P < 0.01). This resulted in Doppler mode having similar high values as B mode for Negative Predictive Value (99.9 vs. 99.6 %; P = 0.85) and Sensitivity (99.8 vs. 99.7 %; P = 0.86) but there were differences in Specificity (86 vs. 80.9 %; P < 0.01), Positive Predictive Value (88 vs. 84.3 %; P < 0.01), and Accuracy (93.0 vs. 90.4 %; P < 0.01). In conclusion, evaluation of CL blood perfusion by Doppler produced greater accuracy in the early identification of nonpregnant heifers and cows on D21 after TAI than measurement of CL diameter with B mode ultrasound; although both had over 90 % accuracy in identifying pregnant and nonpregnant females.

Influence of commercial inactivated or modified-live virus vaccination at time of AI on corpus luteum development and function in beef cattle.

Our objective was to evaluate the effect of vaccination with an inactivated virus vaccine (IVV) or modified-live virus (MLV) vaccine on the corpus luteum (CL). On d0, synchronized beef cows were treated with MLV (n = 70; BoviShield Gold FP5VL5), IVV (n = 16; ViraShield 6VL5HB), or were unvaccinated controls (n = 5). Plasma was collected from treated animals on d0 and every other day through d22. Plasma was analyzed for concentrations of progesterone and 15 cytokines. Between d10 and d13, selected females (n = 13) were ovariectomized; controls were slaughtered on d15/16 to obtain CL for histological evaluation. There were reduced numbers of large luteal cells (LLC) in MLV compared to IVV and controls (P < 0.0001), but IVV were similar to controls (P = 0.11). MLV had decreased LLC percentage compared to controls, and IVV were intermediate (P < 0.0001, MLV: 1.57 ± 0.33 %, IVV: 2.99 ± 0.30 %, Control: 6.45 ± 0.33 %). Based on progesterone concentrations, 24 % MLV and 0 % IVV had an abnormal cycle following vaccination. Overall, MLV had reduced progesterone concentrations (P = 0.02; MLV: 3.61 ± 0.22; IVV: 4.81 ± 0.46 ng/mL). The new CL that formed following an abnormal cycle in MLV had the greatest percentage (35.56 ± 5.5 %) of apoptotic cells. Treatment by cycle status interaction, and time significantly affected IFN-γ, IP-10, MIP-1ß, and MCP-1 (P < 0.03), with several time points having elevated concentrations in abnormally cycling MLV animals. Collectively, this demonstrates MLV vaccination around estrus negatively influenced LLC, progesterone, and increased luteal apoptosis and pro-inflammatory cytokines.

Neuregulin 1 Signaling Attenuates Tumor Necrosis Factor α-Induced Female Rat Luteal Cell Death.

The corpus luteum (CL) is a transient ovarian endocrine structure that maintains pregnancy in primates during the first trimester and in rodents during the entire pregnancy by producing steroid hormone progesterone (P4). CL lifespan, growth, and differentiation are tightly regulated by survival and cell death signals through luteotrophic and luteolytic factors, including the epidermal growth factor (EGF)-like factor family. Neuregulin 1 (NRG1), a member of the EGF family, mediates its effect through ErbB2/3 receptors. However, the functional role of NRG1 in luteal cells (LCs) is unknown. Thus, this study investigated the role of NRG1 and its molecular mechanism of action in rat LC. Our experimental results suggest a strong positive correlation between steroidogenic acute regulatory protein (StAR) and NRG1 expression in mid-CL and serum P4 and estrogen (E2) production. In contrast, there was a decrease in StAR and NRG1 expression and P4 and E2 production with an increase in tumor necrosis factor α (TNFα) expression in regressing CL. Further in vitro studies in LCs showed that the knockdown of endogenous Nrg1 promoted the expression of proinflammatory and proapoptotic factors and decreased prosurvival factor expression. Subsequently, treatment with exogenous TNFα under these experimental conditions profoundly elevated proinflammatory and proapoptotic factors. Further analysis demonstrated that the phosphorylation status of ErbB2/3, PI3K, Ak strain transforming or protein kinase B (Akt), and ErK1/2 was significantly inhibited under these experimental conditions, whereas the treatment of TNFα further inhibited the phosphorylation of ErbB2/3, PI3K, Akt, and ErK1/2. Collectively, these studies provide new insights into the NRG1-mediated immunomodulatory and prosurvival role in LCs, which may maintain the function of CL.

Sexual pleasure with ruptured corpus luteum cyst that ends in emergency room: A case report.

Rupture of corpus luteum cyst from sexual pleasure is an uncommon event that has not been reported in our environment (Africa). The patient is a 30-year-old primiparous woman who developed severe lower abdominal pain thirty minutes after sex; the pain was unrelenting despite the use of over-the-counter analgesic drugs and local herbs. Twelve hours after sex, when the problem persisted and she complained of dizziness and weakness, she was rushed to the emergency room of our facility for medical treatment. An initial diagnosis of ruptured ectopic gestation was made. She had an emergency laparotomy with repair of the ruptured cyst and blood transfusion. Incisional biopsy was taken from the cyst and pathological examination revealed a ruptured corpus luteum cyst. She did well post-surgery and was discharged to follow up in the clinic on the third day after surgery. Ruptured corpus luteum cyst from sexual pleasure is a rare event. Thus, a high index of suspicion is necessary to elicit a history of sex, which patients are often unwilling to disclose.

Optimisation of Early Pregnancy Diagnosis and Resynchronisation in Beef Cows.

The objectives of this study were (1) to investigate the effects of the preovulatory follicle (POF) size on the accuracy of Doppler-based early pregnancy detection, and (2) to determine whether the removal of PGF2α (PGF) treatment during the resynchronisation protocol would affect fertility in beef cows. In Experiment 1, Nelore suckling cows (n = 224) were enrolled in an estradiol-progesterone-based timed artificial insemination (TAI) protocol. At TAI, cows were separated based on the range of POF diameters, as follows: ≤11.0 mm (n = 50), 11.1-12.9 mm (n = 64), 13.0-14.4 mm (n = 62) and ≥14.5 mm (n = 48). On day 22 after TAI, the corpus luteum (CL) blood flow (CLBF) of all cows was examined by colour Doppler ultrasonography to diagnose nonpregnant cows. The cows with the largest POF had the greatest positive predictive value (88.6%; 31 of 35) and diagnostic accuracy (91.7%; 44 of 48). In Experiment 2, Nelore cows (n = 233) were subjected to the same TAI protocol. Fourteen days after TAI, all cows were started on a resynchronisation protocol. Cows diagnosed as nonpregnant based on CLBF, on day 22, received 0.5 mg estradiol cypionate intramuscular (im) and were assigned to receive either 150 µg of PGF (PGF; n = 50) or 2 mL of saline (control; n = 47). Cows treated with PGF had a P/AI of 30.0% compared with a 48.9% P/AI in controls (p = 0.06). Our findings demonstrate that the POF size affects the accuracy of a CLBF-based early pregnancy diagnosis and that the removal of PGF treatment from the resynchronisation protocol tended to increase P/AI of the second TAI.

Neutral effect of Zishen Yutai Pill on frozen-thawed embryo transfer: a propensity score matching study.

Objective: To investigate whether using Zishen Yutai Pills (ZYP) following embryo transfer would affect the live birth rate in frozen-thawed embryo transfer (FET) cycles. Methods: A retrospective analysis was performed on 15044 FET cycles in the Reproductive Medicine Center of The Affiliated Chenggong Hospital of Xiamen University from January 2013 to December 2020. Patients who used Zishen Yutai Pills were defined as Zishen Yutai Pills Group (ZYP, n=2735), while patients who did not use them were defined as Non- Zishen Yutai Pills Group (Non-ZYP, n=12309). The propensity score matching method was used to control for potential confounders between the two groups, and logistic regression analysis was also used to assess whether using ZYP would affect the live birth rate. Results: After propensity score matching, basic characteristics were similar between the two groups. Using ZYP did not increase the pregnancy rate (51.5% vs. 52.7%, P=0.372), and live birth rate (43.0% vs. 44.7%, P=0.354). This was also confirmed by the logistic regression analysis results (OR=0.95, 95%CI=0.85-1.06). In the subgroup analysis of the endometrial preparation protocols, however, it was found that the use of ZYP in patients with natural cycles increased the live birth rate (47.4% vs. 41.5%, P=0.004). A significant interaction between endometrial preparation and ZYP was found (OR=1.38, 95%CI=1.07-1.79) in the multivariate model. Conclusion: The use of ZYP may not improve the live birth rate of unselected patients in FET cycles. However, a future study is needed on the effect of ZYP in natural cycles for endometrial preparation.

Ruptured Ovarian Cyst Leading to Massive Spontaneous Intraperitoneal Hemorrhage in Chronic Immune Thrombocytopenic Purpura (ITP): Successful Nonsurgical Intervention.

Immune thrombocytopenic purpura (ITP) patients typically exhibit mild mucocutaneous bleeding. Although corpus luteum hemorrhage (CLH) is usually asymptomatic, it can rarely result in serious intraperitoneal bleeding, usually in patients with coagulation abnormalities. In cases of severe thrombocytopenia, hemoperitoneum during ovulation during an ITP flare-up is an extremely unusual and potentially fatal event. Our patient, a 25-year-old multiparous female with a known case of ITP, presented with a spontaneous petechial rash all over the body, abdominal pain, and dizzy spells. Hemoglobin was 6.3 g/L with a platelet count of 0.04 × 109/L and a negative urine pregnancy test. An abdominopelvic ultrasound revealed free fluid in the peritoneal cavity, and a contrast-enhanced computed tomography (CECT) confirmed hemoperitoneum with a ruptured ovarian cyst. We report the case of a young female with ITP relapse who landed into massive intraperitoneal hemorrhage and was managed conservatively with platelet transfusion, steroids, and romiplostim.

Knockdown of Gonadotropin-Releasing Hormone II Receptor Impairs Ovulation Rate, Corpus Luteum Development, and Progesterone Production in Gilts.

Reproduction is classically controlled by gonadotropin-releasing hormone (GnRH-I) and its receptor (GnRHR-I) within the brain. In pigs, a second form (GnRH-II) and its specific receptor (GnRHR-II) are also produced, with greater abundance in peripheral vs. central reproductive tissues. The binding of GnRH-II to GnRHR-II has been implicated in the autocrine/paracrine regulation of gonadal steroidogenesis rather than gonadotropin secretion. Blood samples were collected from transgenic gilts, with the ubiquitous knockdown of GnRHR-II (GnRHR-II KD; n = 8) and littermate controls (n = 7) at the onset of estrus (follicular) and 10 days later (luteal); serum concentrations of 16 steroid hormones were quantified by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Upon euthanasia, ovarian weight (OWT), ovulation rate (OR), and the weight of each excised Corpus luteum (CLWT) were recorded; HPLC-MS/MS was performed on CL homogenates. During the luteal phase, serum progesterone concentration was reduced by 18% in GnRHR-II KD versus control gilts (p = 0.0329). Age and weight at puberty, estrous cycle length, and OWT were similar between lines (p > 0.05). Interestingly, OR was reduced (p = 0.0123), and total CLWT tended to be reduced (p = 0.0958) in GnRHR-II KD compared with control females. Luteal cells in CL sections from GnRHR-II KD gilts were hypotrophic (p < 0.0001). Therefore, GnRH-II and its receptor may help regulate OR, CL development, and progesterone production in gilts.

Improving Milk Yield, Milk Quality, and Follicular Functionality Behavior in Dairy Cows from the Implementation of Microencapsulated Chili Pepper Supplements in Their Diets.

The present study evaluates the effect of including microencapsulated hot chili pepper (MHCP) in the diet of crossbred dairy cows on the volume and quality of milk and on ovarian morphofunctionality. Twenty-four crossbred females in their lactating period were used. The cows were divided into two experimental groups, a control (CT) and an MHCP -supplemented group (CP) given 1 g a day per animal of microencapsulated hot chili in concentrate for 42 days. Over seven weeks of daily milk production was measured, and sample milk was collected weekly for composition analysis. Animals were subject to an ovulation synchronization protocol on day 0 (D0), and an intravaginal progesterone (P4) implant, estradiol benzoate, and prostaglandin (PGF2α) were administered. On D8, the P4 implant was removed and PGF2α, equine chorionic gonadotropin, and estradiol cypionate were administered to the animals. The ovarian dynamics were evaluated in B mode and color Doppler. There were significant differences (p < 0.05) in the group X time interaction, the volume of milk produced, and the amount in kg/day of milk components. There was a higher percentage of vascularization in the preovulatory follicle in the CP group (p ≥ 0.10). The findings show that the inclusion of MHCP in the diet of dairy cows does influence their milk production and reproduction.

Activation of P2X7 Receptor Mediates the Abnormal Ovulation Induced by Chronic Restraint Stress and Chronic Cold Stress.

Chronic stress has become a major problem that endangers people's physical and mental health. Studies have shown that chronic stress impairs female reproduction. However, the related mechanism is not fully understood. P2X7 receptor (P2X7R) is involved in a variety of pathological changes induced by chronic stress. Whether P2X7R is involved in the effect of chronic stress on female reproduction has not been studied. In this study, we established a chronic restraint stress mouse model and chronic cold stress mouse model. We found that the number of corpora lutea was significantly reduced in the two chronic stress models. The number of corpora lutea indirectly reflects the ovulation, suggesting that chronic stress influences ovulation. P2X7R expression was significantly increased in ovaries of the two chronic stress models. A superovulation experiment showed that P2X7R inhibitor A-438079 HCL partially rescued the ovulation rate of the two chronic stress models. Further studies showed that activation of P2X7R signaling inhibited the cumulus expansion and promoted the expression of NPPC in granulosa cells, one key negative factor of cumulus expansion. Moreover, sirius red staining showed that the ovarian fibrosis was increased in the two chronic stress models. For the fibrosis-related factors, TGF-ß1 was increased and MMP2 was decreased. In vitro studies also showed that activation of P2X7R signaling upregulated the expression of TGF-ß1 and downregulated the expression of MMP2 in granulosa cells. In conclusion, P2X7R expression was increased in the ovaries of the chronic restraint-stress and chronic cold-stress mouse models. Activation of P2X7R signaling promoted NPPC expression and cumulus expansion disorder, which contributed to the abnormal ovulation of the chronic stress model. Activation of P2X7R signaling is also associated with the ovarian fibrosis changes in the chronic stress model.

From the laboratory to the field: how to mitigate pregnancy losses in embryo transfer programs?

Pregnancy losses negatively affect the cattle industry, impacting economic indices and consequently the entire production chain. Early embryonic failure has been an important challenge in the embryo industry because proper identification of embryo death at the beginning of gestation is difficult. This review aimed to provide a better understanding on reproductive failure and the relationship between early embryonic loss and different reproductive biotechniques. This review also considers insights and possible strategies for reducing early embryonic loss. The strategies addressed are as follows: i) great impact of rigorous embryo evaluation on reducing embryo losses; ii) selection of recipients at the time of transfer, taking into account health and nutritional status, and classification of the corpus luteum using ultrasound, either in area or vascularization; and iii) paternal effect as one of the factors that contribute to pregnancy losses, with a focus on embryo transfer.

Autophagy in the corpus luteum correlates with tissue growth in pregnant rats.

The developmental activation of the corpus luteum (CL) structurally and functionally is critical for the temporally regulated establishment, maintenance, and termination of pregnancy in rats. In this study, we have investigated the possible involvement of autophagy in the regulation of the CL during pregnancy in rats. The expression ratio of microtubule-associated protein light chain 3 (LC3)-II/-I, a widely used indicator of autophagic activity, in the CL remained relatively stable until day 15 of pregnancy. Subsequently, it progressively increased until day 21, and then declined until day 3 postpartum. This fluctuation was closely associated with the tissue weight of the CL rather than progesterone (P4) production activity. Light and electron microscopy revealed the presence of immunoreactive LC3 aggregates and irregularly shaped autolysosome-like microstructures in the cytoplasm of luteal cells during late pregnancy. Notably, a bolus intrabursal injection of the autophagy inhibitor bafilomycin A1 on day 15 of pregnancy resulted in a significant reduction in luteal cell size and disrupted the normal alteration of circulating P4 levels. Consequently, treatment with this inhibitor increased the likelihood of the varied timing (both advanced and delayed) of delivery and led to reduced body weight in neonates when compared with the vehicle-treated control group. Our findings suggest that autophagy in the rat CL contributes to luteal tissue growth, influences P4 production, and thereby fine-tunes the regulation of gestation length in rats.

Effect of the gonadotropin surge on steroid receptor regulation in preovulatory follicles and newly formed corpora lutea in the cow.

The objective of the study was to characterize the mRNA expression patterns of specific steroid hormone receptors namely, estrogen receptors (ESRRA-estrogen related receptor alpha and ESRRB-estrogen related receptor beta) and progesterone receptors (PGR) in superovulation-induced bovine follicles during the periovulation and subsequent corpus luteum (CL) formation. The bovine ovaries (n = 5 cow / group), containing preovulatory follicles or early CL, were collected relative to injection of the gonadotropin-releasing hormone (GnRH) at (I) 0 h, (II) 4 h, (III) 10 h, (IV) 20 h, (V) 25 h (preovulatory follicles) and (VI) 60 h (CL, 2-3 days after induced ovulation). In this experiment, we analyzed the steroid receptor mRNA expression and their localization in the follicle and CL tissue. The high mRNA expression of ESRRA, ESRRB, and PGR analyzed in the follicles before ovulation is significantly reduced in the group of follicles during ovulation (25 h after GnRH), rising again significantly after ovulation in newly formed CL, only for ESRRA and PGR (P < 0.05). Immunohistochemically, the nuclei of antral follicles' granulosa cells showed a positive staining for ESRRA, followed by higher activity in the large luteal cells just after ovulation (early CL). In contrast, the lower PGR immunopresence in preovulatory follicles increased in both small and large luteal cell nuclei after follicle ovulation. Our results of steroid receptor mRNA expression in this experimentally induced gonadotropin surge provide insight into the molecular mechanisms of the effects of steroid hormones on follicular-luteal tissue in the period close to the ovulation and subsequent CL formation in the cow.

Validation of Selected MicroRNA Transcriptome Data in the Bovine Corpus Luteum during Early Pregnancy by RT-qPCR.

In cattle, the corpus luteum (CL) is pivotal in maintaining early pregnancy by secreting progesterone. To establish pregnancy, the conceptus produces interferon-τ, preventing luteolysis and initiating the transformation of the CL spurium into a CL verum. Although this transformation is tightly regulated, limited data are available on the expression of microRNAs (miRNAs) during and after this process. To address this gap, we re-analyzed previously published RNA-Seq data of CL from pregnant cows and regressed CL from non-pregnant cows. This analysis identified 44 differentially expressed miRNAs. From this pool, three miRNAs-bta-miR-222-3p, bta-miR-29c, and bta-miR-2411-3p-were randomly selected for relative quantification. Using bovine ovaries (n = 14) obtained from an abattoir, total RNA (including miRNAs) was extracted and converted to cDNA for RT-qPCR. The results revealed that bta-miR-222-3p was downregulated (p = 0.016) in pregnant females compared to non-pregnant cows with regressed CL. However, no differences in miRNA expression were observed between CL of pregnant and non-pregnant cows for bta-miR-29c (p > 0.32) or bta-miR-2411-3p (p > 0.60). In silico prediction approaches indicated that these miRNAs are involved in pathways regulating pregnancy maintenance, such as the VEGF- and FoxO-signaling pathways. Additionally, their biogenesis is regulated by GABPA and E2F4 transcription factors. The validation of selected miRNA expression in the CL during pregnancy by RT-qPCR provides novel insights that could potentially lead to the identification of biomarkers related to CL physiology and pregnancy outcome.

Unraveling the role of lipid droplets and perilipin 2 in bovine luteal cells.

Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus-mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone-induced progesterone synthesis in these cells. Lastly, in vivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.

Interferon-tau infusion into the ovine corpus luteum delays luteolysis†.

Conceptus-derived interferon-tau (IFNT) initiates maternal recognition of pregnancy in ewes by paracrine actions on the endometrium and endocrine action on the corpus luteum (CL). To examine the effect of IFNT on the CL without inducing IFN-stimulated genes (ISGs) in the endometrium, recombinant ovine IFNT (roIFNT) or bovine serum albumin was delivered directly into CLs via osmotic pumps at a rate of 10, 50, or 100 ng/h from days 9 to 12 of the estrous cycle. Endometrial and CL samples were collected on day 12. 50 ng/h of roIFNT induced ISG15 in the CL on day 12 without affecting endometrial ISG15 concentrations. In a second experiment, roIFNT (50 ng/h) was infused into the CL from days 10 to 17 of the estrous cycle and serum samples were collected daily. Serum progesterone concentrations were significantly higher from days 15 to 17 in roIFNT-infused ewes compared to controls. Levels of LHCGR, STAR, CYP11A1, HSL, OPA1, and protein kinase A mRNA and proteins were higher in the roIFNT-infused CLs compared to the controls. Levels of ISG15 and MX1 mRNA increased in the CLs of roIFNT-infused ewes but not in the endometrium. Endometrial ESR1 mRNA and protein concentrations were higher in the controls compared to roIFNT-infused ewes. In conclusion, intra-luteal delivery of roIFNT induced ISGs, stabilized steroidogenesis in the CL, and delayed luteolysis without inducing endometrial IFN-stimulated genes. Inhibition of ESR1 in the endometrium of roIFNT-infused ewes was observed suggesting that direct delivery of IFNT to the CL has an additional anti-luteolytic effect on the endometrium.

The regulation of the expression of prokineticin 1 and its receptors and its mechanism of action in the porcine corpus luteum.

Prokineticin 1 (PROK1) is an important factor in pregnancy establishment in pigs, acting at the embryo-maternal interface and the corpus luteum (CL). Estradiol-17ß (E2) is the primary pregnancy recognition signal in pigs, and its effects are augmented by luteotropic prostaglandin E2 (PGE2). On the contrary, prostaglandin F2α (PGF2α) exerts mainly a luteolytic effect. The present study aimed to elucidate whether E2, PGE2, and PGF2α regulate the expression of PROK1 and its receptors in the porcine CL and to determine the PROK1 effect on luteal endothelial cells and pathways that may be involved in this regulation. The effects of E2, PGE2, and PGF2α on the expressions of PROK1 and its receptors in the CL were studied using an in vitro model of ultrathin luteal tissue explants model. Additionally, the effects of E2 and PGE2 on the PROK1 system were determined using an in vivo approach, in which the hormones were administered into the uterine lumen to imitate their secretion by embryos. Endothelial cell proliferation was measured using the colorimetric method. E2 acting via estrogen receptors simulated the mRNA and protein expressions of PROK1 and PROKR1 in CL explants in vitro (p < 0.05). The simultaneous action of E2 with PGE2 enhanced the expression of luteal PROK1 mRNA in vitro (p < 0.05). Estradiol-17ß acting alone significantly increased PROK1 mRNA levels in vivo, whereas E2 simultaneously administered with PGE2 significantly elevated the PROK1 mRNA expression and PROKR1 mRNA and protein contents in CLs adjacent to uterine horns receiving hormonal infusion compared with CLs adjacent to placebo-treated uterine horns (p < 0.01). The PROK1 protein expression was significantly higher in the CLs of pigs treated with E2, PGE2, and E2 together with PGE2 than in the control group. PGF2α increased the PROK1 mRNA content in CLs on days 12 and 14 of the estrous cycle (p < 0.05). The expression of PROKR2 at the mRNA and protein levels remained unchanged in response to in vitro and in vivo treatments. PROK1 stimulated the proliferation of luteal endothelial cells by activating the MAPK, AKT, and mTOR pathways (p < 0.05). In summary, the luteal expressions of PROK1 and PROKR1 in early pregnancy are regulated by E2 and PGE2. PROK1 stimulates luteal angiogenesis by activating the MAPK, AKT, and mTOR pathways. The regulation of luteal PROK1 expression by PGF2α indicates PROK1's putative role during luteolysis. We conclude that PROK1-PROKR1 signaling supports luteal function during CL rescue in pregnancy in pigs.

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

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

Is prolonged luteal phase a problem in lactating Holstein cows?

In this study, the main objective was to assess if long luteal phases could have causes other than pregnancy loss. We enrolled Holstein dairy cows ≥50 DIM from a commercial herd in Brazil from October 2016 to August 2017. All cows received an estradiol-based synchronization protocol, and, on the day of insemination (d 0), were randomly assigned either an AI or a placebo insemination (PBO) in a 3:1 ratio. An ultrasound was used to assess the presence of a corpus luteum (CL) on d 17, 24, and 31, which, combined to the information from patches for the detection of estrus, was used to determine the length of the luteal phase following AI or PBO. Pregnancy was assessed by ultrasound on d 31 and cows that were pregnant were excluded from the analyses. The length of the estrous cycles was categorized as short (<17 d), normal (17-23 d), long (24-30 d), and very long (≥31 d). We compared the proportion of cows in each category between the AI and PBO groups using a cumulative ordinal mixed model. We define prolonged luteal phase as estrous cycles ≥24 d and tested its association with potential risk factors (parity, season, DIM, uterine size and position score, milk production, BCS, and the presence of a CL at enrollment to the synchronization protocol) using mixed logistic regression models. Results are presented as odds ratio (OR) and 95% Bayesian credible intervals (BCI). Data from 876 inseminations (AI: n = 616, PBO: n = 260) was collected. Overall, 12% of estrous cycles were short, 31% were normal, 19% were long, and 38% were very long. There was no difference in the odds of being in longer estrous cycle categories for the AI compared with the PBO group (OR = 0.92; 95% BCI = 0.76-1.10). Season and presence of a CL at enrollment were associated with prolonged luteal phase. In the AI group, there was a possible effect of early pregnancy losses on the lifespan of the CL, but not the PBO group, which led us to conclude that long and very long estrous cycles were not all caused by the embryonic loss. In fact, the high prevalence of cows with an extended CL lifespan in the present study suggests this could be an under- or miss-reported characteristic of high-producing lactating Holstein cows. This finding may have important repercussions in the understanding of the CL function physiology of lactating Holstein cows.