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Despite the negative environmental and biologic effects, organophosphates have currently been widely used. We aimed to examine the possible negative effects of diazinon, a type of organophosphate, on rat ovarian tissue. Wistar Albino rats were divided into four groups. No treatment was given to control, olive oil was applied to sham group. Experimental groups were injected intraperitoneally with 30 and 60 mg/kg/day diazinon, respectively. 24 h later, ovarian tissues were extracted, preparated, examined via light and electron microscope. In the experimental groups granulosa and corpus luteum showed degenerative changes. Dilatation of endoplasmic reticulum cisterns and morphological alterations of mitochondria in granulosa cells were detected utrastructurally. Also, accumulation of lipid droplets and autophagic vacuoles was observed in cells of corpus luteum. A statistically significant dose-dependent decrease in superoxide dismutase and catalase reactivity and a statistically significant increase in caspase-3 expression in cells of atretic follicles and corpus luteum were observed. Results show that exposure to a single dose of diazinon may disrupt antioxidant system, trigger atresia in follicles and negatively effect corpus luteum functions. It was concluded that studies applying possible antioxidant treatments should be carried out to reduce and prevent the negative effects of diazinon on the reproductive system., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Scope: Luteal phase deficiency (LPD) is the main cause of infertility without an effective cure. Quercetin (QUE) is a bioactive flavonoid with antioxidant properties, while its role in treating LPD remains unclear. This study aims to investigate the therapeutic effects of QUE on infertility and menstrual disorders induced by LPD, thus further exploring the underlying mechanism., Methods and Results: Mifepristone-induced rats are used to explore the protective effects of QUE against LPD. QUE stimulates the spontaneous secretion of progesterone to improve luteal function and endometrial receptivity in LPD rats by activating the kisspeptin/GPR54 system to facilitate the gonadotropin-releasing hormone (GnRH) pulsatility. Bioinformatics analysis reveals that the core mechanism of QUE in treating LPD is to attenuate the GnRH neuron pyroptosis by inhibiting the NF-κB pathway, which is further verified in LPD rats and lipopolysaccharide (LPS)-treated GT1-7, as QUE significantly reduces the expression of key factors concerning NF-κB pathway and NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome., Conclusion: This study first proposes that neuron pyroptosis-induced GnRH pulsatility disruption accounts for the pathogenesis of LPD, and QUE facilitates the pulse secretion of GnRH to boost the spontaneous progesterone secretion by inhibiting NF-κB/NLRP3-mediated neuron pyroptosis, which provides a new therapeutic target and strategy for LPD., (© 2024 Wiley‐VCH GmbH.)

This study evaluated the use of flunixin meglumine to prevent the occurrence of premature corpus luteum (CL) regression in superovulated ewes, improving embryo recovery and viability. Ewes (n=23) submitted to conventional superovulatory protocol and laparoscopic artificial insemination were treated with 2.2 mg/kg/day of flunixin meglumine (FLU, n=12) or 1.5 mL saline solution (CONT, n=11) on Days 2, 3, and 4 (Day 0 = 48 h after device removal). Serum progesterone (P 4 ) concentrations were measured (Day 1-6). Ultrasound (US, Days 3 and 6) and laparoscopic evaluation (Day 6) were performed to identify luteinized structures. In the US, laparoscopy, and P 4 assessments, the percentage of ewes with premature CL regression differed (P<0.05) between CONT (54.5; 63.6; and 54.5 %) and FLU (0.0; 0.0; and 0.0 %), respectively. The US exams revealed the effect (P<0.05) of treatment on the number of regressing CL between CONT (1.4 ± 0.6) and FLU (0.0 ± 0.0). Greater (P<0.05) number of normal CLs (10.5 ± 1.8 vs. 4.4 ± 1.5), ova/embryos (9.1 ± 2.1 vs. 3.7 ± 1.3), viable embryos (5.1 ± 1.1 vs. 2.6 ± 1.2), and recovery rate (79.5 ± 9.6 vs. 41.3 ± 15.0 %) were observed in FLU compared to CONT, respectively. The embryo viability rate did not differ (P>0.05) between FLU (60.7 ± 10.5 %) and CONT (45.5 ± 16.1 %). In conclusion, the flunixin meglumine protocol was able to prevent the occurrence of premature CL regression in superovulated ewes, increasing the recovery rate and embryo production., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cervical relaxation (CR) was performed in ewes during diestrus, to prospect a feasible protocol for non-surgical embryo transfer (NSET). In Trial 1, naturally mated ewes (n=13) received CR protocols with estradiol benzoate (EB, 1 mg on D6) and oxytocin (OT, 50 IU on D7) only (G-EB+OT) or associated with human chorionic gonadotropin (hCG, 300 IU on D7, G-EB+OT+hCG) and were compared to non-hormonally treated (G-control) ewes. Estradiol concentration increased (P<0.05) from D6 to D7 in G-EB+OT and G-EB+OT+hCG, then decreased (P<0.05) on D8. The G-EB+OT had reduced (P<0.05) progesterone on D8, and 0% (0/5) pregnancy, while G-control had 100% (3/3) conception rate. The treatment with hCG mitigated the progesterone drop on D8 and resulted in 50% (2/4) conception rate. In Trial 2, cervical transposing success after treatment with EB, OT, and hCG on D6 and D7 was assessed in nulliparous (n=25) and multiparous (n=30) ewes. The EB+OT+hCG protocol allowed efficient cervical transposition (~78% of cervical transposition success into ~5 min) in nulliparous and multiparous females and did not alter total and blood perfusion areas of corpora lutea (CL). The use of EB+OT+hCG was efficient to ensure effective cervical transposing in nulliparous and multiparous ewes, with no apparent deleterious effect on CL function, and may contribute to animal welfare, allowing a less invasive NSET procedure.

The corpus luteum is a temporary endocrine gland that is crucial for pregnancy, as it produces the progesterone needed to maintain optimal uterine conditions for implantation. In the absence of a conceptus, the corpus luteum becomes non-functional and undergoes rapid tissue remodeling to regress into a fibrotic corpus albicans. Early luteal regression is characterized by increased cytokine release. Because the role of fibroblasts in the bovine corpus luteum remains to be elucidated, the aim of this study was to elucidate the response of bovine luteal fibroblasts to inflammatory cytokines, tumor necrosis factor α (TNFα), and interleukin 1β (IL1β). Both cytokines induced canonical mitogen activated protein kinase (MAPK) signaling in luteal fibroblasts by phosphorylation of ERK1/2, p38 MAPK, and JNK. IL1β elevated expression and phosphorylation of cytosolic phospholipase A2 (cPLA 2 ), an enzyme that mobilizes arachidonic acid for prostanoid synthesis. IL1β also elevated expression of prostaglandin-endoperoxide synthase 2 (PTGS2), another enzyme needed to synthesize prostanoids. IL1β increased PGF2α and PGE 2 levels in the culture medium over 20-fold. Inhibition of MAPKs with small-molecule inhibitors abrogated the stimulatory effects of IL1β. IL1β also induced prostaglandin production in steroidogenic cells; however, there was no elevation in cPLA 2 . Therefore, actions of IL1β differ based on ovarian cell type. All together, we have identified luteal fibroblasts as potential inflammatory mediators during luteal regression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

The capacity of recipients to support transferred embryos is a challenge for dairy cattle production systems. This review explores methods for increasing P4 to improve early pregnancy establishment in dairy cattle. A key aspect of P4 supplementation during the critical first weeks of pregnancy is its correlation with successful embryo elongation. Moreover, P4 was correlated to an increased chance of embryo survival after maternal recognition of pregnancy. Internal releasing devices increase circulating P4 concentrations but may negatively impact fertility possibly due to unwanted health pathologies such as vaginitis and the still undefined mechanism of haste luteolysis. The accessory corpus luteum (CL) induction in recipient dairy cattle increases endogenous P4 output increasing circulating P4 concentrations. Two ovulatory inducers are commonly used: gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG). Despite their distinct structures, GnRH and hCG are equally capable of inducing ovulation in cattle. The P4 supplementation by an accessory CL formation was widely used in fixed-time artificial insemination breeding programs, but still not exhaustively studied in embryo transfer (ET) programs. The induction of an accessory CL in ET programs has yielded inconsistent results depending on parity, embryo type production, embryo stage of development, and type of hormone used. Although maternal physiological mechanisms support the idea of increasing P4 for higher fertility and better embryo development, the actual results in creating a high-P4 maternal environment are still uncertain., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Prokineticin 1 (PROK1) is an important factor in pregnancy establishment in pigs, acting at the embryo-maternal interface and the corpus luteum (CL). Estradiol-17β (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-17β 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., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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 Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Nanoplastics (NPs) affect fertility. We evaluated the effects of NPs treatment on luteal and endothelial cells. We examined crucial markers of growth and redox status. NPs treatment did not induce changes in ATP levels in luteal cells, while it increased (p< 0.05) their proliferation. In endothelial cells, no change in proliferation was detected, while an increase (p<0.05) in ATP levels was observed. The increase of reactive oxygen species, superoxide anion (p<0.05) and nitric oxide (p<0.001) was detected in both cell types, which also showed changes in superoxide dismutase enzyme activity as well as an increase of non-enzymatic antioxidant power (p<0.05). A decrease (p<0.05) in progesterone production as well as an increase of vascular endothelial growth factor A levels were detected (p<0.05). In addition, a dose-dependent accumulation of NPs in endothelial cells was shown, that likely occurred through adhesion and internalization. Results underline potential risk of NPs for corpus luteum functionality., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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, 3β-HSD, and aromatase proteins and decreasing the BAX/BCL2 ratio. Exogenous adropin treatment increased progesterone production by increasing the expression of GPR19, StAR and 3β-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., Competing Interests: Declaration of Competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Commercial embryo flushing of horses has required hormonal management of both the donor and recipient mares throughout the breeding season., Aim: This study aimed to find out the effect of using human chorionic gonadotropin (hCG) and prostaglandin F2α (PG) on the ovarian and uterine dynamics and hemodynamics, estradiol (E2), progesterone, oxidants-antioxidants, and blood biochemicals in embryo donor mares during the hottest months of the year in a subtropical climate., Methods: Three Control estrous cycles of native mares (10-20 years; N = 10) followed by two treated cycles with hCG and PGF2α were examined daily from May to August using Doppler ultrasound with blood sampling. Circulating, progesterone (P4), total cholesterol, total proteins, albumin, haptoglobin, nitric oxide (NO), catalase, alkaline phosphatase, lactate dehydrogenase (LDH), and myeloperoxidase were measured in blood serum., Results: Days during the control estrous cycle impacted the dominant follicle (DF) diameter ( p < 0.0001), antrum diameter ( p < 0.0001), area ( p < 0.0001), antral area ( p < 0.0001), and color area % ( p > 0.05), and corpus luteum (CL) diameter ( p < 0.0001). PG tended to impact DF diameter ( p > 0.05) but influenced its antrum diameter ( p < 0.05), color area ( p < 0.05), CL diameter ( p < 0.01), and area ( p = 0.013). Days after hCG tended to impact DF antrum diameter ( p > 0.05) and the antrum area ( p > 0.05), but influenced CL diameter ( p < 0.0001). PGF2α and hCG increased uterine horn area ( p = 0.016) and color area ( p = 0.023), total cholesterol ( p < 0.0001), and NO ( p < 0.0001) levels but hCG increased the levels of myeloperoxidase ( p < 0.005), total proteins ( p < 0.001), and albumin ( p < 0.0001). Globulins achieved the highest level ( p = 0.054) but the Albumin/globulin ratio reached a minimum value on Day 0 of the control mares ( p < 0.0001). PGF2α increased LDH ( p < 0.0001) and sharply declined ( p = 0.028) progesterone., Conclusion: In conclusion, the treatment protocols of hCG and PGF2α showed minimal effects on the produced ovulating follicles and can be used during the summer season to manage embryo donor mares., Competing Interests: The authors declare that they do not have any conflict of interest.

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 PGF 2α 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., Competing Interests: Declaration of Competing Interest The authors declare that they hold no financial or personal affiliations with anyone or organizations capable of improperly influencing or biasing this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

Background: PGF 2α 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 PGF 2α ., 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). PGF 2α 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 PGF 2α was unaffected; however, there is a considerable risk of unwanted pregnancy (5/27 = 18.5%) in donors from which an embryo was not recovered., (© 2024 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

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., (© 2024. The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE).)

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 (P 4 , E 2 ) and prostaglandin (PGE 2 , PGF 2α ), 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 P 4 , while on days 10-12 it stimulated P 4 . Visfatin increased secretion of E 2 on days 2-3, PGE 2 on days 2-3 and 10-12, reduced PGF 2α 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., (© 2024. The Author(s).)

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 (PGF 2α ) on estrus behaviour, follicular and luteal morphometry, and serum estradiol (E 2 ) and progesterone (P 4 ) 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 PGF 2α 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 (E 2 ) concentration (66.01 ± 11.92 vs. 104.9 ± 13.21 pg/mL, p = .003) observed at the post-treatment period in the PGF 2α treatment group. At the same time, CL diameter was reduced significantly at a higher regression rate in the PGF 2α 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 PGF 2α 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 E 2 and P 4 concentration would be useful to determine the right time of insemination in sub-estrus buffaloes during non-breeding season., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

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 P 4 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 P 4 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 P 4 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 P 4 concentration and improved luteal function., Competing Interests: Declaration of Competing Interest This research paper is submitted to be consider for publication as a full text. It has not been published before or simultaneously submitted for publication elsewhere. All authors have approved the final version of the manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

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 (PGF 2α ) alone and in combination with trace minerals on the follicular and corpus luteum (CL) dynamics, serum estradiol (E 2 ) and progesterone (P 4 ) 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 PGF 2α , Inj. Cloprostenol 500 μg, i.m, n = 17), T2 (Synthetic PGF 2α  + 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 P 4 concentration were significantly lower at post-treatment, whereas the pre-ovulatory follicle (POF) size and serum E 2 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 E 2 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 E 2 concentration were relatively higher in the buffaloes conceived as compared to those not conceived. It is concluded that synthetic PGF 2α 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 E 2 and P 4 concentration could be used to optimise the breeding time for augmenting the conception rate in sub-estrus buffaloes., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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 H 2 O 2 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2021. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the effects of tubal ligation (TL) via modified Pomeroy method on ovarian reserve and to determine the role of curcumin (Curcuma longa [Indian saffron]) against ovarian reserve decrement after TL. Forty-eight albino Wistar rats were randomly divided into four groups: (1) Control group: a sham operation was performed (n = 12), (2) Tubal ligation group: TL was performed (n = 12), (3) TL+DMSO group: 1 mL/day dimethyl sulfoxide was used for 50 days after TL, (4) TL+Curc group: 100 mg/kg/day curcumin dissolved in DMSO was administrated for 50 days after TL. Pre-operatively and on post-operative day 50, blood samples were collected for AMH evaluation, and oophorectomy was performed for histological and immunohistochemical examinations of ovaries in all groups. No difference in the basal AMH levels was found among the groups (p = 0.249). Compared to the basal, AMH levels were lower in the control, TL, and TL+DMSO groups (p = 0.003, p = 0.004, and p < 0.001, respectively) but not different in the TL+Curc group (p = 0.503) on post-operative day 50. No significant differences in the number of primary, preantral, antral, atretic follicles, and corpus luteum among the groups (p > 0.05) were found. The percentage of granulosa cells stained for caspase-3 in antral follicles and the corpus luteum was higher in the TL+Curc group than in the control and TL groups ([antral follicles; p < 0.01 for both groups], [corpus leteum; p = 0.009 and 0.002 for the control and TL groups, respectively]). It seems that TL does not decrease ovarian reserve and curcumin might have a positive effect on ovarian reserve in the setting of TL., (© 2021. Society for Reproductive Investigation.)

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 β-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 β-arrestin 1 expression, while luteal expression of MAP kinases remained unaffected. Further, siRNA-mediated depletion of β-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 β-arrestin 1 and 20α-hsd expression. These findings have implications for our understanding of the molecular pathways that regulate luteolysis.

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.

When women with small ovarian reserves are subjected to assisted reproductive technologies, high doses of gonadotropins are linked to high oocyte and embryo wastage and low live birth rates. We hypothesized that excessive follicle-stimulating hormone (FSH) doses during superovulation are detrimental to ovulatory follicle function in individuals with a small ovarian reserve. To test this hypothesis, heifers with small ovarian reserves were injected twice daily for 4 days, beginning on Day 1 of the estrous cycle with 35, 70, 140, or 210 IU doses of Folltropin-V (FSH). Each heifer (n = 8) was superovulated using a Williams Latin Square Design. During each superovulation regimen, three prostaglandin F2α injections were given at 12-h interval, starting at the seventh FSH injection to regress the newly formed corpus luteum (CL). Human chorionic gonadotropin was injected 12 h after the last (8th) FSH injection to induce ovulation. Daily ultrasonography and blood sampling were used to determine the number and size of follicles and corpora lutea, uterine thickness, and circulating concentrations of estradiol, progesterone, and anti-Müllerian hormone (AMH). The highest doses of FSH did not increase AMH, progesterone, number of ovulatory-size follicles, uterine thickness, or number of CL. However, estradiol production and ovulation rate were lower for heifers given high FSH doses compared to lower doses, indicating detrimental effects on ovulatory follicle function., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 E 2 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., (© 2021 Wiley Periodicals LLC.)

This study hypothesizes that melatonin with exogenous progesterone (CIDR) can improve follicular, luteal, ovarian and uterine haemodynamic of heat-stressed cows. Holstein cows (N = 12) studied for two spontaneous oestrous cycles during winter then divided equally during summer into the CIDR group received CIDR for 7 days and the melatonin group (Mel) received three injections of melatonin (75 mg/head) at the CIDR insertion, removal and ovulation days. Blood samples were collected to assay oestradiol (E2), progesterone (P4) and nitric oxide (NO). On day 0 (Ovulation), Mel had more small follicles (p < .05), higher ipsilateral and contralateral ovarian arteries (Ov.A.) peak systolic velocity (PSV), higher ipsilateral uterine artery (Ut.A.) PSV (p = .031) and blood flow volume (BFV), also Mel elevated contralateral Ut.A. PSV and BFV (p < .0001) but lowered contra Ut.A. pulsatility index (PI, p < .0001), E2 (p < .01) and NO (p < .0001). Mel increased the corpus luteum diameter (CL, p < .001), coloured area (p < .007) and P4 (p < .0001) on day 5 and reduced them (p < .05; p < .01) on Day 14. On day 10, Mel obtained CL diameter (p < .03) and coloured area (p < .002) of spontaneous that was higher than CIDR and decreased P4 (p < .003). Mel increased CL diameter, area and coloured area and decreased them thereafter. Mel increased the ipsilateral ovarian and uterine arteries PSV and BFV before ovulation and until day 8. Mel increased P4 and decreased NO until days 6 and 14. In conclusion, the improvement in follicular, luteal, ovarian and uterine haemodynamic and the decrease of NO production proved our hypothesis Melatonin doses higher than 75 mg/head is recommended to improve the heat-stressed cow's fertility., (© 2021 Wiley-VCH GmbH.)

Based on our previous study in follicles, the first aim of this work was to evaluate the effect of melatonin in the swine corpus luteum (CL). Luteal cells were exposed to 10 and 20pg mL-1 melatonin. We evaluated the effect on proliferation (bromo-deoxy-uridine uptake), steroidogenesis (progesterone) and redox status by means of Griess test (nitric oxide production), WST-1 test (superoxide anion generation) and FRAP test (non-enzymatic antioxidant power). The results showed a significant increase in antioxidant power, as well as a reduction in the other parameters analysed. These data and the expression of MT2 observed in luteal cells allow us to hypothesise a physiological role of melatonin in the regulation of CL functionality. The reproductive function is dependent on energy reserves stored in adipose tissue. Therefore, we sought to verify the effect of melatonin on adipose stromal cells (ASCs). MT2 receptor expression was detected in ASCs and the presence of gene markers (PPARγ and leptin) before and after adipogenic differentiation was verified. The differentiation was significantly inhibited by melatonin, as well as cell viability. In conclusion, present results suggest that melatonin exerts a potential inhibitory action on luteal function and adipogenesis, possibly mediated by MT2.

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.

The objective of this study was to compare the effects of different lengths of ovulation synchronization protocols using 2 controlled internal drug release (CIDR) devices on ovarian dynamics and pregnancy outcomes in lactating dairy cows. Lactating Holstein cows (n = 1,979) were randomly assigned to receive timed artificial insemination (TAI; d 0) following 1 of 2 treatments: (1) 9-d protocol (n = 988; 9D) with 2 intravaginal devices containing 1.9 g of progesterone (CIDR) and 2.0 mg of estradiol benzoate on day -11; 25 mg (i.m.) of dinoprost tromethamine (PG) and withdrawal of 1 CIDR on d -4; 1.0 mg (i.m.) of estradiol cypionate, second CIDR withdrawal, and PG on d -2; and TAI on d 0 and (2) 10-d protocol (n = 991; 10D) with 2 CIDR and 2.0 mg of estradiol benzoate on d -12; 25 mg of PG and withdrawal of 1 CIDR on d -4; 1.0 mg of estradiol, second CIDR withdrawal, and PG on d -2; and TAI on d 0. There was no effect of protocol on estrus detection, whereas a greater percentage of cows from 10D had ovulated close to TAI [no corpus luteum (CL) at AI and a CL at d 7] versus cows assigned to 9D protocol. A protocol × heat stress (average cow temperature ≥39.1°C on day of AI and d 7) interaction was observed in a manner that pregnancy per AI (P/AI) was greater in non-heat-stressed 10D versus 9D cows, whereas P/AI did not differ when cows were under heat stress. Furthermore, 10D protocol did not increase P/AI when all cows that received AI were included in the analysis or in cows that ovulated near TAI. However, animals assigned to 9D without any event of heat stress had a reduced P/AI when compared with cows assigned to 10D without heat stress. A protocol × CL presence at the beginning of the protocol interaction was observed and cows with a CL at the beginning of the protocol had a greater P/AI in 10D versus 9D; meanwhile, in cows without a CL, no differences on P/AI were observed. The protocol × CL presence at the beginning of the protocol interaction on P/AI was also observed for cows that ovulated near TAI. A greater percentage of cows assigned to 9D had follicles of medium size (13-15.9 mm), and greater percentage of cows assigned to 10D had larger follicles (>16 mm). Increasing the length of an estradiol with progesterone-based ovulation synchronization protocol (10D vs. 9D) increased the proportion of cows with larger follicles (>16 mm) and increased P/AI in cows without heat stress and in cows with a CL at beginning of the protocol. Moreover, the 10D protocol increased the proportion of cows with ovulation near TAI, demonstrating the effectiveness of this protocol in improving the reproductive performance of lactating Holstein cows., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The specific changes in antral follicle numbers and wave-like development have remained unrevealed in cyclic ewes fed high-protein, high-energy lupin grain for 6 days during the luteal phase of the estrous cycle (i.e., short-term nutritional flushing). This study was mainly conducted to determine ovarian effects of the 6-day lupin grain feeding in non-prolific Polish Mountain ewes, using transrectal ovarian ultrasonography and abdominal videoendoscopy. Estrus and ovulations were synchronized in 24 ewes with progestin-releasing intravaginal sponges for 12 days during the middle portion of the breeding season (September-October; 50.0458&deg;N, 19.8406&deg;E). Twenty-four ewes were assigned to three equal groups (n=8 each), including the Control group being fed the maintenance diet (i.e., hay-only), Treatment 1 receiving 500 g of lupin grain once a day, and Treatment 2 receiving 250 g of lupin grain twice a day, from days 9-14 of the synchronized estrous cycle (day 0=first ovulation of the interovulatory period studied). No differences were observed in the mean ovulation rate among the three groups of Polish Mountain ewes (P&gt;0.05). Ovarian antral follicles emerging in the penultimate wave of the estrous cycle in Treatment 2 ewes had a longer growth phase (p &lt;0.05) and attained a greater diameter (p &lt;0.05) before ovulation, in comparison to those in the other two groups. A final wave of the interovulatory interval emerged ~1 day earlier in Treatment 2 than in Treatment 1 ewes (p &lt;0.05). Nutritional supplementation with lupin grain increased the number of 3-mm follicles in Treatment 2 ewes (p &lt;0.05). The results of this study indicated that short-term nutritional flushing with lupin grain from mid- to late luteal phase did not consistently enhance ovulatory responses in non-prolific genotypes of ewes. Although the administration of lupins altered the timing of wave emergence, ovulatory follicle diameter, or duration of different stages of the follicular lifespan, it failed to increase the number of ovulatory follicles emerging in the penultimate and final waves of the estrous cycle in non-prolific Polish Mountain sheep., (Copyright © 2021, Author(s). Published by Kowsar.)

Benzo(a)pyrene (B(a)P) is a widespread persistent organic pollutant (POP) and a well-known endocrine disruptor. Exposure to BaP is known to disrupt the steroid balance and impair embryo implantation, but the mechanism under it remains unclear. The corpus luteum (CL), the primary source of progesterone during early pregnancy, plays a pivotal role in embryo implantation and pregnancy maintenance. The inappropriate luteal function may result in implantation failure and spontaneous abortions. Therefore, this study was conducted to assess the effects and potential mechanisms of B(a)P on the CL function. Our results showed that pregnant mice received B(a)P displayed impaired embryo implantation and dysfunction of ovarian CL. The estrogen and progesterone levels decreased by B(a)P. In vitro, exposure to BPDE, which is the metabolite of B(a)P, affected the luteinization of granular cell KK-1. Additionally, melatonin and its receptors, which are important for ovarian function and anti-oxidative damage, were affected by B(a)P or BPDE. B(a)P or BPDE-treated alone impaired antioxidant capacity of ovarian granulosa cells, caused an increasing of ROS and cell apoptosis, and disrupted the PI3K/AKT/GSK3β signaling pathway in vivo and in vitro. Co-treatment with melatonin alleviated B(a)P or BPDE-induced CL dysfunction by ameliorating oxidative stress, counteracting phosphorylation of PI3K/AKT/GSK3β signaling pathway, decreasing the apoptosis of the ovarian cells. Moreover, activation of the melatonin receptor by ramelteon in KK-1 cells exhibits an analogous protective effect as melatonin. In conclusion, our findings not only firstly clarify the potential mechanisms of BaP-induced CL dysfunction, but also extend the understanding about the ovarian protection of melatonin and its receptors against B(a)P exposure., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

This study was conducted to assess effects of different doses of pFSH on follicular recruitment, superovulatory response, ova/embryo recovery, and embryo yield in lactating ewes. Ewes (n = 24) had a superovulation treatment regimen imposed. All ewes were implanted with a progesterone intravaginal device for 9 d, and administered either 100 (G-100) or 200 (G-200) mg pFSH, proportioned into six doses administered at 12-h intervals, starting 60 h before device removal. At 7 days subsequent to progesterone device removal, there were non-surgical embryo recoveries (NSER) from ewes having three or more corpora lutea. At the time of the first pFSH injection, number of antral follicles were similar (P < 0.05) between ewes in the G-100 and G-200 group, however, there were more 3.1-4.0 mm follicles in ewes of the G-200 than G-100 group at the time of the second pFSH administration. Estrous response and CL number were less (P < 0.05) in ewes of the G-100 (66.7 % and 2.6 ± 0.7) than G-200 (91.7 % and 11.6 ± 1.2) group. There were embryo collections from 100 % and 90.9 % of ewes in the G-100 and G-200 groups, respectively (P > 0.05). Viable embryo numbers and ova/embryo recovery rate were greater (P < 0.05) in ewes of the G-200 (6.9 ± 1.1 and 67.8 %) than G-100 (1.0 ± 0.5 and 27.6 %) group. A dose of 200 mg pFSH was more effective in inducing a superovulatory response and embryo yield after NSER in ewes, however, the 100 mg dose was insufficient for these purposes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Visfatin is a crucial adipokine, which also regulates ovarian functions in many animals. Mice estrous cycle is characterized by a dynamic complex physiological process in the reproductive system. Expression of various factors changes during the estrous cycle in the ovary. To the best of our knowledge, no previous study has been conducted on the expression of visfatin in mice ovaries during the estrous cycle. Therefore, we investigated the localization and expression of visfatin protein in the ovary of mice during the estrous cycle. Western blot analysis showed the elevated expression of visfatin in proestrus and lowest in diestrus. Immunohistochemical localization of visfatin showed intense staining in the corpus luteum of proestrus and diestrus ovaries. Thecal cells, granulosa cells, and oocytes also showed the presence of visfatin. Expression of ovarian visfatin was correlated to BCL2 and active caspase3 expression and exhibited a significant positive correlation. Furthermore, in vivo inhibition of visfatin by FK866 in the proestrus ovary down-regulated active caspase3 and PCNA expression, and up-regulated the BCL2 expression. These results suggest the role of visfatin in the proliferation and apoptosis of the follicles and specific localization of visfatin in the corpus luteum also indicate its role in corpus luteum function, which may be in progesterone biosynthesis and regression of old corpus luteum. However, further study is required to support these findings. In conclusion, visfatin may also be regulating follicular growth during the estrous cycle by regulating proliferation and apoptosis., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

This study was conducted in ewes to assess effects of human chorionic gonadotropin (hCG) administration after imposing an estrous induction treatment regimen. Ewes (n = 115) were treated with a 60 mg medroxyprogesterone-intravaginal-sponge for 6 d plus 200 IU of equine chorionic gonadotropin (eCG) im and 37.5 μg d-cloprostenol im 36 h before sponge removal (Day 0). After natural mating, ewes having at least one corpus luteum (CL; n = 108) were administered either 1 mL of saline (G-Control; n = 53) or 300 IU of hCG (G-hCG; n = 55) on Day 7.5 after sponge removal (Day 0). Ovarian ultrasonography and blood collection were performed on Days 7.5, 13.5, 17.5, 21.5, and 30.5. Accessory CL (aCL) were observed in 81.5 % (G-hCG) and 0.0 % (G-Control) of ewes (P = 0.0001). Diameter, area, and volume of luteal tissue were greater (P < 0.05) in G-hCG from Day 13.5 to 30.5. Progesterone (P 4 ) concentrations were greater (P < 0.05) on Days 13.5, 17.5, 21.5 and 30.5 for ewes of the G-hCG group. Pregnancy percentage was similar (P = 0.25) between groups [47.1 % (G-control) compared with 60.0 % (G-hCG)], although total number of lambs produced by estrous synchronized ewes was greater (P = 0.005) in ewes of the G-hCG group (90.9 % compared with 66.0 %). In conclusion, hCG administration 7.5 days after sponge removal from Morada Nova ewes during the non-breeding season is an effective treatment to induce aCL formation, improve luteal tissue biometry and P4 concentrations, and to enhance the total number of lambs born., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives were to evaluate effects of a smaller than typically used dose of equine chorionic gonadotropin (eCG) during a fixed-time artificial insemination (FTAI) treatment regimen. Transrectal ultrasonic (US) examinations were conducted on dairy cows on Day 0 (D0) and the treatment regimen was initiated with administrations of an intravaginal progesterone (P 4 ) implant, estradiol benzoate (im), and prostaglandin F 2α (PGF 2α; im). On D8, the P 4 implant was removed and PGF 2α and estradiol cypionate were administered to all animals. Subsequently, cows were randomly assigned to three groups and eCG was administered to Groups 1, 2, and 3 in doses of 300 (im); 100 (im); and 100 (Baihui acupoint) IUs, respectively. The B-mode and power-flow US cineloops were performed to assess follicular dynamics and evaluate various morphological and vascular characteristics of the corpus luteum. Blood samples were collected to quantify serum P 4 concentrations. There were no differences between the ovulation synchronization treatment regimens for all follicular dynamic variables tested; however, cows in Group 3 differed from Group 2 having a larger follicle diameter (FD) on D10 (P = 0.06) and larger preovulatory FD (P = 0.09), as well as a blood perfusion area of the dominant follicle wall on D8 (P = 0.07). There were no differences in responses to the ovulation synchronization treatment regimens for the luteal variables evaluated subsequent to ovulation. In conclusion, the Baihui acupoint was effective as an alternative route for eCG dose reduction when FTAI treatment regimens were imposed without detrimentally affecting values for reproductive variables., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Vaspin, visceral adipose tissue-derived serine protease inhibitor, plays important roles in inflammation, obesity, and glucose metabolism. Our recent research has shown the expression and role of vaspin in the function of ovarian follicles. However, whether vaspin regulates steroidogenesis and luteolysis in the corpus luteum (CL) is still unknown. The aim of this study was first to determine the expression of vaspin and its receptor GRP78 in porcine CL at the early, middle, and late stages of the luteal phase. Next, we investigated the hormonal regulation of vaspin levels in luteal cells in response to luteinizing hormone (LH), progesterone (P4), and prostaglandin PGE2 and PGF2α. Finally, we determined vaspin's direct impact on luteal cells steroidogenesis, luteolysis and kinases phosphorylation. Our results are the first to show higher vaspin/GRP78 expression in middle and late vs early stages; immunohistochemistry showed cytoplasmic vaspin/GRP78 localization in small and large luteal cells. In vitro, we found that LH, P4, PGE2, and PGF2α significantly decreased vaspin levels. Furthermore, vaspin stimulated steroidogenesis by the activation of the GRP78 receptor and protein kinase A (PKA). Also, vaspin increased the ratio of luteotropic PGE2 to luteolytic PGF2α secretion via GRP78 and mitogen-activated kinase (MAP3/1). Moreover, vaspin, in a dose-dependent manner, decreased GRP78 expression, while it, in a time-dependent manner, increased kinases PKA and MAPK3/1 phosphorylation. Taken together, we found that vaspin/GRP78 expression depends on the luteal phase stage and vaspin affects luteal cells endocrinology, indicating that vaspin is a new regulator of luteal cells steroidogenesis and CL formation.

This study aimed to evaluate the exogenous progesterone (P4) effect on the luteal function from Day 16 to Day 21 of the oestrous cycle in inseminated goats with unknown pregnancy status. A total of 54 does passed through a short progestin-based synchronization protocol and, on Day 16 of the following oestrous cycle, 27 does received a new P4 device which was retained until Day 21. Blood samples were collected daily from all does during this period, as well as on Day 24. Pregnancy diagnoses were performed on Day 30. Serum P4 values from 26 animals (G NPSP : Group of non-pregnant does with second sponge: n = 8; G NPNSP : Group of non-pregnant does without second sponge: n = 6; G PSP : Group of pregnant does with second sponge: n = 5; G PNSP : Group of pregnant does without second sponge: n = 7) were determined by radioimmunoassay commercial kits. No P4 differences were found between groups (G NPSP : 3.1 ± 2.8; 1.7 ± 1.8; 0.4 ± 1.0; and 0.0 ± 0.0 vs. G NPNSP : 4.4 ± 1.8; 3.0 ± 2.2; 0.8 ± 0.8; and 0.0 ± 0.0 or G PSP : 4.2 ± 1.0; 3.4 ± 0.6; 3.3 ± 1.6; 3.2 ± 0.9; 3.6 ± 1.2; 3.5 ± 1.3; 2.7 ± 1.3 vs. G PNSP : 4.4 ± 1.6; 3.6 ± 1.5; 3.7 ± 1.5; 3.8 ± 1.4; 3.2 ± 1.2; 3.1 ± 1.2; 3.6 ± 1.1; D16, D17, D18, D19, D20, D21, D24, respectively) or for the interaction of group and time. In conclusion, a second progestogen device had no effect on luteolysis or early pregnancy in the following oestrous cycle., (© 2020 Wiley-VCH GmbH.)

There was investigation of whether there were ovulations from co-dominant follicles following eCG administration. In all experiments, there was GnRH injection and CIDR insertion on day 0 (D0), CIDR withdrawal on D8, and cloprostenol administration on D8 (Exp. I and II) or D7 and D8 (Exp. III). Females in the control group were not administered any further treatment. Females in other group(s) were treated with eCG (500 IU) on Day 2 in Exp. I, Day 2 (eCG-2) or 8 (eCG-8) in Exp. II and Day 2 (eCG-2) or Days 2 and 6 (eCG-2-6) in Exp. III. Ovaries were examined using ultrasonography. In Experiments I and II, females had follicle emergence on Day 2. At the time of CIDR removal, more eCG-treated heifers (8/9; Exp. I) and cows (5/6; eCG-2; Exp. II) had co-dominant follicles compared to those in the control group (P < 0.05). Occurrence of ovulations from co-dominant for individual cows was minimal. In Experiment III, the time period from CIDR removal to estrus in cows treated with eCG-2 (68 ± 13 h) was longer compared to cows in the control (37±2 h) and eCG-2-6-treated group (38 ± 5 h; P < 0.05). There was a greater proportion of heifers having ovulations and thus greater progesterone concentration in the eCG-2-6 than eCG-2 group (P < 0.05). Administering eCG twice 4 days apart with the initial administration being two days after GnRH administration, at the time of follicle wave emergence, could induce growth of and ovulation from co-dominant follicles and enhance progesterone production in cattle., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: The main goal of the present study is to investigate the effects of retinoic acid and fibroblast growth factor-2 on serum levels of FSH and LH, histology, and apoptosis in the mouse model of Poly Cystic Ovary Syndrome (PCOS)., Materials and Methods: 80 female NMRI mice have been randomly divided into eight groups. Group 1 received normal saline as a control, and Group 2 received estradiol valerate (EV) at 4 mg/100 g of body weight. Moreover, Groups 3-4 were administered with RA (a dose of 0.05 μg/μl) and FGF2 (a dose of 0.01 μg/kg), respectively. Groups 5 and 6 respectively received the EV plus the RA (0.05 μg/μl) and FGF2 (0.01 μg/kg). Group 7 received the RA and FGF2 at doses corresponding to healthy mice, and Group 8 received the EV plus the RA + FGF2 (similar to previous doses). RA and FGF2 were injected three times per week for four weeks. Finally, histological and immunohistological parameters of the ovary were evaluated., Results: The study revealed that both single and combined injection of fibroblast growth factor-2 (FGF2) and retinoic acid (RA) in groups 5, 6, and 8 significantly reduced follicular diameters compared to group 2. Measurements confirmed that simultaneous injection of RA and FGF2 into polycystic mice significantly increased antral follicles, corpus luteum (CL), epithelial thickness, and oocyte diameter as well as decreased cystic follicles. Positive TUNEL cells that were considerably increased in the antral follicle of group 2 significantly decreased in the RA and FGF2 recipient groups, either alone or in combination. Besides, the injection of FGF2 increased preantral follicles and CL., Conclusion: The findings of the present investigation reveal that injection of RA and FGF2 has both protective and ameliorative effects that can promise new therapies for women with PCOS., Competing Interests: Declaration of Competing Interest There is no conflict of interest, and all authors have read and approved the manuscript being submitted., (Copyright © 2020. Published by Elsevier B.V.)

We examined the effects of a single or 2.5-fold dose of dephereline [a gonadotropin-releasing hormone (GnRH) analogue] as well as the drainage of the smaller follicle at the time of insemination on ovulation in dairy cows with two follicles of pre-ovulatory size in the same ovary. The three study groups included 220 monovular cows (control), 110 non-drained cows with two follicles, and 110 cows with two follicles, of which one was drained. In each group, cows treated with a single dose or 2.5-fold dose of dephereline showed similar results following treatment. Ovulation failure of the non-drained follicle occurred in 29.1% of the drained cows, whereas ovulation occurred in 96% of the non-drained and control cows. Twin pregnancy was recorded in 19.4% of the pregnant non-drained cows with two follicles. In conclusion, the increased dephereline dose did not improve the ovulation rate in any group. Follicular drainage, however, prevented twin pregnancy in cows with two follicles, but also resulted in an increase in the non-drained follicle's rate of ovulation failure.

Pentraxin 3 (PTX3), a multimeric glycoprotein, is implicated in various biological functions. PTX3 was shown to be elevated in the corpus luteum (CL) of early pregnant ewes; however, its role in sheep or other ruminants' CL during this reproductive stage or how it is regulated remain unknown. Here we explored the role of PTX3 and its relationship with interferon-tau (IFNT; the pregnancy recognition signaling molecule during early pregnancy in domestic ruminants) in bovine luteinized granulosa cells (LGCs). IFNT robustly elevated PTX3 expression in bovine LGCs, and significantly stimulated its expression in luteal endothelial cells, along with CL slices; yet, LGCs were the most responsive and sensitive among these luteal models. ALK2/ALK3/ALK6 kinase inhibitor, dorsomorphin, dose-dependently inhibited basal and IFNT-elevated PTX3 expression in LGCs. In contrast, ALK4/5/7 inhibitor, SB431542, did not alter basal and TGFB1-induced PTX3. We found that recombinant human PTX3 itself moderately but significantly increases LGC numbers. Because PTX3 is highly expressed in bovine LGCs, we next examined the impact of lowering endogenous PTX3 levels with siRNA. PTX3 silencing decreased the viable cell numbers and reversed IFNT actions on cell viability, percentage of proliferating cells, and on two key survival/death genes: BIRC5 encoding surviving protein, and FASL - a death-inducing signal. Interestingly, thrombospondin-1, a known luteal proapoptotic factor, was inversely related to PTX3 in LGCs. Together, these findings suggest a novel role for PTX3 during early pregnancy, as mediator of IFNT prosurvival actions supporting CL maintenance during this reproductive stage.

Supplementation of progesterone (P 4 ) in pregnant gilts increases concentrations of circulating P 4 and stimulates the secretory activity of the endometrium. In this study, there was examination of the consequences of exogenous P 4 administration on luteal P 4 content and the expression of genes related to the corpus luteum (CL) function. Gilts with gonadotropin-induced estrus were administered daily injections of corn oil (n = 8) or P 4 (n = 8) on days 3 through 10 after insemination. The animals were slaughtered on day 12 of pregnancy to obtain corpora lutea for real-time polymerase chain reaction analyses of selected genes and for enzyme immunoassay of P 4 . Injections with P 4 had no effect on the concentration of P 4 and the relative abundance of mRNA transcripts of cholesterol transport-related proteins, steroidogenic enzymes, and receptors for luteotropic factors in the luteal tissue. The abundance of prostaglandin (PG) endoperoxide synthase 2, PGI2 synthase, PGI2 receptor, fibroblast growth factor 2, peroxisome proliferator-activated receptor γ, and tumor necrosis factor α receptor type I transcripts increased after P 4 treatment. In contrast, the relative abundance of angiopoietin 2 mRNA decreased in response to P 4 administration. In summary, P 4 supplementation in pregnant gilts does not affect luteal steroidogenesis but modulates the abundance of factors related to vascular function. Given that the endometrium is the main target tissue for P 4 , an indirect uterine-mediated effect of exogenous P 4 on CL function is likely., (Copyright © 2020 Elsevier B.V. All rights reserved.)