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During an ovulatory follicular wave in the monovulatory species of heifers, mares, and women, the two largest follicles deviate in diameter at the end of a common follicle growth phase. The largest follicle before deviation becomes the future ovulatory follicle in most ovulatory waves. In 10-30% of the ovulatory waves, the destiny of the two follicles switches just before or at deviation so that the second-largest follicle becomes the future ovulatory follicle, and the largest follicle becomes a subordinate. In FSH-driven switching in heifers, mares, and women, the wave-stimulating FSH surge decreases to a low concentration before the largest follicle has developed the ability to utilize the low concentrations. The concentrations of FSH then increase (mares, women) or cease to decrease (heifers), and the next largest follicle acquires the capability of becoming the future ovulatory follicle. Luteolysis-driven switching has been reported in heifers but not in mares and women. The switching in heifers occurs during ovulatory wave 3 of three wave interovulatory intervals (IOI) when the wave of follicles is in the common growth phase in synchrony with the time of luteolysis. Regression of the CL during the common growth phase of ovulatory wave 3 is accompanied by decreased activity of follicles that are adjacent to the regressing CL but not when follicles and CL are separated or in opposite ovaries. The role of luteolysis in switching in heifers has been tested by treating with PGF2α when the largest follicle of wave 2 was near the end of the common growth phase. Switching in destiny of the largest follicle from the expected future dominant to a future subordinate occurred in most waves (10 of 17) when the largest follicle and regressing CL were in the same ovary and adjacent but not when separated in the same ovary or when in opposite ovaries (0 of 11). The newly selected future ovulatory follicle may develop in the opposite ovary. Thereby, frequency of the contralateral vs ipsilateral relationship between the preovulatory follicle and CL in heifers is greater in three-wave IOI than in two-wave IOI. In summary, the second largest predeviation follicle becomes the postdeviation dominant follicle when the decreasing FSH is out of phase with the largest predeviation follicle in heifers, mares, and women or when luteolysis and predeviation are in synchrony in heifers.

The increase of oxidative stress is one of the important characteristics of mammalian luteal regression. Previous investigations have revealed the essential role of reactive oxygen species (ROS) in luteal cell death during luteolysis, while it is unknown how ROS is regulated in this process. Considering the decrease of blood flow and increase of PGF2α during luteolysis, we hypothesized that the HIF-1α pathway may be involved in the regulation of ROS in the luteal cell of the late corpus luteum (CL). Here, by using a pseudopregnant rat model, we showed that the level of both HIF-1α and its downstream BNIP3 was increased during luteal regression. Consistently, we observed the increase of autophagy level during luteolysis, which is regulated in a Beclin1-independent manner. Comparing with early (Day 7 of pseudopregnancy) and middle CL (Day 14), the level of ROS was significantly increased in late CL, indicating the contribution of oxidative stress in luteolysis. Inhibition of HIF-1α by echinomycin (Ech), a potent HIF-1α inhibitor, ameliorated the upregulation of BNIP3 and NIX, as well as the induction of autophagy and the accumulation of ROS in luteal cells on Day 21 of pseudopregnancy. Morphologically, Ech treatment delayed the atrophy of the luteal structure at the late-luteal stage. An in vitro study indicated that inhibition of HIF-1α can also attenuate PGF2α-induced ROS and luteal cell apoptosis. Furthermore, the decrease of cell apoptosis can also be observed by ROS inhibition under PGF2α treatment. Taken together, our results indicated that HIF-1α signaling is involved in the regression of CL by modulating ROS production via orchestrating autophagy. Inhibition of HIF-1α could obviously hamper the apoptosis of luteal cells and the process of luteal regression.

Kisspeptins (KISSs) and RFamide-related peptide-3 (RFRP-3) affect the synthesis and secretion of luteinizing hormone (LH) and modulate female reproductive processes. The presence of KISS and RFRP-3 in the porcine pituitary gland and their contribution to the regulation of follicle-stimulating hormone (FSH) synthesis and secretion is unknown. This study analyzed the presence of KISS and RFRP-3 in the pituitary of estrous-cyclic pigs on days 2 to 3, 10 to 11, 12 to 13, 15 to 16 and 19 to 20 and early pregnant pigs on days 10 to 11, 12 to 13 and 15 to 16, and evaluated the effect of KISS and RFRP-3 on β-Fsh mRNA expression and FSH secretion in vitro by pituitary cells collected on selected days of the estrous cycle. The cells were cultured in vitro and treated with KISS (10−6 M, 10−7 M) and RFRP-3 (10−6 M, 10−7 M) or gonadotropin-releasing hormone (GnRH; 100 ng/mL) alone and in combinations (4 h or 24 h). The relative abundance of Kiss and Rfrp-3 and their receptor mRNA transcripts, as well as the KISS and RFRP-3 proteins, were found in the pituitaries of estrous-cyclic and early pregnant pigs. KISS after 4 h increased the secretion of FSH in estrous cyclic pigs mostly during the early-luteal phase and luteolysis. RFRP-3 inhibited the synthesis and secretion of FSH in estrous-cyclic pigs on days 19 to 20 and the secretion of FSH on days 2 to 3 and 10 to 12 of the estrous cycle compared with GnRH-treated cells. KISS in co-treatment with GnRH after 24 h enhanced FSH release on days 2 to 3 and 15 to 16 of the estrous cycle. In conclusion, KISS and RFRP-3 systems are present in the pituitary of estrous-cyclic and pregnant pigs. In estrous-cyclic pigs, KISS and RFRP-3 may affect the synthesis and secretion of FSH by pituitary cells.

Establishment of a vascular system within the corpus luteum (CL) is critical for progesterone (P4) secretion. Measurement of luteal blood flow (LBF) may be a feasible way to determine luteolysis induced with cloprostenol sodium (CLO). Our overall objective was to establish timelines to assess luteolysis via Doppler ultrasonography. Estrous cycles were synchronized in 11- to 12-mo-old Holstein heifers (n = 37). Heifers were injected CLO at a random stage of the estrous cycle 12 d from treatment. Gonadotropin-releasing hormone (GnRH) was administered 2 (d −10) and 8 d (d −4) after the initial CLO. This satisfied the study objective of inducing simultaneous presence of CL at d 4 of development (D4 CL) and CL at d 10 of development (D10 CL) on the day of treatment with different CLO doses (hereafter referred to as d 0). Heifers were randomly assigned to 1 of 5 treatments on d 0: negative control (NC) consisting of no treatment with CLO (n = 8); a quarter dose of CLO (0.125 mg; n = 8); half dose of CLO (0.25 mg; n = 8); full dose of CLO (0.5 mg; n = 8); or positive control (PC) consisting of 4 doses of 0.5 mg of CLO at 24-h intervals starting at d 0 (n = 5). Data collection was performed at d 0 (before and 1 h after treatment) and 2, 4, 6, and 8 d following treatment, to determine luteal volume (LV), LBF, and circulating concentrations of P4. Both NC and PC were efficient in mimicking physiological scenarios that occur during normal luteal development and luteolysis. Heifers that received PC had complete LBF disappearance of both D4 and D10 CL between d 2 and 4 after the first of 4 CLO treatments given 24 h apart (average 4.0 ± 0.0 and 3.2 ± 0.7 d, respectively). Complete LBF disappearance was used as a luteolysis marker. Treatment with different doses of CLO did not impair luteal development of the D4 CL. However, concurrent complete LBF disappearance for D10 CL in heifers treated with half (5/8 heifers) and full doses of CLO (8/8 heifers) resulted in less LBF in the half dose, and less LV and LBF in the full-dose treatment, in D4 CL at d 8 post-treatment, compared with NC. Treatment with various doses of CLO induced an acute increase in LBF 1 h after treatment, regardless of dose in D10 but not in D4 CL. We found a lack of dose response in LV reduction of D4 and D10 CL. Interestingly, LV of the D10 CL decreased in untreated NC between d 0 and 8 after treatment (d 10–18 of luteal development). Assessment with color Doppler ultrasound was sensitive enough to identify dose-response patterns in Holstein heifers (absence, partial, or complete luteolysis) following various doses of CLO. Variability in time to complete LBF disappearance of mature D10 CL following a full dose of CLO limits the use of Doppler ultrasonography to detect luteolysis at a single time point following treatment.

The pulsatile pattern of prostaglandin F2alpha (PGF) secretion during spontaneous luteolysis is well documented, with multiple pulses of exogenous PGF necessary to induce regression using physiologic concentrations of PGF. However, during spontaneous regression, the earliest pulses of PGF are small and not associated with detectable changes in circulating progesterone (P4), bringing into question what, if any, role these early, subluteolytic PGF pulses have during physiologic regression. To investigate the effect of small PGF pulses, luteal biopsies were collected throughout natural luteolysis in conjunction with bihourly blood samples to determine circulating P4 and PGF metabolite to retrospectively assign biopsies to early and later regression. Whole transcriptome analysis was conducted on CL biopsies. Early PGF pulses altered the luteal transcriptome, inducing differential expression of 210 genes (Q < 0.05) during early regression, compared with 4615 differentially expressed genes during later regression. In early regression, few of these differentially expressed genes were directly associated with luteolysis, rather there were changes in local steroid and glutathione metabolism. Most (94%) differentially expressed genes from early regression were also differentially expressed during later regression, with 98% of these continuing to be altered in the same direction compared with CL at a similar stage of the cycle that had not yet been exposed to PGF. Thus, early, subluteolytic PGF pulses impact the luteal transcriptome, though not by altering steroidogenesis or causing direct inhibition of cellular function. Rather, small pulses alter pathways resulting in the removal of cellular support systems, which may sensitize the CL to later pulses of PGF. Summary sentence Early stages of spontaneous luteolysis involve prostaglandin F2alpha pulses too small to impact steroidogenesis; nonetheless, these pulses are capable of removing cellular support systems such as local progesterone signaling and glutathione-based anti-oxidant metabolism that may sensitize the CL to later pulses of PGF.

It is well known that corpus luteum normal functioning is crucial for the luteal phase, which determines the embryo implantation and the progression of pregnancy. Luteal phase deficiency (LPD), associated with impaired progesterone secretion by the corpus luteum, is considered as a significant factor of infertility and early pregnancy loss, both in the natural cycle and in assisted reproductive technology (ART) programs. The LPD formation is associated with hypothalamic-pituitary-ovarian axis dysregulation, which leads to abnormal secretion of FSH, LH, ovulation and luteinization disorders, premature luteolysis. The most significant problem in the study of LPD is the lack of reliable and reproducible methods of its verification. This review summarizes the available data on the methods and issues of LPD diagnosing, including the duration of the luteal phase, the level of progesterone secretion, and endometrial biopsy. LPD is an important factor in reproductive failures during IVF, which is caused by suppression of the physiological FSH, LH secretion and requires mandatory progesterone support in the luteal phase of the cycle. It’s hard to define the contribution of LPD to miscarriage, however, empirical progestogen therapy may increase the live births rate in women with recurrent pregnancy loss. Currently, there is no evidence of the LPD role and progesterone support effectiveness in infertility management, so the diagnosis and therapy of LPD among these patients should not be considered.

Research question Can luteolysis-targeted drugs, gonadotrophin-releasing hormone antagonist (GnRH-ant), mifepristone and letrozole, administered separately or in combination, prevent the progression of ovarian hyperstimulation syndrome (OHSS) in a rat model? Design Thirty-six female Wistar rats were randomly divided into six groups, including control group (OHSS group, ovarian hyperstimulation-induced OHSS); GnRH-ant group (OHSS with GnRH-ant treatment); mifepristone group (OHSS with mifepristone treatment); letrozole group (OHSS with letrozole treatment); combination group (OHSS with GnRH-ant, mifepristone and letrozole treatment in combination). The main outcomes were the alterations in OHSS-related indices, including ovarian weight, vascular permeability, serum oestradiol and progesterone levels, corpus luteum proportion and diameter, ovarian vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), caspase-3 and cleaved caspase-3 levels. Results No significant difference was found in body weight gain among the six groups. Compared with the control group, the OHSS group showed significant increases in all OHSS-related indices. GnRH-ant treatment showed decreases in vascular permeability, serum oestradiol level, corpus luteum diameter, ovarian VEGF /IL-6 mRNA levels, and increases in ovarian caspase-3 and cleaved caspase-3 levels. Mifepristone treatment demonstrated reduction in serum progesterone level and corpus luteum diameter, and elevation in ovarian caspase-3 and cleaved caspase-3 levels. Letrozole treatment displayed a decline in serum oestradiol level and corpus luteum diameter, and up-regulation in ovarian caspase-3 and cleaved caspase-3 levels. The combination treatment by GnRH-ant, mifepristone and letrozole showed enhanced synergistic effect on reducing OHSS-related indices. Conclusions GnRH-ant, mifepristone and letrozole are beneficial in preventing the progression of OHSS through different luteolytic mechanisms. Cocktail style treatment shows enhanced synergistic effect on preventing the progression of OHSS.

Made available in DSpace on 2021-06-25T10:33:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Our objectives were to investigate potential changes in the size of steroidogenic large luteal cells (LLC) during partial luteolysis induced by a sub-dose of cloprostenol in early diestrus and to determine transcriptional variations in genes involved in corpus luteum (CL) functions. Cows were subjected to an Ovsynch protocol, with the time of the second GnRH treatment defined as Day 0 (D0). On D6, cows were randomly allocated into three treatments: Control (2 mL saline, im; n = 10), 2XPGF (two doses of 500 μg of cloprostenol, im, 2 h apart; n = 8) or 1/6PGF (single dose of 83.3 μg of cloprostenol, im; n = 10). Before treatments and every 8 h during the 48-h experimental period, blood samples were collected and CL volumes measured. Furthermore, two CL biopsies were obtained at 24 and 40 h post-treatment. The 1/6PGF treatment caused partial luteolysis, characterized by sudden decreases in plasma progesterone (P4) concentrations, luteal volume and LLC size, followed by increases (to pretreatment values) in P4 and luteal volume at 24 and 40 h post-treatment, respectively. However, at the end of the study, P4, luteal volume and LLC size were all significantly smaller than in Control cows. Temporally associated with these phenotypes, there was a lower mRNA abundance of VEGFA at 24 and 40 h, and ABCA1 at 24 h (P < 0.05). In conclusion, a sudden reduction in CL size during partial luteolysis induced by a sub-dose of PGF2α analog on day 6 of the estrous cycle was attributed to a reduction in LLC size, although these changes did not account for the entire phenomenon. In addition to its involvement in reducing CL size, decreased VEGFA mRNA abundance impaired CL development, resulting in a smaller luteal gland and lower plasma P4 concentrations compared to Control cows. Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Institute of Animal Husbandry APTA – SAA Department of Veterinary Clinical Science School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Department of Structural and Functional Biology Institute of Biosciences São Paulo State University (UNESP) Department of Pharmacology Institute of Biosciences São Paulo State University (UNESP) University of Western São Paulo (Unoeste) Department of Production Animal Health Faculty of Veterinary Medicine University of Calgary Department of Veterinary Surgery and Animal Reproduction School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Department of Veterinary Clinical Science School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Department of Structural and Functional Biology Institute of Biosciences São Paulo State University (UNESP) Department of Pharmacology Institute of Biosciences São Paulo State University (UNESP) FAPESP: #2011/20449–7 FAPESP: 2012/18297–7

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 (GNPSP : Group of non-pregnant does with second sponge: n = 8; GNPNSP : Group of non-pregnant does without second sponge: n = 6; GPSP : Group of pregnant does with second sponge: n = 5; GPNSP : Group of pregnant does without second sponge: n = 7) were determined by radioimmunoassay commercial kits. No P4 differences were found between groups (GNPSP : 3.1 ± 2.8; 1.7 ± 1.8; 0.4 ± 1.0; and 0.0 ± 0.0 vs. GNPNSP : 4.4 ± 1.8; 3.0 ± 2.2; 0.8 ± 0.8; and 0.0 ± 0.0 or GPSP : 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. GPNSP : 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.

Molecular mechanisms underlying luteinization (terminal differentiation of granulosa and theca cells after ovulation) and luteolysis (demise of corpus luteum) are poorly understood in human ovary. Here we report that activin-A, after binding to its cognate receptors induces a functional luteolytic state and reverses luteinization phenotype by downregulating the expression of the steroidogenic enzymes, LH receptor and VEGF and reducing estradiol (E2) progesterone (P4) production and upregulating FSH receptor and cyclin D1 expression in human primary luteinized granulosa cells. Further, this action of activin-A involves downregulation of JNK signaling pathway and is opposite to that of human chorionic gonadotropin (hCG), which acts as a luteotropic hormone and improves luteal function through the activation of JNK pathway in the same cell type. Reversal of luteinization phenotype in luteal granulosa cells by activin-A potentially makes this hormone an attractive candidate for use under certain clinical situations, where induction of luteolysis and rapid reduction of endogenous sex steroid levels are beneficial such as ovarian hyperstimulation syndrome (OHSS), in which the ovaries hyper-respond to gonadotropin stimulation by producing too many growing follicles along with development of ascites, pleural effusion, and hemo-concentrations as a result of increased vascular permeability and leakage of intravascular volume into third spaces. Our work unveils a previously undefined role for activin-A and JNK signaling pathway in human corpus luteum biology, that might have a direct clinical impact in assisted reproductive technologies., School of Medicine; the Graduate School of Health Sciences of Koç University; Koç University Research Center for Translational Medicine (KUTTAM)

Among domestic animal species, the reproductive biology of the dog belongs to the most peculiar. This includes the conceptus-maternal communication and endocrine mechanisms involved in maintenance of pregnancy. Dogs fully depend on luteal progesterone (P4) throughout pregnancy, with similar steroid secretion patterns in pregnant and non-pregnant bitches until prepartum luteolysis. Thus, dogs lack the classical recognition of pregnancy. The luteal P4 is the most important hormone regulating the onset and maintenance of pregnancy in previously estrogenized bitches. Although the canine uterus is exposed to high P4 levels, decidualization is not spontaneous but induced by the presence of embryos. Following implantation, decidualization continues, associated with development of the invasive endotheliochorial placenta, leading to establishment of maternal decidual cells expressing the nuclear P4 receptor (PGR). Consequently, although not producing steroids, the canine placenta remains highly sensitive to circulating ovarian steroids. The placental conceptus-maternal communication is responsible for the maintenance of pregnancy, with functional withdrawal of PGR evoking a luteolytic cascade with prepartum PGF2α release. The fetal trophoblast is the major source of prepartum placental prostaglandins. This conceptus-maternal communication is unique to the dog and has clinical implications. Due to luteal steroids, there is no prepartum estradiol increase. Elevated cortisol levels are observed irregularly. This emphasizes the unique character of canine reproductive physiology and the challenges in transferring translational research to the dog. Further research is needed for better understanding of canine reproduction and improving clinical protocols, including the latest results obtained from applying modern laboratory technologies such as the transcriptomic approach.

The process of luteal regression is tightly regulated by the immune system and chemokines-small cytokines responsible mostly for the activation and migration of immune cells. The role of chemokines in porcine corpus luteum (CL) function is still not well understood. The aim of this study was to investigate the expression profile and distribution of CC chemokines in the porcine CL during the natural oestrous cycle and early pregnancy. Additionally, the effect of PGF2α on the expression of selected chemokines and their luteotropic and apoptotic influence on CL cells were studied in vitro. The expression levels of the chemokines CCL2, CCL4, and CCL5 and the chemokine receptor CCR5 were time-dependent (low on Days 8-10 and high on Days 12-14 of the oestrous cycle). Moreover, CCL8 and CCR2 transcript levels were also elevated during the period of luteolysis. The immunolocalization of CCL2, CCL4, CCL5, CCR1, CCR2 and CCR5 was determined using CL sections obtained from cycling and pregnant pigs. The immunofluorescence signals were localized mainly in luteal cells. PGF2α treatment of CL cells caused increased mRNA expression of CCL2 and CCR1. CCL2 treatment alone upregulated the expression of genes BAX, BCL2 and StAR in CL cells in vitro, but additional experiments showed that the chemokines CCL2, CCL4 and CCL5 alone do not cause apoptosis in a mixed population of CL cells. The chemokine CCL4 increased the transcript levels of StAR and HSD3-β1. Additionally, CCL5 led to the inhibition of BAX gene expression. The differential spatiotemporal expression of CCL2, CCL4, CCL5 and CCR5 throughout the oestrous cycle and the direct but aberrant effect of these three chemokines on genes associated with apoptosis and progesterone synthesis indicate the complicated involvement of these factors in the regulation of luteolysis in pigs.

There has been increasing interest in the role of hypoxia in the microenvironment of organs, because of the discovery of hypoxia-inducible factor-1 (HIF1), which acts as a transcription factor for many genes activated specifically under hypoxic conditions. The ovary changes day by day during the estrous cycle as it goes through phases of follicular growth, ovulation, and formation and regression of the corpus luteum (CL). These phenomena are regulated by hypothalamic and pituitary hormones, sex steroids, peptides and cytokines, as well as oxygen conditions. Hypoxia strongly induces angiogenesis via transcription of a potent angiogenic factor, vascular endothelial growth factor (VEGF), that is regulated by HIF1. A CL forms with a rapid increase of angiogenesis that is mainly induced by HIF1-VEGF signaling. Hypoxia also contributes to luteolysis by down-regulating progesterone synthesis and by up-regulating apoptosis of luteal cells. This review focuses on recent studies on the roles of hypoxia- and HIF1-regulated genes in the regulation of bovine CL function.

The estrous cycle in cattle lasts for 18–24 days. It consists of a luteal phase (14–18 days) and a follicular phase (4–6 days). During the cycle, there are generally two (dairy cows) or three (heifers and beef cows) waves of ovarian follicle growth. Each wave of follicle growth consists of a period of emergence of a cohort of follicles, selection of a dominant follicle (DF), and either atresia or ovulation of the DF. These waves of follicle growth occur throughout the entire cycle, with only the DF of the final wave coinciding with the follicular phase that undergoes final maturation and ovulation. Ovarian function (follicle growth, ovulation, luteinization, and luteolysis) are regulated by the endocrine hormones of the hypothalamus (gonadotropin-releasing hormone), anterior pituitary (follicle-stimulating hormone and luteinizing hormone), ovaries (progesterone, estradiol, and inhibins), and the uterus (prostaglandin F 2α ).

Made available in DSpace on 2022-04-28T19:43:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) In cattle, 17β-estradiol (E2) stimulates prostaglandin F2α (PGF2α) synthesis, which causes luteolysis. Except for the well-established upregulation of oxytocin receptor gene (OXTR), molecular mechanisms of E2-induced PGF2α release in vivo remain unknown. We hypothesized that E2-induced PGF2α release requires de novo transcription of components of the PGF2α synthesis machinery. Beef cows (n = 52) were assigned to remain untreated (Control; n = 10), to receive 50% ethanol infusion intravenously (Placebo; n = 21), or 3 mg E2 in 50% ethanol infusion intravenously (Estradiol; n = 21) on day 15 (D15) after estrus. We collected a single endometrial biopsy per animal at the time of the treatment (0h; Control B0h group), 4 hours (4h; Placebo B4h group and Estradiol B4h group), or 7 hours (7h; Placebo B7h group and Estradiol B7h group) post-treatment. Compared to the Placebo group, the Estradiol group presented significantly greater 13,14-dihydro-15-keto-PGF2α concentrations between 4h and 7h and underwent earlier luteolysis. At 4h, the qPCR analysis showed a lower abundance of ESR1, ESR2 and aldo-keto reductase family 1 member B1 (AKR1B1) genes in the Estradiol B4h group, and a greater abundance of OXTR compared to the Placebo B4h group. Similarly, the E2 treatment significantly reduced the abundance of AKR1B1, and AKR1C4 in the Estradiol B7h group, compared to the placebo group. Overall, E2-induced PGF2α release and luteolysis involved an unexpected and transient downregulation of components of the PGF2α-synthesis cascade, except for OXTR, which was upregulated. Collectively, our data suggest that E2 connects newly-synthesized OXTR to pre-existing cellular machinery to synthesize PGF2α and cause luteal regression. Agrarian Sciences Center State University of Maranhão Tocantine Region Department of Animal Reproduction University of São Paulo North Florida Research and Education Center Institute of Food and Agricultural Sciences University of Florida Department of Animal Sciences University of Florida Department of Pharmacology and Biotechnology São Paulo State University Department of Animal Science Luiz de Queiroz College of Agriculture University of São Paulo Department of Animal Sciences São Paulo State University Department of Pharmacology and Biotechnology São Paulo State University Department of Animal Sciences São Paulo State University FAPESP: 2015/03331-3)

The canine corpus luteum (CL) is the main source of reproductive steroids during dioestrus in the dog and remains active even in the absence of pregnancy (non-pregnant dioestrus, physiological pseudopregnancy). Whereas the biological effects of 17β-oestradiol (E2) in the canine CL remain unclear, the transcriptional availability of oestrogen receptors, ESR1 and ESR2, as well as other modulators of local availability of E2, for example, HSD17B7 (converts oestrone into oestradiol), SULT1E1 (inactivates E2 binding capacity to its own receptors through sulphonation) and STS (reverts E2 sulphonation), were previously detected in the CL of non-pregnant bitches. The aim of the present work was to evaluate the mRNA amounts of these factors involved in luteal sensitivity and metabolism of E2 in the canine CL during the course of non-pregnant dioestrus (days 10, 20, 30, 40, 50 and 60 post-ovulation, n = 5/group) and at different stages of pregnancy (n = 4-6/group): pre-implantation (days 8-12), post-implantation (days 18-25), mid-gestation (days 35-40) and prepartum luteolysis. During pregnancy, the availability of ESR1, HSD17B7, SULT1E1 and STS decreased from mid-pregnancy to prepartum luteolysis. The main findings during non-pregnant dioestrus were as follows: increased ESR2:ESR1 ratio on days 40 and 50 after ovulation, decreasing during luteal regression (day 60); increased STS at day 30 when SULT1E1 levels decreased; increased availability of SULT1E1 transcripts during luteal regression; and decreased amounts of HSD17B7 mRNA in early dioestrus, increasing towards later stages. These results suggest that E2 signalling and biologically active local concentrations could diverge in response to time and pregnancy status of the bitch.

The corpus luteum (CL) plays a vital role in regulating the reproductive cycle, fertility, and in maintaining pregnancy. Interferon-tau (IFNT) is the maternal recognition of a pregnancy signal in domestic ruminants; its uterine, paracrine actions, which extend the CL lifespan, are widely established. However, considerable evidence also suggests a direct, endocrine role for IFNT. The purpose of this review is to highlight the importance of IFNT in CL maintenance, acting directly and in a cell-specific manner. A transcriptomic study revealed a distinct molecular profile of IFNT-exposed day 18, pregnant bovine CL, compared to the non-pregnant gland. A substantial fraction of the differentially expressed genes was downregulated, many of which are known to be elevated by prostaglandin F2A (PGF2A). In vitro, IFNT was found to mimic changes observed in the luteal transcriptome of early pregnancy. Key luteolytic genes such as endothelin-1 (EDN1), transforming growth factor-B1 (TGFB1), thrombospondins (THBSs) 1&2 and serpine-1 (SERPINE1) were downregulated in luteal endothelial cells. Luteal steroidogenic large cells (LGCs) were also found to be a target for the antilutelotytic actions of IFNT. IFNT-treated LGCs showed a significant reduction in the expression of the proapoptotic, antiangiogenic THBS1&2, as well as TGFBR1 and 2. Furthermore, IFNT was shown to be a potent survival factor for luteal cells in vivo and in vitro, activating diverse pathways to promote cell survival while suppressing cell death signals. Pentraxin 3 (PTX3), robustly upregulated by IFNT in various luteal cell types, mediated many of the prosurvival effects of IFNT in LGCs. A novel reciprocal inhibitory crosstalk between PTX3 and THBS1 lends further support to their respective survival and apoptotic actions in the CL. Even though IFNT did not directly regulate progesterone synthesis, it could maintain its concentrations, by increasing luteal cell survival and by supporting vascular stabilization. The direct effects of IFNT in the CL, enhancing cell survival and vasculature stabilization while curbing luteolytic activities, may constitute an important complementary branch leading to the extension of the luteal lifespan during early pregnancy.

BACKGROUND Information on the impact of hormonal protocols for cervical dilation on the quality of ovine embryos is scarce. METHODS To compare the quality of embryos after cervical dilation protocol, ewes (n = 64) were allocated into either a treated group (100 μg estradiol benzoate intravenous and 0.12 mg cloprostenol intramuscularly, 12 hours before embryo collection plus 100 iu oxytocin intravenous 15 minutes before the collection procedure) or a control group (saline). Luteal function was analysed using ultrasonography and P4 measurement. Some collected embryos were frozen/thawed for gene expression, others were cultured in vitro, frozen/thawed for gene expression, and the remaining embryos were fixed for the apoptosis test (TUNEL test). RESULTS The treatment reduced fluid (p=0.04) and structure (p=0.03) recovery rates, but the morphological quality, development stage, and apoptosis incidence of the embryos were not affected by treatment. The corpora lutea of the control group had greater blood perfusion (p = 0.002) and greater P4 concentrations at 6, 9, and 12 h after the treatment (p

Prostaglandin (PG) F2α and its analogs (aPGF2α) are used to induce regression of the corpus luteum (CL); their administration during the middle stage of the estrous cycle causes luteolysis in cattle. However, the bovine CL is resistant to the luteolytic actions of aPGF2α in the early stage of the estrous cycle. The mechanisms underlying this differential luteal sensitivity, as well as acquisition of luteolytic sensitivity by the CL, are still not fully understood. Therefore, to characterize possible differences in response to aPGF2α administration, we aimed to determine changes in expression of genes related to (1) angiogenesis-fibroblast growth factor 2 (FGF2), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2); and (2) steroidogenesis-steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (P450scc), and hydroxy-delta-5-steroid dehydrogenase, 3 β- and steroid delta-isomerase 1 (HSD3B) in early- and middle-stage CL that accompany local (intra-CL) versus systemic (i.m.) aPGF2α injection. Cows at d 4 (early stage) or d 10 (middle stage) of the estrous cycle were treated as follows: (1) systemic saline injection, (2) systemic aPGF2α injection (25 mg), (3) local saline injection, and (4) local aPGF2α injection (2.5 mg). Progesterone (P4) concentration was measured in jugular vein blood samples during the entire set of experiments. After 4 h of treatment, CL were collected by ovariectomy, and mRNA and protein expression levels were determined by reverse transcription quantitative-PCR and Western blotting, respectively. Local and systemic aPGF2α injections upregulated FGF2 expression but decreased expression of VEGFA in both CL stages. Both aPGF2α injections increased the expression of STAR in early-stage CL, but downregulated it in middle-stage CL. In the early-stage CL, local administration of aPGF2α upregulated HSD3B, whereas systemic injection decreased its mRNA expression in early- and middle-stage CL. Moreover, we observed a decrease in the P4 level earlier after local aPGF2α injection than after systemic administration. These results indicate that aPGF2α acting locally may play a luteotrophic role in early-stage CL. The systemic effect of aPGF2α on the mRNA expression of genes participating in steroidogenesis seems to be more substantial than its local effect in middle-stage CL.

The objectives of this study were to investigate the carryover effects of differing pre- and postweaning planes of nutrition on prepubertal reproductive tract development and postpubertal estrous cycle characteristics in Holstein heifers. Heifer calves (n = 36) were randomly assigned to receive either a low or a high (5 or 10 L of whole milk/d, respectively) preweaning diet from 1 to 7 wk of age and either a low or a high (70 or 85% of concentrate dry total mixed ration, respectively) postweaning diet from 11 to 25 wk of age. Starting at wk 26 of age, heifers were subjected to weekly transrectal ultrasonography until wk 33 or until first ovulation to assess endometrial thickness and ovarian follicular count and size in the prepubertal phase. In a subset of heifers (n = 28), ovarian ultrasonography continued weekly until at least the second ovulation was confirmed; thereafter, ovarian dynamics (through ultrasonography) and blood progesterone (P4) were assessed every 2 d throughout 1 complete estrous cycle in the postpubertal phase. In the prepubertal phase, endometrial thickness (12.0 ± 0.4 vs. 10.8 ± 0.3 mm) and largest follicle size (11.8 ± 0.3 vs. 10.9 ± 0.2 mm) were greater in heifers fed the high postweaning diet than in those fed the low postweaning diet. Furthermore, the number of class 2 (6-9 mm) follicles was greater in heifers fed the high preweaning diet than in those fed the low preweaning diet (1.6 ± 0.1 vs. 1.1 ± 0.1), whereas the number of class 3 (>9 mm) follicles was greater in heifers fed the high postweaning diet than in those fed the low postweaning diet (1.2 ± 0.1 vs. 1.0 ± 0.1). In the postpubertal phase, overall corpus luteum and P4 dynamics did not differ among pre- or postweaning treatments; however, P4 at 4 d preceding luteolysis was lesser in heifers fed the high postweaning diet than in those fed the low postweaning diet (6.1 ± 0.4 vs. 7.7 ± 0.4 ng/mL). In addition, compared with heifers fed the low postweaning diet, those fed the high postweaning diet had a greater number of antral follicles (31.4 ± 2.2 vs. 21.4 ± 2.3) and tended to have more class 3 follicles (3.6 ± 0.3 vs. 2.7 ± 0.3). Results indicate positive carryover effects of increasing the preweaning plane of nutrition from 5 to 10 L of whole milk/d on prepubertal follicular growth in Holstein heifers. Furthermore, an increased postweaning plane of nutrition (85 vs. 70% of concentrate dry total mixed ration) advanced reproductive development through greater endometrial thickness and follicular growth in the prepubertal phase and increased the population of antral follicles in the postpubertal estrous cycle.

El objetivo del presente estudio fue evaluar la asociación entre los diferentes grados de mastitis subclínica con la pérdida temprana de gestación durante los primeros 90 días posteriores al servicio en vacas lecheras Holstein. La investigación se realizó en una explotación lechera ubicada en Ecuador, provincia de Pichincha, cantón Mejía. Para el estudio se analizaron los datos de 619 vacas durante el período de octubre de 2015 hasta octubre de 2016. Se utilizaron tres grupos experimentales clasificados por la severidad de mastitis subclínica diagnosticada por California Mastitis Test (CMT). El Grupo control (CMT 0) vacas que no presentaron mastitis subclínica hasta los 90 días posteriores a la inseminación artificial (IA). El grupo CMT T-1, vacas diagnosticadas con mastitis subclínica grado trazas y grado 1 hasta los 90 días posteriores a la IA y el grupo CMT 2-3, vacas que presentaron mastitis subclínica grado 2 y 3 hasta los 90 días posteriores a la IA. El diagnóstico de gestación se realizó por ultrasonografía transrectal entre los 28 y 35 días posteriores a la IA y se realizó un seguimiento ecográfico a los 60 y 90 días de gestación. Se encontró una pérdida de gestación entre los 30 a 60 días del 12% y entre los 60 a 90 días del 5%. Con este resultado se evidenció una asociación entre mastitis subclínica grado 2 y 3 con la pérdida temprana de gestación (OR 2,6; p

Made available in DSpace on 2019-10-06T15:47:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-08-27 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) To assist in evaluating and quantifying tissue changes, fractal dimension (FD) is a useful method for assessing the organization in an image from fractals that describes the amount of space and the self-similarity of the structure, once FD detects subtle morphological changes and performs functional quantitative measures. Here, we hypothesized that fractal analysis may be different in functional and regressing bovine corpus luteum (CL) and may be correlated with differential expression of genes involved in extracellular matrix remodeling. CL presents two developmental stages, the functional and regressing CL, according to progesterone levels and morphology. First, we found a lower FD in functional CL using HE staining and picrosirius red approach. Additionally, we found a great amount of total collagen in regressing CL. Regarding gene expression, we showed an up regulation of COL1A1, COL1A2, MMP2, and MMP14 and a down regulation of TIMP1 and TIMP2 in regressing CL compared to the functional one. Thus, we concluded that differential FD observed during luteal regression is an effective method to evaluate the tissue changes observed during luteal development in cattle and is related to differential quantity of genes involved in extracellular matrix remodeling. University of Western São Paulo (Unoeste), São Paulo Department of Pharmacology Institute of Biosciences University of São Paulo State (Unesp), São Paulo Department of Pharmacology Institute of Biosciences University of São Paulo State (Unesp), São Paulo FAPESP: 2013/11480-3 FAPESP: 2018/06674-7

The objective was to determine if the introduction of androgenized steers or females in oestrus has luteolytic effects during the advanced luteal phase (Day 12–13 of the cycle, Day of ovulation = Day 0) in heifers by analysing the changes in corpus luteum (CL) size and perfused area together with progesterone (P4) secretion. Experiment 1 (EXP1) was carried out in May (autumn) with 12 Angus and Angus X Hereford heifers and experiment 2 (EXP2) in September (spring) with 20 heifers of the same breed. Procedures for both experiments were the same. Firstly, oestrus was synchronized in heifers, then, transrectal colour doppler ultrasonography was performed daily from Day 10 to Day 12 of the cycle in all animals. On Day 12 in the afternoon, animals were allocated to two experimental groups (control and biostimulated) and maintained separated (minimum distance: 1000 m) until the end of each experiment. In EXP1, two androgenized steers were introduced into the biostimulated group (BAS) and compared with unstimulated control group (CON1). In EXP2, 20 animals were separated into control group (CON2) and biostimulated group (BHE), in which 4 oestrous heifers were introduced on Day 12 in the afternoon, and 4 more on day 13. The oestrous heifers were injected with 2 mg i.m. of oestradiol benzoate every 12 h until the end of the experiment to maintain the receptiveness. In both experiments, from Day 13 until the day on which detectable luteal blood flow (irrigation) disappeared, colour doppler ultrasonography was performed every 12 h in both groups. Blood samples were collected from all heifers every 12 h from Day 10 to the day in which irrigation disappeared. In EXP1 there was no effect of treatment on CL volume. The BAS had less CL’s perfused area than controls 0.09 ± 0.02 cm2 vs 0.16 ± 0.02 cm2, respectively (p = 0.015), less percentage of perfused area (2.4 ± 0.4% vs 4.2 ± 0.4%; p = 0,011), and lower progesterone (P4) concentration than CON1 (2.7 ± 1.0 ng/mL vs 5.8 ± 0.9 ng/mL respectively; p = 0.046). On Day 14.5 of the cycle, the BAS tended to have a lower concentration of P4 than the CON1 (p = 0.06) and on Days 15.5, 16, 16.5, 17, 17.5, 19.5 the P4 concentration was lower in BAS than in CON1 (p

Toll-like receptors (TLRs) are an important part of the innate immune system and play a role in activating the acquired immune system. TLR4 is the best characterised of the many TLRs; its signalling mechanisms are known to be mediated by many inflammatory pathways and by endogenous substances. The aim of the present study was to elucidate the expression profiles for TLR4 mRNA and protein and determine the cell-specific localisation of TLR4 in the equine endometrium during the oestrous cycle and early pregnancy. Endometrial biopsy samples were obtained from mares during early pregnancy (on day 14, P14, n = 4) and, for comparison, on day 14 of the oestrous cycle (late dioestrus, LD, n = 4) and when luteolysis was completed (after luteolysis, AL, n = 4). Total RNA was extracted and transcribed into cDNA, and the relative expression of TLR4 mRNA was quantified by qPCR. Immunohistochemistry was used to define the spatio-temporal localisation of TLR4 protein in the uterus. Expression of TLR4 mRNA was reduced (P < 0.05) on P14 compared to LD. The expression of TLR4 mRNA was similar for P14 and AL. TLR4 protein was localised mainly in the luminal and glandular epithelia, but was also present in endothelial cells of capillaries. Compared to P14, TLR4 protein was abundant in both luminal and glandular epithelia of uteri of mares in LD. These results suggest that downregulation of TLR4 in the endometrial epithelia in early pregnancy may play a role in the immune tolerance mechanisms that protect the conceptus (embryo/foetus and associated membranes)

The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1−/−) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1−/− embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1−/− embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1−/− conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1−/− embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1−/− gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1−/− embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .

An estradiol metabolite, 2-methoxyestradiol (2ME), has emerged as an important regulator of ovarian physiology. 2ME is recognized as a potent anti-angiogenic agent in clinical trials and laboratory studies. However, little is known about its molecular actions and its endogenous targets. 2ME is produced by human ovarian cells during the normal menstrual cycle, being higher during regression of the corpus luteum, and is postulated to be involved in the anti-angiogenic process that plays out during luteolysis. We utilized cell biology techniques to understand the molecular mechanism of 2ME anti-angiogenic effects on human granulosa luteal cells. The principal effect of 2ME was to alter Hypoxia Inducible Factor 1A (HIF1A) sub-cellular localization. Molecular modelling and multiple bioinformatics tools indicated that 2ME impairs Hypoxia Inducible Factor complex (HIF) nuclear translocation by binding to a buried pocket in the HIF1A Per Arnt Sim (PAS)-B domain. Binding of 2ME to HIF1A protein is predicted to perturb HIF1A-Hypoxia Inducible Factor B (HIFB) interaction, a key step in HIF nuclear translocation, preventing the transcriptional actions of HIF, including Vascular Endotelial Growth Factor (VEGF) gene activation. To our knowledge, 2ME is the first putative HIF endogenous ligand characterized with anti-angiogenic activity. This postulate has important implications for reproduction, because angiogenic processes are critical for ovarian follicular development, ovulation and corpus luteum regression. The present research could contribute to the development of novel pharmacological approaches for controlling HIF activity in human reproductive diseases.

STUDY QUESTION Are there any differences in the molecular characteristics of the luteal granulosa cells (GC) obtained from stimulated versus non-stimulated (natural) IVF cycles that may help explain the defective luteal phase in the former? SUMMARY ANSWER Luteal GC of stimulated IVF cycles, particularly those of agonist-triggered antagonist cycles, are less viable ex vivo, express LH receptor and anti-apoptotic genes at lower levels, undergo apoptosis earlier and fail to maintain their estradiol (E2) and progesterone (P4) production in comparison to natural cycle GC. WHAT IS KNOWN ALREADY Luteal function is defective in stimulated IVF cycles, which necessitates P4 and/or hCG administration (known as luteal phase support) in order to improve clinical pregnancy rates and prevent miscarriage. The luteal phase becomes shorter and menstruation begins earlier than a natural cycle if a pregnancy cannot be achieved, indicative of early demise of corpus luteum (premature luteolysis). Supra-physiological levels of steroids produced by multiple corpora luteae in the stimulated IVF cycles are believed to inhibit LH release directly via negative feedback actions on the hypothalamic-pituitary-ovarian axis resulting in low circulating levels of LH and a defective luteal phase. We hypothesized that some defects in the viability and steroidogenic activity of the luteal GC of the stimulated IVF cycles might contribute to this defective luteal phase in comparison to natural cycle GC. This issue has not been studied in human before. STUDY DESIGN, SIZE, DURATION A comparative translational research study of ex vivo and in vitro models of luteal GC recovered from IVF patients undergoing natural versus stimulated IVF cycles was carried out. Luteinized GC were obtained from 154 IVF patients undergoing either natural (n = 22) or stimulated IVF cycles with recombinant FSH and GnRH agonist (long) (n = 44), or antagonist protocol triggered conventionally either with recombinant hCG (n = 46) or with a GnRH agonist (n = 42). GC were maintained in vitro for up to 6 days. PARTICIPANTS/MATERIALS, SETTING, METHODS Cellular viability (YO-PRO-1 staining), the expression of the steroidogenic enzymes, pro-apoptotic genes [Bcl-2-associated death promoter (BAD), Bcl-2-associated X protein (BAX) and Caspase-3 (CASP3)], anti-apoptotic genes [RAC-alpha serine/threonine-protein kinase (AKT-1) and Bcl-2-like protein 2 (BCL2-L2)], LH receptor, vascular endothelial growth factor (VEGF) (using real-time quantitative PCR at mRNA level and western blot immunoprecipitation assay at protein level) and in vitro E2 and P4 production (electrochemiluminescence immunoassay) were compared in GC among the groups. MAIN RESULTS AND THE ROLE OF CHANCE Natural cycle GC were significantly more viable ex vivo (88%) compared to their counterparts of the stimulated IVF cycles (66, 64 and 37% for agonist and antagonist cycles triggered with hCG and GnRH agonist respectively, P < 0.01). They were also more capable of maintaining their vitality in culture compared to their counterparts from the stimulated IVF cycles: at the end of the 6-day culture period, 74% of the cells were still viable whereas only 48, 43 and 22% of the cells from the agonist and antagonist cycles triggered with hCG and agonist respectively, were viable (P < 0.01). The mRNA expression of anti-apoptotic genes (AKT-1 and BCL2-L2) was significantly lower, while that of pro-apoptotic genes (BAD, BAX and CASP3) was significantly higher in the stimulated cycles, particularly in the agonist-triggered antagonist cycles, compared to natural cycle GC (P < 0.01 for long protocol and antagonist hCG trigger, P < 0.001 for agonist trigger). The expression of steroidogenic enzymes (stAR, SCC, 3β-HSD and aromatase) and VEGF was significantly higher in the agonist and hCG-triggered antagonist cycles compared to natural cycle GC. Therefore, in vitro E2 and P4 production in cells from the stimulated IVF cycles was significantly higher than their counterparts obtained from the natural cycles in the first 2 days of culture. However, after Day 2, their viability and hormone production began to decline very rapidly with the most drastic decrease being observed in the agonist-triggered cycles. By contrast, natural cycle GC maintained their viability and produced E2 and P4 in increasing amounts in culture up to 6 days. In vitro P production and the mRNA and protein expression of LH receptor, VEGF and 3β-HSD were most defective in the agonist-triggered antagonist cycles compared to natural and agonist and hCG-triggered antagonist cycles. In vitro hCG treatment of a subset of the cells from the agonist-triggered cycles improved their viability, increased E2 and P4 production in vitro and up-regulated the mRNA expression of anti-apoptotic gene BCL-L2 together with steroidogenic enzymes stAR, SCC, 3B-HSD, LH receptor and VEGF. LARGE SCALE DATA Not applicable. LIMITATIONS, REASONS FOR CAUTION The limitations include analysis of luteinized GC only might not reflect the in vivo mechanisms involved in survival and function of the whole corpus luteum; GC recovered during oocyte retrieval belong to a very early stage of the luteal phase and might not be representative; effects of ovulation triggered with hCG may not equate to the endogenous LH trigger; the clinical characteristics of the patients may vary among the different groups and it was not possible to correlate stimulation-related molecular alterations in luteal GC with the clinical outcome, as no oocytes have been utilized yet. Therefore, our findings do not conclusively rule out the possibility that some other mechanisms in vivo may also account for defective luteal function observed in stimulated IVF cycles. WIDER IMPLICATIONS OF THE FINDINGS Ovarian stimulation is associated with significant alterations in the viability and steroidogenic activity of luteal GC depending on the stimulation protocol and mode of ovulation trigger. Reduced survival and down-regulated expression of 3B-HSD, LH receptor and VEGF leading to compromised steroid production in stimulated cycles, and particularly in the agonist-triggered cycles, may at least in part help explain why the luteal phase is defective and requires exogenous support in these cycles. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the School of Medicine, the Graduate School of Health Sciences of Koc University and Koc University Research Center for Translational Medicine (KUTTAM), equally funded by the Republic of Turkey Ministry of Development Research Infrastructure Support Program. All authors declare no conflict of interest.

The aim was to compare the early luteal development in ewes superovulated with different doses of pFSH. Twenty-nine Santa Ines ewes received a progesterone device (CIDR®) for 8 days. Gonadotrophic treatment started on Day 6: G200 (control, n = 9, 200 mg); G133 (n = 10, 133 mg); and G100 (n = 10, 100 mg of pFSH). On Day 6, all females received eCG (300 IU). B-mode and spectral Doppler ultrasonography were performed daily during the early luteal phase (Days 11-15) to monitor the development of corpora lutea (CLs; dimensions) and ovarian arteries indices. CLs were also classified as normal or prematurely regressed (PRCL) on Day 15 by videolaparoscopy. Ewes from G100 and G133 showed gradual increase in luteal diameter during the early luteal phase (p

Aim: The present study was designed to study the effect of insulin and insulin-like growth factor-1 (IGF-I) administration during luteal phase of the estrous cycle on the hormonal profile of Sahiwal embryo donor cows during superovulation. Materials and Methods: Cows (n=18) were selected and divided into three groups; control (n=6, untreated), T-I (n=6, insulin-treated), and T-II (n=6, IGF-I treated). Insulin and IGF-I were given S/C on 5th, 6th, 7th, and 8th days of estrous cycle. Superovulatory treatment was started on day 9th of the estrous cycle. With the sixth dose of follicle-stimulating hormone, prostaglandin was injected to induce superovulatory heat. The superovulated cows were bred, and superovulatory response of each animal was recorded. The embryos were collected non-surgically on the 7th day of superovulatory estrus. About 15 ml blood without anticoagulant was collected on days 5, 7, 9, 11, 13, 15, 17, 19, and 21 or day of embryo recovery where day 0 of estrous cycle was taken as day of estrus. Serum was separated, centrifuged, and transferred into sterilized serum vials. All samples were stored at -20°C till analysis. Progesterone, insulin, and IGF-I were estimated in blood serum by radioimmunoassay using radioimmunoassay kits. Results and discussion: The mean concentration of progesterone on days 7 and 11, insulin on days 7 and 9, and IGF-I on days 5, 7, 9, 11, and 15 was significantly higher in insulin-treated T-I groups as compared to untreated control. Conclusions: It may be concluded that exogenous insulin administration during mid-luteal phase may be helpful in follicular and embryonic development through modulation of progesterone, insulin, and IGF-I in indigenous (Bos indicus) Sahiwal embryo donor cows.

Treatment with intrauterine devices (IUD) prolongs luteal phases in mares, but the mechanism for this has not been fully elucidated. The aims of the present study were to examine how IUDs affect the uterus to induce longer luteal phases, particularly the role of cyclooxygenase-2 (COX-2) in the maintenance of the corpus luteum (CL). Twenty-seven reproductively normal mares were included: 12 were inseminated (AI), and 15 were fitted with IUDs. Blood samples for progesterone were obtained on Days 0, 3, 5, 7, 9, 11, 13, 14, and 15 (relative to day of ovulation). The groups were further divided into non-pregnant (AI-N, n = 4), pregnant (AI-P, n = 8), normal (IUD-N, n = 8) and prolonged luteal phase (IUD-P, n = 7) based on ultrasonic examinations and serum progesterone concentrations on Days 14 and 15. Blood sampling to quantify the PGF(2 alpha) metabolite (PGFM) was performed through a catheter hourly from 15:00 to 20:00 h on Day 14, and from 6:00 until 13:00 h on Day 15. On Day 15, a low-volume uterine lavage followed by an endometrial biopsy was performed. Estradiol concentration in the Day 15 serum and lavage fluid was determined, while the abundance of COX-2 was evaluated in the biopsy specimens using western blotting (WB) and irnmunohistochemistry (IHC). All pregnant mares were negative for COX-2 in IHC samples and 5 of 8 were negative in WB samples while all mares of the IUD-N group were positive for COX-2. Of the seven mares in the IUD-P group, five and four were negative for COX-2 with the IHC and WB samples, respectively. The results from this study indicate that IUDs, when effective, suppress COX-2, leading to the inhibition of PGF2 alpha release and maintenance of CL.

The objective of this study was to evaluate the effect of accessory corpus luteum (CL) induction on fertility in dairy cows. On day 5 after artificial insemination (AI), lactating Holstein cows were assigned unequally to receive gonadotrophin-releasing hormone treatment (GnRH) (n = 641) or no treatment (control; n = 289). Cows had their blood sampled for progesterone (P4), and ovaries were scanned by ultrasound on days 5, 12, 19, 26, 33, 47, and 61 after AI. Pregnancy diagnosis was performed on days 26, 33, 47, and 61. On day 12, cows treated with GnRH were allocated to ipsilateral (n = 239) or contralateral (n = 241) groups based on the side of accessory CL formation relative to previous ovulation. Accessory CL cows had greater P4 than controls. In total, 52.7% (78/148) of pregnant cows in contralateral group had accessory CL regression earlier (

Inappropriate corpus luteum (CL) regression can produce pregnancy loss. An experimental model was utilized to investigate regression of accessory CL during pregnancy in dairy cows. Cows were bred (day 0) and treated with gonadotrophin-releasing hormone 6 days later to form accessory CL. Transrectal ultrasound (every other days) and blood samples for progesterone (P4; daily) were performed until day 56 of pregnancy. On day 28, 13 cows were confirmed pregnant, and accessory CL were found contralateral (n = 9) or ipsilateral (n = 4) to previous ovulation. On day 18, CL biopsy was performed to analyze mRNA expression for interferon-stimulated genes (ISGs). Luteolysis occurred more frequently in cows that had contralateral accessory CL (88.9% (8/9)) than in cows with ipsilateral accessory CL (0% (0/4)). Luteolysis of contralateral accessory CL occurred either earlier (days 19–23; 2/8) or later (days 48–53; 6/8) in pregnancy and occurred rapidly (24 h), based on daily P4. After onset of earlier or later accessory CL regression, circulating P4 decreased by 41.2%. There was no difference in luteal tissue mRNA expression for ISGs on day 18 between accessory and original CL and between CL that subsequently regressed or did not regress. On day 56, an oxytocin challenge dramatically increased prostaglandin F2α metabolite (PGFM) in all cows but produced no pregnancy losses, although cows with previous accessory CL regression had greater PGFM. In summary, ipsilateral accessory CL did not regress during pregnancy, whereas most contralateral CL regressed by 63 days of pregnancy, providing evidence for local mechanisms in regression of accessory CL and protection of CL during pregnancy.

OBJECTIVES: To evaluates the effect of different modes of final follicular maturation triggering on the degree of apoptosis of granulosa cells (GCs) and the potential effect on progesterone secretion. METHODS: Thirty patients undergoing controlled ovarian hyperstimulation for IVF who received hCG, GnRH agonist, or dual trigger for final follicular maturation were included in the study. Granulosa cells were obtained at the time of oocyte retrieval. The proportion of apoptotic cells was evaluated via TUNEL and immunohistochemistry. RESULTS: The proportion of apoptotic cells was significantly higher in the GnRH agonist–alone group compared to hCG-alone and the dual trigger groups (13.5 ± 1.5% vs. 7.8% ± 1.8 vs. 10.1% ± 2, respectively, P < 0.01). Moreover, the expression of active-caspase-3 was also significantly increased in the GnRH agonist–alone group compared with the hCG-alone and the dual trigger groups (15.5% ± 2.9 vs. 8.4% ± 1.6 vs. 12.7% ± 2.6, respectively, P < 0.01). The progesterone levels measured in the granulosa-luteal cell culture medium after 24 h of incubation were similar between the three groups. CONCLUSIONS: The levels of apoptosis are increased after GnRH agonist/dual trigger. The increased apoptosis might be one of the culprit of the subsequent premature demise of the corpus luteum post GnRH agonist trigger.

Understanding luteal maintenance during early pregnancy is of substantial biological and practical importance. Characterizing effects of early pregnancy, however, has historically been confounded by use of controls with potential exposure to early Prostaglandin F2-alpha (PGF) pulses or differences in Corpus Luteum (CL) age. To avoid this, the present study utilized bihourly blood sampling to ensure control CL (n = 6) were of a similar age to CL from pregnant animals (n = 5), yet without exposure to PGF pulses. Additionally, CL from second month of pregnancy (n = 4) were analyzed to track fate of altered genes after cessation of embryonic interferon tau (IFNT) secretion. The major alteration in gene expression in first month of pregnancy occurred in interferon-stimulated genes (ISGs), with immune/interferon signaling pathways enriched in three independent over-representation analyses. Most ISGs decreased during second month of pregnancy, though, surprisingly, some ISGs remained elevated in the second month even after cessation of IFNT secretion. Investigation of luteolytic genes found few altered transcripts, in contrast to previous reports, likely due to removal of controls exposed to PGF pulses. An exception to this trend was decreased expression of transcription factor NR4A1. Beyond luteolytic genes and ISGs, over representation analyses highlighted the prevalence of altered genes within the extracellular matrix and regulation of Insulin-like growth factor (IGF) availability, confirming results of other studies independent of luteolytic genes. These results support the idea that CL maintenance in early pregnancy is related to lack of PGF exposure, although potential roles for CL expression of diverse ISGs and other pathways activated during early pregnancy remain undefined.Summary sentenceComparison of Day 20 CL from pregnant and cycling animals without initiation of luteolysis found 94 mRNA transcripts with differential expression, including many ISGs, some acutely regulated and others with elevated expression into second month of pregnancy, although no major alterations in luteolytic or steroidogenic genes indicate luteal maintenance during pregnancy is not transcriptomically opposite to CL regression.

Background Maintenance of the corpus luteum (CL) beyond the time of luteolysis is essential for establishing pregnancy. Identifying the distinct features of early pregnancy CL remains unresolved, hence we analyzed here the transcriptome of CL on day 18 pregnant (P) and non-pregnant (NP) cows using RNA-Seq. CL of P cows expressed ISGs, verifying exposure to the pregnancy recognition signal, interferon-tau (IFNT), whereas the CL of NP cows had elevated luteal progesterone levels, implying that luteolysis had not yet commenced. Results The DEGs, IPA, and metascape canonical pathways, along with GSEA analysis, differed markedly in the CL of P cows from those of NP cows, at the same day of the cycle. Both metascape and IPA identified similar significantly enriched pathways such as interferon alpha/beta, sonic hedgehog pathway, TNFA, EDN1, TGFB1, and PDGF. However, type-1 interferon and sonic hedgehog pathways were positively enriched whereas most of the enriched pathways were downregulated in the P compared to NP samples. Thirty-four % of these pathways are known to be elevated by PGF2A during luteolysis. Notably, selective DEGs in luteinized granulosa cells were modulated by IFNT in vitro in a similar manner to their regulation in the CL of P cows. Conclusion This study unraveled the unique transcriptomic signature of the IFNT-exposed, early pregnancy CL, highlighting the abundance of downregulated pathways known to be otherwise induced during luteolysis. These and IFNT-regulated in vitro pregnancy-specific DEGs suggest that IFNT contributes to the characteristics and maintenance of early pregnancy CL.

The roles of fibroblast growth factor 2 (FGF2) in the corpus luteum (CL) function and its modulatory effect on prostaglandin (PG) F2α during the bovine estrous cycle were studied using the following design of in vivo and in vitro experiments: (1) effects of FGF2 and FGF receptor 1 inhibitor (PD173074) on bovine CL function in the early (PGF2α-resistant) and mid (PGF2α-responsive) luteal stage in vivo, (2) the modulatory effect of FGF2 on PGF2α action during the luteal phase in vivo and (3) effects of FGF2 and PD173074 on bovine CL secretory function in vitro. Cows were treated by injection into the CL with: (1) saline (control), (2) FGF2, (3) PD173074, (4) FGF2 followed by intramuscular (i.m.) PGF2α, (5) PD173074 followed by i.m. PGF2α and (6) i.m. PGF2α as a positive control. For in vitro experiments, CL explants were treated with the aforementioned factors. Progesterone (P4) concentrations of blood samples or culture media were determined by radioimmunoassay. Relative mRNA expressions of the genes involved in angiogenesis and steroidogenesis were determined by quantitative real-time PCR. Although FGF2 treatment on day 4 of the estrous cycle did not change the cycle length, FGF2 with PGF2α decreased the P4 concentrations observed during the estrous cycle compared to the control group (P < 0.001). Moreover, FGF2 treatment on day 10 prolonged CL function as indicated by a significantly greater concentration of P4 on day 21 compared to the control group. In the in vitro study, FGF2 decreased cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase (HSD3B1) mRNA expression (P < 0.01) and decreased P4 production in the early-stage CL (P < 0.001). However, FGF2 + PGF2α or PGF2α alone resulted in an elevation of steroidogenic acute regulatory protein and CYP11A1 mRNA expression and P4 secretion in the early-stage CL (P < 0.01). In the mid-luteal phase, FGF2 upregulated CYP11A1 and HSD3B1 mRNA expression (P < 0.01), while FGF2 + PGF2α increased only HSD3B1 mRNA expression (P < 0.001). In conclusion, FGF2 seems to play a modulatory role in CL development or luteolysis, differentially regulating steroidogenesis and angiogenic factors as well as PGF2α actions.

Altres ajuts: acords transformatius de la UAB Altres ajuts: This work was supported by Finnish Veterinary Research, Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) Intrauterine devices block luteolysis in cyclic mares, but the underlying mechanism is unknown. To clarify the mechanisms, the protein profile of the endometrial secretome was analyzed using two-dimensional difference gel electrophoresis (2D-DIGE). Twenty-seven mares were classified according to whether they were inseminated (AI) or had an intrauterine device (IUD), a water-filled plastic sphere, inserted into the uterus on Day 3 after ovulation. Uterine lavage fluids were collected on Day 15 from pregnant inseminated mares (AI-P; n = 8), non-pregnant inseminated mares (AI-N; n = 4), and mares with IUD (n = 15). The IUD group was further divided into prolonged (IUD-P; n = 7) and normal luteal phase (IUD-N; n = 8) groups on the basis of ultrasound examinations, serum levels of progesterone and PGFM on Days 14 and 15, and COX-2 results on Day 15. Four mares from each group were selected for the 2D-DIGE analyses. Ten proteins had significantly different abundance among the groups, nine of the proteins were identified. Malate dehydrogenase 1, increased sodium tolerance 1, aldehyde dehydrogenase 1A1, prostaglandin reductase 1, albumin and hemoglobin were highest in pregnant mares; T-complex protein 1 was highest in non-pregnant mares; and annexin A1 and 6-phosphogluconolactonase were highest in IUD mares. The results suggest that the mechanism behind the intrauterine devices is likely related to inflammation.

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.

We evaluated if an additional GnRH injection 7 days before pre-synchronization with simultaneous PGF2α and GnRH (PG+G) would improve responses to presynchronization, synchronization, and pregnancy per AI (P/AI). We hypothesized that administering GnRH 7 days before PG+G would increase ovulation and corpus luteum (CL) presence at the PG+G, improve response to OvSynch treatments and P/AI. Holstein cows were blocked by parity and randomly assigned to either a PG+G (Control, n = 205); or to GnRH followed 7 days later by PG+G (ExtG, n = 201). At enrollment, Control was left untreated, whereas ExtG received GnRH. Seven days after enrollment, Control and ExtG received PG+G followed by OvSynch 7 days later (GnRH, 7 days PGF2α, 56 h GnRH, 16 h timed AI). Ovarian dynamics were assessed using ultrasonography in a subset of cows (n = 53 for Control; and n = 50 for ExtG) at each treatment, except the 2nd GnRH of OvSynch. Pregnancy diagnosed at 32- and 67-days post AI. Ovulation at enrollment tended (P = 0.06) to be higher for ExtG, but ovulation was not different at PG+G (P = 0.41) and first GnRH of the OvSynch (P = 0.25). There was a tendency (P = 0.08) for ExtG to have larger CL than Control at PGF2α of the OvSynch. There were no differences in CL and follicle sizes in any other treatment point assessed. There were no differences (P = 0.12) in luteolysis between treatments after PG+G. Overall P/AI was similar between treatments on Day 32 (Control = 33.0% vs. ExtG = 34.6%, P = 0.75) and 67 (Control = 31.8% vs. ExtG = 32.5%, P = 0.29) post AI. There was a tendency for an interaction between treatment and parity (P = 0.09) for P/AI at day 67 post-AI. In multiparous cows, ExtG tended to have greater P/AI than Control, whereas, in primiparous cows Control tended to have greater P/AI than ExtG at day 67 post-AI. In conclusion, the effects of GnRH 7 days before PG+G presynchronization lead to positive and negative tendencies, respectively, in multiparous and primiparous cows for P/AI at day 67 post-AI and needs further investigation.

Although the major source of relaxin in pigs is the corpus luteum of pregnancy, there is now evidence for relaxin gene expression and translation into protein in the theca interna cells of the preovulatory follicle, the corpus luteum of the cycle and the uterus. The theca interna cells retain their ability to express the relaxin gene and protein following ovulation. During the early stages of development of the corpus luteum, the theca-derived small lutein cells are the source of the relaxin transcript. As the corpus luteum becomes fully functional, there is a switch in the site of relaxin synthesis from small theca-derived lutein cells to large granulosa-derived cells. In the absence of luteolysis, this switch is accompanied by a dramatic rise in relaxin synthesis. Relaxin has been identified in boar seminal plasma and can maintain or increase sperm motility. However, a source of relaxin in the boar has not been identified. Relaxin is an important regulator of uterine function during pregnancy acting systemically to suppress myometrial activity and promote cervical dilation at parturition. The changes in thecal relaxin production during follicle development and its ability to promote growth and changes in proteolytic enzyme activity of granulosa cells in vitro have led to the concept of an autocrine or paracrine role for relaxin within the follicle. Uterotrophic effects of relaxin have been reported in rodents and swine and support the hypothesis that relaxin promotes uterine growth and expansion in early pregnancy to accommodate the growing fetuses. Mammotrophic effects of relaxin in rodents have now been extended to pigs, with evidence that relaxin is necessary for normal mammary parenchymal development in late pregnancy. In most instances the mechanisms responsible for, and the physiological significance of, these diverse biological effects remain to be elucidated.

To better understand the molecular basis of corpus luteum (CL) development and function RNA-Seq was utilized to identify differentially expressed genes (DEGs) in porcine CL during different physiological stages of the estrous cycle viz. early (EL), mid (ML), late (LL) and regressed (R) luteal. Stage wise comparisons obtained 717 (EL vs. ML), 568 (EL vs. LL), 527 (EL vs. R), 786 (ML vs. LL), 474 (ML vs. R) and 534 (LL vs. R) DEGs with log2(FC) ≥1 and p

Luteal phase (LP) is the period of time beginning shortly after ovulation and ending either with luteolysis, shortly before menstrual bleeding, or with the establishment of pregnancy. During the LP, the corpus luteum (CL) secretes progesterone and some other hormones that are essential to prepare the uterus for implantation and further development of the embryo, the function known as uterine receptivity. LP deficiency (LPD) can occur when the secretory activity of the CL is deficient, but also in cases of normal CL function, where it is caused by a defective endometrial response to normal levels of progesterone. LPD is particularly frequent in treatments using assisted reproductive technology (ART). Controlled ovarian stimulation usually aims to obtain the highest number possible of good-quality oocytes and requires the use of gonadotropin-releasing hormone (GnRH) analogs, to prevent premature ovulation, as well as an ovulation trigger to achieve timed final oocyte maturation. Altogether, these treatments suppress pituitary secretion of luteinizing hormone (LH), required for the formation and early activity of the CL. In addition to problems of endometrial receptivity for embryos, LPD also leads to dysfunction of the local uterine immune system, with an increased risk of embryo rejection, abnormally high uterine contractility, and restriction of uterine blood flow. There are two alternatives of LPD prevention: a direct administration of exogenous progesterone to restore the physiological progesterone serum concentration independently of the CL function, on the one hand, and treatments aimed to stimulate the CL activity so as to increase endogenous progesterone production, on the other hand. In case of pregnancy, some kind of LP support is often needed until the luteal–placental shift occurs. If LPD is caused by defective response of the endometrium and uterine immune cells to normal concentrations of progesterone, a still poorly defined condition, symptomatic treatments are the only available solution currently available.

Heat stress (HS) reduces reproductive performance of cattle, possibly by disrupting endocrine regulation such as prostaglandin (PG) production from uterus and estradiol 17β production from the dominant follicle. Prostaglandin F2α (PGF2α) secretion from endometrium surges during the luteal phase due to tumor necrosis factor (TNF) α stimulation and a positive-feedback loop with oxytocin (OT) from the corpus luteum, ultimately triggering luteolysis, while interferon τ (IFNT) inhibits upregulation of PGF2α production by TNFα and OT, thereby preventing luteolysis and triggering recognition of pregnancy. In the present study, we investigated the effect of OT, TNFα, and IFNT on PGF2α production in both types of endometrial cells under HS conditions. Stimulation of PGF2α production in endometrial epithelial cells by OT was unaffected by HS, while stimulation of PGF2α production in endometrial stromal cells by TNFα was enhanced by HS, and this increased PGF2α production was not significantly suppressed by IFNT. These results suggest that HS disrupted the regulation of PGF2α production by TNFα and IFNT in bovine endometrial stromal cells and it might be one of causes for low conception rate of cattle in summer.

Chemerin is a recently discovered adipokine that participates in the regulation of many physiological and disorder-related processes in mammals, including metabolism, inflammatory reactions, obesity, and reproduction. We investigated how chemerin affects the transcriptome profile of porcine luteal cells. The luteal cells were acquired from mature gilts. After the in vitro culturing with and without chemerin, the total RNAs were isolated and high-throughput sequencing was performed. Obtained datasets were processed using bioinformatic tools. The study revealed 509 differentially expressed genes under the chemerin influence. Their products take part in many processes, important for the functions of the corpus luteum, such as steroids and prostaglandins synthesis, NF-&kappa, B and JAK/STAT signal transducing pathways, and apoptosis. The expression of the CASP3, HSD3B7, IL1B, and PTGS2 genes, due to their important role in the physiology of the corpus luteum, was validated using the quantitative real-time polymerase chain reaction (qPCR) method. The qPCR confirmed the changes of gene expression. Chemerin in physiological concentrations significantly affects the expression of many genes in luteal cells of pigs, which is likely to result in modification of physiological processes related to reproduction.

Made available in DSpace on 2020-12-12T02:02:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Heat stress (HS) has deleterious effects on bovine reproduction, including prolongation of the luteal phase in Holstein cows, perhaps due to compromised luteolysis. The objective was to characterize effects of HS on luteolytic responses of nonlactating Holstein cows given 25 or 12.5 mg of PGF2α on d 7 of the estrous cycle. Cows were randomly distributed into 2 environments: thermoneutral (n = 12; 25°C) or HS (n = 12; 36°C). In each environment, cows were treated with 2 mL of saline, 25 or 12.5 mg of PGF2α (n = 4 cows per group). The HS environment induced a significant increase in rectal temperature and respiratory rate compared with the thermoneutral environment. Heat stress did not have significant effects on luteolytic responses or circulating progesterone concentrations. Rapid and complete luteolysis occurred in all cows given 25 mg of PGF2α and in 4 of 8 cows given 12.5 mg; the other 4 cows given 12.5 mg had partial luteolysis, with circulating progesterone concentrations initially suppressed, but subsequently rebounding. Therefore, we conclude that HS does not change corpus luteum sensitivity to PGF2α. Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science São Paulo State University (UNESP), Rua Prof. Dr. Walter Maurício Correa s/n 18618-681 Department of Morphology Institute of Biosciences University of Sao Paulo State (UNESP), R. Prof. Dr. Antônio Celso Wagner Zanin, 250, Distrito de Rubião Junior School of Agriculture São Paulo State University (UNESP) Department of Production Animal Health Faculty of Veterinary Medicine University of Calgary Department of Animal Science Luiz de Queiroz College of Agriculture (ESALQ) University of São Paulo, Av. Pádua Dias, 11 Department of Animal and Dairy Sciences University of Wisconsin Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science São Paulo State University (UNESP), Rua Prof. Dr. Walter Maurício Correa s/n 18618-681 Department of Morphology Institute of Biosciences University of Sao Paulo State (UNESP), R. Prof. Dr. Antônio Celso Wagner Zanin, 250, Distrito de Rubião Junior School of Agriculture São Paulo State University (UNESP)

International audience; Inflammation is not only the first line of defense of the organism but is also required in many reproductive processes such as ovulation, corpus luteum development, luteolysis, uterine clearance after insemination and post partum. Nevertheless, if excessive or persistent, inflammation can switch from a positive mechanism to a deleterious process, impairing oocyte quality and embryo development. Not only uterine but also non genital inflammatory sites can depreciate reproductive performances, with a carry over effect of 2 to 4 months. Since the metabolic challenges of the peripartum transition period make difficult for the cow to control inflammation, dairy cows are frequently in a pro-inflammatory stage, suggesting that inflammation, rather than infection, is a limiting factor of fertility in modern dairy cows. Within the first week after calving, cows have to mount an intense inflammatory response to the bacterial invasion of the uterine cavity with the challenge of being able to switch it off in no more than 5-6 weeks. The absence of neutrophils on endometrial smear is associated with the highest success rate at insemination. Since a fine tuning - rather than an absence - of inflammation is required along the reproductive cycle, anti-inflammatory drugs do not allow any improvement of pregnancy rate, except in the specific case of embryo transfer. Appropriate management of the transition period (especially nutritional) and in a long term perspective, genetic selection contribute to improve the aptitude of cows to controls the intensity of inflammatory process.

Although rescue of the corpus luteum (CL) is required for pregnancy, luteal function during maternal recognition of pregnancy remains largely unexplored. CL were collected from pregnant cattle on days 14, 17, 20, and 23, to encompass the maternal recognition of pregnancy period. Next-generation sequencing was used to profile mRNA abundance during this time, while tandem mass spectrometry and nanostring technology were used to profile proteins and miRNA, respectively. A total of 1157 mRNA were differentially abundant, while 27 miRNA changed, and 29 proteins tended to change. mRNA that increased were regulators of interferon signaling and DNA repair, while those that decreased were associated with luteolytic processes, such as calcium signaling and matrix metallopeptidase (MMP) signaling, indicating inhibition of these processes. One of these, MMP12, was regulated by prostaglandin F2A in vitro. mRNA that were maximally abundant on day 20 were primarily associated with immune processes. Two of these, C-C motif chemokine ligand 1 and NFKB inhibitor alpha, were regulated by interferon tau in vitro. MiRNA that increased were predicted to inhibit phosphatidylinositol signaling, while those that decreased may be negative regulators of steroidogenesis. One protein that was greater on day 20 than on day 14 was aldehyde dehydrogenase 1 family member A1 (ALDH1A1), which synthesizes retinoic acid. Pharmacological inhibition of this enzyme, or of retinoic acid receptor signaling, led to suppression of progesterone production in vitro. Overall, these data indicate that there are changes in the CL of pregnancy that are important for continued luteal function.

The fate of the canine corpus luteum (CL) differs from that of other domestic species: beyond the extended luteal regression observed in both pregnant and non-pregnant cycles, active luteolysis is observed only in pregnant dogs. Luteal regression in the absence of pregnancy lacks a luteolytic trigger. The CL lifespan during pregnancy is around 60 days, as long as that of the cyclic CL. Although they are already available in the first half of diestrus, LH and especially prolactin (PRL) play a decisive luteotropic role from approximately day 25 post-ovulation onwards. Nevertheless, many locally-produced factors are orchestrated to ensure a fully functional CL, which in the bitch produces progesterone (P4), 17b-estradiol, and other local regulators. Recently, insulin has been described as another luteotropic factor in this species, able to increase glucose uptake in luteal cells and contribute to steroid biosynthesis. The locally-produced PGE2 is also a potent luteotropic factor in the first half of diestrus, promoting STAR expression, as are also proliferating, vasoactive- and immunomodulatory factors. These, in turn, all contribute to the formation and maintenance of the canine CL. Meanwhile PGF2a, produced by the utero-placental compartment, participates actively in triggering pre-partum luteolysis. Cytokines play different roles, either contributing as luteotropic or as acute inflammation molecules. So far, the one clinically most efficient mechanism of interrupting a pregnancy in the dog is to block P4 receptors, using an antigestagen (e.g., aglepristone) in the second half of diestrus. To enhance the chances of pregnancy, however, several luteotropic factors could be used.

A viable corpus luteum (CL) producing an adequate amount of progesterone is absolutely essential for establishment and maintenance of pregnancy. One function of embryonic signaling to the mother is to ensure that the CL is maintained. In ruminants, the secretion of uterine prostaglandin F2alpha (PGF2A) is the signal that initiates luteolysis. Despite many studies to determine if PGF2A secretion from the uterus is altered in early pregnancy, conflicting interpretations have led to controversy regarding the exact mechanisms by which maternal recognition of pregnancy is achieved. In addition to alteration of uterine prostaglandin metabolism, changes within the CL itself may facilitate the establishment of a successful pregnancy. These changes include alteration of luteal blood flow, prostaglandin metabolism, sensitivity to prostaglandin actions, increased steroidogenic capacity, significant intracellular molecular changes and modification of the immune cells that are within the CL. Whether these intraluteal changes are necessary to establish pregnancy is undetermined. The focus of this review will be to provide a brief historical perspective on the utero-ovarian relationships that regulate luteal lifespan and review current knowledge of the CL of pregnancy in sheep and cattle.