Regression Of Corpus Luteum Research Articles

Review: Comparative of monitoring estrus cycle in livestock: Hormonal features and ultrasound

The estrus cycle is a period in which a female animal shows the ability to become pregnant, and this event will continue over some time. The estrus cycle has two different phases, the follicular phase characterized by the presence of graffian follicles which are classified as proestrus and estrus stages or periods from regression of the corpus luteum to ovulation, and luteal phase which is characterized by the presence of the corpus luteum which is classified as the metestrus and diestrus stage or period of ovulation to the corpus luteum regression. Proestrus is chained by decreasing the concentration of progesterone, followed by increasing the concentration of estradiol. Estrus is generally characterized by a very low concentration of progesterone, an increase in the concentration of LH, and a decrease in the concentration of estrogen. Metestrus is characterized by a low concentration of progesterone. Diestrus is characterized by an increase in the concentration of progesterone and an increase in steroid hormone production along with the maturation of the corpus luteum. The purpose of this review is to discuss the comparative monitoring of the estrus cycle in buffalo, dairy cows, sheep and camels related to hormonal features and ultrasonography.

Identification of stable genes in the corpus luteum of lactating Holstein cows in pregnancy and luteolysis: Implications for selection of reverse-transcription quantitative PCR reference genes

In lactating dairy cattle, the corpus luteum (CL) is a dynamic endocrine tissue vital for pregnancy maintenance, fertility, and cyclicity. Understanding processes underlying luteal physiology is therefore necessary to increase reproductive efficiency in cattle. A common technique for investigating luteal physiology is reverse-transcription quantitative PCR (RT-qPCR), a valuable tool for quantifying gene expression. However, reference-gene-based RT-qPCR quantification methods require utilization of stably expressed genes to accurately assess mRNA expression. Historically, selection of reference genes in cattle has relied on subjective selection of a small pool of reference genes, many of which may have significant expression variation among different tissues or physiologic states. This is particularly concerning in dynamic tissues such as the CL, with its capacity for rapid physiologic changes during luteolysis, and likely in the less characterized period of CL maintenance during pregnancy. Thus, there is a clear need to identify reference genes well suited for the bovine CL over a wide range of physiological states. Whole-transcriptome RNA sequencing stands as an effective method to identify new reference genes by enabling the assessment of the expression profile of the entire pool of mRNA transcripts. We report the identification of 13 novel putative reference genes using RNA sequencing in the bovine CL throughout early pregnancy and luteolysis: RPL4, UQCRFS1, COX4I1, RPS4X, SSR3, CST3, ZNF266, CDC42, CD63, HIF1A, YWHAE, EIF3E, and PPIB. Independent RT-qPCR analyses were conducted confirming expression stability in another set of CL tissues from pregnancy and regression, with analyses performed for 3 groups of samples: (1) all samples, (2) samples from pregnancy alone, and (3) samples throughout the process of CL regression. Seven genes were found to be more stable in all states than 2 traditional reference genes (ACTB and GAPDH): RPS4X, COX4I1, PPIB, SSR3, RPL4, YWHAE, and CDC42. When CL tissues from pregnant animals alone were analyzed, CST3, HIF1A, and CD63 were also identified as more stable than ACTB and GAPDH. Identification of these new reference genes will aid in accurate normalization of RT-qPCR results, contributing to proper interpretation of gene expression relevant to luteal physiology. Furthermore, our analysis sheds light on the effects of luteolysis and pregnancy on the stability of gene expression in the bovine CL.

Clustered microRNAs: The molecular mechanism supporting the maintenance of luteal function during early pregnancy.

MicroRNAs (miRNAs) are recognized as the important regulators of ovarian function. However, little is known about the hormonal regulation of miRNA expression and the role of the specific miRNA-mRNA interactions in corpus luteum. Therefore, the present study was undertaken to determine: (a) the expression of miRNAs in the corpus luteum in early pregnancy vs regression; (b) the effect of conceptus and uterine signals in the expression of selected miRNAs; and (c) the role of specific miRNA-mRNA interactions in the molecular changes and secretory function of the corpus luteum in the pig. The results showed that the majority of miRNAs differentially expressed in the corpus luteum in early pregnancy vs regression belong to independent clusters (eg, miR-99b, miR-532), which are highly conserved among different animal species. The main conceptus signal in the pig (17β-estradiol) elevated the luteal expression of the miR-99b cluster and lowered the expression of NR4A1 and AKR1C1, the genes involved in corpus luteum regression. Furthermore, the delivery of miR-99b cluster mimics to luteal tissue concomitantly decreased NR4A1 and AKR1C1 expression and enhanced progesterone secretion. The present study demonstrated that conceptus signals can support the maintenance of luteal function during pregnancy by clustered miRNA-stimulated pathways, governing the expression of genes involved in luteal regression.

Neutrophils, monocytes and other immune components in the equine endometrium: Friends or foes?

The innate and adaptive immune mechanisms are key components of regulation of reproductive physiological function and uterine disorders in equine uterus. The predominant immunological response in equine endometrium, characterized by an innate immune response, occurs under estrogens influence, in the follicular phase. Although, the increase in immune-related genes in equine endometrium during estrus has been suggested to play a role in uterine clearance after mating, immune cells and their product, i.e. cytokines play also mandatory role in the luteal development and maintenance, regression of equine corpus luteum, as well as in early pregnancy. Innate immune response is nonspecific and acts as the first line of defense against pathogens, foreign stimuli that include constituents of seminal fluid and local infections (endometritis). It has been recently established that a phagocytosis-independent mechanism to restrain bacteria, by means of neutrophil extracellular traps (NETs) formation, is involved in pathogenesis of in mare endometrial fibrosis (endometrosis). Moreover, persistent macrophages and mast cell activation could also have pro-fibrotic roles by secreting great amounts of pro-fibrotic factors and lead to fibrosis. This review will highlight the involvement of immune key components of the innate and adaptive immune system and their products in equine uterus and their contribution to reproductive physiological function and uterine disorders.

Intravaginal instillation of prostaglandin F2α was as effective as intramuscular injection for induction of luteal regression in lactating dairy cows

Our objectives were to test the efficacy of intravaginal (IVG) administration of PGF2α to induce corpus luteum (CL) regression, compare circulating progesterone (P4) profiles in cows receiving IVG versus intramuscular (IM) treatment with PGF2α, and evaluate reproductive outcomes. Lactating Holstein cows were synchronized using a Double-Ovsynch protocol [GnRH, 7 d later PGF2α, 3 d later GnRH, 7 d later GnRH, 7 d later PGF2α, 1 d later PGF2α, 32 h later GnRH, 16 to 20 h timed artificial insemination (TAI)] to receive TAI at 67 ± 3 d in milk. Seven days after the first GnRH treatment (time 0), cows with at least 1 visible CL ≥15 mm were blocked by parity and randomly assigned to a treatment that consisted of IM injection (IM-PGF; n = 201) or IVG instillation (IVG-PGF; n = 201) of PGF2α. Cows in IM-PGF received a single 25-mg dose of PGF2α (dinoprost tromethamine) intramuscularly. Cows in IVG-PGF received two 25-mg doses of PGF2α 12 h apart delivered through a catheter in the cranial portion of the vagina. Blood samples were collected at 0, 12, 48, and 72 h after treatment. Ovulation to the first GnRH of Double-Ovsynch was determined through transrectal ultrasonography. Only cows with P4 ≥1 ng/mL (functional CL) at time 0 (IM-PGF = 169; IVG-PGF = 179) were included in the analyses. Binary and quantitative data were analyzed by logistic regression and ANOVA with repeated measures, respectively. Results are presented as least squares means. Concentrations of P4 and the proportion of cows with a new CL at time 0 did not differ. Overall, the proportion of cows with CL regression using 1 ng of P4/mL (IM-PGF = 89.0%; IVG-PGF = 86.7%) or 0.5 ng of P4/mL (IM-PGF = 82.2%; IVG-PGF = 82.1%) as the cutoff did not differ. Concentrations of P4 were affected by treatment, time, and treatment × time interaction. Cows in IVG-PGF had greater mean P4 at 12 h than cows in IM-PGF. Mean P4 did not differ at 48 or 72 h after treatment. The proportion of cows with estrus recorded within 3 d of treatment (IM-PGF = 45.4%; IVG-PGF = 48.9%), ovulation risk after treatment (IM-PGF = 88.5%; IVG-PGF = 85.1%), and pregnancies per artificial insemination after TAI (IM-PGF = 51.5%; IVG-PGF = 57.8%) did not differ. We concluded that 2 IVG doses of 25 mg of PGF2α 12 h apart were as effective as a single 25-mg IM dose of PGF2α for inducing luteal regression in lactating dairy cattle.

Effects of prostaglandin F2\u03b1 (PGF2\u03b1) on cell-death pathways in the bovine corpus luteum (CL)

BackgroundProstaglandin F2α (PGF2α) may differentially affect viability of luteal cells by inducing either proliferation or cell death (via apoptosis or necroptosis). The diverse effects of PGF2α may depend on its local vs. systemic actions. In our study, we determined changes in expression of genes related to: (i) apoptosis: caspase (CASP) 3, CASP8, BCL2 associated X (BAX), B-cell lymphoma 2 (BCL2) and (ii) necroptosis: receptor-interacting protein kinase (RIPK) 1, RIPK3, cylindromatosis (CYLD), and mixed lineage kinase domain-like (MLKL) in the early and mid-stage corpus luteum (CL) that accompany local (intra-CL) vs. systemic (i.m.) analogue of PGF2α (aPGF2α) actions. Cows at day 4 (n = 24) or day 10 (n = 24) of the estrous cycle were treated by injections as follows: (1) systemic saline, (2) systemic aPGF2α (25 mg; Dinoprost), (3) local saline, (4) local aPGF2α (2.5 mg; Dinoprost). After 4 h, CLs were collected by ovariectomy. Expression levels of mRNA and protein were investigated by RT-q PCR, Western blotting and immunohistochemistry, respectively.ResultsWe found that local and systemic administration of aPGF2α in the early-stage CL resulted in decreased expression of CASP3 (P < 0.01), but CASP8 mRNA expression was up-regulated (P < 0.05). However, the expression of CASP3 was up-regulated after local aPGF2α treatment in the middle-stage CL, whereas systemic aPGF2α administration increased both CASP3 and CASP8 expression (P < 0.01). Moreover, we observed that both local and systemic aPGF2α injections increased RIPK1, RIPK3 and MLKL expression in the middle-stage CL (P < 0.05) while CYLD expression was markedly higher after i.m. aPGF2α injections (P < 0.001). Moreover, we investigated the localization of necroptotic factors (RIPK1, RIPK3, CYLD and MLKL) in bovine CL tissue after local and systemic aPGF2α injections in the bovine CL.ConclusionOur results demonstrated for the first time that genes related to cell death pathways exhibit stage-specific responses to PGF2α administration depending on its local or systemic actions. Locally-acting PGF2α plays a luteoprotective role by inhibiting apoptosis and necroptosis in the early CL. Necroptosis is a potent mechanism responsible for structural CL regression during PGF2α-induced luteolysis in cattle.

Possible Mechanisms for Maintenance and Regression of Corpus Luteum Through the Ubiquitin-Proteasome and Autophagy System Regulated by Transcriptional Factors.

The corpus luteum (CL) is an important tissue of the female reproductive process which is established through ovulation of the mature follicle. Pulsatile release of prostaglandin F2α from the uterus leads to the regression of luteal cells and restarts the estrous cycle in most non-primate species. The rapid functional regression of the CL, which coincides with decrease of progesterone production, is followed by its structural regression. Although we now have a better understanding of how the CL is triggered to undergo programmed cell death, the precise mechanisms governing CL protein degradation in a very short period of luteolysis remains unknown. In this context, activation of ubiquitin-proteasome pathway (UPP), unfolded protein response (UPR) and autophagy are potential subcellular mechanisms involved. The ubiquitin-proteasome pathway (UPP) maintains tissue homeostasis in the face of both internal and external stressors. The UPP also controls physiological processes in many gonadal cells. Emerging evidence suggests that UPP dysfunction is involved in male and female reproductive tract dysfunction. Autophagy is activated when cells are exposed to different types of stressors such as hypoxia, starvation, and oxidative stress. While emerging evidence points to an important role for the UPP and autophagy in the CL, the key underlying transcriptional mechanisms have not been well-documented. In this review, we propose how CL regression may be governed by the ubiquitin-proteasome and autophagy pathways. We will further consider potential transcription factors which may regulate these events in the CL.

Prevention of miscarriage following ovulation induction in women with endocrine infertility in anamnesis

Prescription of adjusted maintenance hormonal therapy during early stages of induced pregnancy is one of the most effective means of reducing reproductive losses.Aim. To improve the maintenance hormonal therapy during early stages of induced pregnancy in patients with endocrine infertility of various genesis in anamnesis.Materials and methods. In total, 44 women with induced singleton pregnancy and anovulatory infertility in anamnesis and 20 healthy pregnant women underwent ultrasound and hormonal monitoring during the first trimester of pregnancy. Depending on the dynamics of hormonal changes, corrective hormonal therapy was prescribed for patients with induced pregnancy. Prolongation of pregnancy up to 12 weeks was considered to be the main criterion for the evaluation of the therapeutic measures effectiveness. Statistical processing of data was performed using the programs StatSoft Statistica v.6.0, Microsoft Excel XP.Results. Despite the exogenous administration of progestins in the group of women with anovulatory infertility in anamnesis, there were detected high risks of miscarriage (47.7 %), chorionic pathology (29.5 %), abortions (27.3 %). Hormonal monitoring in patients with termination of pregnancy and hypothalamic-pituitary insufficiency revealed an initially low level of β-hCG, normal level of progesterone but an inadequate their rise in dynamics; in patients with hyperprolactinemia – a very high concentration of progesterone, normal β-hCG level in early gestation and its gradual decrease during the first trimester; in patients with hyperandrogenemia – a very high concentration of progesterone at the beginning and progressive its decrease during the first trimester, inadequate rise of β-hCG from the beginning of the study combined with increased levels of androgens – dehydroepiandrostenediol sulfate and/or free testosterone.Conclusions. In order to select an appropriate maintenance hormonal therapy, the concentration of progesterone and β-hCG should be determined at the beginning of the first trimester in patients with induced pregnancy and endocrine infertility in anamnesis. The first weeks of gestation (hyperconcentration of hormones) and the middle of the first trimester (corpus luteum regression) are the critical periods for such pregnant women. An adequate rise of β-hCG with normal or increased level of progesterone in women with hyperandrogenism and hyperprolactinemia during the first weeks of pregnancy serves as a reason for withdrawal of exogenous progesterone administration with a possible prolonged therapy after 6–7 weeks of gestation under the dynamic control of progesterone and β-hCG levels. Patients with hypothalamic-pituitary insufficiency should receive constant maintenance hormonal therapy with progestins in high therapeutic doses at the beginning of pregnancy.

A novel strategy for resynchronization of ovulation in Nelore cows using injectable progesterone (P4) and P4 releasing devices to perform two timed inseminations within 22 days.

We aimed to evaluate the reproductive performance of Nelore lactating cows submitted to a resynchronization 12days after timed artificial insemination (TAI) with or without a long-acting progesterone (P4-LA) treatment. Nelore cows were submitted to a P4/oestradiol-based TAI protocol (D0=insemination). On D12, cows in the control group (n=184) received a new P4 intravaginal device (0.96g), whereas cows in the P4-LA group (n=192) received the P4 device and 75mg P4-LA. Cows identified as non-pregnant (n=120) by regression of corpus luteum using colour Doppler ultrasonography on D20 had the P4 device removed and received 500ug of sodium cloprostenol, 1mg of oestradiol cypionate and 300IU of eCG and were re-inseminated on D22. There was no difference (p>0.10) in the pregnancy rate at D20, D30 and D60 after first TAI between the control (69%, 59.7% and 57%, respectively) and P4-LA (67%, 55.7%, and 55.2%, respectively) groups. Pregnancy losses were similar between both groups (p>0.1). For cows submitted to the second TAI, the pre-ovulatory follicle size did not differ (p>0.1), but the oestrous detection and pregnancy rates were greater (p<0.05) in the P4-LA group (92.2% [59/64] and 60.9% [39/64], respectively) than in controls (75% [42/56] and 44.6% [25/56]). The cumulative pregnancy rate after two TAIs did not differ (p>0.1) between control (73.3% [135/184]) and P4-LA (76% [146/192]) groups. The use of P4-LA at 12days after TAI potentially increases the pregnancy rates for a new early resynchronization strategy associated with the Doppler imaging for pregnancy diagnosis and results in an alternative to perform two TAIs in 22days in beef cows.

Hormonal regulation and function of an RNA helicase, Ddx5 in corpus luteum of adult Wistar rats

Corpus luteum (CL) is an endocrine tissue involved in regulation of reproductive cycle and early pregnancy establishment. In the present study DEAD-box helicase-5 (Ddx5), a member of the DEAD box family of RNA helicases was investigated for its expression, regulation and function in CL of Wistar rats. Ddx5 was expressed in adult rat CL. Primary cell culture from supra-ovulated ovaries were established for in vitro studies. Addition of luteinizing hormone (LH; 100 ng/ml), a luteotrophic factor in primary cell culture, decreased Ddx5 RNA expression (foldchange:0.6 ± 0.075) while prostaglandin alpha (PGF2α; 1μM), a luteolytic factor caused an increase (foldchange:2.4 ± 0.4) compared to control group. Under in vivo conditions, the administration of PGF2α or gonadotropin-releasing hormone antagonist; cetrorelix (CET) caused luteolysis as well as an increase in the protein level of Ddx5 (foldchange:1.9 ± 0.27 and 1.4 ± 0.09 viz.; p < 0.05) in CL of adult rats. LH was administered post CET treatment which suppressed Ddx5 protein expression (foldchange:0.8 ± 0.16; p < 0.05) compared to CET treated group. Further, it was observed that the expression of Ddx5 was upregulated (foldchange:1.5 ± 0.23; p < 0.05) in CL during late pregnancy compared to mid pregnancy concomitant to luteolysis in adult rats. Overall, the results suggest for the first time that Ddx5 is expressed in rat CL and regulated by luteolytic and luteotrophic factors in an inverse fashion. Further, the data significantly correlates ddx5 expression to CL regression suggesting involvement of ddx5 in luteolysis. These results suggest a significant role of Ddx5 in female reproduction biology and warrant in depth examination of the function of Ddx5 in CL.

Regulation of follicle waves to maximize fertility in cattle

Cattle have recurrent follicular waves every 7-10 days in most physiological situations; an FSH increase is associated with emergence of the wave and LH pulse frequency determines the fate of the dominant follicle. To control oestrus with hormones it is necessary to ensure that either induced corpus luteum regression or the termination of a progestogen treatment coincides with the selection of the dominant follicle during the wave, to give a precise onset of oestrus and high fertility. The exogenous administration of progesterone or progestagen blocks the normal turnover of the dominant follicle once the corpus luteum regresses. Thus, the effects of duration of dominance of the preovulatory follicle on onset of oestrus and fertility were examined. The variation in onset of oestrus was reduced but occurred 5-9 h later after 4 versus 8 days of dominance; pregnancy rate was also affected with dominance periods of 2-4, 4-8 and > 10 days resulting in 0, 10-15% or 20-50% reduction in pregnancy rates, respectively. The necessity for short duration of dominance of the preovulatory follicle means that to ensure high fertility the follicular wave needs to be regulated when using hormones to control oestrus. Two approaches were examined, namely the use of GnRH or oestradiol at time of progesterone intravaginal releasing device insertion. The effect of 250 micrograms of synthetic GnRH on the fate of an existing follicle wave was to ovulate the dominant follicle (20/20 cows) and a new wave emerged 1.6 +/- 0.3 days later; however, there was no effect of GnRH on the wave if administered before dominant follicle selection. The effect of oestradiol concentrations on suppression of FSH in ovariectomized heifers showed that increasing oestradiol to 10-15 pg ml-1 caused a 37 +/- 6.9% decrease in FSH for 24 h, with a subsequent increase to pretreatment values by 57 +/- 13 h. In cyclic heifers, increasing oestradiol to > 10 pg ml-1 in conjunction with progesterone treatment at emergence of the first wave of the cycle affected the current follicle wave by either preventing dominant follicle selection or decreasing diameter of the dominant follicle, without consistently affecting the interval to new wave emergence. Increase of oestradiol after dominance, however, delayed new wave emergence by 2-5 days. A better understanding of the hormonal control of follicle waves will lead to development of improved hormonal regimens to control oestrus sufficiently to give high pregnancy rates to a single AI without recourse to detection of oestrus.

Inter- and intra-cellular mechanisms of prostaglandin F2a action during corpus luteum regression in cattle

The bovine corpus luteum (CL) grows very fast and regresses within a few days at luteolysis. Mechanisms controlling development and secretory function of the bovine CL may involve many factors that are produced both within and outside the CL. In the cow, luteolysis is initiated by uterine prostaglandin (PG)F2α released at the late luteal stage. It can also be induced by injection of exogenous PGF2α given at the mid luteal stage. Luteolysis consists of a phase of rapid decrease in progesterone (P4) production by the CL, followed by a phase of structural regression. Although uterine PGF2α is known to be the main luteolytic factor, its direct action on the CL is mediated by the products of accessory luteal cells: immune cells, endothelial cells, pericytes and fibroblasts. There are studies showing that beside endothelin-1, cytokines (tumor necrosis factor-α, interferons) and nitric oxide play critical roles in functional and structural luteolysis in cattle by stimulating leukotrienes and PGF2α, decreasing P4 secretion and apoptosis induction. Because of luteal blood flow and P4 concentrations decrease in parallel during both spontaneous and PGF2α-induced luteolysis, a decrease in luteal blood flow resulting in hypoxia has been proposed as one of the main luteolytic mechanisms in the cow. Hypoxia inhibits P4 synthesis in luteal cells by inhibiting the steroidogenic enzymes and promotes apoptosis of luteal cells by increasing pro-apoptotic proteins. Although reduction of luteal blood flow and hypoxia contribute to the late events of luteolysis, little is known about the physiological relevance and the cause of the transient increase in luteal blood flow and reactive oxygen species during the initial step of luteolysis.

Factors that affect fertility during oestrous cycles with short or normal luteal phases in postpartum cows

We have used a model to study infertility in postpartum cows. In mated cows with short luteal phases, daily supplementation with progestagen, beginning on day 3, failed to maintain pregnancy, despite the fact that fertilization, early embryo development and transport of the embryo into the uterus appeared to be normal. Normal embryos were transferred, on day 7 after oestrus, into cows with short luteal phases that received daily supplementation with progestagen, and embryos from cows with short luteal phases were transferred, on day 6 after mating, into cows with normal cycles. Pregnancy rates in each case were about half those achieved using progestagen-pretreated cows with normal luteal phases. In mated cows with short luteal phases supplemented with progestagen, pregnancy rates were markedly improved by removal of the regressing corpus luteum on day 4 or 5 after mating. In cows with normal cycles, fertility was lower when preovulatory concentrations of oestradiol were high for more than 3 days. This may result from persistent follicles, which develop when progesterone is low and frequency of tonic pulses of LH is high. In cows with such follicles, oocytes may resume maturation before the surge of LH, and embryos died in the oviduct before the 16-cell stage. Finally, in both early postpartum cows with transferred embryos and cows with normal cycles, excessive follicular development and high oestradiol during the luteal phase were detrimental to embryo survival. Treatments designed to maximize fertility in early postpartum cows must provide high progesterone and low oestradiol before mating and not yield luteolytic influences such as PGF2 alpha and oestradiol, both early after mating and during maternal recognition of pregnancy.

Thrombospondin-1 at the crossroads of corpus luteum fate decisions.

The multimodular matricellular protein thrombospondin-1 (THBS1) was among the first identified endogenous antiangiogenic molecules. Recent studies have shown THBS1-mediated suppression of angiogenesis and other critical activities for corpus luteum (CL) regression. THBS1 is specifically induced by prostaglandin F2alpha in mature CL undergoing regression, whereas luteinizing signals such as luteinizing hormone and insulin reduced its expression. THBS1 interacts both synergistically and antagonistically with other essential luteal factors, such as fibroblast growth factor 2, transforming growth factor beta1 and serpin family E member 1, to promote vascular instability, apoptosis and matrix remodeling during luteal regression. Expression of THBS1 is also downregulated by pregnancy recognition signals to maintain the CL during early pregnancy. This dynamic pattern of luteal expression, the extensive interactivity with other luteal factors and strong antiangiogenic and proapoptotic activities indicate that THBS1 is a major determinant of CL fate.

Necroptosis in primate luteolysis: a role for ceramide

The corpus luteum (CL) is a transient endocrine organ, yet molecular mechanisms resulting in its demise are not well known. The presence of phosphorylated mixed lineage kinase domain-like pseudokinase pMLKL(T357/S358) in human and nonhuman primate CL samples (Macaca mulatta and Callithrix jacchus) implied that necroptosis of luteal cells may be involved. In M. mulatta CL, pMLKL positive staining became detectable only from the mid-late luteal phase onwards, pointing to necroptosis during regression of the CL. Cell death, including necroptosis, was previously observed in cultures of human luteal granulosa cells (GCs), an apt model for the study of the human CL. To explore mechanisms of necroptotic cell death in GCs during culture, we performed a proteomic analysis. The levels of 50 proteins were significantly altered after 5 days of culture. Interconnectivity analysis and immunocytochemistry implicated specifically the ceramide salvage pathway to be enhanced. M. mulatta CL transcriptome analysis indicated in vivo relevance. Perturbing endogenous ceramide generation by fumonisin B1 (FB1) and addition of soluble ceramide (C2-CER) yielded opposite actions on viability of GCs and therefore supported the significance of the ceramide pathway. Morphological changes indicated necrotic cell death in the C2-CER treated group. Studies with the pan caspase blocker zVAD-fmk or the necroptosis blocker necrosulfonamid (NSA) further supported that C2-CER induced necroptosis. Our data pinpoint necroptosis in a physiological process, namely CL regression. This raises the possibility that the primate CL could be rescued by pharmacological inhibition of necroptosis or by interaction with ceramide metabolism.

Molecular modelling predicts that 2-methoxyestradiol disrupts HIF function by binding to the PAS-B domain

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.

Dihydrotestosterone synthesis in the sheep corpus luteum and its potential mechanism in luteal regression.

Corpus luteum (CL) regression is a complex physiological process. Previous studies have shown that dihydrotestosterone (DHT) may be involved in regulating CL regression, but the mechanism is still unclear. In this study, we evaluated the localization of the two isoforms of DHT synthetase 5α-reductase (5α-red1 and 5α-red2) and androgen receptor (AR) in sheep CL, and investigated 5α-red1, 5α-red2, AR, and DHT levels at different luteal stages of CL (early, middle, and late phase) by immunohistochemistry, quantitative real-time polymerase chain reaction, and western blot analysis. Moreover, we cultured luteal cells from middle phase CL and treated them with different concentrations of DHT (10-10 -10 -6 M) and the AR antagonist flutamide (10 -5 M), to evaluate whether DHT is involved in the regulation of progesterone (P4) secretion and progesterone nuclear receptor (PGR) expression and whether these effects are regulated by the AR pathway. We also investigated the effects of DHT and flutamide on prostaglandin F2α (PGF2α) secretion and apoptotic gene and protein expression. Our results showed that 5α-red1, 5α-red2, and AR were expressed in the CL, and their expression and DHT levels were changed during the luteal phase. DHT was involved in mediating P4 and PGF2α secretion and PGR and apoptotic gene and protein expression. The effects of DHT on CL were at least partially regulated by the AR pathway. This study reveals the mechanism of action of DHT on sheep CL regression and lays the foundation for further exploration of androgen regulation of CL function.

Transforming growth factor-beta family members are regulated during induced luteolysis in cattle

The transforming growth factors beta (TGFβ) are local factors produced by ovarian cells which, after binding to their receptors, regulate follicular deviation and ovulation. However, their regulation and function during corpus luteum (CL) regression has been poorly investigated. The present study evaluated the mRNA regulation of some TGFβ family ligands and their receptors in the bovine CL during induced luteolysis in vivo. On day 10 of the estrous cycle, cows received an injection of prostaglandin F2α (PGF) and luteal samples were obtained from separate groups of cows (n= 4-5 cows per time-point) at 0, 2, 12, 24 or 48 h after treatment. Since TGF beta family comprises more than 30 ligands, we focused in some candidates genes such as activin receptors (ACVR-1A, -1B, -2A, -2B) AMH, AMHR2, BMPs (BMP-1, -2, -3, -4, -6 and -7), BMP receptors (BMPR-1A, -1B and -2), inhibin subunits (INH-A, -BA, -BB) and betaglycan (TGFBR3). The mRNA levels of BMP4, BMP6 and INHBA were higher at 2 h after PGF administration (P<0.05) in comparison to 0 h. The relative mRNA abundance of BMP1, BMP2, BMP3, BMP4, BMP6, ACVR1B, INHBA and INHBB was upregulated up to 12 h post PGF (P<0.05). On the other hand, TGFBR3 mRNA that codes for a reservoir of ligands that bind to TGF-beta receptors, was lower at 48 h. In conclusion, findings from this study demonstrated that genes encoding several TGFβ family members are expressed in a time-specific manner after PGF administration.

Immunohistochemical localization of progesterone receptors alpha (PRA) in ovary of the pseudopregnant rabbit.

Progesterone plays an important role in the reproductive function and follicular development in mammals. The aim of the present study was to examine the localization of progesterone receptor alpha (PRA) in ovary of pseudopregnant rabbit by immunohistochemical methods. Samples were collected from 14 h. to 18 days of pseudopregnancy. At the first stage of pseudopregnancy (14 h.), the rabbit ovary showed moderate immunostaining of PRA in the granulosa cells and theca interna cells of preovulatory follicle and in the stroma cells. At the middle stage of pseudopregnancy (3-7 days), the rabbit ovary showed strong immunostaining of PRA in ovarian surface epithelial cells, follicular cells of the primary follicle, granulosa cells and theca interna cells of the growing and antral follicles. Moderate immunoexpression of PRA were observed in the large lutein cells and endothelial cells of the corpus haemorrhagicum and corpus luteum and in the stroma cells. At the end of pseudopregnancy (18 days) strong PRA reactions were detected in the small lutein cells of the regressed corpus luteum. Moderate to strong PRA immuno-expression were observed in the proliferated theca interna cells of the atretic antral follicles. The atretic large lutein cells of the regressed corpus luteum showed negative immunostaining for PRA. This study showed that the PRA positive small lutein cells of the regressed corpus luteum and the PRA positive proliferated theca interna cells of the atretic antral follicles were transformed into PRA positive interstitial gland cells. In conclusion, the present study had described the distribution of PRA in the ovary of pseudopregnant rabbit, which is not discussed before in the available literature. It also gives more information about follicular dynamic, formation and origin of interstitial glands, mechanism of ovulation, formation and regression of the corpus luteum.

Second prostaglandin F2α treatment during Ovsynch protocol does not improve fertility outcomes in dairy cows.

The objective of this study was to evaluate the effect of a second prostaglandin F2α (PGF2α) treatment during Ovsynch on luteal regression and fertility in dairy cows, compared with standard Ovsynch. The study was conducted on 111 Holstein Friesian multiparous cows on commercial dairy farm. The cows in the experimental group (n=48) received two treatments of PGF2α 24 hours apart during Ovsynch. The cows in the control group (n=63) were synchronized with standard Ovsynch. To assess the progesterone (P4) concentration blood samples were collected at the day of PGF2α treatment and at the 2nd GnRH treatment. Pregnancy was evaluated by ultrasound examination 37-40 days after timed artificial insemination (TAI) by ultrasound. Cows diagnosed pregnant were re-examined between days 70-80 after TAI. The percentage of cows with complete corpus luteum (CL) regression (P4⟨0.5 ng/ml at the time of the 2nd GnRH treatment) was 89.6 % after two PGF2α treatments and 88.9 % after one PGF2α treatment. There were no statistically significant differences (p>0.05) in the pregnancies per artificial insemination (P/AI) between the experimental and control group (P/AI). However, the pregnancy loss rate was lower in cows receiving two PGF2α treatments than in the control animals (0.0 % vs. 6.4 %; p⟨0.05). In conclusion, the second PGF2α treatment during Ovsynch protocol had no significant effect on CL regression and P/AI in dairy cows. The pregnancy losses until days 75-80 after TAI were significantly lower after two PGF2α treatments than after one PGF2α treatment.