Maintenance Of Corpus Luteum Research Articles

P-724 Gene expression profiling of cumulus cells from women with adenomyosis disclose differential pattern to endometriosis: striking the chord for evaluation of a biomarker

Abstract Study question Can metabolic picture of cumulus cell (CC) assess assisted reproductive technology outcome/s in women with adenomyosis/endometriosis and have implications on treatment decisions? Summary answer The differentially expressed genes between uterine pathologies explain the functional gap to identify diagnostic/prognostic markers of CC and further dissect molecular contribution to oocyte maturation. What is known already A healthy intra-follicular environment supports achievement of oocyte developmental competence (ODC) largely via coordinated CC-follicular fluid (FF) crosstalk. Lately, promising studies document CC of endometriosis are involved in important pathways for ODC like oxidative phosphorylation, metabolism, mitochondrial function and acetylation to name a few. Similar presentation/s of adenomyosis to other gynecological diseases including endometriosis makes the diagnosis difficult. Given that the functional integrities of CCs are susceptible to concurrent pathological conditions, understanding the influence of CCs on oocyte quality compared to routine morphological analysis in presence of deleterious systemic or pelvic conditions may impact clinical decisions. Study design, size, duration Twenty-eight consented patients undergoing IVF-ICSI between January to December 2021at Institute of Reproductive Medicine, Kolkata were enrolled. CCs obtained and corresponding oocyte maturation was assessed. They were sub-stratified (n = 9) according to uterine pathologies: adenomyosis (Group A), and endometriosis (Group B) as diagnosed by transvaginal ultrasound (TVUS). Counterpart/s of male sub-fertility, defined by World Health Organization criteria (5th edition) was treated as control (Group C; n = 10). CC from mature MII and immature GV oocytes were compared. Participants/materials, setting, methods CCs collected following oocyte-denudation were evaluated by Trypan Blue viability test. Patient age and embryo development were recorded with respect to each cumulus complex. Real Time-quantitative polymerase-chain-reaction (qRTPCR) was performed for glycolysis (PFKP, PKM2, ALDOA, LDHA, TPI1), cholesterol-biosynthesis (CYP51, MVK, PMVK, EBP) and extracellular-matrix (PTGS2, PTX3, TNFAIP6, VCAN) related gene/s known to have important functions in cumulus expansion and metabolism. Patient characteristics and gene expression were correlated by Spearman’s correlation. P < 0.05 was considered statistically significant. Main results and the role of chance A significant higher (p < 0.001) age (A vs B: 35.77±2.33 vs 25.88± 1.76; A vs C: 35.77±2.33 vs 31.9±1.98) and increased number of oocytes retrieved (A vs B: 11.22±1.56 vs 8.55±0.88) was documented in adenomyosis cohort. Immature oocytes from Group A expressed enhanced cholesterol/steroid biosynthesis, with significant (A vs B; p < 0.01) up-regulation of CYP51, MVK, PMVK, EBP in spite of hyper-estrogenic environment in peripheral blood. On contrary, significant attenuation (p < 0.01) of cholesterol biosynthesis was documented in endometriosis cohort compared to control. Expression of PTGS2, PTX3 and VCAN was down-regulated (p < 0.001) (which together mediate cumulus expansion and oocyte maturation, as well as corpus luteum formation and maintenance and reduce spindle abnormalities) in both Group A and B. Expression/s of CC-PFKP and PKM2 was lower in immature compared to mature oocytes (B vs C; p < 0.01) in endometriosis making the duo responsible for follicular/oocyte growth, and embryonic development. This supports the role in promoting cumulus expansion and oocyte competence. Expression levels of other genes including ALDOA, LDHA, TPI1, and TNFAIP6 were not associated with oocyte maturity, and fertilization in either cohort/s. Interestingly, levels of VCAN, TNFAIP6, PTX3 changed significantly as a function of the subject’s age and embryo development. Limitations, reasons for caution Our findings are limited by the relatively small sample size and inherent biological variability of human samples. Several yet known or unknown factors are presumably involved in the implantation process of adenomyosis and/or endometriosis. Multi-centric systematic studies are needed to corroborate these findings and account for the probable confounding factors. Wider implications of the findings A differential metabolic dependence of CC play essential role in oocyte competence in adenomyosis influencing cumulus expansion and oocyte maturation. This would be useful to do further studies to know how molecular interface of implantation capacity with live-birth rate would be affected to complement embryonic morphological assessment/s. Trial registration number Not Applicable

Recent progress of interferon-tau research and potential direction beyond pregnancy recognition.

Since the discovery of interferon-tau (IFNT) over 30 years ago as the trophectodermal cytokine responsible for the maintenance of the maternal corpus luteum (CL) in ruminants, exhaustive studies have been conducted to identify genes and gene products related to CL maintenance. Recent studies have provided evidence that although CL maintenance, with the up- and down-regulation of IFNT, is important, its regulatory role in the endometrial expression of interferon-stimulated genes (ISGs) is far more important for conditioning the uterine environment for successful conceptus implantation and thereafter. This review initially describes the mammalian implantation process, briefly but focuses on recent findings, as there appears to be a common phenomenon during early to mid-pregnancy among mammalian species.

Effects of carprofen and/or CIDR administration on pregnancies per artificial insemination around pregnancy recognition in lactating dairy cows

Effect of carprofen and/or CIDR on pregnancies per AI (P/AI) 14 days after artificial insemination (AI) in lactating cows were investigated. Following detection of corpus luteum (CL) by ultrasonography (USG) 14 days after AI, cows (n=853) were randomly allocated to CARP (carprofen, 1.4 mg/kg; Rimadyl®XL), CIDR (progesterone, 1.38g, for seven days), CARP+CIDR and CONT (control) groups. CL was re-determined with USG 21 days after AI to monitored the maintenance of CL. Rates of maintenance of CL did not differed among CARP (79.6% [168/211]), CIDR (86.0% [196/228]), CARP+CIDR (80.0% [172/215]) and CONT (74.9% [149/199]) groups; however, the interaction effect of treatment by body condition score (BCS) at AI (P<0.05) were existed. In this matter, among cows with BCS≤2.5, chances of maintenance of CL was lower in CONT (70.7%, [111/157]) group compared to those in CARP (81.7%, [125/153]), CIDR (83.6%, [153/183]) and CARP+CIDR (80.1%, [129/161]) groups. Whereas, among cows with BCS>2.5, chances of maintenance of CL were lower in CARP (74.1%, [43/58]) and CARP+CIDR (79.6%, [43/54]) groups compared to those in CONT (90.5%, [38/42]) and CIDR (95.6%, [43/45]) groups. For the P/AI at 28-32 days after AI, there was no difference among CARP (48.8% [103/211]), CIDR (50.9 [116/228]), CARP+CIDR (47.4% [102/215]) and CONT (44.7% [89/199]) groups. Pregnancy losses between 28-32 and 55-60 days did not differ among CARP (3.9%, [4/103]), CIDR (4.3%, [5/116]), CARP+CIDR (5.9%, [6/102]) and CONT (6.7%, [6/89]) groups. However, there was a significant (P<0.05) interaction effect of treatment by the number of services on pregnancy losses. In this regard, pregnancy losses were higher in cows inseminated thrice and four or more times in CONT (11.8% and 16.7%) group compared to those in CARP (0% and 4.8%), CIDR (0% and 6.9%), CARP+CIDR (0% and 11.1%); respectively. Consequently, no effects of carprofen or CIDR around pregnancy recognition on P/AI were observed despite a higher maintenance rate of CL in lactating cattle. Furthermore, fewer pregnancy losses in cows following three or more services could indicate the beneficial carry-over effects of carprofen and/or CIDR administration around maternal recognition of pregnancy.

Uterine Notch2 facilitates pregnancy recognition and corpus luteum maintenance via upregulating decidual Prl8a2.

The maternal recognition of pregnancy is a necessary prerequisite for gestation maintenance through prolonging the corpus luteum lifespan and ensuring progesterone production. In addition to pituitary prolactin and placental lactogens, decidual derived prolactin family members have been presumed to possess luteotropic effect. However, there was a lack of convincing evidence to support this hypothesis. Here, we unveiled an essential role of uterine Notch2 in pregnancy recognition and corpus luteum maintenance. Uterine-specific deletion of Notch2 did not affect female fertility. Nevertheless, the expression of decidual Prl8a2, a member of the prolactin family, was downregulated due to Notch2 ablation. Subsequently, we interrupted pituitary prolactin function to determine the luteotropic role of the decidua by employing the lipopolysaccharide-induced prolactin resistance model, or blocking the prolactin signaling by prolactin receptor-Fc fusion protein, or repressing pituitary prolactin release by dopamine receptor agonist bromocriptine, and found that Notch2-deficient females were more sensitive to these stresses and ended up in pregnancy loss resulting from abnormal corpus luteum function and insufficient serum progesterone level. Overexpression of Prl8a2 in Notch2 knockout mice rescued lipopolysaccharide-induced abortion, highlighting its luteotropic function. Further investigation adopting Rbpj knockout and DNMAML overexpression mouse models along with chromatin immunoprecipitation assay and luciferase analysis confirmed that Prl8a2 was regulated by the canonical Notch signaling. Collectively, our findings demonstrated that decidual prolactin members, under the control of uterine Notch signaling, assisted pituitary prolactin to sustain corpus luteum function and serum progesterone level during post-implantation phase, which was conducive to pregnancy recognition and maintenance.

Interferon-Tau regulates a plethora of functions in the corpus luteum

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.

Downregulated luteolytic pathways in the transcriptome of early pregnancy bovine corpus luteum are mimicked by interferon-tau in vitro

BackgroundMaintenance 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.ResultsThe 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.ConclusionThis 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.

Pregnancy-induced changes in the transcriptome of the bovine corpus luteum during and after embryonic interferon-tau secretion†.

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.

48 Timing of luteolysis and conceptus expulsion after induced pregnancy loss at three different timepoints after maternal recognition of pregnancy in cattle

Results from previous research indicate that induced pregnancy loss (IPL) on Day 36 of gestation in cattle resulted in luteolysis by gestation Day ∼45. The aim of this study was to test the hypothesis that IPL at 25, 35, or 55 days of gestation will result in luteolysis and whether the interval between IPL and luteolysis is positively associated with days of gestation at time of treatment. Pregnant non-lactating beef cows were randomly assigned to undergo IPL, consisting of an intrauterine infusion of 120mL of hypertonic saline (7.2%), at Day 25 (IPL25, n=15), Day 35 (IPL35, n=14), or Day 55 (IPL55, n=14). Corpus luteum (CL) volume and embryo/fetal viability were evaluated by transrectal B-mode ultrasonography daily for 19 days following treatment and every other day subsequently until luteolysis was identified or gestation Day 91. Blood flow through the CL was evaluated using colour-Doppler ultrasonography, and luteolysis was defined as the day when <25% of the CL was represented with colour pixels. Conceptus expulsion was defined as the absence of a conceptus and fluid accumulation in the uterus was <25%. Data were analysed using Proc Mixed in SAS (v9.4; SAS Institute Inc.). Cessation of conceptus heartbeat occurred in all animals within 24h of administration of treatment. Day of luteolysis occurred earlier (P<0.01) in IPL25 (5.3±0.4d) than IPL55 (8.9±1.4d) cows, and luteolysis tended (P=0.07) to occur earlier in IPL35 (6.6±0.6d) than IPL55 cows; however, values for this variable in IPL25 and IPL35 cows were similar (P>0.2). The variance in timing of luteolysis was greater for IPL55 than for IPL35 cows, whereas variance for IPL35 was greater than that for IPL25 (P<0.05) cows. Conceptus expulsion was detected earlier (P<0.01) in IPL25 (Day 4.2±0.4) and IPL35 (Day 5.8±0.5) than IPL55 (Day 10.4±1.4) cows; however, IPL25 and IPL35 cows had similar (P>0.1) values for this variable. Values for luteolysis and conceptus expulsion were correlated in IPL35 (r=0.84; P<0.01) and IPL55 (r=0.88; P<0.01) cows, whereas there was no correlation among values for these variables in IPL25 (r=0.31; P=0.3) cows. There were effects of treatment (P<0.01), time (P<0.01), and treatment by time interaction (P<0.01) on CL volume. Volume of the CL was less (P<0.04) in IPL25-treated cows by treatment day 5 compared with that in IPL55-treated cows. Additionally, CL volume was less (P<0.03) in IPL25 by treatment day 6 than in IPL35 cows, and IPL35 cows had a lesser (P<0.01) CL volume starting on treatment day 10 than IPL55 cows. Induction of pregnancy loss at different times of gestation resulted in luteolysis; however, the interval between treatment and luteolysis tended to increase as gestation days increased. Furthermore, the variability in the timing of luteolysis increased as gestation days increased. Results from the present study support the working hypothesis of presence of a continuous mechanism for CL maintenance during pregnancy beyond the classical maternal recognition period in cattle.

Embryo–uterineinteractions in pigs during week 2 of pregnancy

The second week of pregnancy is a particularly critical period for embryonic survival in pigs. Within that time, conceptus oestrogen synthesis is initiated, spacing and final placement of conceptuses is completed, and the signal for extending the functional lifespan of the corpora lutea is received by the mother. There is also a marked increase in blood flow to the uterus and the uterine endometrium produces and secretes nutrient histotrophe. Coneeptus-derived oestrogen has been implicated in many of these events. It is also during this period that the trophoblast elongates dramatically and the inner cell mass starts to differentiate into the embryo proper. Here, we critically review the evidence that oestrogen is the sole factor initiating long-term corpus luteum maintenance in pigs. We also review the functions and general properties of the major secretory proteins in histotrophe and the role of oestrogen in controlling their expression. It is now generally accepted that asynchrony within a litter underlies much of the losses of conceptuses that are otherwise genetically normal, but which are lagging in their development; however, the underlying mechanisms remain unclear. Here we hypothesize that oestrogenic compounds derived from more advanced conceptuses or provided prematurely, either by injection or in the diet, trigger a massive increase in uterine expression and secretion of retinol-binding protein laden with retinol. We propose that less developed, smaller conceptuses are least able to contend with the sudden exposure to this potential teratogen at a time when they are particularly susceptible to imbalance in retinol supply. Hence, even though their growth proceeds for a few days, their developmental potential is irrevocably compromised.

Novel Insights on the Corpus Luteum Function: Role of Vaspin on Porcine Luteal Cell Angiogenesis, Proliferation and Apoptosis by Activation of GRP78 Receptor and MAP3/1 Kinase Pathways.

Formation and limited lifespan of corpus luteum (CL) are important for proper ovarian periodicity and fertility. Failed vascularization, imbalance between proliferation and apoptosis leads to luteal phase deficiency and infertility. The aim of this study was to examine the effect of vaspin on angiogenesis, apoptosis and proliferation as well as the involvement of 78-kDa glucose-regulated protein receptor (GRP78) and mitogen-activated kinase (MAP3/1) in these processes. Porcine luteal cells were incubated with vaspin (0.1–10 ng/mL) for 24 h to 72 h and then mRNA and protein expression of angiogenesis: vascular endothelial growth factor (VEGFA), fibroblast growth factor 2 (FGF2), angiopoietin 1 (ANGPT1), VEGFA receptors (VEGFR1, VEGFR2), apoptosis: caspase 3, bcl-2-like protein 4 (BAX), B-cell lymphoma (BCL2), and proliferation: proliferating cells nuclear antigen (PCNA), cyclin A factors as well as secretion of VEGFA, FGF2, ANGT1 were measured by real-time polymerase chain reaction (PCR), immunoblotting and enzyme-linked immunosorbent assay (ELISA), respectively. Moreover, apoptosis was assessed by caspase activity using the Caspase-Glo 3/7 assay, while proliferation was by alamarBlue. We found that vaspin enhanced luteal cell angiogenesis, proliferation, and significantly decreased apoptosis. Additionally, using GRP78 siRNA and the pharmacological inhibitor of MAP3/1 (PD98059), we observed that the effect of vaspin was reversed to the control level in all investigated processes. Taken together, our results suggest that vaspin is a new regulator of female fertility by direct regulation of CL formation and maintenance of luteal cell function.

Pentraxin-3 mediates prosurvival actions of interferon tau in bovine luteinized granulosa cells.

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

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.

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.

Interferon-Tau Exerts Direct Prosurvival and Antiapoptotic Actions in Luteinized Bovine Granulosa Cells.

Interferon-tau (IFNT), serves as a signal to maintain the corpus luteum (CL) during early pregnancy in domestic ruminants. We investigated here whether IFNT directly affects the function of luteinized bovine granulosa cells (LGCs), a model for large-luteal cells. Recombinant ovine IFNT (roIFNT) induced the IFN-stimulated genes (ISGs; MX2, ISG15, and OAS1Y). IFNT induced a rapid and transient (15–45 min) phosphorylation of STAT1, while total STAT1 protein was higher only after 24 h. IFNT treatment elevated viable LGCs numbers and decreased dead/apoptotic cell counts. Consistent with these effects on cell viability, IFNT upregulated cell survival proteins (MCL1, BCL-xL, and XIAP) and reduced the levels of gamma-H2AX, cleaved caspase-3, and thrombospondin-2 (THBS2) implicated in apoptosis. Notably, IFNT reversed the actions of THBS1 on cell viability, XIAP, and cleaved caspase-3. Furthermore, roIFNT stimulated proangiogenic genes, including FGF2, PDGFB, and PDGFAR. Corroborating the in vitro observations, CL collected from day 18 pregnant cows comprised higher ISGs together with elevated FGF2, PDGFB, and XIAP, compared with CL derived from day 18 cyclic cows. This study reveals that IFNT activates diverse pathways in LGCs, promoting survival and blood vessel stabilization while suppressing cell death signals. These mechanisms might contribute to CL maintenance during early pregnancy.

Bone morphogenetic protein 6 affects cell-cell communication by altering the expression of Connexin43 in human granulosa-lutein cells

Connexin 43 (Cx43)-coupled gap junctions in granulosa cells play an important role in follicular development, oocyte maturation, and corpus luteum maintenance. Bone morphogenetic protein 6 (BMP6) is highly expressed in human oocytes and granulosa cells and is involved in the regulation of female reproduction. Currently, whether oocyte- and granulosa cell-derived BMP6 affects the expression of Cx43 and its related gap junction intercellular communication (GJIC) activity in human granulosa cells remains unknown. In this study, we demonstrate that BMP6 treatment significantly suppressed the expression of Cx43 in both primary and immortalized (SVOG) human granulosa-lutein cells. Using both pharmacological inhibitors and small interfering RNA-mediated knockdown approaches, we demonstrate that ALK2 and ALK3 BMP type I receptors are involved in BMP6-induced suppressive effects on Cx43 expression and GJIC activity in SVOG cells. Furthermore, these cellular activities are most likely mediated by the SMAD1/SMAD5-SMAD4-dependent signaling pathway. Notably, the ChIP analyses demonstrated that phosphorylated SMADs could bind to human Cx43 promoter. Our findings provide new insight into the molecular mechanisms by which an intrafollicular growth factor regulates cell-cell communication in human granulosa cells.

Spatio-temporal expression profile of matrix metalloproteinase (Mmp) modulators Reck and Sparc during the rat ovarian dynamics

BackgroundMatrix metalloproteinases (Mmps) and their tissue inhibitors (Timps) are widely recognized as crucial factors for extracellular matrix remodeling in the ovary and are involved in follicular growth, ovulation, luteinization, and luteolysis during the estrous cycle. Recently, several genes have been associated to the modulation of Mmps activity, including Basigin (Bsg), which induces the expression of Mmps in rat ovaries; Sparc, a TGF-β modulator that is related to increased expression of Mmps in cancer; and Reck, which is associated with Mmps inhibition. However, the expression pattern of Mmp modulators in ovary dynamics is still largely uncharacterized.MethodsTo characterize the expression pattern of Mmps network members in ovary dynamics, we analyzed the spatio-temporal expression pattern of Reck and Sparc, as well as of Mmp2, Mmp9 and Mmp14 proteins, by immunohistochemistry (IHC), in pre-pubertal rat ovaries obtained from an artificial cycle induced by eCG/hCG, in the different phases of the hormone-induced estrous cycle. We also determined the gene expression profiles of Mmps (2, 9, 13 14), Timps (1, 2, 3), Sparc, Bsg, and Reck to complement this panel.ResultsIHC analysis revealed that Mmp protein expression peaks at the early stages of folliculogenesis and ovulation, decreases during ovulation-luteogenesis transition and luteogenesis, increasing again during corpus luteum maintenance and luteolysis. The protein expression patterns of these metalloproteinases and Sparc were inverse relative to the pattern displayed by Reck. We observed that the gene expression peaks of Mmps inhibitors Reck and Timp2 were closely paraleled by Mmp2 and Mmp9 suppression. The opposite was also true: increased Mmp2 and Mmp9 expression was concomitant to reduced Reck and Timp2 levels.ConclusionTherefore, our results generate a spatio-temporal expression profile panel of Mmps and their regulators, suggesting that Reck and Sparc seem to play a role during ovarian dynamics: Reck as a possible inhibitor and Sparc as an inducer of Mmps.

Cyclooxygenase-2 is inhibited in prolonged luteal maintenance induced by intrauterine devices in mares

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 PGF2α 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 immunohistochemistry (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 PGF2α release and maintenance of CL.

Periconceptional undernutrition affects the levels of DNA methylation in the peri-implantation pig endometrium and in embryos

Maternal undernutrition during the periconceptional period alters the transcriptomic profile of pig endometrium and embryos. Herein, we tested the hypothesis that restricted maternal consumption by females during the periconceptional period impairs the pattern of DNA methylation in both the endometrium and embryos during the peri-implantation period (Day 15–16 of gestation). Affected genes in restricted-diet-fed pig endometrium and embryos were identified using quantitative methylation-specific PCR and comprised those genes which are known to be important in reproductive, metabolic and epigenetic function, thereby exhibiting altered transcriptomic expression in endometrium and embryos of restricted-diet-fed gilts. Specifically, levels of DNA methylation of selected genes with altered expression in the endometrium included acid phosphatase type 2C (PPAP2C), salivary lipocalin (SAL1), endothelin receptor type B (EDNRB), regulator of G-protein signalling 12 (RGS12), type 4 17β-hydroxysteroid dehydrogenase (HSD17B4), toll-like receptor 3 (TLR3), and adiponectin receptor 1 (ADIPOR1). In embryos, adiponectin receptor 2 (ADIPOR2), prostaglandin-endoperoxide synthase 2 (PTGS2), arachidonate 12-lipoxygenase (ALOX12), progestin and adipoQ receptor family member 7 (PAQR7), progesterone receptor membrane component 2 (PGRMC2), steroidogenic acute regulatory protein (STAR), and serpin family A member 1 (SERPINA1) were altered. Finally, 5 acid phosphatase tartrate resistant (ACP5), high mobility group box 2 (HMGB2), and DNA (cytosine-5)-methyltransferase 1 (DNMT1) were altered in both the endometrium and in embryos. In the endometrium, the methylation levels of ACP5 (regulation of endometrial-conceptus iron transport), RGS12 (protein-coupled receptor signalling), and TLR3 (immune response) were increased, while that of EDNRB (corpus luteum maintenance) was decreased. In embryos, the methylation levels of ADIPOR2 (metabolic homeostasis) and DNMT1 (DNA methylation maintenance) were increased. The levels of methylation in other studied endometrial and embryonic genes were unchanged. DNA methylation levels in both the peri-implantation pig endometrium and embryos may be altered in response to female nutritional restriction.

Regulation of the porcine corpus luteum during pregnancy.

The new corpora lutea (CLs) in pigs are formed from the preovulatory follicles after the luteinizing hormone (LH) surge. However, total autonomy and independence of CLs from LH up to Day 12 of cycle has recently been questioned. Transformation of estrous cycle CL to CL of pregnancy initiated by embryonic signals requires not only the cessation of prostaglandin F2 (PGF2α) supply to the luteal tissue but also needs the CL to overcome luteolytic acquisition and/or changing its sensitivity to PGF2α during Days 12-14 of pregnancy. The luteolytic cascade is prevented by inhibition of lymphocyte infiltration and leucocyte recruitment, limitation of cell apoptosis, upregulation of pregnancy-associated genes and an enhanced antiluteolytic role of PGE2 Our 'two-signal switch hypothesis' highlights the importance of post PGF2α and PGE2 receptor signaling pathways activation in CLs during luteolysis and rescue. The 'luteolytic switch' involves increased expression of many regression mediators and activation of the post PTGFR signaling pathway. The 'rescue switch' initiated by embryonic signals - estradiol 17β and PGE2 - induces post PTGER2/4 pathway, turning the 'luteolytic switch' off and triggering activity of genes responsible for CL maintenance. In mid and late pregnancy, CLs are maintained by LH and the synergistic action of metabolic hormones. This paper provides an outline of recent views on CL regression, rescue and maintenance during pregnancy in pigs that conflict with previous paradigms and highlights new findings regarding the actions of prostaglandins, role of microRNAs (miRNA) and immune system and signaling pathways governing the life cycle of porcine CL.

Effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation implantation dysfunction.

The effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation (COH) implantation dysfunction were studied. The COH implantation dysfunction model in mice was established by intraperitoneal injection of 7.5 IU pregnant mare's serum gonadotrophin (PMSG), followed by 7.5 IU human chorionic gonadotrophin (HCG) 48 h later. Then the female mice were mated with male at a ratio of 2:1 in the same cage at 6:00 p.m. The female mice from normal group were injected intraperitoneally with normal saline and mated at the corresponding time. Day 1 of pregnancy was recorded by examining its vaginal smears at 8:00 a.m. of the next day. Fifty successfully pregnant mice were equally randomly divided into 5 groups: normal control pregnant group (NC), COH implantation dysfunction model group (COH), low dosage of Bu-Shen-An-Tai recipe group (LOW), middle dosage of Bu-Shen-An-Tai recipe group (MID) and high dosage of Bu-Shen-An-Tai recipe group (HIGH). Then from day 1, the mice in different groups were respectively intragastrically given corresponding treatments at 9:00 a.m. for 5 consecutive days. The concentrations of 17β-estradiol (E2) and progesterone (P4) were determined by radioimmunoassay (RIA). The ultrastructural changes of ovarian tissues were observed by transmission electron microscope (TEM). The histopathological changes of ovarian tissues were observed by HE staining. The number of atretic follicles and pregnant corpus luteum were also recorded. TUNEL was applied to measure apoptotic cells of ovarian tissues. Western blotting was used to detect the protein expression of apoptosis- related factors like Bax, Bcl-2 and cleaved-caspase-3 in ovarian tissue of mice. The results showed that ovarian weight, the concentrations of E2 and P4, the number of atretic follicles and pregnant corpus luteum, as well as the apoptosis of granulosa cells were significantly increased in the COH group. The ultrastructures of ovarian tissues in the COH group showed that chromatin in granulosa cells was increased, agglutinated, aggregated or crescent-shaped. The focal cavitation and the typical apoptotic bodies could be seen in granulosa cells in the late stage of apoptosis. After the treatment with different doses of Bu-Shen-An-Tai recipe, the ultrastructural changes of ovarian granulosa cells apoptosis were dramatically improved and even disappeared under TEM. Visible mitochondria and mitochondrial cristae were increased and vacuoles were significantly reduced. The lipid dropltes were shown in a circluar or oval shape. The protein expression levels of Bax and cleaved-caspase-3 were decreased, and the expression of Bcl-2 protein was increased after treatment. It was concluded that Bu-Shen-An-Tai recipe can inhibit the apoptosis of ovarian granulosa cells, probably by up-regulating the protein expression of Bcl-2 and down-regulating Bax and cleaved-caspase-3, which contributes to the formation and maintenance of ovarian corpus luteum. It's helpful to promote the embryonic implantation, to reduce embryo loss and ultimately to improve the success rate of pregnancy.