Cumulus Cell-oocyte Complexes Research Articles

Perfluorohexane sulfonate (PFHxS) disturbs the estrous cycle, ovulation rate, oocyte cell communication and calcium homeostasis in mice

Perfluoroalkyl substances are man-made chemicals with ample consumer and industrial applications. They are widely used and are resistant to environmental and metabolic degradation. Several studies have evaluated the effects of Perfluorohexane sulfonate on reproduction. However, there are few reports exploring the cell and molecular mechanisms of its toxicity in the ovary. The aim of this study was to investigate the effects of PFHxS exposure on the estrous cycle, ovulation rate, and the underlying mechanisms of action in female mice in vivo. The animals received a single sub-lethal dose of PFHxS (25.1 mg/kg, 62.5 mg/kg) or vehicle and were stimulated to obtain immature cumulus cell-oocyte complexes (COCs) from the ovaries, or superovulated to develop mature COCs. To evaluate oocyte physiology, Gap-junction intercellular communication (GJIC) was analyzed in immature COCs and calcium homeostasis was evaluated in mature oocytes. PFHxS exposure prolonged the estrous cycle and decreased ovulation rate in female mice. Connexins, Cx43 and Cx37, were downregulated and GJIC was impaired in immature COCs, providing a possible mechanism for the alterations in the estrous cycle and ovulation. No morphological abnormalities were observed in the mature PFHxS-exposed oocytes, but calcium homeostasis was affected. This effect is probably due, at least partially, to deregulation of the endoplasmic reticulum calcium modulator, Stim1. These mechanisms of ovarian injury could explain the reported correlation among PFHxS levels and subfertility in women undergoing fertility treatments.

A Simple and Efficient Method for Generating KO Rats Using In Vitro Fertilized Oocytes.

The development of ZFN, TALEN, and CRISPR/Cas9 systems has simplified the process of generating knockout (KO) and knock-in (KI) rats in addition to mice. However, in rats, an efficient genome editing technique that uses in vitro fertilized oocytes has not been established. Recently, we reported the stable generation of offspring from five standard strains of rats by superovulation and in vitro fertilization (IVF). Furthermore, genome-edited rats can be easily generated by electroporation. First, juvenile female rats are administered LHRH (luteinizing hormone-releasing hormone) to synchronize the estrous cycle and then AIS (Automatic Identification System) with PMSG (pregnant mare serum gonadotropin) before hCG (human chorionic gonadotropin) for superovulation. Sperm collected from a sexually mature male rat the following morning is then pre-cultured. Cumulus cell-oocyte complexes (COCs) are collected from female rats under anesthesia, and COCs are induced into a medium containing concentration-adjusted sperm. Thereafter, oocytes with two pronucleus are selected as fertilized oocytes. Next, fertilized oocytes are transferred into a glass chamber containing CRISPR ribonucleoprotein (RNP) complexes formed from gRNA and Cas9 protein. After electroporation, fertilized oocytes are then immediately transferred to culture medium. The next day, embryos are transferred into the oviduct of pseudopregnant female rats. Using the above method, offspring can be obtained 22days after the day of embryo transfer. In this paper, we outline a method allowing simple and efficient generation of genetically modified rats without the need for technically difficult micromanipulation techniques.

Efficient editing BMP15 in porcine oocytes through microinjection of CRISPR ctRNP

Bone morphogenetic protein 15 (BMP15) is an X-linked gene encoding an oocyte secreted factor, which plays varied functions in the female fertility between mono-ovulatory and poly-ovulatory mammalian species. We previously found that knockout of BMP15 completely blocked porcine follicular development at preantral stages. However, the specific function of BMP15 on porcine oocytes in vitro maturation remains largely unknown. Here, we injected the pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complex into the cytoplasm of germinal vesicle stage porcine oocytes to disrupt BMP15. The ctRNP composed of Cas9 nuclease and crRNA-tracrRNA complex at 1.2/1 content ratio. The tested crRNA-tracrRNA complex concentration ranging from 50 to 200 ng/μL, all presented effective editing of BMP15 in porcine oocytes, and the 125 ng/μL crRNA-tracrRNA complex presented the highest editing efficiency (39.23 ± 3.33%). Surprisingly, we found approximately 95% edited oocytes presented monoallelic mutations, and only 5% edited oocytes harbored biallelic mutations. Interestingly, the coinjected two crRNAs guided the ctRNP complex to concurrently cut within a 10 bp window of the PAM (protospacer adjacent motif), resulting in a precise deletion within BMP15 in 85.9% edited oocytes, and additional deletion happened in 14.1% edited oocytes, which resulted in large fragment deletions in BMP15. Most deletions caused frameshift and introduced premature stop codon in BMP15, resulting in the disruption of BMP15 protein expression, which was confirmed by the Western blot analysis showing the reduced BMP15 protein expression in ctRNP injected oocytes. The disruption of BMP15 attenuated the activation of SMAD1/5/8 signaling, and impaired cumulus expansion of porcine cumulus cell-oocyte complexes (COCs). Our study proved that delivering CRISPR ctRNP into porcine oocytes by microinjection was able to edit BMP15 efficiently, providing a new strategy to investigate the functions of oocyte-specific secreted factors in oocyte in vitro maturation.

Oocyte-specific disruption of adrenomedullin 2 gene enhances ovarian follicle growth after superovulation.

Adrenomedullin 2 (ADM2), adrenomedullin (ADM), and calcitonin gene-related peptides (α- and β-CGRPs) signal through heterodimeric calcitonin receptor-like receptor/receptor activity-modifying protein 1, 2 and 3 (CLR/RAMP1, 2 and 3) complexes. These peptides are important regulators of neurotransmission, vasotone, cardiovascular development, and metabolic homeostasis. In rodents, ADM is essential for regulating embryo implantation, fetal-placental development, and hemodynamic adaptation during pregnancy. On the other hand, ADM2 was shown to affect vascular lumen enlargement, and cumulus cell-oocyte complex (COC) communication in rodent and bovine ovarian follicles. To investigate whether oocyte-derived ADM2 plays a physiological role in regulating ovarian folliculogenesis, we generated mice with oocyte-specific disruption of the Adm2 gene using a LoxP-flanked Adm2 transgene (Adm2 loxP/loxP) and crossed them with Zp3-Cre mice which carry a zona pellucida 3 (Zp3) promoter-Cre recombinase transgene. While heterozygous Adm2 +/-/Zp3-Cre and homozygous Adm2 -/-/Zp3-Cre mice were fertile, Adm2 disruption in oocytes significantly increased the number of ovulated oocytes following a superovulation treatment. Oocyte-specific Adm2 disruption also significantly impaired the developmental capacity of fertilized eggs and decreased the size of the corpus luteum following superovulation, perhaps due to a reduction of ovarian cyclin D2-associated signaling. The disruption of intrafollicular ADM2 signaling leads to follicular dysfunction. These data suggested that oocyte-derived ADM2 plays a facilitative role in the regulation of hormonal response and follicle growth independent of the closely related ADM and CGRP peptides, albeit in a subtle manner.

The secretion and metabolism of cumulus cells support fertilization in the bovine model

The cumulus oophorus is a structure that surrounds the mammalian egg and plays a key role in fertilization. However, very little is known with regards to how secretions from the cumulus cells can specifically promote fertilization. We hypothesized that secretions from bovine cumulus cells, and the reduction of oxygen stress by metabolic change, would enhance the fertilization capacity of sperm during in vitro fertilization (IVF) procedures. To prove our hypothesis, sperm were pre-incubated in chemically defined capacitation media containing methyl-beta-cyclodextrin and used to inseminate cumulus cell oocyte complexes, or denuded oocytes, with some components. While sperm capacitation was induced in capacitation media, fertilization was impeded by the removal of cumulus cells from cumulus cell oocyte complexes. Secretions from cumulus cells promoted the formation of two pronuclei via a filter and the fertilization of denuded oocytes was dramatically enhanced with hyaluronate, low oxygen concentration, or progesterone in fertilization media (P < 0.05). This demonstrates that these factors-maintained sperm motility and capacitation or enhanced the hyper-activation of capacitated sperm (P < 0.05). We conclude that cumulus cells secrete progesterone, hyaluronate and undergo metabolic events to reduce oxidative stress in fertilization media. These phenomenons help to improve the fertilization capacity of sperm. We believe that this study makes a significant contribution to our understanding of the function of cumulus cells during fertilization in animals and humans.

Peroxiredoxin 4, a new favorable regulator, can protect oocytes against oxidative stress damage during in vitro maturation

BackgroundFinding an effective regulator to avoid harmful effects caused by excessive reactive oxygen species (ROS) is a bottleneck during oocyte in vitro maturation (IVM). Previously, we found that peroxiredoxin 4 (Prdx4) expression is significantly higher in mature cumulus cell–oocyte complexes (COCs) than in immature COCs. Prdx4 belongs to the antioxidant enzyme family and can catalyze the reduction of H2O2. ResultsIn this study, we established an oxidative stress model with mouse COCs cultured in vitro. Treatment with H2O2 decreased cumulus expansion indexes and oocyte maturation in a concentration-dependent manner, indicating follicular development dysplasia. Infection with a Prdx4-overexpressing adenovirus significantly attenuated H2O2-induced changes, exhibiting effects similar to those of the intracellular ROS scavenger tiron (the positive control). Furthermore, the results confirmed that the protective effect of Prdx4 on oocyte maturation may be due to reductions in ROS levels and apoptosis. However, when the gap junctions between cumulus cells (CCs) and oocytes were destroyed, Prdx4 overexpression did not exert antiapoptotic effects. The expression levels of the gap junction marker protein CX43 were significantly recovered in the Prdx4-overexpressing group. ConclusionsThese results demonstrate that Prdx4 in CCs may be a new favorable regulator that improves in vitro-matured oocyte quality and enhances oocyte developmental competence by preventing CC apoptosis caused by oxidative stress through gap junctions. The findings expand the body of knowledge regarding follicle development, and the identification of Prdx4 as a new favorable regulator will aid in immature oocyte IVM.

Excessive follicle-stimulating hormone during ovarian stimulation of cattle may induce premature luteinization of most ovulatory-size follicles†.

High follicle-stimulating hormone (FSH) doses during ovarian stimulation are detrimental to ovulatory follicle function and decrease live birth rate in cattle and women. However, the mechanism whereby excessive FSH causes ovarian dysfunction is unknown. This study tested the hypothesis that excessive FSH during ovarian stimulation induces premature luteinization of ovulatory-size follicles. Small ovarian reserve heifers were injected twice daily for 4 days with 70IU (N = 7 heifers) or 210IU (N = 6 heifers) Folltropin-V [commercial FSH-enriched preparation of porcine pituitary glands with minor (<1%) luteinizing hormone (LH) contamination, cpFSH]. Ovulatory-size (≥10mm) follicles were excised from ovaries after the last cpFSH injection and hormone concentrations in follicular fluid (FF) were determined using ELISA. Luteinization was monitored by assessing cumulus cell-oocyte complex (COC) morphology and measuring concentrations of estradiol (E), progesterone (P), and oxytocin (O) in FF. COCs were classified as having compact (cCOC) or expanded (eCOC) cumulus cell layers, and as estrogen-active (E:P in FF ≥1), estrogen-inactive (EI, E:P in FF ≤1 > 0.1), or extreme-estrogen-inactive (EEI, E:P in FF ≤0.1). A high proportion (72%) of ovulatory-size follicles in 210IU, but not 70IU, dose heifers displayed eCOCs. The high doses also produced higher proportions of EI or EEI follicles which had lower E:P ratio and/or E but higher P and/or O concentrations compared with the 70IU dose heifers. In conclusion, excessive cpFSH doses during ovarian stimulation may induce premature luteinization of most ovulatory-size follicles in heifers with small ovarian reserves.

Expression and regulation of estrogen receptor 2 and its coregulators in mouse granulosa cells.

The cooperative effects of estrogen and oocyte-derived paracrine factors (ODPFs) play critical roles in the normal development of ovarian follicles; however, the mechanism underlying this cooperation has not been well studied. The present study aimed to determine whether ODPFs affect estrogen signaling by regulating the expression of estrogen receptor (ESR) and its coregulators in mouse granulosa cells. Some transcripts encoding ESR coregulators were differentially expressed between cumulus and mural granulosa cells (MGCs). The transcript levels of ESR coregulators, including nuclear receptor corepressor 1 and activator 2, in cumulus cells were significantly suppressed by ODPFs; however, they increased when cumulus cell-oocyte complexes were treated with the transforming growth factor beta receptor I inhibitor, SB431542. Moreover, MGCs exhibited significantly higher ESR2 protein and transcript levels than those in cumulus cells. ODPFs promoted Esr2 expression in cumulus cells but had no effect on that in MGCs. Overall, regulation of the expression of ESR2 and its coregulators in cumulus cells by oocytes seems to be one of the mechanisms underlying estrogen-oocyte cooperation in well-developed antral follicles in mice.

The microenvironment of ovarian follicles in fertile dairy cows is associated with high oocyte quality

We hypothesised that heifers and cows with positive genetic merit for fertility would have a follicular microenvironment that resulted in better quality oocytes. To test this, we compared cumulus cell-oocyte complexes (COC) and follicular fluid from preovulatory follicles of 36 Holstein-Friesian nulliparous heifers and 50 primiparous lactating cows with either positive (POS, +5%) or negative (NEG, −5%) fertility breeding values (FertBV). Established gene markers of oocyte quality were measured in individual cumulus cell masses and oocytes, and concentrations of amino acids, steroids, and metabolites were quantified in corresponding follicular fluid and plasma. The timing of visually detectable oestrus in NEG FertBV heifers was inconsistent with their stage of COC maturation. Retrospective analyses of oestrous activity data indicated that NEG FertBV heifers were sampled earlier. Their recovered COC were morphologically less mature and exhibited differential expression of genes that are associated with follicular maturation (lower levels of BMPR2) and protein processing (higher levels of HSP90B1). Despite consistent sampling times being achieved in the lactating cows, lower concentrations of serine, proline, methionine, isoleucine, and non-esterified fatty acids were present in follicular fluid from POS FertBV cows. This was associated with higher expression of gene biomarkers of good oocyte quality (VCAN, PDE8A) in COC recovered from POS FertBV cows. This study supports our hypothesis that the follicular microenvironment in lactating dairy cows with high genetic merit leads to COC with higher metabolic rates and oocytes of superior quality. Moreover, an additional stressor such as lactation is required for this difference to be pronounced.

Silica encapsulation of ZnO nanoparticles reduces their toxicity for cumulus cell-oocyte-complex expansion

BackgroundMetal oxide nanoparticles (NPs) are increasingly used in many industrial and biomedical applications, hence their impact on occupational and public health has become a concern. In recent years, interest on the effect that exposure to NPs may exert on human reproduction has grown, however data are still scant. In the present work, we investigated whether different metal oxide NPs interfere with mouse cumulus cell-oocyte complex (COC) expansion.MethodsMouse COCs from pre-ovulatory follicles were cultured in vitro in the presence of various concentrations of two types of TiO2 NPs (JRC NM-103 and NM-104) and four types of ZnO NPs (JRC NM-110, NM-111, and in-house prepared uncoated and SiO2-coated NPs) and the organization of a muco-elastic extracellular matrix by cumulus cells during the process named cumulus expansion was investigated.ResultsWe show that COC expansion was not affected by the presence of both types of TiO2 NPs at all tested doses, while ZnO NM-110 and NM-111 induced strong toxicity and inhibited COCs expansion at relatively low concentration. Medium conditioned by these NPs showed lower toxicity, suggesting that, beside ion release, inhibition of COC expansion also depends on NPs per se. To further elucidate this, we compared COC expansion in the presence of uncoated or SiO2-coated NPs. Differently from the uncoated NPs, SiO2-coated NPs underwent slower dissolution, were not internalized by the cells, and showed an overall lower toxicity. Gene expression analysis demonstrated that ZnO NPs, but not SiO2-coated ZnO NPs, affected the expression of genes fundamental for COC expansion. Dosimetry analysis revealed that the delivered-to-cell mass fractions for both NPs was very low.ConclusionsAltogether, these results suggest that chemical composition, dissolution, and cell internalization are all responsible for the adverse effects of the tested NPs and support the importance of a tailored, safer-by-design production of NPs to reduce toxicity.

Morphology and Biochemistry of Ovulation.

The process of ovulation involves multiple and iterrelated genetic, biochemical, and morphological events: cessation of the proliferation of granulosa cells, resumption of oocyte meiosis, expansion of cumulus cell-oocyte complexes, digestion of the follicle wall, and extrusion of the metaphase-II oocyte. The present narrative review examines these interrelated steps in detail. The combined or isolated roles of the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are highlighted. Genes indiced by the FSH genes are relevant in the cumulus expansion, and LH-induced genes are critical for the resumption of meiosis and digestion of the follicle wall. A non-human model for follicle-wall digestion and oocyte release was provided.

Human bone morphogenetic protein 8A promotes expansion and prevents apoptosis of cumulus cells in vitro

Cumulus expansion is essential for ovulation and oocyte maturation in mammals. Previous studies suggest that this process requires certain cumulus expansion enabling factors, induced by LH surge, that activate SMAD signaling locally. However, their identities remain uncertain. Using a superovulated rat model, we showed that Bmp8 transcripts were abundant in cumulus cell-oocyte complexes (COCs) and their levels can be further induced during ovulation. By analyzing human COC-related transcriptomic datasets, BMP8 transcripts in cumulus cells were also found to be significantly elevated along with the maturation status and developmental competence of enclosed oocytes. In cultured rat COCs, treatment with recombinant BMP8A protein activated both SMAD1/5/8 and SMAD2/3 pathways; the resulting SMAD2/3 signaling induced COC expansion as well as the expression of COC expansion-related genes, whereas the resulting SMAD2/3 and SMAD1/5/8 activations were both required for protecting expanded cumulus cells from apoptosis. Taken together, our data demonstrated that addition of BMP8 protein in the in vitro rat COC cultures not only promotes cumulus expansion but also sustains survival of expanded cumulus cells via different SMAD downstreams. With these capabilities, BMP8 may have clinical applications to ameliorate the fertilizability and subsequent developmental competence of the enclosed oocytes when doing in vitro COC maturation.

MiR-29 regulates the function of goat granulosa cell by targeting PTX3 via the PI3K/AKT/mTOR and Erk1/2 signaling pathways

PTX3, a member of the pentraxin protein family, plays important roles in ovulation as a marker of cumulus cell–oocyte complex expansion. However, the expression and function of PTX3 in goat ovarian GCs remain unclear. We isolated GCs from small and large follicles and found that PTX3 expression was significantly decreased and miR-29 mRNA expression was significantly increased during the growth of antral follicles. MiR-29 decreased PTX3 expression by targeting its 3′ untranslated. Furthermore, miR-29 promoted GC proliferation, suppressed steroidogenesis and apoptosis by targeting PTX3 via the activation of the PI3K/AKT/mTOR and Erk1/2 signaling pathways. Treatment with inhibitors also verified these results. Meanwhile, we found that PI3K/AKT/mTOR and Erk1/2 signaling pathways had different role in secretion of E2 and P4 by regulating differently various steroidogenic enzyme (CYP19A1, CYP11A1, StAR and HSD3B) expression. These outcomes indicate the potential role of PTX3 in goat follicular growth and atresia.

Functional Characterization of Fusion Proteins of the Tumor Necrosis Factor Alpha‐Induced Protein 6 (TNFAIP6) Domains

TNFAIP6 is an extracellular protein that is essential for cumulus cell extracellular matrix formation and female fertility in mice. A mammalian expression system was used to generate recombinant proteins of the full length TNFAIP6 and its individual domains, Link and CUB. These proteins contained a short amino terminal sequence essential for catalytic function and a carboxy‐terminal Fc region of mouse IgG2a for easy detection, and were termed TNFAIP6Fc, LinkFc and CUBFc, respectively. Hyaluronan‐binding of the fusion proteins was measured with an enzyme‐linked immunosorbent assay. The catalytic activity of the proteins was assessed by their ability to remove heavy chain 1 (HC1) from inter‐alpha‐trypsin inhibitor (IαI) using western‐blot. The ability of the fusion proteins to restore cumulus cell extracellular matrix formation was tested in a cell culture assay of Tnfaip6−/− cumulus cell‐oocyte complexes (COCs). Both TNFAIP6Fc and LinkFc were able to bind hyaluronan, with LinkFc showing much stronger binding at equimolar concentrations. CUBFc showed no significant binding to hyaluronan even at high concentrations. TNFAIP6Fc catalysed the removal of HC1 from IαI in a concentration dependent manner, indicating that the Fc tag did not interfere with catalytic function. LinkFc, CUBFc, and their combination were unable to remove the HC1 from IαI at high concentrations even though they contained the essential short amino‐terminal region. TNFAIP6Fc successfully restored cumulus cell extracellular matrix formation in Tnfaip6−/− COCs, while LinkFc, CUBFc, and their combination were unable to do so. LinkFc, CUBFc, and their combination did not disrupt normal cumulus cell extracellular matrix formation of Tnfaip6+/− COCs. Our results suggest that the catalytic function of TNFAIP6 requires both of its domains in addition to the previously identified serine residue in the amino‐terminal region, because neither domain alone is able to remove HC1 from IαI or support cumulus cell extracellular matrix formation in Tnfaip6−/− COCs. Furthermore, the absence of catalytic activity of both domains when combined together could imply a pivotal conformational interaction normally achieved by their covalent attachment. The finding that the LinkFc has stronger hyaluronan binding than TNFAIP6Fc could support the presence of such a conformational interaction that weakens the hyaluronan‐binding of the full‐length protein compared to the Link domain alone.Support or Funding InformationSupported by internal funding of Touro University Nevada

Involvement of GJA1 and Gap Junctional Intercellular Communication between Cumulus Cells and Oocytes from Women with PCOS.

Polycystic ovary syndrome (PCOS) is a common female endocrine system disease that affects 17.8% of women of reproductive age and leads to infertility, obesity, glucose metabolic disorders, cardiovascular disease, and body-mind problems. However, the etiology of PCOS remains unclear. Follicular growth is disrupted as a result of ovarian hyperandrogenism and distorted intraovarian paracrine signaling in women with PCOS. Microcommunication between oocytes and cumulus cells plays a critical role in folliculogenesis. Gap junction alpha 1 (GJA1) plays a crucial role in the developing follicles by forming communication channels between cumulus cells and oocytes, but this has not yet been reported in women with PCOS. Therefore, we aimed to study the role of GJA1 in the microcommunication between oocytes and cumulus cells in women with PCOS. In our study, cumulus cell-oocyte complexes (COCs) from women were isolated via ultrasound-guided vaginal puncture, and oocytes were selected from COCs and categorized based on 3 oocyte maturation stages. Then, RT-qPCR and immunofluorescence analysis were performed to detect both the gene expression and protein of GJA1 in oocytes from women with and without PCOS. There was no statistically significant difference in age and BMI (body mass index), but patients with PCOS had a higher ratio of basic LH/FSH (luteinizing hormone/follicle-stimulating hormone), androstenedione, and total ovarian volume. The qRT-PCR results showed higher gene expression of GJA1 in oocytes without PCOS at the germinal vesicle (GV) stage compared with that of oocytes from women with PCOS. Immunofluorescence analysis showed that the expression level of GJA1 in oocytes from women with PCOS was very weak compared with that of oocytes from women without PCOS. In conclusion, GJA1 may play a critical role in the development of oogenesis arrest in women with PCOS throughout the oogenesis processes, including oogenesis and oocyte maturation.

Gonadotropin Releasing Hormone Agonists Have an Anti-apoptotic Effect on Cumulus Cells.

Background: Ovaries are sensitive to chemotherapy, which may lead to early depletion of primordial follicle reserve. One strategy for gonadal function preservation is temporary ovarian suppression with Gonadotropin Releasing Hormone agonists (GnRHa) during chemotherapy. To date, GnRHa protective mechanism of action remains not fully elucidated. Methods: We collected 260 immature cumulus cell-oocyte complexes (COC) from 111 women < 38 years old, with a normal ovarian reserve. The COC were randomly assigned to the following groups: (a) control; culture with the addition of (b) GnRHa; (c) cyclophosphamide; (d) cyclophosphamide plus GnRHa. After in vitro treatments, RNA and proteins were extracted from oocytes and cumulus cells (CC), separately. Potential effects of drugs were evaluated on GnRH receptors, apoptosis pathways, ceramide pathway, and glutathione synthesis by quantitative PCR and, whenever possible, by Western blot. Results: Cyclophosphamide triggered activation of the extrinsic pathway of apoptosis mediated by BAX in CC. The co-administration of GnRHa inhibited the apoptosis pathway in CC. According to our model, the GnRHa does not directly act on oocytes, which do not express GnRH receptors. Moreover, glutathione synthesis was decreased after GnRHa treatment both in CC and oocytes. Conclusion: Our data suggest that the protective mechanisms induced by GnRHa is mediated by an anti-apoptotic effect on CC.

Effect of cumulus cells of cumulus-oocyte complexes on in vitro maturation, embryonic developmental and expression pattern of apoptotic genes after in vitro fertilization in water buffalo (Bubalus bubalis)

In the present study, the potential of different grades of cumulus-oocyte complexes (COCs) for in vitro maturation (IVM) and embryonic development was assessed. Further, the association of the expression pattern of anti-apoptotic Mcl-1 and pro-apoptotic Bax genes in embryonic development was analyzed. Abattoir derived oocytes were graded into grade A and B based on surrounding cumulus rings. Out of 1050 ovaries, a total number of 770 and 1360, were of grade A and B COCs, respectively, were aspirated. After IVM, grade A COCs had a significantly higher number of polar bodies (92.04 ± 0.60%) as compared to grade B (85.88 ± 0.46%). On IVF and embryo culture, grade A COCs produced the significantly higher rate of cleavage and blastocyst (90.44 ± 0.71% and 41.55 ± 0.96%) as compared to grade B COCs (79.77 ± 0.76% and 30.44 ± 0.96%). The transcriptional analysis of apoptotic genes expression by Real-time PCR revealed a significantly higher expression of Mcl-1 gene in embryos of grade A as compared to grade B, whereas, the relative expression of Bax gene was down-regulated in grade A than grade B embryos. Thus it was concluded that the pattern of apoptotic genes expression in early-stage embryos can be used as a marker gene to predict the developmental competence of COCs.

Follicular factors determining granulosa cell number and developmental competence of porcine oocytes.

Granulosa cell (GC) number in follicles is a simple characteristic of follicles. The present study examined the hypothesis that follicular fluid (FF) determines GC number and oocyte developmental ability and revealed the molecular background determining the number of follicular GCs. FF was collected from antral follicles (3-5mm in diameter), after which the number of GCs per follicle was determined for each donor gilt using real time PCR targeting single copy gene. GCs were analyzed by next-generation RNA sequencing and IPA pathway analysis. When oocyte cumulus cell-oocyte-complexes (COCs) were cultured in maturation medium containing 10% of each individual FF, the rate of development to the blastocyst stage was significantly correlated with the number of GCs in the donor gilt. In addition, when GCs were cultured in medium containing FF, the proliferative activity of the GCs was also significantly correlated to the number of GCs in the donor gilt. Moreover, when the FFs were categorized based on the number of GCs in the follicle, it was found that supplementation of culture media with GC-rich FF improved the developmental ability of oocytes compared to those supplemented with GC-poor FF. RNA sequencing and a pathway analysis of GCs from GC-rich and -poor follicles revealed the key regulatory pathway determining GC number in follicles. GC number may be a useful marker for "good" follicles and oocytes, and the characteristics of the FFs determine granulosa cell number and oocyte developmental ability.

MicroRNA-21 up-regulates metalloprotease by down-regulating TIMP3 during cumulus cell-oocyte complex in vitro maturation.

Cumulus cells and the remodeling of their extracellular matrix around oocytes are essential for oocyte maturation and ovulation in the ovary. An important extracellular metalloprotease, ADAMTS1, and its partner VERSICAN, mediate essential cumulus-oocyte-complex (COC) structural remodeling. However, how the expression of these proteinases is regulated during oocyte maturation is unclear. Here we report that both ADAMTS1 and VERSICAN significantly increased in porcine cumulus cells during COC in vitro maturation (IVM). Interestingly, one of the tissue inhibitors of the metalloproteinase family member, TIMP3, was found to be significantly decreased in cumulus cells during this process. Down-regulation of TIMP3 using specific small interfering RNA decreased TIMP3 expression, while increased the levels of ADAMTS1 and VERSICAN, suggesting an inverse relationship between TIMP3, the metalloprotease, and the breakdown product of its substrate. MiR-21 significantly increased in cumulus cells during COC maturation. Knockdown of miR-21 in cumulus cells during COC maturation resulted in increased TIMP3 and decreased ADAMTS1 and VERSICAN expression, which is accompanied by a decrease in cumulus cell expansion and the ratio of oocytes that reached MII stage. In contrast, over-expression of miR-21 decreased TIMP3 and increased ADAMTS1, and enhanced cumulus cell expansion and oocyte maturation. Moreover, in silico prediction revealed that a miR-21 binding site is present at the 3-untranslated region (3-UTR) of the TIMP3 mRNA, which was further confirmed to be the target site of miR-21 by luciferase gene reporter assays. Our findings revealed that miR-21 promotes cumulus expansion and oocyte maturation via down-regulating TIMP3, and subsequent increase of ADAMTS1 and VERSICAN during in vitro COC maturation.

Short-term heat stress induces mitochondrial degradation and biogenesis and enhances mitochondrial quality in porcine oocytes

Mitochondria in oocytes play important roles in many processes, including early embryo development. Promotion of mitochondrial degradation and biogenesis through Sirtuin 1 (SIRT1) activation enhances mitochondrial function and oocyte quality. Previous studies that used somatic cells have shown that short-term heat stress (SHS) induces SIRT1-regulated mitochondrial biogenesis. In this study, we examined whether SHS can induce mitochondrial degradation and biogenesis in porcine oocytes. We collected cumulus cell-oocyte complexes (COCs) from prepubertal gilt ovaries acquired from a slaughterhouse. COCs were treated at 41.5 °C (vehicle: 38.5 °C) for the first one hour of in vitro maturation, and the mitochondrial kinetics, oocyte function, and developmental competence of oocytes were examined. SHS increased the expression level of heat shock protein 72, which induced the high expression of SIRT1 and the phosphorylation of AMP-activated protein kinase. SHS did not alter the mitochondrial DNA copy number in oocytes, but induced mitochondrial degradation and biogenesis, which enhanced the mitochondrial membrane potential and ATP content in oocytes, and improved the ability of the oocytes to develop into blastocysts.