Intact Cumulus-oocyte Complexes Research Articles

Oocyte secreted factors control genes regulating FSH signaling and the maturation cascade in cumulus cells: the oocyte is not in a hurry.

To assess the effects of the oocyte on mRNA abundance of FSHR, AMH and major genes of the maturation cascade (AREG, EREG, ADAM17, EGFR, PTGS2, TNFAIP6, PTX3, and HAS2) in bovine cumulus cells. (1) Intact cumulus-oocyte complexes, (2) microsurgically oocytectomized cumulus-oolema complexes (OOX), and (3) OOX + denuded oocytes (OOX+DO) were subjected to in vitro maturation (IVM) stimulated with FSH for 22 h or with AREG for 4 and 22 h. After IVM, cumulus cells were separated and relative mRNA abundance was measured by RT-qPCR. After 22 h of FSH-stimulated IVM, oocytectomy increased FSHR mRNA levels (p=0.005) while decreasing those of AMH (p=0.0004). In parallel, oocytectomy increased mRNA abundance of AREG, EREG, ADAM17, PTGS2, TNFAIP6, and PTX3, while decreasing that of HAS2 (p<0.02). All these effects were abrogated in OOX+DO. Oocytectomy also reduced EGFR mRNA levels (p=0.009), which was not reverted in OOX+DO. The stimulatory effect of oocytectomy on AREG mRNA abundance (p=0.01) and its neutralization in OOX+DO was again observed after 4 h of AREG-stimulated IVM. After 22 h of AREG-stimulated IVM, oocytectomy and addition of DOs to OOX caused the same effects on gene expression observed after 22 h of FSH-stimulated IVM, except for ADAM17 (p<0.025). These findings suggest that oocyte-secreted factors inhibit FSH signaling and the expression of major genes of the maturation cascade in cumulus cells. These may be important actions of the oocyte favoring its communication with cumulus cells and preventing premature activation of the maturation cascade.

Morphokinetic parameters of mouse oocyte meiotic maturation and cumulus expansion are not affected by reproductive age or ploidy status

IntroductionMorphokinetic analysis using a closed time-lapse monitoring system (EmbryoScope + ™) provides quantitative metrics of meiotic progression and cumulus expansion. The goal of this study was to use a physiologic aging mouse model, in which egg aneuploidy levels increase, to determine whether there are age-dependent differences in morphokinetic parameters of oocyte maturation.MethodsDenuded oocytes and intact cumulus-oocyte complexes (COCs) were isolated from reproductively young and old mice and in vitro matured in the EmbryoScope + ™. Morphokinetic parameters of meiotic progression and cumulus expansion were evaluated, compared between reproductively young and old mice, and correlated with egg ploidy status.ResultsOocytes from reproductively old mice were smaller than young counterparts in terms of GV area (446.42 ± 4.15 vs. 416.79 ± 5.24 µm2, p < 0.0001) and oocyte area (4195.71 ± 33.10 vs. 4081.62 ± 41.04 µm2, p < 0.05). In addition, the aneuploidy incidence was higher in eggs with advanced reproductive age (24–27% vs. 8–9%, p < 0.05). There were no differences in the morphokinetic parameters of oocyte maturation between oocytes from reproductively young and old mice with respect to time to germinal vesicle breakdown (GVBD) (1.03 ± 0.03 vs. 1.01 ± 0.04 h), polar body extrusion (PBE) (8.56 ± 0.11 vs. 8.52 ± 0.15 h), duration of meiosis I (7.58 ± 0.10 vs. 7.48 ± 0.11 h), and kinetics of cumulus expansion (0.093 ± 0.002 vs. 0.089 ± 0.003 µm/min). All morphokinetic parameters of oocyte maturation were similar between euploid and aneuploid eggs irrespective of age.ConclusionThere is no association between age or ploidy and the morphokinetics of mouse oocyte in vitro maturation (IVM). Future studies are needed to evaluate whether there is an association between morphokinetic dynamics of mouse IVM and embryo developmental competence.

Application of Ex-Vivo In Vitro Maturation (IVM) to Fertility Preservation in Cancer Patients

Background: Oocyte in vitro maturation (IVM) is a reproductive technology that generates mature oocytes from patients who have received minimal or no ovarian stimulation (OS). The minimal-stimulation feature of IVM makes it a valuable option for cancer patients requiring fertility preservation (FP), before chemo- or radiotherapy. When ovarian tissue is collected for cryopreservation (OTC), only the ovarian cortex is preserved whereas the ovarian medulla containing growing follicles is discarded. To maximise the future fertility prospects for cancer patients, OTC should be coupled with a modified form of IVM, termed ex-vivo IVM, in which immature cumulus-oocyte complexes (COCs) are collected from antral follicles. Aim: To examine the utility of emergency ex-vivo IVM for FP. Method: A 31-year-old woman diagnosed with locally invasive appendiceal cancer required urgent FP as there was no time for conventional OS egg freeze before surgery, and OTC was contra-indicated due to the possibility of later regrafting of cancer cells. She underwent peritonectomy with bilateral oophorectomy with no prior ovarian stimulation or cycle management. Visible antral follicles were aspirated ex-vivo, the ovaries were then manually dissected, antral follicles punctured and intact COCs collected. COCs were washed and matured for 30-46h in G-2 PLUS medium supplemented with 0.1IU/mL rec-FSH, 0.5IU/mL rec-hCG, and 10% (v/v) autologous heat-inactivated patient serum. Results: 40 COCs were collected and 18 (45%) reached metaphase II, 1 was cryopreserved and 17 were inseminated by ICSI. 13/17 oocytes fertilised (2PN; 76%) and 7/13 (54%) blastocysts were generated (D5: 1× HB-1-1, 3× B-1-1, 1× B-2-1. D6: 1× HB-1-2, 1× B-2-2). Seven blastocysts and one oocyte were vitrified for future use by the patient. Conclusion: Ex-vivo IVM is a viable FP option for patients diagnosed with cancer. It can readily be coupled with OTC and/or a regular IVM oocyte pickup to maximise a cancer patient’s future fertility prospects.

Quantitative morphokinetic parameters identify novel dynamics of oocyte meiotic maturation and cumulus expansion†.

Meiotic maturation and cumulus expansion are essential for the generation of a developmentally competent gamete, and both processes can be recapitulated in vitro. We used a closed time-lapse incubator (EmbryoScope+™) to establish morphokinetic parameters of meiotic progression and cumulus expansion in mice and correlated these outcomes with egg ploidy. The average time to germinal vesicle breakdown (GVBD), time to first polar body extrusion (PBE), and duration of meiosis I were 0.91 ± 0.01, 8.82 ± 0.06, and 7.93 ± 0.06 h, respectively. The overall rate of cumulus layer expansion was 0.091 ± 0.002 μm/min, and the velocity of expansion peaked during the first 8 h of in vitro maturation (IVM) and then slowed. IVM of oocytes exposed to Nocodazole, a microtubule disrupting agent, and cumulus oocyte complexes (COCs) to 4-methylumbelliferone, a hyaluronan synthesis inhibitor, resulted in a dose-dependent perturbation of morphokinetics, thereby validating the system. The incidence of euploidy following IVM was >90% for both denuded oocytes and intact COCs. No differences were observed between euploid and aneuploid eggs with respect to time to GVBD (0.90 ± 0.22 vs. 0.97 ± 0.19 h), time to PBE (8.89 ± 0.98 vs. 9.10 ± 1.42 h), duration of meiosis I (8.01 ± 0.91 vs. 8.13 ± 1.38 h), and overall rate and kinetics of cumulus expansion (0.089 ± 0.02 vs 0.088 ± 0.03 μm/min) (P > 0.05). These morphokinetic parameters provide novel quantitative and non-invasive metrics for the evaluation of meiotic maturation and cumulus expansion and will enable screening compounds that modulate these processes.

P-651 Oocyte secreted factors supress the expression of prostaglandin synthase-2 (PTGS2), a key maturation promoter in cumulus cells: the oocyte is not in a hurry

Abstract Study question Does the oocyte control the expression of genes regulating the ovulatory cascade and the formation of the extracellular matrix in cumulus cells? Summary answer Oocyte secreted factors (OSFs) suppress the expression of prostaglandin synthase-2 (PTGS2) in cumulus cells, a key mediator of the maturation cascade. What is known already Oocyte quality depends on cytoplasmic and nuclear maturation synchrony. OSFs modulate maturation dynamics by inhibiting the expression of AREG (amphiregulin) and EREG (epiregulin), key mediators of the ovulatory cascade. AREG and EREG stimulate PTGS2 expression in cumulus cells, which catalyses the synthesis of prostaglandins, that amplify and translate AREG/EREG signals leading to meiotic completion and expression of genes required for cumulus expansion, among which HAS2 (hyaluronan synthase-2), essential for the synthesis of hyaluronan, the backbone of cumulus extracellular matrix, as well as PTX3 (pentraxin 3) and TNFAIP6 (tumor necrosis factor alpha-induced protein 6), genes encoding for matrix structural proteins. Study design, size, duration Relative mRNA levels of PTGS2, HAS2, PTX3 and TNFAIP6 were compared in cumulus cells from 3 treatment-groups: 1-COC: Intact cumulus-oocyte complexes subjected to in vitro maturation (IVM); 2-OOX: oocytectomized COC subjected to IVM; 3-OOX+DO: OOX subjected to IVM with the addition of denuded oocytes (1 DO/µL). Four experimental replicates including all three treatment-groups were performed. The inclusion of OOX+DO aimed to assess whether the effects of the oocyte on gene expression are mediated by OSFs. Participants/materials, setting, methods COC were aspirated from 3-8mm follicles of bovine ovaries. Oocytectomy was achieved by ooplasm aspiration with a micromanipulator. Pools of 20 COCs/OOX/OOX+DO were cultured for 22 hours in TCM199 with 100ng/mL AREG and 0.4% BSA. Subsequently, cumulus cells were separated, total RNA extracted, and mRNA abundance assessed by real-time RT-PCR with oligo-dT primers and two internal controls for relative quantification. Treatment effects were tested by ANOVA and groups were compared with the Tukey test. Main results and the role of chance Oocyctectomy drastically increased PTGS2 mRNA abundance, which was reversed by the addition of denuded oocytes (DO) to culture. Relative PTGS2 mRNA levels (mean ± EPM) were 1.02 ± 0.12, 10.40 ± 1.65 and 0.88 ± 0.08 for COC, OOX and OOX+DO, respectively (P = 0.02). Consistently, although in a lower magnitude, oocytectomy increased mRNA levels of PTX3(1.00 ± 0.04, 2.50 ± 0.18 and 0.75 ± 0.15 for COC, OOX and OOX+DO, respectively. P &amp;lt; 0.001) and TNFAIP6 (1.00 ± 0.07, 2.04 ± 0.17 and 0.67 ± 0.04 for COC, OOX and OOX+DO, respectively. P &amp;lt; 0.001), an effect again reversed by the presence of DO in culture. In contrast, oocytectomy decreased the expression of HAS2, which was neutralised in the presence of DO (1.01 ± 0.09, 0.69 ± 0.02 and 1.11 ± 0.08 for COC, OOX and OOX+DO, respectively. P = 0.005). Our data indicate that the oocyte modulates its own maturation pace by inhibiting the expression of PTGS2, which is accompanied by similar negative impacts on genes that strongly respond to prostaglandin signalling such as PTX3 and TNFAIP6. In parallel, our data suggest that OSFs exert a potent stimulatory effect on HAS2 transcription, capable to overcome their concomitant inhibitory influence via supressed AREG/EREG and PTGS2 expression. Limitations, reasons for caution Our study utilised animal and culture models that may not exactly represent human COC physiological maturation. In addition, our data are restricted to mRNA levels. Nevertheless, somatic cells predominantly transcribe poly A-mRNA that is promptly exported and translated. Accordingly, our RT-PCR strategy utilised poly-A reverse transcription with oligo-dT primers. Wider implications of the findings Our data indicate that OSFs inhibit the expression of PTGS2, a key gene for activation and amplification of the maturation cascade in cumulus cells. We speculate that this may represent an effort of the oocyte to prevent precocious meiotic resumption, thus favouring nuclear and cytoplasmic maturation synchrony and developmental competence. Trial registration number Not applicable

Multiyear outcomes using sibling oocytes demonstrates safety and efficacy of a simplified culture system consistent with use in a low-cost IVF setting

Research questionCan a novel closed simplified IVF culture system be used to achieve outcomes comparable to those obtained with intracytoplasmic sperm injection (ICSI) followed by conventional culturing? DesignThis analysis is part of a non-inferiority prospective study comparing ICSI and a simplified culture system (SCS) for gamete fertilization in a selected group of patients. According to protocol, sibling oocytes in intact cumulus–oocyte complexes were randomly distributed between ICSI and conventional insemination in the SCS. For women, selection criteria included being under 43 years of age and at least six eggs at retrieval. An inseminating motile sperm count ≥1 million was required. The primary outcome measure was ongoing pregnancy rate (>12 weeks) per cycle; secondary outcome measures included fertilization rate, miscarriage rate and implantation rate (ongoing pregnancy rate per embryo). ResultsFrom January 2016 until December 2019, 653 SCS/ICSI cycles were performed yielding a total of 7915 oocytes. The fertilization rate was 61.1% and 50.4% for SCS and ICSI (P < 0.0001), respectively. The ongoing pregnancy rate was 32.0% for SCS and 36.7% for ICSI (P = 0.27). Implantation rate was 30.6% for SCS and 34.4% for ICSI (P = 0.35). The miscarriage rate was 7.5% and 6.5% for SCS and ICSI, respectively (P = 0.75). ConclusionNo difference was found in ongoing pregnancy rate, implantation rate and the miscarriage rate between SCS and ICSI in this selected patient cohort.

A cohort study on safety for oocyte retrieval in IVF using the same 20G needle for local analgesia and retrieval. Vaginal cleaning did not add to the safety of the procedure

Aim of study: What is the effectiveness and safety of using a 20G single lumen needle (SLN) for both applying local analgesia (LA) in the vaginal vault and ovarian capsule and oocyte retrieval (OR) using a simple syringe? And will thoroughly cleaning (TC) of the vagina before reducing the risk of the puncture procedure? Using a thin SLN has been postulated to jeopardize cumulus oocyte complexes (COC). Further applying LA in the same needle without retracting the needle before OR has been claimed to harm oocyte development. Also, many clinics extensively clean the vagina before OR. Methods: We performed a retrospective cohort study including 4983 women. 877 underwent no cleaning (nonTC) of the vagina and 4106 underwent TC using NaCl. All had OR with a 20G SLN attached to a syringe for local anaesthesia and egg collection. The treatments were done in one centre during the period from January 2016 to June 2019. We studied women undergoing IVF treatment aged 18–45 years. Women had either thoroughly vaginal cleaning before OR or no cleaning depending on the physicians preferences. All women had LA using Citanest Dental Octapressin 2ml. placed in the vaginal vault in the direction of the intended puncture including in the ovarian capsule. Therefore, only one puncture was needed in both sides, left and right. All punctures were hereafter followed by OR using the same 20G SLN. Aspiration was done by a 20ml syringe handled manually by the physician. All women underwent a conventional antagonist protocol with FSH stimulation and Ovitrelle 250IU for induction or Mild Stimulation using Tamoxifen, FSH and Ovitrelle. Outcome measurement were bleeding measured by the necessity of applying compression after the puncture or infection observed up to 1 month. Results: Baseline characteristics including age, BMI and type of stimulation were comparable between the groups. In the nonTC group no infections, bleedings or abscesses were found. In the TC group there were 1 abscess observed. Intact COC was similar in the two groups as well as pregnancy rates. Conclusion: During OR in ART the use of a 20G SLN using the same needle for LA and hereafter, without changing needle in the puncture channel, continue with the egg collection (EC) is simple and safe. In this situation TC is not necessary before puncture, making the procedure more patients friendly. In women undergoing oocyte retrieval for IVF, we found the use of a 20G SLN for both applying LA and retrieving COC safe, effective and simple. Additional TC of the vagina before puncture did not add more to the safety of the procedure.

L-Serine transport in growing and maturing mouse oocytes.

Serine has roles in cell metabolism besides protein synthesis including providing one-carbon units to the folate cycle. Since growing mouse oocytes undergo a burst of folate accumulation as they near full size, we have investigated whether oocytes transport serine. Substantial serine transport appeared in oocytes near the end of their growth. Serine transport continued when oocytes resumed meiosis but ceased partway through first meiotic metaphase, remaining quiescent in mature eggs in second meiotic metaphase. The serine transporter was sodium dependent and inhibited by alanine, cysteine, leucine, or histidine, and had a Michaelis-Menten constant (Km ) for serine of 200 µM. Unexpectedly, exposing cumulus cell-enclosed oocytes to the physiological mediator of meiotic arrest, natriuretic peptide precursor Type C, substantially stimulated serine transport by the enclosed oocyte. Finally, in addition to transport by the oocyte itself, cumulus cells also supply serine to the enclosed oocyte via gap junctions within intact cumulus-oocyte complexes.

Ultrastructural Imaging Analysis of the Zona Pellucida Surface in Bovine Oocytes.

The aims of the present study were to: (i) evaluate the ultrastructural differences in the zona pellucida (ZP) surface between immature and mature bovine oocytes, and (ii) describe a new objective technique to measure the pores in the outer ZP. Intact cumulus-oocyte complexes (COCs) obtained from a local abattoir were immediately fixed (immature group) or submitted to in vitro maturation (IVM) at 38.5 °C for 24 h in a humidified atmosphere of 5% CO2 in air (mature group). Oocytes from both groups were morphologically evaluated via Scanning Electron Microscopy (SEM) and the images were processed in the Fiji/ImageJ software using a new objective methodology through the Trainable Weka Segmentation plugin. The average number of pores in ZP was greater (p 0.05) between groups. In conclusion, it has been shown that the number of pores highlighted the main ultrastructural change in the morphology of the ZP surface of bovine oocytes during the IVM process. We have described an objective method that can be used to evaluate ultrastructural modifications of the ZP surface during oocyte maturation and early embryo development.

Buffalo oocyte-secreted factors promote cumulus cells apoptosis and the rate of cGMP production but not steroidogenesis.

The objectives of this study were to investigate the effect of buffalo oocyte-secreted factors (OSFs) on cumulus cells (CCs) functions, apoptosis and cGMP generation, and whether the direct contact between oocyte and CCs is essential for oocyte-mediated regulation of CCs functions. Buffalo CCs were cultured during IVM within three groups: (a) intact cumulus-oocyte complexes (COCs), (b) CCs cocultured with denuded oocytes (DOs) (CCs+DOs) and (c) CCs monolayer cultured alone (CCsM). After 24hr of IVM, CCs were harvested for evaluation of the relative mRNA abundance of the genes encoding gap junction (GJA1), glycolysis (PFKP and LDHA), apoptosis (CASPASE-3 and BCL-2) and steroidogenesis (ER-β and PGR) by QRT-PCR, and CASPASE-3 proteins, using western blot. Intracellular cGMP content was also assessed by ELISA. Results showed that the relative abundance of LDHA, PFKP and BCL-2 significantly increased (p<0.05) in COCs, whereas GJA1 and CASPASE-3 exhibited lower expression (p<0.05) compared to CCs+DOs and CCsM groups. However, the expression levels of CASPASE-3, both mRNA and protein, were significantly (p<0.05) downregulated in CCs+DOs compared to CCsM. There was no significant difference in the expression level of PGR and ER-β between the groups. The intracellular content of cGMP was notably (p<0.05) higher in COCs compared to CCs+DOs and CCsM groups. In conclusion, this study demonstrated, for the first time, that buffalo OSFs protect CCs against apoptosis and stimulate their cGMP production; however, the regulation of cumulus glycolysis and gap junction is confined to those in close contact with the oocyte. Neither OSFs from COCs nor those from DOs have any effect on CCs steroidogenesis.

Developmental competence of buffalo (Bubalus bubalis) denuded oocytes cocultured with cumulus cells: Protective role of cumulus cells

The objectives of the present study were to evaluate the developmental competence of buffalo denuded oocytes (DOs) cocultured with cumulus cells (CCs) during in vitro maturation, and to investigate the mechanisms by which CCs promote oocyte maturation and development. Buffalo oocytes were matured in vitro for 24 h in three groups: (1) intact cumulus–oocyte complexes (COCs) (2) DOs cocultured with CCs (DOsCC), and (3) DOs cultured alone (DOs). Matured oocytes were used to determine the relative mRNA abundance of Gdf-9, Bmp15, Zar1, Caspase-3, Bcl-2, Zp2, Zp3, Cd9 and Pde3a by Rt-qPCR and CASPASE-3 protein expression by immunofluorescence. The intracellular content of cGMP, cAMP and MPF activity and the rate of embryonic development were also assessed. Results of the present study showed that in DOs, the relative mRNA abundance of Gdf-9, Bmp15, and Cd9 significantly (P < 0.05) decreased, whereas Caspase-3 (mRNA and protein levels), Bcl-2, and Pde3a exhibited higher expression than DOsCC and COCs. However, there was no significant difference among the groups in the expression level of Zar-1, Zp2, and Zp3. The intracellular content of cAMP and MPF activity was notably higher (P < 0.05) in DOs compared to COCs and DOsCC. There was no significant difference between COCs and DOsCC in cGMP content, which was significantly lower (P < 0.05) in DOs. Moreover, the cleavage and blastocyst rates were 58.4 ± 1.8%, 43.7 ± 1.1%, 18.4 ± 0.9% and 18.0 ± 1.3%, 11.0 ± 0.9% and 4.5 ± 0.6% in COCs, DOsCC and DOs groups, respectively. In conclusion, the presence of CCs protects buffalo DOs from apoptosis and promotes maturation through regulation of the intracellular content of cAMP and MPF activity and improves the fertilizing capacity of oocytes through modulation of the gamete fusion gene, Cd9.

Glucose metabolism during in vitro maturation of mouse oocytes: An study using RNA interference

In previous studies on glucose metabolism during in vitro maturation, intact cumulus-oocyte complexes (COCs) were treated with enzyme inhibitors/activators. Because inhibitors/activators may have non-specificity and/or toxicity, and culture of COCs cannot differentiate whether glucose metabolism of cumulus cells (CCs) or that of the oocyte supports oocyte maturation, results from the previous studies must be verified by silencing genes in either CCs or cumulus-denuded oocytes (DOs). In this study, RNAi was adopted to specify the effects of glucose metabolism in CCs or DOs on oocyte maturation. Although silencing either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or glucose-6-phosphate dehydrogenase (G6PD) genes in CCs significantly decreased competence of the cocultured DOs, silencing G6PD impaired competence to a greater extent. While silencing G6PD or GAPDH of CCs decreased glutathione and ATP contents of cocultured DOs to similar extents, silencing G6PD increased oxidative stress as well. Analysis on metabolite contents and oxidative stress index and culture of DOs in medium conditioned with gene-silenced CCs indicated that CCs supported oocyte maturation by releasing glucose metabolites. Silencing mitochondrial pyruvate carrier 1 or NADH dehydrogenase (ubiquintone) flavoprotein 1 of DOs significantly impaired their maturation. The results have unequivocally confirmed that CCs promote oocyte maturation by releasing glucose metabolites from both pentose phosphate pathway (PPP) and glycolysis. Pyruvate is transferred into DOs by mitochondrial pyruvate carrier (MPC) and utilized through mitochondrial electron transport to support maturation.

Heat stress impairs gap junction communication and cumulus function of bovine oocytes.

The intimate association of cumulus cells with one another and with the oocyte is important for regulating oocyte meiotic arrest and resumption. The objective of this study was to determine the effects of heat stress on cumulus cell communication and functions that may be related to accelerated oocyte meiosis during early maturation. Bovine cumulus-oocyte complexes underwent in vitro maturation for up to 6 h at thermoneutral control (38.5°C) or elevated (40.0, 41.0 or 42.0°C) temperatures. Gap junction communication between the cumulus cells and the oocyte was assessed using the fluorescent dye calcein after 4 h of in vitro maturation. Dye transfer was reduced in cumulus-oocyte complexes matured at 41.0°C or 42.0°C; transfer at 40.0°C was similar to control (P < 0.0001). Subsequent staining of oocytes with Hoechst revealed that oocytes matured at 41.0 or 42.0°C contained chromatin at more advanced stages of condensation. Maturation of cumulus-oocyte complexes at elevated temperatures reduced levels of active 5’ adenosine monophosphate activated kinase (P = 0.03). Heat stress exposure had no effect on active extracellular-regulated kinase 1/2 in oocytes (P = 0.67), associated cumulus cells (P = 0.60) or intact cumulus-oocyte complexes (P = 0.44). Heat-induced increases in progesterone production by cumulus-oocyte complexes were detected during the first 6 h of maturation (P = 0.001). Heat-induced alterations in gap junction communication and other cumulus-cell functions likely cooperate to accelerate bovine oocyte meiotic progression.

Role of cumulus cells during vitrification and fertilization of mature bovine oocytes: Effects on survival, fertilization, and blastocyst development

This study was designed to determine the role of cumulus cells during vitrification of bovine oocytes. Mature cumulus-oocyte complexes (COCs) with many layers of cumulus cells, corona radiata oocytes (CRs), with a few layers of cumulus cells, and denuded oocytes (DOs) without cumulus cells were vitrified in 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.5-M sucrose. Oocytes that survived the vitrification process were fertilized. Denuded oocytes were fertilized with or without supplementation of intact COCs (DOsCOCs). First, survival and embryo development rates were studied. Higher survival rates were obtained for DOs and DOsCOCs (94% and 95%, respectively) compared with COCs (82.7%, P < 0.05). Corona radiata oocytes showed similar survival rates when compared with DOs. The cleavage and blastocyst rates of vitrified DOs were compromised because cumulus cells were not present during the fertilization (34% and 2.7%, respectively). However, the situation could be reverted when DOs were supplemented with intact COCs (DOsCOCs; 62.7% and 12.7%, respectively, P < 0.05). Vitrified CRs showed similar cleavage and blastocyst rate (49.3% and 7.7%, respectively) compared with COCs (54.8% and 4.9%, respectively). In the second experiment, the penetration rate was analyzed. Removing cumulus cells before fertilization reduced the fertilization of vitrified DOs compared with COCs (24.3% vs. 52.8%, P < 0.05). The supplementation of DOs with intact COCs (DOsCOCs) improved the fertilization rate though (49.6%, P < 0.05). No differences in the fertilization rate were found between CRs and COCs. In the third experiment, parthenogenetic activation was examined. Interestingly, the CRs group showed higher cleavage and blastocyst rates (76.8% and 29.6%, respectively) than the COCs (39.1% and 7.5%, respectively, P < 0.05). Furthermore, oocytes from vitrified CRs had the same odds to become a blastocyst as fresh oocytes (1.1 vs. 1.5, respectively). In conclusion, our data reported that cumulus cells reduce survival after the vitrification of mature bovine oocytes. Because cumulus cells are required for fertilization, the use of partially DOs (CRs) or the addition of intact COCs (DOsCOCs) during fertilization can result in higher survival and embryo development after vitrification.

Cumulus co-culture system does not improve the in-vitro maturation (IVM) of oocytes in mice

Abstract Objective: To examine the effect of co-culture system with CCs on the in vitro maturation (IVM) of immature oocytes retrieved from mice. Design: This is an experimental study. Setting: Research and Clinical Center for Infertility of Yazd. Sample: A total of 144 oocytes from 5 to 6 weeks mice. Material: After dissecting ovaries, denuded GVs (DGVs) were considered as control group and put in IVM medium. In cumulus oocyte complexes (COCs) as exp I, intact COCs were put in IVM medium and observed for maturation after 24 h and 48 h. In exp II, GVs were co-cultured with mature CCs. For exp II, COCs were incubated for 30 min before removing oocytes. Then, denuded mature oocytes were removed and the remaining CCs were used to culture denuded GVs recovered from ovaries. Main outcome measures: Maturation rates of oocytes after 24 h and 48 h. Results: The rate of oocyte maturation was 77.36 ± 14.4 in control group. In exp I and exp II, the rates of maturation were 91.32 ± 22.5 and 63.33 ± 7.4, respectively ( P = 0.04). However, maturation rate did not increase after 24 h. The maturation rate in metaphase I (MI) oocytes was as low as 15% in different groups at 24 h and did not increase by 48 h. Degeneration rate was increased from time 0 to 48 h, but not significant in different times. Conclusion: Presence of intact COCs significantly improved the oocyte maturation rates in animal model. However, co-culture of GVs with mature CCs did not improve IVM program in mice.

255 SONIC HEDGEHOG PROMOTES IN VITRO OOCYTE MATURATION AND EMBRYO DEVELOPMENT IN GOATS

The signalling of the Hh family peptides is mediated through a cell surface receptor system consisting of 2 proteins: patched (Ptc) and smoothened (Smo). In the absence of Hh ligand, the Hh receptor Ptc represses Smo, whereas in the presence of Hh, the suppression of Smo is lifted, leading to the activation of downstream transcriptional factors (Gli1, Gli2, and Gli3) in vertebrates. Previous studies have examined Sonic hedgehog (Shh) signalling pathways in developing and adult mouse ovaries and concluded that the Shh signalling pathway may be involved in granulosa cell proliferation and oocyte maturation. We investigated the effects of Shh protein on caprine oocyte maturation, embryo development, and embryo survival rate after transfer of vitrified/thawed in vitro-produced (IVP) embryos to recipients. Cumulus-oocyte complexes (COC) were collected by slicing ovarian follicles (1–5 mm in diameter). On average, 40 to 50 oocytes were randomly allocated to each well containing 500 μL of IVM medium and supplemented with 0 (control), 0.125, 0.25, 0.5, or 1.0 μg mL–1 recombinant mouse Shh protein. After 24 h of IVM, cumulus cells were partially removed. Oocytes were washed and transferred into a droplet of 80 μL of fertilization medium and were fertilized with frozen-thawed sperm for 18 h at 38.8°C. After IVF, presumptive zygotes were cultured on goat oviduct epithelial monolayers in M199 for 9 days. The 2 frozen-thawed selected embryos were transferred to one recipient. All data were subjected to ANOVA, using the general linear model procedure in SAS (version 9), followed by Tukey's test. Embryo survival rates were compared by using the chi-square test. The RT-PCR analyses showed that the expressions of Shh, SMO, Ptch1, and Gli1 were detected in whole ovaries, granulosa cells, COC, cumulus cells, oocytes, and oviduct epithelia except for Ptch1 in cumulus cells. Supplementation of Shh (0.25 or 0.5 μg mL–1) enhanced oocyte maturation as opposed to the control group (92.4%, n = 67 and 95.0%, n = 62 v. 86.2%, n = 64, respectively, P &lt; 0.05). This effect could be reversed by the simultaneous addition of cyclopamine (0.5–1.0 μm), a Shh inhibitor. Similar to intact COC, denuded oocytes showed enhanced maturation (72.0%, n = 94 v. 60.5%, n = 126) with Shh supplementation. For subsequent embryo development, an improved blastocyst rate (P &lt; 0.05) was 66.3 ± 10.9 (n = 135) when embryos were derived from the oocytes matured in the presence of 0.5 μg mL–1 Shh rather than 41.4 ± 12.9 (n = 137) of the control group. After embryo transfer, the kidding and embryo survival rates of vitrified embryos derived from the Shh-supplemented group were 56 (16 recipients) and 31% (48 embryos) higher than that 38 (16 recipients) and 15% (54 embryos) without Shh supplementation (P &lt; 0.05). The present study suggests that Shh signalling is active in caprine ovaries during folliculogenesis and beneficial to oocyte maturation and subsequent embryo development to the blastocyst stage (in vitro) and to term.

Interactions between oocytes and cumulus cells during in vitro maturation of porcine cumulus-oocyte complexes in a chemically defined medium: Effect of denuded oocytes on cumulus expansion and oocyte maturation

The aim of the present study was to clarify interactions between oocytes and cumulus cells (CCs) on the level of cumulus expansion and oocyte maturation during IVM of cumulus-oocyte complexes (COCs) in a chemically defined medium using a system that allows individual tracking of oocytes. Especially, the influence of oocyte-secreted factors was investigated by the aid of addition of denuded oocytes (DOs) as a possible approach to improve the IVM system. The basic maturation medium was porcine oocyte medium with addition of gonadotropins only during the first 20 hours of IVM. During IVM, COCs were kept fixed to the bottom of culture dish by adhesive Cell-Tak coating, which enabled individual tracking of COCs during IVM. Size changes in COCs during IVM were measured by digital image analysis. Cumulus expansion in a porcine oocyte medium of intact COCs increased in a typical manner until 20 hours and decreased in size subsequently until 48 hours of IVM (P < 0.05). Removal of oocytes from COCs by oocytectomy allowed the expansion of CCs to some extent, although their expansion ability was lower than that of COCs (P < 0.05). Addition of DOs (COCs to DOs ratio of 9:16) did not improve cumulus expansion and oocyte maturation rates of intact COCs (P > 0.05) but did enhance cumulus expansion of oocytectomized complexes (P < 0.05). Furthermore, removal of CCs before IVM increased oocyte maturation rates compared with COCs (52.3% and 32.9%, respectively) (P < 0.05) and a similar effect was observed in COCs when the gap junction inhibitor carbenoxolone was added to the IVM medium: carbenoxolone repressed the expansion of COCs at 20 hours of IVM. In conclusion, the porcine oocyte enhances cumulus expansion both by gap junctional communications and presumably by oocyte-secreted factor production. Nevertheless, the presence of oocytes is not a prerequisite for this process. In return, CCs maintain meiotic arrest in cumulus-enclosed oocytes during the initial culture through gap junctions. On the basis of these findings, future research could investigate if coculture with DOs during IVM is beneficial for fertilization and embryo development.

Leukemia inhibitory factor enhances bovine oocyte maturation and early embryo development

The present study was conducted to examine the effects of leukemia inhibitory factor (LIF) on bovine oocyte maturation and early embryo development in vitro. Results showed that LIF supplementation (25 ng/ml) enhanced nuclear maturation of intact cumulus-oocyte complexes (COCs) compared to the vehicle control. Similar results were observed in denuded oocytes, indicating that LIF directly influences oocyte development. LIF-treated oocytes showed a higher cortical-granule-migration rate and increased expression of CD9, a tetraspanin transmembrane protein essential for fertilization. After in vitro fertilization, oocytes receiving LIF supplementation exhibited a higher cleavage rate and yielded a significantly higher number of blastocysts. To further dissect the molecular mechanism underlying this LIF-induced bovine oocyte maturation phenotype, we examined the involvement of two signaling cascades, mitogen-activated protein kinases (MAPK3/1)- and the signal transducer and activator of transcription 3 (STAT3)-dependent pathways. Western blot results revealed that LIF phosphorylated MAPK3/1 and STAT3. Inhibition of MAPK3/1 activation with MEK inhibitor U0126 only partially blocked LIF-induced nuclear maturation, although it attenuated oocyte cytoplasmic maturation. Inhibition of JAK/STAT3 activation with a specific pharmacological inhibitor completely abolished the LIF-response in bovine oocyte. In summary, these data revealed a novel role for LIF in bovine oocyte maturation subsequent embryonic development.

Uptake of Betaine into Mouse Cumulus-Oocyte Complexes via the SLC7A6 Isoform of y+L Transporter1

Betaine (N,N,N-trimethylglycine) has previously been shown to function in cell volume homeostasis in early mouse embryos and also to be a key donor to the methyl pool in the blastocyst. A betaine transporter (SLC6A20A or SIT1) has been shown to be activated after fertilization, but there is no saturable betaine uptake in mouse oocytes or eggs. Unexpectedly, the same high level of betaine is present in mature metaphase II (MII) eggs as is found in one-cell embryos despite the lack of transport in oocytes or eggs. Significant saturable betaine transport is, however, present in intact cumulus-oocyte complexes (COCs). This transport system has an affinity for betaine of ∼227 μM. The inhibition profile indicates that betaine transport by COCs could be completely blocked by methionine, proline, leucine, lysine, and arginine, and transport is dependent on Na(+) but not Cl(-). This is consistent with transport by a y+L-type amino acid transport system. Both transcripts and protein of one y+L isoform, SLC7A6 (y+LAT2), are present in COCs, with little or no expression in isolated germinal vesicle (GV)-stage oocytes, MII eggs, or one-cell embryos. Betaine accumulated by COCs is transferred into the enclosed GV oocyte, which requires functional gap junctions. Thus, at least a portion of the endogenous betaine in MII eggs could be derived from transport into cumulus cells and subsequent transfer into the enclosed oocyte before gap junction closure during meiotic maturation. The oocyte-derived betaine then could be regulated and supplemented by the SIT1 transporter that arises in the embryo after fertilization.

Effects of differing oocyte-secreted factors during mouse in vitro maturation on subsequent embryo and fetal development

We hypothesised that varying native oocyte-secreted factor (OSF) exposure or using different recombinant OSF peptides would have differential effects on post-in vitro maturation (IVM) embryo and fetal development. Mouse cumulus oocyte complexes (COCs) were treated with the purified mature domain of GDF9 and/or BMP15 or were co-cultured with denuded oocytes (DOs) from 0 h or 3 h of IVM. DOs were matured for 3 h as either intact COCs+/-FSH before denuding, or as DOs + FSH. COCs were fertilised and blastocyst development was assessed on days 5 and 6, and either differentially stained for ICM numbers or vitrified/warmed embryos were transferred to recipients to assess implantation and fetal rates. No improvement in embryo development was observed with the addition of GDF9 and/or BMP15 to IVM. In contrast, embryos derived from COCs co-cultured with DOs had significantly improved blastocyst rates and ICM numbers compared to controls (P < 0.05). The highest response was obtained when DOs were first added to COCs at 3 h of IVM, after being pre-treated (0-3 h) as COCs + FSH. Compared to control, co-culture with DOs from 3 h did not affect implantation rates but more than doubled fetal yield (21% vs 48%; P < 0.05). GDF9 Western blot analysis was unable to detect any differences in quantity or form of GDF9 (17 and 65 kDa) in extracts of DO at 0 h or 3 h. This study provides new knowledge on means to improve oocyte quality in vitro which has the potential to significantly aid human infertility treatment and animal embryo production technologies.