Cumulus Cell Mass Research Articles

The impact of cumulus cell viability and pre-culture with the healthy cell mass on brilliant cresyl blue (BCB) staining assessment and meiotic competence of suboptimal porcine oocytes

Objectives of the present study were to investigate the characteristics including glucose-6-phosphate dehydrogenase activity, as determined by Brilliant Cresyl Blue (BCB) staining, of suboptimal porcine oocytes and to enhance the meiotic competence of those through pre-culture with cumulus cell masses (CCMs). Percentage of oocyte-cumulus complexes (OCCs) derived from small follicles (SF; <3 mm in diameter) containing the oocytes that were assessed as BCB-negative (BCB-) was significantly higher than those derived from medium follicles (MF; 3–6 mm in diameter). Degrees of dead cumulus cells were significantly higher in OCCs containing BCB- oocytes, regardless of the origin of OCCs (MF vs. SF), than those containing BCB-positive (BCB+) ones. Exposing OCCs containing BCB+ oocytes to the apoptosis inducer, carbonyl cyanide m-chlorophenylhydrazone, for 20 h significantly induced the transition to BCB- and meiotic progression of exposed OCCs were significantly reduced in both SF and MF derived ones. Transit of BCB- oocytes to BCB+ was induced when OCCs were pre-cultured with CCMs of MF derived OCCs containing BCB+ oocytes for 20 h before IVM. This pre-culture also significantly increased the meiotic competence of BCB- oocytes, particularly in SF derived ones. However, reactive oxygen species levels were significantly higher in BCB+ oocytes as compared with BCB- ones, regardless of pre-culture with CCMs, whereas no significant differences were found in the ATP contents among the treatment groups. In conclusion, the BCB result of oocytes could be regulated by the healthy status and content of surrounding cumulus cells and the meiotic competence of suboptimal BCB- porcine oocytes is improved by pre-culture with healthy CCMs.

Sexual selection drives the coevolution of male and female reproductive traits in Peromyscus mice.

When females mate with multiple partners within a single reproductive cycle, sperm from rival males may compete for fertilization of a limited number of ova, and females may bias the fertilization of their ova by particular sperm. Over evolutionary timescales, these two forms of selection shape both male and female reproductive physiology when females mate multiply, yet in monogamous systems, post-copulatory sexual selection is weak or absent. Here, we examine how divergent mating strategies within a genus of closely related mice, Peromyscus, have shaped the evolution of reproductive traits. We show that in promiscuous species, males exhibit traits associated with increased sperm production and sperm swimming performance, and females exhibit traits that are predicted to limit sperm access to their ova including increased oviduct length and a larger cumulus cell mass surrounding the ova, compared to monogamous species. Importantly, we found that across species, oviduct length and cumulus cell density are significantly correlated with sperm velocity, but not sperm count or relative testes size, suggesting that these female traits may have coevolved with increased sperm quality rather than quantity. Taken together, our results highlight how male and female traits evolve in concert and respond to changes in the level of post-copulatory sexual selection.

Oocyte diameter predicts the maturation rate of human immature oocytes collected ex vivo

PurposeTo study the impact of oocyte diameter and cumulus cell mass on the potential for final maturation of immature human oocytes in vitro.MethodsImmature oocytes (n = 1563) from 75 women undergoing fertility preservation by ovarian tissue cryopreservation (14–41 years) were collected. After preparation of the ovarian cortex for freezing, immature oocytes were collected from the surplus medulla. After collection, IVM was performed according to standard published methods. The mass of cumulus cell surrounding the immature oocyte was grouped according to size. After IVM, each oocyte was photographed, measured, and the diameter was calculated as a mean of two perpendicular measurements.ResultsThe diameter of the oocytes ranged from 60 to 171 µm with a mean of 115 µm (SD:12.1) and an interquartile range from 107 to 124 µm. The oocyte diameter was positively associated with a higher incidence of MII (p < 0.001). MII oocytes had a significantly larger mean diameter than MI, GV, and degenerated oocytes. The size of the cumulus cell mass was significantly associated with the MII stage (p < 0.001) and larger oocyte diameter (p < 0.001). The results further confirm that the diameter of the fully grown oocyte is reached relatively early in human follicular development and that the factors governing oocyte maturation in vitro are connected to the surrounding cell mass and the oocyte.ConclusionThe diameter of the oocyte is a highly determining factor in the nuclear maturation of the human oocyte during in vitro maturation, and the size of the cumulus cell mass is closely positively associated with a larger diameter.

Automated Denudation of Oocytes.

Denudation is a technique for removal of the cumulus cell mass from oocytes in clinical intracytoplasmic sperm injection (ICSI). Manual oocyte denudation requires long training hours and stringent skills, but still suffers from low yield rate and denudation efficiency due to human fatigue and skill variations across operators. To address these limitations, this paper reports a robotic system for automated oocyte denudation. In this system, several key techniques are proposed, including a vision-based contact detection method for measuring the relative z position between the micropipette tip and the dish substrate, recognition of oocytes and the surrounding cumulus cells, automated calibration algorithm for eliminating the misalignment angle, and automated control of the flow rate based on the model of oocyte dynamics during micropipette aspiration and deposition. Experiments on mouse oocytes demonstrated that the robotic denudation system achieved a high yield rate of 97.0 ± 2.8% and denudation efficiency of 95.0 ± 0.8%. Additionally, oocytes denuded by the robotic system showed comparable fertilization rate and developmental competence compared with manual denudation. Our robotic denudation system represents one step towards the automation and standardization of ICSI procedures.

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.

An Efficient Nitroblue Tetrazolium Staining and Bright-Field Microscopy Based Method for Detecting and Quantifying Intracellular Reactive Oxygen Species in Oocytes, Cumulus Cells and Embryos.

Assessment of intracellular reactive oxygen species (ROS) is important for evaluating the developmental ability of cumulus-oocyte complexes (COC) and embryos. Although, fluorescence-based 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method is used widely for detecting intracellular ROS in COC and embryos, it is associated with several limitations. This study aimed to develop an alternative method for detecting and quantifying intracellular ROS in oocytes, cumulus cells and embryos based on nitroblue tetrazolium (NBT) staining and bright-field microscopy. Nitroblue tetrazolium reacts with ROS and forms formazan precipitate that can be detected as dark purple/blue spots under bright-field microscope. Ovine COC were matured in vitro without (control) or with the supplementation of Interleukin-7 (IL-7; for stimulating intracellular ROS), Tempol (superoxide scavenger) or combination of IL-7 and Tempol. The matured COC were stained with NBT and the formation of intracellular formazan precipitates was assessed. Additionally, the matured COC were stained with DCFH-DA to compare the level of intracellular ROS. Further, ovine embryos (8-cell, morula, and degenerating) were generated in vitro and stained with NBT for assessing intracellular ROS. The level of intracellular ROS was expressed as the proportion (%) of the NBT stained area of oocytes, compact cumulus cell masses or embryos. The proportions of NBT stained area in the matured oocytes and cumulus cells was found significantly lesser in the control as compared to the IL-7 (1 and 5 ng/ml) treated groups. A similar trend in the intracellular ROS level was also observed in the matured COC, when assessed based on the DCFH-DA staining. Following the treatment with Tempol (100 mM), negligible NBT stained area in oocytes and cumulus cells was observed. The NBT staining patterns of the oocytes and cumulus cells following the combined treatment with IL-7 (5 ng/ml) and Tempol (10 and 25 mM) were comparable with that of the control. The proportion of NBT stained area did not differ significantly between the 8-cell embryos and morula, but was found significantly greater in the degenerating embryos. In conclusion, the developed NBT staining method was found effective for detecting and interpreting the level of intracellular ROS in oocytes, cumulus cells and embryos. This method can be used as an alternative to the DCFH-DA staining method.

Versican expression level in cumulus cells is associated with human oocyte developmental competence

ABSTRACT To study the relationship between the expression of 10 selected genes in cumulus cells and the corresponding oocyte development competence, and the effect of patient age and body mass index on gene expression of cumulus cells, we collected 354 cumulus cell masses associated with individual oocyte from 48 women. The expression levels of the genes involved in glucose metabolism (PFKP, PKM2, LDHA and GFPT) and expansion (HAS2, VCAN, TNFAIP6, PTGS2, PTX3 and SDC4) in cumulus cells were detected by reverse transcription polymerase chain reaction. These were compared among oocyte maturity, fertilization, embryo morphology and implantation, and analyzed the effect of the subject’s age and body mass index. Cumulus cell PFKP expression from mature oocytes was higher than those from immature oocytes (P = 0.014), and VCAN expression was higher from oocytes that developed into high-quality embryos (P = 0.024). TNFAIP6 expression in cumulus cells from fertilized oocytes was lower than that from unfertilized oocytes (P = 0.044). The levels of VCAN, TNFAIP6, PTX3 and SDC4 were changed significantly as a function of the subject’s age and body mass index. In conclusion, the level of VCAN expression in cumulus cells is positively correlated with the early embryo morphology score, and with further development could perhaps be used to evaluate oocyte developmental competence to complement embryonic morphological assessment. Abbreviations CCs: cumulus cells; GDF9: growth differentiation factor 9; BMP15: bone morphogenetic protein 15; PTGS2: prostaglandin synthase 2; HAS2: hyaluronic acid synthase 2; VCAN: versican; GREM1: gremlin 1; PFKP: phosphofructokinase, platelet; PKM2: pyruvate kinase isozyme type M2; LDHA: lactic dehydrogenase; GFPT: glucosaminefructo-6-phosphate transaminase; TNFAIP6: tumor necrosis factor 6 protein; PTX3: penetrin 3; SDC4: syndecan-4; BMI: body mass index; MD: median values; IQR: interquartile range; FSH: follicle-stimulating hormone; LH: luteinizing hormone; HCG: human chorionic gonadotropin; ICSI: intracytoplasmic sperm injection; GnRH: gonadotropin-releasing hormone; hMG: human menopausal gonadotropin; GV: germinal vesicle; M I: metaphase I; M II: metaphase II; cDNA: complementary DNA; SD: standard deviation.

Bone morphogenetic protein 6 expression in cumulus cells is negatively associated with oocyte maturation

Bone morphogenetic protein 6 (BMP6) is a regulatory peptide secreted by oocytes and granulosa cells that locally regulates folliculogenesis and follicular development. To determine BMP6 location, we studied BMP6 expression in human follicles using immunohistochemistry, real-time polymerase chain reaction (RT-PCR) and western blot analysis. RT-PCR was performed on 354 individual cumulus cell (CC) masses from 48 women to investigate the relationship between BMP6 mRNA expression in CCs and oocyte developmental potential. Results showed that BMP6 protein was mainly located in oocytes from preantral follicles and in granulosa cells from antral follicles. BMP6 mRNA expression was much higher in oocytes than in CCs and mural granulosa cells (mGCs) from preovulatory follicles (p < 0.01), and BMP6 protein level was higher in CCs than in mGCs (p < 0.05). BMP6 mRNA expression was higher in CCs from immature oocytes than in those from mature oocytes (p < 0.05). However, BMP6 mRNA expression in CCs was not associated with oocyte fertilization, embryo morphological grading, or implantation. In conclusion, BMP6 was mainly expressed in oocytes at all human follicular developmental stages and BMP6 mRNA expression in CCs may be negatively correlated with oocyte maturation. BMP6 expression could therefore be used as a biomarker of oocyte maturation.

Versican (VCAN) mRNA expression in cumulus cells predicts embryo quality, but does not predict pregnancy outcome in patients with reduced ovarian reserve

Cumulus cells surround the oocyte and are pivotal in determining oocyte developmental competence. Several studies have revealed certain genes which have a correlation with oocyte competence, embryo development and pregnancy rates. The rational of this approach is to identify significant predictors of blastocyst quality which could be used for non-invasive embryo selection. A total of 160 cumulus cell masses (CCM) obtained from 40 patients with reduced ovarian reserve (age <43 years; AMH < 1,2ng/ml, FSH>11 IU/ml) were measured for gene expression. 58 CCM were excluded (from immature oocytes, unfertilized oocytes, arrested embryos). The remaining 102 CCM were divided into 3 groups (Gardner & Schoolcraft): I group (n=42) - CCM from oocytes developing into TOP-quality embryos (3BB and higher); II group (n = 43) - CCM from oocytes developing into good quality embryos (2BB, nBC); group III (n = 17) - CCM from oocytes developing into fair quality embryos (nCB, CC). ET was performed in cryo cycle. HAS2, VCAN, ALCAM, PTGS2, GREM1, ITPKA, TRPM7, SDC4,CALM2,SPSB2, TP53I3 genes expression were measured with real-time PCR. Statistical analysis - Kruskal-Wallis test, ROC analysis, p<0.05 was considered to be statistically significant. VCAN and hyaluronan synthase 2 (HAS2) genes had significantly higher expression levels in CCM that surrounded oocytes developing into TOP and good quality embryos (Table 1). Prostaglandin-endoperoxide synthase 2(PTGS2) and inositol-trisphosphate 3-kinase A (ITPKA) genes had significantly higher mRNA expression levels in CCM from oocytes developing into TOP-quality embryos (Table 1). But, according to ROC-analysis, only level of VCAN mRNA expression was associated with good and TOP-quality embryos (the baseline level of expression was set at 12.46, the area under curve - 0,845, sensitivity -90.5%, specificity-70.6%). Clinical pregnancy rate per ET was 37.5% (15/40). There was no correlation found between the expression levels of all tested genes and clinical pregnancy rate. *p<0.05 - differences are significant with group III. VCAN mRNA expression in CCM correlated with embryo quality and could be used to assist the selection of oocytes, but does not predict pregnancy outcome.Tabled 1Table 1CC genes mRNA expressionI groupII groupIII groupVCAN17,63* (14,25-17,63)16,76* (13,38-22,00)8,57 (5,7-14,56)HAS20,44* (0,32-0,52)0,44 (0,36-0,60)0,24 (0,17-0,31)ITPKA0,31* (0,23-0,42)0,311 (0,18-0,53)0,602 (0,22-0,71)PTGS22,61* (0,22-0,41)1,481 (0,75-3,19)0,871 (0,52-1,29) Open table in a new tab

Supplementation with cumulus cell masses improves the in vitro meiotic competence of porcine cumulus-oocytes complexes derived from small follicles.

The present study was conducted to examine the supplemented effect of cumulus cell masses (CCMs) derived from middle follicle (MF; 3-6mm diameter) on the morphology and the meiotic or developmental competence of oocytes from small follicles (SF; 1-2mm diameter). The number of cumulus cells surrounding oocytes just after collection was also lower in cumulus-oocyte complexes (COCs) from SF than MF. The ooplasmic diameter of oocytes was significantly smaller in SF-derived oocytes than MF-derived ones before and after in vitro maturation (IVM), whereas the diameter significantly increased during the culture. Co-culture of SF-derived COCs with MF-derived CCMs during IVM significantly improved the meiotic competence of the oocytes to the metaphase-II stage. Furthermore, the ooplasmic diameter of SF-derived COCs during IVM was increased to the similar size of MF-derived those in the presence of MF-derived CCMs. The abilities of oocytes to be penetrated, to form male pronuclear formation and to cleave or develop to the blastocyst stage were not affected by the co-culture with CCMs. Electrophoretic analysis of CCM secretions clearly showed the presence of more protein(s) approximately 27.6kDa in the conditioned medium when supplemented with MF-derived CCMs. In conclusion, we demonstrate that supplementation with MF-derived CCMs improves the ooplasmic diameter and meiotic competence of SF-derived oocytes.

Heat stress and antioxidant enzyme activity in bubaline (Bubalus bubalis) oocytes during in vitro maturation.

In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5°C for complete 24h of meiotic maturation and heat stress of 40.5 and 41.5°C was applied to COC during the first 12h of maturation and then moved to 38.5°C for rest of the 12h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly (P < 0.05) higher in the oocytes subjected to heat stress (40.5 and 41.5°C) during meiotic maturation compared to the oocytes matured under standard in vitro culture conditions (38.5°C). Also, the antioxidant enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were significantly (P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.

24 EFFECT OF DONOR CELL TYPE ON IN VITRO AND IN VIVO DEVELOPMENTAL COMPETENCE OF CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

Selection of the donor cell type for somatic cell NT is very important based on its capability to be reprogrammed by the oocyte cytoplasm. A very wide variety of donor cells of different origin have been used for somatic cell NT, having differences in the overall efficiency. The aim of this study was to compare the cloning efficiency of donor cells derived from the ventral side of origin of tail skin and seminal plasma of a buffalo bull (age: 3 years old). Somatic cells from skin and seminal plasma were isolated and cultured as described by Selokar et al. (2014 PLOS ONE 9(3), e90755). Cultured seminal plasma cells had classic epithelial morphology, grew in clusters, were hexagonal in outline shape, and were positive for immunocytochemical detection of keratin marker, indicating that they were of epithelial origin, whereas tail-derived cells were spindle in shape and found positive for vimentin expression, indicating the fibroblast origin. To determine their reprogramming potential, these cells between passages 5 to 8 were used for the production of buffalo cloned embryos by handmade cloning as per the method described by Selokar et al. (2012 Theriogenology 78, 930–936). In brief, oocytes were isolated from slaughter-house ovaries and matured in vitro. After 21 h of maturation, cumulus cell mass and zona pellucida were removed by enzymatic treatment, hyaluronidase and pronase, respectively. Zona-free buffalo oocytes were enucleated on the basis of protrusion cone. A single somatic cell was attached to an enucleated oocyte with addition of phytohemagglutinin, followed by sandwich type of electrofusion between the somatic cell-bearing oocyte and enucleated oocyte using BTX electrofusion machine. Fused oocytes were activated by 4 μM calcium ionophore for 5 min and incubated in 2 mM 6-DMAP for 4 h and were cultured in K-RVCL-50® medium for 7 days on a flat surface in a 4-well dish in an incubator (5% CO2 and 38.5°C temperature). The total numbers of embryos reconstructed from tail-derived cells and semen-derived cells were 132 and 158, respectively. Cleavage and blastocyst rate were calculated from total embryos cultured, and data were analysed by Student’s t-test. We found no significant effect on both cleavage (89.30 ± 2.1 v. 94.1 ± 0.6) and blastocyst rate (40.7 ± 4.0 v. 43.1 ± 9.6) for the embryos produced from cells derived from tail and seminal plasma. To study the in vivo developmental competence of embryos derived from the 2 donor cell types, one embryo of each cell type was transferred into 6 recipient animals. Pregnancies were confirmed by ultrasonography at 30 to 35 days after transfer and monitored regularly at 15-day intervals up to 90 days. Three pregnancies were found for tail-derived cells, whereas no pregnancy was obtained for semen-derived cells. Out of 3 pregnancies obtained, 1 embryonic death was observed before 45 days, and 2 are continuing at advance stage. In conclusion, tail-derived cells are the better donor cell choice for buffalo somatic cell NT research. Currently, our focus is on epigenetic reprogramming behaviour of these 2 different cell types to elucidate the possible reprogramming mechanism.

Zona free hamster ova penetration test: fertility indicator of buck spermatozoa

An experiment was conducted to assess the sperm fertility of Sirohi bucks. Ejaculates from three bucks were collected through artificial vagina and evaluated microscopically at 37° C. After final washing with Biggers-Whitten Whittingham (BWW) medium, semen was kept in a CO2 incubator for 20 min (swim up). The hamster oocytes were recovered following superovulation and visualized on the basis of cumulus cell mass. The oocytes were washed in BWW medium and then denuded with 0.1% hyaluronidase, the cumulus-free oocytes were treated with 0.1% protease for 2–3 min for zona lysis. Oocytes and swim up motile spermatozoa were co-incubated at 37° C for 3 h. in already incubated BWW drop (50 μl). At the end of co-incubation, washing of oocyte sperm complexes was done with BWW medium and then kept in hypotonic KCl solution (50 mg/ml), fixed in 2.5% glutaraldehyde for 10 min and stained with 0.1μg/ml DAPI (4,6-di amidino 2-phenyl indole). The number of sperms penetrated into the oocytes was determined. The result of the study indicated that among the three bucks, buck No. 1 had higher penetration rate (100%) as compared to buck No. 2 (93.00%) and buck No 3 (86.66%).

298 CYCLIC AMP AND CYCLIC GMP CONTENTS IN PORCINE OOCYTE&ndash;CUMULUS COMPLEXES, DENUDED OOCYTES, AND CUMULUS CELLS DERIVED FROM SMALL AND MIDDLE FOLLICLES DURING IN VITRO MATURATION

Drastic changes in intracellular cAMP and cGMP levels play a critical role in the regulation of meiotic resumption. The objective of this study was to compare cAMP and cGMP contents in cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell masses (CC) derived from small (SF) and middle follicles (MF) during in vitro maturation (IVM). The COC were aspirated from SF (1–3 mm in diameter) or MF (3–6 mm in diameter) of prepubertal gilt ovaries. The COC were cultured in modified porcine oocyte medium (mPOM) with eCG, hCG, and dibutyryl cAMP for 20 h and then in fresh mPOM without those supplements for 4 h in an atmosphere of 5% CO2 in air at 39°C. At 0, 10, 20, and 24 h of IVM, COC, DO, and CC were collected. The DO were prepared by removal of cumulus cells and zona pellucida. The CC were prepared by puncturing ooplasm by using 18-gauge needle. Intracellular contents of cAMP and cGMP were determined by direct enzyme immunoassay kits. Statistical analyses of 3 to 7 replicated data were performed by ANOVA. There were no significant differences in contents of cAMP and cGMP between DO from SF and MF in all observation points (P &gt; 0.05). Cyclic AMP contents in COC and CC derived from MF were higher than those from SF at 20 h of IVM (MF 33.0 ± 0.5 fmol/COC v. SF 28.4 ± 1.0 fmol/COC, MF 20.9 ± 0.9 fmol/CC v. SF 14.6 ± 0.8 fmol/CC; P &lt; 0.05), whereas there were no significant differences between origins of those (SF v. MF, P &gt; 0.05) at 0, 10, and 24 h of IVM. Furthermore, although cAMP content in CC from MF was not significantly different between 10 and 20 h of IVM (25.4 ± 1.7 and 20.9 ± 0.9 fmol/CC, respectively; P &gt; 0.05), the content in CC from SF significantly decreased between 10 and 20 h (23.1 ± 1.2 , and 14.6 ± 0.8 fmol/CC, respectively; P &lt; 0.05). At 0 and 10 h of IVM, cGMP contents in COC and CC from MF were significantly higher than those from SF (0 h: 81.8 ± 4.5 fmol/COC from MF v. 41.7 ± 10.6 fmol/COC from SF and 82.7 ± 7.5 fmol/CC from MF v. 10.7 ± 2.7 fmol/CC from SF; 10 h: 64.8 ± 8.4 fmol/COC from MF v. 24.8 ± 8.2 fmol/COC from SF, 49.3 ± 14.9 fmol/CC from MF v. 13.5 ± 4.8 fmol/CC from SF; P &lt; 0.05). However, there were no significant differences in cGMP contents in COC and CC between the origins (MF v. SF) at 20 and 24 h of IVM (P &gt; 0.05). From these results, we conclude that cAMP and cGMP contents in cumulus cells are significantly differences between the origins (MF v. SF) during IVM.

Characterization of pig sperm hyaluronidase and improvement of the digestibility of cumulus cell mass by recombinant pSPAM1 hyaluronidase in an in vitro fertilization assay

Although sperm hyaluronidase is thought to play an important role in mammalian fertilization, the molecular function underlying these steps remains largely unknown. In mouse models, sperm-specific SPAM1 and HYAL5 hyaluronidase are believed to function in both sperm penetration of the cumulus matrix and sperm-ZP binding. However, gene-targeting studies for SPAM1 or HYAL5 show that hyaluronidases are not essential for fertilization, despite the fact that exogenous hyaluronidase can disrupt the cumulus matrix. Therefore, to evaluate whether sperm hyaluronidase is essential for mammalian fertilization, it is necessary to generate HYAL5/SPAM1 double-knockout mice. However, generating double-knockout mice is very difficult because these two genes exist on the same chromosome. Recently, investigators have begun to employ the pig model system to study human disease due to its similarities to human anatomy and physiology. In this study, we confirmed that pig SPAM1 exists as a single copy gene on chromosome 18 and is specifically expressed in the testis. In addition, we expressed recombinant pig SPAM1 in human embryonic kidney 293 cells and showed that these enzymes possess hyaluronidase activity. We also demonstrated that a polyclonal antibody against pig sperm hyaluronidase inhibits sperm–egg interactions in an in vitro fertilization (IVF) assay. Our results suggest that pig SPAM1 may play a critical role in pig fertilization and that recombinant SPAM1 can disperse the oocyte–cumulus complex in an IVF assay.

Coculture with autologous cumulus cell mass in patients with previous IVF failure

This study was to evaluate the effect of autologous cumulus cell mass on embryo coculture in patients with a history of previous IVF failure. Retrospective cohort study between April 2013 and March 2014. A total of two hundred and forty-four patients who underwent GnRH long or antagonist protocol with fresh embryo transfer were analyzed. The cycles with severe male factor and single embryo transfer were excluded. Patients were divided into two groups. Patients who had embryos cultured in 20 ul of defined medium with autologous cumulus cell mass (ACC) (n = 101) and without autologous cumulus cell mass (No ACC) (n = 143). Well dispersed autologous cumulus cell mass directly put into the culture medium without hyaluronidase treatment. We compared the rates of good-quality embryos, implantation, multiple pregnancy, clinical pregnancy, and ongoing pregnancy between ACC and No ACC. There were no differences between ACC and No ACC regarding mean female age (35.3 ± 3.3 vs. 34.5 ± 3.3, p = 0.134), mean number of retrieved oocytes (10.1 ± 6.1 vs. 9.8 ± 6.0, p = 0.736), maturation rate (87.8% vs. 92.4%, p = 0.798), fertilization rate (78.5% vs. 77.3%, p = 0.941), and mean number of transferred embryos (2.5 ± 0.6 vs. 2.4 ± 0.6, p = 0.130). We also observed similar rates of good-quality embryos (8.5% vs. 10.1%, p = 0.323), multiple pregnancy (23.4% vs. 30.4%, p = 0.445), and implantation (23.0% vs. 17.9%, p = 0.123) in ACC and No ACC, respectively. Although an increased mean previous IVF failure in ACC (2.4 ± 1.4 vs. 1.7 ± 1.1, p = 0.000), ACC achieved significantly higher rates of clinical pregnancy (46.5% vs. 32.2%, p = 0.023) and ongoing pregnancy (38.6% vs. 25.9%, p = 0.034) than those of No ACC. These results suggested that coculture with autologous cumulus cell mass could improve the rates of clinical pregnancy and ongoing pregnancy in patients with a history of previous IVF failure.

Evaluation of bovine (Bos indicus) ovarian potential for in vitro embryo production in the Adamawa plateau (Cameroon)

An abattoir study was conducted to evaluate the ovarian potential of 201 local zebu cattle from Ngaoundere, Adamawa region (Cameroon) for in vitro embryo production (IVEP). The ovaries were excised, submerged in normal saline solution (0.9%) and transported to the laboratory for a detailed evaluation. Follicles on each ovary were counted, their diameters (F) measured and were grouped into 3 categories: small (F &lt; 3 mm), medium (3 = F = 8 mm) and large (F &gt; 8 mm). Each ovary was then sliced into a petri dish; the oocytes were recovered in Dulbecco’s phosphate buffered saline, examined under a stereoscope (x10) and graded into four groups based on the morphology of cumulus oophorus cells and cytoplasmic changes of the oocytes. Grade I (GI): oocytes with more than 4 layers of bunch of compact cumulus cells mass with evenly granulated cytoplasm; grade II (GII): oocyte with at least 2–4 layers of compact cumulus cell mass with evenly granulated cytoplasm; grade III (GIII): oocyte with at least one layer of compact cumulus cell mass with evenly granulated cytoplasm; grade IV (GIV): denuded oocyte with no cumulus cells or incomplete layer of cumulus cell or expanded cells and having dark or unevenly granulated cytoplasm. The effects of both ovarian (ovarian localization, corpus luteum, size and weight of ovary) and non-ovarian factors (breed, age, body condition score (BCS) and pregnancy status of cow) on the follicular population and oocyte recovery rate were determined. There were an average of 16.75±0.83 follicles per ovary. The small, medium and large follicles were 8.39±0.60, 8.14±0.43 and 0.21±0.02 respectively. Oocyte recovery was 10.97±0.43 per ovary (65%). Oocytes graded I, II, III and IV were 3.53±0.19 (32.21%), 2.72±0.15 (24.82%), 2.24±0.15 (20.43%) and 2.47±0.20 (22.54%) respectively. The oocyte quality index was 2.26. Younger non pregnant cows having BCS of 3 and large ovaries presented higher number of follicles and oocyte quality (P &lt; 0.05) compared with other animals. Oocytes with quality (grade I and II) acceptable for IVEP constituted 57.15% of the harvest. This study indicated that factors such as age, pregnancy status, BCS and ovarian size must be taken into account to increase the potential of the ovary for IVEP.

186 EFFECT OF POLYVINYLPYRROLIDONE SUPPLEMENTATION IN CULTURE MEDIUM ON THE GROWTH OF MOUSE OOCYTES

A high volume of polyvinylpyrrolidone (PVP) supplementation in culture medium has a significant impact on the growth of bovine oocytes. The objective of the present study was to determine whether or not PVP affects oocyte growth in the mouse. Oocyte–granulosa cell complexes were isolated from 11- or 12-day-old mice (ICR) by mechanical isolation of follicles, followed by a collagenase treatment (0.1%; 10 min). Twenty complexes were placed on each insert fit in the 24-well culture plate and cultured for 10 days in an atmosphere of 5% CO2 in air at 37°C. The culture medium was a modified α-MEM supplemented with 5% fetal bovine serum and 1 ng mL–1 FSH. The concentration of PVP (molecular weight of 360 000) was 0%, 1%, 2%, or 3% (w/v). During the first 2 days, only medium with 0% PVP was used. The oocytes recovered on Day 10 were subjected to in vitro maturation, IVF, and embryo culture. In 12 replications, the total numbers of oocytes cultured in medium with 0%, 1%, 2%, and 3% PVP were 235, 233, 233, and 231, respectively. In some additional experiments, oocytes were fixed on Day 10 and processed for transmission electron microscopy (TEM). The oocytes in medium with 0% PVP became located within an enlarged dome-like structure. In medium with 2% PVP and 3% PVP, no such domes were formed, and the oocytes within several granulosa cell layers were exposed to medium; however, the cumulus cell mass specifically became larger than that in medium with 0% PVP. The viabilities of oocytes recovered from medium with 0%, 1%, 2%, and 3% PVP were 83%, 81%, 91%, and 93%, respectively. The survival rate was significantly higher in medium with 3% PVP than in medium with 0% PVP or 1% PVP (P &lt; 0.05). The mean oocyte diameter increased from 59 µm (Day 0) to 72, 71, 71, and 72 µm in medium with 0, 1, 2, and 3% PVP, respectively, but they continued to be smaller than in vivo grown oocytes (81.0 µm; P &lt; 0.01). When maturation was induced, cumulus cell mucification occurred irrespective of PVP concentration during the growth. No significant differences were found between the groups in the percentage of polar body extrusion (ranging from 78 to 88%). Developmental outcomes based on oocytes used for in vitro fertilization were the following: cleavage rates were 67, 78, 74, and 76%; and blastocyst rates were 37, 44, 47, and 36% of oocytes that had been grown in medium with 0, 1, 2, and 3% PVP, respectively. The numbers of oocytes included were 60, 59, 68, and 66, respectively. The TEM observation suggests that more intimate contacts were maintained between the oocyte and cumulus cells in medium with 2% PVP than in medium with 0% PVP. Taken together, PVP supplementation in medium has a considerable influence on the morphology of mouse oocyte–granulosa cell complexes and close contacts within the complexes in the long-term culture, as having been observed with bovine oocytes.

194 IN VITRO MATURATION AND RNA CONTENT AND DISTRIBUTION OF PORCINE OOCYTES DERIVED FROM SMALL AND MEDIUM FOLLICLES AND CLASSIFIED BY BRILLIANT CRESYL BLUE ASSAY

Oocytes collected from the surface of slaughterhouse ovaries clearly have heterogeneous quality. The objective was to characterize a change or difference in RNA distribution and the content of porcine oocytes that were collected from small (SF; 1–2 mm in diameter) and medium follicles (MF; 3–6 mm in diameter) and assessed for glucose-6-phosphate dehydrogenase (G6PD) activity by brilliant cresyl blue (BCB) assay. Following BCB staining, porcine cumulus–oocyte complexes (COC) with dark blue (DB; G6PD-inactive, suggesting good quality) and light blue (LB; G6PD-active, suggesting immaturity) ooplasm were then separately cultured in vitro to evaluate nuclear maturation and analyze the characteristics of the RNA aspect. RNA distributions in cumulus cell mass and ooplasm were labeled with fluorescence, SYTO RNA select green and then examined at different periods of culture for in vitro maturation (IVM; with gonadotropins and dbcAMP for 20 h and then without those for 24 h). Total RNA content of oocytes and cumulus cell mass were measured by using a manufacture's kit (Invitrogen Quant-iT RNA assay kit). Statistical analyses of results from 3 to 5 replicated trials were performed by ANOVA with a Bonferroni/Dunn post-hoc test (significance, P &lt; 0.05). When oocytes were classified by BCB assay, the percentage of oocytes with DB cytoplasm was much higher (P &lt; 0.05) in oocytes from MF (72.5% of 208) compared with those of SF (53.6% of 352). Regardless of the origin of oocytes (SF vs MF), the incidence of mature oocytes following culture was higher in DB than LB (64.0% of 147 and 72.1% of 133 vs 50.9% of 108 and 52.8% of 54, respectively). Twenty hours after the start of IVM, the DB oocytes had a higher proportion of RNA-free zone inside the germinal vesicle compared to the LB oocytes (66.1% of 193 and 26.8% of 159 in MF; 47.9% of 185 and 17.9% of 184 in SF; P &lt; 0.05). When the total content of RNA was examined during IVM, both oocytes from SF and MF contained higher levels of total RNA at the beginning of IVM as compared with 20 and 44 h after the start of IVM, whereas the content (40 denuded oocytes/sample) was higher in oocytes with DB cytoplasm from MF than those from SF (P &lt; 0.05). Regardless of the origin of oocytes from SF and MF, the total content of RNA in oocytes with LB cytoplasm was significantly lower than in oocytes with DB cytoplasm (P &lt; 0.05) before the start of IVM. The total RNA content of cumulus cells (40 COC/sample) before IVM culture was also higher in the cell mass surrounding the DB oocytes than the LB ones and in the cell mass surrounding the oocytes from MF rather than SF (P &lt; 0.05). These results demonstrate a possibility that differences in RNA distribution and content in oocytes, as well as the content in cumulus cells, reflect in the ability of porcine oocytes to mature in vitro.

Improvement in embryo quality and pregnancy rates by using autologous cumulus body during icsi cycles

To determine whether the addition of intact cumulus cell mass (ICM) to both embryo culture (EC) and embryo transfer (ET) improves embryo quality and pregnancy rates. A total of 133 infertile couples were included, of which 67 received ICM (study group) and 66 did not (control group). The ICM was obtained from a simple cutting of the cumulus corona oocyte complex (CCOC). A case control study design was used. The clinical characteristics of the two groups before the embryo culturing step were similar with respect to age, estradiol level on the day of hCG and endometrial thickness on the day of embryo transfer (p>0.05). On the other hand study group with ICM had higher number of high quality embryos (3.1±1.4 vs 2.4±1.1, p=0.03), higher implantation rate (53.7% vs 34.8%, p=0.02) and higher ultrasound confirmation of gestational sac and fetal heart beat as ongoing pregnancy rates (44.7% vs 27.2%, p=0.04) compared to the control group without ICM. Addition of ICM improves embryo quality and pregnancy rates. This is a cost-and time-effective simple procedure that shows great promise for the improvement of infertility treatment.