Synergistic impact of α-linolenic acid and α-tocopherol on in vitro maturation and culture of buffalo oocytes

The objective of the current study was to investigate the synergistic impact of α-Tocopherol and α-Linolenic acid (100 µM) on IVM and IVC of Nili Ravi buffalo oocytes. Oocytes were obtained from the ovaries of slaughtered buffaloes within two hours after slaughter and brought to laboratory. Buffalo cumulus oocyte complexes were placed randomly in the five experimental groups included; GROUP 1: Maturation media (MM) + 100 µM ALA (control), GROUP 2: MM + 100 µM ALA + 50μM α-Tocopherol, GROUP 3: MM + 100 µM ALA + 100μM α-Tocopherol, GROUP 4: MM + 100 µM ALA + 200 μM α-Tocopherol and GROUP 5: MM + 100 µM ALA + 300 μM α-Tocopherol under an atmosphere of 5% CO2 in air at 38.5 °C for 22-24 h. Cumulus expansion and nuclear maturation status was determined (Experiment 1). In experiment 2, oocytes were matured as in experiment 1. The matured oocytes were then fertilized in Tyrode’s Albumin Lactate Pyruvate (TALP) medium for about 20 h and cultured in synthetic oviductal fluid (SOF) medium to determine effect of α-Linolenic acid (100 µM) and α-Tocopherol in IVM medium on IVC of presumptive zygotes. To study the effect of α-Linolenic acid (100 µM) in IVM media and increasing concentration of α-tocopherol in the culture media on early embryo development (Experiment 3), the presumptive zygotes were randomly distributed into the five experimental groups with increasing concentration of α-tocopherol in culture media. Higher percentage of MII stage oocytes in experiment 1(65.2±2.0), embryos at morula stage in experiment 2 (30.4±1.5) and experiment 3 (22.2±2.0) were obtained. However, overall results for cumulus cell expansion, maturation of oocyte to MII stage and subsequent embryo development among treatments remain statistically similar (P > 0.05). Supplementation of α-tocopherol in maturation media having α-Linolenic acid and/or in embryo culture media did not further enhance in vitro maturation of oocyte or embryo production.

In vitro maturation of goat oocytes selected using Brilliant cresyl blue staining

The present study was conducted to assess the developmental competence of goat oocytes selected using Brilliant cresyl blue (BCB) staining. Goat ovaries were collected from the slaughtered animals with unknown reproductive history. The oocytes retrieved by aspiration technique were selected based on morphology and subjected to BCB staining. Brilliant cresyl blue staining is based on the activity of glucose-6-phospahte dehydrogenase (G6PDH) enzyme synthesised by the oocytes. The cytoplasm remains blue in oocytes that have finished the growth phase (BCB+) while the growing oocytes remain colourless (BCB-). The stained and unstained oocytes were subjected to in vitro maturation separately to assess cumulus cell expansion index and polar body extrusion. A total of 206 culture grade oocytes were subjected to study, out of which, 76.75 ± 2.38 per cent of oocytes showed positive to BCB staining and 23.21 ± 2.38 per cent were negatively stained. Significantly higher maturation rate was observed in BCB+(92.89 ± 2.37%) oocytes than BCB-(29.72 ± 2.46%). The present study concluded that BCB staining can be used for selecting goat oocytes with good cytoplasmic maturation for further in vitro embryo production

Cyclic adenosine monophosphate modulator supplementation on in vitro maturation of bovine oocytes

Study evaluated the role of cAMP modulator (Forskolin and 3-isobutyl-1- methyl xanthine) supplementation on developmental competence of bovine oocytes. Cumulus oocyte complexes recovered from bovine ovaries of unknown reproductive status were used for the study. Oocytes retrieved by aspiration method were graded based on cumulus cell distribution and culture grade oocytes were selected for the study. A total of 414 culture grade oocytes were taken and divided into two groups. Group I constituted of 201 oocytes in which pre-maturation was carried out for a period of 2 h. In group II, 213 oocytes were selected in which normal maturation was carried out. Maturation was assessed after 24h of culture in CO2 incubator maintained at 38.5o C in 95 per cent humidified atmosphere of 5 per cent CO2 . Fertilisation was carried out using frozen thawed semen and the presumptive zygotes were then transferred to culture media and cleavage was assessed 48 h after insemination. A significantly higher maturation rate (p ?0.05) was observed in group I compared to group II (86.85 ± 1.19 vs 79.88 ± 2.67). There was a highly significant increase (p ?0.01) in cleavage rate in group I (65.92 ± 1.23) compared to group II (59.29 ± 1.50). A higher fertilisation rate was observed in group I (75.35 ± 1.19) than group II (71.88 ± 2.56). It could be concluded that pre-maturation with cAMP modulators improved the developmental competence of bovine oocytes.

Cumulus Cell DNA Damage as an Index of Human Oocyte Competence

The determination of oocyte quality is crucial for achieving effective syngamy post-sperm injection and embryonic development. Cumulus cells (CCs) have been proposed as biomarkers of oocyte quality because of their close bio-dynamic relationship with the oocyte. To determine the quality of the oocyte, CCs were sampled during oocyte preparation for ICSI to determine a CC DNA fragmentation index (CCDFI) of each individual oocyte using a variant of the chromatin dispersion test. One hundred and thirty oocytes were selected and studied from two Spanish fertility clinics, 90 of which were fertilized and developed to embryos. Significant differences were found between the CCDFI of unfertilized and fertilized oocytes (p < .001) and between the CCDFI of embryos that were discarded and those that developed suitable for transfer or cryopreservation (p < .001). Oocyte quality was negatively correlated with CCDFI (Spearman’s rho = − 0.45; p < .001). Receiver operator characteristics curves (ROC) suggested that a cut-off value of 24% CCDFI was able to discriminate the capacity of the gametes to result in syngamy with a sensitivity and specificity of 75.6% and 65%, respectively. This cut-off supports the application of CCDFI as potential index for the evaluation of the reproductive potential of oocytes prior to fertilization.

Effects of BMP15 and GDF9 on Ovine Oocytes in an In Vitro Maturation System

<p>Oocyte developmental competency is the intrinsic measure of oocyte quality and the capacity for a mature oocyte to support the early stages of embryo development and implantation. Oocyte-secreted factors (OSFs), such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), play a pivotal role in regulating the synchrony of various complex maturation events within the cumulus-oocyte complex (COC) through the induction of paracrine and endocrine signalling. These proteins act synergistically to influence the proliferation and differentiation of granulosa cells (GCs), cumulus cell (CC) expansion, promote survival, ovulation, the attainment of developmental competency and fertility. Species-specific ratios suggest that poly-ovulatory mammals have increased fecundity due to high ratios of GDF9:BMP15, which is directly reflected in their large litter size. Interestingly, it has also been found that higher ratios of GDF9:BMP15 also increased blastocyst rate in sheep implying that these embryos develop from oocytes that are more developmentally competent. In this study, I investigated the hypothesis that supplementing a commercial in vitro maturation (IVM) system with a high ratio of GDF9:BMP15 would increase the developmental competency sheep oocytes; a species with low-moderate litter size. To test this hypothesis, ovine oocytes were matured in a biphasic IVM system containing GDF9 and BMP15 at three divergent ratios (1:6, 1:1, 6:1). The results herein show that the 6:1 ratio resulted in higher levels of reagent transfer to the ovine oocyte through gap junctions (GJs) after 24 hours of incubation. Similarly, it was also observed that at the higher ratio, glutathione (GSH) levels were higher at 7.5 hours of incubation. The high GDF9:BMP15 ratio also facilitated the increased consumption of pyruvate by the COC consistently throughout the culture period. Importantly, the high GDF9:BMP15 ratio showed higher expression of the gene that encodes GJ (CX43) at 24 hours relative to the control. It was also demonstrated through decreased apoptotic factor (BAX:BCL2) ratios, that the addition of OSFs, regardless of ratio, protected against cell death. In summary, this study provides novel results that support the notion that a high GDF9:BMP15 ratio improves oocyte quality by delaying the timing of meiotic resumption. This subsequently improves the transport of key metabolites and antioxidants to protect against oxidative stress and cell death and aid in the completion of maturation, ultimately resulting in the increased developmental competency observed in high fecundity poly-ovulatory species.</p>

Effects of BMP15 and GDF9 on Ovine Oocytes in an In Vitro Maturation System

<p>Oocyte developmental competency is the intrinsic measure of oocyte quality and the capacity for a mature oocyte to support the early stages of embryo development and implantation. Oocyte-secreted factors (OSFs), such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), play a pivotal role in regulating the synchrony of various complex maturation events within the cumulus-oocyte complex (COC) through the induction of paracrine and endocrine signalling. These proteins act synergistically to influence the proliferation and differentiation of granulosa cells (GCs), cumulus cell (CC) expansion, promote survival, ovulation, the attainment of developmental competency and fertility. Species-specific ratios suggest that poly-ovulatory mammals have increased fecundity due to high ratios of GDF9:BMP15, which is directly reflected in their large litter size. Interestingly, it has also been found that higher ratios of GDF9:BMP15 also increased blastocyst rate in sheep implying that these embryos develop from oocytes that are more developmentally competent. In this study, I investigated the hypothesis that supplementing a commercial in vitro maturation (IVM) system with a high ratio of GDF9:BMP15 would increase the developmental competency sheep oocytes; a species with low-moderate litter size. To test this hypothesis, ovine oocytes were matured in a biphasic IVM system containing GDF9 and BMP15 at three divergent ratios (1:6, 1:1, 6:1). The results herein show that the 6:1 ratio resulted in higher levels of reagent transfer to the ovine oocyte through gap junctions (GJs) after 24 hours of incubation. Similarly, it was also observed that at the higher ratio, glutathione (GSH) levels were higher at 7.5 hours of incubation. The high GDF9:BMP15 ratio also facilitated the increased consumption of pyruvate by the COC consistently throughout the culture period. Importantly, the high GDF9:BMP15 ratio showed higher expression of the gene that encodes GJ (CX43) at 24 hours relative to the control. It was also demonstrated through decreased apoptotic factor (BAX:BCL2) ratios, that the addition of OSFs, regardless of ratio, protected against cell death. In summary, this study provides novel results that support the notion that a high GDF9:BMP15 ratio improves oocyte quality by delaying the timing of meiotic resumption. This subsequently improves the transport of key metabolites and antioxidants to protect against oxidative stress and cell death and aid in the completion of maturation, ultimately resulting in the increased developmental competency observed in high fecundity poly-ovulatory species.</p>

Comparison of embryonic competence and clinical outcomes between early and late cumulus cell removal for in vitro fertilization

Objective: The impact of early mechanical removal of cumulus cells on fertilization and embryonic development is not yet precisely known. This study aimed to investigate the effects of early and late cumulus cell removal on fertilization, polyspermy, embryonic development potential, blastocyst development, and clinical outcomes.Methods: A prospective study was conducted of patients who underwent in vitro fertilization between September 2019 and October 2020. Sibling oocytes were randomly allocated after insemination to early cumulus cell removal at 6 hours (group I) and late cumulus cell removal at 16–18 hours (group II). If total fertilization failure (TFF) was determined to have occurred at early cumulus cell removal, rescue intracytoplasmic sperm injection (ICSI) was performed. Fertilization, embryonic development, and pregnancy outcomes were compared.Results: A total of 912 oocytes were assigned to group I (458 oocytes) and group II (454 oocytes). Fertilization, polyspermy, embryo quality, and pregnancy outcomes were not significantly different between both groups. Rescue ICSI enabled fertilization of 79.2% of the TFF oocytes.Conclusion: Early cumulus cell removal at 6 hours had no significant difference in fertilization, polyspermy, embryo development, or obstetric and perinatal outcomes compared to late removal. Early cumulus cell removal combined with early rescue ICSI may have the potential to help couples with TFF.

The role of follicular-secreted factors in the maintenance of meiotic arrest in rats

<p>Meiosis is the process by which diploid germ cells develop into competent haploid gametes. In female mammals, meiosis is characterised by two periods of arrest, the duration of which is species-specific. This study investigated the first period of meiotic arrest which occurs at the diplotene stage of prophase I. This period of arrest has important implications for artificial reproductive technologies as the maintenance of meiotic arrest in the in vitro situation has been correlated with improved embryological outcomes. Despite there being extensive evidence that the somatic cells of the follicle (granulosa and cumulus cells) produce meiosis-inhibiting factors, the factors themselves and the mechanisms through which they act are unclear. Recent evidence implicates C-type natriuretic peptide (CNP) and oestradiol in the regulation of meiotic arrest in mouse oocytes. In this proposed hypothesis, CNP is produced by the granulosa cells and activates its cognate receptor, NPR2, on cumulus cells. This results in the production of cyclic guanosine monophosphate (cGMP) in cumulus cells which is transferred to the oocyte via gap junctions. In the oocyte, cGMP slows the rate of hydrolysis of cyclic adenosine monophosphate (cAMP) by phosphodiesterase 3A resulting in elevated intra-oocyte cAMP levels. By maintaining high levels of cAMP in the oocyte, maturation-promoting factor (MPF) activity is inhibited, preventing re-entry into the cell cycle, thus maintaining meiotic arrest. The overall objective of this study was to investigate the validity of this aforementioned hypothesised regulatory pathway in another mammalian species, the rat. Four fundamental components of this pathway were chosen to be investigated and these framed the four aims of this study. The aims of this study were to investigate in cultured rat cumulus cell-oocyte complexes (COCs) the short and long-term effects of CNP and oestradiol, both alone and in combination on (1) gap junction permeability using a validated gap junction assay, (2) intracellular cGMP levels using a direct competitive immunoassay, (3) mRNA expression levels of key cumulus cell-derived genes (Npr2, the receptor for CNP; and Pde4b and Pde4d, phosphodiesterases) using an optimised multiplex TaqMan qPCR reaction, and (4) duration of meiotic arrest. Overall, the results of this study indicated that the assessed treatments did not alter gap junction permeability in rat COCs in vitro. Whilst treatment with CNP and oestradiol appeared to increase the intracellular levels of cGMP in COCs, this requires further investigation. Notably, this study confirmed the role of steroid hormones in upregulating Npr2 expression. Indirect evidence suggests that PDE4D in particular, is a major regulator of cyclic nucleotide levels in the cumulus cells. Finally, treatment of rat COCs with CNP and oestradiol increased the duration of meiotic arrest in oocytes incubated in vitro. The results of this study provide the first evidence that the hypothesised regulatory pathway proposed above is also relevant in the rat. Nonetheless, further investigation of the effects of CNP and oestradiol on the modulation of intracellular cGMP levels are required to fully validate the model.</p>