Germinal Vesicle Oocytes Research Articles

Oxidative damage in naturally aged mouse oocytes is exacerbated by dysregulation of proteasomal activity

An increase in oxidative protein damage is a leading contributor to the age-associated decline in oocyte quality. By removing such damaged proteins, the proteasome plays an essential role in maintaining the fidelity of oocyte meiosis. In this study, we established that decreased proteasome activity in naturally aged, germinal vesicle (GV) mouse oocytes positively correlates with increased protein modification by the lipid aldehyde 4-hydroxynonenal (4-HNE). Furthermore, attenuation of proteasome activity in GV oocytes of young animals was accompanied by an increase in 4-HNE-modified proteins, including α-tubulin, thereby contributing to a reduction in tubulin polymerization, microtubule stability, and integrity of oocyte meiosis. A decrease in proteasome activity was also recapitulated in the GV oocytes of young animals following exposure to oxidative insults in the form of either hydrogen peroxide (H2O2) or 4-HNE. We also observed that upon oxidative insult, 4-HNE exhibits elevated adduction to multiple proteasomal subunits. Notably, the inclusion of the antioxidant penicillamine, to limit propagation of oxidative stress cascades, led to a complete recovery of proteasome activity and enhanced clearance of 4-HNE-adducted α-tubulin during a 6-h post-treatment recovery period. This strategy also proved effective in reducing the incidence of oxidative stress-induced aneuploidy following in vitro oocyte maturation, but was ineffective for naturally aged oocytes. Taken together, our results implicate proteasome dysfunction as an important factor in the accumulation of oxidatively induced protein damage in the female germline. This discovery holds promise for the design of therapeutic interventions to address the age-dependent decline in oocyte quality.

Abnormal expressions of ADAMTS-1, ADAMTS-9 and progesterone receptors are associated with lower oocyte maturation in women with polycystic ovary syndrome.

ADAMTS-1 and 9 play a crucial role in the ovulation and their altered levels may play a role in the pathogenesis of polycystic ovary syndrome (PCOS). The aim of this study was to assess ADAMTS-1 and 9 expression and their correlation with the oocyte quality and maturity in the cumulus cells (CCs) of PCOS patients and normovulatory women during an IVF procedure. Expression of ADAMTS-1 and 9 and progesterone receptors (PRs) in the CCs containing MII and germinal vesicle (GV) oocytes of 37 PCOS patients and 37 women with normal ovulatory function who underwent IVF treatment was evaluated using qRT-PCR. Moreover, correlation between ADAMTS-1 and 9 expression and oocyte quality were also investigated. mRNA expression levels of ADAMTS-1 and ADAMTS-9 were significantly reduced in the women with PCOS compared to the normovulatory women. ADAMTS-1 and ADAMTS-9 mRNA expression levels in the CCs showed a considerable correlation. Lower expression levels of ADAMTS-1 and ADAMTS-9 in PCOS patients were strongly correlated with diminished oocyte maturation. There was a remarkable association between ADAMTS-1 and ADAMTS-9 mRNA expression levels and oocyte quality. PRs (PRA and PRB) were dramatically decreased in PCOS patients when compared with the control group. The results of the present study indicated that ADAMTS-1 and ADAMTS-9 as well as PRs are downregulated in the human CCs in PCOS patients, which could be associated with impaired oocyte maturation and may result in a lower oocyte recovery and oocyte maturity rates, as well as lower fertilization rate.

Variable IGF1R mRNA Expression in Individual GV and In Vitro Matured M2 Human Oocytes

Objective Characterization of human insulin-like growth factor 1 receptor (IGF1R) mRNA expression in individual human oocytes to better understand the role of biologically significant mRNAs in establishing oocyte quality following ovarian hyperstimulation for in vitro fertilization (IVF) treatment. Design Germinal vesicle oocytes (GV) were identified after removal of all cumulus cells prior to ICSI and collected from consenting patients with IRB-approval. Materials and Methods Single GVs were placed directly into lysis buffer (5μl). Oocytes in vitro matured (IVM) to the M2 stage were placed into individual tubes containing lysis buffer after 24 to 48 hours (h) in culture. Total RNA isolated from cumulus granulosa cells (GC) using the TRIzol reagent (Invitrogen) as stated in the manufacturer’s protocol served as a positive control. Reverse Transcription (RT) was performed using anchored oligo-dT primers (IDT, Coralville, IA) and Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen) on the entire 5μl oocyte lysate at 37°C for 1.5h or 1mg total GC RNA 42°C for 1h. Quantitative polymerase chain reaction (qPCR) was performed combining standards or 1μl cDNA with PCR buffer containing SYBR Green I (Sigma Chemical Co, St. Louis, Missouri), Taq polymerase (Genscript, Piscataway, NJ), and primers for IGF1R. Melting curves were routinely determined to ascertain generation of only the expected product. Results qPCR performed on GC cDNA demonstrated detection of the expected product under the reaction conditions in use. Gel electrophoresis of the PCR product confirmed the size of the DNA fragment generated. qPCR analysis detected a signal for IGF1R mRNA in 9 of the 15 GVs analyzed from 9 patients. Moreover, for 3 patients with multiple GVs, differences in IGF1R rnRNA expression were seen between individual GV oocytes, as well as between oocytes from different patients. Similarly, 8 of 12 IVM M2 oocytes from 11 patients gave a signal for the presence of IGF1R mRNA. Conclusions These results report the use of a direct-lysis method for the extraction and analysis of mRNA from single human oocytes. These data suggest that GV oocytes failing to respond to the hCG trigger during ovarian stimulation for IVF vary in their biological integrity. Furthermore, we observed that GV oocytes advanced to the M2 stage in vitro in the absence of any demonstrable IGF1R mRNA expression. Molecular assessment of single human oocytes can be used to better understand the low per-oocyte success rate in human IVF. Disclosures Nothing to disclose. Funding None.

Comparison of different in vitro differentiation conditions for murine female germline stem cells.

ObjectivesIn vitro differentiation of oocytes from female germline stem cells (FGSCs) has exciting potential applications for reproductive medicine. Some researchers have attempted to reveal the in vitro differentiation capacity of FGSCs. However, no systematic comparative study of in vitro differentiation conditions has been performed for murine FGSCs (mFGSCs).Materials and MethodsmFGSCs line was cultured under five different conditions for in vitro differentiation. RT‐PCR was performed to detect the expression of Oct4, Fragilis, Blimp1, Mvh, Scp3 and Zp3. Immunofluorescence was carried out to test the expression of Mvh, Fragilis and Zp3. Two‐photon laser‐scanning microscope was used to analyze nucleus‐plasma ratio, and the proportion of chromatin of GV oocytes differentiated from mFGSCs in vitro (IVD‐GVO), GV oocytes from in vivo (GVO) and mFGSCs.ResultsRT‐PCR and immunofluorescence showed that mFGSC line expressed germ cell‐specific markers, but not a meiosis‐specific marker. By evaluating five different in vitro differentiation conditions, condition 5, which included a hanging drop procedure and co‐culture of mFGSCs with granulosa cells, was shown to be optimal. mFGSCs could be successfully differentiated into germinal vesicle (GV) ‐stage oocytes under this condition. 3D observation revealed that both the nucleus‐plasma ratio and proportion of chromatin were not significantly different between IVD‐GVO and GVO.ConclusionWe evaluated five in vitro differentiation conditions for mFGSCs and successfully differentiate mFGSCs into GV‐stage oocytes using a three‐step differentiation process.

Embryonic poly(A)-binding proteinis differently expressed and interacts with the messenger RNAs in the mouse oocytes and early embryos.

The embryonic poly(A)-binding protein (EPAB) functions in the translational regulation of the maternal messenger RNAs (mRNAs) required during oocyte maturation, fertilization, and early embryo development. Since there is no antibody specific to mammalian EPAB protein, all studies related to the Epab gene could be performed at the mRNA levels except for the investigations in the Xenopus. In this study, we have produced an EPAB-specific antibody. When we examined its expressional distribution in the mouse gonadal and somatic tissues, the EPAB protein was found to be expressed only in the mouse ovary and testis tissues, but it is undetectable level in the somatic tissues including stomach, liver, heart, small intestine, and kidney. Additionally, the spatial and temporal expression patterns of the EPAB and poly(A)-binding protein cytoplasmic 1 (PABPC1) proteins were analyzed in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, one-cell, and two-cell embryos. While EPAB expression gradually decreased from GV oocytes to two-cell embryos, the PABPC1 protein level progressively increased from GV oocytes to one-cell embryos and remarkably declined in the two-cell embryos ( P < 0.05). We have also described herein that the EPAB protein interacted with Epab, Pabpc1, Ccnb1, Gdf9, and Bmp15 mRNAs dependent upon the developmental stages of the mouse oocytes and early embryos. As a result, we have first produced an EPAB-specific antibody and characterized its expression patterns and interacting mRNAs in the mouse oocytes and early embryos. The findings suggest that EPAB in cooperation with PABPC1 implicate in the translational control of maternal mRNAs during oogenesis and early embryo development.

Do immature and mature sibling oocytes recovered from stimulated cycles have the same reproductive potential?

How can laboratory and clinical outcomes of spontaneously, early maturing germinal-vesicle oocytes and sibling in-vivo-matured (metaphase II [MII]) oocytes be quantified and compared? A prospective, non-randomized intra-cohort study of oocytes from women aged 38 years or younger, with six or fewer MII oocytes and four or more germinal vesicles retrieved. No indication was identified for genetic tests or oocyte or embryo cryopreservation. The study was carried out at IVIRMA-Valencia. Early maturing germinal vesicles were selected for reproductive purposes. In vitro- and in-vivo MII oocytes were fertilized. After time-lapse culture, hatching blastocysts from germinal vesicles were biopsied for aneuploidy screening and vitrified. Laboratory and clinical outcomes were compared according to oocyte origin. Almost 70% of germinal vesicles had matured early and spontaneously, and had comparable in vitro-outcomes and morphokinetics to sibling in vivo-matured oocytes. Fifty per cent of biopsied blastocysts were euploid. Germinal-vesicle rescue increased the number of MII oocytes per cycle to 3.9, finally adding one extra-blastocyst per cycle. A live birth confirmed the feasibility of this approach. Further data, however, are needed to quantify its real contribution to standard intracytoplasmic sperm injection cycles. Nevertheless, 40% of patients obtained either an immediate advantage (reduction of cancellation rate) or long-term benefit (availability of extra blastocysts of attempts). Germinal-vesicle rescue can be considered as a complementary approach when folliculometry (expected) and number of MII (observed) are unequal.

Does rescue in vitro maturation of germinal vesicle stage oocytes impair embryo morphokinetics development?

SummaryCurrently, rescue in vitro maturation (IVM) is not a routine method in assisted reproductive treatment (ART) programmes but is a promising procedure for ART to improve IVM. The aim of this study was to compare embryo morphokinetics of germinal vesicles (GV) with metaphase II (MII) oocytes from controlled ovarian hyperstimulation (COH) cycles by time-lapse photography monitoring (TLM). Morphokinetics of the same number of embryos derived from the in vivo (group I) and rescue of in vitro matured oocytes (group II) from 310 patients were analyzed and compared retrospectively. The time to form second PB extrusion (tPB2), time of pronuclei appearance (tPNa), time of pronuclei fading (tPNf) and time of two to eight discrete cells (t2-t8) were assessed. Abnormal cleavage patterns such as uneven blastomeres at the two-cell stage, cell fusion (Fu), trichotomous mitoses (TM), and the rates of embryo arrest were assessed. These data showed that tPB2, tPNa, tPNf, t2, t3 and t4 stages took place later in group II compared with group I (P<0.001, P=0.017, P<0.001, P<0.001, P<0.001, P<0.001, respectively). The rates of uneven blastomeres, Fu, TM, and embryo arrest were increased significantly in group II compared with group I (P=0.001, P<0.001, P=0.003, P<0.001, respectively). Based on the exact annotation of timing parameters and cleavage patterns, the present data agreed with the concept that rescue IVM of oocytes negatively influences embryo morphokinetics. Therefore, cautious use of embryos derived from rescue IVM of GV oocytes should be made.

Separation and Loss of Centrioles From Primordidal Germ Cells To Mature Oocytes In The Mouse

Oocytes, including from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM) and a rise in single CETN2 pairs is identified, mostly at meiotic metaphase-I and -II spindle poles. Partial CETN2 foci dissolution occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, later than previously thought.

Age-related gene expression profiles of immature human oocytes.

What is the difference between the gene expression profiles of single human germinal vesicle (GV) oocytes from women of different ages? There were no statistically significant differences in gene expression profiles of human GV oocytes from women of different ages (range: 25-43). It is well established that reproductive capacity declines as women age, which is attributed to oocyte quality since this decline is counterbalanced in older women receiving young donor oocytes. Altered gene expression of human oocytes at different stages of development in relation to female age is one of the suggested mechanisms that could explain the decrease in oocyte quality. Between 2012 and 2014, 40 human GV oocytes of 40 women were obtained during follicular aspiration as part of routine ICSI treatment. Gene expression profiles of 38 GV oocytes were determined in four different age groups: 25-30, 31-35, 36-38 and 39-43 years of age. GV oocytes were donated for research and frozen between 3.5 and 7.5 h after follicular aspiration. Subsequently, GV oocytes were thawed and prepared for gene expression profile analysis using Agilent microarrays containing ~42 000 Human Gene Expression probe-sets. Gene expression profiles were visualized by hierarchical clustering and the top 500 most differing genes were determined by multidimensional scaling (MDS). Transcripts were analysed in a class comparison between the four age groups and for indicators of biological age: antral follicle count (AFC) and the total dosage of FSH needed for ovarian stimulation. Individual transcripts were analysed using linear regression. A false discovery rate <0.05 was considered statistically significant. Visualization of gene expression profiles of GV oocytes with hierarchal clustering and MDS demonstrated no clear grouping of samples based on female age, AFC or FSH dosage. The gene expression profile of GV oocytes classified in four age groups revealed no significantly differentially expressed genes between the four different age groups. There were also no significantly differentially expressed genes in the linear regression analysis for individual transcripts against age. Not applicable. Immature (GV) oocytes obtained from ovarian stimulation cycles were used. Findings may therefore differ for oocytes at other developmental stages and for in-vivo matured oocytes under physiological conditions. Due to our relatively large, but still limited study sample (40 GV oocytes), we cannot exclude that there might be smaller age-related gene-expression differences, i.e. due to a lack of power. We did not find an effect of female age on gene expression profiles of individual human GV oocytes. Other studies have suggested that gene-expression profiles are affected in mature oocytes, which might imply that female age affects oocyte maturation. Alternatively, other mechanisms in human oocytes might cause the age-related fertility decline. This study received no external funding and there are no competing interests.

SRSF3 maintains transcriptome integrity in oocytes by regulation of alternative splicing and transposable elements

The RNA-binding protein SRSF3 (also known as SRp20) has critical roles in the regulation of pre-mRNA splicing. Zygotic knockout of Srsf3 results in embryo arrest at the blastocyst stage. However, SRSF3 is also present in oocytes, suggesting that it might be critical as a maternally inherited factor. Here we identify SRSF3 as an essential regulator of alternative splicing and of transposable elements to maintain transcriptome integrity in mouse oocyte. Using 3D time-lapse confocal live imaging, we show that conditional deletion of Srsf3 in fully grown germinal vesicle oocytes substantially compromises the capacity of germinal vesicle breakdown (GVBD), and consequently entry into meiosis. By combining single cell RNA-seq, and oocyte micromanipulation with steric blocking antisense oligonucleotides and RNAse-H inducing gapmers, we found that the GVBD defect in mutant oocytes is due to both aberrant alternative splicing and derepression of B2 SINE transposable elements. Together, our study highlights how control of transcriptional identity of the maternal transcriptome by the RNA-binding protein SRSF3 is essential to the development of fertilized-competent oocytes.

Primary Culture of Human Cumulus Cells Requires Stearoyl-Coenzyme A Desaturase 1 Activity for Steroidogenesis and Enhancing Oocyte In Vitro Maturation

Stearoyl-coenzyme A desaturase 1 (SCD1) is a key enzyme in lipid metabolism and is expressed in cumulus cells. The objective of the present study was to evaluate the effect of SCD1 inhibition in human cumulus cells on triglyceride content, steroidogenesis, and oocyte in vitro maturation. Human cumulus cells were exposed to SCD1 inhibitor CAY10566 (SCDinhib) alone or in combination with oleic acid in primary culture. The SCDinhib markedly suppressed triglyceride accumulation (-47%, P = .01), aromatase gene expression (-36%, P = .02), and estradiol production (-49%, P = .01) even at a dose not affecting cell viability and apoptosis. Human immature oocytes at the germinal vesicle (GV) stage were cocultured with pretreated cumulus cells. The rate of oocytes reaching the metaphase II stage was significantly lower in coculture with SCDinhib-treated cumulus cells than with control cumulus cells (-18%, P < .01), which recovered by oleic acid supplementation. This finding on in vitro maturation rate was also reproducible with mouse GV oocytes. The results suggest that SCD1 activity is required for cumulus cell lipid storage and steroidogenesis. In addition, oocyte maturation is negatively affected by SCD1 inhibition in cumulus cells, possibly due to a deficient lipid-mediated paracrine support.

Comparison of RNA extraction and microRNA detection protocols for a small amount of germinal vesicle oocytes in bovine

RT-qPCR is a widely used method to detect miRNA expression. Compared with mRNA, miRNA has a shorter length and lower abundance which hinders the acquisition of reliable results. Thus, miRNA detection requires a method with high sensitivity and accuracy. Collecting large amounts of material is particularly difficult for oocytes and pre-implantation embryos of domestic animals. Establishing a set of miRNA detection methods that are suitable to detect trace amounts of such materials is urgently needed. In this study, the total RNA in 50 germinal vesicle (GV) oocytes was isolated through direct lysis and by using mirVana miRNA Isolation Kit and miRNeasy Micro Kit. The OD260/280 values and concentrations of the RNA in these three groups were compared to identify a superior RNA isolation method. In addition, the specificity and sensitivity of common DNA and LNA primers were compared by real-time quantitative polymerase chain reaction for miRNA detection. Results show that the RNA concentration of in the direct lysis group was significantly higher than that in the other two groups. The specificity between the DNA primers and LNA primers was identical, whereas the sensitivity of LNA primers was superior to that of DNA primers. These results suggest that direct lysis combined with LNA primers might be a suitable protocol for the miRNA detection of a small amounts of GV oocytes and pre-implantation embryos in cattle.

Novel mutations in PATL2 cause female infertility with oocyte germinal vesicle arrest.

Do PATL2 mutations account for female infertility with oocyte germinal vesicle (GV) arrest? Four of nine independent families with oocyte GV arrest were identified with biallelic PATL2 mutations, suggesting that these mutations may be responsible for oocyte maturation arrest in primary infertile women. Recently, two independent studies have demonstrated that infertility in some women with oocyte maturation arrest at the GV stage was caused by biallelic mutations in PATL2. PATL2 encodes protein PAT1 homolog 2, an RNA-binding protein that may act as a translational repressor. In this study, nine unrelated primary infertile females presenting with oocyte GV arrest were recruited during the treatment of early rescue ICSI or ICSI from January 2013 to December 2016. Genomic DNA was isolated from blood samples obtained from all nine affected individuals and all of their available family members. All the coding regions of PATL2 were sequenced by Sanger sequencing. The pathogenicity of the identified variants and their possible effects on the protein were evaluated in silico. Five novel point mutations and one recurrent splicing mutation in PATL2 were identified in four of nine (44.4%) unrelated patients. We found a consanguineous family with a homozygous missense mutation in two affected sisters, and their fertile brother. There were no clear phenotypic differences in oocytes between the patient with the homozygous missense mutation, patients with nonsense mutations and undiagnosed patients. n/a. The function of PATL2 remains largely unknown. Both the exact pathogenic mechanism(s) of mutated PATL2 causing human oocyte maturation arrest and the strategies to overcome this condition should be further investigated in the future. According to our data, mutations in PATL2 account for 44.4% of the individuals with oocyte GV arrest. Our study further confirms that PATL2 is required for human oocyte maturation and female fertility, which indicates a potential prognostic value of testing for PATL2 mutations in primary infertile women with oocyte maturation arrest. Natural Science Foundation of Anhui Province (1808085MH241), National Natural Science Foundation of China (81401251 and 81370757) and Central Guided Local Development of Science and Technology Special Fund (2016080802D114) supported this study. None of the authors have any competing interests.

Expression of CD9 and CD81 in bovine germinal vesicle oocytes after vitrification followed by in vitro maturation

The present study aimed to investigate the effect of vitrification on the expression of fertilization related genes (CD9 and CD81) and DNA methyl transferases (DNMT1 and DNMT3b) in bovine germinal vesicle (GV) oocytes and their resulting metaphase Ⅱ (MⅡ) stages after in vitro maturation culture. GV oocytes were vitrified using the open-pulled straw method; after warming, they were cultured in vitro. The vitrified-warmed GV oocytes and more developed MII oocytes were used to calculate the maturation rates (first polar body extrusion under a stereomicroscopy), and to detect mRNA expression (qRT-PCR). Fresh GV oocytes and their in vitro-derived MII oocytes served as controls. The results showed that both the maturation rate (54.23% vs. 42.93%) and the relative abundance of CD9 mRNA decreased significantly (p < 0.05) in bovine GV oocytes after vitrification, but the expression of CD81 and DNMT3b increased significantly. After in vitro maturation of vitrified GV oocytes, the resulting MII oocytes showed lower (p < 0.05) mRNA expression of genes (CD9, CD81, DNMT1 and DNMT3b) when compared to the control group (MII oocytes). Altogether, vitrification decreased the maturation rate of bovine GV oocytes and changed the expression of fertilization related genes and DNA methyl transferases during in vitro maturation.

Single-cell transcriptome sequencing reveals that cell division cycle 5-like protein is essential for porcine oocyte maturation

The brilliant cresyl blue (BCB) test is used in both basic biological research and assisted reproduction to identify oocytes likely to be developmentally competent. However, the underlying molecular mechanism targeted by the BCB test is still unclear. To explore this question, we first confirmed that BCB-positive porcine oocytes had higher rates of meiotic maturation, better rates of cleavage and development into blastocysts, and lower death rates. Subsequent single-cell transcriptome sequencing on porcine germinal vesicle (GV)-stage oocytes identified 155 genes that were significantly differentially expressed between BCB-negative and BCB-positive oocytes. These included genes such as cdc5l, ldha, spata22, rgs2, paip1, wee1b, and hsp27, which are enriched in functionally important signaling pathways including cell cycle regulation, oocyte meiosis, spliceosome formation, and nucleotide excision repair. In BCB-positive GV oocytes that additionally had a lower frequency of DNA double-strand breaks, the CDC5L protein was significantly more abundant. cdc5l/CDC5L inhibition by short interference (si)RNA or antibody microinjection significantly impaired porcine oocyte meiotic maturation and subsequent parthenote development. Taken together, our single-oocyte sequencing data point to a potential new role for CDC5L in porcine oocyte meiosis and early embryo development, and supports further analysis of this protein in the context of the BCB test.

Dynamic changes in the global transcriptome of bovine germinal vesicle oocytes after vitrification followed by in vitro maturation.

This study was conducted to investigate the effect of vitrification on the dynamics of the global transcriptome in bovine germinal vesicle (GV) oocytes and their in vitro-derived metaphase II (MII) oocytes. The GV oocytes were vitrified using the open-pulled straw method. After warming, GV oocytes and the resulting MII-stage oocytes were cultured in vitro for 2h and 24h respectively and were then collected. The fresh GV oocytes and their in vitro-derived MII oocytes were used as controls. Then, each pool (fresh GV, n=3; vitrified GV, n=4; fresh MII, n=1 and MII derived from vitrified GV, n=2) from the different stages was used for mRNA transcriptome sequencing. The results showed that the in vitro maturation rates of GV oocytes were significantly decreased (32.36% vs 53.14%) after vitrification. Bovine GV oocyte vitrification leads to 12 significantly upregulated and 19 downregulated genes. After culturing in vitro, the vitrification-derived MII oocytes showed 47 significantly upregulated and six downregulated genes when compared with those from fresh GV oocytes. Based on molecular function-gene ontology terms analysis and the Kyoto encyclopaedia of genes (KEGG) pathway database, the differentially expressed genes were associated with the pathways of cell differentiation and mitosis, transcription regulation, regulation of actin cytoskeleton, apoptosis and so on, which potentially result in the lower in vitro development of GV bovine oocytes.

Combined Effect of Retinoic Acid and Basic Fibroblast Growth Factor on Maturation of Mouse Oocyte and Subsequent Fertilization and Development.

BackgroundMany autocrine and paracrine elements that are produced within follicular niche have been the focus of much in vitro maturation (IVM) research. The present study was carried out to compare retinoic acid (RA) and basic fibroblast growth factor (bFGF) efficacy on IVM of mouse oocytes, and their further dual consumption to reach an optimal protocol.Materials and MethodsIn this experimental study, germinal vesicle (GV) oocytes obtained from two-months-old NMRI mice were randomly divided into control, sham and three experimental groups. The basic culture medium was α-MEM supplemented with 10% fetal bovine serum (FBS), 50 mg/l streptomycin, 60 mg/l penicillin and 10 ng/ ml epidermal growth factors. Each of the experimental groups received one of the following treatments: RA (2 µM), bFGF (20 ng/ml) or combination of RA and bFGF with the indicated concentrations. After 24 hours, capacitated spermatozoa were added to in vitro matured oocytes. Five hours later, the oocytes were cultured in fresh droplets of M2 medium for 24 hours and assessed for cleavage to the two-cells stage.ResultsAs compared with the control group, the rate of maturation was significantly increased in the RA (P<0.001) and bFGF+RA (P<0.02) groups with 58 ± 10 and 57 ± 3.46, respectively. The rate of maturation was significant in the RA (P<0.02) and bFGF+RA (P<0.03) groups, in comparison with the bFGF group. The bFGF+RA group had higher rate (83 ± 1.52) of two-cells development, than control (33 ± 1, P<0.001).ConclusionOur findings showed beneficial effects of 2 µM RA and 20 ng/ml bFGF combination on mouse oocyte IVM.

In Vitro Maturation and Culture of Human Oocytes.

We describe the collection, culture, and ex vivo, in vitro maturation of germinal vesicle (GV) oocytes obtained from human small antral follicles (hSAFs). hSAFs contain fully grown GV oocytes and have the advantages that they are more numerous than large or mature follicles, which are used in IVF treatment. hSAFs can be obtained directly from human ovarian tissue without exogenous gonadotrophin stimulation and therefore allows studies of oocytes even from young women and girls. The method described here was developed to study human female meiosis but could in theory also be used for fertility treatment.

The Effects of In Vitro Maturation Technique on The Expression of Genes Involved in Embryonic Genome Activation of Human Embryos.

Objective In vitro maturation technique (IVM) is shown to have an effect on full maturation of immature oocytes and the subsequent embryo development. Embryonic genome activation (EGA) is considered as a crucial and the first process after fertilization. EGA failure leads to embryo arrest and possible implantation failure. This study aimed to determine the role of IVM in EGA-related genes expression in human embryo originated from immature oocytes and recovered from women receiving gonadotrophin treatment for assisted reproduction. Materials and Methods In this experimental study, germinal vesicle (GV) oocytes were cultured in vitro. After intracytoplasmic sperm injection of the oocytes, fertilization, cleavage and embryo quality score were assessed in vitro and in vivo. After 3-4 days, a single blastomere was biopsied from the embryos and then frozen. Afterwards, the expression of EGA-related genes in embryos was assayed using quantitative reverse transcriptase-polymerase chain reaction (PCR). Results The in vitro study showed reduced quality of embryos. No significant difference was found between embryo quality scores for the two groups (P=0.754). The in vitro group exhibited a relatively reduced expression of the EGA- related genes, when compared to the in vivo group (all of them showed P=0.0001). ConclusionAlthough displaying the normal morphology, the IVM process appeared to have a negative influence on developmental gene expression levels of human preimplanted embryos. Based on our results, the embryo normal morphology cannot be considered as an ideal scale for the successful growth of embryo at implantation and downstream processes.

The Effects of Sodium Selenite on Mitochondrial DNA Copy Number and Reactive Oxygen Species Levels of In Vitro Matured Mouse Oocytes.

Objective The aim of present study is to determine the effects of supplementation of oocyte maturation medium with sodium selenite (SS) on oocyte mitochondrial DNA (mtDNA) copy number and reactive oxygen species (ROS) levels. Materials and Methods In this experimental study germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) stage oocytes were recovered from 6-8 week old female mice after superovulation. Some of the GV oocytes were cultured and matured in the presence and absence of SS. Then in vivo and in vitro matured (IVM) oocytes were subjected to mitochondria staining by MitoTracker green, ROS analysis, and mtDNA copy number determination using absolute real-time polymerase chain reaction (PCR). Results The maturation rate of GV oocytes to the MII stage significantly increased in the SS supplemented group (79.25%) compared to the control group (72.46%, P<0.05). The intensity of mitochondrial staining was not different among the studied groups, whereas the mitochondria distribution in the cytoplasm of the IVM oocytes showed some aggregation pattern. The in vivo obtained MII oocytes had lower ROS levels and higher mtDNA copy numbers than IVM-MII oocytes (P<0.05). The SS supplemented group had significantly lower ROS levels and higher mtDNA copy numbers than the non-treated group (P<0.05). ConclusionSS increased oocyte mtDNA copy number by decreasing oxidative stress. SS had an association with better oocyte developmental competence.