Mouse Oocytes, A Complex Single Cell Transcriptome.

Germinal vesicle (GV) stage is a critical transition point from growth to maturation in mammalian oocyte development. During the following meiotic maturation, active RNA degradation and absence of transcription significantly reprofile the oocyte transcriptome to determine oocyte quality. Oocyte RNA-seq has revealed transcriptome differences between two defined phases of GV stage, namely non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) phases. In addition, oocyte RNA-seq has identified a variety of dysregulated genes upon genetic mutation or environmental perturbation. Historically, due to the low amount of RNA per oocyte, a few (20-200) oocytes were needed for a regular library construction in bulk RNA-seq. In recent years, development of single cell sequencing allows detailing the transcriptome of individual oocytes. Here in this study, different RNA-seq datasets from single and bulk of mouse oocytes are compared, and single oocyte RNA-seq (soRNA-seq) shows higher reproducibility. In addition, soRNA-seq better illustrates developmental progression of GV oocytes, revealing more complex gene changes than traditional views. Specially, an elevated level of ribosomal RNA 5'-ETS (5' external transcribed spacer) has been shown to highly correlate with SN property. This study further demonstrates that UMI (unique molecular identifiers) based and other deduplication methods are limited in their ability to improve the precision of the soRNA-seq datasets. Finally, this study proposes that external spike-in molecules are useful for normalizing samples of different transcriptome sizes. A list of stable genes has been identified during oocyte maturation that are comparable to external spike-in molecules. These findings highlight the advantage of soRNA-seq, and have established ways for better clustering and cross-stage normalization, which can provide more insight into the biological features of oocyte maturation.

A Comparison of Embryonic Development and Clinical Outcomes between In vitro Oocytes Maturation Using Micro-Vibration System and In vivo Oocytes Maturation in Polycystic Ovarian Syndrome Patients.

BACKGROUND/OBJECTIVES: Mechanical micro-vibration remains insufficient for improving embryo culture conditions in human immature oocytes. This study compared the clinical outcomes and embryo development between germinal vesicle (GV) oocytes with the micro-vibration culture (MVC) system in in vitro maturation (IVM) cycles and in vivo-matured oocytes in controlled ovarian hyperstimulation (COH) cycles in polycystic ovarian syndrome (PCOS) patients. METHODS: This study investigated 152 PCOS patients who underwent 159 fresh embryo transfer cycles, including IVM cycles with embryos derived from GV oocytes and the COH cycles with embryos derived from in vivo-matured oocytes. The IVM cycles were divided into groups according to the culture system used: static culture (SC) and MVC: In the IVM-S group (n = 47), SC was applied during both IVM and in vitro culture (IVC), whereas in the IVM-MV group (n = 44), MVC was applied during both IVM and IVC. For the COH cycles, in the COH-S group (n = 68), SC was applied during IVC. RESULTS: The number of in vitro-matured oocytes was similar in the IVM-S and IVM-MV groups, but the good-quality embryo (GQE; ≥6-cells) rate was significantly higher in the IVM-MV group (p < 0.01). The GQE rate and clinical outcomes of the COH-S group were significantly better than those of the IVM-S group (p < 0.05) but similar to those of the IVM-MV group. CONCLUSION: Compared with the SC system, the MVC system in IVM cycles improves the embryonic quality of GV oocytes and clinical outcomes, resulting in development of potential equivalent to in vivo-matured oocytes.

The effect of sodium selenite on apoptotic gene expression and development of in vitro cultured mouse oocytes in comparison with in vivo obtained oocytes.

In vitro maturation (IVM) of oocytes is widely used in assisted reproduction technologies. The present study aimed to improve the in vitro oocyte maturation and its development through enriching the culture media with sodium selenite (SS). Moreover, the effects of SS on the expression of the oocytes apoptosis-related genes were assessed. In this study, male and female NMRI mice were used and after collecting their germinal vesicle (GV) oocytes, they were cultured with SS (experimental group) and without SS (control group). Collected metaphase II oocytes (MII) from the fallopian tube were considered as in vivo group. After in vitro culture, the oocytes were assessed in terms of nuclear maturation. The MII oocytes were inseminated and the development was examined until the blastocyst stage. Also, oocytes were subjected to the molecular analysis for evaluating the expression of BAX, BCL2, P53, and BAD genes using the real-time RT-PCR. The maturation rate was significantly increased in the SS supplemented group compared to the control one. The developmental rate of the embryos was significantly higher for both of the in vivo and SS supplemented groups rather than the control one, however, no significant difference was seen between these rates of the experimental and in vivo groups. Real-time RT-PCR did not show any significant differences in the expression of the apoptosis-related genes for all of the studied groups. The p53 gene was not expressed in any of groups. Sodium selenite improved the oocyte developmental competence but did not change the expression of the apoptosis-related genes in MII oocytes.

Improvement of embryonic development and clinical outcomes of germinal vesicle stage oocytes using a microvibration culture system.

In vitro maturation (IVM) has evolved as a clinical treatment option in assisted reproductive technology. However, the poor developmental potential of germinal vesicle (GV)-stage oocytes is still suboptimal. This study's objective was to evaluate the effect of a microvibration culture system (MVC) during IVM and/or in vitro culture (IVC) on the clinical outcomes and the embryonic development potential of human GV-stage oocytes collected from human chorionic gonadotropin (HCG)-primed IVM and fertilization-embryo transfer (IVM/F-ET) cycles of patients with polycystic ovaries (PCO). A total of 206 HCG-primed IVM/F-ET cycles were divided into four groups according to the microvibration and static culture system applied during IVM and/or IVC: Group SS (static system during both IVM and IVC); Group SV (static system during IVM alternated with microvibration system during IVC); Group VS (microvibration system during IVM alternated with static system during IVC), and Group VV (microvibration system during both IVM and IVC). The results indicate that the rates of in vitro MII oocytes per cycle, fertilization, and cleavage were not significantly different between the groups. The rate of good-quality embryos in Group SV tended to be higher than the rate in Groups SS and VS, but there was no significant difference between Group SS and Group SV. Clinical pregnancy, implantation, and live birth rates of Groups SV and VS were slightly higher than those of Group SS. However, the rate of good-quality embryos with at least six cells on day 4, the clinical pregnancy, implantation, and live births in Group VV were significantly higher than those in Group SS. These results indicate that, compared with the static culture system, the MVC system applied for both IVM and IVC seems to improve the clinical outcomes and the quality of embryos of GV oocytes derived from HCG-primed IVM/F-ET cycles in PCO patients. Abbreviations: PCO: polycystic ovaries; HCG: human chorionic gonadotropin; GV: germinal vesicle; MII: metaphase II; IVM: in vitro maturation; IVF: in vitro fertilization; IVC: in vitro culture: MVC: microvibration culture; SC: static culture; ICSI: intracytoplasmic sperm injection; IVM/F-ET: IVM and fertilization-embryo transfer; AMH: anti-Mullerian hormone; OHSS: ovarian hyperstimulation syndrome.

Dynamic changes of histone H3 lysine 9 following trimethylation in bovine oocytes and pre-implantation embryos.

OBJECTIVES: We have examined dynamic changes of histone H3 lysine 9 following trimethylation (H3K9me3), the mRNA expression levels of SUV39H1 and SUV39H2 in bovine oocytes and the role in the development of in vitro fertilization (IVF) pre-implantation embryos. RESULTS: There were strong H3K9me3 signals in germinal vesicle (GV) oocytes but no signals in MII oocytes. H3K9me3 signals were maintained during IVF pre-implantation embryo development. SUV39H1 and SUV39H2 showed significantly higher mRNA expression levels in GV oocytes than MII oocytes (P < 0.01). SUV39H1 showed high mRNA expression level in two-cell embryos, however, SUV39H2 showed high mRNA expression level in four-cell embryos. In other development stage, SUV39H1 and SUV39H2 showed low expression levels. CONCLUSION: Bovine IVF pre-implantation embryos maintain strong H3K9me3 signals and SUV39H1 and SUV39H2 are highly expressed at the early development stage of pre-implantation embryos.

A stereological study on organelle distribution in human oocytes at prophase I.

The ultrastructural analysis of human oocytes at different maturation stages has only been descriptive. The aim of this study was to use a stereological approach to quantify the distribution of organelles in oocytes at prophase I (GV). Seven immature GV oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. The Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction were used to compare the means of the relative volumes occupied by organelles in oocyte regions: cortex (C), subcortex (SC) and inner cytoplasm (IC). Here we first describe in GV oocytes very large vesicles of the smooth endoplasmic reticulum (SER), vesicles containing zona pellucida-like materials and coated vesicles. The most abundant organelles were the very large vesicles of the SER (6.9%), mitochondria (6.3%) and other SER vesicles (6.1%). Significant differences in organelle distribution were observed between ooplasm regions: cortical vesicles (C: 1.3% versus SC: 0.1%, IC: 0.1%, P = 0.001) and medium-sized vesicles containing zona pellucida-like materials (C: 0.2% versus SC: 0.02%, IC: 0%, P = 0.004) were mostly observed at the oocyte cortex, whereas mitochondria (C: 3.6% versus SC: 6.0%, IC: 7.2%, P = 0.005) were preferentially located in the subcortex and inner cytoplasm, and SER very large vesicles (IC: 10.1% versus C: 0.9%, SC: 1.67%, P = 0.001) in the oocyte inner cytoplasm. Further quantitative studies are needed in immature metaphase-I and mature metaphase-II oocytes, as well as analysis of correlations between ultrastructural and molecular data, to better understand human oocyte in vitro maturation.

Ultrastructure of in vitro Matured Human Oocytes.

BACKGROUND: Approximately 20% of recovered oocytes are immature and discarded in intracytoplasmic sperm injection (ICSI) procedures. These oocytes represent a potential resource for both clinical and basic science application. OBJECTIVE: The aim of this study was to evaluate the ultrastructure architecture of in vitro matured human oocytes using transmission electron microscopy (TEM). MATERIALS AND METHODS: A total of 204 immature oocytes from infertile patients who underwent ICSI cycles were included in this prospective study. Immature oocytes were divided into two groups: (i) GV oocytes (n = 101); and (ii) MI oocytes (n = 103). Supernumerary fresh in vivo matured oocytes (n = 10) were used as control. RESULTS: The rates of maturations were 61.38% for GV and 73.78% for MI oocytes in IVM medium (P = 0.07). However, the rate of oocyte arrest was significant between groups (P <0 .05). Ultrastructurally; in vitro and in vivo matured oocytes appeared round, with a homogeneous cytoplasm, an intact oolemma and an intact zona pellucida. However, immature oocytes indicated numerous large mitochondria-vesicle complexes (M-VC). CONCLUSIONS: Ultrastructural changes of M-VC in IVM groups emphasize the need for further research in order to refine culture conditions and improve the implantation rate of in-vitro matured oocytes.