Mitochondrial Stress Response Gene Clpp Is Not Required for Granulosa Cell Function

Mitochondrial unfolded protein response (UPRmt) is a highly conserved mechanism, which is activated upon cellular or metabolic stress and aims to help cells maintain homeostasis. CLPP (caseinolytic peptidase P) plays a crucial factor for UPRmt; it promotes the degradation of unfolded mitochondrial proteins. Global germline deletion of Clpp in mice results in female infertility and accelerated follicular depletion. Here, we asked whether CLPP is necessary for granulosa/cumulus cell function. Clppflox/flox mice were generated and crossbred with Cyp19a1-Cre mice to generate mice with granulosa/cumulus cell-specific Clpp deletion (Clpp−/−). Mature (8-week-old) Clpp−/− female mice (8-week-old) were compared to same age wild type (WT) mice. We found that mature Clpp−/− female mice were fertile and produced a similar number of pups per litter compared to WT. Folliculogenesis was not affected by the loss of CLPP in granulosa/cumulus cells as Clpp−/− and WT mice had a similar number of primordial, primary, secondary, early antral, and antral follicles. The number of germinal vesicles (GV) and MII oocytes collected from Clpp−/− and WT female mice were also similar. Our findings demonstrate that fertility in female mice is not affected by granulosa/cumulus cell-specific UPRmt disruption through CLPP deletion.

2 The landscape of accessible chromatin in bovine oocytes and early embryos

Chromatin reorganization governs gene expression regulation during pre-implantation development. However, the global chromatin landscape and its dynamics in this period remain unexplored in bovine. In this study, we constructed a genome-wide map of accessible chromatin in bovine oocytes and early embryos using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). We analysed pools of 20 germinal vesicles or MII oocytes or 2-, 4-, 8-, 16-cell, morula, and blastocyst stage invitro-produced embryos. We conducted ATAC-seq on six pools for each stage and an additional four pools of invivo-derived morula and blastocysts and six replicates using individual Day 14 elongating embryos. We obtained ~110 million paired end reads uniquely mapped to the bovine reference genome for each stage. Hierarchical clustering, t-distributed stochastic neighbour embedding, and principal component analysis showed four distinct patterns for open chromatin status: (1) low accessibility in germinal vesicles and MII oocytes and in 2- and 4-cell embryos; (2) significantly elevated accessibility in 8-cell, 16-cell, and morula embryos; (3) less accessibility in blastocysts; and (4) extremely high accessibility in elongating embryos. This dynamic and sequential chromatin remodelling is consistent with transcription activation during the bovine minor embryonic genome activation from fertilization to 4-cell, major embryonic genome activation at 8-cell, first differentiation at blastocyst and drastic transcription initiation for embryo elongation. Genome-wide characteristics of accessible chromatin showed (1) accessible chromatin near the transcription start sites of active genes and CpG-rich promoters; (2) widespread accessible chromatin regions extensively overlapped with transposable elements; (3) distal peaks preferentially enriched for repeats including LINE, SINE, and LTR from 8-cell to morula embryos, especially for LTR, whereas enrichment in simple repeats were found from oocytes to 4-cell and in elongating embryos; and (4) highly stage-specific transcription factor motifs in distal peaks were unveiled. By integrating the maps of chromatin accessibility with bovine embryo transcriptomes and DNA methylomes, we found promoter accessibility and DNA methylation in bovine embryos correlated with both gene activities and CpG densities. Most importantly, we constructed the regulatory networks of stage-specific expressed genes and stage-specific activated genes with three omics datasets in bovine early embryos and revealed conserved and distinctive transcriptional regulatory networks between invivo- and invitro-derived embryos. This comprehensive analysis revealed critical features of the chromatin landscape and epigenetic reprogramming during bovine early embryo development.

Enucleolation and nucleolus transfer in mammalian oocytes and zygotes

The oocyte GV/GVs (germinal vesicle/germinal vesicles) and zygot PN/PNs (pronucleus/pronuclei) of some mammals contain clearly visible nucleoli which exhibit an atypical morphological structure. These nucleoli (NCLs) can be relatively easily manipulated, i.e. removed from GVs/PNs or eventually transferred into another oocyte/zygote. Thus, with the help of micromanipulation techniques it was possible to uncover the real function(s) they play in processes of oocyte maturation and early embryonic development. The purpose of our review is to describe briefly the micromanipulation techniques that can be used for oocyte/zygote nucleoli manipulation. Moreover, we present some examples of results that were obtained in nucleolus manipulation experiments.

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.

173 Size Matters: a Follicle-of-Origin Effect on the Preovulatory Epigenetic Constitution of Germinal Vesicle-Stage Porcine Oocytes

The goal of this study was to evaluate global levels of a variety of histone modifications at different lysine (K) residues on Histone 3 (H3) within the chromatin of porcine germinal vesicle (GV)-stage oocytes that were aspirated from follicles of different sizes. We hypothesised that we would see evidence of a transition from open, transcriptionally active chromatin (in oocytes from smaller, growing follicles) to more closed, transcriptionally silent chromatin associated with fully grown oocytes (aspirated from large, preovulatory follicles). Cumulus-enclosed oocytes were aspirated from small (<3 mm) or large (>7 mm) follicles from abattoir-derived pig ovaries. Oocytes were denuded immediately after aspiration and then immunoprobed with antibodies specific for trimethylated (me3) H3K4, H3K9me3, and H3K27me3. Background-corrected nuclear fluorescence levels for each histone mark were collected from multiple oocytes from each of at least three experimental replicates (aspiration days). Data were subjected to one-way ANOVA with a Bonferroni multiple testing correction to determine whether there were differences in fluorescence intensities in the nuclei (germinal vesicles) of oocytes from small v. large follicles. Oocytes from large follicles displayed more intense nuclear staining for all 3 histone marks: average nuclear H3K4me3 intensity was 31.4% higher (P = 0.0004), H3K9me3 was 70.3% higher (P = 0.0218), and H3K27me3 was 32.0% higher (P = 0.0231) in oocytes from large follicles. An ancillary analysis of the data revealed no effect (P > 0.1) of pubertal status (i.e. whether small and large follicles were aspirated from pre- v. post-pubertal ovaries) on the intensity of nuclear fluorescence for any of the marks evaluated. In continuation, 3 oocytes from both follicle types were collected on each of 6 aspiration days (i.e. 18 individual oocytes from each follicle type), and the mRNA from these were used for an RT-qPCR experiment to detect the relative abundance of transcripts from 21 different genes coding for histone methyltransferase or demethylase enzymes in oocytes from large v. small follicles. Of the 21 genes tested, 5 genes (KDM4C, KDM4D, KDM5B, KDM5C, and SETD7) were not detectable in our individual oocyte samples, but transcripts from 6 of the 16 remaining genes (KDM6A, KMT2B, MLL3, SETD1B, SETDB1, and SUV39H2) were shown to be significantly more abundant in oocytes from large follicles (at least 2-fold greater abundance and P < 0.05). Although our expectation-that histone marks (and related transcripts) would consistently reflect a globally “repressive” chromatin configuration in oocytes from large follicles and a more “open” configuration in oocytes from small follicles-turned out to be untrue, the evidence suggests that the epigenetic constitution of oocytes from small follicles may indeed vary from that of oocytes from large, preovulatory follicles, and this phenomenon warrants further investigation.