Relationship of quantitative reverse transcription polymerase chain reaction (RT-PCR) to RNA Sequencing (RNAseq) transcriptome identifies mouse preimplantation embryo reference genes†.

Numerous reference genes for use with quantitative reverse transcription polymerase chain reaction (RT-qPCR) have been used for oocytes, eggs, and preimplantation embryos. However, none are actually suitable because of their large variations in expression between developmental stages. To address this, we produced a standardized and merged RNA sequencing (RNAseq) data set by combining multiple publicly available RNAseq data sets that spanned mouse GV oocytes, MII eggs, and 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stage embryos to identify transcripts with essentially constant expression across all stages. Their expression was then measured using RT-qPCR, with which they did not exhibit constant expression but instead revealed a fixed quantitative relationship between measurements by the two techniques. From this, the relative amounts of total messenger RNA at each stage from the GV oocyte through blastocyst stages were calculated. The quantitative relationship between measurements by RNAseq and RT-qPCR was then used to find genes predicted to have constant expression across stages in RT-qPCR. Candidates were assessed by RT-qPCR to confirm constant expression, identifying Hmgb3 and Rb1cc1 or the geometric mean of those plus either Taf1d or Cd320 as suitable reference genes. This work not only identified transcripts with constant expression from mouse GV oocytes to blastocysts, but also determined a general quantitative relationship between expression measured by RNAseq and RT-qPCR across stages that revealed the relative levels of total mRNA at each stage. The standardized and merged RNA data set should also prove useful in determining transcript expression in mouse oocytes, eggs, and embryos.

Initial detachment of the mouse oocyte from the zona pellucida is mediated by metallopeptidase activity†.

The fully grown mammalian oocyte is tightly attached to its extracellular matrix shell, the zona pellucida (ZP), but the oocyte detaches from the ZP shortly after ovulation is signaled. The mechanism by which the oocyte detaches from the ZP is unknown. Because ZP proteins are initially secreted as transmembrane proteins, we hypothesized that attachment of the oocyte to the ZP is mediated by transmembrane ZP proteins and that detachment occurs when these proteins are cleaved by peptidases. To identify potential candidates for the type of peptidase, we used mouse oocyte transcriptome data sets to identify candidate peptidases localized to the exterior of the oocyte. Screening with a set of small molecule inhibitors that broadly target the families of peptidases represented by the candidates, we found that only inhibitors of the M10 and M12 families of metallopeptidases prevented detachment. Using more selective inhibitors indicated that detachment was prevented by an inhibitor, GI254023X, developed to be selective for ADAM10 in the M12 family but not by those considered selective for the M10 family or for other M12 metallopeptidases expressed in oocytes. Using an antibody that binds to an epitope just distal to the likely cleavage site of murine ZP3 showed that this site was gradually lost from the oocyte surface during the period when detachment occurs and that inhibiting metallopeptidase activity prevented the loss of this epitope. Taken together, these results indicate that detachment of the oocyte from the ZP is mediated by a metallopeptidase.

Roles of the cumulus-oocyte transzonal network and the Fragile X protein family in oocyte competence.

In brief: RNA granules travel through the cumulus cell network of transzonal projections which is associated with oocyte developmental competence, and RNA packaging involves RNA-binding proteins of the Fragile X protein family. Abstract: The determinants of oocyte developmental competence have puzzled scientists for decades. It is known that follicular conditions can nurture the production of a high-quality oocyte, but the underlying mechanisms remain unknown. Somatic cumulus cells most proximal to the oocyte are known to have cellular extensions that reach across the zona pellucida and contact with the oocyte plasma membrane. Herein, it was found that transzonal projections (TZPs) network quality is associated with developmental competence. Knowing that ribonucleoparticles are abundant within TZPs, the distribution of RNA-binding proteins was studied. The Fragile X-related proteins (FXR1P and FXR2P) and two partnering protein families, namely cytoplasmic FMRP-interacting protein and nuclear FMRP-interacting protein, exhibited distinctive patterns consistent with roles in regulating mRNA packaging, transport, and translation. The expression of green fluorescent protein (GFP)-FMRP fusion protein in cumulus cells showed active granule formation and their transport and transfer through filipodia connecting with neighboring cells. Near the projections' ends was found the cytoskeletal anchoring protein Filamin A and active protein synthesis sites. This study highlights key proteins involved in delivering mRNA to the oocyte. Thus, cumulus cells appear to indeed support the development of high-quality oocytes via the transzonal network.

SWI/SNF chromatin remodeling subunit Smarca4/BRG1 is essential for female fertility†.

Mammalian folliculogenesis is a complex process that involves the regulation of chromatin structure for gene expression and oocyte meiotic resumption. The SWI/SNF complex is a chromatin remodeler using either Brahma-regulated gene 1 (BRG1) or BRM (encoded by Smarca4 and Smarca2, respectively) as its catalytic subunit. SMARCA4 loss of expression is associated with a rare type of ovarian cancer; however, its function during folliculogenesis remains poorly understood. In this study, we describe the phenotype of BRG1 mutant mice to better understand its role in female fertility. Although no tumor emerged from BRG1 mutant mice, conditional depletion of Brg1 in the granulosa cells (GCs) of Brg1fl/fl;Amhr2-Cre mice caused sterility, whereas conditional depletion of Brg1 in the oocytes of Brg1fl/fl;Gdf9-Cre mice resulted in subfertility. Recovery of cumulus-oocyte complexes after natural mating or superovulation showed no significant difference in the Brg1fl/fl;Amhr2-Cre mutant mice and significantly fewer oocytes in the Brg1fl/fl;Gdf9-Cre mutant mice compared with controls, which may account for the subfertility. Interestingly, the evaluation of oocyte developmental competence by in vitro culture of retrieved two-cell embryos indicated that oocytes originating from the Brg1fl/fl;Amhr2-Cre mice did not reach the blastocyst stage and had higher rates of mitotic defects, including micronuclei. Together, these results indicate that BRG1 plays an important role in female fertility by regulating granulosa and oocyte functions during follicle growth and is needed for the acquisition of oocyte developmental competence.

Initiation of cell volume regulation and unique cell volume regulatory mechanisms in mammalian oocytes and embryos.

The period beginning with the signal for ovulation, when a fully-grown oocyte progresses through meiosis to become a mature egg that is fertilized and develops as a preimplantation embryo, is crucial for healthy development. The early preimplantation embryo is unusually sensitive to cell volume perturbations, with even moderate decreases in volume or dysregulation of volume-regulatory mechanisms resulting in developmental arrest. To prevent this, early embryos possess mechanisms of cell volume control that are apparently unique to them. These rely on the accumulation of glycine and betaine (N, N, N-trimethylglycine) as organic osmolytes-compounds that can provide intracellular osmotic support without the deleterious effects of inorganic ions. Preimplantation embryos also have the same mechanisms as somatic cells that mediate rapid responses to deviations in cell volume, which rely on inorganic ion transport. Both the unique, embryo-specific mechanisms that use glycine and betaine and the inorganic ion-dependent mechanisms undergo major changes during meiotic maturation and preimplantation development. The most profound changes occur immediately after ovulation is triggered. Before this, oocytes cannot regulate their volume, since they are strongly attached to their rigid extracellular matrix shell, the zona pellucida. After ovulation is triggered, the oocyte detaches from the zona pellucida and first becomes capable of independent volume regulation. A complex set of developmental changes in each cell volume-regulatory mechanism continues through egg maturation and preimplantation development. The unique cell volume-regulatory mechanisms in eggs and preimplantation embryos and the developmental changes they undergo appear critical for normal healthy embryo development.

Chromosome dynamics and spindle microtubule establishment in mouse embryos.

Chromosome segregation errors in mammalian embryos are common and jeopardize embryo health. Here, we perform for the first time 4-Dimensional imaging and tracking of chromosomes and centromeres through each preimplantation mitotic cell division in mouse embryos to define the normal dynamics of chromosome segregation. We show that a microtubule (MT)-dependent inward movement of chromosomes occurs at the time of nuclear envelope breakdown (NEBD), particularly in the earliest cell divisions, to position chromosomes prior to spindle assembly. Establishment of a rudimentary metaphase plate occurs immediately after NEBD, and is followed by a progressive alignment and biorientation of mitotic chromosomes. Stable end-on kinetochore-MT attachments form rapidly and attachment errors are uncommon. Altogether our data describe a rapid and efficient spindle assembly pathway that apparently minimizes the need for canonical MT attachment error correction in normally dividing embryos.

Cumulus Cell Transcripts Transit to the Bovine Oocyte in Preparation for Maturation.

So far, the characteristics of a good quality egg have been elusive, similar to the nature of the physiological, cellular, and molecular cues leading to its production both in vivo and in vitro. Current understanding highlights a strong and complex interdependence between the follicular cells and the gamete. Secreted factors induce cellular responses in the follicular cells, and direct exchange of small molecules from the cumulus cells to the oocyte through gap junctions controls meiotic arrest. Studying the interconnection between the cumulus cells and the oocyte, we previously demonstrated that the somatic cells also contribute transcripts to the gamete. Here, we show that these transcripts can be visualized moving down the transzonal projections (TZPs) to the oocyte, and that a time course analysis revealed progressive RNA accumulation in the TZPs, indicating that RNA transfer occurs before the initiation of meiosis resumption under a timetable fitting with the acquisition of developmental competence. A comparison of the identity of the nascent transcripts trafficking in the TZPs, with those in the oocyte increasing in abundance during maturation, and that are present on the oocyte's polyribosomes, revealed transcripts common to all three fractions, suggesting the use of transferred transcripts for translation. Furthermore, the removal of potential RNA trafficking by stripping the cumulus cells caused a significant reduction in maturation rates, indicating the need for the cumulus cell RNA transfer to the oocyte. These results offer a new perspective to the determinants of oocyte quality and female fertility, as well as provide insight that may eventually be used to improve in vitro maturation conditions.

The adenosine salvage pathway as an alternative to mitochondrial production of ATP in maturing mammalian oocytes.

Although the oocyte is the largest cell in the body and an unavoidable phase in life, its physiology is still poorly understood, and other cell types provide little insight into its unique nature. Even basic cellular functions in the oocyte such as energy metabolism are not yet fully understood. It is known that the mitochondria of the female gamete exhibit an immature form characterized by limited energy production from glucose and oxidative phosphorylation. We show that the bovine oocyte uses alternative means to maintain ATP production during maturation, namely, the adenosine salvage pathway. Meiosis resumption is triggered by destruction of cyclic AMP by phosphodiesterases producing adenosine monophosphate that is converted into ATP by adenylate kinases and creatine kinases. Inhibition of these enzymes decreased ATP production, and addition of their substrates restored ATP production in denuded oocytes. Addition of phosphocreatine to the oocyte maturation medium influenced the phenotype of the resulting blastocysts. We propose a model in which adenylate kinases and creatine kinases act as drivers of ATP production from added AMP during oocyte maturation.

The gametic synapse: RNA transfer to the bovine oocyte.

Even after several decades of quiescent storage in the ovary, the female germ cell is capable of reinitiating transcription to build the reserves that are essential to support early embryonic development. In the current model of mammalian oogenesis, there exists bilateral communication between the gamete and the surrounding cells that is limited to paracrine signaling and direct transfer of small molecules via gap junctions existing at the end of the somatic cells' projections that are in contact with the oolemma. The purpose of this work was to explore the role of cumulus cell projections as a means of conductance of large molecules, including RNA, to the mammalian oocyte. By studying nascent RNA with confocal and transmission electron microscopy in combination with transcript detection, we show that the somatic cells surrounding the fully grown bovine oocyte contribute to the maternal reserves by actively transferring large cargo, including mRNA and long noncoding RNA. This occurrence was further demonstrated by the reconstruction of cumulus-oocyte complexes with transfected cumulus cells transferring a synthetic transcript. We propose selective transfer of transcripts occurs, the delivery of which is supported by a remarkable synapselike vesicular trafficking connection between the cumulus cells and the gamete. This unexpected exogenous contribution to the maternal stores offers a new perspective on the determinants of female fertility.

Influence of physiological concentrations of androgens on the developmental competence and sex ratio of in vitro produced bovine embryos.

This study was designed to determine if the addition of androgens at ovarian follicular fluid (FF) concentrations to oocyte maturation media would alter the development and sex ratio of bovine embryos. To maximize hormone bioavailability, oil was removed and glass culture dishes were used during in vitro maturation (IVM) phase; this modified system was then used in the present experiment along with the standard IVM system utilizing plastic containers and incubation under oil. Ethanol (0.2%) was the vector for steroid hormone delivery. Oocytes were incubated for 22h in the presence of two doses ("low" and "high") of androstenedione (A4) or testosterone (T); the doses were based on the concentrations of both androgens in preovulatory bovine follicles (A4: 337.5 and 562.5ng/ml; T: 22.2 and 42.6ng/ml). The results of hormone assays indicated that bioavailability of steroid hormones remained relatively constant, regardless of the IVM system used. The plasticware with the addition of T resulted in significantly higher cleavage rates (80.0±2.1%) than any other combination of treatments (plasticware×A4: 71.5±2.6%; glassware×T: 71.2±1.9%; and glassware×A4: 71.4±2.4%). The blastocyst formation rate for the plasticware×T treatment (39.7±2.5%) was significantly greater than for all other combinations (glassware×T: 28.7±2.2%; glassware×A4: 24.0±2.8%; and plasticware×A4: 19.8±3.0%) and the low dose of T (37.1±2.5%) resulted in higher (p<0.05) blastocyst formation rates than all other treatments (T high dose: 29.2±2.5%; A4 high dose: 27.1±2.9%; and A4 low dose: 20.2±3.0%). The proportion of male embryos was greater (p<0.05) in plastic than glass dishes in the low-dose A4 group (59.1±8.7% vs. 38.2±5.5%, plasticware vs. glassware, respectively) and it tended to be greater (p<0.08) in the control groups and high-dose A4 group, but not in the T groups. There was a moderate positive correlation between blastocyst formation rates across all treatment and control groups, and the percentage of male bovine embryos (r=0.38, p<0.05). In summary, specific combinations of androgen and glassware/plasticware treatments did alter early bovine embryo development and sex ratio. The addition of T to IVM media increased the cleavage and blastocyst formation rates in plasticware and may be employed to improve the efficiency of the standard in vitro embryo production systems. Androstenedione appeared to enhance whereas testosterone nullified the deviation in sex ratio (pro-femaleness) associated with the use of glass IVM dishes.