The origin of betaine in mouse oocytes and preimplantation embryos†.

Betaine has important roles in preimplantation mouse embryos, including as an organic osmolyte that functions in cell volume regulation in the early preimplantation stages and as a donor to the methyl pool in blastocysts. The origin of betaine in oocytes and embryos was largely unknown. Here, we found that betaine was present from the earliest stage of growing oocytes.

Oocyte-Specific Deletion of Encoding the GLYT1 Glycine Transporter Eliminates Glycine Transport in Mouse Preimplantation Embryos and Their Ability to Counter Hypertonic Stress.

Early preimplantation mouse embryos are sensitive to increased osmolarity, which can block their development. To overcome this, they accumulate organic osmolytes to maintain cell volume. The main organic osmolyte used by early mouse embryos is glycine.

The cell volume-regulatory glycine transporter GLYT1 is activated following metallopeptidase-mediated detachment of the oocyte from the zona pellucida.

Independent cell volume regulation is first acquired by the oocyte in two steps that occur during meiotic maturation: (1) activation of the glycine transporter GLYT1 (Slc6a9) that mediates the intracellular accumulation of glycine to provide osmotic support in the mature egg and early preimplantation embryo, and (2) release of the oocyte from the strong attachment to its rigid extracellular matrix shell, the zona pellucida (ZP). It was recently shown that oocyte-ZP detachment requires metallopeptidase activity that is proposed to cleave transmembrane ZP proteins connecting the oocyte to the ZP. It is unknown, however, how GLYT1 is activated.

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.

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.

5,10-Methylenetetrahydrofolate reductase becomes phosphorylated during meiotic maturation in mouse oocytes.

The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) links the folate cycle that produces one-carbon units with the methionine cycle that converts these into -adenosylmethionine (SAM), the universal methyl donor for almost all methyltransferases. Previously, MTHFR has been shown to be regulated by phosphorylation, which suppresses its activity. SAM levels have been shown to increase substantially soon after initiation of meiotic maturation of the mouse germinal vesicle (GV) stage oocyte and then decrease back to their original low level in mature second meiotic metaphase (MII) eggs.

Paternal MTHFR deficiency leads to hypomethylation of young retrotransposons and reproductive decline across two successive generations.

5,10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in the folate metabolic pathway with a key role in generating methyl groups. As MTHFR deficiency impacts male fertility and sperm DNA methylation, there is the potential for epimutations to be passed to the next generation. Here, we assessed whether the impact of MTHFR deficiency on testis morphology and sperm DNA methylation is exacerbated across generations in mouse.

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.

l-Serine transport in growing and maturing mouse oocytes.

Serine has roles in cell metabolism besides protein synthesis including providing one-carbon units to the folate cycle. Since growing mouse oocytes undergo a burst of folate accumulation as they near full size, we have investigated whether oocytes transport serine. Substantial serine transport appeared in oocytes near the end of their growth.

Focal adhesion kinase PTK2 autophosphorylation is not required for the activation of sodium-hydrogen exchange by decreased cell volume in the preimplantation mouse embryo.

SummaryRecovery from decreased cell volume is accomplished by a regulated increase of intracellular osmolarity. The acute response is activation of inorganic ion transport into the cell, the main effector of which is the Na+/H+ exchanger NHE1. NHE1 is rapidly activated by a cell volume decrease in early embryos, but how this occurs is incompletely understood.

Betaine is accumulated via transient choline dehydrogenase activation during mouse oocyte meiotic maturation.

Betaine (-trimethylglycine) plays key roles in mouse eggs and preimplantation embryos first in a novel mechanism of cell volume regulation and second as a major methyl donor in blastocysts, but its origin is unknown. Here, we determined that endogenous betaine was present at low levels in germinal vesicle (GV) stage mouse oocytes before ovulation and reached high levels in the mature, ovulated egg. However, no betaine transport into oocytes was detected during meiotic maturation.

Preovulatory suppression of mouse oocyte cell volume-regulatory mechanisms is via signalling that is distinct from meiotic arrest.

GLYT1-mediated glycine transport is the main cell volume-homeostatic mechanism in mouse eggs and early preimplantation embryos. It is unique to these developmental stages and key to their healthy development. GLYT1 first becomes activated in oocytes only after ovulation is triggered, when meiotic arrest of the oocyte is released, but how this occurs was unknown.

Acute cell volume regulation by Janus kinase 2-mediated sodium/hydrogen exchange activation develops at the late one-cell stage in mouse preimplantation embryos.

Early preimplantation embryos are extremely sensitive to dysregulation of cell volume, which can lead to developmental arrest. It was previously shown that mouse embryos at the two-cell stage respond to a cell volume decrease by quickly activating Na+/H+ exchange via a signaling mechanism that involves the tyrosine kinase Janus kinase 2 (JAK2). However, it was not known whether this mechanism is active at the one-cell stage, when embryos are most sensitive to perturbed cell volume.

Mouse Oocytes Acquire Mechanisms That Permit Independent Cell Volume Regulation at the End of Oogenesis.

Mouse embryos employ a unique mechanism of cell volume regulation in which glycine is imported via the GLYT1 transporter to regulate intracellular osmotic pressure. Independent cell volume regulation normally becomes active in the oocyte after ovulation is triggered. This involves two steps: the first is the release of the strong adhesion between the oocyte and zona pellucida (ZP) while the second is the activation of GLYT1.

Growing Mouse Oocytes Transiently Activate Folate Transport via Folate Receptors As They Approach Full Size.

The folate cycle is central to cellular one-carbon metabolism, where folates are carriers of one-carbon units that are critical for synthesis of purines, thymidylate, and S-adenosylmethionine, the universal methyl donor that forms the cellular methyl pool. Although folates are well-known to be important for early embryo and fetal development, their role in oogenesis has not been clearly established. Here, folate transport proteins were detected in developing neonatal ovaries and growing oocytes by immunohistochemistry, Western blot, and immunofluorescence.

Both the folate cycle and betaine-homocysteine methyltransferase contribute methyl groups for DNA methylation in mouse blastocysts.

The embryonic pattern of global DNA methylation is first established in the inner cell mass (ICM) of the mouse blastocyst. The methyl donor S-adenosylmethionine (SAM) is produced in most cells through the folate cycle, but only a few cell types generate SAM from betaine (N,N,N-trimethylglycine) via betaine-homocysteine methyltransferase (BHMT), which is expressed in the mouse ICM. Here, mean ICM cell numbers decreased from 18-19 in controls to 11-13 when the folate cycle was inhibited by the antifolate methotrexate and to 12-14 when BHMT expression was knocked down by antisense morpholinos.

Prophase I arrest of mouse oocytes mediated by natriuretic peptide precursor C requires GJA1 (connexin-43) and GJA4 (connexin-37) gap junctions in the antral follicle and cumulus-oocyte complex.

Fully grown germinal vesicle stage mouse oocytes remain arrested in meiotic prophase I until ovulation. This arrest is maintained by cGMP produced in cumulus granulosa cells surrounding the oocyte. Recently, it was found that cGMP production in cumulus cells depends on NPR2 guanylate cyclase activated by its ligand natriuretic peptide precursor C (NPPC).

Identifiability and privacy in pluripotent stem cell research.

Data sharing is an essential element of research; however, recent scientific and social developments have challenged conventional methods for protecting privacy. Here we provide guidance for determining data sharing thresholds for human pluripotent stem cell research aimed at a wide range of stakeholders, including research consortia, biorepositories, policy-makers, and funders.

Uptake of betaine into mouse cumulus-oocyte complexes via the SLC7A6 isoform of y+L transporter.

Betaine (N,N,N-trimethylglycine) has previously been shown to function in cell volume homeostasis in early mouse embryos and also to be a key donor to the methyl pool in the blastocyst. A betaine transporter (SLC6A20A or SIT1) has been shown to be activated after fertilization, but there is no saturable betaine uptake in mouse oocytes or eggs. Unexpectedly, the same high level of betaine is present in mature metaphase II (MII) eggs as is found in one-cell embryos despite the lack of transport in oocytes or eggs.

The REDIH experience: an emerging design to develop an effective training program for graduate students in reproductive science.

Background: A training program in Reproduction, Early Development, and the Impact on Health (REDIH) was initiated in 2009 by researchers specializing in biomedical, clinical, population health, and ethics research from seven collaborating universities in Quebec and Ontario, and Health Canada. This paper reports the findings from the first three years of the 6-year program.

Objectives: The objective of the REDIH program is to provide increased opportunities for excellent training in reproduction and early development for graduate students and fellows, in order to build research, clinical, regulatory, decision-making, and industry capacity in Canada.

Connections between preimplantation embryo physiology and culture.

Purpose: To review the history of experimental embryo culture and how culture media that permitted complete preimplantation development in vitro were first discovered, and the physiological insights gained.

Methods: This article reviews the history of in vitro mammalian embryo culture, in particular the efforts that led to the current generation of successful culture media and how these reflect embryo physiology, highlighting the contributions of Dr. John D.

Folate transport in mouse cumulus-oocyte complexes and preimplantation embryos.

Endogenous folate stores are required in preimplantation embryos of several species, but how folates are accumulated and whether they can be replenished has not been determined. Folates are generally taken up into cells by specific transporters, mainly the reduced folate carrier RFC1 (SLC19A1 protein) and the high-affinity folate receptors FOLR1 and FOLR2. Quantitative RT-PCR showed that Slc19a1 mRNA was expressed in mouse cumulus-oocyte complexes (COCs) and oocytes, whereas Folr1 showed expression only in preimplantation embryos, increasing from the 2-cell stage onward.

NHE1 is the sodium-hydrogen exchanger active in acute intracellular pH regulation in preimplantation mouse embryos.

Sodium-hydrogen exchangers (NHE) of the Slc9 gene family are the major regulators of intracellular pH against acidosis in mammalian cells. Of five plasma membrane NHE isoforms, mouse oocytes and preimplantation embryos express mRNAs encoding NHE1 (SLC9A1), NHE3 (SLC9A3), and NHE4 (SLC9A4), with higher mRNA levels for each in oocytes through one-cell stage embryos and lower levels after the two-cell stage. NHE2 (SLC9A2) and NHE5 (SLC9A5) are not expressed.

Na+/H+ exchange is inactivated during mouse oocyte meiosis, facilitating glycine accumulation that maintains embryo cell volume.

The coupled action of the Na(+)/H(+) exchanger NHE1 and the HCO3(-)/Cl(-) exchanger AE2 constitutes the principal mechanism for acute correction of decreased cell volume in mammalian somatic cells, while, when acting separately, they regulate intracellular pH. It was previously found that AE2 becomes inactivated during meiosis in mouse oocytes. Similarly, NHE1 activity stimulated by intracellular acidosis was present in preovulatory germinal vesicle stage (GV) mouse oocytes and then decreased during meiotic maturation.