Design of novel oocyte activation methods: The role of zinc

Oocyte activation occurs at the time of fertilization and is a series of cellular events initiated by intracellular Ca2+ increases. Consequently, oocytes are alleviated from their arrested state in meiotic metaphase II (MII), allowing for the completion of meiosis. Oocyte activation is also an essential step for somatic cell nuclear transfer (SCNT) and an important tool to overcome clinical infertility. Traditional artificial activation methods aim to mimic the intracellular Ca2+ changes which occur during fertilization. Recent studies emphasize the importance of cytoplasmic Zn2+ on oocyte maturation and the completion of meiosis, thus suggesting artificial oocyte activation approaches that are centered around the concentration of available Zn2+in oocytes. Depletion of intracellular Zn2+ in oocytes with heavy metal chelators leads to successful oocyte activation in the absence of cellular Ca2+ changes, indicating that successful oocyte activation does not always depends on intracellular Ca2+ increases. Current findings lead to new approaches to artificially activate mammalian oocytes by reducing available Zn2+ contents, and the approaches improve the outcome of oocyte activation when combined with existing Ca2+ based oocyte activation methods. Here, we review the important role of Ca2+ and Zn2+ in mammalian oocyte activation and development of novel oocyte activation approaches based on Zn2+ availability.

Poly(A) tail length is a major regulator of maternal gene expression during the mammalian oocyte-to-embryo transition

Transcription is silent during the mammalian oocyte-to-embryo transition (OET) until zygotic genome activation (ZGA). Therefore, the OET relies on post-transcriptional regulation of maternal mRNA, among which poly(A) tail lengths have been found to regulate translation for a small number of genes1–3. However, transcriptome-wide poly(A) tail length dynamics and their role in gene expression during the mammalian OET remain unknown. Here, we quantified transcriptome-wide mRNA poly(A) tail length dynamics during the mammalian OET using PAIso-seq1 and PAIso-seq24,5, two methods with different underlying principles that preserve the poly(A) tail information. We revealed that poly(A) tail length was highly dynamic during the mouse OET, and Btg4 is responsible for global maternal mRNA deadenylation. We found that the poly(A) tail length positively associated with translational efficiency transcriptome-wide in mouse oocytes. In addition, genes with different alternative polyadenylation isoforms show longer poly(A) tails for isoforms with distal polyadenylation sites compared to those with proximal polyadenylation sites in mouse, rat, pig and human oocytes after meiotic resumption, which is not seen in cultured cell lines. Surprisingly, mammalian embryos, namely mouse, rat, pig, and human embryos, all experience highly conserved global mRNA re-polyadenylation after fertilization, providing molecular evidence that the early embryo development before ZGA is driven by re-polyadenylated maternal mRNAs rather than newly transcribed mRNAs. Together, our study reveals the conserved mRNA poly(A) tail length landscape. This resource can be used for exploring spatiotemporal post-transcriptional regulation throughout the mammalian OET.

Conservation and divergence of poly(A) tail regulation during the mammalian oocyte-to-embryo transition

SUMMARYPoly(A) tail length and non-A residues are vital for oocyte-to-embryo transition (OET) in mice and humans1–5. However, the role of poly(A) tail length and non-A residues during OET in other commonly used mammalian animal models for human diseases remains unexplored. In addition, the degree of conservation in maternal mRNA poly(A) tail dynamics during OET across different mammal species is unknown. Here, we conduct a comparative analysis of the poly(A) tails during OET across four species: mice, rats, pigs, and humans. Dynamics during OET found to be conserved across all four species include: maternal mRNA deadenylation during oocyte maturation and re-polyadenylation after fertilization; a fall-rise trend in poly(A) tail length distribution; a rise-fall trend in the ratio of poly(A) tails with non-A residues; higher abundance of non-A residues in poly(A) tails of maternal mRNA than in zygotic genome activation (ZGA) mRNA; maternal mRNA with U residues degrades faster than those without U residues at the stage when ZGA takes place. While in mice and rats maternal mRNA deadenylation is impaired in parthenogenetic embryos and ZGA inhibition leads to blocked maternal mRNA deadenylation in mice and humans. In contrast, the length of consecutive U residues and the duration time of U residues in poly(A) tail diverges across the four species. Together, these findings reveal that the poly(A) tail mediated maternal mRNA post-transcriptional regulation is highly conserved in mammals with unique divergences in the length and life-span of U residues, providing new insights for the further understanding of OET across different mammals.

Re-polyadenylation occurs predominantly on maternal mRNA degradation intermediates during mammalian oocyte-to-embryo transition

The nascent mRNA transcribed in the nucleus is cleaved and polyadenylated before it is transported to the cytoplasm for translation. Polyadenylation can also occur in the cytoplasm for post-transcriptional regulation, especially in neurons, oocytes and early embryos. Recently, we revealed transcriptome-wide maternal mRNA cytoplasmic re-polyadenylation during the mammalian oocyte-to-embryo transition (OET). However, the mechanism of re-polyadenylation during mammalian OET, including the sites to be re-polyadenylated and the enzymes involved, is still poorly understood. Here, by analyzing the PAIso-seq1 and PAIso-seq2 poly(A) inclusive transcriptome data during OET in mice, rats, pigs, and humans, we reveal conserved re-polyadenylation of mRNA degradation intermediates. These re-polyadenylated mRNA degradation intermediates account for over half of the polyadenylated mRNA during OET in all four species. We find that mRNA degradation intermediates for re-polyadenylation are generated through Btg4-mediated deadenylation in both mouse and human. Interestingly, the poly(A) tails on the re-polyadenylated mRNA degradation intermediates are of different lengths and contain different levels of non-A residues compared to regular polyadenylation sites, suggesting specific regulation and function of these poly(A) tails in mammalian OET. Together, our findings reveal the maternal mRNA degradation intermediates as substrates for conserved cytoplasmic dominant re-polyadenylation during mammalian OET, and uncover the mechanism of production of these mRNA degradation intermediates. These findings provide new insights into mRNA post-transcriptional regulation, and a new direction for the study of mammalian OET.

Sperm-binding regions on bovine egg zona pellucida glycoprotein ZP4 studied in a solid supported form on plastic plate

The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm–oocyte interactions. The bovine ZP consists of the glycoproteins ZP2, ZP3, and ZP4. Sperm-binding mechanisms of the bovine ZP are not yet fully elucidated. In a previous report, we established the expression system of bovine ZP glycoproteins using Sf9 insect cells and found that the ZP3/ZP4 heterocomplex inhibits the binding of sperm to the ZP in a competitive inhibition assay, while ZP2, ZP3, ZP4, the ZP2/ZP3 complex, and the ZP2/ZP4 complex do not exhibit this activity. Here, we show that bovine sperm binds to plastic plates coated with ZP4 in the absence of ZP3. We made a series of ZP4 deletion mutants to study the sperm-binding sites. The N-terminal region, Lys-25 to Asp-136, and the middle region, Ser-290 to Lys-340, of ZP4 exhibit sperm-binding activity. These results suggest that among the three components of bovine ZP glycoproteins, ZP4 contains the major potential sperm-binding sites, and the formation of a multivalent complex is necessary for the sperm-binding activity of ZP4.

Post-Translational Modifications in Oocyte Maturation and Embryo Development

Mammalian oocyte maturation and embryo development are unique biological processes regulated by various modifications. Since de novo mRNA transcription is absent during oocyte meiosis, protein-level regulation, especially post-translational modification (PTM), is crucial. It is known that PTM plays key roles in diverse cellular events such as DNA damage response, chromosome condensation, and cytoskeletal organization during oocyte maturation and embryo development. However, most previous reviews on PTM in oocytes and embryos have only focused on studies of Xenopus laevis or Caenorhabditis elegans eggs. In this review, we will discuss the latest discoveries regarding PTM in mammalian oocytes maturation and embryo development, focusing on phosphorylation, ubiquitination, SUMOylation and Poly(ADP-ribosyl)ation (PARylation). Phosphorylation functions in chromosome condensation and spindle alignment by regulating histone H3, mitogen-activated protein kinases, and some other pathways during mammalian oocyte maturation. Ubiquitination is a three-step enzymatic cascade that facilitates the degradation of proteins, and numerous E3 ubiquitin ligases are involved in modifying substrates and thus regulating oocyte maturation, oocyte-sperm binding, and early embryo development. Through the reversible addition and removal of SUMO (small ubiquitin-related modifier) on lysine residues, SUMOylation affects the cell cycle and DNA damage response in oocytes. As an emerging PTM, PARlation has been shown to not only participate in DNA damage repair, but also mediate asymmetric division of oocyte meiosis. Each of these PTMs and external environments is versatile and contributes to distinct phases during oocyte maturation and embryo development.

Essential shared and species-specific features of mammalian oocyte maturation-associated transcriptome changes impacting oocyte physiology

Oogenesis is a complex process resulting in the production of a truly remarkable cell-the oocyte. Oocytes execute many unique processes and functions such as meiotic segregation of maternal genetic material, and essential life-generating functions after fertilization including post-transcriptional support of essential homeostatic and metabolic processes, and activation and reprogramming of the embryonic genome. An essential goal for understanding female fertility and infertility in mammals is to discover critical features driving the production of quality oocytes, particularly the complex regulation of oocyte maternal mRNAs. We report here the first in-depth meta-analysis of oocyte maturation-associated transcriptome changes, using eight data sets encompassing 94 RNAseq libraries for human, rhesus monkey, mouse, and cow. A majority of maternal mRNAs are regulated in a species-restricted manner, highlighting considerable divergence in oocyte transcriptome handling during maturation. We identified 121 mRNAs changing in relative abundance similarly across all four species (92 of high homology), and 993 (670 high homology) mRNAs regulated similarly in at least three of the four species, corresponding to just 0.84% and 6.9% of mRNAs analyzed. Ingenuity Pathway Analysis (IPA) revealed an association of these shared mRNAs with many shared pathways and functions, most prominently oxidative phosphorylation and mitochondrial function. These shared functions were reinforced further by primate-specific and species-specific DEGs. Thus, correct down-regulation of mRNAs related to oxidative phosphorylation and mitochondrial function is a major shared feature of mammalian oocyte maturation.

Creating an Artificial 3-Dimensional Ovarian Follicle Culture System Using a Microfluidic System

We hypothesized that the creation of a 3-dimensional ovarian follicle, with embedded granulosa and theca cells, would better mimic the environment necessary to support early oocytes, both structurally and hormonally. Using a microfluidic system with controlled flow rates, 3-dimensional two-layer (core and shell) capsules were created. The core consists of murine granulosa cells in 0.8 mg/mL collagen + 0.05% alginate, while the shell is composed of murine theca cells suspended in 2% alginate. Somatic cell viability tests and hormonal assessments (estradiol, progesterone, and androstenedione) were performed on days 1, 6, 13, 20, and 27. Confocal microscopy confirmed appropriate compartmentalization of fluorescently-labeled murine granulosa cells to the inner capsule and theca cells to the outer shell. Greater than 78% of cells present in capsules were alive up to 27 days after collection. Artificially constructed ovarian follicles exhibited intact endocrine function as evidenced by the production of estradiol, progesterone, and androstenedione. Oocytes from primary and early secondary follicles were successfully encapsulated, which maintained size and cellular compartmentalization. This novel microfluidic system successfully encapsulated oocytes from primary and secondary follicles, recapitulating the two-compartment system necessary for the development of the mammalian oocyte. Importantly, this microfluidic system can be easily adapted for sterile, high throughput applications.