RNA helicase D1PAS1 resolves R-loops and forms a complex for mouse pachytene piRNA biogenesis required for male fertility.

During meiosis, RNA polymerase II transcribes pachytene piRNA precursors with unusually long and unspliced transcripts from discrete autosomal loci in the mouse genome. Despite the importance of piRNA for male fertility and a well-defined maturation process, the transcriptional machinery remains poorly understood. Here, we document that D1PAS1, an ATP-dependent RNA helicase, is critical for pachytene piRNA expression from multiple genomic loci and subsequent translocation into the cytoplasm to ensure mature piRNA biogenesis.

DIS3 ribonuclease is essential for spermatogenesis and male fertility in mice.

Spermatogonial stem cell (SSC) self-renewal and differentiation provide foundational support for long-term, steady-state spermatogenesis in mammals. Here, we have investigated the essential role of RNA exosome associated DIS3 ribonuclease in maintaining spermatogonial homeostasis and facilitating germ cell differentiation. We have established male germ-cell Dis3 conditional knockout (cKO) mice in which the first and subsequent waves of spermatogenesis are disrupted.

Stromal Pbrm1 mediates chromatin remodeling necessary for embryo implantation in the mouse uterus.

Early gestational loss occurs in approximately 20% of all clinically recognized human pregnancies and is an important cause of morbidity. Either embryonic or maternal defects can cause loss, but a functioning and receptive uterine endometrium is crucial for embryo implantation. We report that the switch/sucrose nonfermentable (SWI/SNF) remodeling complex containing polybromo-1 (PBRM1) and Brahma-related gene 1 (BRG1) is essential for implantation of the embryonic blastocyst on the wall of the uterus in mice.

Degradation and translation of maternal mRNA for embryogenesis.

Maternal mRNAs accumulate during egg growth and must be judiciously degraded or translated to ensure successful development of mammalian embryos. In this review we integrate recent investigations into pathways controlling rapid degradation of maternal mRNAs during the maternal-to-zygotic transition. Degradation is not indiscriminate, and some mRNAs are selectively protected and rapidly translated after fertilization for reprogramming the zygotic genome during early embryogenesis.

Reduced female fertility due to sequestration of RNA Pol II by pervasive transcription in exosome RNase-depleted oocytes.

Perturbing the transcriptome of mammalian oocytes results in meiotic failure. We previously reported that RNA-exosome-associated RNase, EXOSC10, degrades unwanted protein-coding RNA and processes ribosomal RNA to ensure proper oocyte maturation. Here, we establish oocyte-specific knockout mice of another RNA-exosome-associated RNase, DIS3.

Chromatin remodeling of prostaglandin signaling in smooth muscle enables mouse embryo passage through the female reproductive tract.

Early mammalian development occurs during embryo transit of the female reproductive tract. Transport is orchestrated by secreted oviduct fluid, unidirectional beating of epithelial cilia, and smooth muscle contractions. Using gene-edited mice, we document that conditional disruption of a component of the SWI/SNF chromatin remodeling complex in smooth muscle cells prevents transport through the oviduct without perturbing embryogenesis.

Germ cell-specific eIF4E1b regulates maternal mRNA translation to ensure zygotic genome activation.

Translation of maternal mRNAs is detected before transcription of zygotic genes and is essential for mammalian embryo development. How certain maternal mRNAs are selected for translation instead of degradation and how this burst of translation affects zygotic genome activation remain unknown. Using gene-edited mice, we document that the oocyte-specific eukaryotic translation initiation factor 4E family member 1b (eIF4E1b) is the regulator of maternal mRNA expression that ensures subsequent reprogramming of the zygotic genome.

DIS3L2 ribonuclease degrades terminal-uridylated RNA to ensure oocyte maturation and female fertility.

During oocyte development in mice, transcripts accumulate in the growth phase and are subsequently degraded during maturation. At the transition point between growth and maturation, oocytes have an intact nucleus or germinal vesicle (GV), and terminal uridylation labels RNA for degradation in meiosis I. By profiling the transcriptome using single-oocyte long-read PacBio RNA sequencing, we document that a small cohort of mRNAs are polyadenylated after terminal uridylation in GV oocytes [designated uridylated-poly(A) RNA].

RNA exosome ribonuclease DIS3 degrades Pou6f1 to promote mouse pre-implantation cell differentiation.

Mammalian development is precisely controlled by cell differentiation. Identifying new regulators and investigating their interactions provide insight into genetic networks defining pre-implantation development. We established a knockout mouse model of Dis3, an exosome associated ribonuclease.

Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA.

piRNAs are small non-coding RNAs required to maintain genome integrity and preserve RNA homeostasis during male gametogenesis. In murine adult testes, the highest levels of piRNAs are present in the pachytene stage of meiosis, but their mode of action and function remain incompletely understood. We previously reported that BTBD18 binds to 50 pachytene piRNA-producing loci.

Maternal factors regulating preimplantation development in mice.

Mammalian embryogenesis depends on maternal factors accumulated in eggs prior to fertilization and on placental transfers later in gestation. In this review, we focus on initial events when the organism has insufficient newly synthesized embryonic factors to sustain development. These maternal factors regulate preimplantation embryogenesis both uniquely in pronuclear formation, genome reprogramming and cell fate determination and more universally in regulating cell division, transcription and RNA metabolism.

EXOSC10 sculpts the transcriptome during the growth-to-maturation transition in mouse oocytes.

Growing mammalian oocytes accumulate substantial amounts of RNA, most of which is degraded during subsequent meiotic maturation. The growth-to-maturation transition begins with germinal vesicle or nuclear envelope breakdown (GVBD) and is critical for oocyte quality and early development. The molecular machinery responsible for the oocyte transcriptome transition remains unclear.

FIGLA, LHX8 and SOHLH1 transcription factor networks regulate mouse oocyte growth and differentiation.

Germ-cell transcription factors control gene networks that regulate oocyte differentiation and primordial follicle formation during early, postnatal mouse oogenesis. Taking advantage of gene-edited mice lacking transcription factors expressed in female germ cells, we analyzed global gene expression profiles in perinatal ovaries from wildtype, FiglaNull, Lhx8Null and Sohlh1Null mice. Figla deficiency dysregulates expression of meiosis-related genes (e.

Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice.

Spermatogonial stem cells (SSCs) have the dual capacity to self-renew and differentiate into progenitor spermatogonia that develop into mature spermatozoa. Here, we document that preferentially expressed antigen of melanoma family member 12 (PRAMEF12) plays a key role in maintenance of the spermatogenic lineage. In male mice, genetic ablation of Pramef12 arrests spermatogenesis and results in sterility which can be rescued by transgenic expression of Pramef12.

Anchoring cortical granules in the cortex ensures trafficking to the plasma membrane for post-fertilization exocytosis.

Following fertilization, cortical granules exocytose ovastacin, a metalloendopeptidase that cleaves ZP2 in the zona pellucida surrounding mouse eggs to prevent additional sperm binding. Using high- and super-resolution imaging with ovastacin as a fluorescent marker, we characterize cortical granule dynamics at single granule resolution in transgenic mouse eggs. Newly-developed imaging protocols provide an unprecedented view of vesicular dynamics near the plasma membrane in mouse eggs.

Glycan-Independent Gamete Recognition Triggers Egg Zinc Sparks and ZP2 Cleavage to Prevent Polyspermy.

The zona pellucida surrounding ovulated eggs regulates monospermic fertilization necessary for successful development. Using mouse transgenesis, we document that the N terminus of ZP2 is sufficient for sperm binding to the zona matrix and for in vivo fertility. Sperm binding is independent of ZP2 glycans and does not occur after complete cleavage of ZP2 by ovastacin, a zinc metalloendopeptidase stored in egg cortical granules.

Cytoplasmic cleavage of DPPA3 is required for intracellular trafficking and cleavage-stage development in mice.

Degradation of maternal proteins by the ubiquitin-proteasome system (UPS) accompanies the maternal-to-zygotic transition. DPPA3/Stella/PGC7, encoded by a maternal effect gene, is present in the nucleus and cytoplasm of zygotes and has been associated with protecting the female pronucleus from TET3-mediated demethylation. We now report that cytoplasmic DPPA3 is partially cleaved by the ubiquitin-proteasome system and an N-terminus fragment remains in the cytoplasm where it associates with early and re-cycling endosomes.

BTBD18 Regulates a Subset of piRNA-Generating Loci through Transcription Elongation in Mice.

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs essential for animal germ cell development. Despite intense investigation of post-transcriptional processing, chromatin regulators for piRNA biogenesis in mammals remain largely unexplored. Here we document that BTBD18 is a pachytene nuclear protein in mouse testes that occupies a subset of pachytene piRNA-producing loci.

A Unique Egg Cortical Granule Localization Motif Is Required for Ovastacin Sequestration to Prevent Premature ZP2 Cleavage and Ensure Female Fertility in Mice.

Monospermic fertilization is mediated by the extracellular zona pellucida composed of ZP1, ZP2 and ZP3. Sperm bind to the N-terminus of ZP2 which is cleaved after fertilization by ovastacin (encoded by Astl) exocytosed from egg cortical granules to prevent sperm binding. AstlNull mice lack the post-fertilization block to sperm binding and the ability to rescue this phenotype with AstlmCherry transgenic mice confirms the role of ovastacin in providing a definitive block to polyspermy.

Genetic mosaics and time-lapse imaging identify functions of histone H3.3 residues in mouse oocytes and embryos.

During development from oocyte to embryo, genetic programs in mouse germ cells are reshaped by chromatin remodeling to orchestrate the onset of development. Epigenetic modifications of specific amino acid residues of core histones and their isoforms can dramatically alter activation and suppression of gene expression. H3.

ZP2 peptide beads select human sperm in vitro, decoy mouse sperm in vivo, and provide reversible contraception.

Gamete recognition in the female reproductive tract occurs at the surface of the zona pellucida surrounding ovulated eggs. The acellular zona matrix is composed of three (mouse) or four (human) proteins (ZP1 to ZP4), and the amino terminus of ZP2 is the primary sperm-binding ligand. Mouse and human sperm bind, respectively, to recombinant moZP2(35-149) and huZP2(39-154) peptides attached to agarose beads.

Exacting Requirements for Development of the Egg.

The hallmark of successful human reproduction is the fusion between a haploid spermatozoon and a metaphase II oocyte. The generation of such an oocyte involves a series of steps whereby germinal-vesicle oocytes (in which the nuclei are intact) at prophase I are stimulated to resume meiosis and mature to metaphase II, a sequence of events that prepares the oocyte for fertilization. Meiosis in mammalian females entails two reductive divisions of DNA to produce ovulated oocytes that can be fertilized in the oviduct (Figure 1).