The establishment of cell type-specific chromatin landscapes is essential for cellular identity, but how these landscapes are generated remains poorly understood. Here, we demonstrate that the chromatin remodeler SMARCA5 establishes epigenetic priming that is required for retinoic acid (RA)-induced differentiation in the male germline. Germ cell-specific deletion of Smarca5 results in a complete loss of differentiating spermatogonia, phenocopying vitamin A-deficient mice that lack RA signaling. During the perinatal transition from prospermatogonia to undifferentiated spermatogonia, SMARCA5 is recruited to binding sites of the transcription factor DMRT1, which are located at distal putative enhancers and promoters of germline genes. The SMARCA5-DMRT1 pioneer complex establishes chromatin accessibility at these loci, generating poised enhancers and promoters that serve as RA receptor (RAR)-binding sites. Thus, SMARCA5 licenses transcriptional responses to RA that enable spermatogenic differentiation. Our findings uncover a mechanism linking pioneer factor activity to external signal responsiveness.

In vitro fertilization (IVF) is a noncoital method of conception used to treat human infertility. Although IVF is viewed as largely safe, it is associated with adverse outcomes in the fetus, placenta, and adult offspring. Because studies focusing on the effect of IVF on the male reproductive system are limited, we used a mouse model to assess the morphological and molecular effects of IVF on male offspring. We evaluated 3 developmental stages: 18.5-day fetuses and 12- and 39-week-old adults. Regardless of age, we observed changes in testicular-to-body weight ratios, serum testosterone levels, testicular morphology, gene expression, and DNA methylation. Also, sperm showed changes in morphology and DNA methylation. To assess multigenerational phenotypes, we mated IVF-conceived and naturally conceived males with wild-type females. Offspring from IVF males exhibited decreased fetal-to-placental weight ratios and changes in placenta gene expression and morphology regardless of sex. At 12 weeks of age, offspring showed higher body weights and differences in glucose, triglyceride, insulin, total cholesterol, HDL-C, and LDL/VLDL-C levels. Both sexes showed changes in gene expression in liver, testes, and ovaries and decreased global DNA methylation. Collectively, our findings demonstrate that male IVF offspring exhibit abnormal testicular and sperm morphology and molecular alterations with a multigenerational impact.

Female mammals are born with a finite pool of non-growing oocytes (NGOs) housed in primordial follicles, which form the ovarian reserve that determines reproductive lifespan. Mechanisms underlying the reserve's long-term maintenance and subsequent follicular activation remain elusive. Using total RNA sequencing and de novo transcriptome assembly, we first captured the comprehensive oocyte transcriptome across perinatal oogenesis in mice. We show that NGOs establish accessible chromatin at gene regulatory elements-including promoters and enhancers-partly driven by newly evolved endogenous retroviruses (ERVs). In NGOs, epigenetic priming for follicular activation involves prior loading of transcription factors TCF3 and TCF12 and non-phosphorylated form of RNA polymerase II at these sites. This primed state is counteracted by repression via Polycomb Repressive Complex 1-mediated H2AK119 ubiquitylation. We propose that ERV-mediated epigenetic priming underlies the ovarian reserve's long-term maintenance and establishes a transcriptionally competent yet repressive configuration that enables rapid gene activation upon oocyte growth.

As transposable elements (TEs) coevolved with the host genome, the host genome exploited TEs as functional regulatory elements. What remains largely unknown are how the activity of TEs, namely, endogenous retroviruses (ERVs), are regulated and how TEs evolved in the germline. Here we show that KRAB domain-containing zinc-finger proteins (KZFPs), which are highly expressed in mitotically dividing spermatogonia, bind to suppressed ERVs that function following entry into meiosis as active enhancers. These features are observed for independently evolved KZFPs and ERVs in mice and humans, i.e., are evolutionarily conserved in mammals. Further, we show that meiotic sex chromosome inactivation (MSCI) antagonizes the coevolution of KZFPs and ERVs in mammals. Our study uncovers a mechanism by which KZFPs regulate ERVs to sculpt germline transcriptomes. We propose that epigenetic programming in the mammalian germline during the mitosis-to-meiosis transition facilitates coevolution of KZFPs and TEs on autosomes and is antagonized by MSCI.

Female reproductive lifespan is defined by long-lived, non-growing oocytes (NGOs) that comprise the ovarian reserve. NGOs are assumed to acquire the epigenetic marks that will define the early embryo only after they exit the ovarian reserve and become activated for growth. Contrary to this dogma, we show that mouse NGOs possess abundant histone modifications that both underlie maintenance of the ovarian reserve and prime the epigenome of growing oocytes for early embryogenesis. As NGOs are established around birth, Polycomb Repressive Complex 1 (PRC1) mediates abundant H2AK119 ubiquitylation and reprograms the H3K27 acetylation landscape, which is essential to maintain the ovarian reserve. Importantly, the PRC1-driven epigenetic state of NGOs provides a blueprint for subsequent generation of a PRC2-catalyzed H3K27 trimethylation profile in growing oocytes that is characterized by broad domains and DNA methylation-independent imprints that are transmitted to the embryo. Thus, Polycomb complexes play pivotal roles in priming the NGO epigenome for oocyte maturation and early embryogenesis.

The ovarian reserve defines female reproductive lifespan, which in humans spans decades due to the maintenance of meiotic arrest in non-growing oocytes (NGOs) residing in primordial follicles. Unknown is how the chromatin state of NGOs is established to enable long-term maintenance of the ovarian reserve. Here, we show that a chromatin remodeler, CHD4, a member of the Nucleosome Remodeling and Deacetylase (NuRD) complex, establishes chromatin states required for formation and maintenance of the ovarian reserve. Conditional loss of CHD4 in perinatal mouse oocytes results in acute death of NGOs and depletion of the ovarian reserve. CHD4 establishes closed chromatin at regulatory elements of pro-apoptotic genes to prevent cell death and at specific genes required for meiotic prophase I to facilitate the transition from meiotic prophase I oocytes to meiotically-arrested NGOs. In male germ cells, CHD4 establishes closed chromatin at the regulatory elements of pro-apoptotic genes, allowing germ cell survival. These results demonstrate a role for CHD4 in defining a chromatin state that ensures germ cell survival, thereby enabling the long-term maintenance of both female and male germ cells.

STUDY QUESTION Does trophectoderm biopsy (TEBx) of blastocysts for preimplantation genetic testing in the clinic affect normal placental and embryo development and offspring metabolic outcomes in a mouse model? SUMMARY ANSWER TEBx impacts placental and embryonic health during early development, with some alterations resolving and others worsening later in development and triggering metabolic changes in adult offspring. WHAT IS KNOWN ALREADY Previous studies have not assessed the epigenetic and morphological impacts of TEBx either in human populations or in animal models. STUDY DESIGN, SIZE, DURATION We employed a mouse model to identify the effects of TEBx during IVF. Three groups were assessed: naturally conceived (Naturals), IVF, and IVF + TEBx, at two developmental timepoints: embryonic day (E)12.5 (n = 40/Naturals, n = 36/IVF, and n = 36/IVF + TEBx) and E18.5 (n = 42/Naturals, n = 30/IVF, and n = 35/IVF + TEBx). Additionally, to mimic clinical practice, we assessed a fourth group: IVF + TEBx + Vitrification (Vit) at E12.5 (n = 29) that combines TEBx and vitrification. To assess the effect of TEBx in offspring health, we characterized a 12-week-old cohort (n = 24/Naturals, n = 25/IVF and n = 25/IVF + TEBx). PARTICIPANTS/MATERIALS, SETTING, METHODS Our mouse model used CF-1 females as egg donors and SJL/B6 males as sperm donors. IVF, TEBx, and vitrification were performed using standardized methods. Placenta morphology was evaluated by hematoxylin-eosin staining, in situ hybridization using Tpbpa as a junctional zone marker and immunohistochemistry using CD34 fetal endothelial cell markers. For molecular analysis of placentas and embryos, DNA methylation was analyzed using pyrosequencing, luminometric methylation assay, and chip array technology. Expression patterns were ascertained by RNA sequencing. Triglycerides, total cholesterol, high-, low-, and very low-density lipoprotein, insulin, and glucose were determined in the 12-week-old cohort using commercially available kits. MAIN RESULTS AND THE ROLE OF CHANCE We observed that at E12.5, IVF + TEBx had a worse outcome in terms of changes in DNA methylation and differential gene expression in placentas and whole embryos compared with IVF alone and compared with Naturals. These changes were reflected in alterations in placental morphology and blood vessel density. At E18.5, early molecular changes in fetuses were maintained or exacerbated. With respect to placentas, the molecular and morphological changes, although different compared to Naturals, were equivalent to the IVF group, except for changes in blood vessel density, which persisted. Of note is that most differences were sex specific. We conclude that TEBx has more detrimental effects in mid-gestation placental and embryonic tissues, with alterations in embryonic tissues persisting or worsening in later developmental stages compared to IVF alone, and the addition of vitrification after TEBx results in more pronounced and potentially detrimental epigenetic effects: these changes are significantly different compared to Naturals. Finally, we observed that 12-week IVF + TEBx offspring, regardless of sex, showed higher glucose, insulin, triglycerides, lower total cholesterol, and lower high-density lipoprotein compared to IVF and Naturals, with only males having higher body weight compared to IVF and Naturals. Our findings in a mouse model additionally support the need for more studies to assess the impact of new procedures in ART to ensure healthy pregnancies and offspring outcomes. LARGE SCALE DATA Data reported in this work have been deposited in the NCBI Gene Expression Omnibus under accession number GSE225318. LIMITATIONS, REASONS FOR CAUTION This study was performed using a mouse model that mimics many clinical IVF procedures and outcomes observed in humans, where studies on early embryos are not possible. WIDER IMPLICATIONS OF THE FINDINGS This study highlights the importance of assaying new procedures used in ART to assess their impact on placenta and embryo development, and offspring metabolic outcomes. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by a National Centers for Translational Research in Reproduction and Infertility grant P50 HD068157-06A1 (M.S.B., C.C., M.M.), Ruth L. Kirschstein National Service Award Individual Postdoctoral Fellowship F32 HD107914 (E.A.R.-C.) and F32 HD089623 (L.A.V.), and National Institutes of Health Training program in Cell and Molecular Biology T32 GM007229 (C.N.H.). No conflict of interest.

AbstractZygotic genome activation (ZGA), the first transcription event following fertilization, kickstarts the embryonic program that takes over the control of early development from the maternal products. How ZGA occurs, especially in mammals, is poorly understood due to the limited amount of research materials. With the rapid development of single-cell and low-input technologies, remarkable progress made in the past decade has unveiled dramatic transitions of the epigenomes, transcriptomes, proteomes, and metabolomes associated with ZGA. Moreover, functional investigations are yielding insights into the key regulators of ZGA, among which two major classes of players are emerging: licensors and specifiers. Licensors would control the permission of transcription and its timing during ZGA. Accumulating evidence suggests that such licensors of ZGA include regulators of the transcription apparatus and nuclear gatekeepers. Specifiers would instruct the activation of specific genes during ZGA. These specifiers include key transcription factors present at this stage, often facilitated by epigenetic regulators. Based on data primarily from mammals but also results from other species, we discuss in this review how recent research sheds light on the molecular regulation of ZGA and its executors, including the licensors and specifiers.

Zygotic genome activation (ZGA) activates the quiescent genome to enable the maternal-to-zygotic transition 1 , 2 . However, the identity of transcription factors that underlie mammalian ZGA in vivo remains elusive. Here we show that OBOX, a PRD-like homeobox domain transcription factor family (OBOX1–OBOX8) 3 – 5 , are key regulators of mouse ZGA. Mice deficient for maternally transcribed Obox1 / 2 / 5 / 7 and zygotically expressed Obox3 / 4 had a two-cell to four-cell arrest, accompanied by impaired ZGA. The Obox knockout defects could be rescued by restoring either maternal and zygotic OBOX, which suggests that maternal and zygotic OBOX redundantly support embryonic development. Chromatin-binding analysis showed that Obox knockout preferentially affected OBOX-binding targets. Mechanistically, OBOX facilitated the ‘preconfiguration’ of RNA polymerase II, as the polymerase relocated from the initial one-cell binding targets to ZGA gene promoters and distal enhancers. Impaired polymerase II preconfiguration in Obox mutants was accompanied by defective ZGA and chromatin accessibility transition, as well as aberrant activation of one-cell polymerase II targets. Finally, ectopic expression of OBOX activated ZGA genes and MERVL repeats in mouse embryonic stem cells. These data thus demonstrate that OBOX regulates mouse ZGA and early embryogenesis.

How histone modifications regulate changes in gene expression during preimplantation development in any species remains poorly understood. Using CUT&Tag to overcome limiting amounts of biological material, we profiled two activating (H3K4me3 and H3K27ac) and two repressive (H3K9me3 and H3K27me3) marks in bovine oocytes, 2-, 4-, and 8-cell embryos, morula, blastocysts, inner cell mass, and trophectoderm. In oocytes, broad bivalent domains mark developmental genes, and prior to embryonic genome activation (EGA), H3K9me3 and H3K27me3 co-occupy gene bodies, suggesting a global mechanism for transcription repression. During EGA, chromatin accessibility is established before canonical H3K4me3 and H3K27ac signatures. Embryonic transcription is required for this remodeling, indicating that maternally provided products alone are insufficient for reprogramming. Last, H3K27me3 plays a major role in restriction of cellular potency, as blastocyst lineages are defined by differential polycomb repression and transcription factor activity. Notably, inferred regulators of EGA and blastocyst formation strongly resemble those described in humans, as opposed to mice. These similarities suggest that cattle are a better model than rodents to investigate the molecular basis of human preimplantation development.

After fertilization, remodeling of the oocyte and sperm genomes is essential to convert these highly differentiated and transcriptionally quiescent cells into early cleavage-stage blastomeres that are transcriptionally active and totipotent. This developmental transition is accompanied by cell cycle adaptation, such as lengthening or shortening of the gap phases G1 and G2. However, regulation of these cell cycle changes is poorly understood, especially in mammals. Checkpoint kinase 1 (CHK1) is a protein kinase that regulates cell cycle progression in somatic cells. Here, we show that CHK1 regulates cell cycle progression in early mouse embryos by restraining CDK1 kinase activity due to CDC25A phosphatase degradation. CHK1 kinase also ensures the long G2 phase needed for genome activation and reprogramming gene expression in two-cell stage mouse embryos. Finally, Chk1 depletion leads to DNA damage and chromosome segregation errors that result in aneuploidy and infertility.

Enhancer activation by the MLR family of H3K4 mono-methyltransferases requires proper recognition of histones for the deposition of the mono-methyl mark. MLR proteins contain two clusters of PHD zinc finger domains implicated in chromatin regulation. The second cluster is the most highly conserved, preserved as an ancient three finger functional unit throughout evolution. Studies of the isolated 3rd PHD finger within this cluster suggested specificity for the H4 [aa16-20] tail region. We determined the histone binding properties of the full three PHD finger cluster b module (PHDb) from the Drosophila Cmi protein which revealed unexpected recognition of an extended region of H3. Importantly, the zinc finger spacer separating the first two PHDb fingers from the third is critical for proper alignment and coordination among fingers for maximal histone engagement. Human homologs, MLL3 and MLL4, also show conservation of H3 binding, expanding current views of histone recognition for this class of proteins. We further implicate chromatin remodeling by the SWI/SNF complex as a possible mechanism for the accessibility of PHDb to globular regions of histone H3 beyond the tail region. Our results suggest a two-tail histone recognition mechanism by the conserved PHDb domain involving a flexible hinge to promote interdomain coordination.

The ovarian reserve defines female reproductive lifespan, which in humans spans decades. The ovarian reserve consists of oocytes residing in primordial follicles arrested in meiotic prophase I and is maintained independent of DNA replication and cell proliferation, thereby lacking stem cell-based maintenance. Largely unknown is how cellular states of the ovarian reserve are established and maintained for decades. Our recent study revealed that a distinct chromatin state is established during ovarian reserve formation in mice, uncovering a novel window of epigenetic programming in female germline development. We showed that an epigenetic regulator, Polycomb Repressive Complex 1 (PRC1), establishes a repressive chromatin state in perinatal mouse oocytes that is essential for prophase I-arrested oocytes to form the ovarian reserve. Here we discuss the biological roles and mechanisms underlying epigenetic programming in ovarian reserve formation, highlighting current knowledge gaps and emerging research areas in female reproductive biology.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression after transcription. miRNAs are present in transcriptionally quiescent full-grown oocytes and preimplantation embryos that display a low level of transcription prior to embryonic genome activation. The role of miRNAs, if any, in preimplantation development is not known. The temporal pattern of expression of miRNAs during bovine preimplantation development was determined by small RNA-sequencing using eggs and preimplantation embryos (1-cell, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst). Embryos cultured in the presence of α-amanitin, which permitted the distinguishing of maternal miRNAs from embryonic miRNAs, indicated that embryonic miRNA expression was first detected at the two-cell stage but dramatically increased during the morula and blastocyst stages. Targeting DGCR8 by a small-interfering RNA/morpholino approach revealed a role for miRNAs in the morula-to-blastocyst transition. Knockdown of DGCR8 not only inhibited expression of embryonically expressed miRNAs but also inhibited the morula-to-blastocyst transition. In addition, RNA-sequencing identified an increased relative abundance of messenger RNAs potentially targeted by embryonic miRNAs in DGCR8-knockdown embryos when compared with controls. Results from these experiments implicate an essential role for miRNAs in bovine preimplantation embryo development.

The ovarian reserve defines the female reproductive lifespan, which in humans spans decades due to robust maintenance of meiotic arrest in oocytes residing in primordial follicles. Epigenetic reprogramming, including DNA demethylation, accompanies meiotic entry, but the chromatin changes that underpin the generation and preservation of ovarian reserves are poorly defined. We report that the Polycomb Repressive Complex 1 (PRC1) establishes repressive chromatin states in perinatal mouse oocytes that directly suppress the gene expression program of meiotic prophase-I and thereby enable the transition to dictyate arrest. PRC1 dysfuction causes depletion of the ovarian reserve and leads to premature ovarian failure. Our study demonstrates a fundamental role for PRC1-mediated gene silencing in female reproductive lifespan, and reveals a critical window of epigenetic programming required to establish ovarian reserve.

It remains poorly understood how histone modifications regulate changes in gene expression during preimplantation development. Using a bovine model, we profiled changes in two activating (H3K4me3 and H3K27ac) and two repressive (H3K9me3 and H3K27me3) marks in oocytes, 2-, 4- and 8-cell embryos (that developed in the presence or absence of the transcription inhibitor a-amanitin), morula, blastocysts, inner cell mass cells and trophectoderm. In oocytes, we find that broad bivalent domains of H3K4me3 and H3K27me3 mark developmental genes, and that prior to genome activation, H3K9me3 and H3K27me3 co-occupy gene bodies. During genome activation, chromatin accessibility is established before canonical H3K4me3 and H3K27ac, and although embryonic transcription is required for this active remodeling, it is dispensable for maintenance of pre-established histone marks. Finally, blastocyst lineages are defined by differential Polycomb repression and transcription factor activity. Overall, these results further support the use of bovine as a more appropriate model system than the mouse to study genome activation and cell lineage specification during human preimplantation development.

Mammalian oocytes are arrested at meiotic prophase I. The dual-specificity phosphatase CDC25B is essential for cyclin-dependent kinase 1 (CDK1) activation that drives resumption of meiosis. CDC25B reverses the inhibitory effect of the protein kinases WEE1 and MYT1 on CDK1 activation. Cdc25b-/- female mice are infertile because oocytes cannot activate CDK1. To identify a role for CDC25B following resumption of meiosis, we restored CDK1 activation in Cdc25b-/- oocytes by inhibiting WEE1 and MYT1, or expressing EGFP-CDC25A or constitutively active EGFP-CDK1 from microinjected complementary RNAs. Forced CDK1 activation in Cdc25b-/- oocytes allowed resumption of meiosis, but oocytes mostly arrested at metaphase I (MI) with intact spindles. Similarly, approximately a third of Cdc25b+/- oocytes with a reduced amount of CDC25B arrested in MI. MI-arrested Cdc25b-/- oocytes also displayed a transient decrease in CDK1 activity similar to Cdc25b+/+ oocytes during the MI-MII transition, whereas Cdc25b+/- oocytes exhibited only a partial anaphase-promoting complex/cyclosome activation and anaphase I entry. Thus, CDC25B is necessary for the resumption of meiosis and the MI-MII transition.

Assisted Reproductive Technologies (ART) employ gamete/embryo handling and culture in vitro to produce offspring. ART pregnancies have an increased risk of low birth weight, abnormal placentation, pregnancy complications, and imprinting disorders. Embryo culture induces low birth weight, abnormal placental morphology, and lower levels of DNA methylation in placentas in a mouse model of ART. Whether preimplantation embryos at specific stages of development are more susceptible to these perturbations remains unresolved. Accordingly, we performed embryo culture for several discrete periods of preimplantation development and following embryo transfer, assessed fetal and placental outcomes at term. We observed a reduction in fetal:placental ratio associated with two distinct windows of preimplantation embryo development, one prior to the morula stage and the other from the morula to blastocyst stage, whereas placental morphological abnormalities and reduced imprinting control region methylation were only associated with culture prior to the morula stage. Extended culture to the blastocyst stage also induces additional placental DNA methylation changes compared to embryos transferred at the morula stage, and female concepti exhibited a higher loss of DNA methylation than males. By identifying specific developmental windows of susceptibility, this study provides a framework to optimize further culture conditions to minimize risks associated with ART pregnancies.

Assembling egg cells involves cytoplasmic and nuclear processes that together enable embryonic development. A study now defines a set of transcription factors required for cytoplasmic, but not key nuclear, processes.