Role of Mammalian DNA Methyltransferases in Development.

DNA methylation at the 5-position of cytosine (5mC) plays vital roles in mammalian development. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), and the two DNMT families, DNMT3 and DNMT1, are responsible for methylation establishment and maintenance, respectively. Since their discovery, biochemical and structural studies have revealed the key mechanisms underlying how DNMTs catalyze de novo and maintenance DNA methylation. In particular, recent development of low-input genomic and epigenomic technologies has deepened our understanding of DNA methylation regulation in germ lines and early stage embryos. In this review, we first describe the methylation machinery including the DNMTs and their essential cofactors. We then discuss how DNMTs are recruited to or excluded from certain genomic elements. Lastly, we summarize recent understanding of the regulation of DNA methylation dynamics in mammalian germ lines and early embryos with a focus on both mice and humans.

Molecular Mechanisms of Facultative Heterochromatin Formation: An X-Chromosome Perspective.

Facultative heterochromatin (fHC) concerns the developmentally regulated heterochromatinization of different regions of the genome and, in the case of the mammalian X chromosome and imprinted loci, of only one allele of a homologous pair. The formation of fHC participates in the timely repression of genes, by resisting strong trans activators. In this review, we discuss the molecular mechanisms underlying the establishment and maintenance of fHC in mammals using a mouse model. We focus on X-chromosome inactivation (XCI) as a paradigm for fHC but also relate it to genomic imprinting and homeobox (Hox) gene cluster repression. A vital role for noncoding transcription and/or transcripts emerges as the general principle of triggering XCI and canonical imprinting. However, other types of fHC are established through an unknown mechanism, independent of noncoding transcription (Hox clusters and noncanonical imprinting). We also extensively discuss polycomb-group repressive complexes (PRCs), which frequently play a vital role in fHC maintenance.

Sperm bauplan and function and underlying processes of sperm formation and selection.

The spermatozoon is a highly differentiated and polarized cell, with two main structures: the head, containing a haploid nucleus and the acrosomal exocytotic granule, and the flagellum, which generates energy and propels the cell; both structures are connected by the neck. The sperm's main aim is to participate in fertilization, thus activating development. Despite this common bauplan and function, there is an enormous diversity in structure and performance of sperm cells. For example, mammalian spermatozoa may exhibit several head patterns and overall sperm lengths ranging from ∼30 to 350 µm. Mechanisms of transport in the female tract, preparation for fertilization, and recognition of and interaction with the oocyte also show considerable variation. There has been much interest in understanding the origin of this diversity, both in evolutionary terms and in relation to mechanisms underlying sperm differentiation in the testis. Here, relationships between sperm bauplan and function are examined at two levels: first, by analyzing the selective forces that drive changes in sperm structure and physiology to understand the adaptive values of this variation and impact on male reproductive success and second, by examining cellular and molecular mechanisms of sperm formation in the testis that may explain how differentiation can give rise to such a wide array of sperm forms and functions.

A male germ-cell-specific ribosome controls male fertility.

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis1,2. Male germ cell development involves complex translational regulation during sperm formation3. However, it remains unclear whether translation during sperm formation is performed by a specific ribosome. Here we report a ribosome with a specialized nascent polypeptide exit tunnel, RibosomeST, that is assembled with the male germ-cell-specific protein RPL39L, the paralogue of core ribosome (RibosomeCore) protein RPL39. Deletion of RibosomeST in mice causes defective sperm formation, resulting in substantially reduced fertility. Our comparison of single-particle cryo-electron microscopy structures of ribosomes from mouse kidneys and testes indicates that RibosomeST features a ribosomal polypeptide exit tunnel of distinct size and charge states compared with RibosomeCore. RibosomeST predominantly cotranslationally regulates the folding of a subset of male germ-cell-specific proteins that are essential for the formation of sperm. Moreover, we found that specialized functions of RibosomeST were not replaceable by RibosomeCore. Taken together, identification of this sperm-specific ribosome should greatly expand our understanding of ribosome function and tissue-specific regulation of protein expression pattern in mammals.

Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm.

Meiotic recombination and de novo mutation are the two main contributions toward gamete genome diversity, and many questions remain about how an individual human's genome is edited by these two processes. Here, we describe a high-throughput method for single-cell whole-genome analysis that was used to measure the genomic diversity in one individual's gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize nonspecific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High-throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics.

Maintenance of CTCF- and Transcription Factor-Mediated Interactions from the Gametes to the Early Mouse Embryo.

The epigenetic information present in mammalian gametes and whether it is transmitted to the progeny are relatively unknown. We find that many promoters in mouse sperm are occupied by RNA polymerase II (Pol II) and Mediator. The same promoters are accessible in GV and MII oocytes and preimplantation embryos. Sperm distal ATAC-seq sites containing motifs for various transcription factors are conserved in monkeys and humans. ChIP-seq analyses confirm that Foxa1, ERα, and AR occupy distal enhancers in sperm. Accessible sperm enhancers containing H3.3 and H2A.Z are also accessible in oocytes and preimplantation embryos. Furthermore, their interactions with promoters in the gametes persist during early development. Sperm- or oocyte-specific interactions mediated by CTCF and cohesin are only present in the paternal or maternal chromosomes, respectively, in the zygote and 2-cell stages. These interactions converge in both chromosomes by the 8-cell stage. Thus, mammalian gametes contain complex patterns of 3D interactions that can be transmitted to the zygote after fertilization.

Insights into variation in meiosis from 31,228 human sperm genomes.

Meiosis, although essential for reproduction, is also variable and error-prone: rates of chromosome crossover vary among gametes, between the sexes, and among humans of the same sex, and chromosome missegregation leads to abnormal chromosome numbers (aneuploidy)1-8. To study diverse meiotic outcomes and how they covary across chromosomes, gametes and humans, we developed Sperm-seq, a way of simultaneously analysing the genomes of thousands of individual sperm. Here we analyse the genomes of 31,228 human gametes from 20 sperm donors, identifying 813,122 crossovers and 787 aneuploid chromosomes. Sperm donors had aneuploidy rates ranging from 0.01 to 0.05 aneuploidies per gamete; crossovers partially protected chromosomes from nondisjunction at the meiosis I cell division. Some chromosomes and donors underwent more-frequent nondisjunction during meiosis I, and others showed more meiosis II segregation failures. Sperm genomes also manifested many genomic anomalies that could not be explained by simple nondisjunction. Diverse recombination phenotypes-from crossover rates to crossover location and separation, a measure of crossover interference-covaried strongly across individuals and cells. Our results can be incorporated with earlier observations into a unified model in which a core mechanism, the variable physical compaction of meiotic chromosomes, generates interindividual and cell-to-cell variation in diverse meiotic phenotypes.

Sex Determination in the Mammalian Germline.

Sexual reproduction crucially depends on the production of sperm in males and oocytes in females. Both types of gamete arise from the same precursor, the germ cells. We review the events that characterize the development of germ cells during fetal life as they commit to, and prepare for, oogenesis or spermatogenesis. In females, fetal germ cells enter meiosis, whereas in males they delay meiosis and instead lose pluripotency, activate an irreversible program of prospermatogonial differentiation, and temporarily cease dividing. Both pathways involve sex-specific molecular signals from the somatic cells of the developing gonads and a suite of intrinsic receptors, signal transducers, transcription factors, RNA stability factors, and epigenetic modulators that act in complex, interconnected positive and negative regulatory networks. Understanding these networks is important in the contexts of the etiology, diagnosis, and treatment of infertility and gonadal cancers, and in efforts to augment human and animal fertility using stem cell approaches.

Restarting life: fertilization and the transition from meiosis to mitosis.

Fertilization triggers a complex cellular programme that transforms two highly specialized meiotic germ cells, the oocyte and the sperm, into a totipotent mitotic embryo. Linkages between sister chromatids are remodelled to support the switch from reductional meiotic to equational mitotic divisions; the centrosome, which is absent from the egg, is reintroduced; cell division shifts from being extremely asymmetric to symmetric; genomic imprinting is selectively erased and re-established; and protein expression shifts from translational control to transcriptional control. Recent work has started to reveal how this remarkable transition from meiosis to mitosis is achieved.

Shedding light on sperm pHertility.

The acquisition of fertilization capacity by sperm is regulated by intracellular pH (pH(i)), but the transport pathways that regulate pH(i) are not well understood. Lishko et al. (2010) now report that Hv1, the voltage-sensitive proton channel, is present in human sperm and is an important regulator of the functional maturation of sperm.

Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel.

Human spermatozoa are quiescent in the male reproductive system and must undergo activation once introduced into the female reproductive tract. This process is known to require alkalinization of sperm cytoplasm, but the mechanism responsible for transmembrane proton extrusion has remained unknown because of the inability to measure membrane conductance in human sperm. Here, by successfully patch clamping human spermatozoa, we show that proton channel Hv1 is their dominant proton conductance. Hv1 is confined to the principal piece of the sperm flagellum, where it is expressed at unusually high density. Robust flagellar Hv1-dependent proton conductance is activated by membrane depolarization, an alkaline extracellular environment, endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. Hv1 allows only outward transport of protons and is therefore dedicated to inducing intracellular alkalinization and activating spermatozoa. The importance of Hv1 for sperm activation makes it an attractive target for controlling male fertility.

C9orf131 and C10orf120 are not essential for male fertility in humans or mice.

Male infertility affects approximately 7% of childbearing couples and is a major health issue. Although nearly 50% idiopathic infertile men are assumed to have a genetic basis, the underlying causes remain largely unknown in most infertility cases. Here, we report two rare homozygous variants in two previously uncharacterized genes, C9orf131 and C10orf120, identified in two unrelated men with asthenozoospermia. Both genes were predominantly expressed in the testes. Furthermore, C9orf131 and C10orf120 knockout mice were successfully generated using the CRISPR-Cas9 technology. However, both C9orf131-/- and C10orf120-/- adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type mice. No overt differences were found between wild-type, C9orf131-/-, and C10orf120-/- mice regarding testicular/epididymal tissue morphology, sperm count, sperm motility, or sperm morphology. Moreover, TUNEL assays indicated that the number of apoptotic germ cells in testes was not significantly different between the three groups. In summary, these findings suggest that C9orf131 and C10orf120 are redundant genes in male infertility.

Differentiation-linked changes in the biosynthesis and turnover of sphingomyelins in rat male germ cells: Genes involved and effects of testosterone.

In rodents, sphingomyelins (SMs) species with very-long-chain polyunsaturated fatty acid (VLCPUFA) are required for normal spermatogenesis. Data on the expression of enzymes with roles in their biosynthesis and turnover during germ cell differentiation and on possible effects on such expression of testosterone (Tes), known to promote this biological process, were lacking. Here we quantified, in isolated pachytene spermatocytes (PtS), round spermatids (RS), and later spermatids (LS), the mRNA levels from genes encoding ceramide (Cer), glucosylceramide (GlcCer), and SM synthases (Cers3, Gcs, Sms1, and Sms2) and sphingomyelinases (aSmase, nSmase) and assessed products of their activity in cells in culture using nitrobenzoxadiazole (NBD)-labeled substrates and [3H]palmitate as precursor. Transcript levels from Cers3 and Gcs were maximal in PtS. While mRNA levels from Sms1 increased with differentiation in the direction PtS→RS→LS, those from Sms2 increased between PtS and RS but decreased in LS. In turn, the nSmase transcript increased in the PtS→RS→LS order. During incubations with NBD-Cer, spermatocytes produced more GlcCer and SM than did spermatids. In total germ cells cultured for up to 25 h with NBD-SM, not only abundant NBD-Cer but also NBD-GlcCer were formed, demonstrating SM→Cer turnover and Cer recycling. After 20 h with [3H]palmitate, PtS produced [3H]SM and RS formed [3H]SM and [3H]Cer, all containing VLCPUFA, and Tes increased their labeling. In total germ cells, Tes augmented in 5 h the expression of genes with roles in VLCPUFA synthesis, decreased the mRNA from Sms2, and increased that from nSmase. Thus, Tes enhanced or accelerated the metabolic changes occurring to VLCPUFA-SM during germ cell differentiation.

Association between embryo morphokinetic development and intracytoplasmic sperm injection with epididymal sperm via time-lapse imaging.

The objective of this study was to investigate the impact of sperm source on embryo morphokinetics and the clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles by considering the clustering of data (multiple embryos per patient that share a comparable developmental timing). This matched cohort study was performed at a private university-affiliated in vitro fertilization center. Women who underwent ICSI with epididymal sperm between January 2019 and December 2020 (the percutaneous epididymal sperm aspiration group, n = 32 cycles) were matched with women who underwent ICSI with ejaculated sperm because of idiopathic male factor infertility (the male factor infertility [MFI] group, n = 32 cycles) or female infertility (the control group, n = 32 cycles). Embryos were cultured in a time-lapse imaging incubator, and morphokinetic development was recorded and compared among the groups. Significantly slower divisions were observed in embryos derived from epididymal sperm than in those derived from the MFI and control groups. Embryos derived from epididymal sperm had a significantly lower KIDScore (3.1 ± 0.2) than did those derived from ejaculated spermatozoa from the MFI (5.4 ± 0.1) and control (5.6 ± 0.2, p < 0.001) groups. Epididymal sperm-derived embryos showed a significantly greater occurrence of multinucleation (23.2%) than did those derived from ejaculated sperm from the MFI and control groups (2.8% and 3.7%, p < 0.001, respectively). Epididymal sperm-derived embryos were significantly more likely to undergo direct or reverse cleavage (11.1%) than ejaculated sperm-derived embryos in the control group (4.3%, p = 0.001). In conclusion, delayed cell cleavage and increased incidences of blastomere multinucleation and abnormal cleavage patterns are observed when epididymal-derived sperm are used for ICSI.

Microfluidics as an emerging paradigm for assisted reproductive technology: A sperm separation perspective.

Millions of people are subject to infertility worldwide and one in every six people, regardless of gender, experiences infertility at some period in their life, according to the World Health Organization. Assisted reproductive technologies are defined as a set of procedures that can address the infertility issue among couples, culminating in the alleviation of the condition. However, the costly conventional procedures of assisted reproduction and the inherent vagaries of the processes involved represent a setback for its successful implementation. Microfluidics, an emerging tool for processing low-volume samples, have recently started to play a role in infertility diagnosis and treatment. Given its host of benefits, including manipulating cells at the microscale, repeatability, automation, and superior biocompatibility, microfluidics have been adopted for various procedures in assisted reproduction, ranging from sperm sorting and analysis to more advanced processes such as IVF-on-a-chip. In this review, we try to adopt a more holistic approach and cover different uses of microfluidics for a variety of applications, specifically aimed at sperm separation and analysis. We present various sperm separation microfluidic techniques, categorized as natural and non-natural methods. A few of the recent developments in on-chip fertilization are also discussed.

Embryonic development through in vitro fertilization using high-quality bovine sperm separated in a biomimetic cervix environment.

This study is the first to identify bovine blastocysts through in vitro fertilization (IVF) of matured oocytes with a large quantity of high-quality sperm separated from a biomimetic cervix environment. We obtained high-quality sperm in large quantities using an IVF sperm sorting chip (SSC), which could mimic the viscous environment of the bovine cervix during ovulation and facilitates isolation of progressively motile sperm from semen. The viscous environment-on-a-chip was realized by formulating and implementing polyvinylpyrrolidone (PVP)-based solutions for the SSC medium. Sperm separated from the IVF-SSC containing PVP 1.5% showed high motility, normal morphology and high DNA integrity. As a result of IVF, a higher rate of hatching blastocysts, which is the pre-implantation stage, were observed, compared to the conventional swim-up method. Our results may significantly contribute to improving livestock with superior male and female genetic traits, thus overcoming the limitation of artificial insemination based on the superior genetic traits of existing males.

Advances in microfluidic technology for sperm screening and in vitro fertilization.

About 18% of reproductive-age adults worldwide are affected by infertility. In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are widely used assisted reproductive technologies (ARTs) aimed at improving clinical outcomes. Efficient and noninvasive selection and isolation of highly motile sperm with intact DNA are essential for the success of IVF and ICSI and can potentially impact the therapeutic efficacy and the health of the offspring. Compared to traditional methods, microfluidic technology offers significant advantages such as low sample consumption, high efficiency, minimal damage, high integration, similar microenvironment, and high automation, providing a new platform for ARTs. Here, we review the current situation of microfluidic technology in the field of sperm motility screening and evaluation and IVF research. First, we focus on the working principle, structural design, and screening results of sperm selection microfluidic platforms. We then highlight how the multiple steps of the IVF process can be facilitated and integrated into a microfluidic chip, including oocyte capture, sperm collection and isolation, sperm sorting, fertilization, and embryo culture. Ultimately, we summarize how microfluidics can complement and optimize current sperm sorting and IVF protocols, and challenges and possible solutions are discussed.

Resetting Epigenetic Memory by Reprogramming of Histone Modifications in Mammals.

Polycomb group proteins and the related histone modification H3K27me3 can maintain the silencing of key developmental regulators and provide cellular memory. However, how such an epigenetic state is reprogrammed and inherited between generations is poorly understood. Using an ultra-sensitive approach, STAR ChIP-seq, we investigated H3K27me3 across 14 developmental stages along mouse gametogenesis and early development. Interestingly, highly pervasive H3K27me3 is found in regions depleted of transcription and DNA methylation in oocytes. Unexpectedly, we observed extensive loss of promoter H3K27me3 at Hox and other developmental genes upon fertilization. This is accompanied by global erasure of sperm H3K27me3 but inheritance of distal H3K27me3 from oocytes. The resulting allele-specific H3K27me3 patterns persist to blastocysts before being converted to canonical forms in postimplantation embryos, where both H3K4me3/H3K27me3 bivalent promoter marks are restored at developmental genes. Together, these data revealed widespread resetting of epigenetic memory and striking plasticity of epigenome during gametogenesis and early development.

Isoform Switch of TET1 Regulates DNA Demethylation and Mouse Development.

The methylcytosine oxidase TET proteins play important roles in DNA demethylation and development. However, it remains elusive how exactly they target substrates and execute oxidation. Interestingly, we found that, in mice, the full-length TET1 isoform (TET1e) is restricted to early embryos, embryonic stem cells (ESCs), and primordial germ cells (PGCs). By contrast, a short isoform (TET1s) is preferentially expressed in somatic cells, which lacks the N terminus including the CXXC domain, a DNA-binding module that often recognizes CpG islands (CGIs) where TET1 predominantly occupies. Unexpectedly, TET1s can still bind CGIs despite the fact that its global chromatin binding is significantly reduced. Interestingly, global chromatin binding, but not targeted binding at CGIs, is correlated with TET1-mediated demethylation. Finally, mice with exclusive expression of Tet1s failed to erase imprints in PGCs and displayed developmental defects in progeny. These data show that isoform switch of TET1 regulates epigenetic memory erasure and mouse development.