Paternal obesity increases metabolic risk in offspring, but whether this risk can be reduced by restoring paternal health before conception remains unresolved. We developed a within-sire induction-and-reversal model in outbred CD1 mice in which high-fat diet (HFD)-exposed males generated offspring before and after transition to an ingredient-matched control diet with voluntary exercise. HFD caused obesity, glucose intolerance, insulin resistance, and extensive remodeling of sperm mRNA, lncRNA, and sncRNA profiles, together with transcriptomic changes in metabolic tissues. Diet and exercise reversal normalized paternal metabolic indices and broadly restored tissue RNA profiles, although sperm retained a limited transcriptional memory of prior HFD exposure. Offspring sired before reversal developed sex-dependent metabolic dysfunction despite control-diet rearing, whereas offspring sired after reversal showed substantial improvement. These findings show that paternal metabolic risk is modifiable before conception and that this reversibility is linked to remodeling of sperm RNA. ( 140 words ). Highlights:Paternal HFD-Ex induces obesity, glucose intolerance and insulin resistance in CD1 malesSperm shows much stronger RNA response than four metabolic organs profiledDiet and exercise reversal restores metabolism and RNA profiles in sperm and four metabolic organs analyzedOffspring metabolic risk is reduced when sires conceive after reversal through diet and exercise intervention. eTOC Blurb:Chen, Magalhaes, et al. show that paternal metabolic recovery before conception remodels sperm RNA and reduces transmission of HFD-associated metabolic risk to offspring in a within-sire mouse model.

Inheritance is the transmission of traits or information from one generation of individuals or cells to the next. Inheritance can occur via two different overall mechanisms—genetic inheritance or epigenetic inheritance. Genetic inheritance refers to the transmission of the genomic sequence on the basis of DNA replication. Epigenetic inheritance refers to the transmission of secondary modifications of the genome or chromatin including DNA methylation or histone modifications on the basis of a well described mechanism of maintenance methylation or a yet to described process of propagation of histone modification patterns. Thus, genetic inheritance and epigenetic inheritance differ with respect to (1) the chemical nature of the heritable information being transmitted (DNA sequence for genetic inheritance versus secondary DNA or chromatin modifications for epigenetic inheritance), and (2) the mechanisms by which the relevant heritable information is propagated (DNA replication for genetic inheritance versus propagation of DNA methylation and/or histone modification patterns for epigenetic inheritance). Together, genetic and epigenetic inheritance provide the mechanisms by which heritable information is transmitted from one generation to the next or from parent to daughter cells.

This article provides a brief overview of Volume 3 of this encyclopedia entitled, “Gametogenesis, Fertilization and Development”—three processes that occur sequentially and are central to reproduction. Together these processes include the production of gametes in each sex (gametogenesis), the union of a male and female gamete (fertilization), and the initial development of the resulting new embryo (development).

The common marmoset (Callithrix jacchus) is a promising animal model for preclinical biomedical research due to genetic, anatomic, and physiological similarity to humans. Assisted reproductive technologies can maximize the potential of captive research marmoset colonies, including preservation and propagation of genetically desirable or modified individuals. The literature on marmoset sperm cryopreservation is sparse, and limited assessments have been applied to the existing protocols that have yet to be fully optimized. In this study, we refined and standardized established cryopreservation components for common marmoset sperm, performed a comprehensive evaluation of frozen-thawed sperm quality, and identified accessible components for widespread adoption. While sperm were significantly less motile following cryopreservation compared to fresh sperm, no significant differences were detected across different cryostorage durations up to 6 months. Moreover, the average post-thaw recovery rates were 66% (total motility) and 36% (progressive motility), exceeding previously reported outcomes. Fresh and frozen-thawed sperm exhibited no significant differences in key structural parameters, including acrosome integrity and DNA fragmentation. The improved marmoset sperm cryopreservation protocol reported here will facilitate the sharing of genetically diverse and/or gene-edited sperm for research and colony management and provide a robust foundation for future studies aimed at enhancing the outcomes of frozen-thawed sperm in this and other NHP species.

Nonhuman primates (NHPs) are in increasing demand as models for preclinical research. The availability of a highly efficient cryopreservation protocol for use with NHP sperm has potential to significantly alleviate this demand and contribute to rapid increases in overall number of NHPs available for biomedical research. Thus, this systematic review assesses the state-of-the-art in NHP sperm cryopreservation and uses meta-analyses to describe the impact of varied cryopreservation approaches on NHP sperm. We searched the literature deposited in PubMed, Scopus, and Web of Science databases through June 2024 to identify data relevant to the effect of sperm cryopreservation on quality of thawed sperm compared to fresh sperm across NHP species. All original NHP studies reporting sperm parameters both before cryopreservation and after thawing were included for analysis. In total, 32 articles were included for qualitative analysis and not surprisingly, all 32 studies demonstrated negative effects of cryopreservation on sperm parameters, Specifically, motility and viability were significantly decreased in thawed NHP sperm. While the method of sperm collection did not have a significant impact on recovery of motile sperm, significant variability was evident between species. Importantly, recovery of motile NHP sperm was greatest following rapid sperm freezing methods. This analysis provides critical insights into future research directions to enhance and optimize cryopreservation protocols in NHPs.

Spermatogenesis is an ongoing differentiation process that occurs in the seminiferous epithelium in the testis in males to produce spermatozoa (sperm) and is sustained by a tissue-specific stem cell termed the "spermatogonial stem cell." Spermatogonial stem cells (SSCs) must maintain the spermatogenic stem cell population via self-renewal while also providing an ongoing supply of cells that enter the terminal differentiation process of spermatogenesis. This article describes current evidence and theories regarding the manner in which a unique subset of developing spermatogonia become allocated to form the SSCs.

Introduction: Retention of source cell-type epigenetic memory may mitigate the potential for induced pluripotent stem cells (iPSCs) to fully achieve transitions in cell fate in vitro. While this may not preclude the use of iPSC-derived somatic cell types for therapeutic applications, it becomes a major concern impacting the potential use of iPSC-derived germline cell types for reproductive applications. The transition from a source somatic cell type to iPSCs and then on to germ-cell like cells (GCLCs) recapitulates two major epigenetic reprogramming events that normally occur during development in vivo—embryonic reprogramming in the epiblast and germline reprogramming in primordial germ cells (PGCs). We examined the extent of epigenetic and transcriptomic memory persisting first during the transition from differentiated source cell types to iPSCs, and then during the transition from iPSCs to PGC-like cells (PGCLCs).Methods: We derived iPSCs from four differentiated mouse cell types including two somatic and two germ cell types and tested the extent to which each resulting iPSC line resembled a) a validated ES cell reference line, and b) their respective source cell types, on the basis of genome-wide gene expression and DNA methylation patterns. We then induced each iPSC line to form PGCLCs, and assessed epigenomic and transcriptomic memory in each compared to endogenous PGCs/M-prospermatogonia.Results: In each iPSC line, we found residual gene expression and epigenetic programming patterns characteristic of the corresponding source differentiated cell type from which each was derived. However, upon deriving PGCLCs, we found very little evidence of lingering epigenetic or transcriptomic memory of the original source cell type.Discussion: This result indicates that derivation of iPSCs and then GCLCs from differentiated source cell types in vitro recapitulates the two-phase epigenetic reprogramming that normally occurs in vivo, and that, to a significant extent, germline cell types derived in vitro from pluripotent cells accurately recapitulate epigenetic programming and gene expression patterns corresponding to equivalent endogenous germ cell types, suggesting that they have the potential to form the basis of in vitro gametogenesis as a useful therapeutic strategy for treatment of infertility.

Endocrine disrupting chemicals (EDCs) such as bisphenol S (BPS) are xenobiotic compounds that can disrupt endocrine signaling due to steric similarities to endogenous hormones. EDCs have been shown to induce disruptions in normal epigenetic programming (epimutations) and differentially expressed genes (DEGs) that predispose disease states. Most interestingly, the prevalence of epimutations following exposure to many EDCs persists over multiple generations. Many studies have described direct and prolonged effects of EDC exposure in animal models, but many questions remain about molecular mechanisms by which EDC-induced epimutations are introduced or subsequently propagated, whether there are cell type-specific susceptibilities to the same EDC, and whether this correlates with differential expression of relevant hormone receptors. We exposed cultured pluripotent (iPS), somatic (Sertoli and granulosa), and primordial germ cell-like (PGCLC) cells to BPS and found that differential incidences of BPS-induced epimutations and DEGs correlated with differential expression of relevant hormone receptors inducing epimutations near relevant hormone response elements in somatic and pluripotent, but not germ cell types. Most interestingly, we found that when iPS cells were exposed to BPS and then induced to differentiate into PGCLCs, the prevalence of epimutations and DEGs was largely retained, however, >90% of the specific epimutations and DEGs were replaced by novel epimutations and DEGs. These results suggest a unique mechanism by which an EDC-induced epimutated state may be propagated transgenerationally.

Despite rapid evolution across eutherian mammals, the X-linked MIR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes ( SLITRK2 and FMR1 ) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked MIR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernible defects, but simultaneous ablation of five clusters containing 19 members of the MIR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility, and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked MIR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the MIR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.

Background: Common marmosets (Callithrix jacchus) are increasingly recognized as valuable nonhuman primates (NHPs) for biomedical research due to their small size and short reproductive cycle and lifespan relative to other NHP species. Maximizing the utility of captive research marmosets, including genetically manipulated animals, will require the use of assisted reproductive techniques (ART) including manipulation, storage, and sharing of marmoset sperm. Here, we identify characteristics of high-quality semen samples and validate a simple method for selecting high-quality sperm. Methods: Computer-assisted sperm analysis (CASA) was used to evaluate sperm quality in semen samples collected from 44 marmosets and assessed the use of the swim-up method for the selection of high-quality sperm was also tested in half the samples as a potential means to optimize in vitro fertilization or intrauterine insemination. Results: For each reference parameter, samples at or below the 5th percentile were categorized as abnormal sperm, while those above the 5th percentile were considered to be normal. Among normal samples, those at or above the 50th percentile were categorized as high-quality. High-quality semen samples exhibited the following characteristics: semen volume >= 30 mu L; sperm count >= 10(7)/ejaculate; total motility >= 35%; and normal morphology >= 5%. Sperm isolated by swim-up exhibited superior sperm progressive motility (19.7% +/- 4.5 vs. 5.6% +/- 2.1; P = 0.01) and normal morphology (13.1 +/- 1.59 vs. 7.65 +/- 1.1; P < 0.001) compared with unselected sperm. Conclusion: This study defines robust, statistically supported reference values for evaluating marmoset semen samples to assist with the identification of optimal sperm donors and the selection of high-quality sperm samples for assisted reproduction. Ultimately, these reference values combined with a validated selection method will contribute to consistent standards for the international sharing of genetically diverse and/or gene-edited marmoset sperm for research and reproduction.

Epigenomics encompasses analyses of a variety of different epigenetic parameters which, collectively, make up the epigenetic programming that dictates cell fate and function. Here, protocols are provided for four different epigenomic methods including whole-genome bisulfite sequencing (WGBS) to assess DNA methylation patterns, chromatin immunoprecipitation-sequencing (ChIP-seq) to assess genomic patterns of either specific histone modifications or bound transcription factors, the assay for transposase-accessible chromatin-sequencing (ATAC-seq) to assess genomic patterns of chromatin accessibility, and high-throughput chromosome conformation capture-sequencing (Hi-C-seq) to assess three-dimensional interactions among distant genomic regions, plus computational methodology to integrate data from those four methodologies using Chromatin State Discovery and Characterization (ChromHMM) to obtain the most comprehensive overall assessment of epigenetic programming.

Analyzing whole-genome bisulfite and related sequencing datasets is a time-intensive process due to the complexity and size of the input raw sequencing files and lengthy read alignment step requiring correction for conversion of all unmethylated Cs to Ts genome-wide. The objective of this study was to modify the read alignment algorithm associated with the whole-genome bisulfite sequencing methylation analysis pipeline (wg-blimp) to shorten the time required to complete this phase while retaining overall read alignment accuracy. Here, we report an update to the recently published pipeline wg-blimp achieved by replacing the use of the bwa-meth aligner with the faster gemBS aligner. This improvement to the wg-blimp pipeline has led to a more than ×7 acceleration in the processing speed of samples when scaled to larger publicly available FASTQ datasets containing 80-160 million reads while maintaining nearly identical accuracy of properly mapped reads when compared with data from the previous pipeline. The modifications to the wg-blimp pipeline reported here merge the speed and accuracy of the gemBS aligner with the comprehensive analysis and data visualization assets of the wg-blimp pipeline to provide a significantly accelerated workflow that can produce high-quality data much more rapidly without compromising read accuracy at the expense of increasing RAM requirements up to 48 GB.

Reconstitution of germ cell fate from pluripotent stem cells provides an opportunity to understand the molecular underpinnings of germ cell development. Here, we established robust methods for induced pluripotent stem cell (iPSC) culture in the common marmoset ( Callithrix jacchus [cj]), allowing stable propagation in an undifferentiated state. Notably, iPSCs cultured on a feeder layer in the presence of a WNT signaling inhibitor upregulated genes related to ubiquitin-dependent protein catabolic processes and enter a permissive state that enables differentiation into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs in vivo. Induction of cjPGCLCs is accompanied by transient upregulation of mesodermal genes, culminating in the establishment of a primate-specific germline transcriptional network. Moreover, cjPGCLCs can be expanded in monolayer while retaining the germline state. Upon co-culture with mouse testicular somatic cells, these cells acquire an early prospermatogonia-like phenotype. Our findings provide a framework for understanding and reconstituting marmoset germ cell development in vitro, thus providing a comparative tool and foundation for a preclinical modeling of human in vitro gametogenesis.

AbstractDespite rapid evolution across eutherian mammals, the X-linkedmiR-506family miRNAs are located in a region flanked by two highly conserved protein-coding genes (Slitrk2andFmr1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linkedmiR-506family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernable defects, but simultaneous ablation of five clusters containing nineteen members of themiR-506family led to reduced male fertility in mice. Despite normal sperm counts, motility and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linkedmiR-506family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that themiR-506family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.Significance StatementThe X-linkedmiR-506family has rapidly evolved in mammals, but their physiological significance remains elusive. Given their abundant and preferential expression in the testis and sperm, these X-linked miRNAs likely play a functional role in spermatogenesis and/or early embryonic development. However, the deletion of either individual miRNA genes or all of the five miRNA clusters encoding 38 mature miRNAs did not cause major fertility defects in mice. When these mutant males were subjected to conditions resembling polyandrous mating, the mutant sperm were much less competitive than the wild-type sperm, rendering the mutant males “functionally sub-fertile”. Our data suggest that themiR-506family of miRNAs regulates sperm competition and the reproductive fitness of the male.

The final data-generation step of genome-wide profiling of any epigenetic parameter typically involves DNA deep sequencing which yields large datasets that must then be computationally analyzed both individually and collectively to comprehensively describe the epigenetic programming that dictates cell fate and function. Here, we describe computational pipelines for analysis of bulk mepigenomic profiling data, including whole-genome bisulfite sequencing (WGBS) to detect DNA methylation patterns, chromatin immunoprecipitation-sequencing (ChIP-seq) to detect genomic patterns of either specific histone modifications or bound transcription factors, the assay for transposase-accessible chromatin-sequencing (ATAC-seq) to detect genomic patterns of chromatin accessibility, and high-throughput chromosome conformation capture-sequencing (Hi-C-seq) to detect 3-dimensional interactions among distant genomic regions. In addition, we describe Chromatin State Discovery and Characterization (ChromHMM) methodology to integrate data from these individual analyses, plus that from RNA-seq analysis of gene expression, to obtain the most comprehensive overall assessment of epigenetic programming associated with gene expression.

New evidence in mice suggests that cells expressing the transcription factor FOXC2 may form a reservoir of quiescent stem cells that contributes to sperm formation.

Because epigenetics is a critical component for gene expression, the hypothesis was tested that DNA methylation alterations are dynamic and continually change throughout gametogenesis to generate the mature sperm. Developmental alterations and stage-specific DNA methylation during gametogenesis from primordial germ cells (PGCs) to mature sperm are investigated. Individual developmental stage germ cells were isolated and analyzed for differential DNA methylation regions (DMRs). The number of DMRs was highest in the first three comparisons with mature PGCs, prospermatogonia, and spermatogonia. The most statistically significant DMRs were present at all stages of development and had variations involving both increases or decreases in DNA methylation. DMR-associated genes were identified and correlated with gene functional categories, pathways, and cellular processes. Observations identified a dynamic cascade of epigenetic changes during development that is dramatic during the early developmental stages. Complex epigenetic alterations are required to regulate genome biology and gene expression during gametogenesis.

The events occurring before and soon after birth appear to impact an individual's health throughout adulthood significantly.The pioneering work of David J.P.Barker studied the nutritional status of 2414 pregnant women and their offspring during the Dutch famine between 1944 and 1945,and convincingly connected undernutrition during pregnancy to the predisposition of offspring to many adult-onset diseases,including cardiovascular disease,aberrant glucose metabolism,hypertension,central obesity,and dyslipidemia.This not only attracted global attention but also con-tributed to the genesis of the term"developmental origins of health and disease"(DOHaD),which emphasizes the contribution of prenatal and perinatal exposure to adverse environments to the etiology of diseases in later childhood and adult life[1].A large number of subsequent clinical epidemiology and animal studies proved that both fetal exposure and infant growth patterns can sig-nificantly affect the occurrence of adult-onset diseases.Given that the phenotypic transmission is often limited to one generation and the phenotypic distribution does not follow Mendel's laws,the underlying mechanism(s)has/have been hypothesized to be epige-netic rather than genetic.

In the developing mammalian testis, only a small proportion of fetal and neonatal prospermatogonia give rise to the foundational pool of spermatogonial stem cells (SSCs). Multiple lines of evidence have suggested the determination of which prospermatogonia give rise to foundational SSCs is not random, but is rather predetermined, such that foundational SSCs are ensured to develop advantageous characteristics such as enhanced genetic integrity. Here I suggest that differential epigenetic programing contributes to the molecular mechanisms by which an early subset of developing prospermatogonia becomes predetermined to form the foundational pool of SSCs. This would include epigenetic programing that promotes active expression of genes needed to develop advantageous characteristics, as well as differential epigenetic priming, which bookmarks genes that comprise the SSC-specific transcriptome to become activated when foundational SSCs appear in the postnatal testis. I suggest that, together, differential epigenetic programing and epigenetic priming contribute to the molecular mechanisms by which an early subset of developing prospermatogonia becomes predetermined to form the foundational pool of SSCs.

Translation of stem cell therapies to the clinic will be most successful following optimization of efficacy and safety in appropriate preclinical model systems. Among available models, nonhuman primates (NHPs) provide the most accurate recapitulation of human anatomy, physiology, genetics and epigenetics. Here, we show that baboon pluripotent cells (PSCs) recapitulate key molecular features of human PSCs with greater accuracy than that found in PSCs from non-primate species such as mice. Specifically, baboon and human PSCs exhibit greater conservation of gene expression patterns, higher sequence and structural homology among pluripotency factors, more equivalent genome-wide patterns of histone and DNA methylation modifications, and similar maintenance of bivalent programming of developmental genes than that found between human and non-primate PSCs.