Male infertility is an intricate multifactorial disease involving the interplay between genetic and environmental factors. Genetic anomalies account for more than 15% of all male infertility cases; however, diagnosing them exhibits enormous challenges due to variable symptomatic presentations and limited knowledge of gene functions. Therefore, a thorough investigation into gene regulatory networks underlying male reproduction is demanded to improve patient counseling and infertility treatment. In this study, we aimed to identify testis-expressed genes essential for male fertility. We searched public databases, such as the National Center for Biotechnology Information (NCBI), Ensembl genome browser, the Human Protein Atlas (HPA), and the Mammalian Reproductive Genetics Database V2 (MRGDv2), to identify genes predominantly expressed in male reproductive tissues. Genetically engineered mouse lines lacking individual genes of interest were generated using either targeted gene replacement or the CRISPR/Cas9 system. To determine the gene functions, we analyzed fertility, testis weight, testis and epididymis histology, and sperm motility and morphology in adult knockout (KO) male mice. Through the in silico screen, we identified 18 testis-expressed genes, including coiled-coil domain containing 182 (Ccdc182), EF-hand calcium-binding domain 15 (Efcab15), family with sequence similarity 187, member B (Fam187b), family with sequence similarity 24, member A (Fam24a), family with sequence similarity 24, member B (Fam24b), glial cell line derived neurotrophic factor family receptor alpha 2 (Gfra2), GLI pathogenesis-related 1 like 1, 2, and 3 (Glipr1l1-3), interleukin 3 (Il3), IZUMO family member 4 (Izumo4), peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1, retrogene 1 (Pin1rt1), solute carrier family 22 (organic cation transporter), member 16 (Slc22a16), sperm microtubule inner protein 2 (Spmip2), testis expressed 51 (Tex51), transmembrane and coiled-coil domains 2 (Tmco2), and tripartite motif family-like 1 and 2 (Triml1/2). The KO males displayed no obvious health problems, and normal mating behavior, fecundity, testis and epididymis histology, and sperm morphology and motility. Our findings indicate that these 18 testis-expressed genes are individually dispensable for male reproduction in mice. Disseminating such genes would promote our understanding of male reproduction and expedite the discovery of novel key male factors. Although we anticipate that mutations in these genes may not impair fertility in men, their enrichment in male germ cells makes them potential biomarkers for sperm count, quality, and morphological anomalies.

Abstract Freezing has proven to be an ideal means for preserving tissues for molecular research on mammals. Here, we trace the origins, early practices, and rapid growth of frozen tissue collections to better understand the history, science, and people that led to the early development and recent growth of these internationally valuable research resources. Frozen tissue collections grew gradually from rudimentary beginnings in the early 1970s but have expanded rapidly in recent years, providing data for studies on biodiversity, phylogenetics, emerging pathogens, isotopes, and contaminants, among other avenues of research. Over the decades, field collecting and tissue storage have changed significantly. In the mid-1980s, for example, the development of polymerase chain reaction—which allowed researchers to amplify a specific segment of DNA many times over—reinforced the importance of frozen tissue resources to the burgeoning field of molecular genetics. Ultracold mechanical freezers that hold tissues at −80 °C became, and remain, popular because of their ease of use and delayed DNA degradation. Portable liquid nitrogen dewars have evolved from rudimentary metal tanks to compact “dry shippers” with an absorbent liner that can maintain tissues at low temperatures for 30 d without being refilled and are commonplace in the field. Larger, vapor-phase nitrogen cryotanks are becoming increasingly popular for long-term tissue preservation because they safeguard tissues from thermal fluctuation that may be induced by temporary electrical failures. More than 1.5 million tissue samples associated with museum voucher preparations are now preserved in research collections worldwide, allowing for in-depth study of mammalian genetics and investigation of research questions that were unimaginable even a few years ago.

The T/t complex of the mouse attracted many of the major figures of mouse genetics to perform genetic, cytogenetic, physiological, biochemical and molecular biological studies of it. These studies started with the discovery of short tailed mutants (Ts) and recessive lethal developmental mutations (ts) which mapped to the same "locus" in the early 1920s in France. However, due to the non-receptivity of French scientists to genetics, they continued to be studied in mostly Anglophone countries to be joined by a wider international community in the 1970s. These discoveries led to developmental studies of the lethal mutants which provided the origin of mammalian developmental genetics. The fascinating property of transmission ratio distortion (non-50/50 segregation of alleles in offspring of males) elicited tremendous interest. There were false leads (that the region consisted of unusual DNA, that the alleles controlled cell surface antigens on embryonic cells and spermatozoa) and exciting discoveries. This historical review provides a review of this extensive area of research and some of the individuals involved in it.

Antibiotic resistance is a priority public health problem resulting from eco-evolutionary dynamics within microbial communities and their interaction at a mammalian host interface or geographical scale. The links between mammalian host genetics, bacterial gut community, and antimicrobial resistance gene (ARG) content must be better understood in natural populations inhabiting heterogeneous environments. Hybridization, the interbreeding of genetically divergent populations, influences different components of the gut microbial communities. However, its impact on bacterial traits such as antibiotic resistance is unknown. Here, we present that hybridization might shape bacterial communities and ARG occurrence. We used amplicon sequencing to study the gut microbiome and to predict ARG composition in natural populations of house mice (Mus musculus). We compared gastrointestinal bacterial and ARG diversity, composition, and abundance across a gradient of pure and hybrid genotypes in the European House Mouse Hybrid Zone. We observed an increased overall predicted richness of ARG in hybrid mice. We found bacteria-ARG interactions by their co-abundance and detected phenotypes of extreme abundances in hybrid mice at the level of specific bacterial taxa and ARGs, mainly multidrug resistance genes. Our work suggests that mammalian host genetic variation impacts the gut microbiome and chromosomal ARGs. However, it raises further questions on how the mammalian host genetics impact ARGs via microbiome dynamics or environmental covariates.

Genetic analyses of mammalian gametogenesis and fertility have the potential to inform about two important and interrelated clinical areas: infertility and contraception. Here, we address the genetics and genomics underlying gamete formation, productivity and function in the context of reproductive success in mammalian systems, primarily mouse and human. Although much is known about the specific genes and proteins required for meiotic processes and sperm function, we know relatively little about other gametic determinants of overall fertility, such as regulation of gamete numbers, duration of gamete production, and gamete selection and function in fertilization. As fertility is not a binary trait, attention is now appropriately focused on the oligogenic, quantitative aspects of reproduction. Multiparent mouse populations, created by complex crossing strategies, exhibit genetic diversity similar to human populations and will be valuable resources for genetic discovery, helping to overcome current limitations to our knowledge of mammalian reproductive genetics. Finally, we discuss how what we know about the genomics of reproduction can ultimately be brought to the clinic, informing our concepts of human fertility and infertility, and improving assisted reproductive technologies.

Abstract Obesity and adult-onset diabetes result from a complex interaction of multiple genetic, environmental, and behavioral factors. The hypothalamus contains multiple nuclei required for the regulation of metabolic homeostasis. One nucleus, termed the ventromedial hypothalamus (VMH), facilitates glucose homeostasis and metabolic adaptations to challenges such as high fat diet (HFD) and exercise. Using a combination of Drosophila and mouse genetics, we uncovered a novel gene network whose function is required for VMH-regulated whole body metabolism. Using Drosophila to rapidly screen orthologs of genes enriched in the mouse VMH, we identified the gene encoding the Ecdysone Induced Protein 93F (E93; human ortholog, Ligand dependent corepressor-like, Lcorl). Adult flies with neural knockdown of E93 are obese and hyperphagic, with increased energy stores, reduced exercise endurance, and dampened circadian amplitude. These findings reveal a novel role of E93 in metabolism. We found that the knockdown of E93 specifically in myoinhibitory peptide (MIP) and GABAergic neurons are sufficient to recapitulate the phenotype seen in a pan-neuronal E93 knockdown. | In mice, we found that Lcorl, the mammalian orthologue of E93, is highly expressed throughout the entire VMH. We used CRISPR/Cas9 to generate mice harboring both a Lcorl KO and a floxed allele to investigate its function in the whole-body and in a tissue specific manner. Lcorl KO mice are viable and fertile. However, they have disruptions in growth, lipid metabolism, and circadian rhythm. Taken together these data reveal that Lcorl is as a novel regulator of energy metabolism and circadian rhythm. Additionally, this study shows the power of combined drosophila and mammalian genetics in uncovering novel regulators of metabolism. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Global biodiversity is organised into biogeographic regions that comprise distinct biotas. The contemporary factors maintaining differences in species composition between regions are poorly understood. Given evidence that populations with sufficient genetic variation can adapt to fill new habitats, it is surprising that more homogenisation of species assemblages across regions has not occurred. Theory suggests that expansion across biogeographic regions could be limited by reduced adaptive capacity due to demographic variation along environmental gradients, but this possibility has not been empirically explored. Using three independently curated data sets describing continental patterns of mammalian demography and population genetics, we show that populations near biogeographic boundaries have lower effective population sizes and genetic diversity, and are more genetically differentiated. These patterns are consistent with reduced adaptive capacity in areas where one biogeographic region transitions into the next. That these patterns are replicated across mammals suggests they are stable and generalisable in their contribution to long-term limits on biodiversity homogenisation. Understanding the contemporary processes that maintain compositional differences among regional biotas is crucial for our understanding of the current and future organisation of global biodiversity.

PD36-10 THE MAMMALIAN REPRODUCTIVE GENETICS DATABASE, VERSION 2 (MRGDv2)

Animal models are essential tools for modern scientists to conduct biological experiments and investigate their hypotheses in vivo. However, for the past decade, raising the throughput of such animal experiments has been a great challenge. Conventionally, in vivo high-throughput assay was achieved through large-scale mutagen-driven forward genetic screening, which took years to find causal genes. In contrast, reverse genetics accelerated the causal gene identification process, but its throughput was also limited by 2 barriers, that is, the genome modification step and the time-consuming crossing step. Defined as genetics without crossing, next-generation genetics is able to produce gene-modified animals that can be analyzed at the founder generation (F0). This method is or can be accomplished through recent technological advances in gene editing and virus-based efficient gene modifications. Notably, next-generation genetics has accelerated the process of cross-species studies, and it will be a useful technique during animal experiments as it can provide genetic perturbation at an individual level without crossing. In this review, we begin by introducing the history of animal-based high-throughput analysis, with a specific focus on chronobiology. We then describe ways that gene modification efficiency during animal experiments was enhanced and why crossing remained a barrier to reaching higher efficiency. Moreover, we mention the Triple CRISPR as a critical technique for achieving next-generation genetics. Finally, we discuss the potential applications and limitations of next-generation mammalian genetics.

The system-level identification and analysis of molecular and cellular networks in mammals can be accelerated by "next-generation" genetics, which is defined as genetics that can achieve desired genetic makeup in a single generation without any animal crossing. We recently established a highly efficient procedure for producing knock-out (KO) mice using the "Triple-CRISPR" method, which targets a single gene by triple gRNAs in the CRISPR/Cas9 system. This procedure achieved an almost perfect KO efficiency (96-100%). We also established a highly efficient procedure, the "ES-mouse" method, for producing knock-in (KI) mice within a single generation. In this method, ES cells were treated with three inhibitors to keep their potency and then injected into 8-cell-stage embryos. These procedures dramatically shortened the time required to produce KO or KI mice from years down to about 3months. The produced KO and KI mice can also be systematically profiled at a single-cell resolution by the "whole-organ cell profiling," which was realized by tissue-clearing methods, such as CUBIC, and an advanced light-sheet microscopy. The review describes the establishment and application of these technologies above in analyzing the three states (NREM sleep, REM sleep, and awake) of mammalian brains. It also discusses the role of calcium and muscarinic receptors in these states as well as the current challenges and future opportunities in the next-generation mammalian genetics and whole-organ cell profiling for organism-level systems biology.

Rhodococcus equi is a major cause of foal pneumonia and an opportunistic pathogen in immunocompromised humans. While alveolar macrophages constitute the primary replicative niche for R. equi, little is known about how intracellular R. equi is sensed by macrophages. Here, we discovered that in addition to previously characterized pro-inflammatory cytokines (e.g., Tnfa, Il6, Il1b), macrophages infected with R. equi induce a robust type I IFN response, including Ifnb and interferon-stimulated genes (ISGs), similar to the evolutionarily related pathogen, Mycobacterium tuberculosis. Follow up studies using a combination of mammalian and bacterial genetics demonstrated that induction of this type I IFN expression program is largely dependent on the cGAS/STING/TBK1 axis of the cytosolic DNA sensing pathway, suggesting that R. equi perturbs the phagosomal membrane and causes DNA release into the cytosol following phagocytosis. Consistent with this, we found that a population of ~12% of R. equi phagosomes recruits the galectin-3,-8 and -9 danger receptors. Interestingly, neither phagosomal damage nor induction of type I IFN require the R. equi's virulence-associated plasmid. Importantly, R. equi infection of both mice and foals stimulates ISG expression, in organs (mice) and circulating monocytes (foals). By demonstrating that R. equi activates cytosolic DNA sensing in macrophages and elicits type I IFN responses in animal models, our work provides novel insights into how R. equi engages the innate immune system and furthers our understanding how this zoonotic pathogen causes inflammation and disease.

The Short Course in Human and Mammalian Genetics and Genomics (aka the "Short Course" or the "Bar Harbor course") is one of Victor McKusick's landmark contributions to medical genetics. Conceived in 1959 as a way to increase the contribution of genetic advances to medicine, it has directly affected more than 7000 students and 600 participating faculty from around the world. Now, more than 10 years after his death, it continues to be a vibrant disseminator of genetics, and genomics knowledge for medicine, a catalytic agent for ongoing research and a source of collegiality in our field. What an extraordinary gift!

Marsupials represent one of three extant mammalian subclasses with very unique characteristics not shared by other mammals. Most notably, much of the development of neonates immaturely born after a relatively short gestation takes place in the external environment. Among marsupials, the gray short-tailed opossum (Monodelphis domestica; hereafter "the opossum") is one of very few established laboratory models. Due to many biologically unique characteristics and experimentally advantageous features, the opossum is used as a prototype species for basic research on marsupial biology.1,2 However, invivo studies of gene function in the opossum, and thus marsupials in general, lag far behind those of eutherian mammals due to the lack of reliable means to manipulate their genomes. In this study, we describe the successful generation of genome edited opossums by a combination of refined methodologies in reproductive biology and embryo manipulation. We took advantage of the opossum's resemblance to popular rodent models, such as the mouse and rat, in body size and breeding characteristics. First, we established a tractable pipeline of reproductive technologies, from induction of ovulation, timed copulation, and zygote collection to embryo transfer to pseudopregnant females, that warrant an essential platform to manipulate opossum zygotes. Further, we successfully demonstrated the generation of gene knockout opossums at the Tyr locus by microinjection of pronuclear stage zygotes using CRISPR/Cas9 genome editing, along with germline transmission of the edited alleles to the F1 generation. This study provides a critical foundation for venues to expand mammalian reverse genetics into the metatherian subclass.

Long transgenes are often used in mammalian genetics, e.g., to rescue mutations in large genes. In the course of experiments addressing the genetic basis of hybrid sterility caused by meiotic defects in mice bearing different alleles of Prdm9, we discovered that introduction of copy-number variation (CNV) via two independent insertions of long transgenes containing incomplete Prdm9 decreased testicular weight and epididymal sperm count. Transgenic animals displayed increased occurrence of seminiferous tubules with apoptotic cells at 18days postpartum (dpp) corresponding to late meiotic prophase I, but not at 21 dpp. We hypothesized that long transgene insertions could cause asynapsis, but the immunocytochemical data revealed that the adult transgenic testes carried a similar percentage of asynaptic pachytene spermatocytes as the controls. These transgenic spermatocytes displayed less crossovers but similar numbers of unrepaired meiotic breaks. Despite slightly increased frequency of metaphase I spermatocytes with univalent chromosome(s) and reduced numbers of metaphase II spermatocytes, cytological studies did not reveal increased apoptosis in tubules containing the metaphase spermatocytes, but found an increased percentage of tubules carrying apoptotic spermatids. Sperm counts of subfertile animals inversely correlated with the transcription levels of the Psmb1 gene encoded within these two transgenes. The effect of the transgenes was dependent on sex and genetic background. Our results imply that the fertility of transgenic hybrid animals is not compromised by the impaired meiotic synapsis of homologous chromosomes, but can be negatively influenced by the increased expression of the introduced genes.

Scientists from 12 countries met at the International Mammalian Genome Conference (IMGC) to share advances in mammalian genetics and genomics research. The event was held in Strasbourg, France and represents the city’s second time hosting the IMGC. A diverse attendance of pre-doctoral and post-doctoral trainees, young investigators, established researchers, clinicians, bioinformaticians, and computational biologists enjoyed a rich scientific program of 63 oral presentations, 65 posters, and 5 workshops in the fields of epigenetics, system genetics, developmental biology, cancer, human disease modeling, technical advances, and bioinformatics. This report presents selected highlights of this meeting which illustrate how recent advances in mammalian genetic approaches have improved our ability to decipher complex biological mechanisms.

Editorial

Salome Gluecksohn-Waelsch was a pioneer in establishing the field of mammalian developmental genetics, bringing together experimental embryology and genetics at a time when the role of genes in development was far from accepted. She studied in Germany in the 1930s with the renowned experimental embryologist Hans Spemann and then moved to New York City where she spent her entire professional career at Columbia University and the Albert Einstein College of Medicine of Yeshiva University. Her career was remarkable not only for its longevity—she continued experiments well into her 90s—but also for ushering in new ways of approaching developmental biology in mammals. In her studies of theT-complex in mice, she made use of naturally occurring mutations as nature's own experiments that allowed the investigation of the normal role of the genes in the events of morphogenesis. In her later work with the albino chromosomal deletions, she extended her studies to the genetics of physiological traits. Throughout the decades that saw a blossoming of the entire field of genetics, Salome Gluecksohn-Waelsch's work tackling some of the most perplexing problems in mammalian genetics firmly established the mouse as model organism, not only for studying development, but also for the eventual application of molecular biology techniques to development. Her published work is a beautifully coherent and rigorous opus, for which she received many honours. Her influence on a generation of geneticists, developmental biologists and the field of developmental genetics was profound.The life of Salome Gluecksohn–Waelsch spanned a century that suffered the destructive upheaval of two world wars but also saw phenomenal progress in the sciences, including embryology and genetics. At the start of Salome's career, these two fields were far apart and developmental genetics was barely a concept. Along with a few other pioneers, Salome was instrumental in establishing that genes actually had roles in development and in founding the field of mammalian developmental genetics. Her career laid the ground work for the eventual integration of genetic and developmental studies through molecular biology.Salome Gluecksohn–Waelsch published under four different names at different stages of her life and career: Salome Glücksohn, Salome Gluecksohn–Schoenheimer, Salome Gluecksohn–Waelsch, and Salome G. Waelsch. Among her colleagues and friends, she was almost universally known as Salome and so for the purpose of this biographical memoir, I have chosen to refer to her by her first name, out of friendship and respect.

ABSTRACTThe power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.

s of papers presented at the 29th Genetic Society's Mammalian Genetics and Development Workshop held at the UCL Great Ormond Street Institute of Child Health, University College London on Thursday 29th November 2018 - Volume 101

Over 150 scientists from more than 50 research institutions and eight countries attended the 32nd annual meeting of the International Mammalian Genome Society (IMGS) held in Rio Mar, Puerto Rico. Attendees included predoctoral and postdoctoral trainees, junior investigators, clinicians, industry professionals, and established leaders in mammalian genetics and genomics. From November 11–14, major scientific advances in the fields of systems genetics, developmental biology, cancer, human disease modeling, and bioinformatics were showcased in a series of 66 poster and 54 platform presentations. Here we provide an overview of the meeting’s proceedings and summarize the exciting, novel research findings communicated by conference participants that, collectively, are advancing the frontiers of mammalian genetics and genomics.