Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis.

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the "metabolism" and the "metabolism of lipids" pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction.

Trio-binning of a hinny refines the comparative organization of the horse and donkey X chromosomes and reveals novel species-specific features.

We generated single haplotype assemblies from a hinny hybrid which significantly improved the gapless contiguity for horse and donkey autosomal genomes and the X chromosomes. We added over 15 Mb of missing sequence to both X chromosomes, 60 Mb to donkey autosomes and corrected numerous errors in donkey and some in horse reference genomes. We resolved functionally important X-linked repeats: the DXZ4 macrosatellite and ampliconic Equine Testis Specific Transcript Y7 (ETSTY7). We pinpointed the location of the pseudoautosomal boundaries (PAB) and determined the size of the horse (1.8 Mb) and donkey (1.88 Mb) pseudoautosomal regions (PARs). We discovered distinct differences in horse and donkey PABs: a testis-expressed gene, XKR3Y, spans horse PAB with exons1-2 located in Y and exon3 in the X-Y PAR, whereas the donkey XKR3Y is Y-specific. DXZ4 had a similar ~ 8 kb monomer in both species with 10 copies in horse and 20 in donkey. We assigned hundreds of copies of ETSTY7, a sequence horizontally transferred from Parascaris and massively amplified in equids, to horse and donkey X chromosomes and three autosomes. The findings and products contribute to molecular studies of equid biology and advance research on X-linked conditions, sex chromosome regulation and evolution in equids.

Single-haplotype comparative genomics provides insights into lineage-specific structural variation during cat evolution.

The role of structurally dynamic genomic regions in speciation is poorly understood due to challenges inherent in diploid genome assembly. Here we reconstructed the evolutionary dynamics of structural variation in five cat species by phasing the genomes of three interspecies F1 hybrids to generate near-gapless single-haplotype assemblies. We discerned that cat genomes have a paucity of segmental duplications relative to great apes, explaining their remarkable karyotypic stability. X chromosomes were hotspots of structural variation, including enrichment with inversions in a large recombination desert with characteristics of a supergene. The X-linked macrosatellite DXZ4 evolves more rapidly than 99.5% of the genome clarifying its role in felid hybrid incompatibility. Resolved sensory gene repertoires revealed functional copy number changes associated with ecomorphological adaptations, sociality and domestication. This study highlights the value of gapless genomes to reveal structural mechanisms underpinning karyotypic evolution, reproductive isolation and ecological niche adaptation.

Centuries of genome instability and evolution in soft-shell clam, Mya arenaria, bivalve transmissible neoplasia.

Transmissible cancers are infectious parasitic clones that metastasize to new hosts, living past the death of the founder animal in which the cancer initiated. We investigated the evolutionary history of a cancer lineage that has spread though the soft-shell clam (Mya arenaria) population by assembling a chromosome-scale soft-shell clam reference genome and characterizing somatic mutations in transmissible cancer. We observe high mutation density, widespread copy-number gain, structural rearrangement, loss of heterozygosity, variable telomere lengths, mitochondrial genome expansion and transposable element activity, all indicative of an unstable cancer genome. We also discover a previously unreported mutational signature associated with overexpression of an error-prone polymerase and use this to estimate the lineage to be >200 years old. Our study reveals the ability for an invertebrate cancer lineage to survive for centuries while its genome continues to structurally mutate, likely contributing to the evolution of this lineage as a parasitic cancer.

Genetic architecture and polygenic risk score prediction of degenerative suspensory ligament desmitis (DSLD) in the Peruvian Horse.

Introduction: Spontaneous rupture of tendons and ligaments is common in several species including humans. In horses, degenerative suspensory ligament desmitis (DSLD) is an important acquired idiopathic disease of a major energy-storing tendon-like structure. DSLD risk is increased in several breeds, including the Peruvian Horse. Affected horses have often been used for breeding before the disease is apparent. Breed predisposition suggests a substantial genetic contribution, but heritability and genetic architecture of DSLD have not been determined. Methods: To identify genomic regions associated with DSLD, we recruited a reference population of 183 Peruvian Horses, phenotyped as DSLD cases or controls, and undertook a genome-wide association study (GWAS), a regional window variance analysis using local genomic partitioning, a signatures of selection (SOS) analysis, and polygenic risk score (PRS) prediction of DSLD risk. We also estimated trait heritability from pedigrees. Results: Heritability was estimated in a population of 1,927 Peruvian horses at 0.22 ± 0.08. After establishing a permutation-based threshold for genome-wide significance, 151 DSLD risk single nucleotide polymorphisms (SNPs) were identified by GWAS. Multiple regions of enriched local heritability were identified across the genome, with strong enrichment signals on chromosomes 1, 2, 6, 10, 13, 16, 18, 22, and the X chromosome. With SOS analysis, there were 66 genes with a selection signature in DSLD cases that was not present in the control group that included the TGFB3 gene. Pathways enriched in DSLD cases included proteoglycan metabolism, extracellular matrix homeostasis, and signal transduction pathways that included the hedgehog signaling pathway. The best PRS predictive performance was obtained when we fitted 1% of top SNPs using a Bayesian Ridge Regression model which achieved the highest mean of R2 on both the probit and logit liability scales, indicating a strong predictive performance. Discussion: We conclude that within-breed GWAS of DSLD in the Peruvian Horse has further confirmed that moderate heritability and a polygenic architecture underlies the trait and identified multiple DSLD SNP associations in novel tendinopathy candidate genes influencing disease risk. Pathways enriched with DSLD risk variants include ones that influence glycosaminoglycan metabolism, extracellular matrix homeostasis, signal transduction pathways.

Genome sequencing of 2000 canids by the Dog10K consortium advances the understanding of demography, genome function and architecture.

BACKGROUND: The international Dog10K project aims to sequence and analyze several thousand canine genomes. Incorporating 20 × data from 1987 individuals, including 1611 dogs (321 breeds), 309 village dogs, 63 wolves, and four coyotes, we identify genomic variation across the canid family, setting the stage for detailed studies of domestication, behavior, morphology, disease susceptibility, and genome architecture and function. RESULTS: We report the analysis of > 48 M single-nucleotide, indel, and structural variants spanning the autosomes, X chromosome, and mitochondria. We discover more than 75% of variation for 239 sampled breeds. Allele sharing analysis indicates that 94.9% of breeds form monophyletic clusters and 25 major clades. German Shepherd Dogs and related breeds show the highest allele sharing with independent breeds from multiple clades. On average, each breed dog differs from the UU_Cfam_GSD_1.0 reference at 26,960 deletions and 14,034 insertions greater than 50 bp, with wolves having 14% more variants. Discovered variants include retrogene insertions from 926 parent genes. To aid functional prioritization, single-nucleotide variants were annotated with SnpEff and Zoonomia phyloP constraint scores. Constrained positions were negatively correlated with allele frequency. Finally, the utility of the Dog10K data as an imputation reference panel is assessed, generating high-confidence calls across varied genotyping platform densities including for breeds not included in the Dog10K collection. CONCLUSIONS: We have developed a dense dataset of 1987 sequenced canids that reveals patterns of allele sharing, identifies likely functional variants, informs breed structure, and enables accurate imputation. Dog10K data are publicly available.

Chromosome-level reference genome for North American bison (Bison bison) and variant database aids in identifying albino mutation.

We developed a highly contiguous chromosome-level reference genome for North American bison to provide a platform to evaluate the conservation, ecological, evolutionary, and population genomics of this species. Generated from a F1 hybrid between a North American bison dam and a domestic cattle bull, completeness and contiguity exceed that of other published bison genome assemblies. To demonstrate the utility for genome-wide variant frequency estimation, we compiled a genomic variant database consisting of 3 true albino bison and 44 wild-type pelage color bison. Through the examination of genomic variants fixed in the albino cohort and absent in the controls, we identified a nonsynonymous single nucleotide polymorphism (SNP) mutation on chromosome 29 in exon 3 of the tyrosinase gene (c.1114C>T). A TaqMan SNP Genotyping Assay was developed to genotype this SNP in a total of 283 animals across 29 herds. This assay confirmed the absence of homozygous variants in all animals except 7 true albino bison included in this study. In addition, the only heterozygous animals identified were 2 wild-type pelage color dams of albino offspring. Therefore, we propose that this new high-quality bison genome assembly and incipient variant database provides a highly robust and informative resource for genomics investigations for this iconic North American species.

Evolution of immunogenetic components encoding ultralong CDR H3.

The genomes of most vertebrates contain many V, D, and J gene segments within their Ig loci to construct highly variable CDR3 sequences through combinatorial diversity. This nucleotide variability translates into an antibody population containing extensive paratope diversity. Cattle have relatively few functional VDJ gene segments, requiring innovative approaches for generating diversity like the use of ultralong-encoding IGHV and IGHD gene segments that yield dramatically elongated CDR H3. Unique knob and stalk microdomains create protracted paratopes, where the antigen-binding knob sits atop a long stalk, allowing the antibody to bind both surface and recessed antigen epitopes. We examined genomes of twelve species of Bovidae to determine when ultralong-encoding IGHV and IGHD gene segments evolved. We located the 8-bp duplication encoding the unique TTVHQ motif in ultralong IGHV segments in six Bovid species (cattle, zebu, wild yak, domestic yak, American bison, and domestic gayal), but we did not find evidence of the duplication in species beyond the Bos and Bison genera. Additionally, we analyzed mRNA from bison spleen and identified a rich repertoire of expressed ultralong CDR H3 antibody mRNA, suggesting that bison use ultralong IGHV transcripts in their host defense. We found ultralong-encoding IGHD gene segments in all the same species except domestic yak, but again not beyond the Bos and Bison clade. Thus, the duplication event leading to this ultralong-encoding IGHV gene segment and the emergence of the ultralong-encoding IGHD gene segment appears to have evolved in a common ancestor of the Bos and Bison genera 5-10 million years ago.

Cellular Transcriptomics of Carboplatin Resistance in a Metastatic Canine Osteosarcoma Cell Line.

Osteosarcoma prognosis has remained unchanged for the past three decades. In both humans and canines, treatment is limited to excision, radiation, and chemotherapy. Chemoresistance is the primary cause of treatment failure, and the trajectory of tumor evolution while under selective pressure from treatment is thought to be the major contributing factor in both species. We sought to understand the nature of platinum-based chemotherapy resistance by investigating cells that were subjected to repeated treatment and recovery cycles with increased carboplatin concentrations. Three HMPOS-derived cell lines, two resistant and one naïve, underwent single-cell RNA sequencing to examine transcriptomic perturbation and identify pathways leading to resistance and phenotypic changes. We identified the mechanisms of acquired chemoresistance and inferred the induced cellular trajectory that evolved with repeated exposure. The gene expression patterns indicated that acquired chemoresistance was strongly associated with a process similar to epithelial-mesenchymal transition (EMT), a phenomenon associated with the acquisition of migratory and invasive properties associated with metastatic disease. We conclude that the observed trajectory of tumor adaptability is directly correlated with chemoresistance and the phase of the EMT-like phenotype is directly affected by the level of chemoresistance. We infer that the EMT-like phenotype is a critical component of tumor evolution under treatment pressure and is vital to understanding the mechanisms of chemoresistance and to improving osteosarcoma prognosis.

Across-breed genetic investigation of canine hip dysplasia, elbow dysplasia, and anterior cruciate ligament rupture using whole-genome sequencing.

Here, we report the use of genome-wide association study (GWAS) for the analysis of canine whole-genome sequencing (WGS) repository data using breed phenotypes. Single-nucleotide polymorphisms (SNPs) were called from WGS data from 648 dogs that included 119 breeds from the Dog10K Genomes Project. Next, we assigned breed phenotypes for hip dysplasia (Orthopedic Foundation for Animals (OFA) HD, n = 230 dogs from 27 breeds; hospital HD, n = 279 dogs from 38 breeds), elbow dysplasia (ED, n = 230 dogs from 27 breeds), and anterior cruciate ligament rupture (ACL rupture, n = 279 dogs from 38 breeds), the three most important canine spontaneous complex orthopedic diseases. Substantial morbidity is common with these diseases. Previous within- and between-breed GWAS for HD, ED, and ACL rupture using array SNPs have identified disease-associated loci. Individual disease phenotypes are lacking in repository data. There is a critical knowledge gap regarding the optimal approach to undertake categorical GWAS without individual phenotypes. We considered four GWAS approaches: a classical linear mixed model, a haplotype-based model, a binary case-control model, and a weighted least squares model using SNP average allelic frequency. We found that categorical GWAS was able to validate HD candidate loci. Additionally, we discovered novel candidate loci and genes for all three diseases, including FBX025, IL1A, IL1B, COL27A1, SPRED2 (HD), UGDH, FAF1 (ED), TGIF2 (ED & ACL rupture), and IL22, IL26, CSMD1, LDHA, and TNS1 (ACL rupture). Therefore, categorical GWAS of ancestral dog populations may contribute to the understanding of any disease for which breed epidemiological risk data are available, including diseases for which GWAS has not been performed and candidate loci remain elusive.

ABCG2 Polymorphisms and Predictive Fluoroquinolone Phototoxicity in Nondomestic Felids.

Fluoroquinolones are a widely used class of chemotherapeutics within veterinary medicine, prized for their broad-spectrum bactericidal activity. These drugs present a known risk of retinal phototoxicity in domestic cats (Felis catus); therefore, using lower doses and alternative antibiotic classes is encouraged in this species. This adverse drug effect of fluoroquinolones, and enrofloxacin specifically, has been determined to be species-specific in domestic felids. Four feline-specific missense variants in ABCG2 result in four amino acid changes (E159M, S279L, H283Q, and T644I) that are unique to the domestic cat compared with multiple other nonfeline mammalian species. These changes alter the ABCG2 protein involved with the cellular transmembrane transport of drugs, including fluoroquinolones, making the protein functionally defective in domestic cats. The predisposition to fluoroquinolone-mediated phototoxicity in nondomestic felids was explored in this study. At least eight nondomestic felids share the four ABCG2 missense variants with domestic cats, and eleven other felids shared at least three of the four domestic cat variants. Taken together, these results suggest the genetic potential for nondomestic felids to also experience fluoroquinolone-induced retinal phototoxicity; therefore, cautions similar to those for domestic cats should be followed for these drugs in the entire feline taxon.

Copy number variation of horse Y chromosome genes in normal equine populations and in horses with abnormal sex development and subfertility: relationship of copy number variations with Y haplogroups.

Structural rearrangements like copy number variations in the male-specific Y chromosome have been associated with male fertility phenotypes in human and mouse but have been sparsely studied in other mammalian species. Here, we designed digital droplet PCR assays for 7 horse male-specific Y chromosome multicopy genes and SRY and evaluated their absolute copy numbers in 209 normal male horses of 22 breeds, 73 XY horses with disorders of sex development and/or infertility, 5 Przewalski's horses and 2 kulans. This established baseline copy number for these genes in horses. The TSPY gene showed the highest copy number and was the most copy number variable between individuals and breeds. SRY was a single-copy gene in most horses but had 2-3 copies in some indigenous breeds. Since SRY is flanked by 2 copies of RBMY, their copy number variations were interrelated and may lead to SRY-negative XY disorders of sex development. The Przewalski's horse and kulan had 1 copy of SRY and RBMY. TSPY and ETSTY2 showed significant copy number variations between cryptorchid and normal males (P < 0.05). No significant copy number variations were observed in subfertile/infertile males. Notably, copy number of TSPY and ETSTY5 differed between successive male generations and between cloned horses, indicating germline and somatic mechanisms for copy number variations. We observed no correlation between male-specific Y chromosome gene copy number variations and male-specific Y chromosome haplotypes. We conclude that the ampliconic male-specific Y chromosome reference assembly has deficiencies and further studies with an improved male-specific Y chromosome assembly are needed to determine selective constraints over horse male-specific Y chromosome gene copy number and their relation to stallion reproduction and male biology.

Genomic Assessment of Cancer Susceptibility in the Threatened Catalina Island Fox (Urocyon littoralis catalinae).

Small effective population sizes raise the probability of extinction by increasing the frequency of potentially deleterious alleles and reducing fitness. However, the extent to which cancers play a role in the fitness reduction of genetically depauperate wildlife populations is unknown. Santa Catalina island foxes (Urocyon littoralis catalinae) sampled in 2007-2008 have a high prevalence of ceruminous gland tumors, which was not detected in the population prior to a recent bottleneck caused by a canine distemper epidemic. The disease appears to be associated with inflammation from chronic ear mite (Otodectes) infections and secondary elevated levels of Staphyloccus pseudointermedius bacterial infections. However, no other environmental factors to date have been found to be associated with elevated cancer risk in this population. Here, we used whole genome sequencing of the case and control individuals from two islands to identify candidate loci associated with cancer based on genetic divergence, nucleotide diversity, allele frequency spectrum, and runs of homozygosity. We identified several candidate loci based on genomic signatures and putative gene functions, suggesting that cancer susceptibility in this population may be polygenic. Due to the efforts of a recovery program and weak fitness effects of late-onset disease, the population size has increased, which may allow selection to be more effective in removing these presumably slightly deleterious alleles. Long-term monitoring of the disease alleles, as well as overall genetic diversity, will provide crucial information for the long-term persistence of this threatened population.

Selection signature analyses and genome-wide association reveal genomic hotspot regions that reflect differences between breeds of horse with contrasting risk of degenerative suspensory ligament desmitis.

Degenerative suspensory ligament desmitis is a progressive idiopathic condition that leads to scarring and rupture of suspensory ligament fibers in multiple limbs in horses. The prevalence of degenerative suspensory ligament desmitis is breed related. Risk is high in the Peruvian Horse, whereas pony and draft breeds have low breed risk. Degenerative suspensory ligament desmitis occurs in families of Peruvian Horses, but its genetic architecture has not been definitively determined. We investigated contrasts between breeds with differing risk of degenerative suspensory ligament desmitis and identified associated risk variants and candidate genes. We analyzed 670k single nucleotide polymorphisms from 10 breeds, each of which was assigned one of the four breed degenerative suspensory ligament desmitis risk categories: control (Belgian, Icelandic Horse, Shetland Pony, and Welsh Pony), low risk (Lusitano, Arabian), medium risk (Standardbred, Thoroughbred, Quarter Horse), and high risk (Peruvian Horse). Single nucleotide polymorphisms were used for genome-wide association and selection signature analysis using breed-assigned risk levels. We found that the Peruvian Horse is a population with low effective population size and our breed contrasts suggest that degenerative suspensory ligament desmitis is a polygenic disease. Variant frequency exhibited signatures of positive selection across degenerative suspensory ligament desmitis breed risk groups on chromosomes 7, 18, and 23. Our results suggest degenerative suspensory ligament desmitis breed risk is associated with disturbances to suspensory ligament homeostasis where matrix responses to mechanical loading are perturbed through disturbances to aging in tendon (PIN1), mechanotransduction (KANK1, KANK2, JUNB, SEMA7A), collagen synthesis (COL4A1, COL5A2, COL5A3, COL6A5), matrix responses to hypoxia (PRDX2), lipid metabolism (LDLR, VLDLR), and BMP signaling (GREM2). Our results do not suggest that suspensory ligament proteoglycan turnover is a primary factor in disease pathogenesis.

Rapid adaptive radiation of Darwin's finches depends on ancestral genetic modules.

Recent adaptive radiations are models for investigating mechanisms contributing to the evolution of biodiversity. An unresolved question is the relative importance of new mutations, ancestral variants, and introgressive hybridization for phenotypic evolution and speciation. Here, we address this issue using Darwin's finches and investigate the genomic architecture underlying their phenotypic diversity. Admixture mapping for beak and body size in the small, medium, and large ground finches revealed 28 loci showing strong genetic differentiation. These loci represent ancestral haplotype blocks with origins predating speciation events during the Darwin's finch radiation. Genes expressed in the developing beak are overrepresented in these genomic regions. Ancestral haplotypes constitute genetic modules for selection and act as key determinants of the unusual phenotypic diversity of Darwin's finches. Such ancestral haplotype blocks can be critical for how species adapt to environmental variability and change.

Genomic evaluation of hybridization in historic and modern North American Bison (Bison bison).

During the late nineteenth century North American bison underwent a significant population bottleneck resulting in a reduction in population size of over 99% and a species-level near-extinction event. Factors responsible for this destruction included indiscriminate killing, loss of access to suitable habitat, and diseases. At the nadir of this population crash, very few wild plains bison survived and were restricted to Yellowstone National Park, USA and a small number of wild wood bison remained in Wood Buffalo National Park, Canada. However, most surviving bison in the late 1800's were maintained by cattle ranchers in private herds where hybridization between bison with various breeds of domestic cattle was often encouraged. Over the last 20 years, the legacy of this introgression has been identified using mitochondrial DNA and limited nuclear microsatellite analyses. However, no genome-wide assessment has been performed, and some herds were believed to be free of introgression based on current genetic testing strategies. Herein, we report detailed analyses using whole genome sequencing from nineteen modern and six historical bison, chosen to represent the major lineages of bison, to identify and quantitate signatures of nuclear introgression in their recent (within 200 years) history. Both low and high coverage genomes provided evidence for recent introgression, including animals from Yellowstone, Wind Cave, and Elk Island National Parks which were previously thought to be free from hybridization with domestic cattle. We employed multiple approaches, including one developed for this work, to identify putative cattle haplotypes in each bison genome. These regions vary greatly in size and frequency by sample and herd, though we detected domestic cattle introgression in all bison genomes tested. Since our sampling strategy spanned across the diversity of modern bison populations, these finding are best explained by multiple historical hybridization events between these two species with significant genetic recombination over the last 200 years. Our results demonstrate that whole genome sequencing approaches are required to accurately quantitate cattle introgression in bison.

Evolutionary Biology and Gene Editing of Cat Allergen, Fel d 1.

Allergy to domestic cat affects up to 15% of the population, and sensitization to cat allergen is associated with asthma. Despite the pervasiveness of cat allergic disease, current treatments have limited impact. Here, we present a bioinformatics analysis of the major cat allergen, Fel d 1, and demonstrate proof of principle for CRISPR gene editing of the allergen. Sequence and structural analyses of Fel d 1 from 50 domestic cats identified conserved coding regions in genes CH1 and CH2 suitable for CRISPR editing. Comparative analyses of Fel d 1 and orthologous sequences from eight exotic felid species determined relatively low-sequence identities for CH1 and CH2, and implied that the allergen may be nonessential for cats, given the apparent lack of evolutionary conservation. In vitro knockouts of domestic cat Fel d 1 using CRISPR-Cas9 yielded editing efficiencies of up to 55% and found no evidence of editing at predicted potential off-target sites. Taken together, our data indicate that Fel d 1 is both a rational and viable candidate for gene deletion, which may profoundly benefit cat allergy sufferers by removing the major allergen at the source.

Hmga2 deficiency is associated with allometric growth retardation, infertility, and behavioral abnormalities in mice.

The high mobility group AT-hook 2 (HMGA2) protein works as an architectural regulator by binding AT-rich DNA sequences to induce conformational changes affecting transcription. Genomic deletions disrupting HMGA2 coding sequences and flanking noncoding sequences cause dwarfism in mice and rabbits. Here, CRISPR/Cas9 was used in mice to generate an Hmga2 null allele that specifically disrupts only the coding sequence. The loss of one or both alleles of Hmga2 resulted in reduced body size of 20% and 60%, respectively, compared to wild-type littermates as well as an allometric reduction in skull length in Hmga2-/- mice. Both male and female Hmga2-/- mice are infertile, whereas Hmga2+/- mice are fertile. Examination of reproductive tissues of Hmga2-/- males revealed a significantly reduced size of testis, epididymis, and seminal vesicle compared to controls, and 70% of knock-out males showed externalized penis, but no cryptorchidism was observed. Sperm analyses revealed severe oligospermia in mutant males and slightly decreased sperm viability, increased DNA damage but normal sperm chromatin compaction. Testis histology surprisingly revealed a normal seminiferous epithelium, despite the significant reduction in testis size. In addition, Hmga2-/- mice showed a significantly reduced exploratory behavior. In summary, the phenotypic effects in mouse using targeted mutagenesis confirmed that Hmga2 is affecting prenatal and postnatal growth regulation, male reproductive tissue development, and presents the first indication that Hmga2 function is required for normal mouse behavior. No specific effect, despite an allometric reduction, on craniofacial development was noted in contrast to previous reports of an altered craniofacial development in mice and rabbits carrying deletions of both coding and noncoding sequences at the 5' part of Hmga2.