Uterine defects and estradiol-dependent development of oviductal diverticula in mice lacking the SMAD4 C-terminal Mad homology 2 domain.

In female mammals, the oviduct and uterus are essential sites for female and male gamete transport, fertilization, implantation, and maintenance of a successful pregnancy. To delineate the reproductive function of Mothers against decapentaplegic homolog 4 (Smad4), we specifically inactivated Smad4 in ovarian granulosa cells and, oviduct and uterine mesenchymal cells using the Amhr2-cre mouse line. Deletion of exon 8 of Smad4 results in the production of an MH2-truncated SMAD4 protein. These mutant mice are infertile due to the development of oviductal diverticula and defects during the implantation process. The ovaries are fully functional as demonstrated in an ovary transfer experiment. The development of oviductal diverticula occurs shortly after puberty and is dependent on estradiol. The diverticula interfere with sperm migration and embryo transit to the uterus, reducing the number of implantation sites. Analysis of the uterus shows that, even if implantation occurs, decidualization and vascularization are defective resulting in embryo resorption as early as the seventh day of pregnancy. Thus, Smad4 plays an important function in female reproduction by controlling the structural and functional integrity of the oviduct and uterus.

Analysis of Polycerate Mutants Reveals the Evolutionary Co-option of HOXD1 for Horn Patterning in Bovidae.

In the course of evolution, pecorans (i.e., higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as "headgear," which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.

Dual role of DMXL2 in olfactory information transmission and the first wave of spermatogenesis.

Gonad differentiation is a crucial step conditioning the future fertility of individuals and most of the master genes involved in this process have been investigated in detail. However, transcriptomic analyses of developing gonads from different animal models have revealed that hundreds of genes present sexually dimorphic expression patterns. DMXL2 was one of these genes and its function in mammalian gonads was unknown. We therefore investigated the phenotypes of total and gonad-specific Dmxl2 knockout mouse lines. The total loss-of-function of Dmxl2 was lethal in neonates, with death occurring within 12 hours of birth. Dmxl2-knockout neonates were weak and did not feed. They also presented defects of olfactory information transmission and severe hypoglycemia, suggesting that their premature death might be due to global neuronal and/or metabolic deficiencies. Dmxl2 expression in the gonads increased after birth, during follicle formation in females and spermatogenesis in males. DMXL2 was detected in both the supporting and germinal cells of both sexes. As Dmxl2 loss-of-function was lethal, only limited investigations of the gonads of Dmxl2 KO pups were possible. They revealed no major defects at birth. The gonadal function of Dmxl2 was then assessed by conditional deletions of the gene in gonadal supporting cells, germinal cells, or both. Conditional Dmxl2 ablation in the gonads did not impair fertility in males or females. By contrast, male mice with Dmxl2 deletions, either throughout the testes or exclusively in germ cells, presented a subtle testicular phenotype during the first wave of spermatogenesis that was clearly detectable at puberty. Indeed, Dmxl2 loss-of-function throughout the testes or in germ cells only, led to sperm counts more than 60% lower than normal and defective seminiferous tubule architecture. Transcriptomic and immunohistochemichal analyses on these abnormal testes revealed a deregulation of Sertoli cell phagocytic activity related to germ cell apoptosis augmentation. In conclusion, we show that Dmxl2 exerts its principal function in the testes at the onset of puberty, although its absence does not compromise male fertility in mice.

An initiator codon mutation in SDE2 causes recessive embryonic lethality in Holstein cattle.

Researching depletions in homozygous genotypes for specific haplotypes among the large cohorts of animals genotyped for genomic selection is a very efficient strategy to map recessive lethal mutations. In this study, by analyzing real or imputed Illumina BovineSNP50 (Illumina Inc., San Diego, CA) genotypes from more than 250,000 Holstein animals, we identified a new locus called HH6 showing significant negative effects on conception rate and nonreturn rate at 56 d in at-risk versus control mating. We fine-mapped this locus in a 1.1-Mb interval and analyzed genome sequence data from 12 carrier and 284 noncarrier Holstein bulls. We report the identification of a strong candidate mutation in the gene encoding SDE2 telomere maintenance homolog (SDE2), a protein essential for genomic stability in eukaryotes. This A-to-G transition changes the initiator ATG (methionine) codon to ACG because the gene is transcribed on the reverse strand. Using RNA sequencing and quantitative reverse-transcription PCR, we demonstrated that this mutation does not significantly affect SDE2 splicing and expression level in heterozygous carriers compared with control animals. Initiation of translation at the closest in-frame methionine codon would truncate the SDE2 precursor by 83 amino acids, including the cleavage site necessary for its activation. Finally, no homozygote for the G allele was observed in a large population of nearly 29,000 individuals genotyped for the mutation. The low frequency (1.3%) of the derived allele in the French population and the availability of a diagnostic test on the Illumina EuroG10K SNP chip routinely used for genomic evaluation will enable rapid and efficient selection against this deleterious mutation.

A reverse genetic approach identifies an ancestral frameshift mutation in RP1 causing recessive progressive retinal degeneration in European cattle breeds.

BACKGROUND: Domestication and artificial selection have resulted in strong genetic drift, relaxation of purifying selection and accumulation of deleterious mutations. As a consequence, bovine breeds experience regular outbreaks of recessive genetic defects which might represent only the tip of the iceberg since their detection depends on the observation of affected animals with distinctive symptoms. Thus, recessive mutations resulting in embryonic mortality or in non-specific symptoms are likely to be missed. The increasing availability of whole-genome sequences has opened new research avenues such as reverse genetics for their investigation. Our aim was to characterize the genetic load of 15 European breeds using data from the 1000 bull genomes consortium and prove that widespread harmful mutations remain to be detected. RESULTS: We listed 2489 putative deleterious variants (in 1923 genes) segregating at a minimal frequency of 5 % in at least one of the breeds studied. Gene enrichment analysis showed major enrichment for genes related to nervous, visual and auditory systems, and moderate enrichment for genes related to cardiovascular and musculoskeletal systems. For verification purposes, we investigated the phenotypic consequences of a frameshift variant in the retinitis pigmentosa-1 gene segregating in several breeds and at a high frequency (27 %) in Normande cattle. As described in certain human patients, clinical and histological examination revealed that this mutation causes progressive degeneration of photoreceptors leading to complete blindness in homozygotes. We established that the deleterious allele was even more frequent in the Normande breed before 1975 (>40 %) and has been progressively counter-selected likely because of its associated negative effect on udder morphology. Finally, using identity-by-descent analysis we demonstrated that this mutation resulted from a unique ancestral event that dates back to ~2800 to 4000 years. CONCLUSIONS: We provide a list of mutations that likely represent a substantial part of the genetic load of domestication in European cattle. We demonstrate that they accumulated non-randomly and that genes related to cognition and sensory functions are particularly affected. Finally, we describe an ancestral deleterious variant segregating in different breeds causing progressive retinal degeneration and irreversible blindness in adult animals.

A specific role for PRND in goat foetal Leydig cells is suggested by prion family gene expression during gonad development in goats and mice.

Three genes of the prion protein gene family are expressed in gonads. Comparative analyses of their expression patterns in mice and goats revealed constant expression of PRNP and SPRN in both species and in both male and female gonads, but with a weaker expression of SPRN. By contrast, expression of PRND was found to be sex-dimorphic, in agreement with its role in spermatogenesis. More importantly, our study revealed that PRND seems to be a key marker of foetal Leydig cells specifically in goats, suggesting a yet unknown role for its encoded protein Doppel during gonadal differentiation in nonrodent mammals.

Whole-genome sequencing identifies a homozygous deletion encompassing exons 17 to 23 of the integrin beta 4 gene in a Charolais calf with junctional epidermolysis bullosa.

BACKGROUND: Since 2010, four Charolais calves with a congenital mechanobullous skin disorder that were born in the same herd from consanguineous matings were reported to us. Clinical and histopathological examination revealed lesions that are compatible with junctional epidermolysis bullosa (JEB). RESULTS: Fifty-four extended regions of homozygosity (>1 Mb) were identified after analysing the whole-genome sequencing (WGS) data from the only case available for DNA sampling at the beginning of the study. Filtering of variants located in these regions for (i) homozygous polymorphisms observed in the WGS data from eight healthy Charolais animals and (ii) homozygous or heterozygous polymorphisms found in the genomes of 234 animals from different breeds did not reveal any deleterious candidate SNPs (single nucleotide polymorphisms) or small indels. Subsequent screening for structural variants in candidate genes located in the same regions identified a homozygous deletion that includes exons 17 to 23 of the integrin beta 4 (ITGB4), a gene that was previously associated with the same defect in humans. Genotyping of a second case and of six parents of affected calves (two sires and four dams) revealed a perfect association between this mutation and the assumed genotypes of the individuals. Mining of Illumina BovineSNP50 Beadchip genotyping data from 6870 Charolais cattle detected only 44 heterozygous animals for a 5.6-Mb haplotype around ITGB4 that was shared with the carriers of the mutation. Interestingly, none of the 16 animals genotyped for the deletion carried the mutation, which suggests a rather recent origin for the mutation. CONCLUSIONS: In conclusion, we successfully identified the causative mutation for a very rare autosomal recessive mutation with only one case by exploiting the most recent DNA sequencing technologies.

Role of the prion protein family in the gonads.

The prion-gene family comprises four members named PRNP (PRP(c)), PRND (Doppel), PRNT (PRT), and SPRN (Shadoo). According to species, PRND is located 16-52 kb downstream from the PRNP locus, whereas SPRN is located on another chromosome. The fourth prion-family gene, PRNT, belongs to the same genomic cluster as PRNP and PRND in humans and bovidae. PRNT and PRND possibly resulted from a duplication event of PRND and PRNP, respectively, that occurred early during eutherian species divergence. Although most of the studies concerning the prion-family has been done on PRP(c) and its involvement in transmissible neurodegenerative disorders, different works report some potential roles of these proteins in the reproductive function of both sexes. Among them, a clear role of PRND, that encodes for the Doppel protein, in male fertility has been demonstrated through gene targeting studies in mice. In other species, Doppel seems to play a role in testis and ovary development but its cellular localization is variable according to the gonadal developmental stage and to the mammalian species considered. For the other three genes, their roles in reproductive function appear ill-defined and/or controversial. The present review aimed to synthesize all the available data on these prion-family members and their relations with reproductive processes, mainly in the gonad of both sexes.

High-throughput sequencing analyses of XX genital ridges lacking FOXL2 reveal DMRT1 up-regulation before SOX9 expression during the sex-reversal process in goats.

FOXL2 loss of function in goats leads to the early transdifferentiation of ovaries into testes, then to the full sex reversal of XX homozygous mutants. By contrast, Foxl2 loss of function in mice induces an arrest of follicle formation after birth, followed by complete female sterility. In order to understand the molecular role of FOXL2 during ovarian differentiation in the goat species, putative FOXL2 target genes were determined at the earliest stage of gonadal sex-specific differentiation by comparing the mRNA profiles of XX gonads expressing the FOXL2 protein or not. Of these 163 deregulated genes, around two-thirds corresponded to testicular genes that were up-regulated when FOXL2 was absent, and only 19 represented female-associated genes, down-regulated in the absence of FOXL2. FOXL2 should therefore be viewed as an antitestis gene rather than as a female-promoting gene. In particular, the key testis-determining gene DMRT1 was found to be up-regulated ahead of SOX9, thus suggesting in goats that SOX9 primary up-regulation may require DMRT1. Overall, our results equated to FOXL2 being an antitestis gene, allowing us to propose an alternative model for the sex-determination process in goats that differs slightly from that demonstrated in mice.

FOXL2 is a female sex-determining gene in the goat.

The origin of sex reversal in XX goats homozygous for the polled intersex syndrome (PIS) mutation was unclear because of the complexity of the mutation that affects the transcription of both FOXL2 and several long noncoding RNAs (lncRNAs). Accumulating evidence suggested that FOXL2 could be the sole gene of the PIS locus responsible for XX sex reversal, the lncRNAs being involved in transcriptional regulation of FOXL2. In this study, using zinc-finger nuclease-directed mutagenesis, we generated several fetuses, of which one XX individual bears biallelic mutations of FOXL2. Our analysis demonstrates that FOXL2 loss of function dissociated from loss of lncRNA expression is sufficient to cause an XX female-to-male sex reversal in the goat model and, as in the mouse model, an agenesis of eyelids. Both developmental defects were reproduced in two newborn animals cloned from the XX FOXL2(-/-) fibroblasts. These results therefore identify FOXL2 as a bona fide female sex-determining gene in the goat. They also highlight a stage-dependent role of FOXL2 in the ovary, different between goats and mice, being important for fetal development in the former but for postnatal maintenance in the latter.

Novel insights into the bovine polled phenotype and horn ontogenesis in Bovidae.

Despite massive research efforts, the molecular etiology of bovine polledness and the developmental pathways involved in horn ontogenesis are still poorly understood. In a recent article, we provided evidence for the existence of at least two different alleles at the Polled locus and identified candidate mutations for each of them. None of these mutations was located in known coding or regulatory regions, thus adding to the complexity of understanding the molecular basis of polledness. We confirm previous results here and exhaustively identify the causative mutation for the Celtic allele (PC) and four candidate mutations for the Friesian allele (PF). We describe a previously unreported eyelash-and-eyelid phenotype associated with regular polledness, and present unique histological and gene expression data on bovine horn bud differentiation in fetuses affected by three different horn defect syndromes, as well as in wild-type controls. We propose the ectopic expression of a lincRNA in PC/p horn buds as a probable cause of horn bud agenesis. In addition, we provide evidence for an involvement of OLIG2, FOXL2 and RXFP2 in horn bud differentiation, and draw a first link between bovine, ovine and caprine Polled loci. Our results represent a first and important step in understanding the genetic pathways and key process involved in horn bud differentiation in Bovidae.

A 3.7 Mb deletion encompassing ZEB2 causes a novel polled and multisystemic syndrome in the progeny of a somatic mosaic bull.

Polled and Multisystemic Syndrome (PMS) is a novel developmental disorder occurring in the progeny of a single bull. Its clinical spectrum includes polledness (complete agenesis of horns), facial dysmorphism, growth delay, chronic diarrhea, premature ovarian failure, and variable neurological and cardiac anomalies. PMS is also characterized by a deviation of the sex-ratio, suggesting male lethality during pregnancy. Using Mendelian error mapping and whole-genome sequencing, we identified a 3.7 Mb deletion on the paternal bovine chromosome 2 encompassing ARHGAP15, GTDC1 and ZEB2 genes. We then produced control and affected 90-day old fetuses to characterize this syndrome by histological and expression analyses. Compared to wild type individuals, affected animals showed a decreased expression of the three deleted genes. Based on a comparison with human Mowat-Wilson syndrome, we suggest that deletion of ZEB2, is responsible for most of the effects of the mutation. Finally sperm-FISH, embryo genotyping and analysis of reproduction records confirmed somatic mosaicism in the founder bull and male-specific lethality during the first third of gestation. In conclusion, we identified a novel locus involved in bovid horn ontogenesis and suggest that epithelial-to-mesenchymal transition plays a critical role in horn bud differentiation. We also provide new insights into the pathogenicity of ZEB2 loss of heterozygosity in bovine and humans and describe the first case of male-specific lethality associated with an autosomal locus in a non-murine mammalian species. This result sets PMS as a unique model to study sex-specific gene expression/regulation.