α/ß-Hydrolase D16B Truncation Results in Premature Sperm Capacitation in Cattle.

Recently it was shown that a specific form of male infertility in Holstein cattle was caused by a nonsense variant in the α/ß-hydrolase domain-containing 16B (ABHD16B) gene resulting in a protein truncation at amino acid position 218 (p.218Q*) and loss of function. Lipidomics showed that the absence of ABHD16B influenced the content of phosphatidylcholine (PC), ceramide (Cer), diacylglycerol (DAG), and sphingomyelin (SM) in variant carrier sperm membranes. However, the exact cause of infertility in affected sires has remained unclear until now. To elucidate the cause of infertility, we analyzed (i) standard sperm parameters (i.e., total sperm number, morphological intact sperm, total sperm motility), (ii) in vitro fertilizability and effects on early embryonic development, and (iii) sperm survival rates (i.e., capacitation time). The affected spermatozoa showed no changes in the usual sperm parameters and were also capable of fertilization in vitro. Furthermore, the absence of ABHD16B did not affect early embryonic development. Based on these results, it was concluded that the affected spermatozoa appeared to be fertilizable per se. Consequently, the actual cause of the inability to fertilize could only be due to a time- and/or place-dependent process after artificial insemination and before fertilization. A process fundamental to the ability to fertilize after insemination is capacitation. Capacitation is a biochemical maturation process that spermatozoa undergo in the female genital tract and is inevitable for the successful fertilization of the oocyte. It is known that the presence and concentration of certain sperm membrane lipids are essential for the correct course of capacitation. However, precisely these lipids are absent in the membrane of spermatozoa affected by the ABHD16B truncation. Since all other causes of fertilization inability were excluded in the previous experiments, consequently, the only remaining hypothesis was that the loss of function of ABHD16B leads to a capacitation disruption. We were able to show that heterozygous and homozygous affected spermatozoa exhibit premature capacitation and therefore decay before fertilization. This effect of the loss of function of ABHD16B has not been described before and our studies now revealed why sires harboring the variant in the ABHD16B gene are infertile.

Genome-Wide Association Studies Reveal Neurological Genes for Dog Herding, Predation, Temperament, and Trainability Traits.

Genome-wide association study (GWAS) using dog breed standard values as phenotypic measurements is an efficient way to identify genes associated with morphological and behavioral traits. As a result of strong human purposeful selections, several specialized behavioral traits such as herding and hunting have been formed in different modern dog breeds. However, genetic analyses on this topic are rather limited due to the accurate phenotyping difficulty for these complex behavioral traits. Here, 268 dog whole-genome sequences from 130 modern breeds were used to investigate candidate genes underlying dog herding, predation, temperament, and trainability by GWAS. Behavioral phenotypes were obtained from the American Kennel Club based on dog breed standard descriptions or groups (conventional categorization of dog historical roles). The GWAS results of herding behavior (without body size as a covariate) revealed 44 significantly associated sites within five chromosomes. Significantly associated sites on CFA7, 9, 10, and 20 were located either in or near neuropathological or neuronal genes including THOC1, ASIC2, MSRB3, LLPH, RFX8, and CHL1. MSRB3 and CHL1 genes were reported to be associated with dog fear. Since herding is a restricted hunting behavior by removing killing instinct, 36 hounds and 55 herding dogs were used to analyze predation behavior. Three neuronal-related genes (JAK2, MEIS1, and LRRTM4) were revealed as candidates for predation behavior. The significantly associated variant of temperament GWAS was located within ACSS3 gene. The highest associated variant in trainability GWAS is located on CFA22, with no variants detected above the Bonferroni threshold. Since dog behaviors are correlated with body size, we next incorporate body mass as covariates into GWAS; and significant signals around THOC1, MSRB3, LLPH, RFX8, CHL1, LRRTM4, and ACSS3 genes were still detected for dog herding, predation, and temperament behaviors. In humans, these candidate genes are either involved in nervous system development or associated with mental disorders. In conclusion, our results imply that these neuronal or psychiatric genes might be involved in biological processes underlying dog herding, predation, and temperament behavioral traits.

Sperm Lipid Markers of Male Fertility in Mammals.

Sperm plasma membrane lipids are essential for the function and integrity of mammalian spermatozoa. Various lipid types are involved in each key step within the fertilization process in their own yet coordinated way. The balance between lipid metabolism is tightly regulated to ensure physiological cellular processes, especially referring to crucial steps such as sperm motility, capacitation, acrosome reaction or fusion. At the same time, it has been shown that male reproductive function depends on the homeostasis of sperm lipids. Here, we review the effects of phospholipid, neutral lipid and glycolipid homeostasis on sperm fertilization function and male fertility in mammals.

A Missense Mutation in the KLF7 Gene Is a Potential Candidate Variant for Congenital Deafness in Australian Stumpy Tail Cattle Dogs.

Congenital deafness is prevalent among modern dog breeds, including Australian Stumpy Tail Cattle Dogs (ASCD). However, in ASCD, no causative gene has been identified so far. Therefore, we performed a genome-wide association study (GWAS) and whole genome sequencing (WGS) of affected and normal individuals. For GWAS, 3 bilateral deaf ASCDs, 43 herding dogs, and one unaffected ASCD were used, resulting in 13 significantly associated loci on 6 chromosomes, i.e., CFA3, 8, 17, 23, 28, and 37. CFA37 harbored a region with the most significant association (-log10(9.54 × 10-21) = 20.02) as well as 7 of the 13 associated loci. For whole genome sequencing, the same three affected ASCDs and one unaffected ASCD were used. The WGS data were compared with 722 canine controls and filtered for protein coding and non-synonymous variants, resulting in four missense variants present only in the affected dogs. Using effect prediction tools, two variants remained with predicted deleterious effects within the Heart development protein with EGF like domains 1 (HEG1) gene (NC_006615.3: g.28028412G>C; XP_022269716.1: p.His531Asp) and Kruppel-like factor 7 (KLF7) gene (NC_006619.3: g.15562684G>A; XP_022270984.1: p.Leu173Phe). Due to its function as a regulator in heart and vessel formation and cardiovascular development, HEG1 was excluded as a candidate gene. On the other hand, KLF7 plays a crucial role in the nervous system, is expressed in the otic placode, and is reported to be involved in inner ear development. 55 additional ASCD samples (28 deaf and 27 normal hearing dogs) were genotyped for the KLF7 variant, and the variant remained significantly associated with deafness in ASCD (p = 0.014). Furthermore, 24 dogs with heterozygous or homozygous mutations were detected, including 18 deaf dogs. The penetrance was calculated to be 0.75, which is in agreement with previous reports. In conclusion, KLF7 is a promising candidate gene causative for ASCD deafness.

Association of α/ß-Hydrolase D16B with Bovine Conception Rate and Sperm Plasma Membrane Lipid Composition.

We have identified a Holstein sire named Tarantino who had been approved for artificial insemination that is based on normal semen characteristics (i.e., morphology, thermoresistance, motility, sperm concentration), but had no progeny after 412 first inseminations, resulting in a non-return rate (NRdev) of -29. Using whole genome association analysis and next generation sequencing, an associated nonsense variant in the α/ß-hydrolase domain-containing 16B gene (ABHD16B) on bovine chromosome 13 was identified. The frequency of the mutant allele in the German Holstein population was determined to be 0.0018 in 222,645 investigated cattle specimens. The mutant allele was traced back to Whirlhill Kingpin (bornFeb. 13th, 1959) as potential founder. The expression of ABHD16B was detected by Western blotting and immunohistochemistry in testis and epididymis of control bulls. A lipidome comparison of the plasma membrane of fresh semen from carriers and controls showed significant differences in the concentration of phosphatidylcholine (PC), diacylglycerol (DAG), ceramide (Cer), sphingomyelin (SM), and phosphatidylcholine (-ether) (PC O-), indicating that ABHD16B plays a role in lipid biosynthesis. The altered lipid contents may explain the reduced fertilization ability of mutated sperms.

Christmas disease in a Hovawart family resembling human hemophilia B Leyden is caused by a single nucleotide deletion in a highly conserved transcription factor binding site of the F9 gene promoter.

Hemophilia B is a classical monogenic, X-chromosomal, recessively transmitted bleeding disorder caused by genetic variants within the coagulation factor IX gene (F9). Although hemophilia B has been described in dogs, it has not yet been reported in the Hovawart breed. Here we describe the identification of a Hovawart family transmitting typical signs of an X-linked bleeding disorder. Five males were reported to suffer from recurrent hemorrhagic episodes. A blood sample from one of these males with only 2% of the normal concentration of plasma factor IX together with samples from seven relatives were provided. Next-generation sequencing of the mother and grandmother revealed a single nucleotide deletion in the F9 promoter. Genotyping of the deletion in 1,298 dog specimens including 720 Hovawarts revealed that the mutant allele was only present in the aforementioned Hovawart family. The deletion is located 73 bp upstream of the F9 start codon in the conserved overlapping DNA binding sites of hepatocyte nuclear factor 4α (HNF-4α) and androgen receptor (AR). The deletion only abolished binding of HNF-4α, while AR binding was unaffected as demonstrated by electrophoretic mobility shift assay using human HNF-4α and AR with double-stranded DNA probes encompassing the mutant promoter region. Luciferase reporter assays using wildtype and mutated promoter fragment constructs transfected into Hep G2 cells showed a significant reduction in expression from the mutant promoter. The data provide evidence that the deletion in the Hovawart family caused a rare type of hemophilia B resembling human hemophilia B Leyden.