Rodriguez-Martinez, H. | bioRxiv Science

bioRxiv2021

Sperm cryopreservation impacts the early development of equine embryos by downregulating specific transcription factors

Equine embryos were obtained by insemination with either fresh or frozen-thawed spermatozoa at 8, 10 and 12 h post spontaneous ovulation, maintaining the pairs mare-stallion for the type of semen used. Next generation sequencing (NGS) was performed in all embryos and bioinformatic and enrichment analysis performed on the 21,058 identified transcripts. A total of 165 transcripts were downregulated in embryos obtained with cryopreserved spermatozoa respect embryos resulting from an insemination with fresh spermatozoa (p=0.021, q=0.1). The enrichment analysis using human orthologs using g:profiler on the downregulated transcripts marked an enrichment in transcription factors (TFs) in mRNAs downregulated in embryos obtained after insemination with cryopreserved spermatozoa. The 12 mRNAs (discriminant variables) most significantly downregulated in these embryos included among others, the chromatin-remodeling ATPase INO80, Lipase maturation factor 1 LMF1, the mitochondrial mRNA pseudouridine synthase RPUSD3, LIM and cysteine-rich domains protein 1, LMCD1. Sperm cryopreservation also caused a significant impact on the embryos at 8 to 10 days of development, but especially in the transition from 10 to 12 days. Overall, our findings provide strong evidence that insemination with cryopreserved spermatozoa poses a major impact in embryo development that may compromise its growth and viability, probably due to modifications in sperm proteins induced by cryopreservation. Identification of specific factors in the spermatozoa causing these changes may improve cryopreservation.

developmental biology↗

bioRxiv2018

A splice donor variant in CCDC189 is associated with asthenospermia in Nordic Red dairy cattle

BackgroundCattle populations are highly amenable to the genetic mapping of male reproductive traits because longitudinal data on ejaculate quality and dense microarray-derived genotypes are available for many artificial insemination bulls. Two young Nordic Red bulls delivered sperm with low progressive motility (i.e., asthenospermia) during a semen collection period of more than four months. The bulls were related through a common ancestor on both their paternal and maternal ancestry. Thus, a recessive mode of inheritance of asthenospermia was suspected. ResultsBoth bulls were genotyped at 54,001 SNPs using the Illumina BovineSNP50 Bead chip. A scan for autozygosity revealed that they were identical by descent for a 2.98 Mb segment located on bovine chromosome 25. This haplotype was not found in the homozygous state in 8,557 fertile bulls although five homozygous haplotype carriers were expected (P=0.018). Whole genome-sequencing uncovered that both asthenospermic bulls were homozygous for a mutation that disrupts a canonical 5 splice donor site of CCDC189 encoding the coiled-coil domain containing protein 189. Transcription analysis showed that the derived allele activates a cryptic splice site resulting in a frameshift and premature termination of translation. The mutated CCDC189 protein is truncated by more than 40%, thus lacking the flagellar C1a complex subunit C1a-32 that is supposed to modulate the physiological movement of the sperm flagella. The mutant allele occurs at a frequency of 2.5% in Nordic Red cattle. ConclusionsOur study in cattle uncovered that CCDC189 is required for physiological movement of sperm flagella thus enabling active progression of spermatozoa and fertilization. A direct gene test may be implemented to monitor the asthenospermia-associated allele and prevent the birth of homozygous bulls that are infertile. Our results have been integrated in the Online Mendelian Inheritance in Animals (OMIA) database (https://omia.org/OMIA002167/9913/).

genomics↗