(275) Investigating the Genetics of Stump Tail Defect in Livestock.

Tasrin Sultana¹, Filip Tirpak¹, Lauren E. Hamilton¹, Jacob Rissman¹, Zhihua Jiang², Dietrich Volkmann³, Robert D. Schnabel¹, Peter Sutovsky^1,4.

1. Division of Animal Sciences, University of Missouri, Columbia MO, USA.

2. Animal Sciences, Washington State University, Pullman WA, USA.

3. College of Veterinary Medicine, University of Missouri, Columbia MO, USA.

4. Department of Obstetrics, Gynecology and Women’s health, University of Missouri, Columbia MO, USA.


Abstract Text:

The stump tail abnormality is a function-limiting spermatozoan morphological defect, that precludes sperm motility and contributes to male infertility in humans and reduced reproductive success in livestock. Identification of the heritable molecular factors of sperm tail malformation may therefore enhance the reproductive efficiency and genetic selection in animal breeding programs. Based on rare, deleterious fertility-affecting gene mutations identified from a cohort of 85 genome-sequenced subfertile bulls enrolled in our ongoing genome-to-phenome (G2P) study, we hypothesize that POC1, CEP152, KIAA0586, MICAL1, and DCDC2C are crucial to sperm tail development. Deleterious mutations or altered expression of these proteins contribute to stump tail phenotype due to axoneme and periaxonemal structure disruption. These proteins are expected to have vital roles in spermatid differentiation. POC1A maintains centriolar integrity, while DCDC2C has been reported to be localized in the endpiece of human spermatozoa. Mutations in centrosomal proteins like CEP128, CEP135, CEP70, and CEP112 have been previously linked to male infertility. Dysfunction of CEP152 might affect cytoskeletal organization. MICAL1 regulates F-actin structures and KIAA0586 is required for ciliogenesis. This study utilizes semen and spermatid samples from previously sequenced bull with stump tail phenotype that carry these candidate gene mutations. We employ immunofluorescence microscopy, Western blotting, image-based flow cytometry, and gene expression analyses to study the localization and functional significance of these candidate proteins. Web-based protein structure modeling is used to predict how such mutations affect protein folding and stability. By correlating genetic variants with protein expression and localization, we aim to link individual gene mutations with the stump tail phenotype. We have confirmed KIAA0586 localization in the midpiece of wildtype bovine spermatozoa, while CEP152 localizes in the acrosomal region, supporting their roles in the morphological development of spermatozoa. Further investigations will determine the expression and localization pattern of POC1, MICAL1, and DCDC2C in both wild type and stump-tailed spermatozoa. We have also observed seminiferous tubule disorganization, and an altered localization of microtubules and microtubule-associated proteins KATNAL2, EML5, and EML4 in stump tail spermatozoa and spermatids of the mutation carrier. Adding to the complexity of genomic influences on sperm quality, three of the five target genes in this study carry alternative polyadenylation (APA) sites, opening a new line of investigation into alternative transcripts in wild-type and mutant sires. Next steps include expanding protein characterization efforts with additional AI sire samples, enhancing our genotype to phenotype correlation analysis, and exploring potential protein-protein interactions within sperm tail assembly pathways. In summary, the biomarker-based sperm phenotyping approach provides a high-throughput, systemic method to analyze sperm defects at molecular level. This study will advance our understanding of the molecular mechanisms of sperm tail development, providing further applications in diagnosing male infertility, assisting in AI sire selection and improving reproductive efficiency in livestock. This work is supported by the grant 2023-67015-39262 from USDA-NIFA Animal Breeding, Genomics and seed funding from MU College of Agriculture.