(224) TDP-43 is Required for Postnatal Differentiation of Epididymis and Epididymal Maturation of Sperm

Abstract Authors: TDP-43 is Required for Postnatal Differentiation of Epididymis and Epididymal Maturation of Sperm

Irene I. Joseph1, Prabhakara P. Reddi1

1. Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois Urbana-Champaign, Urbana, IL, USA.

Abstract Text:

Epididymal maturation is a crucial post-testicular process that enables sperm to acquire motility and fertilizing potential. During epididymal transit, proteins secreted by epididymal epithelial cells modify the sperm plasma membrane, facilitating capacitation and the acrosome reaction necessary for fertilization. While sperm acquire several RNAs during this process, the specific RNA-binding proteins involved remain largely unknown. TDP-43, a ubiquitously expressed, essential DNA/RNA-binding protein, has been implicated in mRNA processing as well as microRNA biogenesis. TDP-43 is expressed by the epididymal epithelial cells.  Aberrant TDP-43 expression has been observed in sperm from infertile men, but its role in epididymal maturation remains unclear. This study investigates whether TDP-43 expression is critical for epididymal function and sperm maturation.
We hypothesize that TDP-43 plays a crucial role in epididymal gene expression, sperm maturation, and male fertility. Our objective is to determine the impact of TDP-43 deletion in the principal cells of the initial segment and caput of the epididymis on epididymal morphology, sperm maturation, and fertility outcomes.
Immunofluorescence analysis was performed on squash preparations (n=3) of seminiferous tubules using C-terminal and N-terminal TDP-43 antibodies to examine its expression in sperms that are leaving the testis i.e. Step 16 spermatids and was compared to Caudal sperms that have undergone epidydimal maturation. Immunohistochemical analysis of epididymal sections identified TDP-43 localization across different cell types and segments. To determine its functional relevance, we generated a Defb41-iCre-mediated knockout (cKO) of TDP-43 in the principal cells of the Initial Segment and Caput. To assess morphological changes, immunohistochemistry was performed at postnatal day 35 (PND 35, n=1) and adult (three months, n=3). Immunofluorescence analysis of caudal sperm between control and cKO sperm were examined for TDP-43 expression patterns, and preliminary sperm motility assays (n=1) were conducted. Fertility trials (n=2) evaluated reproductive outcomes.
TDP-43 was detected in the nuclei of Step 1-8 spermatids (95%), with reduced expression in Step 9-10, where it localized to the manchette and centriole in Step 10-16 spermatids. In caudal sperm, TDP-43 was present in the acrosome (98%), post-acrosomal region (35-60%), and tail (98%). Immunohistochemistry revealed TDP-43 expression in principal, basal, clear, and myoid cells across all epididymal segments. In cKO mice, TDP-43 deletion was confirmed in principal cells of the Initial Segment and Caput, while basal and clear cells retained expression. PND 35 cKO mice exhibited a reduced number of caudal tubules, suggesting altered epididymal morphogenesis. In adult cKO mice, caudal tubules appeared normal, but sperm displayed diminished TDP-43 expression in the acrosome, post-acrosomal region, and tail. Preliminary motility analysis revealed reduced total and progressive motility. Fertility trials demonstrated smaller litter sizes, with perinatal fatality before PND 3.
TDP-43 plays a critical role in epididymal function and sperm maturation. Its deletion in the proximal epididymis disrupts epididymal morphology and sperm localization, leading to impaired motility and reduced fertility. These findings highlight the importance of TDP-43 in regulating sperm maturation and underscore its potential role in male reproductive health.