AGING-RELATED CHANGES IN PORCINE TESTIS FUNCTION

All eukaryotic organisms undergo changes over time, during aging, at molecular, cellular, tissue and organ levels [1]. As for reproductive physiology, the progressive decrease in sexual activity from puberty to old age is the most obvious effect in males [2]. Testicular aging is a heterogenous process characterized by histo-morphological changes, including depletion of germ cells and reduction in Sertoli and Leydig cells. These changes contribute to age-associated decline in spermatogenesis and steroidogenesis [3]. In this context, tight junction (TJs) proteins are essential for maintaining the integrity of the blood-testis barrier and regulating spermatogenesis. The aim of this study is to evaluate the aging-related changes in porcine testis starting from the pre-puberal phase, focusing on testicular morphology and the behavior of specific TJ proteins and Matrix Metalloproteinase (MMPs), involved in testicular function. Sixteen (n=16) boar testes were collected and categorized according to age: pre-puberty (<4 months), post-puberty (10 - 12 months), and older animals at the end of reproductive career (> 25 months). Testes were isolated from the scrota and, after epididymis ablation, were weighed and measured for min and max circumferences: both weights and circumferences of the two gonads were averaged. Parenchyma from the central area was collected and opportunely stored for immunostaining, MMPs and testosterone quantification, and gene expression. Testicular sections were immunostained for Claudin-11 (Cla-11), Zonula Occludens-1 (ZO-1), and SRY–Box Transcription Factor 9 (SOX9) to localize and quantify TJs and Sertoli cells. The expression and distribution of these proteins were analyzed, and signal intensity was measured to assess age-related differences. MMP2 and MMP9 activities were detected by means of gelatin zymography according to product protocol. In addition, a preliminary gene expression study was also settled to evaluate kisspeptin, a molecule correlated to puberty. All parametric data were analyzed with ANOVA test, while non-parametric one with Kruskal-Wallis test; regardless, significance was set at P<0.05. The quantification of TJs revealed a progressive reduction in the expression of Cla-11 and ZO-1 indicating a loss of the integrity of the blood-testis barrier. The decline of TJs expression and Sertoli cell numbers suggests that the structural and functional integrity of the barrier becomes compromised with age. Testicular circumferences significantly differed across all 3 groups, while weights only between the aged group against the other two. MMPs analyses highlighted a higher activity of proMMP2 in pre-puberty animals with decreasing levels in the other groups, as confirmed by the ANOVA (pre-puberty VS older group P=0.0096). Kisspeptin RNA was extracted with RNeasy mini kit according to manufacturer’s instructions and qRT-PCR was performed with SYBR green for target and housekeeping genes. Higher expression was detected in post-puberty group, confirming what described in literature. Future analyses will include kisspeptin receptor Kiss1R, to better investigate the kisspeptinergic system. Our results reveal key alterations in testicular homeostasis that underlie aging-related reproductive decline in boars, with potential relevance across species and laying the groundwork for potential therapeutic strategies to extend reproductive lifespan. [1] S. Gunes et al., 2016, doi: 10.1007/s10815-016-0663-y. [2] I. Kim et al., 2002, doi: 10.1095/biolreprod66.5.1359. [3] D. Huang et al. 2022, doi: 10.1093/procel/pwac057.