ROLE OF CRISP1 IN EPIDIDYMAL EPITHELIUM FUNCTION

CARVAJAL G; REBAGLIATI, ABRIL; SULZYK, VALERIA; BATTISTONE A; BRETON, SYLVIE; WEIGE MUÑOZ, M; CUASNICU P S

Jornada; Jornadas Anuales de la Sociedad Argentina de Biología (SAB; 2022

Epididymal proteins CRISP1 and CRISP4 associate with the sperm surface during epididymal maturation and participate in the fertilization process. Double knockout (KO) males for these proteins are subfertile and show clear defects in the differentiation of the epididymal epithelium as well as an alteration in luminal pH acidification critical for sperm storage in the organ. Based on this, the aim of the present work has been to investigate the role of CRISP proteins in epididymal epithelium function. For this purpose, we used immortalized epididymal epithelial cell lines from different regions of the epididymis called PC1 and DC2. Initially, we analyzed the expression of CRISP1 and CRISP4 by RT-PCR in the cells, observing expression of Crisp1 messenger in both lines and the absence of that corresponding to Crisp4. The presence of CRISP1 protein was neither detected in cell extracts nor in culture supernatants, indicating that Crisp1 messenger was not being expressed in the cells in culture. Based on this, the following studies were carried out adding CRISP1 to the cells in culture, mimicking what occurs in vivo. Considering that CRISP1 KO sperm show alterations in the cAMP-PKA signaling cascade, we decided to evaluate whether CRISP1 protein was also involved in this cascade at the epididymal epithelial level. Purified native CRISP1 was then added to the cells in culture and the levels of phosphorylation in PKA substrates were evaluated by Western Blot. Results showed an increase in phosphorylation levels similar to that observed when the cells were exposed to cAMP and not detected in the presence of a PKA inhibitor. On the other hand, no significant differences were observed in the phosphorylation of PKC or Src substrates. Given that one of the targets of PKA phosphorylation, the CFTR channel, is involved in ATP release to the epididymal lumen, and that extracellular ATP is essential for both epididymal and sperm function, we evaluated whether exposure of epithelial cells to CRISP1 was capable of modulating ATP release into the medium, observing an increase in ATP levels within the media. Altogether, these studies indicate the ability of CRISP1 to regulate not only sperm function but also the functionality of epididymal epithelial cells through the modulation of the cAMP-PKA signaling cascade.