WBP2NL/PAWP mRNA and protein expression in sperm cells are not related to semen parameters, fertilization rate, or reproductive outcome

Abstract

Purpose: WBP2NL/PAWP, a protein found in the post-acrosomal region of mammalian spermatozoa, has been proposed as a sperm-borne oocyte-activating factor (SOAF) contributing to Ca²⁺ release within the oocyte and subsequent fertilization and embryo development. However, its relevance as either a diagnostic or a prognostic marker of fertilization failure has been questioned in the recent literature. We analyzed WBP2NL/PAWP gene and protein expression level and localization in patients without previous intracytoplasmic sperm injection (ICSI) cycles in order to assess its association with both sperm characteristics and ability to fertilize.

Methods: Raw frozen-thawed semen samples from 33 couples referred for oocyte donation were included in the study during 2015. Relative protein expression versus α-tubulin (western blot, WB), proportion of post-acrosomal WBP2NL/PAWP-positive spermatozoa over the total number of sperm cells (immunofluorescence), and WBP2NL/PAWP gene expression (RT-qPCR) were analyzed and correlated with semen analysis parameters (number, motility, and morphology) and with reproductive outcomes.

Results: WBP2NL/PAWP protein was expressed in all samples with high variability: relative protein expression (1.77 ± 0.8, range [0.4-3.7]), proportion of positive cells (49.6% ± 16.1, range [22-89]), and relative gene expression (7.3 ± 8.2). No significant correlation (R ² < 0.1) was found between gene and protein expression, neither between WBP2NL/PAWP gene or protein expression, and fertilization rate or other reproductive outcomes (i.e., pregnancy). In contrast, we found significant correlation between sperm morphology and WBP2NL/PAWP semiquantitative analysis in WB (r = -0.42, p < 0.05) and for sperm motility and WBP2NL/PAWP expression in IF (r = 0.52, p < 0.05).

Conclusion: Taken into account that WBP2NL/PAWP gene and protein levels and distribution did not correlate with fertilization rates, this study questions the interest of WBP2NL/PAWP protein and gene expression analysis in sperm cells as a prognostic factor for the outcome of ICSI cycles. Larger studies focusing on WBP2NL/PAWP protein and gene expression are needed in order to evaluate the role of WBP2NL/PAWP as a prognostic factor for ART.

Keywords: Fertilization; Gene expression; ICSI; Oocyte donation; Sperm; WBP2NL/PAWP.

Fig. 1

Acrosomal status of sperm cells according to PNA labeling and classified as intact acrosome, reacted acrosome, or unlabeled acrosome, and localization pattern of WBP2NL/PAWP in the sperm head. Cells counterstained with Hoechst (nuclei) and PNA (acrosome)

Fig. 2

Correlation of WBP2NL/PAWP protein expression measured with immunofluorescence (R ² = 0.05) (a) or western blot (R ² = 0.03) (b), and WBP2NL/PAWP gene expression (R ² = 0.05) (c) with fertilization rate in oocyte donation cycle. Individual values are presented as black diamond dots and gray triangle dots for patients with normozoospermia and abnormal sperm analysis, respectively

Fig. 3

Correlation of WBP2NL/PAWP protein expression measured with immunofluorescence (a) or western blot (b), and WBP2NL/PAWP gene expression (c) with average embryo quality score in oocyte donation cycle. Individual values are presented as diamond dots and triangle dots for patients with normozoospermia and abnormal sperm analysis, respectively

Fig. 4

Comparison of the respective proportion of WBP2NL/PAWP positive cells (a), WBP2NL/PAWP protein expression in WB (b), and WBP2NL/PAWP gene expression (c) in implantation failure and pregnancy groups. Results are presented as individual values (black diamond and gray triangle for patients with normozoospermia and abnormal sperm analysis, respectively) and mean (red line). All comparisons were not statistically significant (p > 0.05)