From the epididymis to the egg: participation of CRISP proteins in mammalian fertilization

Abstract

Mammalian fertilization is a complex process that involves different steps of interaction between the male and female gametes. In spite of its relevance, the molecular mechanisms underlying this process still remain to be elucidated. The present review describes the contribution of our laboratory to the understanding of mammalian fertilization using Cysteine-RIch Secretory Proteins (CRISP) as model molecules. Substantial evidence obtained from in vitro assays and knockout models shows that epididymal CRISP1 associates with the sperm surface with two different affinities during maturation, and participates in the regulation of signaling pathways during capacitation as well as in both sperm-zona pellucida interaction and gamete fusion. These observations can be extended to humans as judged by our findings showing that the human homolog of the rodent protein (hCRISP1) is also involved in both stages of fertilization. Evidence supports that other members of the CRISP family secreted in the testis (CRISP2), epididymis (CRISP3-4) or during ejaculation (CRISP3) are also involved in sperm-egg interaction, supporting the existence of a functional redundancy and cooperation between homolog proteins ensuring the success of fertilization. Together, our observations indicate that CRISP proteins accompany spermatozoa along their transit through both the male and female reproductive tracts. We believe these results not only contribute to a better mechanistic understanding of fertilization but also support CRISP proteins as excellent candidates for future research on infertility and contraception.

Figure 1

(a) Fate of CRISP1 during mouse sperm capacitation. Total protein extracts from equal amounts of fresh (F) and capacitated (C) epididymal spermatozoa collected from either Crisp1^+/− or Crisp1^−/− animals were analyzed by SDS-PAGE and Western blots using an anti-CRISP1 antibody (α-CRISP1). β-tubulin was used as control of loading (α-tub). (b) Evaluation of tyrosine phosphorylation in CRISP1-null spermatozoa. Fresh (F) and capacitated (C) epididymal spermatozoa from Crisp1^+/− or Crisp1^−/− animals were subjected to flow cytometric analysis with an anti-phospho tyrosine antibody. The figure shows representative histograms for each genotype. The gray histograms correspond to sperm cells incubated with normal IgG as primary antibody (negative control). The mean fluorescence intensity (MFI) ± s.e.m. of three experiments is shown as an inset, *P < 0.05.

Figure 2

(a) Behavior of CRISP3 during human sperm capacitation and the acrosome reaction. Total protein extracts of fresh (F), capacitated (C) and ionophore-treated (C + I) sperm were analyzed by SDS-PAGE and Western blots with an anti-hCRISP3 antibody (α-CRISP3). (b) Localization of CRISP3 in human spermatozoa. Representative phase contrast (upper panels) and fluorescence (lower panels) microphotographs of human capacitated (C) and ionophore-treated (C + I) spermatozoa subjected to immunofluorescence labeling with α-CRISP3 or normal IgG as primary antibodies. (c) Relevance of CRISP3 for human sperm fusion ability. Capacitated spermatozoa were incubated for 15 min in medium containing either normal IgG, α-CRISP3-, α-CRISP1- or α-CRISP2-antibodies, then co-incubated with ZP-free hamster oocytes for 2.5 h and the percentage of penetrated oocytes was determined. Results represent the mean ± s.e.m. of at least three independent experiments, *P < 0.05 versus IgG.

Figure 3

CRISP proteins in spermatozoa during their transit along the male and female reproductive tracts. Spermatozoa leaving the testis carry CRISP2 inside the head and tail. During epididymal transit, CRISP1, CRISP3, and CRISP4, soluble or associated with epididymosomes, bind loosely or strongly to the sperm surface. CRISP3 can also bind to the cells during ejaculation. In the female tract, the loosely associated proteins are partially released from sperm cells during capacitation whereas the intracellular or tightly-bound population remain in the cells and relocalize to the equatorial segment of acrosome-reacted spermatozoa. Each sperm CRISP protein participates in more than one step of fertilization and cooperates with other CRISP homologs in each fertilization step.