ABSTRACT
Reversible phosphorylation has been implicated in many developmental processes. Dephosphorylation is mediated by several families of phosphatases, including type 1 serine/threonine phosphatases (protein phosphatase-1 or PP1). The loss of the murine Ppp1cc gene causes male infertility as a result of impaired spermatogenesis. Ppp1cc encodes two splice isoforms, PPP1CC1 and PPP1CC2, with the latter being the most abundant isoform in the testis. However, the details of PPP1CC2's involvement in spermatogenesis are still unknown. As a phosphatase has been removed from the mutant mouse, a search for hyperphosphorylated proteins in the mutant testis may reveal the direct downstream targets of PPP1CC2. Using a whole tissue proteomics approach to identify testis-specific dephosphorylation targets of PPP1CC2, we found that two-dimensional electrophoresis identified 10 potential targets in the Ppp1cc null testis several of which are factors known to be important for spermatogenesis, such as HSPA2. Another potential target, tubulin, was found to be misregulated during Ppp1cc(-/-) spermatogenesis, disrupting manchette development. This work represents the first survey of the testicular phosphoproteome under pathological conditions.
Subject(s)
Phosphoproteins/metabolism , Protein Phosphatase 1/metabolism , Proteome , Testis/enzymology , Animals , Blotting, Western , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Isoelectric Focusing , Male , Mice , Reverse Transcriptase Polymerase Chain Reaction , Tandem Mass SpectrometryABSTRACT
BACKGROUND: It is now possible for infertile males to father their own genetic children through the technique of ICSI. This prospect has consequently prompted several investigations into the quality of sperm being retrieved from infertile males. One potential risk is the use of aneuploid sperm or spermatids, which might then be transferred to the fertilized oocyte. METHODS: In this investigation, aneuploidy of spermatids was assessed through immunocytochemistry using antibodies directed against chromosome centromeric regions and complexes. Three different types of infertile male mice with phenotypes closely resembling those described in human non-obstructive azoospermia [PP1cgamma-deficient mice, CREM-deficient mice and C57BL/6J.MAC-17(0--23) mice] were examined for chromosome numbers by counting the number of kinetochores in round spermatids using a CREST antiserum. RESULTS: PP1cgamma(-/-) and CREM(-/-) spermatids from infertile mice showed highly significant elevated levels in the rate of aneuploidy compared with wild-type animals (P < 0.0001). Thus infertile males with independent genetic mutations resulting in different histopathologies showed a high risk in the level of aneuploidy in their spermatids. CONCLUSIONS: These results suggest that impaired spermatogenesis may lead to production of aneuploid gametes. Analysis of aneuploidy in gametes from infertile men, coupled with appropriate genetic counselling, is recommended prior to ICSI.
