Sperm Motility Development in the Epididymis is Associated with Decreased Glycogen Synthase Kinase-3 and Protein Phosphatase 1 Activity1

Abstract

Immotile bovine caput epididymal sperm contain levels of protein phosphatase activity twofold higher than do mature motile caudal sperm. Comparison of the inhibition profiles of endogenous phosphatase activities detected by okadaic acid (OA) and calyculin A (CA) revealed a pattern consistent with the predominance of a type 1 protein phosphatase (PP1). Immunoblot analysis identified PP1 gamma 2 (the testis-specific isoform of PP1) as the only PP1 isoform in sperm and showed little protein phosphatase 2A (PP2A). In addition, of the known PP1 inhibitors, i.e., DARPP-32, inhibitor 1 (I1), and inhibitor 2 (I2), only I2-like activity was detected in sperm. Inhibition of PP1 by the heat-stable I2-like activity purified from sperm could be reversed with purified glycogen synthase kinase-3 (GSK-3). Furthermore, sperm extracts contain an inactive complex of PP1 and I2 (termed PP1I) that could also be activated by purified GSK-3. The presence of GSK-3 in sperm was demonstrated by activation of purified PP1I, and quantitation revealed that immotile caput sperm contained sixfold higher GSK-3 activity than motile caudal sperm. Immunoblot analysis confirmed the expression of GSK-3 in sperm and revealed the occurrence of both the alpha and beta isoforms. Our findings suggest that the higher PP1 activity measured in immotile sperm, presumably due to higher GSK-3 activity, is responsible for holding motility in check. This conclusion was supported by the observation that the phosphatase inhibitors OA and CA, at micromolar and nanomolar levels, respectively, were able to induce motility in completely immotile bovine caput epididymal sperm and to stimulate the kinetic activity of mature caudal sperm. The intrasperm levels of cAMP, pH, and calcium were unaltered by treatment with these inhibitors. The results suggest a biochemical basis for the development and regulation of sperm motility and a possible physiological role for the PP1/I2/GSK-3 system.

Taf7l cooperates with Trf2 to regulate spermiogenesis

“…S3), providing additional evidence that Taf7l regulates a subset of spermatogenic and metabolic genes in testis. These findings also provide some insight into the relationship between metabolism and reproduction that has been reported in the study of various animal models (37)(38)(39)(40) linking dysregulated metabolism with sterility. Our genome-wide expression data thus point to the likely involvement of Taf7l in regulating both metabolic and spermatogenesis genes in testis as being at least partly responsible for the observed infertility.…”

Calyculin A-Sensitive Protein Phosphatases are Involved in Maintenance of Progressive Movement in Mouse Spermatozoa In Vitro by Suppression of Autophosphorylation of Protein Kinase A

“…PP1γ2 is a unique isoform of PP1 that is included mainly in the testes and spermatozoa [28,29]. In this study, this isoform was immunodetected in the principal piece, terminal piece and head (Fig.…”

“…gressive movement. This hypothesis is supported by the facts that other isoforms of PP1 (PP1α, PP1β and PP1γ1) are expressed in the mouse testis [28,29] and that flagellar proteins are dephosphorylated by PP2A in spermatozoa from the rainbow trout and chum salmon [50]. However, greater enhancement of the protein Ser/Thr phosphorylation states in the middle pieces was induced with cBiMPS than with calyculin A, whereas the phosphorylation state of PKA catalytic subunit (Thr-197; indicating the level of full activation in PKA) in the middle piece was almost same in the cBiMPS-incubated spermatozoa as in the calyculin A-incubated spermatozoa (Fig.…”

“…Moreover, PP1 has four kinds of isoform, PP1α, PP1β, PP1γ1 and PP1γ2 [23,26,27]. In particular, PP1γ2 is expressed mainly in the testis and is also present in spermatozoa, although the other isoforms are ubiquitously distributed [28,29]. Moreover, PP1 is likely involved in regulation of the production, maturation, flagellar movement and acrosome reaction of mammalian spermatozoa [30][31][32][33].…”

Moving to the beat: a review of mammalian sperm motility regulation

“…The resulting net amount of protein phosphorylation is one factor influencing the status of sperm motility (Tash 1989;Tash and Bracho 1994). In this regard, immotile primate sperm contain higher levels of protein phosphatase 1γ2 (the implication being less net phosphorylation) than do motile sperm and motility can be initiated in bovine caput epididymal sperm by inhibition of phosphatase activity (the implication being an increase in net phosphorylation; Vijayaraghavan et al 1996).…”