Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility.

Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function

Expression of transgenic PPP1CC2 in the testis of Ppp1cc-null mice rescues spermatid viability and spermiation but does not restore normal sperm tail ultrastructure, sperm motility, or fertility

Two isoforms of phosphoprotein phosphatase 1, PPP1CC1 and PPP1CC2, are translated from alternatively spliced transcripts of a single gene, Ppp1cc, and differ only at their extreme C-termini. While PPP1CC1 expression is almost ubiquitous, PPP1CC2 is largely restricted to testicular germ cells and mature spermatozoa. Targeted deletion of Ppp1cc leads to sterility of -/- males due to a combination of gross structural defects in developing spermatids resulting in apoptosis and faulty spermiation. Because PPP1CC2 is the only PP1 isoform that demonstrates high-level expression in wild-type meiotic and postmeiotic male germ cells, we have tested whether its loss in Ppp1cc-/- males is largely responsible for manifestation of this phenotype by expressing PPP1CC2 transgenically in the testis of Ppp1cc-/- mice (rescue mice). Herein, we demonstrate that PPP1CC2 expression in the Ppp1cc-/- testis is antiapoptotic, thus reestablishing spermatid development and spermiation. However, because aberrant flagellar morphogenesis is incompletely ameliorated, rescue males remain infertile. Because these results suggest that expression of PPP1CC2 in developing germ cells is essential but insufficient for normal spermatogenesis to occur, appropriate spatial and temporal expression of both PPP1CC isoforms in the testis during spermatogenesis appears to be necessary to produce structurally normal fertility-competent spermatozoa. © 2009 by the Society for the Study of Reproduction, Inc.Supported by NIH grants HD 38520 to S.V. and HD31164 to S.H.P.Peer Reviewe

Western blot analysis of PP2A in sperm.

<p><b>(A)</b> Presence of immunoreactive PP2A in sperm (2X10⁶sperm/lane) from different regions of epididymis. The blot was probed with PP2A antibodies that recognize PP2A irrespective of its methylation or tyrosine phosphorylation. The same blot was re-probed with anti β-Tubulin antibody as loading control. (<b>B)</b> Western blot with the same extracts used in panel A showing the methylation status of PP2A in sperm extracts (2X10⁶sperm/lane) from different regions of the epididymis, using anti-demethyl PP2A antibody. The blot was re-probed with anti β-Tubulin antibody as loading control for both B and C panels. (<b>C)</b> A duplicate blot of panel A and B (same extracts) but with higher sperm number (4X10⁶) loaded in each lane showing tyrosine phosphorylation determined by reactivity with anti-PP2A Y307 antibody.</p

Catalytic activity of PP2A following its demethylation by L-homocysteine and adenosine treatment.

<p>Caudal sperm incubated with 1 mM L-homocysteine and adenosine were sonicated and the soluble protein fraction was collected. Protein phosphatase activity in this fraction was measured with phosphorylase <i>a</i> as the substrate. PP2A activity was measured as the activity that can be inhibited by 2nM OA. Demethylation of sperm PP2A by L-homocysteine and adenosine results in decreased total phosphatase and PP2A catalytic activity. The mean phosphatase activities from five sets of experiments are represented as mmol of PO4 released/minute/2x10⁵ sperm ± SEM. ‘*’ denotes significant difference with P< 0.05. The demethylation in each experiment was confirmed by western blot analysis of the sperm extract.</p

LCMT1 and PME1 in sperm.

<p><b>A)</b> Western blot showing detectable levels of LCMT1 in mouse whole sperm extracts prepared in 1%SDS and testis extracts. Mouse brain extract was loaded as positive control for Anti-LCMT1 antibody. <b>B)</b> Presence of immunoreactive PME1 at ~43kd in HB+ sonicated soluble bull sperm extracts from all three regions of epididymis is shown with Anti-PME1 antibody. The blot was stripped and probed with Anti-PP2A antibody to show equal protein loading. <b>(C)</b> Sperm isolated from the three regions of the epididymis were incubated with DMSO (control) or 500 nM ABL127 for 1hr. Following this incubation, sperm extracts were prepared and analyzed by Western blot with anti-demethyl PP2A antibody. The first panel shows 10⁶ proximal caput sperm/lane at 10 seconds exposure. The remaining panels show 2X10⁶ distal caput or caudal sperm/lane at 60 seconds exposure. A duplicate blot probed with Anti-PP2A is used as loading control.</p

Serine phosphorylation of GSK3α/β.

<p><b>(A)</b> Western blot analysis with anti-phospho GSK3α/β (pSer 21/9) antibody of freshly prepared sperm extract (4X10⁶ sperm/lane) from sperm of proximal caput, distal caput and caudal regions of epididymis. <b>(B)</b> Sperm incubated at 37°C in 1 mM L-homocysteine and adenosine or 5 nM okadaic acid in HEPES buffer supplemented with glucose and BSA show increased immunoreactivity at 55Kd and 47Kd corresponding to phosphorylated GSK3α and GSK3β respectively (2X10⁶ sperm/lane). The same blot was re-probed with Anti-PP2A antibody showing equal protein loading.</p

<i>In vivo</i> demethylation of sperm PP2A.

<p><b>(A)</b> Caudal sperm were incubated with 1 mM each of L-Homocysteine and Adenosine (L-Hcy + Ado), 1 mM L- Homocysteine (L-Hcy) or 1 mM adenosine (Ado) for 10 min. Sperm extracts were prepared by sonication and analyzed by western blot (2X10⁶ sperm/lane) with anti-demethyl PP2A antibody and a duplicate blot probed with anti-PP2A antibody. Significant increase in levels of demethyl PP2A is observed on treatment with L-Homocysteine and adenosine (L-Hcy + Ado) compared to the untreated control sperm. (<b>B)</b> PP2A in extracts of L-Homocysteine and adenosine treated sperm was concentrated by microcystin pull down followed by western blot analysis of with anti-phosphotyrosine-PP2A (Y307) antibody shows increased levels of phosphorylated PP2A. (<b>C)</b> Caudal sperm incubated with 5 nM okadaic acid (OA), a concentration to specifically inhibit PP2A, also elevates demethyl PP2A and tyrosine phosphorylated PP2A as seen in <b>(D)</b> with 10⁷ sperm/lane.</p

Demethylation of PP2A induced by alkali treatment.

<p>Microcystin beads incubated with sperm extracts from proximal caput, distal caput and caudal regions of epididymis were subjected to NaOH treatment (+). Equal amounts incubated without NaOH are untreated controls (-). Details of the procedure are described in Materials and Methods. Different volumes were loaded in Western blot for each epididymal region, hence shown as separate panels. Duplicate blots were processed, one was probed with anti-demethyl PP2A antibody and the other was probed with anti-PP2A antibody.</p