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

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

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

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

Microcystin pulldown of sperm PP2A.

<p><b>(A</b>) Caudal sperm extracts (2X10⁸ sperm/ml) were incubated with microcystin- sepharose beads. The microcystin- bound proteins were eluted by boiling with Laemmli buffer and analyzed by Western blot with Anti-PP2A antibody (MC). 10μl of the input caudal sperm extract was loaded as a positive control (Input). Flow through (FT) obtained following the pull down, <i>i</i>.<i>e</i>. sperm extract left behind after incubation with microcystin sepharose beads, shows negligible levels of PP2A at the same control input loading volume. The same extracts were run as a duplicate blot and probed with Anti-PP1γ2 as a control for microcystin pull down. (<b>B)</b> Extracts from 5X10⁷ sperm from each region of epididymis were subjected to microcystin pull down. Equal amounts of the microcystin bound proteins boiled in SDS- sample buffer were loaded in each lane and probed with anti-demethyl PP2A antibody. A duplicate blot probed with anti-PP2A antibody as control.</p

Effect of PP2A or PP1 inhibition of serine phosphorylation of GSK3.

<p>Sperm isolated from proximal caput, distal caput and cauda were treated with either 5nM OA to specifically inhibit PP2A or 1uM OA to inhibit both PP1 and PP2A. The extracts were analyzed by western blot analysis with anti-phospho GSK3α/β (pSer 21/9) antibody. 10⁶ proximal caput sperm/lane and 4X10⁶ distal caput or caudal sperm/lane was loaded.</p

Computer assisted motility analysis of sperm.

<p><b>(A)</b> Caudal sperm treated with 1 mM L-homocysteine and adenosine or 5 nM okadaic acid show increased percentage motility and progressive motility after 10 min of incubation. <b>(B, C)</b> Sperm velocity parameters such as path velocity (VAP), Straight line velocity (VSL), Track speed (VCL) and lateral amplitude (ALH) are also increased compared to the control. The data shown is a representation of nine similar experiments.</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