Signaling in sperm: toward a molecular understanding of the acquisition of sperm motility in the mouse epididymis

Abstract

Sperm motility encompasses a wide range of events involving epididymal maturation and activation of biochemical pathways, most notably cyclic AMP (cAMP)-protein kinase A (PKA) activation. Following the discovery of guanine-nucleotide exchange factors (RAPGEFs), also known as exchange proteins activated by cAMP, we investigated the separate roles of PKA and RAPGEFs in sperm motility. RT-PCR showed the presence of Rapgef3, Rapgef4, and Rapgef5, as well as several known RAPGEF partner mRNAs, in spermatogenic cells. However, Rapgef3 and Rapgef4 appeared to be less abundant in condensing spermatids versus pachytene spermatocytes. Similarly, many of these proteins were detected by immunoblotting. RAPGEF5 was detected in germ cells and murine epididymal sperm. Indirect immunofluorescence localized SGK1, SGK3, AKT1 pT(308), and RAPGEF5 to the acrosome, while PDPK1 was found in the postacrosomal region. SGK3 was present throughout the tail, while PDPK1 and AKT1 pT(308) were in the midpiece. When motility was assessed in demembranated cauda epididymal sperm, addition of ATP and the selective ligand for RAPGEFs, 8-pCPT-2'-O-Me-cAMP, resulted in motility, but the sperm were unable to undergo hyperactivated-like motility. In contrast, when demembranated cauda epididymal sperm were incubated with ATP plus dibutyryl cAMP, sperm became motile and progressed to hyperactivated-like motility. However, no significant difference was observed when intact sperm were examined. GSK3 phosphorylation was altered in the presence of H89, a PKA inhibitor. Significantly, intact caput epididymal sperm became motile when incubated in the presence of extracellular ATP. These results provide evidence for a new pathway involved in endowing sperm with the capacity to swim.

Keywords: epididymis; rodents (rats, mice, guinea pigs, voles); sperm; sperm maturation; sperm motility and transport.

FIG. 1

Expression of mRNAs encoding various signaling molecules in spermatogenic cells. RT-PCR was performed on spermatogenic cells: pachytene spermatocytes (P), round spermatids (R), condensing spermatids (C), and mixed germ cells (MGC). One microgram of total RNA was used as the initial template for the RT-PCR reaction. The amplicons were then separated by agarose gel electrophoresis and stained with ethidium bromide. Shown are the signals for Akt1 (423 bp), Pdpk1 (467 bp), Rapgef3 (594 bp), Rapgef4 (527 bp), Rapgef5 (497 bp), Sgk1 (488 bp), Sgk2 (456 bp), and Sgk3 (475 bp) amplicons.

FIG. 2

Immunoblotting of caput and cauda epididymal sperm extracts to detect signaling proteins. Caput epididymal sperm (CpS) and cauda epididymal sperm (CdS) were purified and extracted immediately for immunoblot analysis. Blots were probed with antibodies against AKT1, AKT1 pS⁴⁷³, AKT1 pT³⁰⁸, RAPGEF3, RAPGEF4, RAPGEF5, PDPK1, SGK1, and SGK3. Immunoblotting for RAPGEF3 and RAPGEF4 did not recognize any bands, so the data are not shown. RAPGEF5 was also examined in spermatogenic cells: pachytene spermatocytes (P), round spermatids (R), and condensing spermatids (C). Immunoblots for AKT1, AKT1 pS⁴⁷³, and AKT1 pT³⁰⁸ are displayed in A. Immunoblots for PDPK1 and RAPGEF5 are displayed in B. Immunoblots for SGK1 and SGK3 are displayed in C. The predicted size of each protein is as follows: AKT1, 60 kD; AKT1 pS⁴⁷³, 60 kD; AKT1 pT³⁰⁸, 60 kD; PDPK1, 60 kD; RAPGEF5, 68 kD; SGK1, 50 kD; SGK3, 55 kD.

FIG. 3

Immunofluorescence of signaling proteins in mouse sperm. Antibodies against SGK1 (A), SGK3 (B), PDPK1 (C), AKT1 pT³⁰⁸ (D), AKT1, and AKT1 pS⁴⁷³ were used to detect the proteins in cauda epididymal sperm. RAPGEF5 (E) was detected in caput (data not shown) and cauda epididymal sperm. No staining was observed with AKT1 or AKT1 pS⁴⁷³; therefore, the data are not shown. The panels to the right include the fluorescence and Nomarski images of negative controls using normal sera (IgG). Bars = 10 μm (phase images).

FIG. 4

Extracellular ATP stimulates caput epididymal sperm to become motile. Sperm were recovered from the caput epididymis and incubated in MW medium in the absence (A, still image from Supplemental Video 1) or presence (B, still image from Supplemental Video 2) of extracellular ATP. Caput epididymal sperm freshly isolated from the caput epididymis were completely immotile (Supplemental Video 1). However, caput epididymal sperm placed into medium containing ATP display a flagellar beat (Supplemental Video 2). Bars = 10 μm.

FIG. 5

CASA analysis of sperm motility in the presence of various agents. The motility of cauda epididymal sperm with or without H89 in the presence of no additional agent (A), 8pCPT (B), dbcAMP (C), or N6-benzol-cAMP (D) was assessed using CASA. The percent motility is displayed along the y-axis. The time points are displayed along the x-axis. CASA measurements were taken every 15 min for 2 h. Independent experiments were performed on pooled samples of caput and cauda epididymal sperm from five mice and replicated three times. Means and SDs were calculated. Statistical difference was determined with a Student t-test.

FIG. 6

Phosphorylation states of GSKα and GSKβ in the presence of various agents. SDS-PAGE and immunoblotting with an antibody against the phosphorylated states of GSK3α and GSK3β were performed on cauda epididymal sperm after 1 h in MMW. Eight treatments were performed: MMW without additives (MMW), H89, dbcAMP, dbcAMP + H89, 8pCPT, 8pCPT + H89, N6-benzol-cAMP (N6Benzol), and N6Benzol + H89. Freshly isolated cauda epididymal sperm (CdS) from the epididymis was also included on the blot.

FIG. 7

Proposed model of RAPGEF involvement in sperm motility. We propose that RAPGEFs act to initiate basal motility cascade through the PDPK1-AKT1-GSK3 pathway of protein kinases. After sperm are released from the seminiferous tubules, they are immotile but are carried by fluid flow into the epididymis, eventually accumulating in the cauda epididymis. Here they encounter ATPe secreted by the cauda epididymal epithelium. ATPe, acting through purinergic receptors, causes a calcium influx that stimulates adenylyl cyclase ADCY10. The resulting cAMP causes a cascade of events, leading to the eventual acquisition of basal motility.