Sperm-specific protein kinase A catalytic subunit Cα ₂ orchestrates cAMP signaling for male fertility

Abstract

An unusual cAMP signaling system mediates many of the events that prepare spermatozoa to meet the egg. Its components include the atypical, bicarbonate-stimulated, sperm adenylyl cyclase and a cAMP-dependent protein kinase (PKA) with the unique catalytic subunit termed C␣ 2 or Cs. We generated mice that lack C␣2 to determine its importance in the events downstream of cAMP production. Male C␣ 2 null mice produce normal numbers of sperm that swim spontaneously in vitro. Thus, C␣ 2 has no required role in formation of a functional flagellum or the initiation of motility. In contrast, we find that C␣ 2 is required for bicarbonate to speed the flagellar beat and facilitate Ca 2؉ entry channels. In addition, C␣2 is needed for the protein tyrosine phosphorylation that occurs late in the sequence of sperm maturation and for a negative feedback control of cAMP production, revealed here. Consistent with these specific defects in several important sperm functions, C␣ 2 null males are infertile despite normal mating behavior. These results define several crucial roles of PKA in sperm cell biology, bringing together both known and unique PKA-mediated events that are necessary for male fertility. T he terminally differentiated, transcriptionally dormant, and translationally inactive posttesticular spermatozoan has a limited ability to respond to environmental cues encountered as it progresses through the male and female reproductive tracts. During this passage, the bicarbonate anion present in the reproductive fluids (1, 2) has an unexpectedly prominent role in promoting several of the events (3-5), collectively called capacitation, which transform sperm to readiness for fertilization.Much evidence indicates that bicarbonate directly increases production of cAMP by atypical sperm adenylyl cyclase (sAC) (6, 7), and recent work finds that sAC is required for male fertility and normal sperm motility (8). Although cAMP might open ion channels or activate guanine nucleotide exchange factors, most evidence places cAMP-dependent protein kinase (PKA) as the major downstream effector of cAMP signals in sperm. However, only a small number of sperm proteins have been identified as phospho-substrates of PKA (9, 10). We now apply phenotypic analysis of loss-of-function mutants to examine downstream effects of the PKA subunit C␣ 2 . Materials and MethodsTargeted Disruption of C␣2. C␣ 2 null mice were generated from the targeted disruption of exon 1b of the C␣ gene by homologous recombination in embryonic stem (ES) cells. The targeting vector was constructed from a 13.2-kb genomic fragment containing exons 1-3 of C␣. A loxP-flanked neomycin phosphotransferase (NEO) cassette and a mutation of the translational initiation codon were inserted into exon 1b. The linearized targeting vector was electroporated into ES cells derived from 129SV͞J mice as described (11). Germ-line chimeras were bred to C57BL͞6 mice. Pups carrying the NEO were crossed to heterozygous ROSA26-Cre recombinase transgenic mice (from P. Sorriano, Fred Hutchinson Cancer Rese...

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

“…Our results in this study (Fig. 3) and previous reports [53] show that PKA regulatory subunit II is localized mainly in the flagella of mouse spermatozoa. This regulatory subunit is usually settled on the AKAPs that have the potential to bind to various molecules, including glycolytic enzymes.…”

Characterization of 3-hydroxyisobutyrate dehydrogenase, HIBADH, as a sperm-motility marker

“…Reverse transcription-polymerase chain reactions (RT-PCR) for the synthesis of complementary DNA (cDNA), 12 μL aliquots of master mixture containing 100 ng of human testes RNA, 1 μL of 500 ng/μL oligo(dT) [12][13][14][15][16][17][18] primer (Invetrogen, Grand Island, NY, USA), and 9 μL of diethylpyrocarbonate-treated water were heated to 70°C for 10 min and put on ice. RT reactions were performed in 20 μL containing master mixture, 4 μL of 5X first-strand synthesis buffer, 0.1 M dithiothreitol, 10 mM of each dNTP, and 200 units of Superscript TM II RNase H -reverse transcriptase (Invetrogen, Grand Island, NY, USA).…”

“…One of the downstream targets of cAMP is proteinkinase A (PK-A), a serine/threonine kinase [11]. Mutations of one isoform of PK-A disrupt sperm motility [12]. Calcium signaling is also involved in sperm motility.…”

Cl− Channels and Transporters in Sperm Physiology

“…The stimulation of the sperm soluble adenylate cyclase by HCO 3 − and the consequent cAMP/PKA stimulation is required for the activated motility (Carlson et al 2007 ;Esposito et al 2004 ;Hess et al 2005 ;Nolan et al 2004 ;Xie et al 2006 ). After some time (varying according to species) in the female tract, spermatozoa become hyperactivated, displaying vigorous asymmetrical fl agellar beating with large amplitude and high curvature.…”

“…Motility is one of the most important functions carried out by the sperm because it is essential to achieve fertilization. Indeed, several sperm motility defects can cause male sterility (e.g., sAC, PKA, sNHE, GAPDHs, CatSper, PMCA4, SLO3) (Esposito et al 2004 ;Miki et al 2004 ;Nolan et al 2004 ;Okunade et al 2004 ;Quill et al 2001 ;Ren et al 2001 ;Santi et al 2010 ;Wang et al 2007 ;Zeng et al 2011 ). Sperm motility is driven by the fl agellum, an appendage with an ultrastructure very similar to that of cilia.…”