Biogenesis of the mammalian sperm flagellum is a complex sequential process, starting early during spermiogen-esis with the elaboration of a primary flagellum, a simple axoneme enveloped by a plasma membrane. The primary flagellum is very thin, and ribosomes and other organelles are excluded from it. The proteins destined for the maturing flagellum were assumed to be synthesized in the spermatid cell body and then transported to their sites of assembly. Early in spermiogenesis, anlagen of the fibrous sheath begins to be deposited near the distal terminal of the presumptive principal piece, and assembly proceeds proximally toward the cell body. Outer dense fibers, on the other hand, begin to assemble at the head-tail junction and continue to be laid down in a proximal-to-distal direction. The postmeiotically expressed Akap4 gene is on the mouse X chromosome; this requires the AKAP4 mRNA and/or protein to be shared among conjoined X- and Y-bearing spermatids of a clonal syncytium via the intercellular bridges. The precursor of AKAP4 must be synthesized in the cell body, transported down the axoneme to the distal portion of the principal piece, associated to form the transverse ribs and longitudinal columns, and stabilized into a detergent-resistant structure.
Akap4 Gene Knockout and Testes-Weight Comparison
Western blot assay was used to check the presence of AKAP4 in mice spermatozoa. As expected, AKAP4 was not detected in Akap4 gene knockout spermatozoa but was detected in wild-type spermatozoa. Furthermore, AKAP4 was only present in the sperm pellets of wild-type mice (Fig. 1A). This confirmed the previous article showing that AKAP4 was insoluble and it was present in pellets of sperm homogenates. Testes weights were not different between knockout and wild-type mice (Fig. 1B).
Changes in the Distribution of PKA in Akap4 Gene Knockout Mice
The RI a and RII a subunits of PKA are found in the flagellum of spermatozoa in different species. PKA bound to the flagellum is generally insoluble and is presumably bound to AKAPs. Both RI a and RII a subunits of PKA are able to bind AKAP4 in vitro.
Preparation of Mouse Testes and Sperm-Soluble Extracts
The testes of Akap4 gene knockout and wild-type mice were isolated and weighed. They were homogenized in homogenization buffer (buffer A: 10 mM Tris [pH 7.2] containing 1 mM EDTA, 1 mM EGTA, 10 mM benzamidine-HCl, 1 mM phenylmethylsulfonyl fluoride, 0.01 mM Na-to-syl-phenylalanine-chloromethylketone, and 5 mM p-mercapto-ethanol) using 1 ml buffer for 0.1 g tissue. The homogenized testes were centrifuged at 16 000 X g at 4°C for 10 min; the supernatant is referred to as testes extracts. The testes extracts were supplemented with 10% (v/v) glycerol and stored at —20°C until further use. Caudal epididymal spermatozoa were isolated from mutant and wild-type mice epididymis and washed twice with Whittingham media (buffer B): 99.3 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2-2H2O, 0.5 mM MgCl2-6H2O, 0.36 mM NaH2PO4, 25 mM NaHCO3, 25 mM sodium lactate, 0.50 mM sodium pyruvate, 5.55 mM glucose, 100 U/ml penicillin G-K salt, 50 |xg/ml streptomycin sulfate. Spermatozoa were collected by centrifugation and the pelleted spermatozoa were suspended in buffer A. The sperm suspension was sonicated with three 5-sec bursts of a Biosonic П sonicator (Bronwell Scientific, Rochester, NY) at maximum setting. The sperm sonicate was centrifuged at 16 000 X g at 4°C for 10 min. The 16 000 X g supernatants were supplemented with 10% (v/v) glycerol and stored at —20°C until further use.
The cyclic AMP (cAMP) pathway is a well-studied sig-nal-transduction cascade. The second messenger cAMP mediates its intracellular effects through PKA (cAMP-dependent kinase, formally known as PRKACA). The holoen-zyme of PKA is a tetramer of two catalytic subunits and two regulatory subunits. The catalytic subunits are encoded by three genes, Ca, Cp, and C7, while the regulatory subunits are encoded by four genes, RIa, RIp, RIIa, and RIIp. Each regulatory subunit contains an N-terminal dimeriza-tion domain, which is an autophosphorylation site and also the principal contact site for the catalytic subunit, and two cAMP-binding sites. Binding of two cAMP molecules to each regulatory subunit relieves the autoinhibitory contact and allows the dissociation and activation of the catalytic subunits, resulting in phosphorylation of protein substrates. There are two forms of the heterotetrameric PKA holoen-zyme: type I (RIa and RIp dimers) and type II (RIIa and RIIp dimers). Type I PKA is predominantly cytoplasmic and more sensitive to cAMP than type II. Type II PKA usually associates with specific cellular structures and organelles. The intracellular organization of PKA is controlled through the association with AKAPs (A-kinase-anchoring proteins). AKAPs are a structurally diverse but functionally similar family of proteins of over 50 members. They usually have the following functional motifs: a conserved binding domain interacting with the PKA regulatory subunit dimer, a targeting domain directing the AKAP-PKA complex to specific subcellular locations, and docking sites for other signaling enzymes, such as kinases and phosphatases. buy zyrtec online