Changes in Intracellular Distribution: DISCUSSION
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.
In spermatozoa of Akap4 gene knockout mice, the principal piece was reduced in diameter compared with wild-type spermatozoa. In addition, the flagellum was shortened and the tip was sometimes curled or splayed apart into fine filaments, the outer dense fibers and axoneme were intact, and the smaller diameter and splaying of the flagel-lum was assumed to be caused by a reduction in size and integrity of the fibrous sheath. Formation of the fibrous sheath is incomplete without AKAP4. It would be instructive to determine the fate of signaling proteins in mutant spermatozoa lacking the fibrous sheath.
This article examined the presence, distribution, and/or activities of PKA catalytic subunit and regulatory subunits, the signaling proteins PP172, PI 3-kinase, GSK-3, and other regulatory proteins in spermatozoa lacking a fibrous sheath. The activities and phosphorylation of PP172 were studied in some detail. The presence and distribution of these proteins in testes extracts, in sperm-soluble extracts, and in insoluble sperm pellets were also determined. It is assumed that, if a protein were associated with the fibrous sheath, the disruption of the fibrous sheath due to the absence of AKAP4 most likely would change the presence and/or distribution of the protein in mutant spermatozoa.
Disruption of the fibrous sheath caused by Akap4 gene knockout significantly increases the amount of PKA RII a regulatory subunit and a catalytic subunit in sperm-soluble extracts of mutant mice (Fig. 2). This increase could be the portion of PKA normally bound to AKAP4 in wild-type spermatozoa. Previous studies have shown that the catalytic and regulatory subunits of PKA are mainly insoluble proteins, perhaps because they are bound to AKAPs. The fact that a significant portion of PKA is insoluble in spermatozoa lacking AKAP4 may be a reflection of the portion of the enzyme bound to AKAP3. On the other hand, disruption of the fibrous sheath did not affect the presence and subcellular distribution of PP172 in testes and spermatozoa (Fig. 3A). In addition, phosphorylation and catalytic activity of PP172 did not change in the testis of mutant mice (Fig. 3B). However, phosphorylation of PP172 and the enzymatic activity of PP^2 changed significantly for the mutant mice in sperm-soluble extracts (Fig. 3, A and C). It is known that phosphorylation inhibits PP1 activity, which is consistent with data in this article. The reason for this decrease in PP172 phosphorylation is yet unknown. One explanation is that low levels of ATP might be a contributory factor. In fact, ATP levels in mutant spermatozoa is about one half the levels in wild-type spermatozoa (unpublished data). Because the fibrous sheath anchors glycolytic enzymes, such as GAPDS and HK1-S, disruption of the fibrous sheath might disrupt glycolysis and thus lower local ATP levels. With regard to the PP172 regulators sds22, 14-3-3 protein, and hsp90, there was no change in their presence and subcellular distribution either in testes or spermatozoa. These data suggest that PP172, sds22, 14-3-3 protein, and hsp90 are not bound to the fibrous sheath. One noteworthy fact is that, unlike bovine spermatozoa, which have hsp90 in both soluble sperm extracts and pellet preparations (unpublished data), spermatozoa from mice have hsp90 only in sperm pellet preparations. The underlying reason is still unknown. Indirect immunofluorescence determined that PP172, sds22, 14-3-3 protein, and hsp90 (data not shown) are all flagellar proteins in spermatozoa of mice (Fig. 7A).
The presence of PI 3-kinase in supernatant fraction of mutant sperm extracts suggests that PI 3-kinase may be an enzyme associated with the fibrous sheath. There is no difference in PKB and GSK-3 between mutant and wild-type mice, suggesting that these proteins are not associated with the fibrous sheath.
SP17 was first isolated in rabbit as the 17-kDa member of the rabbit serum antigen family of rabbit testis/sperm autoantigens. SP17 was originally proposed to be a sperm-specific protein and thought to play a role in sperm-egg interactions by binding to the zona pellucida via two conserved heparin-binding motifs. However, more recent data indicate that it may be expressed more widely. SP17 is not expressed on the sperm surface but is localized within the cytoplasm of the sperm head and throughout the tail regions. Transcripts for SP17 have been cloned and sequenced from a wide range of mammalian species, including rabbits, mice, rats, humans, macaques, baboons, and sheep and were found to be highly conserved. Analysis of the primary amino acid sequence of SP17 shows that the protein has several interesting features. In particular, the first 74 amino acid residues are almost totally conserved among all studied species and contain a region showing high sequence similarity to the N-terminus of the PKA regulatory subunit II. The N-terminus of the PKA regulatory subunit is critical for PKA dimerization and interaction with AKAPs. The presence of SP17 in the fraction of soluble extracts from spermatozoa suggests that SP17 may be associated with the fibrous sheath. But whether SP17 directly binds to AKAP4 remains to be determined.
A logical question is whether motility can be induced in mutant spermatozoa. A number of motility stimulants, such as cAMP analogs [5 and unpublished data], cAMP phosphodiesterase inhibitors, and protein phosphatase 1 inhibitor calyculin A [unpublished data] could not induce increase of motility in mutant spermatozoa. Thus, the lack of motility in spermatozoa lacking AKAP4 is not merely due to insufficient protein phosphorylation. Disruption of the fibrous sheath could be a serious structure defect. Further studies are needed to determine the reasons why spermatozoa lacking the fibrous sheath are immotile.
In summary, this article suggests that PI 3-kinase and SP17 may be bound to the fibrous sheath whereas PP172, GSK-3, hsp90, sds22, protein 14-3-3, and PKB are not. A change in the activity and phosphorylation of PP172 in spermatozoa lacking a fibrous sheath is intriguing. This suggests that the fibrous sheath is very important to maintain the phosphorylation level of PP172 in spermatozoa. Further research is required to ascertain the reason for this observation.