Jeffrey E. Welch

Mouse spermatogenic cell–specific type 1 hexokinase (mHk1-s) transcripts are expressed by alternative splicing from the mHk1 gene and the HK1-S protein is localized mainly in the sperm tail

Unique type 1 hexokinase (HK1) mRNAs are present in mouse spermatogenic cells (mHk1‐s). They encode a spermatogenic cell–specific sequence region (SSR) but not the porin‐binding domain (PBD) necessary for HK1 binding to porin on the outer mitochondrial membrane. This study determined the origin of the multiple Hk1‐s transcripts in mouse spermatogenic cells and verified that they are translated in mouse spermatogenic cells. It also showed that a single mHk1 gene encodes the mHk1 transcripts of somatic cells and the mHk1‐sa and mHk1‐sb transcripts of spermatogenic cells, that alternative exons are used during mHk1 gene expression in mouse spermatogenic cells, and that mHK1‐S is translated in mouse spermatogenic cells and is localized mainly with the fibrous sheath in the tail region, not with the mitochondria in the midpiece of mouse sperm. Mol. Reprod. Dev. 49:374–385, 1998.

The GRK4 Subfamily of G Protein-coupled Receptor Kinases ALTERNATIVE SPLICING, GENE ORGANIZATION, AND SEQUENCE CONSERVATION

G protein-coupled receptor kinases (GRKs) desensitize G protein-coupled receptors by phosphorylating activated receptors. The six known GRKs have been classified into three subfamilies based on sequence and functional similarities. Examination of the mouse GRK4 subfamily (GRKs 4, 5, and 6) suggests that mouse GRK4 is not alternatively spliced in a manner analogous to human or rat GRK4, whereas GRK6 undergoes extensive alternative splicing to generate three variants with distinct carboxyl termini. Characterization of the mouse GRK 5 and 6 genes reveals that all members of the GRK4 subfamily share an identical gene structure, in which 15 introns interrupt the coding sequence at equivalent positions in all three genes. Surprisingly, none of the three GRK subgroups (GRK1, GRK2/3, and GRK4/5/6) shares even a single intron in common, indicating that these three subfamilies are distinct gene lineages that have been maintained since their divergence over 1 billion years ago. Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates over the past 100 million years. The divergence of individual GRKs at vastly different rates reveals that strikingly different evolutionary pressures apply to the function of the individual GRKs.

Testis-specific expression of mRNAs for a unique human type 1 hexokinase lacking the porin-binding domain

Several enzymes in the glycolytic pathway are reported to have spermatogenic cell‐specific isozymes. We reported recently the cloning of cDNAs representing three unique type 1 hexokinase mRNAs (mHk1‐sa, mHk1‐sb, and mHk1‐sc) present only in mouse spermatogenic cells and the patterns of expression of these mRNAs (Mori et al., 1993: Biol Reprod 49:191–203). The mRNAs contain a spermatogenic cell‐specific sequence, but lack the sequence for the porin‐binding domain that somatic cell hexokinases use to bind to a pore‐forming protein in the outer mitochondrial membrane. We now report the cloning of cDNAs representing three unique human type 1 hexokinase mRNAs (hHK1‐ta, hHK1‐tb, and hHK1‐tc) expressed in testis, but not detected by Northern analysis in other human tissues. These mRNAs also contain a testis‐specific sequence not present in somatic cell type 1 hexokinase, but lack the sequence for the porin‐binding domain. The hHK1‐tb and hHK1‐tc mRNAs each contain an additional unique sequence. The testis‐specific sequence of the human mRNAs is similar to the spermatogenic cell‐specific sequence of the mouse mRNAs. Furthermore, Northern analysis of RNA from mouse, hamster, guinea pig, rabbit, ram, human, and rat demonstrated expression of type 1 hexokinase mRNAs lacking the porin‐binding domain in the testes of these mammals. These results suggest that hexokinase may have unique structural or functional features in spermatogenic cells and support a model proposed by others for hexokinase gene evolution in mammals.

Mannose 6-Phosphate Receptors: Potential Mediators of Germ Cell-Sertoli Cell Interactions

These studies have demonstrated that mouse pachytene spermatocytes, round spermatids, and Sertoli cells synthesize mannose 6-phosphate receptors and that the proportions of the CI- and CD-MPRs vary markedly between cell types. Isolated spermatogenic cells synthesize predominantly the CD-MPR and lower levels of the CI-MPR. In contrast, cultured Sertoli cells selectively synthesize the CI-MPR, even though transcripts for the CD-MPR have been detected in these cells. These striking differences in the expression of MPRs suggest that these receptors may serve multiple roles during germ cell differentiation. We have hypothesized that MPRs in the seminiferous epithelium mediate interactions between germ cells and Sertoli cells, and participate in the targeting of hydrolytic enzymes to the acrosome. In support of the first hypothesis, we have shown that functional MPRs are localized on the surface of spermatogenic cells and Sertoli cells where they mediate the endocytosis of M6P-containing ligands. As in other somatic cells, the CI-MPR is likely to be responsible for M6P receptor-mediated endocytosis in the seminiferous epithelium. Recent studies have shown that Sertoli cells in culture synthesize and secrete at least ten M6P-containing glycoproteins. Furthermore, pachytene spermatocytes and round spermatids endocytose these Sertoli M6P-glycoproteins and process them to lower molecular weight forms that persist during 17 h culture periods. The identification of relevant ligands for mannose 6-phosphate receptors in the seminiferous epithelium may help define new regulatory mechanisms in cell differentiation. Current efforts to determine if Sertoli M6P-glycoproteins modulate germ cell function should confirm the significance of surface MPRs and clarify their roles in signal transduction and/or the endocytosis of Sertoli cell products.