Human glyceraldehyde 3-phosphate dehydrogenase-2 gene is expressed specifically in spermatogenic cells.

Although the process of glycolysis is highly conserved in eukaryotes, several glycolytic enzymes have unique structural or functional features in spermatogenic cells. We previously identified and characterized the mouse complementary DNA (cDNA) and a gene for 1 of these enzymes, glyceraldehyde 3-phosphate dehydrogenase-s (Gapds). This gene is expressed only in spermatids. The enzyme appears to have an essential role in energy production required for fertilization, and it is reported to be susceptible to inhibition by certain environmental chemicals. We have now cloned and sequenced the cDNA for the human homologue of glyceraldehyde 3-phosphate dehydrogenase (GAPD2) and determined the structure of the gene. The messenger RNA (mRNA) was detected in testis, but not in 15 other human tissues analyzed by Northern blot technique. The deduced GAPD2 protein contains 408 amino acids and is 68% identical with somatic cell GAPD. GAPD2 has a 72-amino acid segment at the amino terminal end that is not present in somatic cell GAPD. This segment is proline-rich but contains smaller stretches of polyproline and is 30 amino acids shorter than the comparable segment of mouse GAPDS. The structure of the human GAPD2 gene was determined by polymerase chain reaction (PCR) to identify exon-intron junctions in a genomic clone and in total genomic DNA. The locations of these junctions in the GAPD2 gene corresponded precisely to those of the 11 exon-intron junctions in the mouse Gapds gene. Immunohistochemical studies found that GAPD2 is located in the principal piece of the flagellum of human spermatozoa, as are GAPDS in mouse and rat spermatozoa. GAPD2 extracted from human spermatozoa and analyzed by Western blot technique migrated with an apparent molecular weight of approximately 56,000, although the calculated molecular weight is 44 501. The conserved nature of the mouse, rat, and human enzymes suggests that they serve similar roles in these and other mammalian species.

Angiotensin-converting enzyme and male fertility.

The angiotensin-converting enzyme (ACE; EC 3.4.15.1) gene (Ace) encodes both a somatic isozyme found in blood and several other tissues, including the epididymis, and a testis-specific isozyme (testis ACE) found only in developing spermatids and mature sperm. We recently used gene targeting to disrupt the gene coding for both ACE isozymes in mice and reported that male homozygous mutants mate normally but have reduced fertility; the mutant females are fertile. Here we explore the male fertility defect. We demonstrate that ACE is important for achieving in vivo fertilization and that sperm from mice lacking both ACE isozymes show defects in transport within the oviducts and in binding to zonae pellucidae. Males generated by gene targeting that lack somatic ACE but retain testis ACE are normally fertile, establishing that somatic ACE in males is not essential for their fertility. Furthermore, male and female mice lacking angiotensinogen have normal fertility, indicating that angiotensin I is not a necessary substrate for testis ACE. Males heterozygous for the mutation inactivating both ACE isozymes sire wild-type and heterozygous offspring at an indistinguishable frequency, indicating no selection against sperm carrying the mutation.

Glyceraldehyde 3-phosphate dehydrogenase-S protein distribution during mouse spermatogenesis.

The spermatogenic cell-specific isoform of glyceraldehyde 3-phosphate dehydrogenase (GAPD-S) may regulate glycolysis and energy production required for sperm motility. Although the steady-state level of Gapd-s mRNA is maximal at step 9 of mouse spermatogenesis, GAPD-S protein was not detected by immunohistochemistry until steps 12-13. This result suggests that Gapd-s is translationally regulated. Western blot analysis of isolated germ cells confirmed that GAPD-S is not detected in pachytene spermatocytes or round spermatids. A major immunoreactive protein migrating with a molecular weight (M(r)) of 69,200 was observed in condensing spermatids and cauda sperm. Additional minor proteins that migrated at M(r) 55,200, 32,500, and 27,500 were detected in sperm. The molecular weight of GAPD-S is higher than the predicted molecular weight of 47,445, apparently due to a proline-rich 105-amino acid domain at the N-terminus. Recombinant GAPD-S protein lacking the proline-rich region migrated at M(r) 38,250, comparably to somatic GAPD, which also lacks the proline-rich domain. Indirect immunofluorescence demonstrated that GAPD-S is restricted to the principal piece in the sperm flagellum. Western blot analysis indicated that GAPD-S is tightly associated with the fibrous sheath of the flagellum, consistent with a potential role in regulating sperm motility.