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.Angiotensin-converting enzyme (ACE; EC 3.4.15.1) catalyzes the cleavage of C-terminal dipeptides from several substrates including angiotensin I and bradykinin (1). The gene for ACE (ACE in humans, Ace in mice) codes for both a somatic and a smaller testis-specific isozyme. Somatic ACE is anchored to the plasma membranes of vascular endothelial cells and several epithelia, including cells in the epididymis, and a soluble form is present in blood. The testis isozyme is found only in developing spermatids and in mature sperm (2-5).Somatic ACE encoded by the entire gene is composed of two homologous amino acid domains (6, 7). The testis isozyme is encoded by the second half of the gene under the control of a testis-specific promoter located within intron 12 (8). The testis isozyme has a unique N-terminal sequence determined by a testis-specific exon; its remaining sequence is identical to the C-terminal domain of somatic ACE (9-11). Transcription of testis ACE in mouse spermatogenic cells begins in late pachytene spermatocytes (3) or after meiosis (4), and ACE protein is first detected in haploid spermatids (2-4). The tissue specificity of testis ACE is achieved with a promoter sequence of 91 base pairs (12). The functions of the ACE isozymes in male reproduction are unknown.We recently generated mice carrying an insertional disruption of exon 14 of the murine Ace gene, which prevents the synthesis of both testis and somatic ACE (13). Intercrossing of heterozygous mice gave Ϸ11% homozygous mutant mice compared with the expected 25%. Compared with wild-type mice (which we designate Ace ST ͞Ace ST , hereafter abbreviated STST to indicate the presence of both the somatic and testis isozymes), the homozygous mutant (stst) mice lacking both isozymes have blood pressures reduced about 3...

The fibrous sheath is a cytoskeletal structure located in the principal piece of mammalian sperm flagella. Previous studies showed that glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS), a germ cell-specific glycolytic isozyme that is required for sperm motility, is tightly bound to the fibrous sheath. To determine if other glycolytic enzymes are also bound to this cytoskeletal structure, we isolated highly purified fibrous sheath preparations from mouse epididymal sperm using a sequential extraction procedure. The isolated fibrous sheaths retain typical ultrastructural features and exhibit little contamination by axonemal or outer dense fiber proteins in Western blot analyses. Proteomic analysis using peptide-mass fingerprinting and MS/MS peptide fragment ion matching identified GAPDHS and two additional glycolytic enzyme subunits, the A isoform of aldolase 1 (ALDOA) and lactate dehydrogenase A (LDHA), in isolated fibrous sheaths. The presence of glycolytic enzymes in the fibrous sheath was also examined by Western blotting. In addition to GAPDHS, ALDOA, and LDHA, this method determined that pyruvate kinase is also tightly bound to the fibrous sheath. These data support a role for the fibrous sheath as a scaffold for anchoring multiple glycolytic enzymes along the length of the flagellum to provide a localized source of ATP that is essential for sperm motility.

The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1,417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS (94% identity) and human GAPD2 (83% identity) isozymes. Northern blotting of rat tissue RNAs detected the 1.5 kb Gapds transcript in the testis and not in RNA from liver, spleen, epididymis, heart, skeletal muscle, brain, seminal vesicle, and kidney. The rat Gapds mRNA was first detected at day 29 of postnatal testis development, an age which coincides with the initial post-meiotic differentiation of round spermatids. When isolated rat spermatogenic cell RNA was probed for Gapds expression, transcripts were detected only in round spermatids and condensing spermatids, but not in pachytene spermatocytes, demonstrating haploid expression of the Gapds gene. However, immunohistochemical staining of rat testis sections with anti-GAPDS antisera did not detect GAPDS in round spermatids, but localized the protein only to stage XIII and later condensing spermatids as well as testicular spermatozoa, indicating that Gapds expression is translationally regulated. The current results are similar to those previously obtained for mouse GAPDS and human GAPD2, suggesting that reliable comparisons can be made between these species in toxicant screening and contraceptive development.

The loss of the cyclin-dependent kinase inhibitors (CKIs) p18(Ink4c) and p19(Ink4d) leads to male reproductive defects (Franklin et al., 1998. Genes Dev 12: 2899-2911; Zindy et al., 2000. Mol Cell Biol 20: 372-378; Zindy et al., 2001. Mol Cell Biol 21: 3244-3255). In order to assess whether these inhibitors directly or indirectly affect male germ cell differentiation, we examined the expression of p18(Ink4c) and p19(Ink4d) in spermatogenic and supporting cells in the testis and in pituitary gonadotropes. Both p18(Ink4c) and p19(Ink4d) are most abundant in the testis after 18 days of age and are expressed in purified populations of spermatogenic and testicular somatic cells. Different p18(Ink4c) mRNAs are expressed in isolated spermatogenic and Leydig cells. Spermatogenic cells also express a novel p19(Ink4d) transcript that is distinct from the smaller transcript expressed in Sertoli cells, Leydig cells and in other tissues. Immunohistochemistry detected significant levels of p19(Ink4d) in preleptotene spermatocytes, pachytene spermatocytes, condensing spermatids, and Sertoli cells. Immunoprecipitation-Western analysis detected both CKI proteins in isolated pachytene spermatocytes and round spermatids. CDK4/6-CKI complexes were detected in germ cells by co-immunoprecipitation, although the composition differed by cell type. p19(Ink4d) was also identified in FSH+ gonadotrophs, suggesting that this CKI may be independently required in the pituitary. Possible cell autonomous and paracrine mechanisms for the spermatogenic defects in mice lacking p18(Ink4c) or p19(Ink4d) are supported by expression of these CKIs in spermatogenic cells and in somatic cells of the testis and pituitary.

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 homo‐logue 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 prolinerich 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 exonintron 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 exonintron 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 ∼56 000, although the calculated molecular weight is 44501. The conserved nature of the mouse, rat, and human enzymes suggests that they serve similar roles in these and other mammalian species.

p19(Ink4d) is a member of the INK4 family of cyclin-dependent kinase inhibitors, which are important negative regulators of the G1-phase cyclin-dependent kinases CDK4 and CDK6. On a mixed C57BL/6 x 129P2/OlaHsd background, mice deficient for p19(Ink4d) exhibited defects in male reproductive function including testicular atrophy, alteration in serum follicle stimulating hormone, qualitative increase in germ cell apoptosis, and delayed kinetics of meiotic prophase markers (Zindy et al., 2001. Mol Cell Biol 21:3244-3255; Zindy et al., 2000. Mol Cell Biol 20:372-378). In this study, a quantitative assessment of these aspects of reproductive capacity demonstrated relatively mild deficits in p19(Ink4d-/-) males compared to controls. These effects did not dramatically worsen in older males although some seminiferous tubule defects were observed. Following marker-assisted backcrossing into the C57BL/6 background, p19(Ink4d-/-) males did not display defects in testis weights, sperm numbers, serum FSH, germ cell apoptosis, or kinetics of selected meiotic prophase markers. These studies indicate that a reduction in Ink4 family function by the loss of p19(Ink4d) is sufficient to induce mild reproductive defects in male mice with a mixed genetic background, but not in the C57BL/6 genetic background.