Although glycolysis is highly conserved, it is remarkable that several unique isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like its human ortholog (GAPD2), is the sole GAPDH isozyme in sperm. It is tightly bound to the fibrous sheath, a cytoskeletal structure that extends most of the length of the sperm flagellum. We disrupted Gapds expression by gene targeting to selectively block sperm glycolysis and assess its relative importance for in vivo sperm function. Gapds ؊/؊ males were infertile and had profound defects in sperm motility, exhibiting sluggish movement without forward progression. Although mitochondrial oxygen consumption was unchanged, sperm from Gapds ؊/؊ mice had ATP levels that were only 10.4% of those in sperm from WT mice. These results imply that most of the energy required for sperm motility is generated by glycolysis rather than oxidative phosphorylation. Furthermore, the critical role of glycolysis in sperm and its dependence on this sperm-specific enzyme suggest that GAPDS is a potential contraceptive target, and that mutations or environmental agents that disrupt its activity could lead to male infertility. glycolysis ͉ gene targeting ͉ infertility S perm motility is essential for normal fertilization, and asthenozoospermia, or low sperm motility, is common in infertile men. In a recent study of 1,085 sperm samples from infertile men, 81% had defects in motility, and 19% had asthenozoospermia without other defects in sperm number or morphology (1). Motility is generated by the extremely long flagellum that comprises Ͼ90% of the length of a mammalian sperm. This process requires substantial ATP to support coordinated movement of the central axoneme and surrounding flagellar structures (2). ATP is hydrolyzed by dynein ATPases, which function as force-generating molecular motors along the axoneme. Although quiescent in the epididymis, mammalian sperm display vigorous forward movement, termed activated or progressive motility, immediately upon ejaculation or collection into physiological medium. The motility waveform changes in the female reproductive tract, with increases in both the amplitude and asymmetry of flagellar bending. These changes result in a whiplash-like motion, termed hyperactivated motility, which facilitates sperm transport in the oviduct and penetration of the zona pellucida surrounding the oocyte (3).Potential sources of ATP to support sperm motility are compartmentalized in distinct regions along the length of the flagellum. Oxidative phosphorylation is confined to the proximal segment of the flagellum where the mitochondria are localized (middle piece). In contrast, glycolysis appears to be restricted to the principal piece, which is distal to the middle piece and is the longest segment of the sperm flagellum (4-8). Several glycolytic enzymes in mammalian sperm are distinct from the isozymes present in somatic tissues. Thr...

Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxy-peptidase that generates the vasoconstricting peptide angiotensin II and inactivates the vasodilating peptide bradykinin. The gene encoding ACE is composed of two homologous regions and codes for both a somatic and testis isoenzyme. Experiments with hypertensive rats and some, but not other, studies of humans suggest that sequences at or linked to the gene influence blood pressure. The testis-specific form of ACE has its own promoter within intron 12 (ref. 14), is encoded by the 3' region of the gene, and is found only in postmeiotic spermatogenic cells and sperm. Its function is unknown. Here we investigate the role of the Ace gene in blood pressure control and reproduction using mice generated to carry an insertional mutation that is designed to inactivate both forms of ACE. All homozygous female mutants were found to be fertile, but the fertility of homozygous male mutants was greatly reduced. Heterozygous males but not females had blood pressures that were 15-20 mm Hg less than normal, although both male and female heterozygotes had reduced serum ACE activity.

The Collaborative Cross Consortium reports here on the development of a unique genetic resource population. The Collaborative Cross (CC) is a multiparental recombinant inbred panel derived from eight laboratory mouse inbred strains. Breeding of the CC lines was initiated at multiple international sites using mice from The Jackson Laboratory. Currently, this innovative project is breeding independent CC lines at the University of North Carolina (UNC), at Tel Aviv University (TAU), and at Geniad in Western Australia (GND). These institutions aim to make publicly available the completed CC lines and their genotypes and sequence information. We genotyped, and report here, results from 458 extant lines from UNC, TAU, and GND using a custom genotyping array with 7500 SNPs designed to be maximally informative in the CC and used a novel algorithm to infer inherited haplotypes directly from hybridization intensity patterns. We identified lines with breeding errors and cousin lines generated by splitting incipient lines into two or more cousin lines at early generations of inbreeding. We then characterized the genome architecture of 350 genetically independent CC lines. Results showed that founder haplotypes are inherited at the expected frequency, although we also consistently observed highly significant transmission ratio distortion at specific loci across all three populations. On chromosome 2, there is significant overrepresentation of WSB/EiJ alleles, and on chromosome X, there is a large deficit of CC lines with CAST/EiJ alleles. Linkage disequilibrium decays as expected and we saw no evidence of gametic disequilibrium in the CC population as a whole or in random subsets of the population. Gametic equilibrium in the CC population is in marked contrast to the gametic disequilibrium present in a large panel of classical inbred strains. Finally, we discuss access to the CC population and to the associated raw data describing the genetic structure of individual lines. Integration of rich phenotypic and genomic data over time and across a wide variety of fields will be vital to delivering on one of the key attributes of the CC, a common genetic reference platform for identifying causative variants and genetic networks determining traits in mammals.

The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibers and defines the extent of the principal piece region of the sperm flagellum. It consists of two longitudinal columns connected by closely arrayed semicircular ribs that assemble from distal to proximal throughout spermiogenesis. The fibrous sheath is believed to influence the degree of flexibility, plane of flagellar motion, and the shape of the flagellar beat. Nearly half of the protein in fibrous sheaths isolated from mouse sperm is AKAP4. This protein and two others, AKAP3 and TAKAP-80, have anchoring sites for cAMP-dependent protein kinase. AKAP3 also anchors ropporin, a spermatogenic cell-specific protein that is linked through rhophilin to the small GTPase Rho. Other proteins associated with the fibrous sheath include two enzymes in the glycolytic pathway. Glyceraldehyde 3-phosphate dehydrogenase-s (GAPDS) is the product of a gene expressed only in spermatogenic cells, while hexokinase type 1-s (HK1-S) is derived from alternative transcripts present only in spermatogenic cells. Most of the other glycolytic enzymes in sperm have unique structural or functional properties. The fibrous sheath also contains a spermatogenic cell-specific member of the mu-class glutathione S-transferase family (GSTM5) and an intermediate filament-like protein (FS39). These and other observations indicate that the fibrous sheath functions as a scaffold for proteins in signaling pathways that might be involved in regulating sperm maturation, motility, capacitation, hyperactivation, and/or acrosome reaction and for enzymes in the glycolytic pathway that provide energy for the hyperactivated motility of sperm that allows them to penetrate the zona pellucida.

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...

No abstract

The presence of two families of seven distinct mammalian cyclin-dependent kinase (CDK) inhibitor genes is thought to mediate the complexity of connecting a variety of cellular processes to the cell cycle control pathway. The distinct pattern of tissue expression of CDK inhibitor genes suggests that they may function as tumor suppressors with different tissue specificities. To test this hypothesis, we have characterized two strains of double mutant mice lacking either p18INK4c and p27 KIP1 or p18 INK4c and p21 CIP1/WAF1 . Loss of both p18 and p27 function resulted in the spontaneous development by 3 months of age of at least eight different types of hyperplastic tissues and/or tumors in the pituitary, adrenals, thyroid, parathyroid, testes, pancreas, duodenum, and stomach. Six of these hyperplastic tissues and tumors were in endocrine organs, and several types of tumors routinely developed within the same animal, a phenotype reminiscent of that seen in combined human multiple endocrine neoplasia syndromes. The p18-p21 double null mice, on the other hand, developed pituitary adenomas, multifocal gastric neuroendocrine hyperplasia, and lung bronchioalveolar tumors later in life. G 1 CDK2 and CDK4 kinase activities were increased in both normal and neoplastic tissues derived from mice lacking individual CDK inhibitors and were synergistically stimulated by the simultaneous loss of two CDK inhibitors. This indicates that an increase in G 1 CDK kinase activity is a critical step during but is not sufficient for tumor growth. Our results suggest that functional collaborations between distinct CDK inhibitor genes are tissue specific and confer yet another level of regulation in cell growth control and tumor suppression.

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.