Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration

AbstractCyclooxygenases 1 and 2 (COX-1 and COX-2) are key enzymes in prostaglandin biosynthesis and the target enzymes for the widely used nonsteroidal anti-inflammatory drugs. To study the physiological roles of the individual isoforms, we have disrupted the mouse Ptgs1 gene encoding COX-1. Homozygous Ptgs1 mutant mice survive well, have no gastric pathology, and show less indomethacin-induced gastric ulceration than wild-type mice, even though their gastric prostaglandin E2 levels are about 1% of wild type. The homozygous mutant mice have reduced platelet aggregation and a decreased inflammatory response to arachidonic acid, but not to tetradecanoyl phorbol acetate. Ptgs1 homozygous mutant females mated to homozygous mutant males produce few live offspring. COX-1-deficient mice provide a useful model to distinguish the physiological roles of COX-1 and COX-2

Neonatal Exposure to Genistein Disrupts Ability of Female Mouse Reproductive Tract to Support Preimplantation Embryo Development and Implantation1

Female mice treated neonatally with the phytoestrogen genistein (50 mg/kg/day) have multioocyte follicles, lack regular estrous cyclicity, and are infertile even after superovulation. To determine the cause of their infertility, we examined oocyte developmental competence and timing of embryo loss. Eggs obtained by superovulation of genistein-treated or control females were equally capable of being fertilized in vitro and cultured to the blastocyst stage. However, if eggs were fertilized in vivo, retrieved at the pronucleus stage, and cultured, there was a significant reduction in the percentage of embryos from genistein-treated females reaching the blastocyst stage. When these blastocysts were transferred to pseudopregnant recipients, the number of live pups produced was similar to that in controls. Preimplantation embryo development in vivo was examined by flushing embryos from the oviduct and/or uterus. Similar numbers of one-cell and two-cell embryos were obtained from genistein-treated and control females. However, significantly fewer embryos (<50%) were obtained from genistein-treated females on postcoital Days 3 and 4. To determine if neonatal genistein treatment altered the ability of the uterus to support implantation, blastocysts from control donors were transferred to control and genistein-treated pseudopregnant recipients. These experiments demonstrated that genistein-treated females are not capable of supporting normal implantation of control embryos. Taken together, these results suggest that oocytes from mice treated neonatally with genistein are developmentally competent; however, the oviductal environment and the uterus have abnormalities that contribute to the observed reproductive failure

Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility

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

Phosphoglycerate Kinase 2 (PGK2) Is Essential for Sperm Function and Male Fertility in Mice1

Phosphoglycerate kinase 2 (PGK2), an isozyme that catalyzes the first ATP-generating step in the glycolytic pathway, is encoded by an autosomal retrogene that is expressed only during spermatogenesis. It replaces the ubiquitously expressed phosphoglycerate kinase 1 (PGK1) isozyme following repression of Pgk1 transcription by meiotic sex chromosome inactivation during meiotic prophase and by postmeiotic sex chromatin during spermiogenesis. The targeted disruption of Pgk2 by homologous recombination eliminates PGK activity in sperm and severely impairs male fertility, but does not block spermatogenesis. Mating behavior, reproductive organ weights (testis, excurrent ducts, and seminal vesicles), testis histology, sperm counts, and sperm ultrastructure were indistinguishable between Pgk2−/− and wild-type mice. However, sperm motility and ATP levels were markedly reduced in males lacking PGK2. These defects in sperm function were slightly less severe than observed in males lacking glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS), the isozyme that catalyzes the step preceding PGK2 in the sperm glycolytic pathway. Unlike Gapdhs−/− males, the Pgk2−/− males also sired occasional pups. Alternative pathways that bypass the PGK step of glycolysis exist. We determined that one of these bypass enzymes, acylphosphatase, is active in mouse sperm, perhaps contributing to phenotypic differences between mice lacking GAPDHS or PGK2. This study determined that PGK2 is not required for the completion of spermatogenesis, but is essential for sperm motility and male fertility. In addition to confirming the importance of the glycolytic pathway for sperm function, distinctive phenotypic characteristics of Pgk2−/− mice may provide further insights into the regulation of sperm metabolism

Impaired sperm fertilizing ability in mice lacking Cysteine-RIch Secretory Protein 1 (CRISP1)

Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. Epididymal protein CRISP1, a member of the Cysteine-RIch Secretory Protein (CRISP) family, was identified by our laboratory and postulated to participate in both sperm-zona pellucida (ZP) interaction and gamete fusion by binding to egg-complementary sites. To elucidate the functional role of CRISP1 in vivo, we disrupted the Crisp1 gene and evaluated the effect on animal fertility and several sperm parameters. Male and female Crisp1(-/-) animals exhibited no differences in fertility compared to controls. Sperm motility and the ability to undergo a spontaneous or progesterone-induced acrosome reaction were neither affected in Crisp1(-/-) mice. However, the level of protein tyrosine phosphorylation during capacitation was clearly lower in mutant sperm than in controls. In vitro fertilization assays showed that Crisp1(-/-) sperm also exhibited a significantly reduced ability to penetrate both ZP-intact and ZP-free eggs. Moreover, when ZP-free eggs were simultaneously inseminated with Crisp1(+/+) and Crisp1(-/-) sperm in a competition assay, the mutant sperm exhibited a greater disadvantage in their fusion ability. Finally, the finding that the fusion ability of Crisp1(-/-) sperm was further inhibited by the presence of CRISP1 or CRISP2 during gamete co-incubation, supports that another CRISP cooperates with CRISP1 during fertilization and might compensate for its lack in the mutant mice. Together, these results indicate that CRISP proteins are players in the mammalian fertilization process. To our knowledge this is the first knockout mice generated for a CRISP protein. The information obtained might have important functional implications for other members of the widely distributed and evolutionarily conserved CRISP family.Fil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Maldera, Julieta Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Willis, William D.. National Institutes of Health; Estados UnidosFil: Cohen, Debora Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Goulding, Eugenia H.. National Institutes of Health; Estados UnidosFil: Gelman, Diego Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Eddy, Edward M.. National Institutes of Health; Estados UnidosFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin