The sperm mitochondria-associated cysteine-rich protein (SMCP) is a cysteine-and proline-rich structural protein that is closely associated with the keratinous capsules of sperm mitochondria in the mitochondrial sheath surrounding the outer dense fibers and axoneme. To investigate the function of SMCP, we generated mice with a targeted disruption of the gene Smcp by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J ؋ 129/Sv) are fully fertile, while they are infertile on the 129/Sv background, although spermatogenesis and mating are normal. Homozygous Smcp ؊/؊ female mice are fertile on both genetic backgrounds. Electron microscopical examination demonstrated normal structures of sperm head, mitochondria, and tail. In vivo experiments with sperm of Smcp ؊/؊ 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility as determined by the computer-assisted semen analysis system (CASA) is significantly affected as compared to wild-type spermatozoa. In vitro fertilization assays showed that Smcpdeficient spermatozoa are able to bind to the oocyte but that the number of fertilized eggs is reduced by more than threefold relative to the wild-type control. However, removal of the zona pellucida resulted in an unaffected sperm-egg fusion which was monitored by the presence of pronuclei and generation of blastocyts. These results indicate that the infertility of the male Smcp ؊/؊ mice on the 129/Sv background is due to reduced motility of the spermatozoa and decreased capability of the spermatozoa to penetrate oocytes.Mitochondria are confined to a structure known as the mitochondrial sheath in the midpiece of mammalian spermatozoa. The mitochondria in the sheath are elongated, crescent shaped, and aligned end-to-end in helices wrapped around the outer dense fibers which in turn surround the flagellar axoneme (21). Pallini et al. (22) purified sonication-and sodium dodecyl sulfate (SDS)-resistant structures from sperm mitochondria, known as capsules, that retain the size and shape of the outer membranes of sperm mitochondria. The integrity of capsules is maintained by disulfide bridges, and purified capsules from bull sperm contain three proteins, including a peculiar hydrophilic protein containing a large amount of proline (26%) and cysteine (18%), the sperm mitochondria-associated cysteine-rich protein (SMCP). For many years, SMCP was thought to be a 20-kDa selenoprotein and the predominant capsular protein (7), but recent work demonstrates that those attributes belong to phospholipid hydroxide glutathione peroxidase, which functions as a cytosolic enzyme in somatic cells and an enzymatically inactive structural protein in the mitochondrial capsule (33). Electron microscope immunocytochemistry localizes mouse SMCP to the outer mitochondrial membranes and intermitochondrial spaces of the mitochondrial sheath (8). Since SMCP is neither a selenoprotein nor the major capsule protein...

Summary We have isolated and sequenced the cDNA and gene coding for a novel member of the insulin‐like hormone superfamily. The gene is expressed exclusively in prenatal and postnatal Leydig cells and in postnatal ovary. We have tentatively proposed the name Leydig insulin‐like (Ley I‐L) for the gene and its encoded protein. The porcine Leydig insulin‐like protein is synthesized as a 131‐amino acid preproprotein, which contains a 24 amino acid signal peptide. Comparison of the deduced amino acid sequence of pro‐Leydig insulin‐like protein predicts that the biologically active protein, after proteolytic processing of the C‐peptide, consists of a 32 residue‐long B‐chain and a 26‐residue‐long A‐chain. Western blot analyses with antiserum against a synthetic oligopeptide containing the amino acid sequence of the B‐chain confirm the proteolytic processing of the pro‐Ley I‐L peptide. Furthermore, we describe here the initial analysis of the Ley I‐L gene promoter by transient gene transfer studies. We show that the sequence between −186 and +13 of the porcine Ley I‐L gene contains sufficient information to direct preferential expression of the chloramphenicol‐acetyltransferase (CAT) reporter gene in Leydig cells and sequences important for negative regulation of Ley I‐L promoter activity are contained in the region between −960 and −186. Zusammenfassung Das Insulin‐ähnliche Peptid der Leydigzelle Wir haben die cDNA und das Gen für ein bislang unbekanntes Insulin‐ähnliches Hormon isoliert und charakterisiert. Das Gen ist ausschließlich in pränatalen‐ und postnatalen Leydigzellen sowie im Ovar exprimiert. Das Gen hat die Bezeichnung Ley I‐L (Leydig Insulin Like) erhalten. Das Ley I‐L Protein wird als 131 Aminosäuren großes Präprotein synthetisiert, welches ein Signalpeptid aus 24 Aminosäuren enthält. Es ist anzunehmen, daß das biologisch aktive Ley I‐L Protein, nach Abspaltung des C‐Peptids, aus einer B‐Kette (32 Aminosäuren) und einer A‐Kette (26 Aminosäuren) besteht. Westernblot‐Analysen unter Verwendung eines Antiserums gegen ein synthetisches Oligopeptid der B‐Kette bestätigen diese Annahme. Wir berichten hier auch über unsere vorläufigen Analysen der Promotorregion des Ley I‐L Gens. Die Sequenz zwischen −186 bis +13 des Gens beinhaltet ausreichende Information für die präferentielle Expression des Chloramphenicolacetyltransferase Gens (CAT) als Reportergen in kultivierten Leydigzellen. Ferner sind Sequenzen nachweisbar (−960 bis −186), die für eine negative Regulation des Ley I‐L Gens in Frage kommen.