Ibrahim M. Adham

Spermatozoa lacking acrosin protein show delayed fertilization.

Acrosin (ACR), a serine proteinase located in the acrosome of the sperm, has been presumed to be involved in the recognition and binding of the sperm to the zona pellucida of the ovum and the sperm penetration through the zona pellucida. To examine the function of acrosin in vivo, we have generated mice carrying a mutation at the acrosin locus (Acr) through targeted disruption in embryonic stem (ES) cells. One chimeric male and female transmitted the targeted gene through their germ line. Homozygous Acr+/− mice are fertile and yield litters comparable in number and size to those of Acr+/− mice. These data show that sperm of the homozygous Acr+/− mice are able to penetrate the zona pellucida, fertilize the ovum, and produce viable offspring. However, spermatozoa lacking acrosin protein show a delayed fertilization. One chimeric male which contained the targeted gene in 20% of its sperm transmitted only the Acr+ allele to its progeny. Furthermore, in vitro fertilization with equally mixed sperm cells of Acr+/− and Acr+/− mice resulted in fertilization only with the Acr+ sperm cells. Incubation of oocytes with Acr+ or Acr− sperm show that the Acr− sperm are faster to fertilize the oocytes than the Acr+ sperm cells. These results suggest that Acr− sperm have a selective disadvantage when they are in competition with Acr+ sperm. Mol. Reprod. Dev. 46:370–376, 1997.

Mouse Leydig insulin-like (Ley I-L) gene: Structure and expression during testis and ovary development

Leydig insulin‐like protein (Ley I‐L) is a novel member of the insulin‐like hormone superfamily. We report here the isolation and expression of the mouse Ley I‐L gene. The gene encodes a polypeptide of 122 amino acids that shows a relatively weak homology (54%) to human and porcine prepro‐Ley I‐L. However, the predicted B and A chain of the mature mouse Ley I‐L exhibit similarities of 73% and 71% with human and porcine Ley I‐L, respectively. Alignment of the 5′ flanking region of the mouse gene with those of human and porcine did not exhibit any significant sequence homology. However, it contains the conserved sequence of the Ad4 binding site that is present in all promoter regions of steroidogenic P‐450 genes and the Müllerian inhibitor substance gene and is recognized by steroidogenic factor 1. The Ley I‐L gene is expressed at a high level in the testis and at a much lower level in the ovary. No transcripts could be detected in placenta prepared between days 10 and 19 of pregnancy. Ley I‐L transcripts were first detected in fetal testis at 13.5 dpc. After birth, transcript levels remain constant during the following 3 weeks, increasing at the stage in which the first wave of round spermatids undergo spermiogenesis suggesting a functional role of the Ley I‐L in early stages of spermatogenesis and germ‐cell maturation. In the ovary, the expression of Ley I‐L was first detected at day 6 after birth. The pattern of Ley I‐L expression at various stages of the estrous cycle and during pregnancy showed a correlation with follicle development. Mol. Reprod. Dev. 47:30–38, 1997.

Targeted deletion of murine coagulation factor XII gene-a model for contact phase activation in vivo

To analyze the biological role of factor XII (FXII, Hageman Factor) in vivo, we generated mice deficient for FXII using a gene targeting approach on two distinct genetic backgrounds, i.e. mixed C57Bl/6J X 129X1/SvJ and inbred 129X1/SvJ. Homozygous FXII knockout (FXII(-)/(-)) mice showed no FXII plasma activity and had a markedly prolonged activated partial thromboplastin time (aPTT). In contrast, coagulation factors XI, VIII, IX, X,VII, V, II and fibrinogen did not differ between FXII(-/-) mice and their wild-type littermates. Heterozygous matings segregated according to the Mendelian inheritance indicating that FXII deficiency does not increase fetal loss. Furthermore, matings of FXII(-/-) males and FXII(-/-) females resulted in normal litter sizes demonstrating that total FXII deficiency in FXII(-/-) females does not affect pregnancy outcome. Also, gross and histological anatomy of FXII(-/-) mice was indistinguishable from that of their wild-type littermates on both genetic backgrounds. Thus it appears that deficiency of murine FXII does not cause thrombophilia or impaired fibrinolysis in vivo. These results indicate that FXII deficiency does not affect hemostasis in vivo and we anticipate that the FXII(-/-) mice will be helpful to elucidate the biological role(s) of FXII in health and disease.

The role of the testicular factor INSL3 in establishing the gonadal position.

INSL3, also designated Leydig insulin-like (Ley I-L) or relaxin-like factor (RLF), belongs to the insulin-like hormone superfamily. It is expressed in pre- and postnatal Leydig cells of the testis and in postnatal theca cells of the ovary. This sexual dimorphic pattern of INSL3 expression during development led us to suggest that the INSL3 factor could play an essential role in sexual differentiation, gonadal function and germ cell development. Key insights into the role of INSL3 came from analyses of INSL3 knockout mice. These mice showed impaired development of the gubernaculum ligament, a structure that is believed to mediate transabdominal descent of the testis during male embryogenesis. In double mutant XY-mice lacking INSL3 and a functional androgen receptor, it was demonstrated that both are essential for establishment of the sexual dimorphic position of the gonads through regulation of gubernaculum development and regression of the cranial suspensory ligament (CSL) during fetal life. Defects in this developmental process can cause cryptorchidism in the male, which is a most common disorder of sexual differentiation in human.

Disruption of ADAM3 Impairs the Migration of Sperm into Oviduct in Mouse

Abstract Sperm from four different gene-disrupted mouse lines (calmegin [Clgn], Adam1a, Adam2, and Ace) are known to have defective zona-binding ability. Moreover, it is also reported that the sperm from all of these mouse lines exhibit another common phenotype of impaired migration into oviduct despite the large number of sperm found in uterus after coitus. On the other hand, the sperm from the Adam3-disrupted mouse line was reported to have defects in binding ability to zona, but were able to move into the oviduct. In order to clarify the difference, we investigated the migration of ADAM3-null sperm into oviduct precisely by visualizing the sperm by using acrosin-green fluorescent protein as a tag. As a result, in contrast to previous observations, it was demonstrated that the Adam3-disrupted sperm were unable to migrate into the oviduct after coitus. It was ultimately shown that, in five out of five different gene-disrupted mouse lines, the phenotype of impaired sperm binding to zona pellucida was accompanied by the loss of ability of sperm to migrate into the oviduct. This indicates a close relationship between the two phenomena, and also that sperm migration into the oviduct is a crucial step for fertilization.

Asthenozoospermia in Mice with Targeted Deletion of the Sperm Mitochondrion-Associated Cysteine-Rich Protein (Smcp) Gene

ABSTRACT 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 Smcp-deficient 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.

Increase in final stages of follicular atresia and premature decay of corpora lutea in Insl3-deficient mice.

The insulin‐like factor 3 (Insl3), a member of the insulin‐like hormone family, is exclusively synthesized in gonads. Our recent analysis of Insl3‐deficient mice revealed the regulating role of the Insl3 factor on the gubernaculum development during the transabdominal descent of the testis. Here we define the role of the Insl3 factor by histometric analysis of wild‐type and Insl3−/− ovaries. Ovaries from 40‐day‐old‐ and 6‐month‐old Insl3−/− mice as well as from wild‐type littermates were serially sectioned. Sections were stained with periodic acid Schiff reaction (PAS) for counting the number of zonae pellucidae which indicated the final stages of follicular atresia. Corpora lutea were also determined. Some sections were processed using either a modified TUNEL method for in situ detection of apoptosis or a lectin labelling technique with Griffonia simplicifolia I agglutinin (GS I) for endothelial cell occurrence. The number of zonae pellucidae was higher in Insl3‐deficient ovaries of both ages than in ovaries of wild‐type sisters (P < 0.05 for 40‐day‐old ovaries; P < 0.01 for 6‐month‐old ovaries). Additionally, the wild‐type mice of both ages possessed threefold more corpora lutea than their Insl3 littermates (P < 0.01 for 40‐day‐old; P < 0.001 for 6‐month‐old). In general, wild‐type corpora lutea looked healthy, showed GS I‐positive endothelial cells and no apoptotic cells. Corpora lutea from mutants were rich in regressing GS I luteal cells, and apoptotic cells appeared. We conclude: Follicular atresia and luteolysis are accelerated in ovaries of Insl3‐deficient mice probably because of increased apoptosis. The Insl3 function thus appears to rescue endocrine cells from the apoptotic pathway. Mol. Reprod. Dev. 58:281–286, 2001.

A human cDNA coding for the Leydig insulin-like peptide (Ley I-L).

AbstractcDNA clones for the human Leydig insulin-like peptide (Ley I-L) have been isolated and characterized. The nucleotide sequence of the 743-bp cDNA includes an incomplete 7-bp 5′-noncoding region, an open reading frame of 393 bp, and a 343-bp 3′-noncoding region. By primer extension analysis, the transcription start site was determined as being 14-bp upstream of the translation start site. The underlying gene is expressed in the testis but not in other organs. From the cDNA sequence, it can be deduced that the Ley I-L protein is synthesized as a 131-amino-acid (aa) preproprotein and that it contains a 24-aa signal peptide. Comparison of the pro Ley I-L protein with members of the insulin-like hormone superfamily predicts that the biologically active hormone, after proteolytic processing of the C peptide, consists of a 31-aa long B chain and a 26-aa long A chain, and that it has a molecular weight of 6.25 kDa.

Determination of bleeding risk using genetic markers in patients taking phenprocoumon

BackgroundIn patients on oral anticoagulation with warfarin, genetic variations of the cytochrome P 450–CYP2C9 have recently been associated with very low warfarin requirements. Patients needing low doses had an increased risk for bleeding complications. In Germany, phenprocoumon (having a similar metabolic pathway) is the most commonly employed vitamin K antagonist. Treatment is usually monitored by general practitioners (GPs).ObjectivesTo determine whether CYP2C9 variant alleles can serve as risk markers in general-practice patients anticoagulated with phenprocoumon.MethodsAll adult anticoagulated patients in 12 teaching general practices and one university outpatient clinic were to be recruited. Blood samples were taken from 185 patients during routine anticoagulation controls and tested for CYP2C9 mutations. Subjects answered a questionnaire concerning bleeding complications, drug intolerance, and personal and family medical history. Phenprocoumon dosages required for stable anticoagulation were recorded. Odds ratios (OR) with 95% confidence intervals (CI) were calculated based on 2-way cross-tabulations and multivariate logistic regression models, t-tests used where appropriate.ResultsBleeding was reported by 19% of the patients, 2.2% of whom had suffered life-threatening bleeding. CYP2C9 variants were carried by 26.3% of 179 patients tested (17.9% *1/*2, 7.8% *1/*3, 0.6% *2/*3). While presence of a *2 allele was not associated with an increased risk (OR 0.35, CI 0.10–1.24), carriers of the rare *3 alleles had a higher risk of bleeding (OR 3.10, CI 1.02–9.40). With regard to bleeding, carrying CYP2C9*3 was highly specific (94%), though sensitivity was low at 17%; post-test probability of bleeding was 40%.ConclusionsCYP2C9*3 variants are associated with an increased bleeding risk in patients anticoagulated with phenprocoumon. Screening can identify patients with a high risk of bleeding. Appropriate clinical consequences (restricted indication for anticoagulation, careful induction, adjustment of target INR, closer monitoring or self-testing of INR) as well as the cost-effectiveness of screening for variant CYP2C9 with regard to patient outcomes should be subject of further research.

The Small Heat Shock Protein ODF1/HSPB10 Is Essential for Tight Linkage of Sperm Head to Tail and Male Fertility in Mice

ABSTRACT Sperm motility and hence male fertility strictly depends on proper development of the sperm tail and its tight anchorage to the head. The main protein of sperm tail outer dense fibers, ODF1/HSPB10, belongs to the family of small heat shock proteins that function as molecular chaperones. However, the impact of ODF1 on sperm tail formation and motility and on male fecundity is unknown. We therefore generated mutant mice in which the Odf1 gene was disrupted. Heterozygous mutant male mice are fertile while sperm motility is reduced, but Odf1-deficient male mice are infertile due to the detachment of the sperm head. Although headless tails are somehow motile, transmission electron microscopy revealed disturbed organization of the mitochondrial sheath, as well as of the outer dense fibers. Our results thus suggest that ODF1, besides being involved in the correct arrangement of mitochondrial sheath and outer dense fibers, is essential for rigid junction of sperm head and tail. Loss of function of ODF1, therefore, might account for some of the cases of human infertility with decapitated sperm heads. In addition, since sperm motility is already affected in heterozygous mice, impairment of ODF1 might even account for some cases of reduced fertility in male patients.