Impairment of gastric acid secretion and increase of embryonic lethality in Foxq1-deficient mice.

The mouse Foxq1 gene, also known as Hfh1, encodes a winged helix/forkhead transcription factor. In adult mice, Foxq1 is highly expressed in kidney and stomach. Here, we report that Foxq1 is expressed during prenatal and postnatal stomach development and the transcripts are restricted to acid secreting parietal cells. Mice homozygous for a deletion of the Foxq1 locus on a 129/Sv x C57BL/6J hybrid genetic background display variable phenotypes consistent with requirement of the gene during embryogenesis. Approximately 50% of Foxq1-/- embryos die in utero. Surviving homozygous mutants are normal and fertile, and have a silky shiny coat. Although the parietal cell development is not affected in the absence of Foxq1, there is a lack of gastric acid secretion in response to various secretagogue stimuli. Ultrastructural analysis suggests that the gastric acid secretion defect in Foxq1-deficient mice might be due to impairment in the fusion of cytoplasmic tubulovesicles to the apical membrane of secretory canaliculi.

The scoliosis (sco) mouse: a new allele of Pax1.

We describe the spontaneous mutant mouse scoliosis (sco) that carries a new allele of Pax1 (un-i, undulated intermediate). The Pax1(un-i) allele is lacking the 5'-flanking region and exon 1 to 4 which is mapped to nt -2636 to -640 and -272 to 4271 of the Pax1 gene. Homozygous mice show a mild form of the known phenotypes of other Pax1 mutants. Adult mice have a lumbar scoliosis and kinky tails. In homozygous embryos the skeleton ossifies early, ossification centers of the vertebral bodies are fused with the ossification centers of the pedicles. Neural arches and spinous processes are underdeveloped but the pedicles and transverse processes are overdeveloped which is in contrast to other Pax1 mutants. In the scapula, the acromion is missing and the deltoid tuberosity of the proximal humerus is shortened and thickened. Among the inner organs the thymus development is affected. In late embryos, the thymus is small and thymocyte numbers are reduced. T-cell development from CD4- and CD8- double negative (DN) to CD4+ and CD8+ double positive (DP) is decelerated. The percentage of CD90+ cells is also reduced but in contrast to other Pax1 mutants no alteration of the expression level of the CD90 (Thy-1) could be found.

Transcription of the Leydig insulin-like gene is mediated by steroidogenic factor-1.

The Leydig insulin-like gene (Ley I-L), a member of the insulin-related gene family, is specifically expressed in pre- and postnatal Leydig cells of the testis and in postnatal theca cells of the ovary. To determine the functional region of the mouse Ley I-L promoter and factors controlling the Ley I-L gene expression, we used 2.1 kb of the 5'-flanking region of the mouse Ley I-L gene to generate chimeric constructs with the chloramphenicol acetyltransferase gene (CAT). Transient transfections of MA10 Leydig cells, LTK- fibroblasts, and F9 embryonic cells by a series of 5'-deleted mouse Ley I-L promoter-CAT constructs revealed that the sequence between nucleotides -157 to +4 directs the transcription of the reporter gene in MA10 but not in LTK- and F9 cells, indicating that the determinants of Leydig cell-specific expression reside within this region. Deoxyribonuclease I (DNase I) footprint analysis revealed that the sequences designated SF-1/1, SF-1/2, and SF-1/3 within three DNase I-protected regions are homologous to the consensus binding site of the steroidogenic factor-1 (SF-1). Competition and antibody studies showed that the three SF-1-binding sites in the Ley I-L promoter have similar binding affinities for SF-1. Furthermore, transient transfections of MA10 cells with mutant reporter constructs, in which SF-1/1 or both SF-1/2 and SF-1/3 were deleted, demonstrated that all three SF-1-binding sites are required for SF-1-mediated stimulation of Ley I-L transcription. Cotransfection of an SF-1-containing expression vector together with a Ley I-L promoter-CAT construct into HeLa cells, which lack the endogenous SF-1 protein, resulted in CAT gene transcription, which indicated that SF-1 can transactivate the Ley I-L promoter. These data demonstrate an essential role of SF-1 in transcriptional activation of the Ley I-L promoter.

Targeted disruption of the Insl3 gene causes bilateral cryptorchidism.

The sexual dimorphic position of the gonads in mammals is dependent on differential development of two ligaments, the cranial suspensory ligament (CSL) and the gubernaculum. During male embryogenesis, outgrowth of the gubernaculum and regression of the CSL result in transabdominal descent of the testes, whereas in the female, development of the CSL in conjunction with failure of the gubernaculum development holds the ovaries in a position lateral to the kidneys. Several lines of evidence suggest that regression of the CSL and induction of gubernaculum development are mediated by testosterone and a yet unidentified testicular factor, respectively. The Insl3 gene (originally designated Ley I-L), a member of the insulin-like superfamily, is specifically expressed in Leydig cells of the fetal and postnatal testis and in theca cells of the postnatal ovary. Here we show that male mice homozygous for a targeted deletion of the Insl3 locus exhibit bilateral cryptorchidism with free moving testes and genital ducts. These malformations are due to failure of gubernaculum development during embryogenesis. In double-mutant male mice for Insl3 and androgen receptor genes, testes are positioned adjacent to the kidneys and steadied in the abdomen by the CSL. These findings demonstrate, that the Insl3 induces gubernaculum development in an androgen-independent way, while androgen-mediated regression of the CSL occurs independently from Insl3.

Hormonal control of gubernaculum development during testis descent: gubernaculum outgrowth in vitro requires both insulin-like factor and androgen.

The gubernaculum connects the gonad to the inguinoscrotal region and is involved in testis descent. It rapidly develops in the male fetus, whereas development in the female fetus is lacking. Possible factors involved in gubernaculum development are androgens, anti-Müllerian hormone (AMH), and insulin-like factor (Insl3). Sexual dimorphism in gubernaculum development correlated with the mitotic activity of cells in the gubernacular bulbs from male and female fetuses. Androgen receptor expression was restricted to the mesenchymal core of the gubernacular bulb, whereas skeletal muscle was detected in its outer layer. In an organ culture system devised to further study gubernaculum development in vitro, morphology of gubernacular explants grown in the presence of testes was comparable with that of gubernacula developed in vivo. Testicular tissue or medium containing R1881, a synthetic androgen, had a growth stimulatory effect on gubernacular explants compared with ovarian tissue or basal medium only. Moreover, Amh-/-, Amh+/-, and Insl3+/- testes stimulated the growth of gubernacular explants to the same extent as control testes. Insl3-/- testes, however, did not produce such an activity. This study reveals an essential role for both androgen and Insl3 in the gubernaculum outgrowth during transabdominal testis descent.

Involvement of insulin-like factor 3 (Insl3) in diethylstilbestrol-induced cryptorchidism.

Recently, it has been shown that targeted inactivation of the Insl3 gene in male mice results in cryptorchidism. The Insl3 gene encodes insulin-like factor 3 (Insl3), which is expressed in fetal Leydig cells. The testicular factor Insl3 appears to play an important role in the transabdominal phase of testis descent, which involves development of the gubernaculum. Other studies have demonstrated that in utero exposure to diethylstilbestrol (DES), a synthetic estrogen, can lead to cryptorchidism both in humans and in animal models. The present study was undertaken to investigate whether prenatal DES-exposure might interfere with testicular Insl3 mRNA expression. Furthermore, the effect of DES on steroidogenic factor 1 (SF-1) mRNA expression level was determined, since it has been shown that SF-1 plays an essential role in transcriptional activation of the Insl3 gene promoter. Timed pregnant mice were treated with DES (100 microg/kg body weight) or vehicle alone on days E9 (gestational day 9) through E17. Control and DES-exposed mouse fetuses were collected at E16, E17 and E18, when transabdominal testis descent is taking place. Lack of gubernaculum development in DES-exposed animals was confirmed by histological analyses at E17. Expression of Insl3 and SF-1 mRNAs was studied in testes of control and DES-exposed fetuses at E16 and E18 by RNase protection assay. Prenatal DES-exposure resulted in a three-fold decrease in Insl3 mRNA expression level (P<0.005), at both E16 and E18. In contrast, DES treatment had no effect on the expression of SF-1 mRNA. These results support our hypothesis that DES may interfere with gubernaculum development by altering Insl3 mRNA expression, providing a possible mechanism by which DES may cause cryptorchidism.

Synergistic effects of germ cell expressed genes on male fertility in mice.

Over 200 genes have been shown to be associated with infertility in mouse models. However, knockout mice reveal unexpected functional redundancy of some germ cell expressed genes. Single null mutations in mouse genes encoding four male germ cell proteins, transition protein 2 (Tnp2), proacrosin (Acr), histone H1.1 (H1.1), histone H1t (H1t) and sperm mitochondria-associated cysteine-rich protein (Smcp) have been generated and analysed. Tnp2 is believed to participate in the removal of the nuclear histones and initial condensation of the spermatid nucleus. Proacrosin is an acrosomal protease synthesized as a proenzyme and activated into acrosin during the acrosome reaction. The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues as well as in germ cells during the S-phase of the cell cycle. The histone gene Hist1h1t is expressed exclusively in spermatocytes and may have a function in establishing an open chromatin structure for the replacement of histones by transition proteins and protamines. Sperm mitochondria-associated cysteine-rich protein (Smcp) is a major structural element of the mitochondria in the midpiece of the sperm tail. Male mutant mice lacking any of these proteins show no apparent defects in spermatogenesis or fertility. To examine the synergistic effects of these proteins in spermatogenesis and during fertilization four lines of double knockout mice Hist1h1a/Mcsp, Hist1h1t/Mcsp, Tnp2/Mcsp and Acr/Mcsp were established. It was found that even when knockout mice are heterozygous for one allele (-/+) and homozygous for the other allele (-/-), mice were subfertile. Homozygous double knockout mice of all four lines are nearly infertile. However, in the four homozygous double knockout mouse lines, different characteristic abnormalities are prominently manifested: In Hist1h1a-/-/Mcsp-/- the migration of spermatozoa is disturbed in female genital tract, in Hist1h1t-/-/Mcsp-/- spermatozoa show morphological head abnormalities, in Tnp2-/-/Mcsp-/- the motility of sperm is affected, and in Acr-/-/Mcsp-/- the sperm-oocyte interaction is impaired. These findings indicate strongly that male germ cell expressed genes have synergistic effects on male fertility.

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.

Cloning, expression, and chromosomal localization of the rat mitochondrial capsule selenoprotein gene (MCS): the reading frame does not contain potential UGA selenocysteine codons.

The mitochondrial capsule selenoprotein (MCS) is a selenium-containing polypeptide. It is one of three proteins that are important for the maintenance and stabilization of the crescent structure of the sperm mitochondria. In this paper, we report the isolation and characterization of the rat MCS cDNA and gene. The cDNA contains a reading frame for a 145-amino-acid protein and it lacks the UGA codons, which have been found in the reading frame of the mouse MCS cDNA and have been presumed to encode the selenocysteine in the amino terminal of the deduced mouse amino acid sequence. The deduced amino acid sequence of the rat and mouse MCS shows a high level of homology (79%). The rat MCS gene contains two exons; the intron sequence interrupts the 5' untranslated sequence at the same position as in the mouse MCS gene. The transcription start site is located 184 bp upstream of the translation start site. Alignment of the 5'-flanking regions of the mouse and rat genes reveals that the first 400 nucleotides upstream of the transcription start site exhibit an overall sequence similarity of 73%. This conserved region contains no TATA or CAAT box motifs. Northern blot analysis indicates that the MCS mRNA is detectable only in the testis after day 30 of postnatal development. Moreover, in situ hybridization revealed that the rat MCS gene is mainly expressed in round spermatids. From the analysis of mouse-rat cell hybrids that segregate rat chromosomes, the MCS gene was assigned to rat chromosome 2.

Molecular cloning, chromosomal localization, and expression analysis of CYRN1 and CYRN2, two human genes coding for cyritestin, a sperm protein involved in gamete interaction.

Germ cell cyritestin is a membrane-anchored protein belonging to the ADAM family of proteins. Sequencing of eight human cyritestin cDNA clones revealed that they are identical at their 5' and 3' ends but differ from each other in the length of an internal deletion, suggesting that the human cyritestin mRNA is alternatively spliced. Internal deletions that are present in some cDNA isoforms do not cause a frameshift in the C-terminal coding region. Analysis of the predicted amino acid sequences demonstrated that the human cyritestin is a polymorphic protein that could include membrane-anchored and soluble forms. Southern blot analysis and characterization of human cyritestin genomic fragments revealed that the human genome contains two copies of the cyritestin gene instead of one as in the mouse. The human CYRN1 and CYRN2 genes were assigned to the region p12-21 of chromosome 8 and q12 of chromosome 16, respectively. Northern blot and RT-PCR analyses revealed that both human genes are expressed in the testis. Amino acid sequence comparisons between cyritestin and other members of the metalloprotease-disintegrin family of proteins suggested that human and mouse cyritestin and monkey tMDCI are homologous molecules.

Exon-intron structure and nucleotide sequence of the rat proacrosin gene.

The nucleotide sequence and exon-intron organization of the proacrosin gene was determined. It consists of 6 exons and 5 introns of which one is located in the 5' untranslated region. The transcription initiation site was determined at position 471 (564 nucleotides upstream of ATG), TATA- and TAAT-boxes were found 588 and 656 bp upstream of the ATG-translation start point, respectively.

Asthenoteratozoospermia in mice lacking testis expressed gene 18 (Tex18).

Testis expressed gene 18 (Tex18) is a small gene with one exon of 240 bp, which is specifically expressed in male germ cells. The gene encodes for a protein of 80 amino acids with unknown domain. To investigate the function of (Tex18) gene, we generated mice with targeted disruption of the (Tex18) gene by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J x 129/Sv) are fertile, while they are subfertile on the 129/Sv background, although mating is normal. We showed that Tex18(-/-) males are subfertile because of abnormal sperm morphology and reduced motility, which is called asthenoteratozoospermia, suggesting that (Tex18) affects sperm characteristics. Maturation of spermatids is unsynchronized and partially impaired in seminiferous tubules of Tex18(-/-) mice. Electron microscopical examination demonstrated abnormal structures of sperm head. In vivo experiments with sperm of Tex18(-/-) 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, is significantly affected, compared to wild-type spermatozoa. Generation of transgenic mice containing Tex18-EGFP fusion construct revealed a high transcriptional activity of (Tex18) during spermiogenesis, a process with morphological changes of haploid germ cells and development to mature spermatozoa. These results indicate that (Tex18) is expressed predominantly during spermatid differentiation and subfertility of the male Tex18(-/-) mice on the 129/Sv background is due to the differentiation arrest, abnormal sperm morphology and reduced sperm motility.

Cloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cells.

We have isolated complementary DNA clones coding for a novel member of the insulin-like hormone super-family from a boar testis cDNA library. Northern blot analysis and in situ hybridization revealed that the gene is expressed exclusively in prenatal and postnatal Leydig cells. We have tentatively proposed the name Leydig insulin-like (Ley I-L) for the gene and its encoded protein. The 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 with members of the insulin-like hormone superfamily 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 and has a molecular size of 6.25 kDa.

[Genetic causes of male infertility]. / Genetische Ursachen der männlichen Infertilität.

7% of all human males suffer from infertility. In at least 10% of these males infertility is due to genetic causes. Because modern reproduction techniques like ICSI (intracytoplasmic sperm injection) can help the couples to overcome infertility, it is mandatory to analyze underlying genetic causes of male infertility. If infertility in a male is due to a genetic defect, the risk of the respective couple for abortuses or malformed children is increased. The main and relevant causes for male infertility known to day are: numerical and structural chromosomal aberrations, meiotic defects, microdeletions in the region q11.21-23 of the Y-chromosome, mutations in the gene for cystic fibrosis and genetically determined syndromes in which infertility is a symptom. The present knowledge concerning these genetic causes of male infertility is pointed out.

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.

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.

Nucleotide sequence and exon-intron organization of the human proacrosin gene.

Acrosin is a serine proteinase and located in a zymogen form, proacrosin, in the acrosome of the sperm. As deduced from the cDNA sequences for human and boar proacrosin, the enzyme is synthesized as a preproenzyme, preproacrosin, which contains a hydrophobic leader sequence. Using cDNA clones as probes, we have isolated the gene coding for human proacrosin from a human leucocyte genomic library and a human cosmid library, respectively. The gene contains four introns between 0.2 kb--4.5 kb in length. Similar to other serine proteinases, the coding sequence of the preproacrosin gene is spread over all the five exons of the gene and the three activesite residues His, Asp and Ser are encoded by three different exons. According to the exon-intron structure, preproacrosin is suggested to be closely related to the serine proteinase subfamily containing trypsin and kallikrein. However, the light chain of proacrosin seems to be similar to that of chymotrypsin. The coding of the serine active-site residue together with the proacrosin-specific proline-rich domain in one exon, namely exon E5, let us assume that the nucleotide sequence for the proline-rich domain was generated during evolution by intron-exon transfer from a foreign gene with subsequent intron excision. By primer extension analysis, the transcription initiation site of the preproacrosin mRNA could be assigned to the residue C at -74 nucleotides upstream from the translation initiation codon ATG. In contrast to most other eucaryotic genes, including the known testis-specific genes, typical TATA and CAAT box sequences in convential distances from the 5' end of the transcription start site could not be evaluated in the proacrosin gene.

Mouse pelota gene (Pelo): cDNA cloning, genomic structure, and chromosomal localization.

The pelota gene of Drosophila melanogaster encodes a protein which is included in cell cycle regulation. Mutations were found to result in spermatogenic arrest, female sterility and disturbances in the patterning of the eye. We have recently isolated and characterized cDNA clones coding for the human pelota gene (PELO). Here we describe the cloning of the murine pelota cDNA and gene (Pelo) that encodes a 385-amino-acid protein. The exon-intron structure of the gene, which contains three exons, was determined. Comparison of the mouse amino acid sequences with the human and Drosophila sequences revealed an overall high identity (96% and 70%, respectively). Northern blot analysis detected a 1.7-kb transcript in all tissues studied. Southern blot analyses revealed that the pelota gene is present as a single copy in the mouse genome. The mouse pelota gene (Pelo) was mapped to the distal end of chromosome 13, in a region that is homologous with a segment of human chromosome 5q11 containing the orthologous human gene. Cloning of the mouse gene is an important step to study the function of the pelota gene in mammals and to create a mouse model for this evolutionarily conserved gene.

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

cDNA 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.

Structural organization of the porcine and human genes coding for a Leydig cell-specific insulin-like peptide (LEY I-L) and chromosomal localization of the human gene (INSL3).

Leydig insulin-like protein (LEY I-L) is a member of the insulin-like hormone superfamily. The LEY I-L gene (designated INSL3) is expressed exclusively in prenatal and postnatal Leydig cells. We report here the cloning and nucleotide sequence of porcine and human LEY I-L genes including the 5' regions. Both genes consist of two exons and one intron. The organization of the LEY I-L gene is similar to that of insulin and relaxin. The transcription start site in the porcine and human LEY I-L gene is localized 13 and 14 bp upstream of the translation start site, respectively. Alignment of the 5' flanking regions of both genes reveals that the first 107 nucleotides upstream of the transcription start site exhibit an overall sequence similarity of 80%. This conserved region contains a consensus TATAA box, a CAAT-like element (GAAT), and a consensus SP1 sequence (GGGCGG) at equivalent positions in both genes and therefore may play a role in regulation of expression of the LEY I-L gene. The porcine and human genome contains a single copy of the LEY I-L gene. By in situ hybridization, the human gene was assigned to bands p13.2-p12 of the short arm of chromosome 19.