Reduced mitotic activity and increased apoptosis of fetal sertoli cells in rat hypogonadic (hgn/hgn) testes

Sterility in male hypogonadic (hgn/hgn) rats results from congenital testicular dysplasia caused by a single recessive gene hgn on rat chromosome 10. We recently identified an insertion mutation in the Spag5/astrin gene of hgn/hgn rats that may cause defective proliferation of immature Sertoli cells in the postnatal hgn/hgn testis. Since the pathological alterations were present in the testes at birth, we examined the involvement of defective mitosis and apoptotic cell death in embryonic development of hgn/hgn testes. Testicular hypoplasia was apparent at embryonic day (ED) 18.5. Immunostaining of hgn/hgn testes at ED 21.5 with antibody to GATA-4, which is specific for fetal Sertoli cells in the seminiferous cords, showed that the significant decrease in the number of fetal Sertoli cells was accompanied by a two fold increase in their mitotic index and abnormal mitosis and apoptosis. Prior to this, we observed a decrease in the number of BrdU-labeled cells, an increase in the number of TUNEL-positive apoptotic cells, and presence of MIS-positive apoptotic cells in hgn/hgn testes on ED 17.5 and 18.5. These results suggest that the Spag5 mutation may cause a reduction in mitotic activity and an increase in apoptosis of fetal Sertoli cells in hgn/hgn testes.     

Identification of a locus for susceptibility to renal cell carcinoma in the Long-Evans Cinnamon rat

The Long-Evans Cinnamon (LEC) mutant rat shows higher incidence of renal cell carcinomas induced by a treatment with the chemical carcinogen N-diethylnitrosamine, as compared to the normal control rat. We performed the first genome-wide scan for genes responsible for susceptibility to chemically induced renal cell carcinoma in an F2 intercross obtained by mating the LEC and Fischer-344 (F344) rats. The genotype of 71 (F344 x LEC) F2 progenies was determined with the use of 338 simple sequence length polymorphisms (SSLPs) spread over the genome. The F2 rats which carried renal cell carcinoma were shown to possess the incidence of homozygosity of the LEC allele which is higher than that of the other genotypes at SSLP markers on chromosome 5 (chi2 = 17.5 for D5Rat21). Our linkage analysis has led to the revelation of a novel gene that influences susceptibility to renal cell carcinoma on rat chromosome 5.     

Further studies with a sex-linked lethal gene in the fowl

1. The ladykiller (lk) gene has been located on the sex chromosome of the fowl. It is 4.2 map units distant from the feathering rate locus (k) and 11.2 map units distant from the dwarf (dw) locus. 2. Most of the embryonic mortalities associated with this gene occurred between 3 and 4 d of incubation.     

Identification of the modifier locus that suppresses neonatal lethality in (♀DDD × ♂DH- Dh/+) F₁-Dh/+ male mice

Most F(1)-Dh/+ male mice resulting from a cross between inbred DDD strain females and DH-Dh/+ strain males exhibit growth retardation and die during the neonatal period. The lethality is caused by a combination of three independent gene loci, namely the Dh locus on chromosome 1, Grdhq1 locus on the X chromosome, and a putative Y chromosome-linked locus in some strains. Among these loci, Grdhq1 was previously mapped to a distal region of the X chromosome using progeny from♀(♀DDD × ♂DH-+/+) F(1) × ♂DH-Dh/+ mice. In this study, fine mapping of Grdhq1 was performed using progeny of ♀(♀DDD × ♂CAST/EiJ) F(1) ♂DH-Dh/+ mice. Contrary to expectation, Dh/+ male pups carrying the DDD allele at DXMit135 (genetic marker nearest to Grdhq1) survived to weaning. The presence of modifier loci that suppressed the lethality by impeding the action of Grdhq1 was suggested; therefore, a genome-wide scan was performed in the surviving Dh/+ males. As a result, a significant modifier locus was identified on proximal chromosome 11. This in turn suggested that Grdhq1 was located more distally than we had expected; that is, the actual location of Grdhq1 appeared to be near and/or distal to the Mid1 locus. Thus, the results revealed that the neonatal lethality in (DDD × DH-Dh/+) F(1)-Dh/+ males was caused by the fourth gene locus on chromosome 11 in addition to the above-mentioned three gene loci on chromosomes 1, X, and Y.     

Characterization of porcine uterine estrogen sulfotransferase

A quantitative trait locus (QTL) for uterine capacity is located on chromosome 8. Comparison of porcine and human genetic maps suggested that the estrogen sulfotransferase (STE) gene may be located near this region. The objectives of this study were to clone the full coding region for STE, compare endometrial STE gene expression between Meishan and White composite pigs during early pregnancy, and map the STE gene. We obtained a clone (1886 bp) containing the full coding region of STE by iterative screening of an expressed sequence tag library. Endometrial STE mRNA expression in White composite gilts was determined by Northern blotting on days 10, 13, and 15 of the estrous cycle; and on days 10, 13, 15, 20, 30, and 40 of pregnancy. STE mRNA expression was elevated (P<0.01) on days 20 and 30 of pregnancy compared to other days of the cycle or pregnancy. Endometrial STE mRNA expression during early pregnancy, determined using real-time RT-PCR, was elevated (P<0.01) on day 20 compared to day 15, decreased (P=0.02) between days 20 and 30, and decreased further (P<0.01) between days 30 and 40 in both Meishan and White composite pigs. Expression of STE mRNA was greater (P=0.01) in White composite pigs compared to Meishan pigs. Using a microsatellite from an STE containing BAC genomic clone, the STE gene was mapped to 65 centimorgans on chromosome 8. Because STE mRNA expression differs between Meishan and White composite pigs, the STE gene may be a candidate for the uterine capacity QTL.     

High-resolution linkage mapping of the rat hooded locus

To identify a gene responsible for the hooded phenotype in the rat, high-resolution linkage mapping for the hooded locus was performed using IS (non-hooded) and LEA (hooded) rats. The map revealed that only Kit gene existed in the critical region, suggesting that the Kit is a strong candidate gene. However, mutation was not found in the coding region of the LEA rat Kit gene. Further, the expressions of Kit mRNA were not different in fetal neural tubes and both neonatal and adult skins between IS and LEA rats. Furthermore, Kit-positive cells, possibly melanocytes, were found in the non-pigmented hair follicles of hooded phenotype rats. Several hypotheses are conceivable to account for mechanisms in the appearance of hooded phenotype.     

A pig–human comparative RH map comprising 20 genes on pig chromosome 13q41 that harbours the ETEC F4ac receptor locus

The enterotoxigenic Escherichia coli (ETEC) F4ac is a major cause of diarrhoea in newborn and young pigs. The locus for the intestinal ETEC F4ac receptor (F4acR) has been mapped to pig chromosome (SSC) 13q41 with known homology to human chromosome (HSA) 3q21 and q29. However, the causative gene and mutation(s) remain unknown. The aim of this study was to characterize gene-derived markers on SSC13q41 for fine mapping of the F4acR locus, and construct a high-resolution pig–human comparative map to select positional candidate genes for F4acR. Pig-specific sequence-tagged site markers were developed for 20 genes that are located in a 6.8-Mb region on HSA3q21 and q29, and a total of 34 single-nucleotide polymorphisms (SNPs) were identified in 14 of 20 markers developed. Eighteen markers were mapped to SSC13q41, while the other two markers ( PLXNA1 and KLF15 ) were assigned to SSC13q32 and SSC7q13, respectively, by radiation hybrid mapping. This result showed that there was a small conserved segment on SSC7 corresponding to HSA3q21. A framework map comprising 18 markers on SSC13q41 was established, refining the synteny breakpoint on SSC13q41 to a region of 12.3 centiRay. The comparative radiation hybrid (RH) map revealed three interesting candidate genes for F4acR from the human genome, viz. MUC4 , MUC13 and MUC20 . Linkage analysis with six marker polymorphisms revealed that MUC4 had the most significant linkage with the F4acR locus.     

Identification of genes responsible for hereditary diseases in Japanese beef cattle

In the breeding of domestic animals, selection of economically desired traits has been the most important consideration for the improvement of animals, but excluding negative factors in animal production, such as causative genes for hereditary diseases, is also required for the genetic improvement of domestic animals. The incidence of various hereditary diseases has been reported in Japanese beef cattle and these diseases have caused serious problems in the breeding and raising of healthy beef cattle. This article reviews the identification of causative genes for the following three hereditary diseases in Japanese beef cattle: (i) Chediak–Higashi syndrome; (ii) renal tubular dysplasia; and (iii) bovine chondrodysplastic dwarfism. Chediak–Higashi syndrome is a hereditary bleeding disorder reported in Japanese black cattle. To identify the cause of this disease, we cloned and sequenced the bovine LYST gene, which has been found to be involved in Chediak–Higashi syndrome in humans, and found that an amino acid substitution of histidine to arginine at amino acid residue 2015 is the causative mutation in the cattle disease. Renal tubular dysplasia is a hereditary disease of Japanese black cattle showing renal failure and growth retardation. We mapped the locus for this disease to the 4 cM region of bovine chromosome 1 by linkage analysis and found a large deletion in this region. The deleted region contained the PCLN1 gene encoding a tight‐junction protein of renal epithelial cells, and we concluded that deletion of the PCLN1 gene is responsible for the disease. Bovine chondrodysplastic dwarfism is a hereditary disease of Japanese brown cattle, displayed by short limbs and joint abnormalities. We mapped the locus for the disease to a region of bovine chromosome 6 by linkage analysis. By constructing YAC and BAC contigs covering this region and sequence analysis, we identified a novel gene (LIMBIN), which plays an essential role in bone formation in this region, and found two mutations responsible for the disease. The identification of these mutations provided the basis for DNA‐based diagnostic systems for these three diseases, and after development of the diagnosis systems, the incidences of these hereditary diseases have dramatically decreased.     

Genetic mapping of the prion protien gene (PRPN) on bovine chromosome 13

The bovine prion protein gene (PRNP) potentially plays a key role in the development of bovine spongiforme encephalopathy (BSE). In species other than cattle, expression of BSE is clearly dependent on polymorphisms in this gene. PRNP has previously been assigned to the bovine chromosome 13 (BTA13). The present study is an attempt to embed PRNP into a grid of published marker maps. A genetic mapping panel consisting of 266 animals has been genotyped with 19 microsatellites and a polymerase chain reaction‐amplified polymorphism within the PRNP coding region. The linear locus order and the relative distances of these loci are presented. Our linkage map spans 111.6c m of BTA13. The results suggest PRNP to be located telomeric of the microsatellite BMS1580 and centromeric of BM9248 with a log‐likelihood of 2. Our findings further characterize the vicinity of PRNP on BTA13.     

The genomic sequence of the bovine T cell receptor gamma TRG loci and localization of the TRGC5 cassette

The bovine and ovine TRG genes have previously been shown to be located in two loci, TRG1 and TRG2, in contrast to human and mouse TRG genes that are located in a single locus. The bovine TRG1 and TRG2 loci are located on chromosome 4 at 4q3.1 and 4q1.5-2.2, respectively. The complete genomic organization of the two bovine loci is described: each locus comprises three cassettes, each one includes one or several variable genes (TRGV) and one or several joining genes (TRGJ) preceding a constant (TRGC) gene. The location of the TRGC5 cassette is conclusively described in 5' of the TRG1 locus. Analysis of 17 TRGV belonging to 10 different subgroups, 8 TRGJ and 6 TRGC genes is conducted which comprises the most comprehensive list to date.     

荧光定量PCR检测牛性别相关基因DAX1表达的标准质粒和标准曲线的构建

本研究根据GenBank中登录的剂量敏感的性别反转－先天性肾上腺发育不良基因1(dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1,DAX1)设计引物,构建包含DAX1基因cDNA片段的质粒,作为中国荷斯坦牛DAX1基因mRNA定量检测的标准品,建立了DAX1基因mRNA表达实时荧光定量PCR检测方法。结果表明,该方法特异性好,检测灵敏度达102拷贝,线性范围为102～106拷贝,阈值循环数(Ct)与PCR体系中起始模板量的对数值间有着良好的线性关系(r=0.99975),扩增效率高(E=100%),可以作为检测牛DAX1基因mRNA定量检测方法。     

A male bovine linkage map for the ADR granddaughter design

The aim of this paper is to present the construction of a male genetic linkage map as a result of the bovine genome mapping project, which is a common effort of the German cattle breeding federation (ADR), four animal breeding institutes, three blood group laboratories and two animal data and breeding value evaluation centres. In total 20 grandsires with 1074 sires were provided from the German cattle population as reference families, 16 of these paternal half‐sib groups are German Holstein families (DH), three are German Simmental (ST) families, and one is a Brown Swiss family (BS). Of 265 markers included in the linkage map, 248 were microsatellite markers, five were bovine blood group systems, eight SSCP markers and four proteins and enzymes. More than 239 000 genotypes resulted from typing the offspring for the respective markers and these were used for the construction of the map. On average 478 informative meioses were provided from each marker of the map. The summarized map length over all chromosomes was 3135.1 cM with an average interval size of 13.34 cM. About 17, 35.7 and 79.1% of the map intervals showed a maximum genetic distance between the adjacent markers of 5, 10 and 20 cM, respectively. The number of loci ranged from two (pseudoautosomal region of the sex chromosome, BTAY) to 15 (BTA23) with an average of 8.8 markers per chromosome. Comparing the length of the chromosomes shows variation from 49.6 cM for BTA26 to 190.5 cM for BTA1 with a mean of 107.7 cM for all autosomes of the genetic linkage map. It was possible to identify chromosomal discrepancies in locus order and map intervals by comparison with other published maps. The map provided sufficient marker density to serve as a useful tool for a scan of segregating quantitative trait loci.     

A single blastomere sexing of caprine embryos by simultaneous amplification of sex chromosome-specific sequence of SRY and amelogenin genes

The primary objective of this study was to develop a simplified, rapid and authenticated protocol for sexing of caprine embryos. Polymerase chain reaction (PCR) is a powerful tool in preimplantation sex diagnosis, using embryo biopsy at the early developmental stage. Based on the amelogenin gene located on the conserved region of the sex chromosome, a primer pair was used and PCR was established to amplify a 262-bp fragment from the Xchromosome in female goat embryos and 262-bp fragments from the X chromosome and 202-bp fragments from the Y chromosome in male embryos. To validate the reliability of PCR, using the sex-determining region Y (SRY) gene located on the conserved region of Y chromosome, a primer pair was used and PCR was established to amplify a 122-bp fragment specific to the Y chromosome in male embryos. The in vitro-produced goat in vitro fertilisation (IVF)-embryos were made zona free by treating with pronase. The cell number in each embryo was counted before sexing. A single blastomere taken from these embryos was directly used as a template in PCR containing SRY and amelogenin gene-specific primers separately. Of 75 pronase-treated and 60 micromanipulated goat IVF embryos, 33 (44%) and 26 (43.33%) were confirmed as male and 42 (56%) and 34 (56.66%) as female, respectively. The sex-diagnosed embryos were kept in research vitro cleavage (RVCL) medium, and developed into 42.66% and 61.66% morulae and 13.33% and 23.33% blastocysts among pronase-treated and micromanipulated embryos, respectively. The AMELX gene-specific primer served as the internal control and did not interfere with amplification of the Y-specific sequence. In conclusion, a single blastomere sexing protocol based on the SRY and the amelogenin gene is simple, rapid, sensitive and efficient for sex determination in caprine early stage embryos.     

Marker-assisted selection for forelimb-girdle muscular anomaly of Japanese Black cattle

Forelimb‐girdle muscular anomaly is a hereditary disorder of Japanese Black cattle characterized by tremors and astasia caused by hypoplasia of the forelimb‐girdle muscles. The locus responsible for this disorder has been mapped on a middle region of bovine chromosome 26. In this study, we applied marker‐assisted selection to identify the carriers of this disorder. Four microsatellite markers, DIK4440, BM4505, MOK2602 and IDVGA‐59, linked to the disorder locus were genotyped in 37 unaffected offspring of a carrier sire. Transmission of the mutant or wild‐type allele of the disorder locus of the sire to the 37 offspring was determined by examining the haplotypes of these markers. The results showed that nine and 18 of the 37 animals possessed the paternally transmitted mutant and wild‐type alleles, respectively, and therefore, the nine animals with the mutant allele were identified as carriers. We concluded that the marker‐assisted selection using these four markers can be applied for the identification of the carriers of forelimb‐girdle muscular anomaly of Japanese Black cattle.     

A new ENU-induced mutant mouse with defective spermatogenesis caused by a nonsense mutation of the syntaxin 2/epimorphin (Stx2/Epim) gene

Repro34 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing male-specific infertility caused by defective spermatogenesis. In the present study, we investigated pathogenesis and molecular lesions in relation to spermatogenesis in the repro34/repro34 homozygous mouse. Histological examination of the testis showed that the seminiferous epithelium of the repro34/repro34 mouse contained spermatogonia and spermatocytes but no round and elongating spermatids. Instead of these haploid cells, multinucleated giant cells occupied the niche of the seminiferous tubules. Immunohistochemical staining for Hsc70t, an elongating spermatid specific protein, confirmed the absence of elongating spermatids. Furthermore, RT-PCR showed that there were significantly reduced expressions of the marker genes specifically expressed in the spermatid and that there was no difference in the expressions of the spermatocyte specific marker genes. These findings indicated interruption of the spermatogenesis during transition from the spermatocyte to spermatid in the repro34/repro34 mouse. The repro34 locus has been mapped on a 7.0-Mb region of mouse chromosome 5 containing the Syntaxin 2/Epimorphin (Stx2/Epim) gene, and targeted disruption of this gene has been reported to cause defective spermatogenesis. We therefore sequenced the entire coding region of the Stx2/Epim gene and found a nucleotide substitution that results in a nonsense mutation of this gene. The expression pattern of the Stx2/Epim gene during the first wave of spermatogenesis, increased expression at later stages of spermatogenesis, was in agreement with the affected phase of spermatogenesis in the adult repro34/repro34 testis. We therefore concluded that the male infertility of the repro34/repro34 mouse is caused by the interruption of spermatogenesis during transition from the spermatocyte to spermatid and that the nonsense mutation of the Stx2/Epim gene is responsible for the interruption of spermatogenesis.     

Booroola羊FecB基因的遗传标记研究进展

从美利奴绵羊中衍生出来的Booroola羊携带增加排卵数和产羔数的一个常染色体突变基因(FecB).基因效应对排卵数是加性的,对产羔数是部分显性的.该位点已被定位到绵羊6号染色体上微卫星标记OarAE101和BM1329之间一个10cM区间内.正在开展限定紧密的侧翼标记的研究工作.     

Localisation of the genomic sequence interval for the blue eggshell gene using an F2 resource population of Dongxiang chickens

1. In order to identify the molecular interval containing the blue shell gene (O locus), linkage analysis was conducted with three microsatellite markers, (TTA)(n), (TG)(n) and (tg)(n), and a SNP in intron 1 of SLCO1C1 (solute carrier organic anion transporter family, member 1C1; A locus) to map the O locus in an F2 resource population of Dongxiang chickens. 2. Linkage analysis based on 98 F2 hens resulted in estimation of the best map order of the O locus with other linked markers as: (TTA)(n)-(TG)(n)-A-O-(tg)(n). 3. Based on these results, we inferred that the O locus was located between the A and (tg)(n) loci, that is, Chr1:67,296,991-69,140,571, which is the first genomic sequence interval to be established for the blue eggshell gene.     

Affected homologous chromosome pairing and phosphorylation of testis specific histone, H2AX, in male meiosis under FKBP6 deficiency

A gene for FK506 binding protein 6 (Fkbp6) expresses during a specific stage of male and female meiosis. Disruption of the gene influences male reproduction, i.e. arrests spermatogenesis, but not female reproduction. Using the mouse model (targeted disruption), the role of the gene in homologous chromosome pairing has been demonstrated in a previous study. For further understanding the function of Fkbp6 in chromosome synapsis, we evaluated chromosome pairings during male meiosis in the as/as rat, a spontaneous null mutation, and compared them with those of the mouse model. Electron microscopy of the pachytene nuclei unveiled several types of abnormal chromosome pairing in the rat model, as shown in the mouse previously. The frequencies of aberrant pairings in the knockout mice and mutant rats were 42 of 67 nuclei (62.7%) and 20 out of 74 nuclei (27.0%), respectively. In order to clarify the mechanism of male specific infertility in Fkbp6 deficiency, the localization of gammaH2AX, a marker protein of XY chromosome inactivation during male meiosis, was examined. Immunostaining of gammaH2AX unveiled normal localization of the molecule to XY chromosomes (XY body) in both models, showing the independency of FKBP6 in sex chromosome inactivation. Besides the XY body, focal localization of gammaH2AX was observed in accordance with the unsynapsed chromosomes in both types of null animal. These results indicate the fundamental role of Fkbp6 in homologous chromosome synapsis during male meiosis. In conclusion, male specific infertility under Fkbp6 deficiency remains unsolved.