• Journal of Embryo Transfer
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• v.11 no.3
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• pp.283-289
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• 1996

This study was carried out to determine the sex of genomic and embryonic DNA using polymerase chain reaction(PCR). Bovine specific(216bp) and Y chromosome speicific DNA primers(l4lbp) were synthesized and tested for sexing. Bovine embryos used in this study were produced by in vitro fertilization. Few blastomeres for PCR were bisected by nicromanipulator and demi -embryos were cultured in TCM 199 medium containing 0.1% of solcoseryl. The results obtained were as follows; 1. Average optical density of genomic DNA extracted from blood of Hanwoo was 1.79$\pm$ 0.14. 2. 2. The ratio of the demi-embryos developed to blastocyst was 62.1 and 81.9% in morula and blastocyst, respectively. 3. When DNA of 2~4, 5~10 and more than 11 blastomeres was amplified with Y chromosome specific DNA primer by PCR, appreance rate of Y specific DNA band was 16.7, 46.2 and 40.0%, respectively. At least 5 to 10 blastomeres were required to determine the sex of embryos. 4. The rate of demi-embryos developed to blastocyst was 73.3% in TCM 199 medium supplemented with 0.1% solcoceryl. but 55.6% in control.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.36-41
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• 1986

These experiments were carried out to obtain basic information necessary for sexing embryos by chromosomal analysis. To observe metaphase chromosomes, all embryos developed to blastocysts were cultured in Ho, pp. & Pitts' medium containing 0.001% Colcemid under the gas phase of 5% CO2 in air at 37$^{\circ}C$ for 2 hours. The sex chromosome of mouse embryos shown normal development after culture in medium containing H-Y antiserum (10%, v/v) and complement (20%, v/v) also was confimed by chromosomal analysis. The results obtained in these experiments were summarized as follows: 1. Among 89 mouse blastocysts, the number of embryos identified to have XX and XY chromosome was 22(25%) and 25(28%), respectively and 42(47%) embryos were not identified. 2. Of total 40 mouse balstocysts cultured in medium containing H-Y antiserum and complement, 23(58%) embryos which were able to be discriminated their sex chromosomes were identified to be XX bearing embryos. 3. Sex chromosomes of a number of embryos subjected to chromosomal analysis were not identified. This result may be due to absence or poor quality of metaphase spreads.

• Journal of Sericultural and Entomological Science
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• v.27 no.1
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• pp.31-36
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• 1985

Using sexual differences between female and male as the indication of the degree of the physiological distrubance in the sex-limited larval marking strains or sex-limited egg colour strains, it was investigated whether the physiological distrubance caused y the translocated autosome fragment on the W-sex determination chromosome, have an effect on expression of the three quantitative characters; cocoon weight, cocoon shell weight, and cocoon shell percentage in the female silkworm. The mean values of the ratios of female to male the above two experimental groups were 127%, 107%, 85%, in cocoon shell weight, and cocoon shell percentage, respectively. On the other hand, those from the three normal stains, namely the basic stock strains, white egg strains, and hybrids were 129% for cocoon weight, 110% for cocoon shell weight, and 85% for cocoon shell percentage, respectively. From the results, it comes to the conclusion that the translocated autosome fragment on the W-chromosome has no influences on the expression of the quantitative characters of the female silkworm because sexual differences of the sex-limited strains were very similar to those of the three normal strains.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.133-137
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• 2004

Sexing from bovine embryos which were fertilized in vitro implicate a possibility of the sex-controlled cattle production. This study was carried out to investigate the possibility of determining of embryo sex by fluorescence in situ hybridization (FISH) technique. FISH was achieved in in vitro fertilized bovine embryos using a bovine Y-specific DNA probe which constructed from the btDYZ-1 sequences. To evaluate Y-chromosome specificity of the FISH probe, metaphase spreads of whole embryos and lymphocytes were prepared and tested. A male-specific signal was detected on 100% of Y chromosome bearing metaphase specimens. Using the FISH technique with a bovine Y-specific probe, 232 whole embryos of 8 cell- to blastocyst-stage were analyzed. Observing the presence of the Y-probe signal on blastomeres, 102 embryos were predicted as male, and 130 embryos as female. The determining rate of embryo sex by FISH technique was about 93% regardless of embryonic stages. In conclusion, the FISH using a bovine Y-specific DNA probe is an accurate, reliable and quick method for determining the sex of bovine embryos.

• Journal of Aquaculture
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• v.20 no.2
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• pp.140-143
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• 2007

Cytogenetic analyses of an endemic species, Coreoleuciscus splendidus (Cyprinidae) was performed including erythrocyte measurement, chromosome count and karyotyping, nucleolar organizing region (NOR) banding and flow cytometric analysis of genome size. C. splendidus had the same modal chromosome number of 2n = 48 between sexes, however, displayed a sex-related dimorphism in their chromosome karyotypes. Males represented a pair of heteromorphic chromosomes which couldn‘t be seen in any female individuals, indicating that the sex determination mechanism of this species should be a typical XX-XY based male heterogamety (female=10M+6SM+8A+XX vs male=10M+6SM+8A+XY). Other cytogenetic features such as Ag-NORs located in a pair of acrocentric chromosomes, estimated nuclear volume ($28{\mu}m^3$) and cellular DNA content (2.4 pg/cell) suggest that genetic recombination might be the main driving force responsible for the evolution of this species rather than the polyploidy-based evolutionary process as in many other Cyprinidae species.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.299-309
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• 2000

This study was performed 1) to establish the optimal PCR condition of sex determination in Hanwoo IVM/IVF embryos, 2) to examine the sex determination and sex ratio to the developmental stages of Hanwoo IVM/IVF embryos by two-step PCR method. The sexing of bovine IVF embryos were accurately determined by PCR methods using Y chromosome specific DNA primer(BOV 97M, 141bp) and bovine specific DNA primer(216bp). The fregment size were shown at 141 and 216 base pairs(bp) in male, and 216 bp in female. Two-steps PCR method in which the samples were amplified by 15 cycles with Y chromosome specific DNA primer and then amplified by additional 30 cycles with bovine specific DNA primer was effective in the sexing of bovine IVF embryos. The zona-free embryos were more effective than zona-intact embryos in bovine IVF embryo sexing. The appearance of Y chromosome specific band was 45.2% in embryos treated with protease K and 53.3% in embryos treated with freezing and thawing repeatedly. The optimun volume of DNA for sexing of Hanwoo IVF embryos were 2 to 10 $\mu$1 in Zona-free embryos and 12 to 13 $\mu$1 in zona-intact embryos. The sexing rate of bovine IVF embryos by PCR was 96.0% and questionable rate not identified sex was 4.0%, respectively. Among the sexed embryos, the percentage of male and female was 49.7% and 46.3%, respectively, the sex ratio was 1: 1.1. The successful rate of embryo sexing was increased to the developmental stages.

• Korean Journal of Plant Resources
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• v.16 no.1
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• pp.55-60
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• 2003

The genetic variation of random amplified polymorphic DNA (RAPD) patterns of genomic DNAs was investigated in dioecious plant Rumex acetosa L., which carries different sex chromosome complements in female (2n=12A+XX) and male (2n=12A+XY$_1$Y$_2$). One hundred and twenty random primers consisted of 10-mer were used for PCR amplification. Polymorphic bands were found in 24 primers. Specific bands for female and male were 16 and 18, respectively. Especially, a band of 1,440 bp from the OPC-10 primer was male specific. These sex specific RAPD markers are used to understanding the sex determination mechanism in plants.

• Journal of Genetic Medicine
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• v.13 no.2
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• pp.78-88
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• 2016

Purpose: To identify the clinical characteristics of SRY-negative male patients and genes related to male sex reversal, we performed a retrospective study using cases of 46,XX testicular disorders of sex development with a review of the literature. Materials and Methods:SRY-negative cases of 46,XX testicular disorders of sex development referred for cytogenetic analysis from 1983 to 2013 were examined using clinical findings, seminal analyses, basal hormone profiles, conventional cytogenetic analysis and polymerase chain reaction. Results: Chromosome analysis of cultured peripheral blood cells of 8,386 individuals found 19 cases (0.23%) with 46,XX testicular disorders of sex development. The SRY gene was confirmed to be absent in three of these 19 cases (15.8%). Conclusion: We report three rare cases of SRY-negative 46,XX testicular disorders of sex development. Genes on autosomes and the X chromosome that may have a role in sex determination were deduced through a literature review. These genes, through differences in gene dosage variation, may have a role in sex reversal in the absence of SRY.

• Journal of Genetic Medicine
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• v.2 no.1
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• pp.11-15
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• 1998

This paper reports 3 cases with 46,XX sex reversed male. Three 46,XX hypogonadal subjects showed complete sex reversal and had normal phallus and azoospermia. We studied them under clinical, cytogenetic and molecular aspects to find out the origin of the sex reversal. Patients had markedly elevated serum follicle-stimulating hormone (FSH) and lutenizing hormone (LH) and decreased or normal range of serum testosterone. The testicular volumes were small (3-8ml). Testicular biopsy showed Leydig cell hyperplasia and atrophy of seminiferous tubules. We obtained the results of normal 46,XX, and the presence of Y chromosome mosaicism was ruled out through XY dual fluorescent in situ hybridization (FISH). By using polymerase chain reaction (PCR), we amplified short arm (SRY, PABY, ZFY and DYS14), centromere (DYZ3), and heterochromatin (DYZ1) region of the Y chromosome. PCR amplification of DNA from these patients showed the presence of the sex-determining region of the Y chromosome (SRY) but didn't show the centromere and heterochromatin region sequence. The SRY gene was detected in all the three patients. Amplification patterns of the other regions were different in these patients; one had four amplified loci (PABY+, SRY+, ZFY+, DYS14+), another had two loci (SRY+, ZFY+) and the other had two loci (PABY+, SRY+). We have found that each patient's translocation elements had different breakpoints at upstream and downstream of the SRY gene region. We conclude that the testicular development in 46,XX male patients were due to insertion or translocation of SRY gene into X chromosome or autosomes.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.4
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• pp.253-260
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• 2004

Objectives: Klinefelter syndrome is the most common genetic cause of male infertility and presents with 47, XXY mainly or 46, XX/47, XXY mosaicism. It is characterized by hypogonadism and azoospermia due to testicular failure, however, sporadic cases of natural pregnancies have been reported. With the development of testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI), sperm can be retrieved successfully and ART is applied in these patients for pregnancy. It has been suggested that the risk of chromosome aneuploidy for both sex chromosome and autosome is increased in the sperms from 47, XXY germ cells. Considering the risk for chromosomal aneuploidy in the offspring, preimplantation genetic diagnosis (PGD) could be applied as a safe and more effective treatment option in Klinefelter syndrome. The aim of this study is to assess the outcome of PGD cycles by using FISH for sex chromosome and autosome in patients with Klinefelter syndrome. Materials and Methods: From Jan. 2001 to Dec. 2003, PGD was attempted in 8 cases of Klinefelter syndrome but TESE was failed to retrieve sperm in the 3 cases, therefore PGD was performed in 8 cycles of 5 cases (four 47, XXY and one 46, XY/47, XXY mosaicism). In one case, ejaculated sperm was used and in 4 cases, TESE sperm was used for ICSI. After fertilization, blastomere biopsy was performed in $6{\sim}7$ cell stage embryo and the chromosome aneuploidy was diagnosed by using FISH with CEP probes for chromosome X, Y and 17 or 18. Results: A total of 127 oocytes were retrieved and ICSI was performed in 113 mature oocytes. The fertilization rate was $65.3{\pm}6.0%$ (mean$\pm$SEM) and 76 embryos were obtained. Blastomere biopsy was performed in 61 developing embryos and FISH analysis was successful in 95.1% of the biopsied blastomeres (58/61). The rate of balanced embryos for chromosome X, Y and 17 or 18 was $39.7{\pm}6.9%$. The rate of aneuploidy for sex chromosome (X and Y) was $45.9{\pm}5.3%$ and $43.2{\pm}5.8%$ for chromosome 17 or 18, respectively. Embryo transfer was performed in all 8 cycles and mean number of transferred embryos was $2.5{\pm}0.5$. In 2 cases, clinical pregnancies were obtained and normal 46, XX and 46, XY karyotypes were confirmed by amniocentesis, respectively. Healthy male and female babies were delivered uneventfully at term. Conclusion: The patients with Klinefelter syndrome can benefit from ART with TESE and ICSI. Considering the risk of aneuploidy for both sex chromosome and autosome in the sperms and embryos of Klinefelter syndrome, PGD could be offered as safe and more effective treatment option.