Characterization of <i>Bovidae</i> sex-determining gene <i>SRY</i>

Cheng, Hanhua; Shi, Huifang; Zhou, Rongjia; Guo, Y. Q.; Liu, Li; Liu, Jiangdong; Jiang, Yong; Kudo, Toshiyuki; Sutou, Shizuyo

doi:10.1051/gse:2001104

Cited by 4 publications

(1 citation statement)

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“…Polymerase chain reaction (PCR) is the most commonly used technique to amplify a specific gene or gene segment and was first used for genetic diagnosis of a single copy gene from bovine blastocysts in 1988 [1]. Upon the identification of SRY as the gene responsible for sex determination in cattle [2], PCR became a widely used method of sexing bovine embryos [3–7]. Although PCR followed by product detection on gel is frequently used, PCR assays for embryo sexing have been optimized to amplify samples with small amounts of DNA and they are also sensitive enough to amplify contamination by a single DNA molecule, consequently providing inconsistent or inaccurate results.…”

Section: Introductionmentioning

confidence: 99%

Use of a Combined Duplex PCR/Dot Blot Assay for more sensitive genetic characterization

et al. 2008

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A reliable and sensitive method of genetic analysis is necessary to detect multiple specific nucleic acid sequences from samples containing limited template. The most widely utilized method of specific gene detection, polymerase chain reaction (PCR), imparts inconsistent results when assessing samples with restricted template, especially in a multiplex reaction when copies of target genes are unequal. This study aimed to compare two methods of PCR product analysis, fluorescent detection following agarose gel electrophoresis or dot blot hybridization with chemiluminescent evaluation, in the detection of a single copy gene (SRY) and a multicopy gene (β-actin). Bovine embryo sex determination was employed to exploit the limited DNA template available and the target genes of unequal copies. Primers were used either independently or together in a duplex reaction with purified bovine genomic DNA or DNA isolated from embryos. When used independently, SRY and β-actin products were detected on a gel at the equivalent of 4-cell or 1-cell of DNA, respectively; however, the duplex reaction produced visible SRY bands at the 256 cell DNA equivalent and β-actin products at the 64 cell DNA equivalent. Upon blotting and hybridization of the duplex PCR reaction, product was visible at the 1-4 cell DNA equivalent. Duplex PCR was also conducted on 186 bovine embryos and product was subjected to gel electrophoresis or dot-blot hybridization in duplicate. Using PCR alone, sex determination was not possible for 22.6% of the samples. Using PCR combined with dot blot hybridization, 100.0% of the samples exhibited either both the male specific and β-actin products or the β-actin signal alone, indicating that the reaction worked in all samples. This study demonstrated that PCR amplification followed by dot blot hybridization provided more conclusive results in the evaluation of samples with low DNA concentrations and target genes of unequal copies.

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Section: Introductionmentioning

confidence: 99%

Use of a Combined Duplex PCR/Dot Blot Assay for more sensitive genetic characterization

et al. 2008

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Two Cases of Bovine Male Pseudohermaphrodites with Different Endocrinological and Pathological Findings

Moriyama

Tani²,

Nibe

et al. 2010

The Journal of Veterinary Medical Science

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ABSTRACT. Two cases of bovine male pseudohermaphrodites (PH) were subjected for clinical investigation with transrectal ultrasonography, endocrinology with adoption of hCG-stimulation test, cytogenetics with analysis of sex chromosome and Y-specific DNA, and finally histological examination. Results were compared with normal calves. Case 1 was a 10-month-old calf with XX/XY chimeras, showing elevation of testosterone (T) levels, but no change in progesterone (P 4 ) after hCG test, and possessed atrophied testes in the cavitas pelvis. Case 2 was an 18-month-old calf with SRY positive-XY chromosome, showing lower level of plasma T and P 4 after hCG test, and possessed atrophied testes and undifferentiated genital ducts. Both cases possessed female-like external genitalia with similar pathological findings, however endocrinological and cytogenetical aspects were different each other.KEY WORDS: atrophied testis, hCG stimulation test, male pseudohermaphrodite, sex chromosome, ultrasonography.J. Vet. Med. Sci. 72(4): 507-510, 2010 Abnormal development of the reproductive organs occurs with different incidence in several species [11,17]. A male or a female pseudohermaphrodite (PH) depends upon the nature of the gonadal tissues. PH has either a testis or an ovary, and possesses external genitalia resembling the opposite sex [8,9,12,15]. PH has been reported in several species, but sporadic in cattle. There are limited cases reports concerning bovine PH, however these often lack general investigation in endocrinological aspect for gonadal function, comparing external and internal genitalia using ultrasonography, or chromosomal examination [5,8,16]. In the present study, we investigated two cases of male PH, which had different nature of endocrinological, cytogenetical and pathophysiological findings. In comparison with PH cases for novel trial, normal male and female calves were examined in the same manner as controls on the stand point for clinical investigation.Case 1: A 10-month old Japanese brown was born to male calf co-twins, and possessed female-like external genitalia showing urination toward upper direction. Coat and skin in neck region seemed to be masculine as the animal had grown. The animal showed a 3 cm in length of labia-like orifice (not vagina), but neither uterus and ovary nor testes were detected in the cavitas pelvis by rectal palpation. In addition, spiral stick-like structure (4  3 cm ) beneath the anal orifice was observed with trasrectal ultrasonography equipped with a 7.5 MHz linear-array transducer (SonoSite Case 2: An 18-month-old Japanese black, which had normal female-like external genitalia, but lacked estrous behavior in life. The vestibule of vagina was 13 cm in length; however neither uterus nor ovary was detected with rectal palpation. With transrectal ultrasonography, a pair of testes like organs (5  3 cm) with mediastinum was depicted (Fig. 1c).In order to investigate endocrinological aspects, 3,000 IU of human chronic gonadotropin (hCG; Veterinary Puberogen ® , Sankyo, Tokyo, Ja...

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Evolution of the Male-Determining GeneSRYWithin the Cat Family Felidae

et al. 2007

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In most placental mammals, SRY is a single-copy gene located on the Y chromosome and is the trigger for male sex determination during embryonic development. Here, we present comparative genomic analyses of SRY (705 bp) along with the adjacent noncoding 59 flank (997 bp) and 39 flank (948 bp) in 36 species of the cat family Felidae. Phylogenetic analyses indicate that the noncoding genomic flanks and SRY closely track species divergence. However, several inconsistencies are observed in SRY. Overall, the gene exhibits purifying selection to maintain function (v ¼ 0.815) yet SRY is under positive selection in two of the eight felid lineages. SRY has low numbers of nucleotide substitutions, yet most encode amino acid changes between species, and four different species have significantly altered SRY due to insertion/ deletions. Moreover, fixation of nonsynonymous substitutions between sister taxa is not consistent and may occur rapidly, as in the case of domestic cat, or not at all over long periods of time, as observed within the Panthera lineage. The former resembles positive selection during speciation, and the latter purifying selection to maintain function. Thus, SRY evolution in cats likely reflects the different phylogeographic histories, selection pressures, and patterns of speciation in modern felids.

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Characterization of Bovidae sex-determining gene SRY

Cited by 4 publications

References 10 publications

Use of a Combined Duplex PCR/Dot Blot Assay for more sensitive genetic characterization

Use of a Combined Duplex PCR/Dot Blot Assay for more sensitive genetic characterization

Two Cases of Bovine Male Pseudohermaphrodites with Different Endocrinological and Pathological Findings

Evolution of the Male-Determining GeneSRYWithin the Cat Family Felidae

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