Sex Determination of Bovine Embryos by Restriction Fragment Polymorphisms of PCR Amplified ZFX/ZFY Loci

Pollevick, Guido D.; Giambiagi, Susana; Mancardi, Sabrina; Luca, Luigi De; Burrone, Óscar R.; Frasch, Alberto C.C.; Ugalde, Rodolfo A.

doi:10.1038/nbt0792-805

Cited by 27 publications

(8 citation statements)

References 9 publications

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“…The basis of our methodology was the description of the ZFX/ZFY gene. The sequences of the ZFX and ZFY loci in this species, which we obtained in collaboration with MediGene and the Royal Free Hospital, are similar to those described in cattle (Pollevick et al, 1992). Only 12 bp of ZFY and 11 bp of ZFX are different between both species (more than 99.9% of similarity).…”

Section: Discussionsupporting

confidence: 69%

Separation of Ram Spermatozoa Bearing X and Y Chromosome by Centrifugal Countercurrent Distribution in an Aqueous Two‐phase System

OLLERO,

PÉREZ‐PÉ,

GARGALLO

et al. 2000

Journal of Andrology

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The availability of reliable quantification techniques of X and Y chromosome‐bearing spermatozoa in a given insemination dose would allow further approaches to their separation, which is a topic of unquestionable interest in animal production. The aim of the current work was the development of a combined approach of polymerase chain reaction and countercurrent distribution to address both objectives. First, using SacI polymorphisms for ZFX/ZFY loci in sheep deoxyribonucleic acid, a linear correlation has been established between the densitometric quantification of the restricted fragment length polymorphisms corresponding to the amplified loci ZFX/ ZFY by polymerase chain reaction and the theoretical proportions of X and Y chromosomes in standard solutions. The method, subsequently applied to semen samples, estimated an equal proportion of spermatozoa bearing each chromosome. Second, by using centrifugal countercurrent distribution in a sensitive‐charge aqueous two‐phase system, we achieved the separation of a sperm population enriched in Y chromosome‐bearing ram spermatozoa (75%) with a high viability rate (57%).

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Section: Discussionsupporting

confidence: 69%

Separation of Ram Spermatozoa Bearing X and Y Chromosome by Centrifugal Countercurrent Distribution in an Aqueous Two‐phase System

OLLERO,

PÉREZ‐PÉ,

GARGALLO

et al. 2000

Journal of Andrology

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“…On a practical basis, the fact that SRY gene sequences show little or no homology between species outside of the HMG box can be exploited in designing species-specific SRY-based primers for genetic sexing of bovine tissues using PCR amplification. These protocols should be useful for bovine embryo sexing, as an adjunct to current PCR methods based on Y chromosome gene or repetitive DNA sequences [25,26].…”

Section: Discussionmentioning

confidence: 99%

Bovine SRY Gene Locus: Cloning and Testicular Expression1

Daneau

Houde

Éthier

et al. 1995

Biology of Reproduction

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The bovine SRY gene was cloned by a combination of anchored polymerase chain reaction (PCR) amplification of genomic restriction fragments and reverse transcription-PCR (RT-PCR) of testicular RNA. We report 1800 bp of combined genomic and cDNA sequences including 911 bp of 5' upstream sequences, an open reading frame of 687 bp, and 202 bp of sequences corresponding to the 3' end of the mRNA. The bovine SRY gene encodes a deduced (predicted on the basis of a cDNA sequence) protein product of 229 amino acids, with sequence conservation between species, notably in the region of the high-mobility group (HMG) domain or HMG box. Outside of the HMG box, the bovine SRY structure shows greater resemblance to the human SRY than to the mouse Sry. As with human SRY promoter sequences, putative binding sites for Sp1 and for SRY itself are seen in the bovine SRY promoter region. Unlike the human SRY promoter, CAAT and TATA box motifs are present in the bovine sequences. Southern analysis and PCR amplification of male and female bovine genomic DNA show that the described sequences are specific to the Y chromosome. Northern analysis of bull testicular RNA demonstrated low levels of expression of the bovine SRY gene in adult testes with a major poly(A) species at 1.9 kb. RT-PCR amplification of bull testicular RNA revealed multiple sites of polyadenylation, but sequencing showed no consensus polyadenylation signal.

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“…In bovine embryos (Peura et al, 1991;Schroder et al, 1990) and ovine embryos (Herr et al, 1990;Appa Rao & Totey, 1992), the PCR method has been standardised to determine sex by simultaneously amplifying malespecific Y-chromosomal repeated sequences and autosomal repeated sequences as control. In horse and porcine embryos, sex was determined by finding a polymorphic restriction site between the ZFY gene (zinc-finger protein Y) located on the Y chromosome and its X-chromosomal homologue, ZFX, amplified by PCR (Bredbacka & Peippo, 1992;Povellick et al, 1992;Hovart et al, 1993;Kirkpatrick & Monson, 1993;Ford & Conley, 1994;Bredbacka et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction

Manna

Neglia

Marino

et al. 2003

Zygote

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The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at −20 °C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).

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Sex Determination of Bovine Embryos by Restriction Fragment Polymorphisms of PCR Amplified ZFX/ZFY Loci

Cited by 27 publications

References 9 publications

Separation of Ram Spermatozoa Bearing X and Y Chromosome by Centrifugal Countercurrent Distribution in an Aqueous Two‐phase System

Separation of Ram Spermatozoa Bearing X and Y Chromosome by Centrifugal Countercurrent Distribution in an Aqueous Two‐phase System

Bovine SRY Gene Locus: Cloning and Testicular Expression1

Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction

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