Evaluation of the expression of a male-specific antigen on cells of equine blastocysts.
Abstract: This experiment was designed (1) to determine if H-Y antigen is expressed on the cell surface of pre-implantation equine blastocyst stage embryos, (2) if so, to identify differences in expression on inner cell mass (ICM) verses trophectoderm cells and (3) to evaluate whether the detection of this glycoprotein would aid in the identification of equine embryonic sex. A total of 33 blastocyst stage horse embryos were collected 6-7 days post-ovulation by trans-cervical flush and were immediately evaluated for the presence of H-Y antigen. Additionally, 17 embryos, collected at similar stages and cultured for 72 h, were similarly evaluated. Embryos were recovered and evaluated by use of a dissecting microscope and then washed for 5 min in phosphate buffered saline supplemented with 1 g/l glucose, 36 mg/l pyruvate, 1% antibiotic-antimycotic and 10% fetal calf serum (FCS) (PBS-2). Embryos were placed in the primary antibody medium and cultured for 60 min. The primary antibody medium consisted of monoclonal antibodies to H-Y antigen (previously determined to have male-specific activity) dilute 1/5 (v/v) with PBS-2 (without FCS, PBS-1). Following an additional wash, embryos were cultured in PBS-1 containing 1/10 (v/v) fluorescein isothiocyanate conjugated goat anti-mouse or rabbit antimouse IgM Fc specific antiserum. Embryos were evaluated at 200-400 x to identify cell specific fluorescence of either trophectoderm or ICM cells. Following evaluation, embryonic sex was independently verified with karyotypes to identify sex chromosomes. Of the 50 embryos evaluated, 29 were evaluated as non-fluorescent and 21 fluorescent. Expression of H-Y antigen was detected on both trophectoderm and ICM cell types in those embryos classified as fluorescent.(ABSTRACT TRUNCATED AT 250 WORDS)
Publication Date: 1988-10-01 PubMed ID: 3058965DOI: 10.1016/0165-0378(88)90031-9Google Scholar: Lookup
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Summary
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This study evaluates whether changes in the expression of a male-specific antigen, referred to as H-Y antigen, can aid in determining the sex of a pre-implantation equine embryo. The team tested this theory on 50 horse embryos collected shortly after ovulation, and analyzed differences in antigen expression across two different cell types: the inner cell mass and trophectoderm cells.
Research Method and Procedure
- The researchers designed a study to examine if equine blastocyst stage embryos, collected 6-7 days post-ovulation, displayed surface expression of H-Y antigen.
- Two groups of embryos were studied. The first group consisted of 33 recovered embryos tested for H-Y antigen immediately after collection. The second group of 17 were similarly tested, but only after further 72 hours of culture.
- The collection process involved a trans-cervical flush, followed by examination under a dissecting microscope. The embryos were then washed using a specific buffer solution (referred to as PBS-2).
- For testing the presence of H-Y antigen, embryos were placed in a primary antibody medium and cultured for one hour. This medium comprised monoclonal antibodies known to react specifically with H-Y antigen.
- Next, the embryos were subjected to another wash before being incubated in a solution containing a conjugated antibody that fluoresces upon binding.
- The cells were then observed again under a microscope to identify any fluorescence in either the trophectoderm or inner cell mass (ICM) cells.
- Finally, to confirm the presence of the antigen, the sex of the embryo was independently verified by analyzing the sex chromosomes in their karyotype.
Key Findings
- A total of 50 embryos were evaluated for H-Y antigen expression. Of these, 29 were evaluated as not fluorescent, suggesting no reaction with H-Y antigen, while 21 did fluoresce, indicating the presence and expression of H-Y antigen.
- The positive fluorescence findings, which were found in both the inner cell mass (ICM) and trophectoderm cells, suggest that the H-Y antigen is expressed on equine blastocyst stage embryos.
Cite This Article
APA
Wood TC, White KL, Thompson DL, Garza F.
(1988).
Evaluation of the expression of a male-specific antigen on cells of equine blastocysts.
J Reprod Immunol, 14(1), 1-8.
https://doi.org/10.1016/0165-0378(88)90031-9 Publication
Researcher Affiliations
- Department of Animal Science, Louisiana State University, Baton Rouge 70803.
MeSH Terms
- Animals
- Antibodies, Monoclonal
- Blastocyst / immunology
- Fluorescent Antibody Technique
- H-Y Antigen / isolation & purification
- Horses
- Male
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