3 TESTIS-SPECIFIC PROTEIN, Y-ENCODED (TSPY) COPY NUMBER AND EXPRESSION IN BOVINE BLASTOCYSTS
Bibliographic record
Abstract
Testis-specific protein, Y-encoded (TSPY) is a gene found on the Y chromosome that, like many genes on the Y chromosome of mammalian species, has multiple copies. Humans have between 20–60 copies, whereas cattle can have up to 200 copies. Genomic copy number of TSPY is of interest because it has been linked to fertility. In previous studies, enormous bull-to-bull variation in TSPY copy number has been found (Hamilton et al. 2009 Sex. Dev. 3, 205–213). The aims of this study were to a) examine the copy number of TSPY in brother embryos to see if variation exists among embryos of a single generation and b) to determine if TSPY mRNA (mRNA) is expressed in the early bovine embryo since there is evidence from transgenic mice studies that human TSPY may play a role in development. 80 Holstein blastocysts were produced using standard in vitro fertilization techniques with the semen of a single bull. Individual blastocysts (n = 50) were lysed and quantitative polymerase chain reaction (qPCR) was used to sex the embryos and to measure TSPY copy number in the embryos that were found to be male. The male-specific TSPY and SRY (sex determining region Y) genes were measured in order to sex the embryos along with ZAR1 (zygote arrest protein 1), as an autosomal control. TSPY copy number was measured with a modified 2??CT method (Hamilton et al. 2009 Sex. Dev. 3, 205–213) and SRY as the single copy reference gene. Messenger RNA was extracted from two pools of blastocysts (n = 20, n = 10) and reverse transcribed into cDNA. The presence of TSPY mRNA in these pools was determined with reverse transcription PCR (RT–PCR) along with the reference gene GAPDH, following standard protocols. Briefly, two sets of TSPY specific primers and one set of GAPDH specific primers were used to amplify the target mRNAs in both pools of embryos using AmpliTaq Gold DNA polymerase (Applied Biosystems) and an annealing temperature of 60°C. PCR products were visualised on a 2% gel using gel electrophoresis and then sequenced to verify specificity of the primers. The results of the embryo sexing experiment showed a 50:50 sex ratio (25 males, 25 females) in the group of individual embryos. TSPY copy number was found to vary significantly (using a one-way ANOVA, P < 0.0001) among brother embryos produced from the semen of the same Hostein bull (n = 25) and ranged from 21.9 ± 7.1 to 159.0 ± 14.0 copies, with an average of 65.9 ± 6.3 copies. Furthermore, the results of the expression experiment showed that TSPY mRNA transcripts were present in both pools of blastocysts. These results provide the first evidence that TSPY is expressed in bovine blastocysts and that copy number can vary within a single generation. This data suggests that the bovine Y chromosome is a dynamic chromosome that is not clonally inherited but can vary its genetic composition within a single generation. The fact that TSPY transcripts were found in the early embryo suggests that it might have another function other than simply a role in spermatogenesis. Supported by National Sciences Engineering and Research Council grant; Canadian Research Chair program; and L’Alliance Boviteq Inc.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".