7a21b93920bc0ca5950a90067ce3fa59 pmc_9959854.pdf 9c86130ce958533b15c4de73a7b9701de77fbac4 pmc_9959854.pdf 85ef94ea1c7b9c7c5295b15102ec7c0928a65af78c0401ab84cfc6a6bac8d705 pmc_9959854.pdf Title: Testis-Specific Protein Y-Encoded (TSPY) Is Required for Male Early Embryo Development in Bos taurus Subject: TSPY is a highly conserved multi-copy gene with copy number variation (CNV) among species, populations, individuals and within families. TSPY has been shown to be involved in male development and fertility. However, information on TSPY in embryonic preimplantation stages is lacking. This study aims to determine whether TSPY CNV plays a role in male early development. Using sex-sorted semen from three different bulls, male embryo groups referred to as 1Y, 2Y and 3Y, were produced by in vitro fertilization (IVF). Developmental competency was assessed by cleavage and blastocyst rates. Embryos at different developmental stages were analyzed for TSPY CN, mRNA and protein levels. Furthermore, TSPY RNA knockdown was performed and embryos were assessed as per above. Development competency was only significantly different at the blastocyst stage, with 3Y being the highest. TSPY CNV and transcripts were detected in the range of 20–75 CN for 1Y, 20–65 CN for 2Y and 20–150 CN for 3Y, with corresponding averages of 30.2 2.5, 33.0 2.4 and 82.3 3.6 copies, respectively. TSPY transcripts exhibited an inverse logarithmic pattern, with 3Y showing significantly higher TSPY. TSPY proteins, detected only in blastocysts, were not significantly different among groups. TSPY knockdown resulted in a significant TSPY depletion (p < 0.05), with no development observed after the eight-cell stage in male embryos, suggesting that TSPY is required for male embryo development. Keywords: copy number variation (CNV); sex-sorted semen; in vitro fertilization; male development; testis-specific protein Y-encoded (TSPY) Author: Na-Young Rho, Teresa Mogas, W. Allan King and Laura A. 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UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Early Embryo Developmental Percentage Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: TSPY Copy Number Variation (CNV) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: TSPY mRNA Expression Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: TSPY Protein Localization and Quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: TSPY Knock-Down Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Unsorted Sex Semen Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Sex-Sorted Semen (X-Bearing) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Sex-Sorted Semen (Y-Bearing) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: TSPY mRNA Expression Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Children: Item: Title: In Vitro Embryo Production (IVP) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Microinjection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: DNA Extraction and Digestion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: RNA Extraction and Reverse Transcription Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Digital Droplet PCR (ddPCR) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Immunocytochemistry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Confocal Imaging and Quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Statistics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Info: Title: Testis-Specific Protein Y-Encoded (TSPY) Is Required for Male Early Embryo Development in Bos taurus Author: Na-Young Rho, Teresa Mogas, W. Allan King and Laura A. Favetta Subject: TSPY is a highly conserved multi-copy gene with copy number variation (CNV) among species, populations, individuals and within families. TSPY has been shown to be involved in male development and fertility. However, information on TSPY in embryonic preimplantation stages is lacking. This study aims to determine whether TSPY CNV plays a role in male early development. Using sex-sorted semen from three different bulls, male embryo groups referred to as 1Y, 2Y and 3Y, were produced by in vitro fertilization (IVF). Developmental competency was assessed by cleavage and blastocyst rates. Embryos at different developmental stages were analyzed for TSPY CN, mRNA and protein levels. Furthermore, TSPY RNA knockdown was performed and embryos were assessed as per above. Development competency was only significantly different at the blastocyst stage, with 3Y being the highest. TSPY CNV and transcripts were detected in the range of 20 75 CN for 1Y, 20 65 CN for 2Y and 20 150 CN for 3Y, with corresponding averages of 30.2 2.5, 33.0 2.4 and 82.3 3.6 copies, respectively. TSPY transcripts exhibited an inverse logarithmic pattern, with 3Y showing significantly higher TSPY. TSPY proteins, detected only in blastocysts, were not significantly different among groups. TSPY knockdown resulted in a significant TSPY depletion (p < 0.05), with no development observed after the eight-cell stage in male embryos, suggesting that TSPY is required for male embryo development. 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