Altered mRNA expression patterns in bovine blastocysts after fertilisation in vitro using flow‐cytometrically sex‐sorted sperm

In this work we analyzed the effects of three culture systems on developmental ability of bovine embryos in vitro produced with sexed sperm, the survival to vitrification (cryologic vitrification method) of such blastocysts, and their pregnancy rates after embryo transfer to recipients, both as fresh and after vitrification/warming. Finally, we measured the accuracy of the sorting protocol by a polymerase chain reaction-based method to validate the embryo sex at blastocyst stages. We confirmed an individual effect of the bull as well as development rates of embryos produced with sorted sperm lower than embryos with unsorted sperm, independent of the culture system used. The cryoresistance to vitrification of embryos produced with sexed sperm did not differ from that of conventionally produced embryos (re-expansion rates at 24 and 48 h: 74.6% vs. 75.5%, and 64.5% vs. 68.1% for embryos produced with conventional and sorted sperm, respectively; hatching rates at 48 h: 63.55% vs. 55.5% for embryos produced with conventional and sorted sperm, respectively). Finally, no significant differences were found in pregnancy rates after the embryo transfer of fresh and vitrified/warmed blastocysts (52.8% vs. 42.0%, respectively; P Ͼ 0.05). Male and female embryos produced with sorted sperm showed the same quality in terms of developmental ability, cryoresistance, and pregnancy rates after transfer. Our culture system, coupled with the vitrification in fiber plugs, provides good quality sex-known embryos which survive vitrification at similar rates than embryos produced with conventional unsorted sperm; also it produces good pregnancy rates after transfer of sexed embryos both fresh and after vitrification and warming.

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“…Also, Morton et al [60] describe a reduced expression of developmentally important genes in embryos produced with sexed sperm.…”

Section: Discussionmentioning

confidence: 99%

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

et al. 2012

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“…Furthermore, the unequal transcriptional level among sexes of X-linked genes may impose a transcriptional regulation upon autosomal genes, broadening the transcriptional effect. In this sense, the transcription level of several candidate genes depends on the embryo sex (Larson et al 2001, Morton et al 2007, Bermejo-Alvarez et al 2008b, 2010a, and microarray studies have shown a large transcriptional dimorphism in mouse (Kobayashi et al 2006) and, particularly, in bovine embryos (BermejoAlvarez et al 2010b).…”

Section: Introductionmentioning

confidence: 99%

Transcriptional sexual dimorphism in elongating bovine embryos: implications for XCI and sex determination genes

Bermejo-Álvarez¹,

Rizos²,

Lonergan³

et al. 2011

Reproduction

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Sex chromosome transcripts can lead to a broad transcriptional sexual dimorphism in the absence of concomitant or previous exposure to sex hormones, especially when X-chromosome inactivation (XCI) is not complete. XCI timing has been suggested to differ greatly among species, and in bovine, most of the X-linked transcripts are upregulated in female blastocysts. To determine the timing of XCI, we analyzed in day 14 bovine embryos the sexual dimorphic transcription of seven X-linked genes known to be upregulated in female blastocysts (X24112, brain-expressed X-linked 2 (BEX2), ubiquitin-conjugating enzyme E2A (UBE2A), glucose-6-phosphate dehydrogenase (G6PD), brain-expressed X-linked 1 (BEX1), calpain 6 (CAPN6), and spermidine/spermine N-acetyltransferase 1 (SAT1)). The transcription of five genes whose expression differs between sexes at the blastocyst stage (DNMT3A, interferon tau (IFNT2), glutathione S-transferase mu 3 (GSTM3), progesterone receptor membrane component 1 (PGRMC1), and laminin alpha 1 (LAMA1)) and four genes related with sex determination (Wilms tumor 1 (WT1), gata binding protein 4 (GATA4), zinc finger protein multitype 2 (ZFPM2), and DMRT1) was also analyzed to determine the evolution of transcriptional sexual dimorphism. The expression level of five X-linked transcripts was effectively equalized among sexes suggesting that, in cattle, a substantial XCI occurs during the period between blastocyst hatching and initiation of elongation, although UBE2A and SAT1 displayed significant transcriptional differences. Similarly, sexual dimorphism was also reduced for autosomal genes with only DNMT3A and IFNT2 exhibiting sex-related differences. Among the genes potentially involved in sex determination, Wilms tumor 1 (WT1) was significantly upregulated in males and GATA4 in females, whereas no differences were observed for ZFPM2 and DMRT1. In conclusion, a major XCI occurred between the blastocyst and early elongation stages leading to a reduction in the transcriptional sexual dimorphism of autosomal genes, which makes the period the most susceptible to sex-specific embryo loss.

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“…However, in comparison with their counterparts derived from unsorted spermatozoa, bovine IVF embryos derived from sex-sorted spermatozoa display a reduction in the relative abundance of developmentally important genes like glucose transporter 3 (Glut3) and glucose-6-phosphate dehydrogenase (G6PD; Morton et al 2007aMorton et al , 2007b, which may be deleterious to the developmental competence of embryos. In other studies cleavage rates after IVF with sex-sorted spermatozoa were 30% below those with unsorted spermatozoa from the same ejaculate.…”

Section: Ivf With Sorted Spermatozoamentioning

confidence: 99%

Sex selection of sperm in farm animals: status report and developmental prospects

Rath¹,

Barcikowski²,

Graaf³

et al. 2013

Reproduction

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Pre-selection of spermatozoa based on the relative DNA difference between X-and Y-chromosome bearing populations by flow cytometry is an established method that has been introduced into commercial cattle production. Although several important improvements have increased the sort efficiency, the fertilising ability of sexed spermatozoa based on offspring per insemination is still behind farmers' expectations. The main stress factors, especially on mitochondria, that reduce the lifespan of spermatozoa are described, and new technical as well as biological solutions to maintain the natural sperm integrity and to increase the sorting efficiency are discussed. Among these methods are the identification of Y-chromosome bearing spermatozoa by bi-functionalised gold nanoparticles and triplex hybridisation in vivo as well as new laser-controlled deflection system that replaces the deflection of spermatozoa in the electrostatic field. Additionally, as well as a new nonsurgical transfer system of spermatozoa into the oviduct of cows has been developed and allows a significant reduction of spermatozoa per transfer. Altogether, the improvements made in the recent years will allow a broader use of sex-sorted spermatozoa even in those species that require more cells than cows and sheep.Reproduction (2013) 145 R15-R30

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Altered mRNA expression patterns in bovine blastocysts after fertilisation in vitro using flow‐cytometrically sex‐sorted sperm

Cited by 91 publications

References 50 publications

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

Transcriptional sexual dimorphism in elongating bovine embryos: implications for XCI and sex determination genes

Sex selection of sperm in farm animals: status report and developmental prospects

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