Rapid detection of male-specific DNA sequence in bovine embryos using fluorescence in situ Hybridization

An accurate, reliable, and quick (less than an hour) method for determining the sex of bovine embryos was developed using a fluorescence in situ hybridization (FISH), with a probe designed from a bovine Y chromosome specific DNA (BC1.2). First, to improve a protocol of FISH and evaluate an accuracy of the method, lymphocyte nuclei prepared from three bulls, two cows, and one freemartin were tested. We found that 5 min was enough for hybridization. The washing solution adequate for posthybridization was 0.5× SSC at 72°C for 5 min. The whole procedure for FISH can be accomplished in less than an hour. A male-specific signal was detected, on average, as 97, 0.5, and 83%, respectively, of lymphocytes in males, females, and a freemartin. Using the rapid FISH protocol developed, 28 embryos were divided. According to the presence of the digoxigenin signal, 16 embryos (57.1%) were predicted as male, and 12 embryos (42.9%), predicted as female. Mol. Reprod. Dev. 51:390–394, 1998. © 1998 Wiley-Liss, Inc.     

Sexing and detection of gene construct in microinjected bovine blastocysts using the polymerase chain reaction

We present a polymerase chain reaction (PCR)-based procedure for rapid bovine embryo sexing and classifying embryos for the presence of exogenous DNA. Fourteen bovine blastocysts microinjected with gene construct DNA at the pronuclear stage were divided into quarters and subjected to amplification with construct-specific and sex gene-specific (ZFY/ZFX) primers in the same initial PCR reaction. Blastocysts carrying microinjected construct DNA could be identified by the presence of construct-specific PCR product in approximately 4 h. Approximately half of the microinjected and two of 16 non-microinjected blastocysts typed PCR-positive for the construct DNA. Owing to erroneous amplifications in the two non-microinjected control blastocysts, and the inability of the system to distinguish integrated from non-integrated copies of the microinjected construct, the number of construct-positive blastocysts determined in our assay most likely overestimates the number of true transgenic embryos. Nevertheless, using this assay, we were able to determine that approximately half of the microinjected embryos were negative for the transgene construct and thus could be eliminated from transfer to a recipient cow. Embryo sexing was achieved in less than 6 h by restriction fragment length polymorphism analysis of nestedZFY/ZFXPCR products reamplified from initial PCR reactions. In 11/14 microinjected blastocysts all sections assayed unambiguously as the same sex. In one embryo, only one section was analysed, while two other blastocysts whowed some discrepancies of sexing results between the sections analysed. The approach employed here to determine the sex and presence of microinjected construct DNA in bovine preimplantation embryos is rapid, accurate among different sections of an embryo and can be used to increase the efficiency of current transgenic cattle production procedures.     

Transformer基因与果蝇和线虫的性别决定

黑腹果蝇(Drosophila melanogaster)和秀丽隐杆线虫（Caeborhabditis elegans）的性别决定的问题已研究得比较详细,且transformer基因是这两种生物性别决定中最重要的基因之一,其有关的性别决定研究在近几年取得了很大的进展。本文就线虫和果蝇的transformer基因及其相关基因的特性与功能进行了特别介绍,并在此基础上对其性别决定的分子机制进行初步的比较和分析。Abstract : Sex determination of Drosophila melanogaster and Caeborhabditis elegans has been known in detail. Great progress, is achieved in recent years, is the research of transformer genes, which are those of most important genes in sex determination in both species. In this paper, molecular character, genetic function and the relative genes of transformer genes are particularly described. On the basis,a primary compariso and analysis between the molecular mechanism of sex determination in C.elegans and D. melanogaster are presented.     

Sex differences in embryo development periods and effects on avian hatching patterns

Competitive interactions among siblings are an important determinantof parental fitness. These are strongly influenced by relativeoffspring size and therefore also by the extent to which parentscan influence offspring size hierarchies. The temporal patternof hatching in an avian clutch has a large effect on size anddevelopmental disparities among chicks. Hatching spread is generallyassumed to be mainly determined by the onset of incubation inrelation to egg laying. However, the extent to which factorsother than incubation onset, such as development rate, alsoinfluence timing of hatching has received little empirical investigation.We compared incubation periods of male and female black guillemot(Cepphus grylle) embryos to ascertain whether the time takenfor an egg to hatch varies with embryo sex. Laying date andegg mass had no significant effect on incubation time, but maleembryos hatched on average a day sooner than did females. Theonset of incubation and hatching spread vary in black guillemots.However, in mixed-sexed clutches in which the first-laid embryois male, a faster development time of males should mean asynchronoushatching regardless of parental incubation regime. This wassupported by empirical investigation. These results demonstratethat factors other than incubation behavior can be importantin establishing avian hatching patterns. Whether these sex differencesin development rate are a result of constraints on the degreeof parental control, or an adaptive strategy to manipulate hatchingpatterns, remains to be established.