344ICSI-MEDIATED GENE TRANSFER SKEWS SEX RATIO AGAINST FEMALE BIRTHS IN MICE

Wild steelhead (Oncorhynchus mykiss) returning to the Hood River in Oregon, USA, show a strongly female-biased sex ratio (average of 63 % female from brood years 1992 to 2004) while first-generation hatchery steelhead (created using local, wild broodstock) display an even sex ratio (51 % female). We considered four hypotheses to explain the difference in sex ratio between populations. First, it is well established that wild male O. mykiss adopt the resident (non-anadromous) life history phenotype at a higher rate than do females, and that the propensity to become resident is under genetic and environmental influence. Therefore, the simplest explanation for the difference in sex ratio between anadromous wild and hatchery fish is that hatchery males adopt the resident life history at a lower rate than do wild males. However, alternative explanations include (1) sex reversal of female hatchery fish to phenotypic males, (2) selection against hatchery females or (3) selection against wild males. The possibility of sex reversal in the hatchery was of particular interest given increased temperature has been shown to skew sex ratios to a male bias. Using a Y-chromosome specific marker (OmyY1 locus) and samples of wild and hatchery fish from various life stages, we were able to reject alternative hypotheses 1, 2 and 3. Hatchery fish were sampled at three life stages (onset of exogenous feeding, 1-year of age and returning adult). Phenotypic and chromosomal sex matched in all 1-year old and adult hatchery samples. Therefore, we see no evidence for sex reversal in the hatchery population. Furthermore, hatchery fish at all three life stages exhibited a 50:50 chromosomal (OmyY1 marker) sex ratio. Therefore, selection against hatchery females while in captivity or after release can be ruled out. The chromosomal sex ratio in a sample of wild smolts was female-biased and matched the sex ratio in returning adults from the same cohort. Therefore, we can also rule out selection against wild males at sea. Given no evidence for sex reversal or selection against either sex it seems most plausible that the greater female bias in wild, compared to hatchery, steelhead from the Hood River results from differential life history expression in males. Wild males appear to become resident (non-anadromous) at a higher rate than do hatchery males.

In vitro post-hatching embryos culture is a procedure that allows the establishment of more accurate tools for evaluating embryo developmental potential without the need of transferring them to recipient animals (Vejlsted et al. 2006 Theriogenology, 65, 153–165). It is well established that in the in vitro embryo production (IVP) technique, the sex ratio is imbalanced in favor of male embryos. The difference in sex ratio observed in the blastocyst stage at day 7 may be attributed to a variety of factors including developmental speed. However, whether or not this difference in sex ratio and speed of development continues after hatching is not known. The objective of this study was to evaluate post-hatching embryonic development until day 11 after in vitro fertilization (day 0) associating embryo size and gender. A total of 468 oocytes, obtained from abattoir-derived ovaries, were used. They were matured, fertilized, and cultured in vitro for 8 days in synthetic oviduct fluid medium (SOF Nutricell�) and incubated at 39�C in 5% CO2 in air. Degenerated embryos on day 8 and non-hatching embryos on day 9 were removed from culture droplets, and only hatched blastocysts were kept. Then, embryos were measured using a graduated ocular and post-hatching development (PHD) medium (Brand�o et al. 2005 Biol. Reprod. 71, 2048–2055) was added in each well, being the final medium 1:1 of SOF:PHD. On day 11, the embryos were evaluated under stereomicroscope and only morphologically normal blastocysts were measured and frozen at –80�C, for gender diagnosis. The DNA from frozen samples was extracted with trizol reagent, sodium citrate solution (0.1 m), and ethanol. Sex embryos determination was performed by PCR and visualized in 2% agarose gel. Data were analyzed using the Mann-Whitney test. The results show that the majority (69%) of the embryos that reached blastocyst stage at day 7 developed in the PHD system until day 11. From the initial oocytes, 144 embryos (30.1%) and 146 (31.1%) embryos had reached the blastocyst stage at days 7 and 8, respectively. At day 9, 89 (19%) embryos were hatched and 65 embryos (13.9%) developed until day 11, of which only 48 embryos (73.8%) had a clear trophoblast. No difference (P > 0.05) in the percentage of male and female embryos was observed when embryos were evaluated at day 11 of culture. In addition the mean size was similar (P > 0.05) for female (467.24 � µm, n = 19) and male (478.84 � 190.21 µm, n = 29) embryos. The results suggest that after post-hatching culture the differences in sex ration and in gender development in IVP bovine embryos are not evident. The development until day 11 showed that post-hatching in vitro culture of bovine blastocyst can be used for embryo evaluation in later phase of development However, several questions still remain to be investigated regarding post-hatching culture of bovine blastocysts before it can be used as a tool to evaluate in vitro embryos. Supported by Embrapa and UnB, Brazil.

BackgroundIt is not clear how foreign DNA molecules insert into the host genome. Recently, we have produced transgenic mice to investigate the role of the fad2 gene in the conversion of oleic acid to linoleic acid. Here we describe an integration mechanism of fad2 transgene by homologous illegitimate random integration.ResultsWe confirmed that one fad2 line had a sole integration site on the X chromosome according to the inheritance patterns. Mapping of insertion sequences with thermal asymmetric interlaced and conventional PCR revealed that the foreign DNA was inserted into the XC1 region of the X chromosome by a homologous illegitimate replacement of an entire 45,556-bp endogenous genomic region, including the ovarian granulosa cell tumourigenesis-4 allele. For 5' and 3' junction sequences, there were very short (3-7 bp) common sequences in the AT-rich domains, which may mediate the recognition of the homologous arms between the transgene and the host genome. In addition, analysis of gene transcription indicated that the transgene was expressed in all tested fad2 tissues and that its transcription level in homozygous female tissues was about twice as high as in the heterozygous female (p < 0.05).ConclusionsTaken together, the results indicated that the foreign fad2 behaved like an X-linked gene and that foreign DNA molecules were inserted into the eukaryotic genome through a homologous illegitimate random integration.

Sex ratios in mule duck embryos at various stages of incubation

The data used in the present study were collected from the records in the Thoroughbred Year-lings Catalogue of Japan (from 1970 to 1973) which was published by the Japan Light-Breed HorseAssociation. The catalogue contained three breeds of horses, i.e., Thoroughbred, Non-Thoroughbred, and Thoroughbred Strain, and data collected from eight districts (see the remarks of Table 1). Thetotal number of offspring and of sires listed in the catalogues were 20, 662 and 493, respectively. The results obtained from a study on the secondary sex ratio of race horses in Japan are summarized as follows.1. Total sex ratio was 49.3 ( ?? %). Its shift was significant at a 5% level. With regard to eachdistrict and each breed, a shift of sex ratio to female was seen only in the Thoroughbred breed in the C district. There was no significant shift of sex ratio in the total of the eight districts for any breed, or in the sum total of the three breeds for any district (Table 1).2. A regional difference in sex ratio was observed most frequently between the H and any other district. It was difficult, however, to find a general rule for regional differences from the present study. Therefore, it cannot be concluded that there is a regional difference in the sex ratio of race horses (Table 2).3. A large number of sires showed a shift of sex ratio to female than a shift to male (Table 3).4. Shifts of sex ratio were found in the Thoroughbred breed in February and March, in the Non-Thoroughbred breed in March, in the Thorughbred Strain in February, and in all the three breeds in February and March. It cannot be concluded, however, that there is a relationship between season and sex ratio (Table 4).5. Shifts of sex ratio were noted in sires at six years of age (to male), and at twelve and eigh-teen years of age (to female). When the sires were divided into three age groups, a significant dif-ference in sex ratio was found only between the two older groups. No permanent shift of sex ratio with the advance in age of sire, however, could be noted in the present study (Table 5 and 6).

Information on genetic variability in cowpea germplasm is important for crop improvement and for efficient utilization of the existing genetic resources. Hence, the objectives of the present investigation were to estimate genetic variability,

We investigated gene transfer in finfish and shellfish via electroporated sperm. The mobility of sperm, the fertilization rate, the hatching rate, gene transfer rate, and abnormality rate of derived embryos were primarily dependent on the voltage level and concentration of DNA during electroporation. Optimal conditions for sperm of each species of aquatic animals can be reached. Genome of the electroporated sperm was analyzed by PCR, and it was shown that an expected-sized product was amplified, corresponding to that of the transgene's amplification. Southern blotting also showed that a positive band located at the same position as the DNA fragment used for the transfer was found in the electroporated sperm after DNase treatment. When the genome isolated from embryos, larvae, juvenile, and adult individuals, all derived from sperm electroporated with foreign DNA molecules, was analyzed by PCR, the existence of foreign DNA was detected in some samples. The integration of the transferred DNA into the genome of transgenic samples was also shown by Southern blot analysis. There was a mosaic distribution of exogenous DNA in a wide variety of tissues analyzed. In addition to CAT activity being positive for the experimental larvae, the transferred GH gene was functional in transgenic finfish and shellfish and resulted in fast-growing transgenic varieties. The overall evidence strongly suggests that the use of electroporated sperm is the simplest yet most efficient approach to perform mass gene transfer in aquacultural animals, including marine mollusks.

Comparison of the offspring sex ratio between fresh and vitrification-thawed blastocyst transfer

Blastocyst transfer does not cause a sex-ratio imbalance

A survey of natural populations of the British ladybird Exochomus quadripustulatus revealed the presence of a single large, acrocentric, supernumerary (B) chromosome in all sites visited. Studies were confined to male meiosis, where more than one B was never found to accompany the six bivalents and neo-XY sex pair. The percentage of males possessing B chromosomes varied from 6.4% to 28.6% in 14 different populations. The sex ratios present in these populations also varied. In some equal numbers of males and females were present, in others there were significant excesses of females. A linear regression was found between the percentage of B chromosomes and the percentages of males and females in those populations. It is suggested that the B chromosomes are not in themselves responsible for the sex ratio differences found for similar differences in sex ratio have been found in related neo-XY species lacking B chromosomes. It is more likely that those factors affecting sex ratio are also responsible for affecting the frequencies of B chromosomes in different populations.

World-wide differences in the sex ratio of bronchial carcinoma

Hepatitis B Virus and Sex Ratio on Kar Kar Island

In an effort to delineate more homogeneous autism subgroups for genetic study, we evaluated 133 consecutive individuals referred to the University of Missouri Autism Center. Each index case underwent a diagnostic evaluation, including a clinical morphology examination, laboratory studies, brain MRI, EEG, and collection of historical, medical, and family data. The 71% (94/133) who fulfilled DSM-IV and CARS autism diagnostic criteria were included in this study. Six of 94 were diagnosed with a known genetic disorder. Of the remaining 88 with apparently "idiopathic autism," 58% (51/88) were phenotypically normal, 22% (19/88) were clearly abnormal, and for 20% (18/88) the clinical morphology examination was equivocal. The percentage of phenotypically abnormal individuals is higher than generally thought and disagrees with the perception that children with autism are usually normally formed. The phenotypically abnormal individuals were 10 times more likely to be diagnosed with a known genetic syndrome (21% vs. 2%) and were more than twice as likely (29% vs. 14%) to have structurally abnormal brain MRIs than the phenotypically normal propositi. Moreover, the male to female ratio correlated with the presence of physical anomalies. The total study group had a male to female ratio of 4.2:1; the morphologically normal subgroup, defined on the basis of a normal physical examination, had a sex ratio of 7.5:1 and the normal subgroup, defined on the basis of both a normal physical examination and a structurally normal brain by MRI had a 23:1 sex ratio. For the phenotypically abnormal subgroup, the sex ratio was 1.7:1. Since differences in sex ratio are presumably a reflection of differences in genetic constitution, we postulate that the phenotypically normal subgroup of individuals with "idiopathic autism" is genetically different from the phenotypically abnormal individuals and that differences in the sex ratio in different autism populations is one indicator of a population's genetic heterogeneity.

Biasing the sex ratio of populations of different organisms, including plants, insects, crustacean, and fish, has been demonstrated by genetic and non-genetic approaches. However, biasing the sex ratio of mammalian populations has not been demonstrated genetically. Here, we provide a first proof of concept for such a genetic system in mammals by crossing two genetically engineered mouse lines. The maternal line encodes a functional Cas9 protein on an autosomal chromosome, whereas the paternal line encodes guide RNAs on the Y chromosome targeting vital mouse genes. After fertilization, the presence of both the Y-encoded guide RNAs from the paternal sperm and the Cas9 protein from the maternal egg targets the vital genes in males. We show that these genes are specifically targeted in males and that this breeding consequently self-destructs solely males. Our results pave the way for a genetic system that allows biased sex production of livestock.

In mammals, male live births exceed female ones. In humans, the ratio of male births to total births is expected to be 0.515. In Europe during 1990-5 this differed significantly...