Molecular dissection of the genetic relationships of source, sink and transport tissue with yield traits in rice.

Source, sink, and translocation capacity of assimilates play important roles during the formation of grain yield. The present study was conducted to characterize the genetic bases of traits representing source, sink and transport tissue, and their relationships with yield traits in rice, by analyzing QTLs for these traits and various ratios among them. The genetic materials were a recombinant inbred population derived from a cross between two indica cultivars Zhenshan 97 and Minghui 63, the parents of the most-widely grown hybrid rice in China. Using a linkage map that covers a total of 1,796 cM based on 221 molecular marker loci, a total of 81 QTLs were identified for the 15 traits studied (three leaf areas as the source, total spikelets per panicle as the sink, the number of large vascular bundles in the stem as transport tissue, three source to sink ratios, three transport tissue to source ratios, one transport tissue to sink ratio and three yield traits). The amount of variation explained by individual QTLs ranged from 1.12% to 24.14%. Five QTLs were identified to show interaction effects with the environment, which explained from 3.19% to 9.15% of the variation. The results showed that close linkage or pleiotropy is the genetic basis for the correlations of grain yield traits with source, sink, transport tissue and the various ratios among them. Of the 25 QTLs identified for source-sink-transport tissue trait, and 43 for various ratios, 8 and 22 QTLs, respectively, were mapped to the similar genomic blocks harboring QTLs for yield traits, especially for grain weight. Co-location of QTLs for yield traits with those for ratios among source, sink and transport tissue may provide a genetic explanation for the physiological expression of yield traits, and also suggest that improvement in ratios among source, sink and transport tissue may result in improvement in yield potential.

Size differences between human X and Y spermatozoa and prefertilization diagnosis.

Normal human spermatozoa carry either the X or the Y chromosome. The differences between X and Y spermatozoa (X and Y haploid cells) may exist in two areas: the different chromosomes (i.e. different kinds and numbers of genes) and the different sperm structures and functions (i.e. different genetic expression). The aim of this study was to determine whether there are any size between X and Y spermatozoa and whether sperm size and shape varies between men. Identification of the Y (and X inferred) status of individual spermatozoa was carried out by polymerase chain reaction (PCR), amplifying the putative testis-determining gene (SRY) together with a control gene (ZP3). PCR amplification of 871 out of 895 (97.3%) single motile spermatozoa showed that 444 (51.0%) were Y and 427 (49.0%) were X-bearing spermatozoa. Of 233 normally-shaped but immobilized spermatozoa, 217 (93.1%) were photographed and measured. Statistically, the length, perimeter and area of the sperm heads, and the length of the sperm necks and tails of X-bearing spermatozoa were significantly larger and longer than those of Y-bearing spermatozoa. Some peculiarities (or variations) in the X and Y sperm shape and size in individual donors were found. The pre-screening by micro-measurement of these specific haploid characteristics of individual spermatozoa in different donors, which may be closely related to their different genetic conditions (or diseases), may be important in human medicine and animal husbandry, especially in sperm prefertilization diagnosis.

Localisation of mRNA for interleukin-1 receptor and interleukin-1 receptor antagonist in the rat ovary.

Interleukin-1 (IL-1) is a multifunctional cytokine with profound effects on ovarian function. The effects of IL-1 on ovarian steroidogenesis have been demonstrated in several species. IL-1 mRNA levels are increased in the thecal layer of the ovulating follicle and IL-1 beta has been shown to induce ovulations in vitro. In this study we have investigated the presence and distribution of the mRNAs for type I IL-1 receptor (IL-1RtI) and for the naturally occurring IL-1 receptor antagonist (IL-1ra) in ovaries of adult cycling rats, to elucidate the target cells for IL-1 action. We have demonstrated the presence of mRNA for both substance by in situ hybridisation and reverse transcription PCR. mRNA for IL-1RtI was not found in primordial follicles but was abundant in the granulosa and thecal layer in developing follicles with stronger signals in the granulosa layer. In the preovulatory and ovulatory follicles, there was a further increase in the signal for IL-1RtI mRNA in the thecal layer compared with the granulosa layer. Corpora lutea were weakly positive at all stages and atretic follicles were largely negative. No mRNA was detected in oocytes of any stage mRNA for IL-1ra showed a similar distribution to that of IL-1RtI. The changes in distribution suggest an action of IL-1 on rat granulosa cells during follicular development and on thecal cells during ovulation.

The effect of stress on semen reduction in the marmoset monkey (Callithrix jacchus).

This study compared a number of semen parameters of two separate groups of the common marmoset monkey (Callithrix jacchus) in order to determine the effect of a continuous potentially stressful situation on these parameters, and thus on the monkey's reproductive ability. The semen from 16 adult male marmoset monkeys was collected and analysed to compare semen parameters between a 'normal' (control) group (n = 9) and a 'blood withdrawn' ('stress') group (n = 7). The semen parameter values observed in the control were: pH 7.51 +/- 0.22, volume 40.2 +/- 27.2 microliters, concentration 27.3 +/- 14.8 x 10(4)/microliters, motility 47.4 +/- 15.9%, grade of velocity 3.5 +/- 1.2, and normal morphological forms 51.8 +/- 13.7%. The 'blood withdrawn' group of marmoset monkeys showed significantly lower semen volume and sperm concentration than the 'normal' group. In addition, total count of spermatozoa, normal spermatozoa, motile spermatozoa and normal motile spermatozoa per ejaculate per monkey was significantly reduced in the 'blood withdrawn' group. The semen of these monkeys also revealed a significantly higher percentage of abnormally-shaped sperm heads than the normal group, and cases of impotence and sham ejaculation were recorded. Our study revealed that the continuous withdrawal of a small volume of blood from a group of marmoset monkeys appeared to be stressful to these monkeys and as a result, influenced their semen parameters, possibly making them less fertile. In addition, electroejaculation was found to be possibly harmful to the monkey's reproductive ability.

Nuclear structural conditions and PCR amplification in human preimplantation diagnosis.

An understanding of the relationship between nuclear morphology and DNA function is important in cytology and preimplantation diagnosis. In this study, direct polymerase chain reaction (PCR) amplification was used to diagnose the common delta F508 mutation of cystic fibrosis in 62 biopsied human embryo cells. The nuclei were photographed and classified into three categories depending on their microscopic appearance; these were further correlated with the results of PCR amplification. The normal nucleus group (42 embryo cells, with clear and regular nuclear membrane, transparent nucleoplasm and prominent nucleoli) showed 100% PCR amplification, with normal amplification results, i.e. bright DNA bands. These were considered to be the living cells. Only half of the cells (10 embryo cells) which contained abnormal nuclei (with abnormal nuclear membranes or nucleoplasm) showed PCR amplification, often with abnormal amplification results, i.e. weak DNA bands. These cells were considered to be either degenerate or to be undergoing degeneration. The anuclear cells (10 embryo cells) were composed of living (metaphase) and degenerated cells and showed about 30% PCR amplification. These results demonstrated that one of the important signs of early visible cell degeneration is the partial or total degeneration of the nucleus. Abnormal morphological changes of the nuclear membrane and nucleoplasm are usually accompanied with functional and structural DNA alteration. It is suggested that base degradation occurs earlier than the breakage of base-sugar bonds and phosphodlester bonds during the course of DNA degradation. The selection of optimal cells with a normal nucleus for single cell embryo biopsy is important for the precision and safety of preimplantation diagnosis.

Optimal polymerase chain reaction amplification for preimplantation diagnosis in cystic fibrosis (delta F508)

OBJECTIVE: To evaluate direct polymerase chain reaction amplification of mutation on single embryo cells for the routine preimplantation diagnosis of cystic fibrosis. DESIGN: Direct polymerase chain reaction amplification of mutation was performed to identify the cystic fibrosis delta F508 mutation in human blood DNA, single lymphocytes, embryos, and embryo cells obtained by biopsy. Preimplantation diagnosis was performed for a couple who were heterozygous carriers of the delta F508 mutation. SETTING: Laboratory for preimplantation diagnosis in a reproductive medicine unit. MAIN OUTCOME MEASURE: Correct diagnosis of homozygous normal, heterozygous, and homozygous abnormal DNA of the cystic fibrosis delta F508 mutation. RESULTS: 45 blood samples (18 homozygous normal, 17 heterozygous, and 10 homozygous abnormal) and 204 single lymphocytes from known sources showed 100% amplification and were diagnosed correctly. 17 human embryos and 52 normal nucleated embryo cells obtained by single cell embryo biopsy also showed 100% amplification. After a miscarriage of the initial pregnancy (diagnosed at preimplantation to be homozygous normal) in the heterozygous carrier couple, fetal tissue was confirmed to be homozygous normal. CONCLUSION: Direct polymerase chain reaction amplification of mutation is a simple, fast, reliable test for the common cystic fibrosis mutation (delta F508) in blood DNA and single cells and should be applicable to routine programmes of general screening, maternal blood examination, and preimplantation diagnosis.

Anatomy of adult female common marmoset (Callithrix jacchus) reproductive system.

Better appreciation of the female reproductive anatomy of the common marmoset (Callithrix jacchus) should improve the prospects for nonsurgical embryo transfer in this model. Vaginal measurements were performed in 8 female adult marmoset monkeys. Four monkeys were measured at laparotomy for gross internal anatomy, and 1 monkey was analysed at autopsy. The vagina of the marmoset monkey was found to be divided into a lower and upper vagina with a marked vaginal isthmus between them. The mean lengths of the lower and upper vagina were 17 mm (34 mm in total vagina). The mean uterine size was 8.4 (length) x 10.0 (width) x 6.4 (thickness) mm, with the ovary measuring 5.3 x 4.3 x 3.8 mm. The mean length of the fallopian tube was 10.5 mm with a width of 1.5 mm. Nonsurgical embryo transfer in this model appears to be feasible, but the proportionally long vagina and short uterine cavity needs to be recognised.

Histopathological analysis of mice born following single cell embryo biopsy.

Single cell embryo biopsy is a useful but invasive technique for preimplantation diagnosis. Biopsy may be performed by physical (direct zona puncture) or chemical methods (zona drilling with acid solution). This study has analysed the safety of a physical method of embryo biopsy in the mouse. Six adult mice (male and female), three from biopsied embryos and three from a control group (non-biopsied) were subjected to histopathological analyses. Macroscopically, the anatomy and morphology of the internal organs in both groups were normal. Microscopic analyses of 15 major organs, which included the brain, heart, lung, liver, kidney, stomach, intestine, voluntary muscle, spleen, pancreas, adrenal, thymus, skin, testis (male) and ovary (female), in both groups were all normal. These results showed that careful single cell embryo biopsy by direct zona puncture performed at the 8-cell embryo stage had no adverse influence on the macroscopic and microscopic structure of the organs. The remaining pluripotential cells of biopsied embryos developed normal microstructure according to the hereditary messages. Ideally, the safety of embryo biopsy requires observation of three stages after embryo biopsy, namely embryonic and fetal development before birth, neonatal assessment and long-term monitoring after birth.

Sex determination of preimplantation embryos by human testis-determining-gene amplification.

When there is a risk of inherited disease, preimplantation diagnosis gives couples an opportunity to avoid having a child with the disease. Sex determination can be used to exclude the likelihood of a sex-linked disorder. Accuracy of the diagnosis is important. We have tested the reliability of sex determination based on the recognition of a testis-determining gene (SRY) sequence. DNA from the blood of 120 men and women and from 38 single lymphocytes was amplified by polymerase chain reaction (PCR) with the SRY and control (ZP3) gene primers. All results confirmed the correct sex of origin (100%). The test was then used to determine the sex of 21 single embryo cells biopsied from 21 (4-8 cell) human polyspermic embryos. 2 embryo cells recognised at biopsy to have degenerated produced negative results. The other 19 single embryo cells showed 100% PCR amplification. 11 (58%) of the embryos were judged to be "male" and 8 (42%) "female". The SRY and ZP3 gene primers selected are highly specific and give accurate results in sex determination and their use provides a new reliable method for routine preimplantation and general prenatal sex determination in man.

Blood analysis of mice born following single-cell embryo biopsy.

Single-cell embryo biopsy is an important technique in preimplantation diagnosis. The development of the mouse embryo and fetus, and the results of some analyses after birth following embryo biopsy, have been demonstrated to be normal. Histopathological analysis of mice born following single-cell embryo biopsy with a physical method (zona puncture) also showed normal organ and cell structure, thus demonstrating the safety of embryo biopsy. This experiment analysed the parameters of blood cells and the blood chemistry of 28 mice born following single-cell embryo biopsy. White cell count, red cell count, haemoglobin level and platelet count of the blood, and plasma sodium, potassium, chloride, bicarbonate, calcium, glucose, albumin, protein, creatinine, urea, bilirubin, aspartate transaminase, creatine kinase and lactate dehydrogenase of the blood were not significantly different between the mice from the biopsied and the control embryo groups, which again demonstrated the safety of single-cell embryo biopsy. The remaining totipotent cells in the biopsied embryos would thus be expected to develop to the correct cell counts and normal organic functions according to the intact hereditary messages. Further studies on the safety of embryo biopsy (including long-term observation after birth) and the improvement of the different biopsy techniques and skills for preimplantation diagnosis are necessary.

Precise sex selected births of mice following single cell embryo biopsy and Y-linked testis-specific gene analysis.

We report on the birth of 39 mice following single cell embryo biopsy and precise sex determination following in-vitro fertilization. Polymerase chain reaction was used to amplify fragments of the mouse testis-specific gene sequence (pYMT2/B) on the Y chromosome and the ovary-specific gene (ZP3) sequence on chromosome 5 from the single biopsied cell. Embryo biopsy was not associated with any deleterious effects.

Hatching rate: an optimal discriminator for the assessment of single-blastomere biopsy.

PURPOSE: In order to determine an optimal marker to discriminate embryo injury following single-blastomere embryo biopsy, mouse embryos were examined for rates of blastocyst formation, hatching, implantation, and fetal development following single-blastomere biopsy. RESULTS: Early studies of single-blastomere biopsy (1-8 series) resulted in similar rates of blastocyst formation (P > 0.05) but a lower rate of hatching of biopsied (n = 140) versus control (nonbiopsied) (n = 145) embryos (78.6 vs 95.2%; p < 0.01). Subsequent experience (9-13 series) eliminated this difference between biopsied (n = 145) and control embryos (n = 133) (95.9 vs 94.0%; P > 0.05). Embryo transfer of hatching blastocysts of biopsied (n = 100) and nonbiopsied control (n = 100) groups resulted in equivalent rate of fetal development (70.0 vs 68.0%; p > 0.05). CONCLUSIONS: The hatching rate appeared to be a simple, sensitive, and reliable method to evaluate the single-blastomere biopsy technique.

Collection and analysis of semen from the common marmoset (Callithrix jacchus).

The authors examined the seminal characteristics of 16 male common marmosets (Callithrix jacchus) to provide baseline data for future studies of the reproductive biology of this species. Semen samples were collected by electroejaculation. There was considerable inter- and intra-male variation in all seminal characteristics. The median seminal volume was 30 microliters (range 8 to 85 microliters), and the median total sperm count was 5.1 x 10(6) sperm (range, 0.1 to 43 x 10(6]. The median progressive sperm motility was 48% (range, 10% to 76%), and 49% of the sperm exhibited normal morphology (range, 24% to 81%). Three types of sperm head abnormalities and eight types of tail defects were noted. Tail defects were common (median, 50%; range, 17% to 76%), whereas head defects were relatively rare (median, 4.5%; range, 0% to 24%). The results indicate that semen samples can be routinely collected from this species, but considerable inter- and intra-male variation can be expected. It is therefore important to examine several semen samples from each male marmoset to obtain an accurate seminal picture.