Developmental asymmetries in experimental animals.

The early embryo orients to the antero-posterior axis and differentiates along this, and the dorso-ventral and lateral axes. From Drosophila melanogaster, detailed knowledge has accrued of how segmentation and dorso-ventral differentiation proceed, and of their genic control, mostly by selector and homeobox (Hox) genes. The study of the control of lateral differentiation, instead, has been largely neglected. Yet handed asymmetry (the "obvious" asymmetries of, for example, heart, lung, anatomical features of the nervous system, etc.) is basic and, possibly, universal. In the mouse, two genes control this: the iv gene which, when mutated, leads to random, in the place of biased, asymmetry and so to random situs inversus viscerum: and the inv mutation which, by contrast, results in 100% situs inversus. Both mutants act as autosomal recessives. Human situs inversus is heterogeneous and may be akin to that produced by the murine iv gene. In spite of situs inversus, there is no shift of hand preference; but there is no information on other lateralization, e.g. of language or of dermatoglyphic patterns. Handed asymmetry is known in Drosophila, but there is no information on its control. In the experimental nematode, Caenorhabditis elegans, asymmetry arises when differently programmed cells arrange themselves to the two body sides, and is present already at the six-cell stage; and even the major sensory neurons chains along the body axis are distributed unequally on the two sides of the worm. Experimentally, by embryonic micro-manipulation or the use of chemical mutagens, the normal and invariate direction of handed asymmetry can be reversed.

Neurodevelopmental disorders and diseases.

Research on cerebral palsy--and related neurodevelopmental disorders--should ultimately aim at primary prevention, and, following examination of existing knowledge of causes and mechanisms of origin, a comprehensive and strategic approach to causes and mechanisms is needed.

A test of the production line hypothesis of mammalian oogenesis.

Germ cells in female mammals become committed to meiosis and enter its prophase sequentially in fetal life and, according to the Production Line Hypothesis, the oocytes thus generated are released after puberty as mature ova in the same sequence as that of meiotic entry in fetu. This hypothesis in its original and complete form has a subordinate proposition that concerns chiasma (Xma) frequency; it postulates that Xma number would decrease with fetal age. Consequently, univalents would increase, leading to errors of chromosome disjunction at the first meiotic division (MI), and thus to maternal age-dependent numerical chromosome anomalies. By using an in vitro/in vivo approach, we radioactively labelled the DNA of germ cells at premeiotic synthesis as they sequentially entered meiosis, while the fetal ovaries were in culture. At the end of this in vitro phase, pachytene/diplotene (P/D) stages were studied to determine their labelled fraction. The ovaries were then transplanted to spayed females and, after the in vivo phase, mature ova were harvested and the proportion of labelled first and second meiotic metaphases (MI/MII) determined. By marking the germ cells with label while in vitro during periods equivalent to early and late gestation, and by comparing the observed proportions of labelled MI/MII with those of oocytes labelled at P/D, we concluded that, in the mouse, ova do not mature at random for release, but are formed according to a production line system in which the time of release after puberty is related to the time of entry into meiosis in fetu.

Prenatal diagnosis of genetic disorders in preimplantation embryos: invasive and non-invasive approaches.

The purpose of this article is to analyse the various non-invasive and invasive methods that offer the opportunity of prenatal diagnosis of selected inherited disorders at the preimplantation stages of human embryonic development and to discuss the advantages and ethical problems associated with such procedures. These investigations should also provide important information on the development of the human embryo and its hormonal and immunological relationship with the mother.

Meiosis in trisomic female mice with Robertsonian translocations. I. Prophase pairing.

The prophase oocytes of two murine Robertsonian translocation (Rb) trisomies of chromosomes 16 and 19 were investigated using electron microscopy and a whole-cell micro-spreading technique after silver staining. About 20% of fetuses of each type were trisomic. They were obtained by mating animals heterozygous for two Rb's, monobrachially homologous for either chromosome 16 or 19, to an entirely acrocentric stock. Because of the almost inevitable prenatal mortality of the trisomic embryos, their fetal ovaries were "rescued" by an in vitro method for prophase studies. Analysis of the recovered oocytes showed frequent, close pairing associations of the three trisomic axes and evidence suggesting that the closely apposed axes coincided with the side-by-side formation of parallel, complete, true synaptonemal complexes; hence, the cytogenetic dogma that pairing is always two-by-two was contradicted. The presence of two parallel complexes has implications for crossing-over recombination. Triple associations of axes were found in almost half the trisomy 19 (Ts19) and in about 70% of the trisomy 16 (Ts16) prophases. The extent of triple associations varied and was greater in Ts16 than in Ts19 oocytes. Other relevant observations concerned the proportions of univalents and of univalence of the trisomic axes (21% in Ts16 and 46% in Ts19) and the distinctive, thickened appearance of all univalent axes. The pairing behaviour observed in balanced heterozygotes confirms what appears to be nonhomologous pairing and synaptic adjustment within the short-arm axes of the Rb trivalents.

Meiosis in trisomic female mice with Robertsonian translocations. II. Chromosome behaviour at first and second meiotic metaphases.

First and second meiotic metaphases (MI and MII, respectively) from female mice of Robertsonian translocation (Rb) stock, trisomic for chromosome 16 (Ts16) or 19 (Ts19), were studied. The mature trisomic oocytes were derived from explanted fetal ovaries that had been cultured and then transplanted so as to mature heterotopically. Multivalent configurations involving the Rb chromosomes and the additional trisomic acrocentric were analysed. Pentavalent configurations occurred in 74.5% of 98 Ts16 MI and 44.2% of 249 Ts19 MI oocytes; quadrivalents (with a univalent acrocentric) were found in 9.2% of Ts16 MI and 10.8% of Ts19 MI oocytes. In 1% of Ts16 MI and 4% of Ts19 MI oocytes, there were two Rb bivalents and a univalent trisomic acrocentric. Rb trivalents and Rb bivalents occurred together in 14.3% of Ts16 MI and 39.4% of Ts19 MI oocytes. Chiasma frequencies were similar in trisomic and chromosomally balanced MI. Chiasma position, distribution, and localization were nearly identical, whether they were found in Rb multivalents or acrocentric bivalents, but one control group (from chromosomally balanced Ts19 littermates) had significantly more terminal chiasmata. Within the triple homologous region of 8% of Rb pentavalents, two chiasmata were observed in the same relative position in the two sister chromatids of one of the three homologs, suggesting a lapse in chiasma position interference. Assortment at MI anaphase was influenced by secondary nondisjunction of the Rb. The ratio of balanced to unbalanced MII oocytes was 1:4 in both trisomies.

Chimaerism of primordial germ cells in the early postimplantation mouse embryo following microsurgical grafting of posterior primitive streak cells in vitro.

A microsurgical grafting technique has been used to introduce primordial germ cell (PGC) precursors into intact primitive-streak-stage mouse embryos in vitro. Operated embryos were cultured for 36-40 h and then analysed by a combined histochemical and autoradiographic method. PGC chimaerism occurred in embryos that received grafts of caudal primitive streak cells but not adjacent embryonic endoderm or anterolateral ectoderm/mesoderm cells. Graft-derived PGCs were found to be migrating through the gut endoderm alongside host-derived PGCs in approximately half of the chimaeric embryos whereas in the other 50% of cases PGCs remained at the site of grafting in association with graft-derived somatic cells. A similar pattern of somatic chimaerism was produced by primitive streak and anterolateral ectoderm/mesoderm grafts: the allantois was colonized predominantly, with, in addition, formation of amnion, surface ectoderm and caudal mesoderm in a few embryos. The majority of embryonic endoderm grafts failed to incorporate into host embryos and formed yolk-sac-like vesicles. The findings of this study indicate that PGCs originate from the embryonic ectoderm via the primitive streak during development of the mouse embryo, and anterolateral ectoderm and mesoderm cells are unable to form PGCs after heterotopic grafting to the posterior primitive streak site. The combined microsurgical and embryo culture methods provide an experimental system for the analysis of PGC development in intact mouse embryos.

Hormonal induction of steroid sulphatase in the mouse.

By comparing steroid sulphatase levels per se, and also ratios to alpha-galactosidase, in 6 sets of mice - normal females, entire and castrated males both with and without exogenous testosterone administration - we obtained support for the contention that induction of this enzyme is in part controlled by male hormones.

Is the sex ratio at birth affected by immune selection?

The suggestion that the sex ratio is distorted following repeated pregnancies raises the possibility that specific immunological reactions may be responsible. In recent years, work on the sex ratio has been carried out both in experimental animals and in man, and this has been interpreted in the light of the discovery (in mice) of a male-specific weak transplantation antigen, the H-Y antigen. The resulting antibodies could, theoretically, operate on sperm selection, but there is no experimental support for this. Experimental data, including our own, do not support the possibility that successive pregnancies may affect the sex ratio through the induction of anti-H-Y antibodies by a male pregnancy. Neither is there evidence that interaction between male antigens and some pathological conditions influences the sex ratio. Nevertheless, there remains the suggestion that the sex ratio in pre-eclampsia shows a male bias. There is evidence that Rh(+) male fetuses are strongly sensitizing to their Rh(-) mothers, and there is a possibility that the sex ratio in the Xg(a) blood group system is unusual. These possibilities require further study.

Neural-tube defects and vitamins: the need for a randomized clinical trial.

It has been suggested that taking extra vitamins around the time of conception may reduce the risk of fetal neural-tube defects. We have examined the evidence for this and conclude that there is considerable doubt about the efficacy of such a regimen. Also, it cannot be assumed that the taking of extra vitamins has no adverse medical effects. We give reasons for our view that a large randomized clinical trial is ethical and the only satisfactory way of resolving the matter. The Medical Research Council is currently conducting such a study in centres in Britain and abroad, involving women who have already had a pregnancy with a fetal neural-tube defect. The design of the Medical Research Council study is described briefly.

Delayed-type hypersensitivity reaction to H-Y antigen in mice. Effects of soluble antigen from Sertoli cells.

The delayed-type hypersensitivity (DTH) response to the H-Y antigen was investigated in female mice which had been pre-treated with soluble or insoluble H-Y antigen. The present results suggest that Sertoli cells, cultured in vitro, release a factor which, if highly soluble, induces tolerance to male spleen cells. Mice showing marked DTH produced high titre H-Y antibodies after further injections of spleen cells from syngeneic males.

Gonadotropins and gonadal steroids in androgen insensitivity (testicular feminization) syndrome: effects of castration and sex steroid administration.

Gonadotropin control mechanisms were examined in 12 subjects with the complete syndrome of androgen insensitivity (testicular feminization). This study confirmed the presence of elevated luteinizing hormone (LH) and normal follicle-stimulating hormone (FSH) values in these subjects and suggests an intact feedback mechanism for FSH but not LH. This gives further credence to the opinion that estrogens and a nonsteroidal inhibin are important in FSH control. Because of an exaggerated pulsatile pattern of gonadotropins in intact and gonadectomized subjects, there were dramatic variations in gonadotropin levels. After gonadectomy, there was a marked rise in FSH and a further rise in LH. Administered estradiol benzoate and, to a lesser degree, testosterone propionate were capable of lowering LH levels. The effect of testosterone could be via conversion to estrogen(s) in the testes or elsewhere.