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Serological Detection of a Cell-Surface Antigen Specified by the T (Brachyury) Mutant Gene in the House Mouse
Abstract
A cell-surface component specified by a mutant gene at the T-locus in the mouse has been detected on sperm by serological methods. The gene product thus recognized is not present on other adult cells.
... To further explore the origin of MSCs in adult BM, we performed the same analyses using a Brachyury-GFPCreER T2 knock-in mouse line (Imuta et al., 2013;Taguchi et al., 2014) to test for a recognized mesoderm and notochord marker at between E7.5 and E9.5 (Herrmann et al., 1990). Brachyury is a transcription factor that belongs to the T-box family of molecules and is required for mesoderm formation (Bennett et al., 1972;Yanagisawa et al., 1981). We treated Brachyury GCE/wt ;Rosa26 Tomato/wt mice with 4-OHT at E6.5, E7.5, E8.5, E9.5, E10.5 and postnatal day (P) 2, P3 and P4, and examined Tomato expression of CFU-F colonies derived from adult BM. ...
... Colony-forming cells have a multipotency for the differentiation into three mesenchymal lineages: adipocytes, chondrocytes and osteocytes. These findings suggest, in turn, that most of BM-MSC originate from both Pdgfrαand brachyury-positive cells at E7.5, which are mesoderm cells (Bennett et al., 1972;Yanagisawa et al., 1981;Herrmann et al., 1990;Takakura et al., 1997;Imuta et al., 2013;Miwa and Era, 2015). In contrast, some CFU-F colonies derived from adult sWAT did not express the Tomato reporter gene. ...
Mesenchymal stem cells (MSCs) are somatic stem cells that can be derived from adult bone marrow (BM) and white adipose tissue (WAT), and that display multipotency and self-renewal capacity. Although MSCs are essential for tissue formation and have already been used in clinical therapy, the origins and markers of these cells remain unknown. In this study, we first investigated the developmental process of MSCs in mouse embryos using the gene encoding platelet-derived growth factor receptor α (Pdgfra) as a marker. We then traced cells expressing Pdgfra and other genes (brachyury, Sox1 and Pmx1) in various mutant mouse embryos until the adult stage. This tracing of MSC origins and destinies indicates that embryonic MSCs emerge in waves and that almost all adult BM MSCs and WAT MSCs originate from mesoderm and embryonic Pdgfrα-positive cells. Furthermore, we demonstrate that adult Pdgfrα-positive cells are involved in some pathological conditions.
... In 1957, Gluecksohn-Waelsch proposed that t-alleles might alter cell surface specificities , and in 1971 Gluecksohn-Waelsch and Erickson refined the immunological hypothesis for T-region effects to state that cell surface antigens present on sperm and embryos might be controlled by this region. Evidence for T-region determined antigens on sperm has been presented from one laboratory (Bennett et al., 1972; Yanagisawa et al., 1974; Artzt and Bennett, 1977 ) but other laboratories have not been able to confirm these findings . In one extensive report, T-region determined antigens could not be found on spermatozoa by numerous antiglobulin tests (Goodfellow et al., 1979). ...
F9 teratocarcinoma antigen is expressed normally on embryos which are homozygous recessive for each of 4 mutations lethal in early development, including t12. Homozygosity for two post-implantation T-region developmental lethal genes, tw5 and TOr1, may alter F9 antigen expression but the results were not definitive. In addition, the time of appearance of the antigen may be affected by some of the genotypes.
... (17) N. Dobrovolskaïa-Zavadskaïa, Sur la modification spontanée de la queue chez la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire «non viable», Comptes rendus de la Société de biologie, 97 (1927) Pour expliquer le biais de transmission en faveur des mutations t, Bennett, qui était une élève de Dunn, proposa que les produits des gènes du complexe T étaient exprimés à la membrane des spermatozoïdes (28), ce qui fut démontré peu de temps après (29). Le développement de ces recherches bénéficia de la collaboration de Bennett avec Boyse, du Sloan-Kettering Institute for Cancer Research de New York. ...
Mouse genetics made many major contributions to immunogenetics—without doubt mice were the experimental animal of choice because of the ease of breeding and genetic manipulation based on all the earlier work that has been described. The start of immunogenetics was with blood groups, using the very accessible red blood cells, and the results allowed blood transfusions. The fact that the antigens of the major histocompatibility complex of mice, so crucial for understanding tissue transplantation, were expressed on red blood cells, while they are not in humans, allowed great advances, which used recombinant inbred lines. As cell surface antigens were implicated in some aspects of development, especially of the immune system, there were claims for their importance in the T/t complex, which were not substantiated.
The analysis of the antigenic structure of murine spermatozoa has been hampered by the lack of a convenient objective serological assay. Spermatozoa, in common with other cells, are capable of concentrating 86rubidium. Antibody mediated complement dependent cytotoxicity of murine spermatozoa has been quantitated by measuring release of 86rubidium from pre-labeled spermatozoa. We have used this assay to study the murine T/t locus.
This paper summarizes the work done in recent years at the Institut Pasteur in an attempt to use cell lines derived from mouse teratocarcinomas to study the immunological properties of the mouse embryo. The cells used throughout this work were derived from a male tumor, OTT6050. A series of multipotential and nullipotential embryonal carcinoma cell lines was isolated. The antigenic properties of a nullipotential line, F9, and of a multipotential line, PCC4, have been studied with respect to detection, distribution among adult and embryonal cells, and relation to H 2 antigens and to the T/t locus. The F9 antigen disappears early from somatic cells and remains only on the germ line, and can therefore elicit an immunological response in all types of immunization: syngeneic, allogeneic, or xenogeneic.
The histocompatibility antigens have been studied in a number of species. The major histocompatibility antigens represent an intermediary group of cell surface glycoproteins. They are present on almost all cells, like most structural proteins, yet they participate in some highly specific biological reactions. Genetic and biological knowledge is for obvious reasons far greater for the murine system than for any other system. The chapter mainly deals with the data pertinent to the mouse major histocompatibility complex (MHC). The knowledge about various aspects of the major histocompatibility complex has expanded rapidly over the past few years. As yet the function(s) of the gene products controlled by the MHC is largely unknown. A common denominator of in vivo or in vitro reactions involving these glycoproteins is, however, their involvement in recognitive processes. Several structural features of the MHC antigens are similar. Polypeptide chains coded for by the K, D, and TL regions are the glycoproteins of similar size. The association of all these polypeptide chains with β2-microglobulin suggests that at least those parts of the MHC and T antigens that are engaged in binding β2-microglobulin may be highly homologous.
An objective antiglobulin radioimmunoassay for the analysis of anti-spermatozoa antisera has been used to characterize more than 30 alloantisera. Each of these antisera was raised against spermatozoa from mice carrying T/t regin mutations. Whilst all of the antisera had considerable activity for mouse spermatozoa, none of them was found to contain antibodies specific for T/t region gene products. A detailed analysis of a single serum revealed that its activity is for antigens which are non-polymorphic, sperm-specific and species-specific. The failure in this study to detect antibodies specific for T/t region gene products is discussed.
Lymphocytes from tx-mutant mice have a definite and consistent pattern of reactivity in MLR. Cells from t-mutant mice within a complementation group usually fail to stimulate each other, while cells from mutants in different complementation groups stimulate each other strongly. This indicates that tx-mutant types are associated with certain H-2 haplotypes, and that members of any give complementation group share the same H-2 haplotype.
Cell surface antigen NS-6 is identified on MUNTAD (mouse undifferentiated neural tumor, adeno virus induced) cells by complement-dependent cytotoxicity with anti-MUNTAD cell antiserum made in C3H/HeJ × C57BL/6J F1 mice. Quantitative absorption shows that this antigen (or antigens) is completely shared by normal brain, kidney, and spermatozoa. It is absent from liver, spleen, thymus, skin, and muscle. NS-6 is present on brain on embryonic Day 9, the earliest stage tested, and the amount of NS-6 on brain and on kidney does not change from birth to adulthood. Brains of many species, from fish to chicken and human, have antigen(s) which cross-react with NS-6. Within the mouse brain, this antigen shows no localization to any region, nor is it apparently restricted to any one cell type. This is consistent with NS-6 being an antigen of pluripotent neural progenitor cells which is passed to both neuronal and glial progeny. Expressed on two mouse and two human nervous system tumors, NS-6 is absent from all other tumors and cell lines tested.
Publisher Summary This chapter discusses a few aspects of ribonucleic acid (RNA) and protein synthesis at different stages of germ cell differentiation in mammals that are relevant to the problem of regulation of spermatogenesis. Spermatogenesis is a highly orderly process that begins with the stem cell and terminates with the release of the mature spermatid into the lumen of the seminiferous tubule. In the mouse, the total duration of spermatogenesis, from the stem cell to the mature spermatid, is about 34.5 days and is subdivided into three phases: (1) the period of multiplication and maturation of spermatogonia or mitotic phase of spermatogenesis that lasts about eight days, (2) meiosis that lasts 13 days, and (3) spermiogenesis, from the early spermatid to the release of the spermatozoon into the lumen, which is about 13.5 days long. Biochemistry of spermatogenesis is one of the most promising areas of research in developmental biology and mammalian reproduction. The development of germ cell is a field, where different experimental approaches, cytology, ultrastructure, cytogenetics, genetics, biochemistry, immunology, endocrinology, can interact in a coordinated view of cell differentiation. The results described provide clear evidence for gene expression during the haploid phase of spermatogenesis in the mouse; the transcriptional activity during early spermiogenesis involves both polyadenylated RNA and ribosomal RNA.
Concanavalin A (ConA) binding to Xenopus oocytes was studied by fluorescent and electron microscopy. ConA binds to the vitelline layer around the oocytes and to the vitelline envelope of mature eggs. However, when oocytes are removed from the follicles with forceps, a regular hexagonal pattern of fluorescence can be seen: the fluorescence is due to the binding of ConA to the vitelline layer and the network is generated by the remaining follicle cells which allow the lectin to reach the vitelline layer only at their junctions or extremities. ConA binding sites have also been detected on the cortical granules and at the edge or the yolk platelets. On the other hand, the plasma membrane of the oocytes does not show appreciable binding of ConA neither during oogenesis nor after maturation. No information on modifications of the membrane during oogenesis or maturation or on any anisotropy at the surface of the egg could be gathered from the study of ConA binding.
The technique of two-dimensional gel electrophoresis was used to identify a major testicular cell protein, p63/6.9, which is specified by a gene (p63) within the mouse T/t complex on chromosome 17. A wild-type gene causes the expression of one form of this protein, p63/6.9b. All lethal and semilethal t haplotypes derived from wild mice cause the expression of an apparently identical alternate allelic form of the p63/6.9 protein. This protein, p63/6.9a, represents the first t haplotype-specific molecule to be biochemically identified. A dominant haplotype (THp) acts as a null allele of the p63 gene; this unique behavior provides additional evidence for the interpretation of THp as a deletion within the T/t region of chromosome 17. Limited proteolysis of viable testicular cells causes selective cleavage of the p63/6.9 proteins, relative to other detergent-soluble testicular cell proteins known to be internal. This result strongly suggests that p63/6.9 proteins are located on the cell surface. Qualitative and quantitative estimates indicate that p63/6.9 is one of the most prominent proteins on the testicular cell surface. p63/6.9 is expressed in all other mouse cell types analyzed but at greatly reduced levels. Partial t haplotypes obtained from infrequent recombination events were used to map the p63 gene close to the dominant mutation T and separate from the lethal factors of t haplotypes. A 100% correlation was observed between the expression of p63/6.9a and the genetic presence of the tail interaction factor of t haplotypes. The significance of this correlation in terms of the evolution of t haplotypes among wild mice is discussed.
Spermatozoa from fertile mice heterozygous for tw32, a recessive lethal allele of the T/t locus, were compared to normal spermatozoa in a fertilization in vitro system. The rate of egg penetration following insemination in vitro was determined for epididymal spermatozoa from C57BL/6-tw32/+ mice and for epididymal spermatozoa from C57BL/6-+/+ mice. At one hour after insemination, the mean of penetration +/- standard deviation for spermatozoa from BL/6-tw32/+ mice was 20% +/- 2.1 (109 eggs observed, 5 experiments), while the mean for spermatozoa from BL/6-+/+ mice was 1% +/- 1.5 (107 eggs observed, 4 experiments). By five hours post-insemination, the levels of egg penetration were not significantly different. These results suggest that tw32 increases the initial rate of egg penetration. Preliminary observations of sperm motility and sperm-egg association at one hour post-insemination in vitro do not support the hypothesis that this earlier penetration is due to improved sperm progress to the egg. Rather, the earlier penetration may be a result of changes in the timing of capacitation, the acrosome reaction, or sperm-egg fusion. It is possible that the earlier penetration may play a role in the distortion of the transmission ratio of tw32.
The rate of RNA synthesis at different stages of spermatogenesis in the mouse, and the preservation of RNA from the diploid to the haploid phase of spermatogenesis were studied in homogeneous germ cell fractions separated by velocity sedimentation at unit gravity. The uridine pool expansion method was used for determining the rate of RNA synthesis: seminiferous tubules were labelled in culture with increasing concentrations of [3H]-uridine and the incorporated radioactivity was estimated in cell fractions separated by velocity sedimentation. The results indicate that before nuclear elongation, round spermatids (steps 1 to 8 of spermiogenesis) synthesize RNA at the same rate per DNA content as middle-late pachytene spermatocytes. The preservation of RNA molecules synthesized in meiosis was investigated by labelling pachytene spermatocytes with T3H]uridine in vivo and collecting samples of germ cells at definite stages of spermatogenesis at various time intervals thereafter. The results show that a considerable proportion the RNA synthesized during the pachytene stage is preserved through spermatid development until late spermiogenesis.
Pre- and postmeiotic stages of male gametogenesis of 10 different vertebrate species belonging to mammals, birds, amphibians, and fishes were subjected to the Ag-AS staining technique (Goodpasture and Bloom, 1975). A uniform pattern of silver-staining is observable during spermatogenesis of the different vertebrate species. Silver-staining is present in spermatogonia and during the whole period of meiotic prophase, but totally absent during diakinesis and metaphase II of meiosis. In early spermatids silver-staining reappears and only disappears around the beginning of elongation of the spermatid nucleus. Since the Ag-AS technique is believed to stain only transcriptionally active nucleolus organizer regions, our findings indicate that ribosomal RNA genes become reactivated in the haploid spermatid.
The presence of H-2-linked gene products on spermatozoa and their time of appearance during spermatogenesis was determined. Thymus leukaemia antigen specificities 1, 2 and 3 could not be detected on spermatozoa by absorption of the antisera. Immunofluorescent studies with anti-Slp sera did not reveal any specific reactivity with target spermatozoa. In contrast, H-2D antigens were present on somatic as well as germ line components in testes so the time of their first appearance during spermatogenesis could not be precisely specified. Cell separation experiments indicate that H-2D antigens are present on pachytene spermatocytes and increased in quantity on spermatids. The sperm-specific isoenzyme of phosphoglycerate kinase, Pgk-2, appears at a later stage of spermatogenesis than do the H-2 region antigens.
The developmental expression of an early embryonic antigen, common to mouse spermatozoa and cleavage embryos as well as to human spermatozoa, has been studied by immunological techniques, during spermatogenesis of both species. The antigen was detected on all diploid precursors of spermatozoa, including primordial gonocytes. It is suggested that this early embryonic antigen might remain expressed, after egg implantation, on those cells which are to give rise to the male germinal line.
The consideration of a differentiation alloantigen complex related to failures of embryogenesis in mice suggests the possibility of differentiation alloantigens as a component of the causation of birth defects in man. This may be of most relevance in birth defect syndromes inherited in Mendelian fashion, where differentiation antigens might be sought by immunization of animals with tissues obtained at surgery or postmortem. Although it is very difficult to detect such antigens as heteroantigens, there are immunolgical 'tricks,' such as making the recipient animal tolerant to pooled normal human tissue for the organ(s) in question, which might make it possible. Such antigens related to birth defects might be of tremendous importance in prenatal diagnosis. However, since many birth defects, including anencephaly and spina bifida, are multifactorial in origin, such differentiation antigens, if present, might only constitute a predisposing factor. It is suggested that the search for differentiation alloantigens associated with birth defects in man, some perhaps linked to the HL A locus, is worthwhile.
Progress has been made in defining cell surface components specified by
the T/t locus in the mouse, both in their expression on sperm and, in
the one case studied, on normal embryonic cells and teratocarcinoma.
Some striking analogies between this putative embryonic cell recognition
system and the adult major histocompatibility complex are presented.
Mitotic activity in the neural tube was examined in +/+, T + and T T mouse embryos. The number of mitotic figures per unit area of ependymal layer (area index) was higher in T + embryos than in +/+ embryos. In +/+ embryos, area index was higher in the dorsal part of the neural tube than in the ventral part, whereas this pattern is disordered in T + embryos. In T T embryos, these values fluctuate. The process of nuclear migration accompanying cell division seems to be normal in the neural tube of T + and T T embryos.
For several years my colleagues and I have been studying the question of how the constitution of cell surfaces may be determined by selective gene action, according to the various pathways of cellular differentiation, in the same way that the specialized functions of differentiated cells are evidently governed by the selective expression of particular genes (4, 8). This has involved a great deal of work on membrane components that are expressed exclusively on the surface of mouse thymocytes, because these cells are especially favorable for immunogenetic studies. While elucidating the details of thymocyte differentiation, it has always been uppermost in our minds that similar principles of differentiation may be involved in developmental processes, and that valuable inferences regarding embryonic development may transpire from the study of cellular differentiation in adult systems.
Serologically defined HLA-A and HLA-B antigens are expressed in a haploid manner on the surface of spermatozoa. This enabled the HLA-genotypes of four men to be determined by using sperm. This technique will mean that the HLA genotype of potential male graft recipients and others can be determined more easily and even when families are not available. These findings also provide strong evidence that some cell products are encoded by a haploid genome.
As described in Chapter 1 the complex cellular interactions of the immune system have been successfully analysed by employing the combined power of genetics and antibodies. A similar approach has been attempted for studying other complex interactions of cells, for example in the early mouse embryo. The starting point for these studies was the theoretical argument that the regulatory products of the MHC might have evolved from a more general system which regulated cellular interactions in metazoan development. These theories seemed to be supported by the discovery that the T/t locus is linked to H-2 on chromosome 17 of the mouse. Mutants at the T/t locus, isolated from wild populations, display a complex phenotype including segregation distortion in males and homozygous lethality at different times in embryogenesis. In 1967, Robert Erickson and Salome Glucksohn-Waelsch proposed that the pleiotropic effects of the T/t locus could be explained by a series of related genes which controlled development through cell surface molecules which in turn regulated cellular interactions.
Recent advances in genetic analysis of the major histocompatibility systems of various species and the first successful attempts at molecular dissection of their products contrast with the limited knowledge concerning the biological function of this particular part of the eukaryotic genome. The major histocompatibility system of the mouse, the H-2 system, adjoins closely the T/t genetic complex in chromosome 17. The T/t complex occupies a region of the chromosome 20 times longer than that pertaining to the H-2 system, and controls a variety of vital functions necessary for the development of the organism and survival of the species.
Cell surface antigens of normal and anemic ( ) mouse erythroid cells have been examined in cytotoxicity assays with two rat antisera. When tested on fetal liver cells, a rat anti-erythroblast serum recognized antigen(s) present on erythroid cells early in development, while rat anti-adult red blood cell serum recognized antigen(s) present on mature erythroid cells. Each of these sera had different activity on normal (+/+ or ) as compared to anemic ( ) erythroid cells.
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Another minor histocompatibility locus, calledH-33, was found on chromosome 17. This locus was revealed by skin graft experiments between BALB/c and a new congenic strain, BALB.TTF.
Cytotoxic antisera were prepared by immunizing wild-type recipient mice with sperm from donors carrying different recessive lethal alleles at theT locus (T/t
0,T/t
w 1
,T/t
w 5
, andT/t
w 32
). After removal of sperm autoantibody by absorption with sperm of recipient type, each antiserum reacted only with sperm from males whose genotype contained at least one of the immunizing alleles. Cytotoxicity was high against sperm populations in which both immunizing alleles were represented and was lower when only one was present. Thus, each allele at theT locus which has so far been tested serologically is recognizable as a discrete antigen on the surface of sperm.
As a first step in the study of the possible relationship between the T/t and H-2 complexes, the H-2 antigenic composition of the strains carrying factors t¹², t/sup w32/, t/sup w2/, t/sup w8/, t¹, t°, tâ¶, t/sup w1/, t/sup w71/, t/sup w73/, t/sup w12/, t/sup w5/, t/sup w75/, and t³â¸, was studied by using a battery of antisera containing antibodies against inbred-derived H-2 antigens. In addition, five t strains (t¹², tâ¶, t/sup w5/, t/sup w1/, and t/sup w2/) were selected for the production of antisera against the H-2 complexes carried by t chromosomes. Spleen, lymph node, and thymus cells from H-2/sup b//t heterozygotes and t/sup w2//t/sup w2/ homozygotes were injected into appropriate Fâ hybrids between two inbred strains that carried the inbred-derived H-2 antigens of the donor. Four new H-2 antigens and one Ia antigen were uncovered and were assigned the symbols H-2.106 through H-2.109, and Ia.101, respectively. Three new H-2 haplotypes were also described, based upon the H-2 antigenic pattern of three t factors, t¹², t/sup w1/, and t/sup w5/. These new haplotypes were given the symbols H-2/sup t12/, H-2/sup tw1/, and H-2/sup tw5/. When the t factors were grouped according to their H-2 haplotypes, their distribution, with certain exceptions, corresponded to the complementation groups. Thus, t chromosomes in the same complementation group carried similar, if not identical, H-2 haplotypes, despite the fact that these chromosomes were derived from widely separated geographic areas. Such an association between the t and H-2 complexes is most unusual in light of what is known of the polymorphism of H-2 halotypes in wild mice populations. It suggests more than a casual relationship, at least at the population level, between the t and H-2 loci. (auth)
Antisera to H-Y (male-specific) antigen were prepared by immunizing female mice with spleen cells from males of the same inbred
strain. These antisera were used in mixed hemadsorption and cytotoxicity tests with cells of rats, guinea pigs, rabbits, and
humans. The results showed that the H-Y components of all four species are antigenically related to H-Y of the mouse.
Observations on the fine structure of mouse embryo cells during chordamesoderm migration and differentiation support the contention that appropriate cellular interactions are essential to normal development, and, further, that the T locus may control cell surface properties that are critical to morphogenesis. Embryos homozygous for the T gene (Brachyury) and for the t9 gene arrest in development with specific groups of cells affected in each of the lethal phenotypes. In both mutants deranged cellular interactions result in organizational defects of major tissues. In T homozygotes at late 8 day to early 9 day stages (8 to 20 somites) the integrity of axial structures is impaired because neuroepithelial cells form close associations, including specialized contact zones, with neighbouring cells identified as notochord and somites. The basal lamina of the neuroepithelium is deficient where contacts between dissimilar cells are seen. Presumptive mesodermal cells of early 8 day t9 homozygotes neither establish typical cell contacts with one another nor form normal microfilament containing filopodia. These structural defects may indicate paralysis of the cellular motility apparatus. It is postulated that components of the cell surface that serve as devices for recognition or response during cell cell interaction are disturbed by the mutant genes and result in abnormal types of cell associations.
The overall picture of mammalian preimplantation development is one of complex but integrated molecular activity. As development progresses from the 1 cell stage, blastomeres acquire properties that differ from those of cells of previous stages and eventually differ from one another even at the same stage. The evidence concerning the role of the embryonic genome during this process can be summarized under two headings:
(1) Expression of embryonic genome
(a) Quantitative and qualitative changes in RNA and protein synthesis begin to occur soon after fertilization.
(b) Changes in structural proteins (enzyme activities, transport systems and intercellular junctions) occur throughout early development.
(c) Paternal gene products can be detected as early as the 2-cell stage.
(2) The need for expression of the embryonic genome
(a) Inhibitors of RNA and protein synthesis result in abnormal and lethal development.
(b) Several mutations (T/t, Ay, Os, c25H, Om, Ts) are lethal during early development.
(c) Chromosomal imbalance (monsomy, haploidy, nullisomy) has adverse effects on early development.
Even though studies in vitro on the inhibition of translation indicate that stable maternal messenger RNAs are present in the developing mouse embryo, the evidence outlined in this article strongly supports the belief that embryonic gene expression occurs very early in development (at least by the 2 cell stage) and that this expression is required for normal development.
The mixed hemadsorption hybrid antibody (MHA.HA) test was applied successfully to the detection of antigens on the surface of testicular cells separated into sub-populations by velocity sedimentation at unit gravity in the staput apparatus. Normal serum from mice of all strains tested, both male and female, was found to contain a natural autoantibody that reacts with testicular cells of all mice tested, but not with sperm or other cells. This autoantibody is detectable at an age of 4–6 weeks in females, and reaches a plateau at about 10 weeks of age. The corresponding antigen is denoted TCDA, because it is evidently a Testicular Cell Differentiation Antigen. Microscopy of the cells forming rosettes in the MHA.HA test confirmed that the TCDA+ cells belong to the gametogenetic series. Because females as well as males produce the autoantibody we presume that TCDA is also present on female gametic cells although it was not feasible to test this adequately. The anti-TCDA autoantibody is not related to the natural autoantibody against sperm, which according to MHA.HA test occurs in the serum of males but not of virgin females.
Direct cytotoxic tests and absorption studies demonstrated thatI-region associated antigens (Ia) are not restricted to lymphocytes. Ia was found on spermatozoa, macrophages, and on epidermal cells, whereas Ia was absent from brain, liver, kidney, and fibroblasts. The possible biological meaning of these observations is discussed.
ThreeT/t-locus haplotypes (t
9,t
0, andt
w2), which specifiy cross-reacting antigens on sperm, were examined by absorption analysis and found to contain a minimum of six specificities. Some specificities were common to more than one haplotype but each haplotype contained a unique specificity. Exceptional recombinant haplotypes fromt
0 andt
w2 retained some of the common specificities but all had lost the unique specificity of the parental haplotype.T/t genes specifying antigens on sperm are apparently complex, and can be separated by recombination into at least two serologically detectable regions.
The prevalence of neoplasia is increased in individuals with certain disorders of sexual differentiation. Etiology and frequency of neoplasia vary with the particular disorder.In uncomplicated cryptorchidism, the testis is at least 10 times more likely to undergo neoplastic transformation than a normal scrotal testis. Neoplasia probably is a function of both testicular location (intraabdominal) and underlying dysgenetic structure. If cryptorchidism is unilateral, and if orchiopexy has not been performed prior to age 6–10 years, orchiectomy should be encouraged.In those forms of gonadal dysgenesis not associated with a Y chromosome (e.g., 45,X; 45,X/46,XX; 46,XX) there is no definite increase in neoplasia, suggesting that elevated gonadotropin levels per se are not carcinogenic. Gonadal tumors are found in at least 30% of individuals with XY gonadal dysgenesis and are particularly frequent (55%) in H-Y antigen-positive patients. These tumors are almost always gonadoblastomas or dysgerminomas. Similar tumors are found in 15%–20% of 45,X/46,XY individuals. In either situation the neoplastic transformation could be a) secondary to the existence of XY gonadal tissue in an inhospitable environment, or b) integrally related to that process—genetic or cytogenetic—producing the dysgenetic gonads. The risk of neoplasia is sufficiently high that most of these patients should be offered early gonadal extirpation.The prevalence of gonadal tumors is not increased in Klinefelter's syndrome, further indicating that gonadotropins are not carcinogenic per se. However, Klinefelter patients are 20 times more likely to develop a carcinoma of the breast than are 46,XY males. Extragonadal germ cell tumors also are more common.In female pseudohermaphrodites there is probably no increased risk of neoplasia, whereas, in true hermaphrodites neoplasia is unusual but does occur. Neoplasia occurs in patients with complete testicular feminization (complete androgen insensitivity) but rarely in those with incomplete testicular feminization/Reifenstein's syndrome, 5α-reductase deficiency, anorchia, agonadia, or testosterone biosynthetic defects. In complete testicular feminization the risk of malignant tumors is small prior to age 25. After age 25, it is about 2%–5%. Orchiectomy is recommended after pubertal feminization.
Genetic analysis of seven dominant short tailed mutations independently induced by radiation of male mice showed that six were allelic to T (Brachyury) but not identical to it. Homozygotes for each mutant die at least 2 days earlier than T/T homozygotes; two that were studied histologically are indistinguishable from one another. The development of these abnormal embryos is arrested by seven days of gestation, when cells of embryonic ectoderm cease proliferation and become pycnotic. Endoderm and extra-embryonic ectoderm do not seem to be primarily affected, and survive and grow for at least 2 days more. Serological studies of one of these mutations suggest that it is a deletion. A review is presented of these and other T-like mutations that have been described; from this it appears that five different categories of T-like mutants are discernible.
Single cells were prepared from mouse tail epidermis by a method which gives high viability counts and so permits their use in cytotoxicity tests.
According to tests with standard alloantisera, the antigen phenotype of mouse epidermal cells is H-2⁺θ⁺Sk⁺H-Y⁺TL⁻Ly-A⁻Ly-B,C⁻PC⁻.
The skin differentiation alloantigen Sk, which is responsible for homograft reactions directed selectively against skin, is expressed also on brain, but not on other cell types; it is present on the transplanted neuroblastoma C1300. Cytotoxicity tests with epidermal cells of H-2 congenic mouse stocks confirm that the Sk locus is not closely linked to H-2.
The lymphoid cell differentiation antigen θ also is present on both epidermal cells and brain. Mice frequently retain θ-incompatible or Sk-incompatible skin grafts although they have formed substantial titers of θ or Sk antibody in response to grafting.
Male (H-Y) antigen is demonstrable on epidermal cells by cytotoxicity tests with H-Y antibody, as it is also on one other type of cell, spermatozoa.
Variant t-alleles, widespread in natural populations, may control differentiation of ectodermal and neural structures.
The structure and behavior of XY bivalent in mice is discussed. The view that XY bivalent in pachytene is embedded within the sex vesicle was fully confirmed. X and Y are paired end-to-end by a nonchiasmatic connection, which is established already in pachytene and persists until first meiotic metaphase. The pachytene complement in mice consists of 19 rod-shaped autosomal bivalents and the XY bivalent embedded within the sex vesicle. A satisfactory identification of individual autosomes in male pachytene has not been found possible.
Patients with reduced renal function commonly require drug therapy for various associated conditions. Most drugs are fully or partially excreted by the kidney; therefore, drug dosage regimens often need to be adjusted in order to provide safe yet effective treatment for patients with renal disease. In addition, certain therapeutic agents have potential nephrotoxicity and pharmacologic actions that may jeopardize already compromised renal function. Understanding of drug pharmacology, the therapeutic dose and the speed of drug elimination in a given patient will lead to correct assessment of the drug regimen.
The present study is an attempt to isolate by a multiple regression analysis some factors probably influencing the birth weight and to utilize the results of the analysis for statistical prediction of birth weight at different gestational ages for various combinations of factors.
Single cells were prepared from mouse tail epidermis by a method which gives high viability counts and so permits their use in cytotoxicity tests. According to tests with standard alloantisera, the antigen phenotype of mouse epidermal cells is H-2(+)theta(+)Sk(+)H-Y(+)TL(-)Ly-A(-)Ly-B,C(-)PC(-). The skin differentiation alloantigen Sk, which is responsible for homograft reactions directed selectively against skin, is expressed also on brain, but not on other cell types; it is present on the transplanted neuroblastoma C1300. Cytotoxicity tests with epidermal cells of H-2 congenic mouse stocks confirm that the Sk locus is not closely linked to H-2. The lymphoid cell differentiation antigen theta also is present on both epidermal cells and brain. Mice frequently retain theta-incompatible or Sk-incompatible skin grafts although they have formed substantial titers of theta or Sk antibody in response to grafting. Male (H-Y) antigen is demonstrable on epidermal cells by cytotoxicity tests with H-Y antibody, as it is also on one other type of cell, spermatozoa.
Mus poschiavinus x M. musculus hybrids, which had seven metacentric chromosomes derived from the poschiavinus complement, were repeatedly backcrossed to M. musculus and selected for the chromosome carrying the H-2 complex. A line called T1/Klj was established which had one metacentric chromosome. It was shown by linkage tests and by cytogenetic studies that one arm of this metacentric chromosome corresponds to the M. musculus acrocentric chromosome carrying linkage group IX, in which the H-2 complex is found. The distance from the H-2 locus to the centromere was tentatively estimated as 14 map units.
1. Study of the association of the somatic and gametic genotypes with the gametic phenotype has been termed the genetics of gametes. The latter is closely associated with problems of fertility and infertility; with the general validity of the postulate of random union of the gametes; with experimental attempts to control sex ratio or other Mendelian segregations; and with the study of chromosomal anomalies. Effects of the post-segregational genotype (after meiosis) are of exceptional interest.
2. The dimensions of mammalian spermatozoa show numerous patterns of genetic behaviour, such as strain and breed differences, additive variation, heterosis, high heritability, maternal effects, and a tentative ‘paternal cytoplasmic effect’. The high degree of additive genetic determination is reflected in the smooth progress of a genetic selection programme that brought into existence strains of mice with either long or short spermatozoan midpieces. The balance sheet of variation in mammalian spermatozoan dimensions is as follows. Apart from the natural variation between individual spermatozoa of a male, the paramount factors are the genetic ones. Variation is extraordinarily independent of other biological sources of variation and of environmental ones. The high heritability of most spermatozoan dimensions suggests that they are not closely associated with reproductive fitness, whereas a measure of fitness (resistance of spermatozoa to eosin staining) shows a low heritability.
3. Post-segregational gene action has been sought but not conclusively demonstrated in attempts to recognize X- and Y-bearing spermatozoa visually, and in attempts to control sex ratio by separating them according to electrophoretic or sedimentation behaviour.
4. Variations in the DNA content of spermatozoa, and gross morphological defects, are associated with infertility and their incidence is often genetically controlled.
5. There is controversy over the balance between specific spermatozoan antigens and ‘coating antigens’ acquired from the seminal fluid. Reports that AB individuals (heterozygous for blood-group antigens) produce phenotypically A and B spermatozoa would indicate post-segregational gene action but are also controversial. Sex ratio has not yet been controlled by subjecting spermatozoa to anti-Y-chromosome antibodies. Genetic (strain) differences exist in spermatozoan antigens.
6. The incidence of non-eosinophil spermatozoa is a measure of semen fertility. Strain and breed differences exist but the heritability is low. An absence of demonstrable genetic variation in spermatozoan motility may be due to a swamping effect of non-genetic factors.
7. Diploid spermatozoa differ genetically and phenotypically from the normal haploid ones. The incidence of diploid spermatozoa is associated with infertility and is controlled genetically. The incidence of polyspermy and of supplementary spermatozoa are also controlled genetically.
8. Heterospermic (mixed) insemination from two or more sires discriminates efficiently between the sires in terms of the relative numbers of offspring produced. One experiment showed a genetic control (strain differences) in heterospermic performance. Heterospermic performance of a sire is constant over periods of time and is a predictor of the relative fertility obtained in ‘ordinary’ fertility tests. Heterospermic insemination per se has no apparent practical value.
9. Species crosses in animals tend to produce diploid oocytes and triploid embryos. ‘Foreign’ mammalian eggs or spermatozoa did not fertilize in the same proportion as native ones.
10. The most recent determination shows a I : I sex ratio in the very early embryo.
11. Mammals are scarcely likely to be exempt from certain unorthodox genetic mechanisms, but positive evidence is only indicative. Micro-organisms have been detected and sometimes have a genetic role in spermatozoa and eggs of animals. There is a theory that mitochondria (and therefore most of the spermatozoan midpiece) are modified bacteria. A tentative ‘paternal cytoplasmic effect’ on mouse spermatozoan dimensions, mediated by mitochondria1 inheritance, has been invoked. Conditions may exist for possible transfer of genetic information from spermatozoa to body cells via leucocytes. There is no confirmation of experiments for transfer of genetic information by injection of DNA of specific genotypes prepared from spermatozoa or other tissues.
12. The behaviour of ‘tailless’ alleles in mice provides a unique example of post-segregational gene action, the t-spermatozoa of heterozygous Tt males yielding more offspring than the T-spermatozoa. This work explains the aberrant transmission of t through the male parent, and also the high frequency of the allele in wild populations. It is the best example of post-segregational gene action in animals, provides an exception to the random union of gametes, and permits a novel way of controlling the transmission of genetic information between generations. There is presumptive post-segregational gene action in other segregations in mouse and man.
13. The study of heteroploidy (abnormal chromosome numbers) provides massive evidence of anomalies in the mammalian egg that are in themselves genetic (e.g. diploidy) and phenotypic (e.g. abnormal numbers of polar bodies or pronuclei); that come into being under genetic control, and that have genetic consequences (e.g. triploidy) and phenotypic ones (e.g. inviability) for the embryo. This is illustrated by suppression of the cytoplasmic second meiotic division of the egg, giving diploid parthenogenetic embryos from unfertilized eggs and triploid ones from fertilized eggs. Triploidy is the dominant form of polyploidy in vertebrates. Triploidy is lethal in the mammalian embryo and is a main cause of the ‘natural’ level of prenatal mortality in man.
14. The groups of cells produced by meiosis in both sexes of animals are virtually syncytial. This has a bearing on post-segregational heterosis and gene effects.
15. The unique and apparently indisputable instance of post-segregational gene action in ‘tailless’ mice is to be contrasted on the one hand with numerous persuasive arguments (especially effective in Drosophila) that such action cannot or does not occur in animals or else is mimicked by another phenomenon, meiotic drive; and on the other hand contrasts with an hypothesis that post-segregational gene action occurs on a gigantic scale in all organisms in which crossing-over takes place. It is concluded for the time being that post-segregational gene action does occur in mammals, but is rare. Because of the rarity, a reasonable first interpretation in all gamete genetics is that gene action is mediated by the somatic genotype, unless there are specific reasons to the contrary. There is urgent need for a technique whereby the biological potential of a given spermatozoon can be recognized by fertilizing a given egg with it.
16. As a series of working hypotheses, it is proposed that the general architecture and course of differentiation of gametes are determined by the genotype of the soma and the same pre-segregational genotype of the germ line, the initial gene action being a synthesis of messenger RNA before meiosis in the germ line or at any time in the soma. These effects of the somatic genotype bring the gamete into existence and are also responsible for most of the widespread variation in its phenotype. The gamete so produced can be varied by a very few genes or sets of genes synthesizing messenger RNA in the germ cells after segregation, or else exerting action in some other way connected with their direct physical and chemical nature or their arrangement. These post segregational effects are more likely to be mediated by whole blocs of genes, especially heterochromatic and nucleolar ones, than by ‘billiard-ball’ single genes in the narrow sense.
1.1. Ribonucleic acid and protein synthesis in germ cells and Sertoli cells of the mouse testis was studied using tritium autoradiography with 3H-uridine and 3H-amino acids, and cytochemical methods.2.2. Spermatogonia.—The rate of RNA and protein syntheses is much higher in the immature type A spermatogonia than in the mature type B spermatogonia. This difference in the synthetic rates is possibly related to a difference in the degree of DNA condensation between the two types of cells. Nuclear and cytoplasmic protein synthesis occurs at all stages of the division cycle, whereas chromosomal RNA synthesis stops during metaphase and anaphase.3.3. Meiosis.—In the autosomes, RNA synthesis ceases or falls to an insignificant level during two periods of the meiotic cycle, early prophase (leptotene to early pachytene) and late diakinesis to metaphase and anaphase I and II. Between these two minima it undergoes a stepwise increase during middle prophase to a peak in middle pachytene, followed by a drop during late pachytene to early diakinesis. In contrast to the autosomes, the heteropycnotic XY bivalent is invariably unlabeled with 3H-uridine throughout meiotic prophase. Protein synthesis, on the contrary, continues during the periods of arrest or depression of RNA synthesis, and is also present in the sex chromosomes.4.4. Spermiogenesis.—RNA synthesis stops very soon after second meiotic division, in very early spermiogenesis. The ribonucleic acids synthesized during meiosis and early spermiogenesis are completely lost from the nucleus both by breakdown and by transfer to the cytoplasm during the course of spermiogenesis, so that middle and late spermatids (from stage 9 to stage 15) do not show any detectable amount of RNA within the nucleus, whereas the cytoplasm exhibits fair amounts of RNA that had been synthesized at least a week earlier during meiotic prophase. The protein synthesis seen to occur in the cytoplasm of spermatid stages 9 to 15 is, then, probably sustained by “templates” produced during meiosis and conserved in the cell to become available during spermiogenesis.5.5. Histone transition during spermiogenesis.—Protein labeling over the nucleus is either absent or very low in spermatids, except in late, elongated, spermatids (stages 11 to 14) which show a marked nuclear labeling as early as 15 min after injection of 3H-arginine. With other amino acids, the nuclear labeling in elongated spermatids is either low or absent. The nuclear incorporation of 3H-arginine is accompanied by changes in the staining properties of nuclear histone (persistance of alkaline fast green stainability after acetylation) which indicate that elongated spermatids synthesize a new nuclear histone very rich in arginine that replaces the previous histone which is a typical histone rich in lysine. A number of evidences suggest that this “arginine-rich” spermatid histone is effectively synthesized within the nucleus, although the alternative interpretation of a cytoplasmic synthesis followed by transfer of the newly synthesized histone into the nucleus cannot be actually rejected.6.6. Sertoli cells.—Nucleoplasm and nucleolus of the supporting Sertoli cells incorporate very rapidly 3H-uridine and 3H-amino acids. The satellite karyosomes (or heteropycnotic bodies) flanking the nucleolus are, however, unlabeled with 3H-uridine and scarcely labeled with 3H-amino acids.7.7. The evidence that the heteropycnotic sex chromosomes during male meiosis and the heteropycnotic karyosomes in Sertoli cells do not incorporate RNA precursors is interpreted to indicate that only dispersed chromatin is active in ribonucleic acid synthesis.
HISTOCOMPATIBILITY antigens of the type H-2 are found on most cells of the mouse, but there are conflicting reports concerning their expression on spermatozoa. Using the indirect immunofluorescence technique, Barth and Russell1 found no H-2 antigen on spermatozoa. Using the techniques of immunogenicity, antibody absorption, and indirect immunofluorescence, Vojtiskova et al.2 concluded that H-2 antigens were probably detectable on spermatozoa by all three procedures. As these authors point out, however, it is difficult to exclude entirely the possibility that contaminating cells were responsible for the positive findings with the first two techniques.
The genetics of the mammalian gameteSynthetic activities during spermato-genesis in the mouse
- R A Beatty
- V Monesi
Beatty, R. A. (1970) "The genetics of the mammalian gamete," Biol. Rev. 45, 73-119. 11.' Monesi, V. (1965) "Synthetic activities during spermato-genesis in the mouse," Exp. Cell Res. 39, 197-224.