Detection of genomic instability in offspring of male mice chronically exposed to gamma-radiation by the "adaptive response" test

The genomic instability (GI) in somatic cells of the progeny (F1 generation) of male mice chronically exposed to low-dose gamma-radiation was studied by comparative analysis of chromosome damage. BALB/C male mice exposed to 0.1 Gy (0.01 Gy/day) and 0.5 Gy (0.01 and 0.05 Gy/day) were mated with unirradiated females 15 days after irradiation. For comparison of radiosensitivity, two-month-old males, the descendants of irradiated and unirradiated animals, were subjected to irradiation with a dose of 1.5 Gy (0.47 Gy/min) from a 60Co source. GI was revealed by the standard scheme of adaptive response. The experiments indicated that, by using the test "adaptive response", it is possible to detect the transition of gamma-radiation-induced genomic instability in sex cells of male parent into somatic cells of mice (F1 generation) either from changes in radiosensitivity or by the absence of the adaptive response induced by a standard scheme.     

Sex determination in the germ line of Drosophila depends on genetic signals and inductive somatic factors

We have analyzed the mechanism of sex determination in the germ line of Drosophila by manipulating three parameters: (1) the ratio of X-chromosomes to sets of autosomes (X:A); (2) the state of activity of the gene Sex-lethal (Sxl), and (3) the sex of the gonadal soma. To this end, animals with a ratio of 2X:2A and 2X:3A were sexually transformed into pseudomales by mutations at the sex-determining genes Sxl (Sex-lethal), tra (transformer), tra-2 (transformer-2), or dsx (double-sex). Animals with the karyotype 2X;3A were also transformed into pseudofemales by the constitutive mutation SxlM1. The sexual phenotype of the gonads and of the germ cells was assessed by phase-contrast microscopy. Confirming the conclusions of Steinmann-Zwicky et al. (Cell 57, 157, 1989), we found that all three parameters affect sex determination in germ cells. In contrast to the soma in which sex determination is completely cell-autonomous, sex determination in the germ line has a non-autonomous component inasmuch as the sex of the soma can influence the sexual pathway of the germ cells. Somatic induction has a clear effect on 2X;2A germ cells that carry a Sxl+ allele. These cells, which form eggs in an ovary, can enter spermatogenesis in testes. Mutations that cause partial loss of function or gain of function of Sxl thwart somatic induction and, independently of the sex of the soma, dictate spermatogenesis or oogenesis, respectively. Somatic induction has a much weaker effect on 2X;3A germ cells. This ratio is essentially a male signal for germ cells which consistently enter spermatogenesis in testes, even when they carry SxlM1. In a female soma, however, SxlM1 enables the 2X;3A germ cells to form almost normal eggs. Our results show that sex determination in the germ line is more complex than in the soma. They provide further evidence that the state of Sxl, the key gene for sex determination and dosage compensation in the soma, also determines the sex of the germ cells, and that, in the germ line, the state of activity of Sxl is regulated not only by the X:A ratio, but also by somatic inductive stimuli.     

Hormonal control of "tissue" transglutaminase induction during programmed cell death in frog liver

In this study, we show that sex hormones (testosterone, estradiol, and progesterone) act as physiological modulators of programmed cell death (PCD) during the frog liver involution observed postvitellogenesis. PCD in parenchymal cells is paralleled by the specific induction of the "tissue" transglutaminase (tTG) gene. tTG protein specifically accumulates in hepatocytes showing the morphological features of apoptosis. The hormone-dependent increase of both PCD and tTG was reproduced in ovariectomized frogs. Treatment of castrated animals with testosterone, estradiol, and progesterone inhibited the induction of both tTG and PCD, thus indicating that in vivo the drop in the circulating sex hormone is the signal favoring the involution phase of the maternal frog liver after mating. Although an affinity-purified polyclonal antibody raised against mammalian transglutaminase reacts in frog liver with a 55- to 60-kDa protein, concomitant with the onset of PCD, tTG cleavage products were detected, suggesting a proteolytic processing of the enzyme protein. These results represent the first evidence indicating that the physiological involution occurring postvitellogenesis of frog liver takes place by programmed cell death and that this, together with the concomitant induction of tTG gene expression, is regulated by sex hormones.     

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka,Oryzias latipes

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.     

Aminergic regulation of pheromone sensillae in the cockroach <Emphasis Type="Italic">Periplaneta americana</Emphasis>

Tremendous adaptability of insects is predominantly provided by fast tuning of physiological functioning of an organism to the permanently changing environmental conditions. One of the mechanisms of plasticity in insects is modulation of performance of their sense organs by neurohormones. Activity of at least three out of four receptor cells located in cockroach pheromonesensitive sensilla is modulated by octopamine. An increase of firing rate of pheromone receptor cells and a decrease of electroantennogram amplitude is accompanied by enhanced behavioral responses of male cockroaches to sex pheromone. The effect of octopamine on reception of a repellent (1,8-cineole, eucalyptol) by an insect is reported for the first time. Simultaneous modulation of responses of receptor cells located in sex specific sensilla to semantically different odorants indicates their cooperation in formation of insect behavior.     

Preliminary Characterization of "Sex Ratio" and Rediscovery and Reinterpretation of "Male Sex Ratio" in DROSOPHILA AFFINIS

In D. affinis "sex ratio" (sr), a form of meiotic drive characterized by the production of mostly or only female progeny by certain males, is associated with two different X chromosome sequences, XS-I XL-II and XS-II XL-IV. The behavior of the two sequences differed, depending on the Y chromosome constitution, being either Y(L) or 0. Males with sequence XS-II XL-IV and Y(L) produced progenies with nearly normal sex ratios; males with the same X chromosome sequence but in the absence of a Y chromosome in some cases gave progenies with nearly normal sex ratios but in other cases gave progenies which tended toward phenotypic sr. Males with sequence XS-I XL-II and Y(L) gave progenies which were characteristically sr (0.97-0.98 females); in the absence of a Y chromosome males with this sequence produced progenies which were virtually all-male. This latter finding is presumably identical to Novitski's (1947) "male sex ratio" (msr). The interpretation offered here attributes msr to an interaction between sr sequence XS-I XL-II and the 0 condition. A general consideration of the available data on sr in D. affinis is presented.     

High temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by increasing cortisol levels during gonadal sex differentiation in Japanese flounder

The Japanese flounder (Paralichthys olivaceus) is a teleost fish with an XX/XY sex determination system. XX flounder can be induced to develop into phenotypic females or males, by rearing them at 18°C or 27°C, respectively, during the sex differentiation period. Therefore, the flounder provides an excellent model to study the molecular mechanisms underlying temperature-dependent sex determination. We previously showed that cortisol, the major glucocorticoid produced by the interrenal cells in teleosts, causes female-to-male sex reversal by directly suppressing mRNA expression of ovary-type aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens in the gonads. Furthermore, an inhibitor of cortisol synthesis prevented masculinization of XX flounder at 27°C, suggesting that masculinization by high temperature is due to the suppression of cyp19a1 mRNA expression by elevated cortisol levels during gonadal sex differentiation in the flounder. In the present study, we found that exposure to high temperature during gonadal sex differentiation upregulates the mRNA expression of retinoid-degrading enzyme (cyp26b1) concomitantly with masculinization of XX gonads and delays meiotic initiation of germ cells. We also found that cortisol induces cyp26b1 mRNA expression and suppresses specific meiotic marker synaptonemal complex protein 3 (sycp3) mRNA expression in gonads during the sexual differentiation. In conclusion, these results suggest that exposure to high temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by elevating cortisol levels during gonadal sex differentiation in Japanese flounder.     

Using upper limits of "bateman gradients" to estimate the opportunity for sexual selection

The widespread use of molecular markers to estimate parentagemakes possible a new index of the opportunity for sexual selection.After demonstrating the need for a new measure, I develop onebased on the upper limit on sexual selection. I describe whatsets the upper limit for each sex by showing how maximum fecundityincreases with number of mates, accounting for the amount ofenergy (or critical resources) available for reproduction andlevels of parental care. For females the upper limit on sexualselection is set by the value of paternal investment that comeswith each mating. For males, the upper limit on sexual selectionis set by the fecundity of their mates (including any boostto female fecundity from paternal investment). Sex-roles aremost likely to reverse (making males choosy and females competitive)when the amount of reproductive energy investment made by eachsex is low, irrespective of the level of paternal investment.Finally, I propose that we use the difference between male andfemale upper limits on sexual selection to quantify sex differencesin the opportunity for sexual selection. Using upper limitsto estimate the opportunity for sexual selection is more intuitivethan older methods (e.g., standardized variance in mating success),it is experimentally measurable, and it is valuable in understandingthe evolution of mating systems.     

Molecular analysis shows differential expression of <Emphasis Type="Italic">R</Emphasis>-<Emphasis Type="Italic">spondin1</Emphasis> in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) gonads

R-spondin1 (RSPO1) is a potential female-determining gene in human (Homo sapiens) and mouse (Mus musculus). Its differential expression in these mammals is correlated with signaling for sex determination. As a way of studying sex determination in fish we cloned and analyzed a RSPO1 gene in zebrafish (Danio rerio). Using real-time PCR, we observed that RSPO1 is expressed more strongly in ovaries than in testes, suggesting that RSPO1 may have a role in gonad differentiation. High RSPO1 expression was detected in some non-gonadal organs like muscle and kidneys. In situ hybridization results demonstrate that RSPO1 is expressed in premature germ cells, in oogonia and primary oocytes in ovaries and in spermatogonia and spermatocytes in testes. It is also expressed in gonad somatic cells during gonadal development: in granulosa cells and theca cells of early and late cortical-alveolar stage follicles in ovaries, and in Leydig cells in testes. This differential expression may indicate that RSPO1 has a role(s) in zebrafish gonad development and differentiation. By fusing zebrafish RSPO1 with a green fluorescent protein gene, we found that RSPO1 is located in the cytosol and Golgi apparatus but not the nucleus of fish epithelioma papulosum cyprinid (EPC) cells. These preliminary findings suggest some aspects of RSPO1 like differential expression linked to sex determination may be conserved in fish while other aspects like subcellular localization differ from the mammalian RSPO1.     

Sex steroid-mediated reprogramming of vascular smooth muscle cells to stem cells and neurons: Possible utilization of sex steroid combinations for regenerative treatment without utilization of in vitro developed stem cells

Previous work from our laboratory demonstrated that sex steroid combinations, but not individual sex steroids alone, cause transdifferentiation of ovarian epithelial cells - ovarian surface epithelium (OSE) and follicular granulosa cells - into neural stem cells (NSC) and differentiating neurons. In the present study we have chosen primary culture of human vascular smooth muscle cells (SMC), a non-epithelial mesenchymal cells in order to test them as a control cell type regarding their morphology and expression of NSC and neuronal markers. Utilization of estradiol (E2), progesterone (PG) or testosterone (TS) alone did not induce the emergence of neurons from the vascular SMC. However, the treatment with sex steroid combinations (PG+TS or E2+PG+TS) caused transdifferentiation into neural/neuronal type cells. By immunohistochemistry, these cells exhibited strong expression of stem cell markers and neural/neuronal glycoconjugates SSEA-1, SSEA-4, Thy-1, NeuN and NCAM. In the Neurobasal/B27 medium both, the OSE and vascular SMC also transdifferentiated into neuronal cells. Western blot analysis has shown significant increase of NeuN 48-kDa species after E2+PG or PG+TS treatment. Secretion of E2 increased significantly in vascular SMC cultures pretreated with TS, PG or TS+PG. Unlike OSE cells, the vascular SMC accompany as pericytes all vessels, including CNS microvasculature. We also observed that sex steroid combinations could produce SMC stem type cells which differentiated within a few days back to mature vascular SMC. This is of potential interest for the vascular regenerative medicine. Altogether, our observations suggest that sex steroid combinations could induce in vivo improvement of neurodegenerative, traumatic and ischemic neurological disorders and vascular diseases via their effect on resident pluripotent vascular SMC, i.e. without a need of in vitro developed stem cells.     

A mammalian somatic "cell cycle" mutant defective in G1

Variants or “mutants” temperature-sensitive (ts) for growth have been isolated by selection from a near-diploid mouse cell line. Thus far. 10 ts mutants which grow normally at 33° C, but not at 39° C, have been isolated. These ts mutants were then studied to determine if any manifested their defect at a unique point or stage in the cell cycle. This type of ts mutant is termed a “cell cycle” mutant. The first screen involves observing individual cells of an asynchronous culture for residual division after a shift from 33° C (permissive temperature) to 39° (nonpermissive temperature). A cell cycle mutant should show some fraction of the cells dividing only once at a normal rate after the shift. The ts variant B54 met this first criterion for a cell cycle mutant (i.e., 50% residual division) and was further analyzed. The second screening technique monitors (1) the rate of entry into S, (2) the length of G₂, and (3) the rate and duration of cells entering mitosis after a shift of an asynchronous culture to 39°. This experiment with B54 revealed that cells in G₁ at the time of the shift to 39° failed to enter S while cells already into S completed the cycle at 39°. These results suggest that B54 is defective in a G₁ function which is required for entry into S, but which is no longer needed once cells have entered S. Other results are presented which also support this hypothesis. In addition the ts function of B54 is apparently required for recovery from a “high density” G₁ arrest.     

Analysis of the Multiple Roles of Gld-1 in Germline Development: Interactions with the Sex Determination Cascade and the Glp-1 Signaling Pathway

The Caenorhabditis elegans gene gld-1 is essential for oocyte development; in gld-1 (null) hermaphrodites, a tumor forms where oogenesis would normally occur. We use genetic epistasis analysis to demonstrate that tumor formation is dependent on the sexual fate of the germline. When the germline sex determination pathway is set in the female mode (terminal fem/fog genes inactive), gld-1 (null) germ cells exit meiotic prophase and proliferate to form a tumor, but when the pathway is set in the male mode, they develop into sperm. We conclude that the gld-1 (null) phenotype is cell-type specific and that gld-1 (+) acts at the end of the cascade to direct oogenesis. We also use cell ablation and epistasis analysis to examine the dependence of tumor formation on the glp-1 signaling pathway. Although glp-1 activity promotes tumor growth, it is not essential for tumor formation by gld-1 (null) germ cells. These data also reveal that gld-1 (+) plays a nonessential (and sex nonspecific) role in regulating germ cell proliferation before their entry into meiosis. Thus gld-1 (+) may negatively regulate proliferation at two distinct points in germ cell development: before entry into meiotic prophase in both sexes (nonessential premeiotic gld-1 function) and during meiotic prophase when the sex determination pathway is set in the female mode (essential meiotic gld-1 function).     

Cell-cell interactions and the control of sex determination in hydra

Sexual phenotype in gonochoristic species of hydra (species having separate males and females) is ultimately determined by cells committed to the sperm pathway. Little is known about the genetic basis for determining sex during embryogenesis, but the sex ratio of offspring is generally 1 : 1, implying a genetic component. Sexual phenotype of adult hydra is labile and sex-reversal in both directions can be induced by experimental manipulations involving addition or loss of the sperm lineage. Loss of the sperm lineage is induced by high temperature, causing males to switch to females. Introduction of the sperm lineage into females by grafting causes sex-reversal to male. These studies provide support for a model of sex determination based on cell-cell interactions.     

Sex-linked AFLP markers indicate a pseudoautosomal region in hemp (<Emphasis Type="Italic">Cannabis sativa</Emphasis> L.)

In dioecious plants of hemp ( Cannabis sativa L.), males are regarded as heterogametic XY and females as homogametic XX, although it is difficult to discriminate the X cytologically from the Y. The Y chromosome is somewhat larger than the X. Our aim was to analyse AFLP markers on X and Y, and to use them to gain some insight into the structure of the sex chromosomes. Markers located on the sex chromosomes can be grouped into different classes, depending on the presence or absence of a fragment on the X and/or the Y. They are detected by separately analysing male and female progenies of a single cross. Five markers were found to be located on both chromosomes. A few recombinants were observed for marker pairs of this class in the male progenies. Two completely linked markers located on the Y chromosome in the male parent show a recombination rate of r = 0.25 with sex. Recombination must have occurred between the sex chromosomes in the male parent. The recombination analysis led to the conclusion that there is a pseudoautosomal region (PAR) on the sex chromosomes, allowing recombination between the X and the Y chromosome. The other regions of the sex chromosomes show only a few recombination events, for the Y as well as for the X. These results are discussed in comparison to other dioecious plants.     

Steroid-mediated differentiation of neural/neuronal cells from epithelial ovarian precursors in vitro

We reported earlier that occasional neurons evolve in human cultures of pluripotent ovarian epithelial stem cells. In subsequent experiments, frequent transdifferentiation into neural stem cells (NSC) and differentiating neurons was observed in human ovarian epithelial stem cells and porcine granulosa cells after exposure to certain combinations of sex steroids. Testosterone (TS), progesterone (PG) or estradiol (E2) alone do not increase the emergence of neurons. However, a mixture of TS+PG after E2 pretreatment converted a majority of ovarian epithelial stem cells or porcine granulosa cells into NSC and differentiating neuronal cells within one to three hours. Cultured neurons manifested an interconnectivity resembling primitive neuronal pathways in culture. These converted cells expressed the cell markers SSEA-1, SSEA-4, NCAM, and Thy-1 glycoconjugates of NSC and neurons, and differentiating cells showed characteristic neuronal morphology. Emergence of NSC and neuronal cells was associated with significant cellular depletion of L-glutamic acid (glutamate), which serves as the major excitatory neurotransmitter in the vertebrate CNS and its fast removal is essential for preventing glutamate excitotoxicity. These observations suggest that certain sequential systemic treatment with common sex steroids and their mixture might be effective in the treatment or prevention of degenerative CNS disorders. The ovarian stem cell cultures readily obtainable from human ovaries regardless of the woman's age have the potential to produce NSC for autologous regenerative treatment of neurologic diseases in aging women. Finally, the proper combination of sex steroids could possibly be employed for transdifferentiation of adult bone marrow stem cells or mobilized peripheral blood cells into autologous NSC and stimulate their neuronal differentiation after homing in the CNS.     

A sex cord‐like structure and some remarkable features in early gonadal sex differentiation in the marine teleost Siganus guttatus(Bloch)

Several notable features of early gonadal sex differentiation in the golden rabbitfish Siganus guttatus are described including the first report among teleosts of a distinctive dual structure, consisting of somatic cells directly enclosing germ cells (sex cord‐like structure, SCS) and outer somatic tissue surrounding the SCS, in both undifferentiated and early differentiated gonads. Germ cells occurred and proliferated exclusively in the SCS during the process of ovarian and testicular differentiation. A second remarkable characteristic was the delayed germinal cell proliferation for oogenesis in the ovary, that commenced simultaneously with that in the testis, a relatively long time after the onset of somatic development. These observations suggest the possibility that sex differentiation of germ cells is preceded by some sex specific changes in somatic components of the SCS that are light‐microscopically indistinguishable between the sexes. The third unique feature was the detachment of gonadal tissue, including both somatic and germ cells, into the ovarian cavity in the ovary and into the seminiferous lobules and main seminal duct in the testis. This phenomenon occurred in the testis, forming the efferent duct network after 73 days post‐hatch (DPH), and in the ovaries, forming the ovigerous lamellae and regulating the number of oocytes attaining full maturation at c . 129 DPH.     

Frequency analysis of the antibody specificity repertoire of mitogen-reactive B cells and "spontaneously" occurring "background" plaque-forming cells in nude mice

The antibody specificity repertoire of lipopolysaccharide (LPS)-reactive B cells has been determined in the spleens and bone marrow (BM) of C57BL/Ka athymic nude mice using a limiting dilution culture system that allows the growth and development of every LPS-reactive B cell into a clone of IgM-secreting cells. In addition, the numbers of "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells as well as the "background" IgM antibody specificity repertoire has been assessed in spleens and BM. The frequencies of antigen-specific LPS-reactive B cells of C57BL/Ka nude and thymus-bearing mice showed a great similarity and ranged from 1 in 1000 to 1 in 2500 for sheep red blood cells (SRBC), horse red blood cells (HRBC), and goat red blood cells (GRBC), from 1 in 10 to 1 in 25 for 5-iodo-3-nitrophenyl-coupled (SRBC), from 1 in 15 to 1 in 150 for 4-hydroxy-3,5-dinitrophenyl-coupled SRBC, and from 1 in 70 to 1 in 140 for 2,4,6-trinitrophenyl-coupled SRBC. The specificity repertoire of the "background" IgM-secreting cells differed from that of age-matched thymus-bearing controls and was different in young and old C57BL/Ka nude mice. Within the limitations of having assessed only a minor fraction of the total B-cell antibody specificity repertoire and supposing that nude mice are largely devoid of functional T cells, the data presented suggest that the generation of the specificity repertoire of newly-formed B cells is hardly or not affected by T cells. On the other hand, T cells do affect the expression of the established repertoire, represented by "background" immunoglobulin-secreting cells.