Somatic damage to the X chromosome of the nematode Caenorhabditis elegans induced by gamma radiation

Summary Wild-type male embryos and young larvae of the nematode Caenorhabditis elegans were more sensitive than wild-type hermaphrodites to inactivation by gamma rays; wild-type males have one X chromosome per cell (XO), whereas wild-type hermaphrodites have two (XX). Furthermore, after transformation into fertile hermaphrodites by a her-1 mutation, XO animals were more radiosensitive than XX her-1 animals; and XX animals transformed into fertile males by a tra-1 mutation did not show increased radiosensitivity. It is concluded that wild-type males are more radiosensitive than wild-type hermaphrodites because they have one X chromosome rather than two, and the predominant mode of inactivation of XO animals involves damage to the single X chromosome. No sex-specific differences in survival were observed after UV irradiation.     

Activity of the Sex-Determining Gene tra-2 Is Modulated to Allow Spermatogenesis in the C. ELEGANS Hermaphrodite

In the nematode C. elegans, there are two sexes, the self-fertilizing hermaphrodite (XX) and the male (XO). The hermaphrodite is essentially a female that makes sperm for a brief period before oogenesis. Sex determination in C. elegans is controlled by a pathway of autosomal regulatory genes, the state of which is determined by the X:A ratio. One of these genes, tra-2, is required for hermaphrodite development, but not for male development, because null mutations in tra-2 masculinize XX animals but have no effect on XO males. Dominant, gain-of-function tra-2 mutations have now been isolated that completely feminize the germline of XX animals so that they make only oocytes and no sperm and, thus, are female. Most of the tra-2(dom) mutations do not correspondingly feminize XO animals, so they do not appear to interfere with control by her-1, a gene thought to negatively regulate tra-2 in XO animals. Thus, these mutations appear to cause gain of tra-2 function in the XX animal only. Dosage studies indicate that 5 of 7 tra-2(dom) alleles are hypomorphic, so they do not simply elevate XX tra-2 activity overall. These properties suggest that in the wild type, tra-2 activity is under two types of control: (1) in males, it is inactivated by her-1 to allow male development to occur, and (2) in hermaphrodites, tra-2 is active but transiently inactivated by another, unknown, regulator to allow hermaphrodite spermatogenesis; this mode of regulation is hindered by the tra-2(dom) mutations, thereby resulting in XX females.     

A sex-determining gene,fem-1, required for both male and hermaphrodite development in Caenorhabditis elegans

Sex in the nematode Caenorhabditis elegans is normally determined by the X chromosome to autosome (X:A) ratio, with XX hermaphrodites and XO males. Previous work has shown that a set of at least four autosomal genes (her-1, tra-2, tra-3, and tra-1) is signaled by the X:A ratio and appears to act in a regulatory pathway to determine sex. Twenty-one new recessive alleles of the gene fem-1(IV) (formerly isx-1) have been isolated. Seven of these may be null alleles; one of these is an amber mutation. The other 14 alleles are temperature sensitive. The putative null mutations cause both XO and XX animals to develop as females when the mother as well as the zygote is fem-1(?). Therefore, fem-1(+) is required (a) for the development of the male body and (b) for spermatogenesis in males and hermaphrodites. In addition, fem-1 shows a maternal effect: wild-type fem-1 product partially rescues the development of fem-1(?) progeny. By analyzing double mutants it has been shown that fem-1(+) is part of the sex-determination pathway and has two distinct functions: (1) in the soma it prevents the action of tra-1, thereby allowing male development to occur, and (2) in the germline it is necessary for spermatogenesis in both sexes.     

Extragenic suppressors of a dominant masculinizing her-1 mutation in C. elegans identify two new genes that affect sex determination in different ways

Summary: The her-1 regulatory switch gene in C. elegans sex determination is normally active in XO animals, resulting in male development, and inactive in XX animals, allowing hermaphrodite development. The her-1(n695gf) mutation results in the incomplete transformation of XX animals into phenotypic males. We describe four extragenic mutations that suppress the masculinized phenotype of her-1(n695gf) XX. They define two previously undescribed genes, sup-26 and sup-27. All four mutations exhibit semidominance of suppression and by themselves have no visible effects on sex determination in otherwise genotypically wild-type XX or XO animals. Analysis of interactions with mutations in the major sex-determining genes show that sup-26 and sup-27 influence sex determination in fundamentally different ways. sup-26 appears to act independently of her-1 to negatively modulate synthesis or function of tra-2 in both XX and XO animals. sup-27 may play a role in X-chromosome dosage compensation and influence sex determination indirectly.     

Sex-Related Differences in Crossing over in Caenorhabditis Elegans

In the nematode Caenorhabditis elegans, hermaphrodite recombination has been characterized and is the basis of the genetic map used in this organism. In this study we have examined male recombination on linkage group I and have found it to be approximately one-third less than that observed in the hermaphrodite. This decrease was interval-dependent and nonuniform. We observed less recombination in the male in 5 out of 6 intervals examined, and no observable difference in one interval on the right end of LG I. Hermaphrodite recombination frequencies are the result of recombination in two germlines; oocyte and hermaphrodite spermatocytes. We have measured recombination in the oocyte and have found it to be approximately twofold lower than that calculated for hermaphrodite spermatocytes and not significantly different from the male spermatocyte frequency. Thus, recombination frequencies appear to be a function of gonad physiology rather than the sex of the germline. Evidence from experiments examining the effect of karyotype on recombination in males sexually transformed by the her-1 mutation into XO hermaphrodites (normally XX), suggests the sexual phenotype rather than genotype determines the recombination frequency characteristic of a particular sex. Hermaphrodite recombination is known to be affected by temperature, maternal age, and the rec-1 mutation. We have examined the effect of these parameters on recombination in the male and have found male recombination frequency increased with elevated temperatures and in the presence of Rec-1, and decreased with paternal age.     

A Novel Dominant Transformer Allele of the Sex-Determining Gene Her-1 of Caenorhabditis Elegans

We have characterized a novel dominant allele of the sex-determining gene her-1 of Caenorhabditis elegans. This allele, called n695, results in the incomplete transformation of XX animals into phenotypic males. Previously characterized recessive her-1 alleles transform XO animals into phenotypic hermaphrodites. We have identified five new recessive her-1 mutations as intragenic suppressors of n695. Three of these suppressors are weak, temperature-sensitive alleles. We show that the recessive her-1 mutations are loss-of-function alleles, and that the her-1(n695) mutation results in a gain-of-function at the her-1 locus. The existence of dominant and recessive alleles that cause opposite phenotypic transformations demonstrates that the her-1 gene acts to control sexual identity in C. elegans.     

Univalent sex chromosomes in spermatocytes of Sxr-carrying mice

Pachytene configurations of the sex chromosomes were studied in whole-mount, silver-stained preparations of spermatocytes in mice with XY,Sxr, XX,Sxr, XO,Sxr, XO,Sxr+5¹² and T(X;4)37H,YSxr chromosomes, and non-Sxr-carrying controls. XY,Sxr males showed an increased number of X and Y univalents and of self-synapsed Y chromosomes. In T(X;4)37H,YSxr males an increased proportion of trivalent+Y configurations was also accompanied by higher numbers of self-paired Y univalents; the proportion of trivalent+X⁴ was not increased, but that of self-synapsed X⁴ univalents was. There was more selfsynapsis in cells containing one univalent than in cells containing two univalents. Spermatocytes of XX,Sxr mice contained single univalent X, which was never seen to be self-synapsed, but self-synapsis of the X occurred in a proportion of cells in XO,Sxr males. There were no self-paired X chromosomes in the XO,Sxr+5¹² mouse although lowlevel pairing of the 5¹² chromosome occurred. All four XX,Sxr and XO,Sxr males contained testicular sperm, and testicular sperm were also present in one T(X;4)37H male, while another such male had sperm in the caput. It is concluded that (1) self-synapsis of univalents is affected by variable conditions in the cell as well as by the DNA sequences of the chromosome, and (2) that the level of achievable spermatogenesis is not always rigidly predetermined by a chromosome anomaly but can be modulated by the genetic background.     

Analysis of the Role of Tra-1 in Germline Sex Determination in the Nematode Caenorhabditis Elegans

In wild-type Caenorhabditis elegans there are two sexes, self-fertilizing hermaphrodites (XX) and males (XO). To investigate the role of tra-1 in controlling sex determination in germline tissue, we have examined germline phenotypes of nine tra-1 loss-of-function (lf) mutations. Previous work has shown that tra-1 is needed for female somatic development as the nongonadal soma of tra-1(lf) XX mutants is masculinized. In contrast, the germline of tra-1(lf) XX and XO animals is often feminized; a brief period of spermatogenesis is followed by oogenesis, rather than the continuous spermatogenesis observed in wild-type males. In addition, abnormal gonadal (germ line and somatic gonad) phenotypes are observed which may reflect defects in development or function of somatic gonad regulatory cells. Analysis of germline feminization and abnormal gonadal phenotypes of the various mutations alone or in trans to a deficiency reveals that they cannot be ordered in an allelic series and they do not converge to a single phenotypic endpoint. These observations lead to the suggestion that tra-1 may produce multiple products and/or is autoregulated. One interpretation of the germline feminization is that tra-1(+) is necessary for continued specification of spermatogenesis in males. We also report the isolation and characterization of tra-1 gain-of-function (gf) mutations with novel phenotypes. These include temperature sensitive, recessive germline feminization, and partial somatic loss-of-function phenotypes.     

tra-2 encodes a membrane protein and may mediate cell communication in the Caenorhabditis elegans sex determination pathway.

The Caenorhabditis elegans sex-determining gene, tra-2, promotes female development in XX animals. In this paper we report the cDNA sequence corresponding to a 4.7 kb tra-2 mRNA and show that it is composed of 23 exons, is trans-spliced to SL2, and contains a perfect direct repeat in the 3' untranslated region. This mRNA is predicted to encode a 1475 amino acid protein, named pTra2A, that has a secretory signal and several potential membrane-spanning domains. The molecular analysis of tra-2 loss-of-function mutations supports our open reading frame identification and suggests that the carboxy-terminal domain is important for tra-2 activity. We propose that in XX animals the carboxy-terminal domain of pTra2A negatively regulates the downstream male promoting fem genes. In XO animals, tra-2 is negatively regulated by her-1, which acts cell nonautonomously. Because hydropathy predictions suggest that pTra2A is an integral membrane protein, pTra2A might act as a receptor for the her-1 protein. We propose that in XO animals, the her-1 protein promotes male development by binding and inactivating pTra2A. The role of cell communication in C. elegans sex determination might be to ensure unified sexual development throughout the animal. If so, then regulation of sexual fate by her-1 and tra-2 might provide a general model for the coordination of groups of cells to follow a single cell fate.     

More Sex-Determination Mutants of CAENORHABDITIS ELEGANS

Sex determination in Caenorhabditis elegans is controlled by the X chromosome: autosome ratio, i.e. 2A;XX animals are hermaphrodite, and 2A;XO animals are male. A procedure for isolating 2A;XO animals that are transformed into hermaphrodites has been developed. Nine mutations causing this transformation have been obtained: eight are recessive, and all of these fall into a new autosomal complementation group, her-1 V. The remaining mutation (her-2) is dominant and has a genetic map location similar to that of tra-1 III. Recessive mutations of tra-1 cause the reverse transformation, transforming 2A;XX animals into males. Therefore, the her-2 mutation may result in constitutive expression of tra-1. Mutations in her-1 are without effect on XX animals, but the her-2 mutation prevents sperm production in both XX and XO animals, in addition to its effect on the sexual phenotype of XO animals. The epistatic relationships between tra and her genes are used to deduce a model for the action of these genes in controlling sex determination.     

The C. elegans sex determination gene laf-1 encodes a putative DEAD-box RNA helicase

The Caenorhabditis elegans gene laf-1 is critical for both embryonic development and sex determination. Laf-1 is thought to promote male cell fates by negatively regulating expression of tra-2 in both hermaphrodites and males. We cloned laf-1 and established that it encodes a putative DEAD-box RNA helicase related to Saccharomyces cerevisiae Ded1p and Drosophila Vasa. Three sequenced laf-1 mutations are missense alleles affecting a small region of the protein in or near helicase motif III. We demonstrate that the phenotypes resulting from laf-1 mutations are due to loss or reduction of laf-1 function, and that both laf-1 and a related helicase vbh-1 function in germline sex determination. Laf-1 mRNA is expressed in both males and hermaphrodites and in both the germline and soma of hermaphrodites. It is expressed at all developmental stages and is most abundant in embryos. LAF-1 is predominantly, if not exclusively, cytoplasmic and colocalizes with PGL-1 in P granules of germline precursor cells. Previous results suggest that laf-1 functions to negatively regulate expression of the sex determination protein TRA-2, and we find that the abundance of TRA-2 is modestly elevated in laf-1/+ females. We discuss potential functions of LAF-1 as a helicase and its roles in sex determination.     

Effects of caffeine on chromosomal loss and nondisjunction in Drosophila melanogaster

Three types of male larvae, normal X males and two types with structurally abnormal X chromosomes (ring X and short X sc ⁴ sc ³, y) were treated during the third instar with 0.5 per cent caffeine in nutrient medium. Upon eclosion, these males were mated to yellow and Oregon-R wild type females. The F₁ generation of each cross was scored for normal (XX and XY) and abnormal (XO and XXY) progeny. Statistical analyses of data demonstrate that caffeine increases chromosomal loss for all genotypes tested. The effect of caffeine on nondisjunction, however, is not clear. There are at least marginal increases in all cases when Oregon-R females are used. Slight increase and decreases noted for offspring of yellow females appear to be dependent upon the genotype of the inseminating male.     

An unusual chromosome system in a leafhopper (Homoptera auchenorrhyncha)

An unidentified species of the genus Austroagalloides is shown to have 7_II+1_IV instead of the normal 11_II+XO configuration at metaphase I of meiosis in males. The quadrivalent manifests two types of pairing which ensures regular disjunction; normal chiasma-type pairing and distance pairing. It is suggested that the 7_II+1_IV form is derived from the 11_II+XO form by a series of fusions.     

Proteins of the Drosophila melanogaster male reproductive system: Two-dimensional gel patterns of proteins synthesized in the XO,XY, and XYY testis and paragonial gland and evidence that the Y chromosome does not code for structural sperm proteins

Testes and paragonial glands of Drosophila melanogaster wild-type males were labeled in vitro using [³⁵S]methionine, and the proteins synthesized were analyzed by 2-dimensional gel electrophoresis. Testes and paragonial glands were also labeled in vivo by feeding male larvae ³⁵S-labeled yeast and then dissecting the adult males. Approximately 1200 proteins were resolved by autoradiography of the gels. The in vitro method was shown to be more sensitive and to allow faithful synthesis of all proteins produced in vivo. [³H]Proline was also used to label testes, and no significant differences from the ³⁵S pattern were noted. Testes and paragonial glands from XO and XYY males were labeled in vitro with [³⁵S]methionine, and the proteins synthesized were compared to those produced by wild-type males of identical autosomal background. No differences attributable to the Y chromosome could be detected in the testes or paragonial gland samples. Pure sperm were dissected manually from in vivo labeled males and the proteins analyzed. Ninety-two proteins were detected, which were all synthesized in comparable amounts by XO, XY, and XYY males, showing that the Y chromosome does not code for any of these structural sperm proteins. It is postulated that no Y chromosome products were detected because they are organizational or regulatory proteins present only in very small amounts in the adult testes. ³⁵S-labeled males were also mated to unlabeled females and the transferred proteins analyzed on two-dimensional PAGE. The contributions of the testis and paragonial gland to the ejaculate were determined.This work was supported by a grant from the Medical Research Council of Canada and by the U.S. National Institutes of Health (Grant No. GM-22753). J. I. B. is the recipient of a Medical Research Council of Canada studentship.     

Characterization of the female-sterile mutant Almondex of Drosophila melanogaster

Drosophila melanogaster females homozygous for the sex-linked recessive mutation almondex (amx) are completely sterile when mated to amx males. Matings of amx X+ yield low numbers of heterozygous female offspring, which frequently show abnormalities of the thorax and abdominal sternites, and an occasional non-disjunctional, non-mutant (XO) male offspring. The results of mating experiments reported here can be explained by assuming that the cytoplasm of eggs produced by amx females is deficient for some material that is necessary for normal development.Homozygous amx females have apparently normal reporductive organs and a high egg yield. Eggs are usually fertilized. In matings to amx males, all zygotes die as embryos; in matings to non-amx males, all ordinary (XY) male zygotes and most female zygotes die as embryos. Survival to the adult stage is more frequent at higher temperatures and, surprisingly, increases also with maternal age.