Normal XX Females Research Articles

Gene Expression and Epigenetic Modification of Aromatase during Sex Reversal and Gonadal Development in Blotched Snakehead (Channa maculata)

The cyp19a1 gene codes aromatase that converts androgen to estrogen, which plays a central role in early female differentiation and ovarian development in teleosts. For the blotched snakehead (Channa maculata), an important aquaculture fish that is susceptible to hormone-induced sex reversal, two aromatase genes were characterized in the present study, cyp19a1a and cyp19a1b. We analyzed gene expression and the epigenetic state of cyp19a1a and cyp19a1b in different adult tissues: the gonad and brain from normal XX females (XX-F), normal XY males (XY-M), sex-reversal females (XY-F) induced by estrogen, and YY super-males (YY-M), and gonads at different development stages. Cyp19a1a exhibited strong female-biased expression patterns in the ovary, and cyp19a1b dominantly expressed in the brain with no sex bias. Cyp19a1a’s expression pattern in the XY-F ovary was similar to that in the XX-F ovary, with a relatively high expression level, which was far higher than that in XY-M and YY-M testis. Meanwhile, CpG methylation levels of cyp19a1a promoter were lower in XX-F and XY-F ovaries compared with XY-M and YY-M testis. A significantly negative correlation between the CpG methylation levels and cyp19a1a expression was elucidated in XX-F, XY-M, XY-F, and YY-M gonads. Furthermore, the strong female-biased cyp19a1a expression was closely related to ovarian differentiation and maturation, and the overall methylation levels of cyp19a1a promoter were inversely correlated with cyp19a1a expression. There were no detectable sexually dimorphic differences in cyp19a1b expression and CpG methylation levels of cyp19a1b promoter in the brain and gonad between sexes in C. maculata, thus the function of cyp19a1b in C. maculata needs further research. Our research illustrates that cyp19a1a is closely related to estrogen production, ovary differentiation/maintenance, and sex reversal, and epigenetic modification plays a crucial part in maintaining the sexual dimorphic expression of cyp19a1a, ovarian differentiation and oogenesis in C. maculata.

Characterization, expression and CpG methylation analysis of Dmrt1 and its response to steroid hormone in blotched snakehead (Channa maculata).

Dmrt1 is an important transcriptional regulator that plays critical role in male gonadogenesis, testicular differentiation and development. In this study, Dmrt1 was cloned from blotched snakehead (Channa maculata), which is designated as CmDmrt1. CmDmrt1 encoded a putative protein with 293 amino acids and presented an extremely conserved DM domain. It was nearly expressed in the gonads, and the expression was more than 15 times higher in the testis than in the ovary. 1851bp promoter sequence of CmDmrt1 was characterized and the methylation levels of the CpG sites were analyzed to detect sex-related differences. A significant negative correlation between CmDmrt1 expression and CpG methylation level of its promoter was found in the testis and ovary. During gonadal development, CmDmrt1 transcription displayed strong male-biased expression patterns, increased with the maturation of testis and reached the peak at 195days after hatching (dah), which indicates a significant role of Dmrt1 in spermatogenesis. Steroid treatment could influence CmDmrt1 expression, and long-term 17β-estradiol (E2) treatment could induce the male-to-female secondary sex reversal (SSR), which resulted in the differentiated testis transformed to ovary or ovotestis. Meanwhile, CmDmrt1 expression was down-regulated to fairly low level in the ovary of the SSR XY fish, which was similar to that in normal XX females ovary. Our research illustrates that Dmrt1 is linked to testis differentiation and spermatogenesis in blotched snakehead, providing information for functional studies on sex differentiation and gonadal development of C. maculata, and scientific basis for the production practice of all-male snakehead breeding.

Comparative transcriptome analysis on four types of gonadal tissues of blotched snakehead (Channa maculata)

Blotched snakehead (Channa maculata) is an economically important freshwater fish in China, of which males grow much faster than females. To illuminate the molecular mechanism of sex differentiation and gonad development, RNA-Sequencing was performed to identify sex-related genes and pathway in gonads of 6-month-old normal XX females (XX-F), normal XY males (XY-M), XY sex reversal females (XY-F) and YY super-males (YY-M). The analysis showed that many differentially expressed genes (DEGs) had similar expression patterns in XY-F and XX-F, which were different from XY-M and YY-M. qRT-PCR indicated that Amh, Dmrt1, and Sox9 had relatively high expression in testes of XY-M and YY-M. Taking Amh as an example, there was a relative fold change of 1.0 in XX-F, 2.1 fold change in XY-F, 36.1 fold change in XY-M, and 26.0 fold change in YY-M. Cyp19a1a, Figla, and Foxl2 were highly expressive in ovaries of XX-F and XY-F. Taking Figla as an example, there was a relative fold change of 557 in XX-F, 304.5 fold change in XY-F, 5.6 fold change in XY-M, and 4.4 fold change in YY-M. KEGG analysis revealed many DEGs distributed in pathways related to sex differentiation, steroid hormone synthesis and growth, etc. Significant variation and trends in relative expression levels tested by qRT-PCR were consistent with those recorded by RNA-Sequencing. This is the first time that transcriptome of snakehead has been investigated systematically and in an integrated way. Large quantities of candidate genes involved in sex differentiation, gonad development and growth dimorphism were identified. The study provides useful resources for understanding sex differentiation and growth dimorphism, potentially assisting mono-sex production of snakehead in aquaculture.

Expression profile and estrogenic regulation of Amh during gonadal sex differentiation in northern snakehead (Channa argus).

Anti-Müllerian hormone (Amh) plays a critical role in both early sex determination and later gonad development in vertebrate species. However, it remains unknown in northern snakehead (Channa argus), which is economically important freshwater fish with sexual dimorphism. This study aimed to identify the expression profiles and estrogenic regulation of CaAmh during gonadal sex differentiation in C. argus. The cDNA and genomic DNA sequences of CaAmh were identified by PCR and RACE techniques. The expression patterns of CaAmh were detected by qRT-PCR during the gonadal sex differentiation and after 17α-ethinyloestradiol (EE2) treatments. CaAmh is composed of seven exons and six introns, and its full-length cDNA is 2413 bp in length, with 1635 bp open reading frame (ORF) that encodes a 544 amino acid protein. Tissues expression patterns revealed that CaAmh display the highest expression in testis of XY males (40.36 folds, p < 0.01). The spatio-temporal expression patterns during gonadal sex differentiation indicated that CaAmh expression differed between XX females and XY males at 30 day after hatching (dah), and reached to the peak (36.03 folds, p < 0.01) at 90 dah in XY gonads. However, CaAmh expression in XX gonads remained low throughout the sampling period. Furthermore, CaAmh expression in the gonads (ovaries) of the sex-reversed XY fish (XY-F) by the administration of estrogen EE2 was downregulated to low level, similar to that in ovaries of normal XX females (XX-F). These results show that Amh plays a critical role in testicular differentiation of C. argus and it is apparently modulated by estrogens in this species.

Effects of Gender and Sex Hormones on Disease Susceptibility of Channel Catfish to Edwardsiella ictaluri

The use of monosex populations for aquaculture is becoming widely used for several species. The current studies determined if there were any differences between male and female channel catfish, Ictalurus punctatus, in disease susceptibility to Edwardsiella ictaluri, one of the most important pathogenic bacteria in catfish culture. Disease challenge experiments were carried out on fingerling channel catfish fed 17β‐estradiol or testosterone before the challenge, and on all male and on sibling all female fingerlings. All male populations were produced by mating YY males with normal XX females. Sibling females were produced by hormonally sex reversing a subpopulation of six of the all male families. Weight gain or specific growth rate did not differ in fish fed testosterone or estrogen. Fish fed the highest dose of estrogen (50 mg/kg) had a significant higher mortality (P &lt; 0.05), while mortality was similar in catfish fed 10 and 50 mg/kg of testosterone compared to controls. There were no differences in mortality between sibling males and females. These data indicate no increased disease susceptibility to E. ictaluri between males and females or due to exogenous sex hormones. Production of all male catfish for culture can proceed without concern for disease susceptibility to E. ictaluri.

Commentary: Unravelling the effects of additional sex chromosomes on cognition and communication – reflections on Lee et al. (2012)

Commentary: Unravelling the effects of additional sex chromosomes on cognition and communication – reflections on Lee et al. (2012)

Up‐regulation of SOX9 in human sex‐determining region on the Y chromosome (SRY)‐negative XX males

In mammals, gonadal sex is normally determined by the presence or absence of the Y chromosome gene SRY. After expression of SRY in the sexually indifferent gonad, a number of genes encoding transcription factors and growth factors implicated in testis differentiation start to show male-specific expression. However, in XX males, these genes must be up-regulated in the absence of SRY, but the aetiology of SRY-negative XX maleness remains unclear. We examined the expression of representative gonad marker genes in SRY-negative XX male testes. RT-PCR and immunohistochemical studies revealed that SOX9, DAX-1, Ad4BP/SF-1, WT-1, GATA-4 and MIS were expressed in testicular tissues of SRY-negative XX males. Expression levels of SOX9 in testes of these patients averaged 1.9-fold higher than in normal XY testes, while expression levels of Ad4BP/SF-1, DAX-1 and MIS were lower in the SRY-negative XX testes than in XY testes. All XX patients were found to carry two copies of the SOX9 gene per diploid genome as do normal XX females and XY males. The XX male patients also carried two copies of the DAX-1 gene as do normal XX females, while normal XY males carry a single DAX-1 gene. Our data suggest that lesions affecting SOX9 expression are the key factor in sex determination in SRY-negative XX males, and that the decreased expression of Ad4BP/SF-1, DAX-1 and MIS contribute to their clinical features.

The mechanism of secondary nondisjunction in Drosophila melanogaster females.

Bridges (1916) observed that X chromosome nondisjunction was much more frequent in XXY females than it was in genetically normal XX females. In addition, virtually all cases of X nondisjunction in XXY females were due to XX <--> Y segregational events in oocytes in which the two X chromosomes had failed to undergo crossing over. He referred to these XX <--> Y segregation events as "secondary nondisjunction." Cooper (1948) proposed that secondary nondisjunction results from the formation of an X-Y-X trivalent, such that the Y chromosome directs the segregation of two achiasmate X chromosomes to opposite poles on the first meiotic spindle. Using in situ hybridization to X and YL chromosomal satellite sequences, we demonstrate that XX <--> Y segregations are indeed presaged by physical associations of the X and Y chromosomal heterochromatin. The physical colocalization of the three sex chromosomes is observed in virtually all oocytes in early prophase and maintained at high frequency until midprophase in all genotypes examined. Although these XXY associations are usually dissolved by late prophase in oocytes that undergo X chromosomal crossing over, they are maintained throughout prophase in oocytes with nonexchange X chromosomes. The persistence of such XXY associations in the absence of exchange presumably facilitates the segregation of the two X chromosomes and the Y chromosome to opposite poles on the developing meiotic spindle. Moreover, the observation that XXY pairings are dissolved at the end of pachytene in oocytes that do undergo X chromosomal crossing over demonstrates that exchanges can alter heterochromatic (and thus presumably centromeric) associations during meiotic prophase.

Sex, Not Genotype, Determines Recombination Levels in Mice

Recombination, the precise physical breakage and rejoining of DNA between homologous chromosomes, plays a central role in mediating the orderly segregation of meiotic chromosomes in most eukaryotes. Despite its importance, the factors that control the number and placement of recombination events within a cell remain poorly defined. The rate of recombination exhibits remarkable species specificity, and, within a species, recombination is affected by the physical size of the chromosome, chromosomal location, proximity to other recombination events (i.e., chiasma interference), and, intriguingly, the sex of the transmitting parent. To distinguish between simple genetic and nongenetic explanations of sex-specific recombination differences in mammals, we compared recombination in meiocytes from XY sex-reversed and XO females with that in meiocytes from XX female and XY male mice. The rate and pattern of recombination in XY and XO oocytes were virtually identical to those in normal XX females, indicating that sex, not genotype, is the primary determinant of meiotic recombination patterns in mammals.

Permanent and functional male-to-female sex reversal in d-rR strain medaka (Oryzias latipes) following egg microinjection of o,p'-DDT.

Complete sex reversal of fish is accomplished routinely in aquaculture practices by exposing fish to exogenous sex steroids during gonadal differentiation. A variety of environmental chemicals are also active at sex steroid receptors and theoretically possess the potential to alter normal sexual differentiation in fish. However, in controlled environmental chemical exposures to date, only partial alterations of fish sexual phenotype have been observed. Here we report complete, permanent, and functional male-to-female sex reversal in the Japanese medaka (Oryzias latipes, d-rR strain) after a onetime embryonic exposure to the xenoestrogen o, p'-DDT. d-rR strain medaka are strict gonochorists that possesses both sex-linked pigmentation, which distinguishes genotypic sex, and sexually dimorphic external secondary sexual characteristics, which distinguish phenotypic sex. We directly microinjected the xenoestrogen o, p'-DDT into the egg yolks of medaka at fertilization to parallel the maternal transfer of lipophilic contaminants to the embryo. At 10 weeks of age, microinjected medaka were examined for mortality and sex reversal. A calculated embryonic dose of 511 +/- 22 ng/egg o, p'-DDT (mean +/- standard error) resulted in 50% mortality. An embryonic exposure of 227 +/- 22 ng/egg o, p'-DDT resulted in 86% (6 of 7) sex reversal of genetic males to a female phenotype (XY females). XY females were distinguished by sex-linked male pigmentation accompanying female secondary sexual characteristics. Histologic examination of the gonads confirmed active ovaries in 100% of the XY females. In 10-day breeding trials in which XY females were paired with normal XY males, 50% of the XY females produced fertilized embryos; this represents a comparable breeding success rate to normal XX females. Fertilized eggs produced from XY females hatched to viable larvae. These results clearly indicate that a weakly estrogenic pesticide, o, p'-DDT, when presented during the critical period of gonadal development, can profoundly alter sexual differentiation.

Lampbrush chromosomes and chiasmata of sex-reversed crested newts.

Triturus cristatus carnifex provides a particularly clear example of sexual dimorphism for chiasma frequency and localisation. Oocytes from normal XX females routinely carry one proximal chiasma on each arm of their lampbrush bivalents. Spermatocytes from normal XY males have more numerous and relatively distal chiasmata. Lampbrush chromosomes from the oocytes of sex-reversed XY neofemales are found to resemble those from normal oocytes in having one proximal chiasma on each bivalent arm. A comparison of particular markers on the heteromorphic long arm of chromosome 1 provides evidence to equate the lampbrush 1A to somatic 1A, and confirms previous reports that lampbrush chromosome 1A is slightly longer than 1B. The XY sex bivalent of neofemales does not show any obvious heteromorphy of recognised marker loops.

Growth performance trials of genetically male tilapia (GMT) derived from YY-males in Oreochromis niloticus L.: On station comparisons with mixed sex and sex reversed male populations

Genetic manipulation of predominantly monofactorial sex determining mechanisms in the Nile tilapia Oreochromis niloticus is proposed as a technology for the mass production of all-male progeny for culture. Existing alternative methods for producing all-male tilapia, principally direct hormonal masculinization, have produced inconsistent results and are largely the preserve of scientists and developed country aquaculturists. Successful adoption of these techniques in developing countries is seriously limited by the number and complexity of factors involved in their effectiveness. Novel YY-male genotypes have been shown to be viable and to produce all- or nearly all-male (XY) progeny in crosses with normal XX females. This paper presents the results from a series of on-station growth performance trials of these genetically male tilapia (GMT) in three environments. Two comprehensive and replicated trials in extensively managed earthen ponds, compared the performance of GMT with sex reversed male (SRT) and normal mixed sex tilapia (MST). Three further, smaller experiments, compared the growth of GMT with that of MST populations in rice-cum-fish culture and under intensive culture in closed recirculating systems. These comparisons were made within the ‘Egypt-UCS’ strain of O. niloticus. Results indicate that culture of GMT populations can improve marketable yields under all environments. In the first experiment in ponds, GMT had significantly ( P = 0.003) higher yields than both MST (+ 58.8%) and SRT (+ 31.03%). The second experiment, following the same design, showed a similar trend, although differences were smaller and not significant. GMT produced yields 11.9% greater than MST in rice-cum-fish culture and 33.6–47.0% greater in recirculating systems. There was no recruitment in GMT pond populations whereas recruits were present in MST and SRT populations. Coefficient of variance for individual weight in ponds was significantly ( P < 0.05) lower for GMT than for MST for both pond experiments. GMT produced from novel YY-males in this strain has considerable potential for increasing yields from tilapia culture.

Fragile-X mutation and Klinefelter syndrome: a reappraisal.

To date the concurrent presence of the fragile-X and the Klinefelter syndromes in the same individual has been found at least 8 times either in the course of screening for the fra(X) condition in mentally retarded males or among the relatives of fra(X) propositi. Given the high frequency of both events in the general population and the heterogeneous approaches with which the above cases were ascertained, it has not been possible to determine unequivocally so far whether the finding is purely coincidental or the expression of some underlying biological relationship. To evaluate the issue, we have screened a large population of institutionalized mentally retarded males for microorchidism, and submitted to a full karyotype analysis and fra(X) testing the patients that were found to have marked bilateral microorchidism. Thus, in a total of 32 microorchidism patients identified among 1115 mentally retarded males, we found 6 to have a 47,XXY chromosome complement in all (or in most) of their cells, with one of them having also the fra(X) marker in 9% of the metaphases examined. In addition, another bearer of the fra(X) marker (but only in 4% of his metaphases) was found among 26 47,XXY mentally normal males ascertained throughout routine cytogenetic analysis of males with microorchidism referred to our genetic counseling unit during the last 10 years. In our laboratory the fra(X) marker has never been observed with such a frequency in a total of several hundred normal XY males and XX females studied as control cases in the course of previously reported family and population studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Müllerian inhibiting substance in sex-reversed dogs.

In normal males, Müllerian Inhibiting Substance (MIS), produced by testes during an embryonic critical period, is thought to induce regression of the Müllerian duct system, including the oviducts and uterus. In XX sex-reversed dogs, an apparent contradiction has been reported: The uterus persists in the presence of testes or ovotestes. The objective of this study is to determine whether testes of XX male and ovotestes of true hermaphrodite dogs produce MIS, and to examine the anatomy of Müllerian duct derivatives of affected dogs for evidence of regression. Gonadal samples were tested for MIS activity in a bioassay. The mean MIS activity score of XX males was similar to that of normal XY males and significantly greater than that of normal XX females. The mean MIS activity score of XX true hermaphrodites was intermediate between normal XX females and XY males. Within the true hermaphrodite group, ovotestes in which the proportion of testicular tissue was greater than or equal to 1/2 had higher MIS scores than those in which the proportion of testicular tissue was less than 1/2. XX males had a well-developed epididymis adjacent to each testis, but no oviducts. In true hermaphrodites, the oviduct regressed and an epididymis was present when greater than or equal to 1/2 of the adjacent ovotestis was testicular, and MIS activity in that gonad was high. A few ovotestes with intermediate levels of MIS activity had both an oviduct and an epididymis. Regression of the oviductal portion of the Müllerian duct system was positively correlated to the amount of testicular tissue and the MIS activity of the gonad, as would be predicted by Jost's original hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)

X-linked genes of the H-Y antigen system in the wood lemming (Myopus schisticolor).

H-Y antigen was investigated in 18 specimens representing six different sex chromosome constitutions of the wood lemming (Myopus schisticolor). The control range of H-Y antigen was defined by the sex difference between normal XX females (H-Y negative per definitionem) and normal XY males (H-Y positive, full titer). H-Y antigen titers of the X*Y and X*0 females were in the male control range, while in the X*X and X0 females the titers were intermediary. Data were obtained with two different H-Y antigen assays: the Raji cell cytotoxicity test and the peroxidase-antiperoxidase (PAP) method. Fibroblasts, gonadal cells, and spleen cells were checked. Presence of full titers of H-Y antigen in the absence of testis differentiation is readily explained by the assumption of a deficiency of the gonad-specific receptor of H-Y antigen. Since sex reversal is inherited as an X-linked trait, genes for this receptor are most likely X-linked. The implications of our findings are discussed in connection with earlier findings concerning H-Y antigen in XY gonadal dysgenesis in man and the X0 situation in man and mouse.

The CBA/N Mouse Strain: An Experimental Model Illustration the Influence of the X-Chromosome on Immunity

Publisher Summary Studies of the mouse strain have identified the gene, or closely linked group of genes, responsible for the immune defect, and it has been named “xid.” This chapter summarizes the known functional immune defects associated with xid and discusses the cellular basis for these defects. The X-chromosomes of mice and men influence the immunological function of these species in a number of interesting ways. In studies of the influence of the murine X-chromosome on serum IgM levels, it was shown that the mean values of serum IgM were higher in female mice than in males of two different mouse strains. However, in contrast to humans with Turner's syndrome (XO), who had lower mean serum IgM values than did normal XX females, the mean serum concentrations of IgM in XO mice were similar to that found in normal female mice. An X-linked form of severe combined immunodeficiency disease is described in the chapter. Children with this disease have variable degrees of lymphopenia, with small foci of lymphocytes in their lymph nodes and spleens. It is unclear if these patients have a primary B- or T-lymphocyte or stem cell defect. Spleens from either CBA/N or defective F1 male mice are considerably smaller than those of normal mice and the number of nucleated cells/spleen reflects this difference. The absence of thymus-independent type 2 (TI-2) responses in immune-defective mice and the apparent B-cell abnormality in this strain is related to an isolated dysfunction in otherwise normal B cells of these mice. It has been detected in studies that the xid defect could impair thymic-dependent (TD) responses by the induction of hapten-specific suppression without altering the development of memory B cells.

Nonexchange alignment: a meiotic process revealed by a synthetic meiotic mutant of Drosophila melanogaster.

mei-S51 is a meiotic mutant of Drosophila melanogaster having the following properties: 1. mei-S51 consists of two recessive genes, located near the centromere regions of the second and third chromosomes, which are both required for the expression of the mutant phenotype. Salivary analysis reveals normal structures for both the second and third chromosomes. 2. mei-S51 has no effect in males but in females it causes a uniform decrease in X chromosome exchange, second chromosome exchange between c and bw, and increased nondisjunction of all chromosomes. Both of these effects appear to result from a defect prior to both exchange and disjunction, rather than one being the cause of the other. 3. Nondisjunction in mei-S51 females having a normal sex chromosome complement is the result of nonhomologous pairing as is the greatly increased nondisjunction found for XXY; mei-S51 females. This nondisjunction is not competitive with exchange; that is, exchange occurs as frequently between the X chromosomes of XXY females as those of XX females in spite of a greatly increased frequency of X chromosome nondisjunction in the former. 4. There is a pronounced increase in the frequency of nondisjunction in both XX and XXY; mei-S51 females if the X chromosomes are heterozygous for the inversion, In(1)dl49, although heterozygosity for In(1)dl49 causes-only a slight increase in nondisjunction in mei-S51+ (normal) XX females. 5. It appears unlikely that, in all instances of nonhomologous pairing, both chromosomes of each bivalent pair nonhomologously. Rather, in many instances one chromosome pairs with a nonhomolog and the other two elements are distributed at random and without loss to the poles. 6. In XX; mei-S51 females, nonhomologous pairing of the fourth chromosomes occurs exclusively with the X chromosomes, implying that the specificity of nonhomologous pairing (perhaps the size recognition mechanism demonstrated by Grell) is not affected by mei-S51.

Effects of 17α-hydroxyprogesterone and androstenedione upon sex differentiation in the medaka, Oryzias latipes

Using the genetically analyzed d- rR strain of the medaka ( Oryzias latipes), an oviparous cyprinodont fish, in which white (X r X r ) fish are normally females and orange-red (X r Y R ) are males, effects of 17 α-hydroxyprogesterone and androstenedione upon gonadal sex differentiation were studied. Daily oral administration of these key steroids in biosynthesis of sex hormones was made from the hatching time (≅4.8 mm) to the 12-mm stage or longer. Thereafter they were reared on a normal diet. Sex distribution was scored when they reached adulthood. The highest dose-level of 17 α-hydroxyprogesterone employed thus far does not affect sex differentiation. Androstenedione acts as a male-inductor(androtermone), i.e., inducing reversal in sex differentiation of XX zygotes. Potency of this steroid as an androtermone is extremely weak as compared with that of methyltestosterone. The range of adequate dose levels to produce functional XX males is limited because higher dose levels yield neuter-like males with extremely atrophied testis. Eleven androstenedione-induced XX males in single crosses with normal XX females sired all-female progenies, totaling 395 fish.

SEX DETERMINATION IN TIPULIDAE (NEMATOCERA, DIPTERA)

1. The heterochromosomes of crane-flies (Tipulidae) are sex chromosomes (XY in males), as could be demonstrated by the sex limited inheritance of Y-autosome translocations in Tipula oleracea, Pales crocata and P. ferruginea. 2. Like the majority of tipulids the species more closely studied in the present contribution, P. ferruginea, possesses 3 pairs of large autosomes and small XY chromosomes, which are heteromorphic. 3. The normal strain of P. ferruginea contains two types of size-different X-chromosomes, Xl and Xk, of a length ratio of 1∶1,4. They show normal mendelian inheritance and are viable in all combinations. X-chromosomes of different length have also been observed in Tipula paludosa and T. oleracea. 4. Triploid larvae with normal spermatogenesis have been found in Tipula paludosa, Pales lunulicornis, and P. ferruginea. The triploid individuals of the two Pales species possess the sex-chromosome combination XXY. 5. In P. ferruginea a strain with supernumerary sex chromosomes has been found. In crosses of XYY males of this strain with normal XX females, besides XX females, XY and XYY males, also XXY males occur which are fertile. Their occurrence proves the existence of an epistatic male determining factor (or factors) in the Y. Presence or absence of Y thus is responsible for sex realisation.