Unusual distribution of Zfy and Zfx sequences on the sex chromosomes of the wood lemming, a species exhibiting XY sex reversal.

Sex reversal occurs naturally in the wood lemming (Myopus schisticolor) due to the presence in populations of this species of a variant (mutated) X chromosome, designated X*. Thus, X*Y animals develop into females, whereas XY animals develop into normal males. Chromosome mapping by in situ hybridization of DNA sequences homologous to the human ZFY gene localized the wood lemming Zfx sequences to region p12----p11 on both the wild-type X and the mutated X* chromosomes, at or proximal to a presumed breakpoint (Xp12) involved in the generation of the X* chromosome from the normal X, and Zfy sequences along the entire short arm of the Y chromosome. Differences between Zfx and Zfx* were readily detected by Southern blot analysis. However, both the Zfx and Zfx* genes expressed similarly sized transcripts in all adult somatic tissues investigated. Although the precise molecular difference between the Zfx and Zfx* genes is still unknown, their chromosomal location suggests that either Zfx or some other closely linked gene(s) on the X chromosome may be a major X-linked sex-determining gene, Tdx, which in the X* chromosome fails to interact properly with the Y-linked testis-determining gene, Tdy, thus causing X*Y embryos to develop into females. At least 15 copies of wood lemming Zfy sequences are distributed along the short arm of the Y chromosome. Northern hybridization analyses of adult tissues and somatic cell lines indicated that these Zfy repeats were transcriptionally inactive. Normally, 3-kb Zfy (ZFY) transcripts are readily detected in mouse and human testes, especially in the germ cells. It has therefore been postulated that expression of the Zfy (ZFY) gene may be important for spermatogenesis. Whether the lack of sufficient Zfy transcripts in the testis of the adult wood lemming has any impact on spermatogenesis in this species is still to be elucidated by further studies.

Steroid sulphatase levels are higher in males than in females of the root vole (Microtus oeconomus). Yet another rodent with an active Y-linked allele?

Steroid sulfatase (STS; EC 3.1.6.2) levels were assayed in cultured fibroblasts of root voles captured in the wild. Four independent experiments were performed using two different substrates (DHEAS and E1S). Evidence is presented that in this species, STS levels are significantly higher in males than in females (ratio 1.6:1). We discuss our findings on a comparative basis and suggest that in the root vole the STS gene(s) is X- and Y-linked (as in the mouse) and that it is subject to X-inactivation, or partially so, on one of the X chromosomes in the female.

Determination of the serological sex-specific (Sxs) antigen ("H-Y antigen") in birds and mammals using high-titer antisera and a sensitive urease ELISA.

A rapid, sensitive, and reproducible enzyme-linked immunosorbent assay (ELISA), for the detection of the serological sex-specific (Sxs) antigen (formerly termed H-Y antigen; see Introduction), is described. This assay uses bovine testes extract as the solid phase antigen, and high-titer anti-Sxs antisera and a urease-conjugated anti rat-IgG as the first and second antibody respectively. The urea containing substrate causes a pH shift in a positive reaction, which in turn is visualized by the use of bromocresol purple as a pH indicator. The method, and some representative applications of it, are described in detail.

Facts and considerations about sex-specific antigens.

The status of the present knowledge on the mammalian sex-specific antigens ("H-Y antigens") is reviewed and critically discussed. Special weight is given to problems related to three major topics, i.e., the immunology, genetics, and biological function(s) of these antigens. Current hypotheses as to the function(s) and the genetic control of the sex-specific antigens are individually scrutinized. Finally, some prospects for further H-Y research which seems especially urgent are briefly suggested.

Evidence for the presence of testicular tissue and Sxs antigen in the absence of Y-derived sequences.

Eleven XX males and seven XX true hermaphrodites have been tested for the presence of Y-derived DNA sequences using six different probes. All eleven XX males were positive with at least one of the probes but none of the seven true hermaphrodites could be shown to possess any Y-DNA. Using a new sensitive test for serologically sex-specific (Sxs) antigen, we found that, despite their apparent lack of Y-DNA, the XX true hermaphrodites were positive for their expression of the Sxs antigen.

Syngeneic male graft rejection by B6 female mice primed with spleen and testes of Sxr and Sxr' mice.

Presence or absence of the mammalian male-specific H-Y transplantation antigen was investigated in Sxr and Sxr' mice. Groups of C57BL/6 (B6) female mice were primed with spleen cells or testicular homogenates of such mice and were subsequently grafted with syngeneic male skin. Median graft survival times (MSTs) were compared with the MSTs of control groups: (i) unprimed B6 females, (ii) B6 females primed with B6 female spleen, and, (iii) B6 females primed with B6 male spleen or testes. The MSTs of the groups primed with B6 male and Sxr spleen were significantly shorter than, and those of the groups primed with B6 female and Sxr' spleen similar to the MST of unprimed females. The MSTs of groups of B6 female mice primed with testicular homogenates of B6, Sxr, and Sxr' males did not differ from the MST of unprimed B6 females. Taken together, this indicates that spleen of B6, and Sxr males carry the H-Y transplantation antigen, while the antigen is lacking on spleen cells of B6 females and Sxr' males. The results of priming with testis are interpreted such that (i) either do testes of B6, Sxr and Sxr' males lack the antigen, or, (ii) priming with testes homogenates renders B6 females unresponsive to H-Y of male grafts.

Serologically H-Y antigen-negative XO mice.

In a series of six independent experiments organ homogenates of 35 mice of the XX, XO or XY sex chromosome constitutions were absorbed using three different anti-H-Y antisera raised in inbred female LEW rats. Residual activities of absorbed antisera were tested in the Raji cell, complement-dependent, cytotoxicity test. Homogenates of various tissues, including the gonads, of XX and XO females were equally unable to absorb H-Y antibodies, indicating that tissues of these mice do not carry the H-Y antigen. In contrast, XY male homogenates fully absorbed H-Y antibodies of antisera at concentrations of 1/2 to 1/4. We discuss our findings with special attention to the problem of the existence of one or more H-Y antigens and, to the genetic regulation of the expression of this antigen.

H-Y transplantation antigen in human XO females.

When sensitized with human cultured fibroblasts of the XY and XO, but not XX, sex chromosomal types C57BL/6 female mice reject syngeneic male grafts accelerated (second set graft reaction). These findings demonstrate that the antigenic determinants of H-Y antigen of man and mouse are homologous and that XO females (at least those tested) carry the H-Y transplantation antigen. The results are discussed in the light of the question of differences between the H-Y antigen as defined by grafting and serology and the chromosomal localization of the H-Y structural gene(s).

Sex determination in the European eel (Anguilla anguilla, L.). A hypothesis based on cytogenetic results, correlated with the findings of skewed sex ratios in eel culture ponds.

Metaphase chromosomes from cultured blood cells of female, male, and hermaphroditic European eels were analyzed. In addition, both gonads from each of the specimens were examined microscopically to ensure correct sexing. The karyological investigation revealed that in some of the specimens a heteromorphic chromosome pair was present. This heteromorphism appeared in both sexes and in the hermaphrodite. C-banding and silver nitrate staining demonstrated that the heteromorphism was due to quantitative differences in constitutive heterochromatin and nucleolar organizing regions in the short arm of chromosome 8. In G-banded preparations it was demonstrated that, except for the heteromorphism mentioned, the karyotypes from both sexes and the hermaphrodite were identical. With the G-band technique it was also easily demonstrated that both the largest metacentric (No. 1) and the smallest metacentric (No. 11) had homologs. Therefore, in contrast to some earlier reports which claimed that these two chromosomes were a heteromorphic pair of sex chromosomes, it is concluded that Anguilla anguilla has no heteromorphic sex chromosomes. The implication of these findings are discussed in relation to the many reports of strongly skewed sex ratios found in commercial eel farms. It is tentatively hypothesized that sex determination in A. anguilla may be metagamic and that sex inversion may occur in this species.