Evolution of ZFY and ZFX Genes

Members of the ZFY and ZNF6 gene families have been cloned from species representing different taxa and different modes of sex determination. Comparisons of these genes show the ZFY-like and ZNF6 sequences to be strongly conserved across marsupials, birds, and lepidosaurians. Sequences analyzed by neighbor-joining indicate that both gene families are monophyletic, with a high bootstrap value. Pairing of sequences from males and females of nonmammalian species show there is no significant difference between male and female sequences from a single species, consistent with autosomal locations. The molecular distances between murine Zfy-1, Zfy-2, and other ZFY-like sequences suggest that Zfy genes have undergone a period of rapid evolutionary change not seen in human ZFY (Johnston, 1998).

Zfy1 and Zfy2 are homologous zinc finger genes on the mouse Y Chromosome. To ask whether these genes are properly classified as members of the ZFY family, their genomic organization has been characterized and compared to that of mouse Zfx, human ZFX, and human ZFY. Zfy1 has 11 exons distributed across at least 56 kb, and Zfy2 has a minimum of 9 exons distributed across at least 52 kb. The Zfy2 locus contains regions similar in size and sequence to all 11 exons of Zfy1, plus an additional 5' UTR exon. All splice sites conform to the GT-AG rule. There are two instances of additional AG dinucleotides immediately 5' of 3' splice sites. Zfy1 and Zfy2 are homologous to other ZFY family members within the coding region, but the untranslated regions show no sequence similarity. Within the coding region, there is conservation of exon length and splice sites, with each splice preceding the second nucleotide of a codon. It is concluded that Zfy1 and Zfy2 are indeed members of the ZFY family, which has evolved from a single common ancestral gene (Mahaffey, 1997).

The entire exon (approximately 1.180 bp) encoding the zinc finger domain of the X-linked and Y-linked zinc finger genes (ZFX and ZFY, respectively) has been sequenced in the orangutan, the baboon, the squirrel monkey, and the rat; a total of 9,442 bp were sequenced. The ratio of the rates of synonymous substitution in the ZFY and ZFX genes is estimated to be 2.1 in primates. This is close to the ratio of 2.3 estimated from primate ZFY and ZFX intron sequences and supports the view that the male-to-female ratio of mutation rate in humans is considerably higher than 1 but not extremely large. The ratio of synonymous substitution rates in ZFY and ZFX is estimated to be 1.3 in the rat lineage but 4.2 in the mouse lineage. The former is close to the estimate (1.4) based on introns. The much higher ratio in the mouse lineage (not statistically significant) might have arisen from relaxation of selective constraints. The synonymous divergence between mouse and rat ZFX is considerably lower than that between mouse and rat autosomal genes, agreeing with previous observations and providing some evidence for stronger selective constraints on synonymous changes in X-linked genes than in autosomal genes. At the protein level ZFX has been highly conserved in all placental mammals studied, while ZFY has been well conserved in primates and foxes but has evolved rapidly in mice and rats, possibly due to relaxation of functional constraints as a result of the development of X-inactivation of ZFX in rodents. The long persistence of the ZFY-ZFX gene pair in mammals provides some insight into the process of degeneration of Y-linked genes (Shimmin, 1994).

A phylogenetic analysis of sex-chromosomal zinc-finger genes (Zfx and Zfy) indicates that the genes have not evolved completely independently since their initial separation. The sequence similarities suggest gene conversion in the last exon between the duplicated Y-chromosomal genes Zfy-1 and Zfy-2 in the mouse. There are also indications of conversion (or recombination) between the X- and Y-chromosomal genes in the crab-eating fox and in the mouse. The method for estimating synonymous and nonsynonymous substitutions is modified by incorporating the substitutions in the twofold-degenerate sites in a novel way. The estimates of synonymous substitutions support the generation-time hypothesis in that the obtained rates are higher in mice (by a factor of 4.7) than in humans and higher in the Y-chromosomal genes (by a factor of 1.9) than in the X-chromosomal genes (Pamilo, 1993).

ZFY-like genes have been observed in a variety of vertebrate species. Although originally implicated as the primary testis-determining gene in humans and other placental mammals, more recent evidence indicates a role(s) outside that of testis determination. In this study, DNA from five species of fish (Carasius auratus, Rivulus marmoratus, Xiphophorus maculatus, X. milleri, and X. nigrensis) was subjected to Southern blot analysis using a PCR-amplified fragment of mouse ZFY-like sequence as a probe. Restriction fragment patterns are not polymorphic between sexes in any one species but show a different pattern for each species. With one exception (Rivulus) a 3.1-kb band from the EcoRI digestion is common to all. Sequence and open reading frame analysis of this fragment shows a strong homology to other known vertebrate ZFY-like genes. Of particular interest in this gene is a novel third finger domain similar to one human and one alligator ZFY-like gene. These studies and others provide evidence for a family of vertebrate ZFY genes, with those having this novel third finger being representative of the ancestral condition (Zimmerer, 1995).

Expression of ZFX and ZFY genes

The candidate testis-determining Y genes of the mouse, Zfy-1 and Zfy-2, encode proteins containing an acidic amino terminus and a carboxyl terminus composed of 13 zinc fingers. The zinc finger domain is conserved among human and mouse zinc finger X and Y genes. There is a 6-amino-acid deletion in the Zfy-2 zinc finger domain of laboratory mice possessing musculus Y chromosomes. The effect of this deletion on the function of Zfy-2 is not known. The reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot techniques were used to study expression of Zfy in adults and fetuses. In adults, the data suggest that Zfy-1 and Zfy-2 transcription is linked to spermatogenesis, that transcription increases with the initiation of meiosis, and that high levels of these mRNAs are found in postmeiotic round spermatid cells. The data also suggest that differential expression of these two genes is present, with the expression of Zfy-2 being slightly greater than that of Zfy-1. In fetuses, Zfy transcripts are detected in several tissues, including the testes. In contrast to the situation in adults, the data suggest that expression of Zfy-1 is greater than that of Zfy-2. The data suggesting that Zfy-1 expression is present in fetal testes supports the hypothesis that this gene plays a role in testis differentiation. However, because the Zfy genes are apparently also expressed during spermatogenesis and in fetal organs other than testes, they may serve other functions, in addition to their postulated role in testis determination (Nagamine, 1990).

Male preimplantation mouse embryos grow faster than female embryos. The transcription of Y chromosomal genes postulated to have a role in sex determination has been studed, using the highly sensitive technique of reverse-transcription polymerase chain reaction at these early stages. Two sex-determining region genes, Sry and Zfy, are transcribed during mouse preimplantation development, while the Zfy homologs Zfx and Zfa and a sex-determining region gene originally called A1s9 (now called Ube1y-1) are not. The anti-Mullerian hormone gene, which contains a Sry consensus binding element in its 5' promoter region, is not transcribed at this time. Developmental curves show that Sry and Zfy are expressed commencing at the two-cell stage. These results suggest that mammalian sex determination starts prior to gonad differentiation (Zwingman, 1993).

The Zfy-1 and Zfy-2 genes, which arose by gene duplication, map to the mouse Y chromosome and encode nearly identical zinc-finger proteins. Zfy-1 is expressed in the genital ridge and adult testis and likely encodes a transcription activator. Although potential roles in sex determination and spermatogenesis have been hotly debated, the biological functions of Zfy-1 remain unknown. To study the gene's regulation, transgenes with 21-28 kb of Zfy-1 5' flanking DNA placed upstream of lacZ were constructed in plasmids or created by homologous recombination of coinjected DNA molecules. The resulting transgenic mice express beta-galactosidase in the genital ridge of both males and females starting between embryonic day 10 and 11 (E10-E11), peaking at E12-E13 and then declining to low levels by E15, a pattern that matches Zfy-1 mRNA as detected by RT-PCR. This lacZ expression in genital ridge is confined to somatic cells as demonstrated by its absence from the alkaline phosphatase-positive germ cells. It had been reported previously that Zfy-1 mRNA is absent from the embryonic gonad of homozygous W(e) embryos which virtually lack germ cells. By contrast, normal expression of the Zfy-1/lacZ transgene is observed when introduced into the W(e) background, suggesting that germ cells are not necessary for expression. In the adult, the Zfy-1/lacZ transgene is expressed abundantly in developing germ cells. Extragonadal (kidney, meninges, arteries, choroid plexus) expression of the transgene is also observed in embryos. A smaller transgene with only 4.3 kb of Zfy-1 5' flanking DNA is only expressed in the germ cells of adult mice. These results suggest that an enhancer for germ cell expression in the adult lies near the Zfy-1 promoter and that an enhancer for expression in the somatic cells of the embryonic gonad is located further 5' (Zambrowicz, 1994).

The zinc finger Y (Zfy) gene is located on the Y chromosome of all placental mammals. Although it is phylogenetically conserved and is expressed in mouse fetal testis, it is not the sex determining Y (Tdy) gene. To address the possible function of the Zfy gene in mice, the distribution of Zfy protein in fetal mice was investigated by immunocytochemical staining using several specific antisera against synthetic peptides of the mouse Zfy protein. Analysis of various fetal tissues at different embryonic stages demonstrates a specific staining only in fetal testis. In particular, reactive protein is initially observed in male fetal gonads at day 11.5 postcoitum (p.c.). In fetal testes the immuno-staining intensifies at day 12 and 12.5 p.c., decreases drastically at day 13 and 14 p.c. and becomes undetectable at day 15 p.c. and beyond. The reactive molecules are distributed mostly within the seminiferous tubules of the embryonic testis. The present observations confirm the previous findings with RT-PCR analysis and indicate that Zfy or Zfy-like protein is expressed in a stage-specific manner during early testis differentiation. Its location in the seminiferous tubules suggests a possible role in early germ cell development (Su, 1992).

ZFX mutation

The zinc-finger proteins ZFX and ZFY, encoded by genes on the mammalian X and Y chromosomes, have been speculated to function in sex differentiation, spermatogenesis, and Turner syndrome. Zfx mutant mice have been derived by targeted mutagenesis. Mutant mice (both males and females) are smaller, less viable, and had fewer germ cells than wild-type mice, features also found in human females with an XO karyotype (Turner syndrome). Mutant XY animals are fully masculinized, with testes and male genitalia, and are fertile, but sperm counts were reduced by one half. Homozygous mutant XX animals are fully feminized, with ovaries and female genitalia, but showed a shortage of oocytes resulting in diminished fertility and shortened reproductive lifespan, as in premature ovarian failure in humans. The number of primordial germ cells is reduced in both XX and XY mutant animals at embryonic day 11.5, prior to gonadal sex differentiation. Zfx mutant animals exhibit a growth deficit evident at embryonic day 12.5, which persists throughout postnatal life and is not complemented by the Zfy genes. These phenotypes provide the first direct evidence of a role forZfx in growth and reproductive development (Luoh, 1997).