InteractiveFly: GeneBrief

hermaphrodite : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Studies of hermaphrodite reinforce the view that there are two classes of genes in the somatic sex determination hierarchy. The first class includes genes with sex-specific expression, such as Sex lethal, transformer, fruitless and doublesex, and also genes that are expressed at higher levels in one sex than the other, such as the X-linked zygotic activators of Sxl [ sisterless-a (sis-a), sis-b (also known as scute), sisterless-c (sis-c) and runt]. Members of this class of genes play instructional roles in sex determination and differentiation.

Members of the second class of genes are not sex specific in terms of their expression, such as the genes that act to facilitate Sxl auto-regulation [sans fille (snf), fl(2)d and virulizer]; the maternal or autosome-linked zygotic regulators of Sxl [daughterless (da), extra machrochaetae (emc), groucho (gro) and deadpan (dpn)], and the gene tra-2. These genes play permissive roles in sex determination and differentiation. Because hermaphrodite's expression is not sex specific, her falls into this second class. Most of the genes in the first class and all of the genes in the second class have functions other than sex determination and differentiation. Thus, of all the genes known to be required for sex determination, only three (Sxl, tra and doublesex) act exclusively in sex determination and/or differentiation, supporting the view that genes participating exclusively in one specific developmental process are rare (Li, 1998a and references).

Where in the sex determination pathway does her fit? Sex lethal is a good place to start looking for an answer: with respect to its role in female somatic sexual differentiation, Sxl is the master regulator of sex determination in Drosophila protein; it directs the splicing of the transformer (tra) pre-mRNA to generate a functional mRNA in females. In males, tra pre-mRNA is spliced in a default pattern that leaves premature stop codons in the mRNA. Downstream of tra, the somatic sex determination pathway splits into two branches: one contains the doublesex (dsx) gene and the other the fruitless (fru) gene. In females, Tra acts together with Transformer-2 (Tra-2) to direct the splicing of the DSX pre-mRNA to generate a female-specific mRNA. Neither gene is expressed sex-specifically in the soma. In males, where functional Tra is present, default splicing of DSX pre-mRNA produces the male-specific DSX mRNA. DSX proteins are sex-specific transcription factors that are required for all aspects of somatic sexual differentiation outside of the CNS. The female-specific DSX protein (DSX F) acts together with the products of the hermaphrodite (her) and the intersex (ix) genes to repress male differentiation and to promote female differentiation in females; conversely, the male-specific DSX protein (DSX M) acts to repress female differentiation and to promote male differentiation in males (Li, 1998a and references).

hermaphrodite and doublesex function both dependently and independently to control various aspects of sexual differentiation in Drosophila

Two proteins function together to regulate sex specific genes: (1) the sex specific transcription factor Doublesex (Dsx), and (2) the non-sex specific transcription factor Hermaphrodite. A study by Li and Baker (1998) analyzes the often complex combinatorial interactions between parallel pathways that intersect in the regulation of even a single gene. The targeted yolk protein (yp) genes are transcriptionally activated by two separate pathways. One is a female-specific pathway, which is positively regulated by the female-specific Doublesex protein (Dsx F). The other is a non-sex-specific pathway, that is positively regulated by Her. The Her pathway is prevented from functioning in males by the action of the male-specific Doublesex protein (Dsx M). The Her and Dsx pathways also function independently to control downstream target genes in the precursor cells that give rise to the vaginal teeth and the dorsal anal plate in females, and the lateral anal plates in males. However, a female-specific pathway that is dependent on both Dsx F and Her controls the female-specific differentiation of the foreleg bristles and tergites 5 and 6, and the male-specific differentiation of these tissues does not require the suppression of Her's function by Dsx M (Li, 1998b).

Since the only characterized target genes of dsx are the yp genes, an investigation was undertaken to see if her also regulates the expression of the yp genes and if so, whether her functions in their regulation in a manner similar to dsx . Northern analysis was used to examine the effects of her on expression of the yp genes. Since the complete loss of her function is lethal, the temperature-sensitive allele her 1 was used. At 25¡C, her 1 flies are intersexual and have severely reduced viability; in contrast to this, at 18¡C, they are morphologically normal, and have wild-type viability and fertility. There is a 10-fold activation of yolk protein 2 (yp2) expression by her +, since mutant her females raised at a non-permissive temperature (25¡C) show a 10-fold reduction of yp2 transcript levels, as compared to wild-type females and their her 1/+ sisters. This is comparable to the activation effect of the dsx + gene in females. Surprisingly, yp2 expression is also reduced 10-fold in the her 1 homozygous females raised at 18¡C. However, when grown at 16¡C, her 1 females have levels of yp2 expression comparable to that seen in wild-type females. These results indicate that yp2 expression is more sensitive to the level of her function than is external sexual morphology. In her males, the yp2 transcript level remains unchanged. This is in striking contrast to dsx males where the yp2 level is increased 20-fold compared to that of wild-type males and dsx/+ brothers, consistent with previous findings that Dsx M (the male Dsx splice variant) functions to repress the transcription of the yp genes. This result reflects a fundamental difference between the her and dsx functions in males. The expression of the yp1 and yp3 genes is regulated the same way by dsx and her as is the yp2 gene. It is concluded that Her is required, like Dsx F, for the activation of the yp genes in female fat body cells. But, in contrast to Dsx M , Her is not required for the inhibition of yp gene expression in males (Li, 1998b).

The reduction of yp2 transcripts in her mutant females could be due to the involvement of her in the regulation of yp2 transcription or yp2 RNA stability. To distinguish between the two possibilities, the yp reporter gene pCR1 was used. In the pCR1 construct, the intergenic regulatory region of the divergently transcribed yp1 and yp2 genes remains intact while the coding sequences of yp1 and yp2 are replaced by the Drosophila Adh and the Escherichia coli lacZ genes, respectively. The effects of her and dsx on the expression of the lacZ gene of pCR1 are in all cases comparable to their effects on yp gene expression as monitored by Northern blots, demonstrating that her, like dsx, controls yp gene expression at the level of transcription, rather than RNA stability. Similarly, her + activity is also required in females for the transcriptional activation of the yp genes, rather than the stability of their transcripts. These results demonstrate that her, like dsx, activates the transcription of the yp genes in females through the intergenic region of yp1 and yp2 (Li, 1998b).

The perceptions that derive from the above experiments concerning the roles of dsx and her in regulating the transcription of the yp genes suggest that both genes function in the activation of the yp genes in females, but that only dsx functions in males, where it acts to repress the yps expression. However, consideration of the quantitative aspects of the data from these experiments indicates that this interpretation is incorrect. In particular, the data with respect to the roles of Dsx M in males and Dsx F in females indicate that dsx function can account for all of the difference between the sexes in the levels of yp gene expression. These findings with regard to dsx clearly contradict the idea that there is a female-specific role for her in the activation of the yp genes. Two alternative views of the role of her in regulating yp gene expression are presented that are consistent with these results. The argument that dsx is the major, if not the only, sex-specific regulator of the yp genes derives from the analysis of the transcriptional regulation of pCR1. The pCR1 lacZ activity in dsx/+ females is about 2000-fold higher than in dsx/+ males (no expression of the yp genes). However, the difference is only about 2.6-fold between the dsx homozygous female and male sibs. Since flies homozygous for the X-linked pCR1 transgene were female, the 2.6-fold difference in the pCR1 activity between the dsx females and males is largely, if not entirely, due to the 2-fold difference in the gene dosage of pCR1 between females (two copies of the pCR1 transgene) and males (one copy of the pCR1 transgene). Therefore, these results demonstrate that in the absence of dsx, the yp genes are expressed at the same levels in both sexes (Li, 1998b).

In considering these results, it is important to note that two factors contribute to making the levels of yp gene expression equivalent in dsx mutant males and females: (1) the expression level of the yp genes is elevated in dsx males (compared to wild-type males), due to the absence of repression by Dsx M, and (2) the expression level of the yp genes in dsx females is reduced, due to the absence of activation by Dsx F. Thus in both dsx mutant males and females, there are significant levels of expression of the yp genes, and these levels are equivalent in the two sexes. There are two ways to reconcile these observations with regard to dsx with the observation that her appears to control the expression of the yp genes female-specifically. One model is that her does function female-specifically, but that its female-specific function is dependent on Dsx F. The second model is that her functions sex-independently to activate the expression of the yp genes, but that its action in males is precluded by Dsx M's repression of any yp gene's expression. These two models make different predictions as to the effects expected of her mutants in dsx mutant backgrounds. If the first model is correct, the presence or absence of her should have no effect on the yp genes when Dsx F is absent. If the second model is correct, her should be able to activate the yp genes in dsx mutant males where Dsx M is absent. To examine the effects of her on yp gene expression in the absence of dsx function, the pCR1 reporter gene was used. To test whether her activates yp gene expression in males in the absence of the inhibition by Dsx M, the responsiveness of pCR1 to her regulation was examined in the absence of Dsx M. In males, when Dsx M is present, pCR1 is not expressed whether or not her is present. However, in males without Dsx M, pCR1 is expressed and the pCR1 activity is 5-fold higher when her is present than when her is absent. This finding suggests that wild-type her function is normally present in males and capable of activating the transcription of the yp genes, but its activity is normally overridden by the inhibitory function of Dsx M. In conclusion, there are two separate pathways for the activation of the yp genes. One is the female-specific activation of yp genes, which is Dsx F-dependent. The other is the non-sex-specific activation of ypgenes, which is Her-dependent, Dsx F-independent and inhibited by Dsx M. These results also suggest that her has the same biological function in both sexes, providing further evidence that the expression of her is independent of the sex determination hierarchy. It is further shown that Dsx and Her can activate the yp genes independently in females (Li, 1998b).

Further dissection of the yp promoter reveals that the fat body enhancer (FBE), the site of Dsx action, is not sufficient to confer her responsiveness and the major her responsive element is located outside of the FBE, in the Her responsive region (HRR). Thus, the HRR is necessary for the Her-dependent non-sex-specific activation of yp1 and yp2 (Li, 1998b).

The fact that her and dsx mutant females have similar external phenotypes raises the possibility that dsx and her may regulate other downstream target genes in a manner similar to how they regulate the yp genes. This predicts that the loss of her should masculinize dsx mutant XX flies and vice versa, since Her and Dsx F regulate the yp genes independently. In addition, the loss of her should also masculinize dsx mutant XY flies, since Dsx M inhibits her's activation of the yp genes. To examine whether these predictions are true, a comparison was made of the phenotypes of five different external cuticular structures (which are sexually dimorphic in wild-type adult flies) among XX and XY sibs of the following four genotypes: (1) her/+; dsx/+, (2) her/her; dsx/+, (3) her/+; dsx/dsx and (4) her/her; dsx/dsx. The first cuticular structure examined was the number of the vaginal teeth. In the precursor cells that give rise to vaginal teeth, her and dsx are shown to act independently as in the case of the regulation of the yp genes in fat body. These results show that the loss of her masculinizes dsx mutant XX flies and vice versa, indicating that her + and dsx + can act in each other's absence in these cells (Li, 1998b).

The second set of cuticular structures examined were the anal plates. The dorsal anal plate of females and the two lateral anal plates of males derive from the same precursor cells. In XX and XY intersex flies, there are a pair of anal plates located dorsolaterally to the anal opening and they are often fused at the dorsoanterior side. This pair of anal plates (referred to as DLAP hereafter) represents the intersexual differentiation of the precursor cells, and they are completely fused to form the dorsal anal plate in wild-type females and are completely separated to form the two lateral anal plates in wild-type males. Loss of her masculinizes dsx mutant XX flies and vice versa. These results indicate that, in the precursor cells of vaginal teeth and DLAP, Her controls downstream female-specific differentiation genes non-sex-specifically, and Her's functioning is independent of Dsx F in females and is inhibited by Dsx M in males, analogous to Her's regulation of the yp genes in fat body cells. However, the results also indicate that this is not the only mechanism by which her and dsx act. In the precursor cells of the last (most distal) transverse row of bristles (LTRB) of the basitarsus of the forelegs (LTRB form sex combs in males), Her functions together with Dsx F. In addition to the cuticular structures already described, the number of the 6th sternite (S6) bristles was also examined on dsx mutant, her mutant, and her; dsx mutant XX and XY flies. The results indicate that (1) in XX flies, S6 differentiation follows a default pathway that is independent of dsx (Dsx F ) and her, and (2) in XY flies, S6 differentiation is dependent on both her and dsx (DSX M) (Li, 1998b).

In summary, analysis of sexual phenotypes of various tissues in the her and dsx single mutants and the her; dsx double mutants demonstrates that there are three ways by which sexual dimorphism is generated. The first utilizes DsxM in males and does not require DsxF in females. The second utilizes DsxF in females and does not require DsxM in males. The third utilizes both DsxM in males and DsxF in females. Her is involved in the last two modes of regulation, and likely also in at least some cases of the first mode of regulation. On theoretical grounds, the most parsimonious way to generate differences between homologous tissues in the two sexes during evolution is to have a regulatory gene product present in the tissues of one sex and absent in the other sex, thus affecting the pre-existing non-sex-specific differentiation in one sex, but not in the other. For example, the default pathway for T6 is full pigmentation. The sexual dimorphism of T6 is solely due to the suppression of the T6 pigmentation by Dsx F in females, in collaboration with Her, and is irrespective of the presence or absence of Dsx M in males. Another example is the formation of sixth sternite (S6) bristles. The default pathway is to form 18 bristles on S6. The sexual dimorphism of S6 is caused by the suppression of bristle formation by Dsx M in males, likely in collaboration with Her, and is irrespective of Dsx F in females. However, in the presence of selective pressures on both sexes in evolution, one way to increase sexual dimorphism is to have female- and male-specific products of regulatory genes that each have active roles in modifying the effects of pre-existing non-sex-specific regulatory systems in opposite ways, thus generating dramatic sex-specific features. For instance, in the absence of Dsx F in females and Dsx M in males, the expression levels of the yp genes are equivalent between the two sexes due to non-sex-specific control by Her. When females have Dsx F and males do not have Dsx M, there is a 30-fold difference between females and males in the expression levels of the yp genes, and when females do not have Dsx F and males have Dsx M, there is a 180-fold difference between females and males. However, a maximum difference (2000-fold) is observed only when Dsx F is present in females and Dsx M is present in males. The sexually dimorphic differentiation of the precursor cells of the vaginal teeth and DLAP is similarly controlled by Her and both Dsx proteins. Thus, her may be viewed as part of a non-sex-specific regulatory system in these tissues, which is subject to sex-specific modification by Dsx F and Dsx M (Li, 1998b).

The dual role of hermaphrodite in the Drosophila sex determination

Hermaphrodite has both maternal and zygotic functions. Maternally, as well as zygotically, her has certain functions that are involved in sex determination/differentiation and other functions that are essential for both sexes. Although the exact nature of the non-sex-specific vital functions of her is unknown, significant insights have been gained into the nature of her's sex determination/differentiation functions (Pultz, 1994 and Pultz, 1995). The maternal sex determination function of her is required for the activation of the early promoter of Sxl. It is unknown whether the her maternal sex-specific function regulates the Sxl early promoter directly, or indirectly, through other regulators of Sxl. With respect to the zygotic sex differentiation function of her in females, it has been shown that her does not regulate the expression of Sxl, tra or dsx at the level of either transcription or the splicing of their pre-mRNAs. These results have led to the suggestion that the female-specific zygotic function of her acts in parallel with, or downstream of, dsx (Pultz, 1995).

A genetic analysis of hermaphrodite, a pleiotropic sex determination gene in Drosophila melanogaster

Zygotically, her+ function is required for female sexual differentiation: when zygotic her+ function is lacking, chromosomal females (females carrying two X chromosomes) are transformed to intersex females. The her female sexual differentiation phenotype is a "true intersex" phenotype similar to that of doublesex and intersex. In "true intersex" individuals, each cell is intersexual; in contrast, "mosaic intersex" individuals have a mixture of cells, some with male-like and others with female-like morphologies. One indicator of a "true intersex" phenotype is seen in the female counterparts of the male sex comb bristles found on the first tarsal segment of mesothoracic and metathoracic legs of adult males. Wild-type males have a row of about 9-14 enlarged, blunt bristles; the row is rotated to an orientation approximately perpendicular to the bristle rows on the metatarsus. In contrast, wild-type females have a row of about 3-8 tapered bristles that is approximately parallel to the other bristle rows. In "true intersexes" the row is partially oriented toward the male orientation, as is seen in chromosomal females with impaired her function. The number of bristles in the rotated row is also increased. The pigmentation of the abdomen and the morphology of genitalia and analia are also intersexual in her females. In wild-type females, the fifth abdominal segment is pigmented only along the posterior margin, whereas in wild-type males, this segment is completely pigmented. In strongly transformed females lacking normal her function, pigmentation extends through the anterior of the fifth abdominal segment. Lack of her function also has effects on males that may indicate a weak transformation to intersexuality. Thus, zygotic her+ function may also play a role in male sexual differentiation (Pultz, 1994).

REGULATION

Targets of Activity

To examine whether maternal her function is needed as a positive regulator of the early female-specific Sxl promoter, a lacZ reporter gene construct of the Sxl promoter was used. Fathers homozygous for the reporter gene construct were crossed to her- mothers and to her + control mothers, and an anti-b-galactosidase monoclonal antibody was used to visualize protein expression. Among the progeny of control mothers, there was a striking difference between a class of darkly staining embryos and a class of lightly staining embryos. All the embryos fell into two equally represented classes: light and dark, although a few embryos showed variable intermediate staining levels. It is assumed that the darkest class of control embryos are females and the lightest are males. In contrast, none of the progeny of her- mothers stained as intensely as the darkest class of control embryos; the staining levels were consistently more uniform among all progeny, as would be expected if daughters of her - mothers were unable to fully activate the early female-specific Sxl promoter. Siblings of these embryos were assayed for viability; daughters of her- mothers survive to adulthood about 4% as often as their brothers, whereas the daughters of control mothers survive as well as their brothers. This experiment suggests a role for maternal her products in the transcriptional activation of Sxl. her- flies also fail to repress Maleless (Male-lethal) X-chromosome binding in some cells of dying daughters, indicating a failure of Sxl directed dosage compensation in mutant daughters (Pultz, 1995).

DEVELOPMENTAL BIOLOGY

Embryonic

Northern analysis shows that her is transcribed throughout development. The high level of transcripts in 0-2 hour early embryos is mostly maternal, since zygotic transcription is negligible in 0-1.5 hour embryos. The level of transcripts drops substantially during 2-4 hours of embryo development, indicating a lag between loss of maternal transcripts and the synthesis of zygotic transcripts. There is a higher level of her expression during 4-8 hours of embryo development. After about 8 hours of embryo development, her is transcribed at a low level. The patterns of her transcripts are similar in adults of both sexes. The level of her transcripts in adult females is slightly higher (less than two-fold) than in males, most likely due to maternal transcription in ovaries. The Northern analysis data strongly suggest that there is no sex-specific regulation of her at the level of transcription (Li, 1998a).

EFFECTS OF MUTATION

The hermaphrodite (her) locus has both maternal and zygotic functions required for normal female development in Drosophila. Maternal her function is needed for the viability of female offspring, while zygotic her function is needed for female sexual differentiation. This study focuses on understanding how her fits into the sex determination regulatory hierarchy. Maternal her function is needed early in the hierarchy: genetic interactions of her with the sisterless genes [sis-a and sis-b (also known as scute)], with function-specific Sex-lethal (Sxl) alleles and with the constitutive allele SxlM#1 suggest that maternal her function is needed for Sxl initiation. When mothers are defective for her function, their daughters fail to activate a reporter gene for the Sxl early promoter and are deficient in Sxl protein expression. Dosage compensation is misregulated in the moribund daughters: some salivary gland cells show binding of the maleless (mle) dosage compensation regulatory protein to the X chromosome, a binding pattern normally seen only in males. Thus maternal her function is needed early in the hierarchy as a positive regulator of Sxl, and the maternal effects of her on female viability probably reflect Sxl's role in regulating dosage compensation (Pultz, 1995).

In contrast to her's maternal function, her's zygotic function in sex determination acts at the end of the hierarchy. Since the maternal effect of her on female viability is due to its role as a positive regulator of Sxl, it seems plausible that the zygotic effects of her on female sexual differentiation might also be through regulation of Sxl. Therefore, a test was carried out of whether the constitutive allele Sxl M#1 can rescue the intersexual phenotype of her females. her- females bearing the Sxl M#1 chromosome have an intersexual phenotype indistinguishable from that of their Sxl +;her - sisters. Since constitutive Sxl expression cannot ameliorate the intersexuality of diplo-X her- individuals, the zygotic aspect of her function must not be acting through Sxlm at least not by controlling any aspect of Sxl expression that occurs constitutively in the Sxl M#1 allele. This suggests that zygotic her function differs from maternal her function in the level at which it is required within the sex determination regulatory hierarchy. The zygotic effect of her is not rescued by constitutive transformer (tra) expression. The expression of doublesex (dsx) transcripts appears normal in her mutant females. It is concluded that the maternal and zygotic functions of her are needed at two distinctly different levels of the sex determination regulatory hierarchy (Pultz, 1995).

Maternally, her+ function is needed to ensure the viability of female progeny: a partial loss of her+ function preferentially kills daughters. In addition, her has both zygotic and maternal functions required for viability in both sexes. All mutant her alleles exhibit some level of temperature sensitivity. After her mothers have been held at 29 degrees for two days (a temperature that is severly restrictive), progeny of both sexes die as embryos, and the lethal mutant phenotype of embryos becomes progressively more severe as mothers continue to be brooded at 29 degrees. The first obvious defect, detected in the majority of dead progeny, is a differentiated but twisted cuticle, with improperly formed mouthparts due to failure of head involution, and with the posterior abdominal segments wrapped around toward the dorsal side, as though having failed in germ band retraction. As mothers are brooded continuously at 29 degrees for more than three days, the cuticle phenotype of the embryos degenerates such that denticles and sclerotized mouthparts are not differentiated and the cuticle appears to contain holes. The eggshells (chorions) of the embryos also become transparent. There are severe gastrulation defects, including irregular buckling of the germband, unusually deep indentation of the cephalic furrow, and failure to complete the normal process of germband extension and retraction. Ventral furrow formation and differentation of the amnioserosa often appears normal in a collection of embryos with uniformly impaired gastrulation, suggesting that the gastrulation defect is not primarily caused by failure to differentiate mesoderm or by misspecification of cell fate in the dorsal region (Pultz, 1994).

Temperature sensitivity prevails for all known her alleles and for all of the her phenotypes described above, suggesting that her may participate in an intrinsically temperature-sensitive process. This analysis of four her alleles also indicates that the zygotic and maternal components of her function are differentially mutable (Pultz, 1994).

EVOLUTIONARY HOMOLOGS

Evolution of ZFY and ZFX Genes

Members of the ZFY and ZNF6 gene families have been cloned from species representing differenttaxa and different modes of sex determination. Comparisons of these genes show the ZFY-like andZNF6 sequences to be strongly conserved across marsupials, birds, and lepidosaurians. Sequencesanalyzed by neighbor-joining indicate that both gene families are monophyletic, with a high bootstrapvalue. Pairing of sequences from males and females of nonmammalian species show there is nosignificant difference between male and female sequences from a single species, consistent withautosomal locations. The molecular distances between murine Zfy-1, Zfy-2, and other ZFY-likesequences suggest that Zfy genes have undergone a period of rapid evolutionary change not seen inhuman ZFY (Johnston, 1998).

Zfy1 and Zfy2 are homologous zinc finger genes on the mouse Y Chromosome. To ask whether thesegenes 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 sequencesimilarity. Within the coding region, there is conservation of exon length and splice sites, with eachsplice preceding the second nucleotide of a codon. It is concluded that Zfy1 and Zfy2 are indeedmembers 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).

Mouse Y-linked Zfy1 and Zfy2 are expressed during the male-specific interphase between meiosis I and meiosis II and promote the 2nd meiotic division

Mouse Zfy1 and Zfy2 encode zinc finger transcription factors that map to the short arm of the Y chromosome (Yp). They have previously been shown to promote meiotic quality control during pachytene (Zfy1 and Zfy2) and at the first meiotic metaphase (Zfy2). However, from these previous studies additional roles for genes encoded on Yp during meiotic progression were inferred. In order to identify these genes and investigate their function in later stages of meiosis, three models were created with diminishing Yp and Zfy gene complements (but lacking the Y-long-arm). Since the Y-long-arm mediates pairing and exchange with the X via their pseudoautosomal regions (PARs), a minute PAR-bearing X chromosome derivative to enable formation of a sex bivalent, thus avoiding Zfy2-mediated meiotic metaphase I (MI) checkpoint responses to the unpaired (univalent) X chromosome. Using these models definitive evidence was obtained that genetic information on Yp promotes meiosis II, and by transgene addition identified Zfy1 and Zfy2 as the genes responsible. Zfy2 was substantially more effective and proved to have a much more potent transactivation domain than Zfy1. Previous work established that only Zfy2 is required for the robust apoptotic elimination of MI spermatocytes in response to a univalent X; the finding that both genes potentiate meiosis II led to the question of whether there was de novo Zfy1 and Zfy2 transcription in the interphase between meiosis I and meiosis II; this proved to be the case. X-encoded Zfx was also expressed at this stage and Zfx over-expression also potentiated meiosis II. An interphase between the meiotic divisions is male-specific and it was previously hypothesised that this allows meiosis II critical X and Y gene reactivation following sex chromosome silencing in meiotic prophase. The interphase transcription and meiosis II function of Zfx, Zfy1 and Zfy2 validate this hypothesis (Vernet, 2014).

Complementary critical functions of Zfy1 and Zfy2 in mouse spermatogenesis and reproduction

The mammalian Y chromosome plays a critical role in spermatogenesis. However, the exact functions of each gene in the Y chromosome have not been completely elucidated, partly owing to difficulties in gene targeting analysis of the Y chromosome. Zfy was first proposed to be a sex determination factor, but its function in spermatogenesis has been recently elucidated. Nevertheless, Zfy gene targeting analysis has not been performed thus far. This study adopted the highly efficient CRISPR/Cas9 system to generate individual Zfy1 or Zfy2 knockout (KO) mice and Zfy1 and Zfy2 double knockout (Zfy1/2-DKO) mice. While individual Zfy1 or Zfy2-KO mice did not show any significant phenotypic alterations in fertility, Zfy1/2-DKO mice were infertile and displayed abnormal sperm morphology, fertilization failure, and early embryonic development failure. Mass spectrometric screening, followed by confirmation with western blot analysis, showed that PLCZ1, PLCD4, PRSS21, and HTT protein expression were significantly deceased in spermatozoa of Zfy1/2-DKO mice compared with those of wild-type mice. These results are consistent with the phenotypic changes seen in the double-mutant mice. Collectively, this strategy and findings revealed that Zfy1 and Zfy2 have redundant functions in spermatogenesis, facilitating a better understanding of fertilization failure and early embryonic development failure (Nakasuji, 2017).

REFERENCES

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