MOLECULAR GENETICS OF MAMMALLVN TESTIS DETERMINATION AND SPERMATOGENESIS 
Yun-Fai Chris Lau, Ph.D., Associate Investigator 
Dr. Lau's laboratory is studying the molecular 
genetics of mammalian sex determination, testic- 
ular differentiation, and spermatogenic regulation. 
For the past year, Dr. Lau and his associates have 
focused on evaluating these biological processes 
through two candidate genes, the zinc finger 
Y (ZFY) and the male-enhanced antigen (MEA) 
genes. 
L Characterization of the ZFV and Related Genes. 
The ZFV gene is a candidate for the testis-deter- 
mining factor gene (TDF) on the human Y chromo- 
some and is postulated to initiate testis differentia- 
tion during embryogenesis. However, ZFX, a gene 
that is highly homologous to the ZFK gene, has also 
been identified and mapped to the p2 1 band of the 
human X chromosome. Southern analysis indicated 
that both ZFY and ZFX genes are present on the sex 
chromosomes of most eutherian mammals. The 
identification of the ZFX gene on the X chromo- 
some has made it difficult to establish the dominant 
role of the ZFV gene in sex determination. 
A. ZFY encodes a protein with two domains in the 
adult human testis. A full-length ZFV cDNA was 
isolated from an adult human testis cDNA library. 
Sequence analysis indicated that the corresponding 
transcript encodes a 90.6 kDa protein of 801 amino 
acids. The ZFY protein is composed of two do- 
mains. The carboxyl-terminal end consists of 13 
zinc finger repeats, similar in structure to other 
DNA-binding proteins involved in regulating the 
transcription of other genes. The amino-terminal 
end of the protein is highly acidic and negatively 
charged. A short stretch of basic amino acids seems 
to define the boundary of these two domains, and 
other investigators have postulated this to be the 
nuclear localization signal. Sequence analysis of a 
partial cDNA from the ZFX gene revealed that ZFV 
and ZFX genes share 1) 95% and 97.4% homology 
at the DNA and protein levels, respectively, at their 
zinc finger domains and 2) a 94% homology at both 
DNA and protein levels at their acidic domains. 
B. Human ZFY and ZFX genes are differentially 
expressed in adult gonadal and somatic tissues. 
Expression analysis indicated that the ZFV gene is 
transcribed primarily as a 3 kb mRNA in adult testis 
and frequently as a 5.7 kb transcript in male tumor 
cell lines of various somatic origins. The ZFX gene 
produces two major transcripts of 6.7 and 8.0 kb in 
ovaries and somatic tissues and cell lines of both 
sexes. In the somatic tissues analyzed, the ZFX tran- 
scripts are the predominant mRNA species; the ZFV 
transcript is scarcely detectable. The differential ex- 
pression of both genes indicates that they may not 
be functionally identical and may serve separate bi- 
ological functions. Significantly, the 3 kb ZFY tran- 
script is also detected in other mammalian adult 
testes, suggesting that it may play an important role 
in the normal physiology of this organ. 
C. Expression of the Zfy genes in adult and fetal 
mice. The mouse harbors two Zfy genes, Zfy-1 and 
Zfy-2, in the sex-determining region of its Y chro- 
mosome. Only Zfy-1 is needed for testis develop- 
ment, and it is considered a candidate gene for 
the testis-determining Y {Tdy) locus. In the adult, 
expression of both Zfy genes can only be detected 
in the testis as a major 3 kb and a minor 2.7 kb 
transcript. The transcription increases with the ini- 
tiation of meiosis and achieves the highest level in 
the round spermatids. Differential expression of 
these two genes was observed: the expression of 
the Zfy-2 gene was slightly greater than that of the 
Zfy-1 gene. In fetuses, low levels ot Zfy expression 
were detected in several tissues, including testes, 
at day 12 of gestation. In contrast, in adults the 
expression of the Zfy-l gene was greater than that 
of the Zfy-2 gene. The data demonstrating the Zfy-1 
gene expression in fetal testes support the hypoth- 
esis that this gene plays a role in testis differen- 
tiation. However, since the Zfy genes are also 
expressed during spermatogenesis and in fetal or- 
gans other than testes, they may play other roles, 
such as regulation of male germ cell development, 
in addition to their postulated role in testis deter- 
mination. 
D. Zbf genes are silent in adult XY ovary. Are high 
levels of Zfy transcription restricted to testes, or 
can these Y-encoded genes also be expressed in XY 
ovaries? To answer this question. Dr. Lau and his 
colleagues performed expression studies of these 
genes on the gonads of the B6.Y^°'^ strain, which 
exhibits XY sex reversal. XY sex reversal occurs 
when the Y chromosome of certain Mus musculus 
domesticus strains is introduced into the B6 strain. 
Half of the XY progeny develop bilateral ovaries. 
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