Smcy transgene does not rescue spermatogenesis in sex-reversed mice
Alexander I. Agulnik,
Wilbur R. Harrison,
Colin E. Bishop
Department of Obstetrics and Gynecology, Baylor College of Medicine, 6550 Fannin Str., Houston, Texas 77030, USA
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA
Received: 16 June 2000 / Accepted: 25 September 2000
Abstract. In mouse, the Sxr
deletion interval (delta Sxr
to the small short arm of the Y chromosome and is known to
contain gene(s) required for normal spermatogenesis; in particular,
Spy, which is essential for the postnatal mitotic proliferation of
spermatogonia. This deletion interval is approximately 1–2 Mb
and contains eight known genes. In this paper we report the con-
struction of YAC transgenic mice containing different regions of
the delta Sxr
interval including Zfy1, Ube1y, Smcy, and Eif2s3.
Two male and one female founder mice, transgenic for all four
genes, were sterile. However, a fertile transgenic, carrying a full-
length copy of the Smcy gene integrated into central Chr 12, was
identified. Smcy is a highly conserved Y chromosome-located
gene, encoding peptides corresponding to epitopes of the male-
specific antigen, H-Y. The Smcy transgene was ubiquitously ex-
pressed in all organs and tissues tested in male and female carriers.
Introduction of the transgene into an X Sxr
/O genetic background
did not rescue the early arrest of spermatogenesis characteristic of
these males. These data indicate that the presence of Smcy is not
sufficient to restore spermatogenesis, making it a highly unlikely
candidate for Spy.
The sex-reversed phenotype of Sxr
mutants (Tp(Y1Ct)/+) is
caused by the translocation of a 5 Mb fragment of the sex-
determining region of Yp to the distal pseudoautosomal region
(PAR). During male meiosis, this fragment is transferred by re-
combination to the X (Chandley and Speed 1987; Evans et al.
1982; Roberts et al. 1988; Singh and Jones 1982), causing chro-
mosomal females (X/X or X/O) to develop as sterile but otherwise
normal males (Cattanach et al. 1971). X/XSxr
males are sterile
with small testes devoid of spermatogonia by 10 days post partum.
Sterility is expected because X/XSxr
males have two active X Chr
in the germ line which has been shown to be incompatible with
mitotic proliferation of spermatogonia (Burgoyne et al. 1992). In
/0 testes contain most stages of spermatogenesis
with even a few abnormally shaped motile sperm. This indicates
that most of the Y-linked genes necessary for spermatogenesis are
located in the small Yp-derived Sxr
fragment (Burgoyne et al.
1992). McLaren et al. (1984) described an Sxr
, which was due toa1to2-Mbinterstitial deletion, the du-
plicated zinc finger genes Zfy1 and Zfy2 defining the deletion
breakpoints (Mardon et al. 1989). An analysis of the germ cells in
/O testis showed them to be blocked prior to the first mei-
otic metaphase. It was, therefore, proposed that the change from
involved the loss of postulated spermatogenesis
gene(s) termed Spy, responsible for the post-natal mitotic prolif-
eration of spermatogonial germ cells.
By using a variety of positional cloning techniques, several
active genes and pseudogenes have been isolated from the delta
interval (Fig. 1; Bishop and Mitchell 1999). These include
Ube1y and its pseudogenes Smcy, Uty, Usp9y, (Dffry), Dby,
Eif2s3y, Tspy pseudogene, and RhoAy1 and 2 pseudogenes. With
the exception of Tspy and RhoAy1,2 these genes have X linked
homologs and produce functional, spliced transcripts.
In mouse and human, the SMCY protein contains peptides
corresponding to epitopes of the male-specific H-Y antigen as
recognized by T-cell assays (Markiewicz et al. 1998; Meadows et
al. 1997; Scott et al. 1995; Wang et al. 1995). As yet, its biological
role is not understood. It shares homology with RBP2 (retinoblas-
toma binding protein 2), contains a zinc-finger and an A/T rich
ARID DNA binding domain, suggesting that the SMCX/Y pro-
teins function through DNA binding and transcriptional regulation
(Agulnik et al. 1999). In the human, SMCY is located on Yq
within the azoospermia factor subregion AZF
, known to contain
genes that play a crucial role in spermatogenesis (Agulnik et al.
1994; Vogt et al. 1996).
In this paper we report the production of transgenic mice car-
rying different fragments of the delta Sxr
interval. A fertile found-
er with a normally expressed full-length copy of Smcy was used to
/O TgN(SmcyGEN) males. TgN(SmcyGEN) is a
transgene of the genomic fragment containing the Smcy gene. His-
tological analysis of spermatogenesis in such males did not reveal
any differences when compared with XSxr
/O control testes.
These data indicate that the presence of Smcy is not sufficient to
restore spermatogonial progression and make it a highly unlikely
candidate for Spy.
Materials and methods
Inbred FVB/NCrlBR mice (Charles River Labs) were used for
transgene YAC microinjection. B6CBACa-A
/O (X/O) females
were obtained from The Jackson Laboratory. X/Y Sxr
males were a kind
gift of Eva Eicher (Jackson Lab, Bar Harbor, ME.)
YAC identification and manipulation.
YAC PG7 was obtained by
screening a C3H male YAC library (Larin et al. 1991) with a Zfy1 cDNA
probe as previously described (Boettger-Tong, et al. 1998). Further analy-
sis with a series of hybridization probes and STSs (see below) correspond-
ing to different Sxr genes and markers showed it to contain complete copies
of the Zfy1, Ube1y, RhoAy2, Smcy, and Eif2s3y genes. PFGE analysis
showed the size of PG7 to be approximately 1.2 Mb. A shorter 400-kb
YAC (PG7.9F), more amenable to transgenic experiments and containing
all these genes, was derived from PG7 by a B1 repeat sequence fragmen-
tation procedure (Lee et al. 1999). FISH analysis of PG7.9F YAC DNA
indicated that it was non-chimeric, hybridizing exclusively with the short
arm of the Y chromosome (data not shown).
Isolation and microinjection of YAC DNA.
PG7.9F cells were grown
to saturation in YPD medium at 30°C. High-density plugs were prepared
according to Birren and Lai (1993). YAC DNA was separated on PFGE
Correspondence to: A. Agulnik; E-mail: email@example.com.
Mammalian Genome 12, 112–116 (2001).
© Springer-Verlag New York Inc. 2001