Submitted January 21, 2005
Returned for revision February 9, 2005
Accepted June 22, 2005
Male Reproductive Tract
Immunolocalization and Regulation of Cystatin 12 in
Mouse Testis and Epididymis
Ying Li ,
Crystal A. Putnam-Lawson ,
Heather Knapp-Hoch ,
Patrick J. Friel ,
Debra Mitchell ,
Randy Hively ,
and
Michael D. Griswold *
* To whom correspondence should be addressed. E-mail: griswold{at}mail.wsu.edu.
Abstract
In previous studies, we identified a new member of the
male reproductive tract subgroup within family 2
cystatins, termed Cystatin 12 (Cst12,
previously known as Cst TE-1 or Cres3). The
mouse Cst12 mRNA was primarily localized to the
Sertoli cells in the testis and to the epithelial cells of
the proximal caput region of the epididymis. In this
report, studies were carried out to characterize the
Cystatin 12 (CST12) protein in mouse testis and
epididymis. A recombinant His-CST12 fusion protein was
expressed in E. coli and purified to generate an
anti-CST12 polyclonal antibody. Western blot analysis
showed little or no cross-reaction between the anti-CST12
antibody and several other known male reproductive tract
cystatins. Immunohistochemistry revealed that CST12
protein was predominantly localized to the cytoplasm of
Sertoli cells in the seminiferous epithelium in a
stage-dependent manner. All stages showed high levels of
expression except stage VII and VIII where very limited
expression of CST12 was observed. In the epididymis,
CST12 was highly expressed in the cytoplasm of the
epithelial cells in the proximal caput and secreted into
the lumen. The mouse CST12 protein was also detected in
other regions of the epididymis, however, the localization
varied greatly along the epididymal tubules. Indirect
immunofluorescence showed that CST12 protein was localized
to the cytoplasmic droplets in both testicular and
epididymal spermatozoa. These observations suggest that
CST12 protein may play a specialized role during
spermatogenesis and sperm maturation. Northern blot
analyses demonstrated that Cst12 transcript levels
in the epididymis decreased after castration, and
testosterone propionate (T) treatment further repressed
the expression of this gene. However, 17-beta estradiol
(E) administration maintained the expression of
Cst12 mRNA after castration while treatment with
both T and E failed to maintain Cst12 mRNA levels in
epididymis. These results suggest that androgen and
estrogen, probably with other testicular factors, are
involved in the regulation of this gene.
Key words:
Testis •
Epididymis •
Estradiol •
Sperm •
Testosterone
