Submitted October 29, 2002
Returned for revision November 14, 2002
Accepted December 16, 2002
Testis
Molecular Weight Forms of Inhibin A and Inhibin B in the
Bovine Testis Change with Age
H. Kaneko *,
J. Noguchi ,
K. Kikuchi ,
Y. Hasegawa
* To whom correspondence should be addressed. E-mail: kaneko{at}nias.affrc.go.jp.
Abstract
To investigate alterations in the molecular weight forms
of inhibin in bull testis from the infantile (4-5 wks of
age) to postpubertal (49-56 wks of age) periods,
testicular homogenates were obtained from animals of
various ages and fractionated by a combination of
immunoaffinity chromatography and SDS-PAGE. Subsequently,
the fractions eluted from the SDS gels were assayed for
total inhibin, inhibin A and inhibin B by
fluoroimmunoassay (FIA) or immunofluorometric assays
(IFMAs) and for inhibin bioactivity by an in vitro
bioassay. The molecular weight patterns of inhibin A and
inhibin B in the testis, as determined by the
dimer-specific IFMAs, showed the presence of a peak of
approximate 47 kDa until 21-26 wks of age. However, the
peak disappeared after 31-32 wks of age. As bulls aged,
especially after 31-32 wks of age, inhibin A and inhibin B
levels increased in the molecular weight region of 27-34
kDa. Total inhibin showed two peaks, of between 20 and 26
kDa and approximately 47 kDa, until 21-26 wks of age and a
single peak between 20 and 30 kDa after 31-32 wks of age.
The eluted fractions corresponding to 29, 31 or 47 kDa
gave a dose-response curve that was parallel to the curve
generated with 32 kDa inhibin A or 29 kDa inhibin B
standard in the IFMA for inhibin A or inhibin B. The
fractions corresponding to 29 and 31 kDa suppressed basal
release of FSH from rat pituitary cells, but the 47 kDa
fraction had a lower FSH-suppressing activity. Immunoblot
analysis revealed the presence of a 29 kDa band
cross-reacting with inhibin 
subunit and
B subunit antibodies, and of a 31 kDa
band cross-reacting with inhibin subunit and
A subunit antibodies, in the testis of
older bulls. The 47 kDa band was recognized by the
, A and B
subunit antibodies. Immunohistochemisty of the testis at
each age showed that inhibin subunits were found
exclusively in Sertoli cells, but the intensity of
immunostaining diminished in older bulls, in parallel with
the decrease in the testicular concentrations of total
inhibin. From the above results, we conclude that: 1)
Bovine Sertoli cells produce both inhibin A and inhibin B;
2) inhibin production in Sertoli cells during the
prepubertal period is characterized by the 47 kDa
inhibin-related material which contains precursor forms of
inhibin A and inhibin B; and 2) the proportion of the
mature forms of inhibin A and inhibin B increases as bulls
age, although total inhibin production in Setroli cells
decrease.
Key words:
Testis •
Inhibin •
Sertoli cells
