AGs, spermatic ducts (SDs), and ejaculatory bulbs (EBs), were
dissected out and fixed overnight in Bouin’s fixative, at 4 ◦C. They
were then washed with 70% ethanol and dehydrated through a
series of ethanol, transferred into toluene, infiltrated with melted
paraffin, and embedded in paraffin blocks. Serial sections of 5m
thick weremadeat the mid-region of the AGs, using a LeicaRM2235
microtome, and then placed on silane coated-slides and air dried
overnight. The paraffin sections were deparaffinized with xylene,
then rehydrated through a series of ethyl alcohol, stained with Harris’s
hematoxylin for 1min and counterstained with eosin for 2min.
They were then dehydrated with a series of ethyl alcohol, cleared
with xylene, and mounted in Permount (Bio-Optica, Milan, Italy).
Sections were examined using a Nikon eclipse E600 microscope
and photographed with a Nikon digital camera DXM1200.
The numbers of cell types present in the AGs were
quantified from uniform cross-sections, using ImageJ software
(http://rsbweb.nih.gov/ij/index.html), then recorded as percentages
of the total cells. In the BrdU assay groups (see Section 2.5),
the numbers of dividing cells per mm2 area were counted by the
same method.