2.3.5.2. Cell numbers. To estimate the number of the different types of
cells that compose the seminiferous epithelium in stage VII, the germ
cell nuclei (spermatogonia A; spermatocyte I in preleptotene/leptotene;
spermatocyte in pachytene; round spermatids) and Sertoli cell nucleolus
were counted in 5 round seminiferous tubule cross-sections, chosen
at random for each animal. For this purpose, the diameters of 10 nuclei
(from germinative cells) or nucleoli (from Sertoli cells) were measured
per animal. These counts were corrected for section thickness and
for nucleus or nucleolus diameter, based on the procedure described
by Abercrombie [37] and modified by Amann & Almquist [38].
Estimates on Sertoli cell number per testicle were performed based
on the Sertoli cell nucleolus corrected number per seminiferous tubule
transversal section in stage VII and the total length of seminiferous
tubule per testicle, following the formula by Hochereau-de Reviers
and Lincoln [39]. The Sertoli cell index (SCI) was obtained from the
ratio between the number of round spermatids per cross section and the number of Sertoli cell nucleoli per cross section of seminiferous
tubules.
The individual volume of the Leydig cells was obtained from nucleus
volume and the proportion between nucleus and cytoplasm. The Leydig
cell nucleus volume was obtained from the knowledge of the mean nuclear
diameter. For this purpose, fifteen nuclei were measured at 1000×
for each animal. Leydig cell nuclear volume (μm3
) was obtained by the
formula 4/3πR3, were R = nuclear diameter / 2. To calculate the
proportion between nucleus and cytoplasm a 100-point square lattice
was placed over the sectioned material at 400× magnification. One
thousand points over Leydig cells were counted for each animal. The
number of Leydig cell per testis was estimated from the Leydig cell
individual volume and the volume occupied by Leydig cell in the testis
parenchyma [40].