Centrisart a 14c

Testicular tissues were subjected to the total RNA extractions by using
TRIzol^Ⓡ Reagent (Invitrogen, Carlsbad, CA, USA) according
to the manufacturer’s protocol. A part of testicular tissue (50-100 mg)
was excised and sonicated (VCX130, Vibra CellTM, Sonics & Materials,
Newtown, CT, USA) with 1 mL of TRIzol^Ⓡ Reagent. The samples
were moved into new microcentrifuge tubes and spun at 12,000 rpm for 5 min at
4°C. The supernatant was transferred into the new tubes and kept at room
temperature for 5 min of incubation, permitting entire dissociation of the
nucleoprotein complex. The tubes had 0.2 mL of chloroform and were capped
securely. After the incubation of 2-3 min, the tubes were spun at 12,000 rpm for
15 min at 4°C. The upper aqueous phase was relocated to the new tubes.
0.5 mL of isopropanol was added and left at room temperature for 10 min. Then
the tubes were rotated 12,000 rpm for 10 min at 4°C (Sartorius,
Centrisart A-14C, Gottingen, Germany). The supernatant was removed and the
pellets were resolved in 1 mL of 75% ethyl alcohol. Following agitation, the
samples were rotated at 7,500 rpm for 5 min at 4°C. The supernatant was
removed and the pellets were dried for at least 5 min. The pellet was resolved
with 20-50 μL of RNase-free water.

The male Syrian hamsters were sacrificed by decapitation. Immediately the testes
were excised and the suitable parts of them were immersed in the physiological
saline. The tissues were cut many times with sterile scissors without delay.
Following 1 min at room temperature, 1 mL of supernatant was transferred into
microcentrifuge tube. The tube was spun at 14,000 rpm for 1 min
(Centrisart^ⓇA-14C, Sartorius, Germany). The supernatant
was removed and 1 mL of saline was added to the tube. The pellet in the tube was
dispersed by sucking and releasing of pipette. One drop of the suspension was
fallen onto the slide glass and smeared uniformly. Then the slide glass was
dried completely and absolute methanol was fully applied. After the entire
dryness of the slide, specific staining solution (hematoxylin) was employed and
dispersed. Ten minutes later, the extra staining solution was cleanly washed
with flowing tap water. Then the presence of spermatozoa was observed by light
microscope (Leica DM500, Leica Microsystems, Switzerland).