USP8, a Regulator of Endosomal Sorting, Is Involved in Mouse Acrosome Biogenesis Through Interaction with the Spermatid ESCRT-0 Complex and Microtubules: MATERIALS AND METHODS

Animals
Testes were collected from 3- to 4-mo-old CD1 male mice (Charles River, Calco, Italy). Handling of mice and experimental procedures were reviewed and approved by the Ethical Committee of the University of Milano and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals promulgated by the Italian Minister of Health, DL 27 January 1992 No. 116.
Antibodies
Primary antibodies used in this study were: rabbit anti-mouse USP8 described previously; rabbit anti-STAM2 (a gift of Dr. Naomi Kitamura, Yokohama, Japan); goat anti-STAM (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-HGS (Santa Cruz Biotechnology); rabbit anti-VPS54 (a gift of Dr. Frank Stenner, Zurich, Switzerland); mouse polyclonal anti-VPS54 (Abnova, Taipei, Taiwan); rabbit or mouse anti-GST (Santa Cruz Biotechnology); mouse monoclonal anti-EEA1 (BD Transduction Laboratories, Mississauga, ON, Canada); mouse monoclonal anti-p-tubulin (Sigma-Aldrich Chemical Company, St. Louis, MO). Secondary anti-rabbit, anti-goat, and anti-mouse horseradish peroxidase-linked antibodies were from GE Healthcare (Buckinghamshire, U.K.); secondary Alexa 488- and Alexa 568-conjugated anti-rabbit IgG, anti-goat IgG and anti-mouse IgG antibodies were from Invitrogen (Leek, The Netherlands).

Isolation of Spermatogenic Cells
Spermatogenic cells were isolated in RPMI-1640 (Sigma) medium from adult mouse testes by sequential enzymatic treatments using standardized methodology. Spermatid-enriched fractions were essentially obtained by mechanical dissociation of seminiferous tubules.
RT-PCR Analysis
Total RNA was extracted from testes and/or isolated spermatogenic cells using TRIzol reagent (Invitrogen) according to the manufacture’s instructions. Complementary DNA was synthesized from 1 ig of total RNA by using Invitrogen SuperScript III First-Strand Synthesis System for RT-PCR. The cDNA was then amplified by using either mouse Staml‘-specific primers (forward 5⁰-CTCCTGGGGCAGTCCCTGGC-3⁰ and reverse 5⁰-TCCAGAAC ACAGCATCCGGTTTGCC-3⁰) that amplify a 795-bp STAM2 fragment or mouse Hgs-specific primers (forward 5 ⁰-AGCGGCCCCTTTAGTGAGTA-3⁰and reverse 5⁰-ATGCTGGGATCTGCTGTTGT-3⁰) that amplify a 725-bp HGS fragment. After 35 cycles of PCR, the reactions were analyzed on a 1.5% agarose-ethidium bromide gel. Images were captured with Polaroid film under ultraviolet light.