ABSTRACT
<p><b>AIM</b>To show whether molecular motor dynein on a microtubule track, molecular motor myosin Va, motor recruiter myosin Va, VIIa-Rab27a/b interacting protein (MyRIP), and vesicle receptor Rab27b on an F-actin track were present during human and monkey spermiogenesis involving intramanchette transport (IMT).</p><p><b>METHODS</b>Spermiogenic cells were obtained from three men with obstructive azoospermia and normal adult cynomolgus monkey (Macaca fascicularis). Immunocytochemical detection and reverse transcription-polymerase chain reaction (RT-PCR) analysis of the proteins were carried out. Samples were analyzed by light microscope.</p><p><b>RESULTS</b>Using RT-PCR, we found that dynein, myosin Va, MyRIP and Rab27b were expressed in monkey testis. These proteins were localized to the manchette, as shown by immunofluorescence, particularly during human and monkey spermiogenesis.</p><p><b>CONCLUSION</b>We speculate that during primate spermiogenesis, those proteins that compose microtubule-based and actin-based vesicle transport systems are actually present in the manchette and might possibly be involved in intramanchette transport.</p>
Subject(s)
Adult , Animals , Humans , Male , Actins , Metabolism , Biological Transport , Physiology , Dyneins , Metabolism , Macaca fascicularis , Microtubules , Metabolism , Myosin Heavy Chains , Metabolism , Myosin Type V , Metabolism , Myosins , Metabolism , Spermatids , Cell Biology , Metabolism , Spermatogenesis , Physiology , Testis , Cell Biology , Metabolism , Transport Vesicles , Physiology , Vesicular Transport Proteins , Metabolism , rab GTP-Binding Proteins , MetabolismABSTRACT
In human fertilization, the sperm centrosome nucleates a radial array of microtubules called the sperm aster. The sperm aster is responsible for apposition of male and female pronuclei, and later gives rise to the first meiotic spindle. The objective of this study was to determine microtubule assembly and chromatin configuration in rabbit oocytes following intracytoplasmic injection with human sperm by piezo-driven pipette. Oocytes were collected from superovulated dose 14-15 h after hCG injection and were fertilized by injection of a single human sperm into the ooplasm of each oocyte without additional activation treatment. Four hours post heterologous intracytoplamic sperm injection [ICSI], rabbit eggs were fixed and microtubule organization and chromatin configuration were examined by immunofluorescence microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle. Following human sperm injection, an aster of microtubules formed adjacent to the sperm head, around mid-piece, and sperm aster was enlarged and assembled around male and female pronuclei. During pronuclear centration, male and female pronuclei were surrounded by a microtubule array without nucleation sites. With fertile human sperm, the sperm aster formation rate was 54.6%. From our data we concluded that human spermatozoa can be injected successfully into rabbit oocytes, resulting in a reasonable survival rate, and that rabbit oocytes provide a reliable tools for assessing human sperm centrosomal function using the Piezo-ICSI system