S. Awata

An investigation of the potential effect of vacuoles in human sperm on DNA damage using a chromosome assay and the TUNEL assay

BACKGROUND The aims of this study were to establish whether individual differences exist in the frequency and size of vacuoles found in human sperm and to ascertain whether such vacuoles are involved in causing DNA damage. METHODS Morphologically normal sperm were obtained from 15 IVF and 2 ICSI patients and 3 fertile donors. (i) Sperm heads were analyzed for the presence of vacuoles under a 1000× differential interference contrast microscope. (ii) In three patients and two donor samples, structural chromosomal damage was evaluated in normal sperm containing large vacuoles and selected at 1000× magnification for injection into mouse oocytes. (iii) In 10 patients and two donor samples, confocal laser microscopy detected DNA damage in sperm-exhibiting large vacuoles and stained with an in situ cell death detection kit. RESULTS (i) Vacuoles were observed in almost all normal sperm from patient and donor ejaculates and were mainly located at the tip or middle area of the sperm heads. However, average incidence of normal sperm exhibiting large vacuoles was 4.6 and 4.2% in the patient and donor groups, respectively. (ii) Sperm chromosome assays did not reveal any differences in the incidence of structural chromosome aberrations between sperm exhibiting large vacuoles and those without them (9.1 versus 4.1%). (iii) No significant difference in frequency of TUNEL-positive cells was found between normal sperm with large vacuoles and those without them in the samples examined. Among 227 sperm exhibiting large vacuoles, only 7 cells were TUNEL positive. CONCLUSION The results showed that large vacuoles were not responsible for DNA damage, suggesting that intra-cytoplasmic injection of morphologically selected sperm may not be required for patients who produce high-quality semen.

Completion of meiosis in human primary spermatocytes through in vitro coculture with Vero cells

OBJECTIVE To examine whether human primary spermatocytes will develop into round spermatids after completing meiosis in an in vitro coculture with Vero cells. DESIGN Prospective, controlled in vitro study. SETTING A private infertility clinic and a university laboratory. PATIENT(S) Five azoospermic men whose spermatogenesis was proved to be arrested at the level of the primary spermatocyte in open biopsies. INTERVENTION(S) In vitro coculture of isolated primary spermatocytes with Vero cells and chromosomal analysis for assessment of the completion of meiosis. MAIN OUTCOME MEASURE(S) Isolated primary spermatocytes were cocultured with Vero cells under various conditions. The number of chromosomes and chromatids in newly generated cells was determined by Giemsa staining after the cells were injected into mouse oocytes. RESULT(S) The generation rates of round spermatids in six types of in vitro culture with Vero cells were 0%-10% (highest rates of division were in minimum essential medium (MEM) + 50% boar rete testicular fluid or in human synthetic oviduct fluid + 10% human serum). The number of chromosomes and chromatids in the newly developed cells was 23. CONCLUSION(S) A single primary spermatocyte was observed to divide into four cells during in vitro coculture with Vero cells. These newly developed cells were proved to be round spermatids by chromosomal analysis. It was verified that a primary spermatocyte developed into round spermatids after completing two cycles of meiosis through in vitro culture.

Are Crater Defects in Human Sperm Heads Physiological Changes during Spermiogenesis and Epididymal Maturation

Abstract We observed the crater defects (CD) of human sperm head at a magnification of×1,000 with an oil immersion lens and using an inverted microscope equipped with Nomarski optics and scanning and transmission electron microscope. Motile and normally shaped sperm with three different sizes of CD were selected and injected into M-II oocytes, donated by a consenting human patient and superovulated mice. On average, 1.54 CDs were confirmed in almost all (95.9%) sperm head, and the incidences of CDs did not change after the various demembraned pretreatments. The CDs were also found in epididymal and testicular sperm and elongating and elongated spermatids, but the incidences were a little fewer than ejaculated sperm. No CDs were found in mouse and boar sperm irrespective of the pretreatments. The fertilization and blastocyst formation rates were not significantly different following the ICSI using human sperm with 3 sizes of CDs. We speculate that CDs are not abnormal findings and physically structural changes during sperm maturation process.