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.

Metaphase II karyoplast transfer from human in-vitro matured oocytes to enucleated mature oocytes.

Metaphase II karyoplast transfer is believed to be a useful method to rescue aged oocytes. This study attempted karyoplast transfer of in-vitro matured metaphase II (MII) oocytes, as a model of aged oocytes, into enucleated freshly ovulated metaphase II oocytes with visualization of their chromosomes under an inverted microscope. Recipient karyoplasts derived from immature oocytes were cultured in-vitro until first polar body extrusion. After 1-2 days culture, 52.1% extruded a polar body, 95.5% had PSC, aneuploidy was very low (4.5%) and none had structural aberrations. Donor oocytes were obtained from IVF or intracytoplasmic sperm injection (ICSI) patients. Chromosomes were easily confirmed in 92.3% and 95.0% of in-vivo and in-vitro matured oocytes respectively. Thirty-one karyoplasts were placed in the perivitelline space of enucleated donor oocytes, and 25 (80.6%) fused to form a reconstituted oocyte. Fertilization, cleavage and blastocyst formation rates following ICSI were 76.0%, 64.0% and 28.0% respectively for reconstructed oocytes and 59.2%, 48.0% and 3.1% respectively for control (in-vitro matured) oocytes. Chromosomal analysis of five embryos developed after karyoplast transfer and ICSI showed normal diploid sets of 46 chromosomes. In conclusion, this metaphase II karyoplast transfer technique can be applied to the solution of chromosomal abnormalities related to oocyte ageing.

Differentiation of human round spermatids into motile spermatozoa through in vitro coculture with Vero cells.

PURPOSE: This study was undertaken to examine whether human early round spermatids will differentiate in an in vitro coculture with Vero cells. METHODS: A total of 1450 and 400 isolated early round spermatids mechanically collected from two non-obstructive and three obstructive azoospermic men with a normal karyotype were cocultured on Vero cell monolayers in minimum essential medium plus 10% fetal bovine serum, with or without 50 or 100 IU/L FSH and 1 or 10 µmol/L testosterone, at 32.5°C, in an environment of 5% CO2 in air. Morphological changes of the spermatids were observed microscopically. RESULTS: After 7 days of coculture, almost half (40-50%) of the round spermatids from both non-obstructive and obstructive azoospermic men resumed spermiogenesis in vitro. Only cells from the latter patients gave rise to spermatozoa, a few of which had a motile flagellum. Low concentrations of FSH and testosterone increased the percentage of in vitro spermiogenesis. CONCLUSIONS: Isolated round spermatids can resume spermiogenesis in vitro when cocultured on a Vero cell monolayer.

Isolated spermatogonia protrude active pseudopodia in vitro.

When dispersed spermatogenic cells obtained by enzymatic digestion from prepuberal mice, adult male mice, nonazoospermic men and normospermic men were observed live using Normarski optics, it was found that, respectively, 47.4%, 1.4%, 5.1%, and 2.4% of them protruded active pseudopodia. These cells were 8 to 10 mum in diameter, had a high N/C ratio, and had one to two prominent nucleoli that were close to a distinct nuclear membrane. They showed low alkaline phosphatase activities and homogeneous nuclear immunoreactive patterns using gamma-H2AX, which suggests that they were spermatogonia.

Preimplantation diagnosis of repeated miscarriage due to chromosomal translocations using metaphase chromosomes of a blastomere biopsied from 4- to 6-cell-stage embryos.

OBJECTIVE: To evaluate the safety and accuracy of karyotyping the blastomere chromosomes at metaphase in the natural cell cycle for preimplantation diagnosis. DESIGN: A pilot study. SETTING: A private infertility clinic and a university laboratory. PATIENT(S): Eleven patients undergoing IVF and preimplantation diagnosis. INTERVENTION(S): Intact human embryos at the 4- to 6-cell stage and human-mouse heterokaryons were cultured and checked hourly for disappearance of the nuclear envelope. After it disappeared, the metaphase chromosomes were analyzed by fluorescence in situ hybridization. MAIN OUTCOME MEASURE(S): Percentage of analyzable metaphase plates and safety and accuracy of the method. RESULT(S): The success rate of electrofusion to form human-mouse heterokaryons was 87.1% (27/31), and analyzable chromosomes were obtained from 77.4% (24/31) of the heterokaryons. On the other hand, disappearance of the nuclear envelope occurred in 89.5% (17/19) of the human embryos and it began earlier than that in the heterokaryons. Analyzable chromosomes were obtained and their translocation sites were identified in all blastomeres biopsied from the 17 embryos. After the biopsy, 67.0% of the embryos could develop to the blastocyst stage. CONCLUSION(S): The natural cell cycle method reported herein requires frequent observation, but it is safe, with no artificial effects on the chromosomes and without loss of or damage to blastomeres, which occurred with the electrofusion method. Using the natural cell cycle method, we could perform preimplantation diagnosis with nearly 100% accuracy.

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.