Chromosomal integrity of freeze-dried karyoplasts in mice.

In mammalian species, there is currently no way to preserve mature oocytes at supra-zero temperatures without cryostorage. Metaphase II (MII) oocytes freeze-dried in any medium or solution cannot be revived after rehydration. Therefore, the injurious effects to chromosomes of freeze-drying MII oocytes have not been reported.

Sperm acrosome status before and during fertilization in the Chinese hamster (Cricetulus griseus), and observation of oviductal vesicles and globules.

The present study was conducted to determine exact location where the acrosome reaction of fertilizing spermatozoa begins in the oviduct of the Chinese hamster. Unlike spermatozoa of other rodent species, Chinese hamster spermatozoa did not spontaneously undergo the acrosome reaction in fertilization-supporting media. In naturally mated females, spermatozoa in the uterus had intact acrosomes, whereas those in the lower oviductal isthmus had visibly thin acrosomal caps.

Production of mouse fetuses using spermatozoa exposed temporarily to high temperature or continuously to room temperature after freeze-drying in Na-free/K-rich EGTA buffer.

Present study aimed to determine to what extent freeze-dried spermatozoa were able to withstand high-temperature conditions: transient increase in storage temperature and long-term exposure to room temperature. Mouse spermatozoa were freeze-dried in EGTA/Tris-HCl buffered solution alkalinized using KOH (K-ETBS, pH 7.7), and then stored for up to 7 months at 4 °C or 25 °C.

Prevention of high-temperature-induced chromosome damage in mouse spermatozoa freeze-dried using Ca chelator-containing buffer alkalinized with NaOH or KOH.

In order to protect sperm chromosomes against degradation when they are being stored for relatively high temperatures, we investigated the optimal pH of the freeze-drying solution, EGTA/Tris-HCl buffered solution alkalinized by NaOH (Na-ETBS) or KOH (K-ETBS). Mouse spermatozoa suspended in Na-ETBS or K-ETBS were freeze-dried at pH 5.0-8.

Chromosomal stability of second polar bodies in mouse embryos.

Purpose: Incorporation of a second polar body (PB2) into one of the blastomeres has been considered as a causal mechanism underlying diploid/triploid mixoploidy in humans. Using a mouse model, we examined whether PB2s can participate in the formation of mixoploidy.

Methods: Uptake of BrdU was examined to determine DNA synthesis in PB2s up to 28 h after fertilization.

Chromosomal integrity and DNA damage in freeze-dried spermatozoa.

Freeze-drying technology may one day be used to preserve mammalian spermatozoa indefinitely without cryopreservation. Freeze-dried mouse spermatozoa stored below 4°C for up to 1 year have maintained the ability to fertilize oocytes and support normal development. The maximum storage period for spermatozoa increases at lower storage temperatures.

Characterization of chromosomal damage accumulated in freeze-dried mouse spermatozoa preserved under ambient and heat stress conditions.

Structural chromosome aberrations and DNA damage generated in freeze-dried mouse spermatozoa were investigated. Freeze-dried sperm samples were preserved at 4, 25 and 50°C for short duration (1 day to 2 months) and at 25°C for long duration (2 years). The spermatozoa were injected into mouse oocytes to analyse the chromosomes of the zygotes at the first cleavage metaphase.

Structural chromosomal aberrations, aneuploidy, and mosaicism in early cleavage mouse embryos derived from spermatozoa exposed to γ-rays.

Purpose: To quantitatively and qualitatively investigate the changes in chromosomal aberrations during early cleavage in mouse embryos derived from γ-irradiated spermatozoa.

Materials And Methods: Mature males were exposed to 2 Gy or 4 Gy of ¹³⁷Cs γ-rays, and their spermatozoa were used to produce embryos via in vitro fertilisation (IVF). The metaphase chromosomes were prepared from one-cell, two-cell, and four-cell embryos.

Shortening of alkaline DNA unwinding time does not interfere with detecting DNA damage to mouse and human spermatozoa in the comet assay.

The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time. The spermatozoa were treated in vitro with the DNA-damaging agents, methyl methanesulfonate (MMS) or hydrogen peroxide (H₂O₂), and then embedded in agarose gel on glass slides. The slides were immersed in alkaline solution (> pH 13) for 1, 5, 10 and 20 min, and then subjected to the electrophoresis under neutral conditions.

Chromosome analysis of mouse zygotes after injecting oocytes with spermatozoa treated in vitro with green tea catechin, (-)-epigallocatechin gallate (EGCG).

The cytogenetic effects of (-)-epigallocatechin gallate (EGCG) on mouse spermatozoa were studied in vitro using an intracytoplasmic sperm injection (ICSI) technique. Spermatozoa were collected by the swim-up method and treated with EGCG at 1 microM and 10 microM. When motile, EGCG-treated spermatozoa were injected into oocytes, structural chromosome aberrations (SCAs) at the first cleavage metaphase did not increase significantly.

Chromosomal integrity of freeze-dried mouse spermatozoa after 137Cs gamma-ray irradiation.

This study demonstrated that freeze-dried mouse spermatozoa possess strong resistance to 137Cs gamma-ray irradiation at doses of up to 8 Gy. Freeze-dried mouse spermatozoa were rehydrated and injected into mouse oocytes with an intracytoplasmic sperm injection (ICSI) technique. Most oocytes can be activated after ICSI by using spermatozoa irradiated with gamma-rays before and after freeze-drying.

Ability to activate oocytes and chromosome integrity of mouse spermatozoa preserved in EGTA Tris-HCl buffered solution supplemented with antioxidants.

Potential methods for cryopreservation of mouse spermatozoa are freeze-drying, desiccation, and suspension in EGTA Tris-HCl buffered solution (ETBS: 50 mM NaCl, 50 mM EGTA, and 10 mM Tris-HCl). To determine the duration that mouse spermatozoa suspended in ETBS-based solutions could retain their normal characteristics without freezing, spermatozoa collected from the cauda epididymis were suspended in ETBS or in ETBS supplemented with the antioxidants, dimethyl sulfoxide (DMSO), or DL-alpha-tocopherol acetate (Vitamin E acetate; VEA) diluted in DMSO, then held at ambient temperature (22-24 degrees C) for up to 9 days. When oocytes were injected with spermatozoa preserved in ETBS alone, activation rates of oocytes and chromosome integrity at the first cleavage metaphase decreased at 1 day (P < 0.

Freeze-dried sperm fertilization leads to full-term development in rabbits.

To date, the laboratory mouse is the only mammal in which freeze-dried spermatozoa have been shown to support full-term development after microinjection into oocytes. Because spermatozoa in mice, unlike in most other mammals, do not contribute centrosomes to zygotes, it is still unknown whether freeze-dried spermatozoa in other mammals are fertile. Rabbit sperm was selected as a model because of its similarity to human sperm (considering the centrosome inheritance pattern).

Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection.

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years.

[Analysis of sperm chromosomes in infertile males with teratozoospermia].

Objectives: The aim of the study was to examine sperm chromosomes from infertile males with teratozoospermia.

Design: Basic research study

Materials And Methods: Spermatozoa were obtained from infertile males with teratozoospermia. Intracytoplasmic sperm injection was used to inject human spermatozoa into mouse oocytes in order to examine sperm chromosomes.

Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa.

Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes and disrupted and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately, mouse spermatozoa appear more vulnerable to freezing than other species, e.

Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains.

Success with in vitro fertilization (IVF) using inbred strains of mice varies considerably and appears to be related to the proportion of motile spermatozoa present in epididymal sperm samples of different strains. In this study, motile spermatozoa were separated from the original samples using a column of Sephadex G25. IVF rates were compared between separated and nonseparated samples of epididymal spermatozoa before and after cryopreservation.

Relevance of chemical structure and cytotoxicity to the induction of chromosome aberrations based on the testing results of 98 high production volume industrial chemicals.

Over a 6-year period (1991-1996), the chromosomal aberration testing of high production volume (HPV) industrial chemicals had been conducted using Chinese hamster lung (CHL/IU) cells according to OECD HPV testing program and the national program in Japan. A total of 98 chemicals were tested for the induction of chromosome aberration (CA), consisting of structural CA and polyploidy. Of the 98 chemicals, structural CA and/or polyploidy were induced by 39 chemicals (40%).