Application of laser-assisted zona drilling to in vitro fertilization of cryopreserved mouse oocytes with spermatozoa from a subfertile transgenic mouse.

Development of assisted reproductive technologies is necessary to obtain fertilized oocytes in a subfertile transgenic mouse strain. Here, we showed the application of laser-assisted drilling of the zona pellucida to in vitro fertilization of cryopreserved mouse oocytes with sperm from subfertile transgenic mice (C57BL/6N-Tg(UCP/FAD2)U8 strain). After cryopreservation by vitrification, the recovery and survival rates of the zona-drilled mouse oocytes were 97% (97/100) and 94% (91/97), respectively. In vitro fertilization of the cryopreserved zona-drilled mouse oocytes with sperm from the subfertile transgenic mice was greatly facilitated (60%, 55/91) compared to that of the cryopreserved zona-intact mouse oocytes (11%, 81/768). In vitro fertilized embryos that developed to the 2-cell stage were again cryopreserved by vitrification, and after warming they were transferred into recipient females. Subsequently, six viable offspring were delivered, and all were confirmed to be transgenic mice. These results indicate that laser-assisted zona drilling of oocytes combined with cryopreservation by vitrification may be a useful approach for large-scale production of in vitro fertilized embryos for managing transgenic mouse strains with reproductive disabilities such as subfertile sperm.

The improvement in fertilizing ability of cryopreserved mouse spermatozoa using laser-microdissected oocytes.

Aim: The C57BL/6 mouse strain is now commonly used for producing transgenic/knockout strains. However, the fertilizing ability of these spermatozoa decreases as a result of cryopreservaion. Although the micromanipulation technique has been established to increase their fertilizing ability, it requires a considerable degree of technical skill. In the present report, we investigate the simple microdissection of zona pellucida by laser to increase the fertilizing ability of cryopreserved spermatozoa. Methods: C57BL/6J spermatozoa were cryopreserved using a solution consisting of 18% raffinose/3% skim milk. Oocytes of the same strain were placed in PB1 medium containing 0, 0.25, 0.50 or 0.75 mol sucrose. The zona pellucida of oocytes was microdissected by laser with different pulse lengths selected from 0.45 to 0.65 ms. Microdissected oocytes were then fertilized with cryopreserved spermatozoa, and the subsequent development of embryos was assessed. Results: When oocytes were microdissected in PB1 medium without sucrose, 81.5% of the oocytes were fertilized. The fertilization rates increased significantly as the pulse length was lengthened when compared with oocytes with intact zona pellucida. Furthermore, normal offspring were obtained in all experiments. Conclusion: The fertilizing ability of cryopreserved spermatozoa is improved when oocytes with their zona pellucida microdissected by laser were used. (Reprod Med Biol 2006; 5: 249-253).

Ovarian follicular development stimulated by leuprorelin acetate plus human menopausal gonadotropin in chimpanzees.

We attempted ovarian stimulation using gonadotropins in 14 chimpanzees. Subjects were given a single administration of leuprorelin acetate, followed by repeated administration of human menopausal gonadotropin (hMG) for 16-21 days. During the dosing period, the ovarian follicle diameter and count were measured by transvaginal ultrasonography. The hormone administration induced the development of multiple follicles, and multiple oocytes were subsequently retrieved. However, the follicle count was decreased, suggesting atresia, in some subjects. Statistically, the final follicle diameter was dependent on the dosing duration and the hMG dose in the late stage, while the maximum follicle count during hMG administration was dependent on age and the hMG dose in the early stage. Five subjects showed mild ovarian hyperstimulation syndrome (OHSS)-like symptoms with a high serum estradiol (E2) concentration. These results suggest that leuprorelin acetate plus hMG administration successfully stimulates the development of multiple ovarian follicles for oocyte retrieval and that the serum E2 concentration is predictive of OHSS-like symptoms in chimpanzees.

Cytoplasmic dysmorphisms in metaphase II chimpanzee oocytes.

Prior to fertilization by intracytoplasmic sperm injection (ICSI), cytoplasmic organization was evaluated in metaphase II chimpanzee oocytes obtained from stimulated ovaries. The findings demonstrate a high frequency of anomalies that are remarkably similar to the types of cytoplasmic dysmorphisms reported for human oocytes used in IVF. Similar to the human, the occurrence of these anomalies was oocyte- and animal-specific and associated with reduced competence as indicated by embryo development in vitro to the blastocyst stage.

Study of low-temperature (4 degrees C) transport of mouse two-cell embryos enclosed in oviducts.

PURPOSE: We examined usefulness of a mouse embryo transportation system for low-temperature transport of oviducts containing mouse two-cell embryos. METHODS: Oviducts containing two-cell mouse embryos were stored at 4 degrees C for 36 h. After that, embryos were collected and cultured for 96 h in Potassium Simplex Optimized Medium (KSOM) medium and evaluated for their rate of development to hatched blastocysts. Embryos were transferred to recipients, and the rate of survival to live young was investigated. The oviducts were then transported from Yamagata to Kumamoto (distance of approx. 1,000 km). At the destination, embryos were implanted in recipient dams and were studied to evaluate their survival to live young. RESULTS: After preservation for 36 h at 4 degrees C, 68.3% of two-cell embryos developed to hatched blastocysts. As a result of transplanting 546 embryos into 25 recipients, 109 normal live young mice were obtained; the rate of development was 20.0%. Results of oviduct transport from Yamagata to Kumamoto indicated that 30.2% of transplanted embryos developed to live young. CONCLUSION: Low-temperature transport of two-cell embryos in oviducts is useful as a method of shipping mouse embryos between institutes.

Experimental evaluation of cross-contamination between cryotubes containing mouse 2-cell embryos and murine pathogens in liquid nitrogen tanks.

It has been suspected that embryos stored in liquid nitrogen tanks may become contaminated with murine pathogens, if some pathogens had been introduced to the tanks accidentally. To examine this, we stored tubes containing embryos with tubes containing mouse hepatitis virus (MHV) or Pasteurella pneumotropica in liquid nitrogen tanks and examined whether progeny mice derived from the embryos were contaminated with the pathogens or not. After storing for 6 months or 1 year the frozen embryos were thawed and implanted into the oviducts of pseudopregnant female mice, and the mice were bred in vinyl isolators. We could not detect serum antibodies to MHV and isolate Pasteurella pneumotropica in the progeny mice, suggesting that cross-contamination between tubes in a liquid nitrogen tank scarcely occurs.

Cryopreservation of spermatozoa from closed colonies, and inbred, spontaneous mutant, and transgenic strains of rats.

We attempted to cryopreserve spermatozoa from closed colonies (Jcl:SD and Jcl:Wistar), and inbred (BN/Crj, F3441 DuCrj, LEW/Crj, Long-Evans and WKY/NCrj), mutant (Zitter [WTC.ZI-zi] and Tremor [TRM]), transgenic (human A-transferase [A], and green fluorescent protein [GFP]) strains of rats. Rat epididymal spermatozoa suspended in cryopreservation solution (23% egg yolk, 8% lactose monohydrate, and 0.7% Equex Stm, pH 7.4, adjusted with 10% Tris [hydroxymethy] aminomethane) were frozen and stored at -196 degrees C. After thawing at 37 degrees C, the spermatozoa were instilled into the tip of each uterine horn of the recipients. A total of five recipient females for each strain were inseminated with cryopreserved spermatozoa, and normal live offspring of all strains (Jcl:SD: 11, Jcl:Wistar: 13, BN/Crj: 9, F344/DuCrj: 28, LEW/Crj: 4, Long-Evans: 6, WKY/NCrj: 8, TRM: 24, WTC.ZI-zi: 27, A: 30 and GFP: 20) were obtained.