Effect of zona incision by piezo-micromanipulator (ZIP) on in vitro fertilization in 21 transgenic mice lines.

Zona incision using a piezo-micromanipulator (ZIP) has been demonstrated to be effective for in vitro fertilization (IVF) using cryopreserved C57BL/6 spermatozoa. In this study, ZIP oocytes inseminated with frozen-thawed genetically modified C57BL/6J or FVB mice spermatozoa (21 lines) showed fertilization rates of 22-75% and live fetus rates of 8-49%. In 6 of the lines, the fertilization rates for oocytes compared with ZIP (42-75%) were significantly higher than that of nontreated oocytes (0-50%). Using only 90 oocytes for IVF with ZIP, 5 breeding pairs were produced from cryopreserved genetically modified mice spermatozoa. Our results indicate that application of the ZIP technique is effective for IVF using cryopreserved genetically modified mouse spermatozoa.

Effect of pressure at primary drying of freeze-drying mouse sperm reproduction ability and preservation potential.

Freeze-dried spermatozoa are capable of participating in normal embryonic development after injection into oocytes and thus useful for the maintenance of genetic materials. We recently reported that long-term preservation of freeze-dried mouse spermatozoa by conventional methods requires temperatures lower than -80 degrees C. Successful permanent preservation of mouse spermatozoa at much higher temperatures requires thorough investigation of the freeze-drying procedure. Thus, we examined the relationship between the pressure at primary drying and the preservation potential of freeze-dried mouse spermatozoa. Three different primary drying pressures were applied to evaluate the effect of pressure on freeze-dried spermatozoa under varying storage conditions and the rate of development measured. The developmental rate of embryos to the blastocyst stage from intracytoplasmic sperm injection by freeze-dried spermatozoa at pressures of 0.04, 0.37, and 1.03 mbar without storage were 59% (337/576), 71% (132/187), and 33% (99/302) respectively. When stored at 4 degrees C for 6 months, the rate was 13% (48/367), 50% (73/145), and 36% (66/182) respectively. These results show that primary drying pressure is an influential factor in the long-term preservation of freeze-dried mouse spermatozoa.

Morphologic and functional analysis of sperm and testes in Aquaporin 7 knockout mice.

OBJECTIVE: To investigate the functional and morphologic role of Aquaporin 7 (AQP7) in testis and sperm. DESIGN: Experimental laboratory study. SETTING: University and research institute units. ANIMAL(S): AQP7 knockout mice (C57BL/6J background). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Morphologic analysis of testis and epididymis, daily sperm production, sperm motility, in vitro fertilization. RESULT(S): There was no difference in the morphology of the testes and epididymis between AQP7 knockout and wild-type mice. The AQP7 knockout male mice and wild-type male mice had similar numbers of offspring. Analysis of the daily sperm production and motility of AQP7 knockout mice did not show any abnormalities. Similarly, the rate of in vitro fertilization using sperm from AQP7 knockout mice was not different from wild-type mice. CONCLUSION(S): Male AQP7 knockout mice were not sterile, and their sperm did not show any morphologic and functional abnormalities.

Fertility of mice receiving vitrified adult mouse ovaries.

Cryopreservation of the ovaries is a useful technology for preservation of germ cells from experimental animals, because if the female founder is infertile or has mutated mitochondrial DNA, preservation of female germ cells is necessary. Although it is possible to cryopreserve immature mouse ovaries with a high degree of viability by vitrification with a mixture of several cryoprotectants, the viability of cryopreserved adult mouse ovaries is still unknown. Here, we investigated the viability of mouse ovaries at various ages after cryopreservation by vitrification techniques. Donor ovaries were collected from 10-day-, 4-week-, 10-week- and 7-month-old, female, nulliparous, green fluorescence protein (GFP)-transgenic mice and cryopreserved by vitrification. The vitrified-warmed ovaries were orthotopically transplanted to 4- or 10-week-old mice. GFP-positive pups were obtained in all experimental groups. In the 4-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 44%, 9%, 12% and 4% respectively. In the 10-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 36%, 16%, 2% and 9% respectively. Furthermore, GFP-positive pups also were obtained from recipients transplanted with ovaries of donors without normal estrous cyclicity. Our results indicate that cryopreservation of mouse ovaries by vitrification is a useful method for the preservation of female germ cells from mice of various ages.

Vitamin E is essential for mouse placentation but not for embryonic development itself.

Vitamin E (alpha-tocopherol) was discovered 80 years ago to be an indispensable nutrient for reproduction in the female. However, it has not been clarified when or where vitamin E is required during pregnancy. We examined the role of alpha-tocopherol in pregnancy using alpha-tocopherol transfer protein (Ttpa)-deficient mice fed specific alpha-tocopherol diets that led to daily, measurable change in plasma alpha-tocopherol levels from nearly normal to almost undetectable levels. A dietary supplement of alpha-tocopherol to pregnant Ttpa-/- (homozygous null) mice was shown to be essential for maintenance of pregnancy from 6.5 to 13.5 days postcoitum but found not to be crucial before or after this time span, which corresponds to initial development and maturation of the placenta. In addition, exposure to a low alpha-tocopherol environment after initiation of placental formation might result in necrosis of placental syncytiotrophoblast cells, followed by necrosis of fetal blood vessel endothelial cells. When Ttpa(-/-)-fertilized eggs were transferred into Ttpa+/+ (wild-type) recipients, plasma alpha-tocopherol concentrations in the Ttpa-/- fetuses were below the detection limit but the fetuses grew normally. These results indicate that alpha-tocopherol is indispensable for the proliferation and/or function of the placenta but not necessary for development of the embryo itself.