Akinori Mitsui

Extended uterine receptivity for blastocyst implantation and full-term fetal development in mice with vitrified–warmed ovarian tissue autotransplantation

PurposeOur previous study demonstrated that vitrified–warmed ovarian tissue autotransplantation (VOAT) into estrus cycle-ceased ovariectomized mice restored fertility to achieve full-term fetal development for transferred embryos, while less steroidogenesis in the corpus luteum was observed in VOAT mice. It has been reported that the window of uterine receptivity for blastocyst implantation is extended at lower estrogen levels. Therefore, we hypothesized that duration of the window in VOAT mice could be extended.MethodsBlastocysts were transferred into VOAT mice on day 5 of pseudopregnancy. Immunohistochemical analysis was performed to examine the potential in VOAT ovarian tissues.ResultsThe rate of live birth pups from embryos transferred on day 5 of pseudopregnant VOAT mice was not different from that of embryos transferred on day 4 of pseudopregnancy in VOAT mice, while embryo transfer on day 5 into intact mice showed no pregnancy. Immunohistochemical analysis of the corpus luteum of day 8 pseudopregnant VOAT mice with uteri having decidualization induced on day 5 showed less steroidogenesis and blood vessel formation as compared to intact mice.ConclusionsUterine receptivity was extended in VOAT mice. Less steroidogenesis and blood vessel formation in the transferred ovarian tissues may be associated with the extended uterine receptivity.

Evaluation of Pre-maturity of Mouse Oocytes Ovulated from Prepubertal Females using an In Vitro Fertilization Technique

ABSTRACT The cytogenetic normality and developmental competence of mouse oocytes derived from prepubertal females were investigated to determine ooplasmic maturity after in vitro fertilization (IVF) and to examine the ability of the resultant blastocysts to develop to term. To estimate the effect of body weight and age on ovulation, prepubertal female (BALB/c × C57BL/6J) F1 mice, 20–30 days of age, were classified into two groups according to body weight as follows: a light group (L) of 8.5–12 g and a heavy (H) group of 13–16 g. The IVF blastocysts were fixed as chromosome samples, and some of the blastocysts were transferred to pseudopregnant recipients. The implantation rates and number of fetuses were subsequently evaluated at 20 days postcoitus. The average number of ovulated oocytes differed significantly, with 46.2, 18.1, and 37.1 in the H, L, and control groups, respectively. All fertilization rates were high, and there were no significant differences. However, 16.8% of zygotes from the L group were arrested at the 1-cell stage, with mostly male premature condensed chromosomes (PCC). The rate of development to the blastocyst stage was significantly low in the L group (36.6%). The rates of implantation and development to newborns were significantly lower in the H group than in the pubertal mice.

Successful pregnancy in ovariectomized mice using a combination of heterotopic autotransplantation of ovarian tissues and embryo transfer

AimThe present report is the first to show that, after ovariectomy, female mice with autotransplanted ovarian sections can maintain pregnancy after embryo transfer (ET) independent of the transplantation site.MethodsThree-month-old ICR females were ovariectomized, and sections from their own ovaries were transplanted either under their kidney capsule (KC group) or into a subcutaneous space (SC group) just after ovariectomy. In vitro fertilized blastocysts were transferred into uterine horns of the pseudopregnant mice that had received the transplanted ovarian tissues. Cesarean sections were carried out 17 days after ET to deliver any live fetuses that were present, and the numbers of implantation sites and fetuses were noted. Transplanted ovarian sections were removed and fixed for histological analysis.ResultsOf the 10 mice in the KC group that received 107 blastocysts, two females (20%) became pregnant; they showed 12 implantation sites (11.2%) and produced four pups (3.7%). In the SC group, 101 blastocysts were transferred to eight females, and three females (37.5%) became pregnant; there were seven implantation sites (6.9%), and three pups (3.0%) were born. There were no statistically significant differences between the two groups in any of the parameters evaluated. On histological examination, luteinization and vascularization of the ovarian sections that were transplanted in the pregnant SC and KC females were noted.ConclusionThe pregnancy and full-term fetal development were obtained in ovariectomized mice using a combination of heterotopic ovarian tissue autotransplantation and transfer of embryos produced by in vitro fertilization.

A Successful Method in Mouse in vitro Fertilization for Beginners

ABSTRACT The procedure of the mouse in-vitro fertilization method which can obtain high rates of fertilization and development to the blastocyst stage was explained in detail using several figures. Some factors affecting the successful rates were also described.

Determination of Appropriate Activation Time and Timing in Round Spermatid Injection into Mouse Oocytes, and Chromosomal Analysis of Resultant Embryos

Abstract: The present study focused on the level of Sr2+ activation required by mouse oocytes in round spermatid injection (ROSI) and analyzed the resultant embryos cytogenetically at the first cleavage and blastocyst stages. Mouse oocytes were divided into 3 groups: Group A, oocytes treated by activation using Sr2+ for 40 min before ROSI; Group B, oocytes treated by activation using Sr2+ for 1 h after ROSI; and Group C, oocytes treated by activation using Sr2+ for 5 h after ROSI. One round spermatid obtained from mature RFM/Ms-Rb(6.15) males was injected into each oocyte individually. The fertilization rate of oocytes in ROSI, the development rate of zygotes to the blastocyst stage and chromosomal normality in the resultant embryos were highest in Group A, suggesting activation using Sr2+ for 40 min before ROSI as the most appropriate treatment. At the first cleavage, many kinds of male-derived abnormalities, such as asynchrony and constitutive chromosome abnormalities etc., were observed in all groups. At the blastocyst stage, many parthenogenetic embryos, showing n, 2n and 2n/n with no translocated chromosome, were typically observed in all groups. Normal offspring were obtained by embryo transfer of the blastocysts derived from Group A, and their fertility after sexual maturity was confirmed by mating.