Hisataka Hasegawa

Successful pregnancy after oocyte activation by a calcium ionophore for a patient with recurrent intracytoplasmic sperm injection failure, with an assessment of oocyte activation and sperm centrosomal function using bovine eggs

OBJECTIVE To report a successful pregnancy after intracytoplasmic sperm injection (ICSI) with artificial oocyte activation (AOA) on a patient whose fertilization rate after ICSI was extremely low; and to report on cytologic analyses of the fertilization failure (FF) eggs after ICSI and a biologic assessment of the sperm of this patient. DESIGN Case report with an assessment of gamete function. SETTING University hospital and an experimental laboratory. PATIENT(S) A couple with severe oligoasthenozoospermia, whose seventh attempt at ICSI ended in the failure. INTERVENTION(S) Cytologic analyses of FF eggs after ICSI, AOA after ICSI, and analyses of human sperm oocyte activation ability and centrosomal function. RESULT(S) Fertilization arrest after ICSI was observed in FF eggs at various stages of fertilization. After artificial oocyte activation by exposure to ionomycin, clinical pregnancy was confirmed, and a healthy baby was born. As assessed by heterologous ICSI of human sperm into bovine oocytes, there was no significant difference in the oocyte activation rates between the patients and control sperm, but the sperm centrosomal function was low in the patients sperm (48.5% vs. 69.6%). CONCLUSION(S) We report a successful pregnancy after ICSI with AOA using a calcium ionophore, after critical cytologic analyses of the FF eggs. Furthermore, sperm centrosomal function was low, indicating that sperms ability to process the events of fertilization after the oocyte activation was poor in this patient.

Aberrant behavior of mouse embryo development after blastomere biopsy as observed through time-lapse cinematography

OBJECTIVE To analyze whether blastomere biopsy affects early embryonal growth as observed through time-lapse cinematography. DESIGN Comparative prospective study between embryos in which a blastomere was removed and embryos in which a blastomere was not removed. SETTING An experimental laboratory of the university. MAIN OUTCOME MEASURE(S) We calculated the time between blastocele formation and the end of hatching, the time between the start and end of hatching, the number of contractions and expansions between blastocyst formation and the end of hatching, and the maximum diameter of the expanded blastocyst. RESULT(S) In blastomere removal embryos, compaction began at the six-cell stage instead of at the eight-cell stage. We also found that hatching was delayed in these embryos as compared with matched controls. Moreover, the frequency of contraction and expansion movements after blastocyst formation was significantly higher in the blastomere removal group as compared with the control group. Finally, the maximum diameter of the expanded blastocyst just before hatching was not significantly different between both groups. CONCLUSION(S) These findings suggested that blastomere removal has an adverse effect on embryonic development around the time of hatching. Thus, future developments in preimplantation genetic diagnosis and screening should involve further consideration and caution in light of the influence of blastomere biopsy on embryonal growth.

Different embryonic development after blastomere biopsy for preimplantation genetic diagnosis, observed by time-lapse imaging

Using time-lapse imaging, we found different behavior of mouse embryonal development after blastomere biopsy for preimplantation genetic diagnosis. Blastomere removal has effects on the developmental behavior of the mouse embryo, including speed of growth, contraction and expansion movements, and hatching.

Microtubule organization during human parthenogenesis.

In human fertilization, the sperm centrosome plays a crucial role as a microtubule organizing center (MTOC). We studied microtubule organization during human parthenogenesis, which occurs when a human egg undergoes cleavage without a sperm centrosome. Multiple cytoplasmic asters were organized in the human oocyte after parthenogenetic activation, indicating that multiple MTOC are present in the human oocyte cytoplasm and function like a human sperm centrosome during parthenogenesis.

The shape of the sperm midpiece in intracytoplasmic morphologically selected sperm injection relates sperm centrosomal function

PurposeTo evaluate whether the morphology of the sperm midpiece observed by high magnification microscopy relates to sperm centrosomal function.MethodsSperm selected by conventional microscopy were defined as controls. By high magnification microscopy, sperm with straight midpieces were defined as Group 1, while those with tapering midpieces were defined as Group 2. Heterologous ICSI of human sperm into bovine oocytes was used to assess human sperm centrosomal function and analysis of sperm aster formation.ResultsThe total rate of sperm aster formation was 80.5% in Group 1, which was significantly higher (P < 0.05) than the rate of 33.3% seen for Group 2. Furthermore, sperm aster formation rates tended to be higher for Group 1 than for the controls.ConclusionsThis study demonstrates improvement of sperm aster formation rates by selecting sperm on the basis of midpiece morphology. The injection of selected sperm bearing morphologically straight midpieces may contribute to improved expression of sperm centrosomal function, providing a positive effect on fertilization after ICSI.

A novel culture system for mouse spermatid maturation which produces elongating spermatids capable of inducing calcium oscillation during fertilization and embryonic development.

PurposeTo establish an in vitro culture system for mouse round spermatids that models spermiogenesis and enables the assessment of oocyte activation ability.MethodsRound spermatids and Sertoli cells were isolated from testicular tissues of B6D2F1 male mice and co-cultured in the presence of testosterone and recombinant FSH. Cultured spermatids were examined for morphology and condensation of nuclei, fertilization and development rate, and Ca2+ oscillation pattern after ICSI.ResultsThe cultured spermatids elongated and resembled normal elongating spermatids in terms of both morphology and nuclear condensation. No significant differences in fertilization and development rates were observed between fresh and cultured elongating spermatids. Moreover, cultured spermatids showed similar Ca2+ oscillation patterns to fresh elongating spermatids during an initial stage in oocyte activation.ConclusionsThese data suggest that a co-culture system of spermatids and Sertoli cells, supplemented with testosterone and recombinant FSH, supports normal differentiation of round spermatids into elongating spermatids, as assessed by their morphology, nuclear condensation, and oocyte activation ability.

Development of human Graafian follicles following transplantation of human ovarian tissue into NOD/SCID/gammac null mice.

Problem  Transplantation of human ovarian cortex into host mice may permit various kinds of challenges in reproductive medicine. A novel immunodeficient mouse strain (NOD/SCID/γcnull: NOG) has been developed as a host of transplantation of human tissue.

ORIGINAL ARTICLE: Development of Human Graafian Follicles Following Transplantation of Human Ovarian Tissue into NOD/SCID/γcnull Mice

Problem  Transplantation of human ovarian cortex into host mice may permit various kinds of challenges in reproductive medicine. A novel immunodeficient mouse strain (NOD/SCID/γcnull: NOG) has been developed as a host of transplantation of human tissue.

Independent Spatial and Temporal Functions of Human Sperm Centrosomes After Dispermic Microinjection Into Bovine Oocytes

During mammalian fertilization, a centrosome is introduced by the sperm during the first cell cycle to organize a radial array of microtubules known as the sperm aster. In nature, multiple human sperm centrosomes may exist in the same egg cytoplasm during polyspermy. However, critical information concerning individual sperm centrosomal function with regards to the latter case remains unknown. We subsequently examined the sperm aster formation after injection of multiple human sperm into a bovine egg. When 2 fertile human sperm were simultaneously microinjected into different regions of the same bovine egg cytoplasm, no difference in sperm aster formation rate was observed compared to cases in which a single sperm was injected. Two human sperm were also microinjected into bovine eggs 30-, 60- and 120-minute intervals apart from one another, and no difference in sperm aster formation rates were observed. Among eggs in which 1 sperm aster was organized, there was no observable bias towards the first or second injected sperm. These findings indicated that when multiple human sperm are present in a single egg cytoplasm, each centrosome can function independently from the other. This fact suggests the possibility of transplanting a normal sperm centrosome into an egg with a sperm known to have centrosomal dysfunction.

Cytoskeletal Dynamics during Oocyte Maturation and Fertilization in Primates with Comparison to Rodents

Abstract Microtubules and microfilaments are elements of the cytoskeleton that are involved in cell motility. Dynamic and proper organization of the cytoskeleton is crucial for completion of oocyte maturation and fertilization. When performing mammalian developmental and reproductive techniques, information concerning the dynamism of the cytoskeleton is necessary and indispensable. Although rodents are widely used for developmental and reproductive technology, there are numerous differences between the cytoskeletal organization of rodent gametogenesis/fertilization and that of primates, including humans. Herein, a review of cytoskeletal organization during oocyte maturation and fertilization involving the differences between rodents, primates and other mammalian species is presented. Furthermore, we also review the function of the centrosome as a microtubule organizing center (MTOC). Proper information on the kinetics of the cytoskeleton is crucial for further expansion of developmental biology.