Publications by authors named "Midori Yoshizawa"

30 Publications

  • Page 1 of 1

Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis).

J Reprod Dev 2021 Aug 9;67(4):265-272. Epub 2021 Jul 9.

Graduate School of Agricultural Science, Utsunomiya University, Tochigi 321-8505, Japan.

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.
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http://dx.doi.org/10.1262/jrd.2021-018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8423609PMC
August 2021

Morphological analyses of the retinal photoreceptor cells in the nocturnally adapted owl monkeys.

J Vet Med Sci 2018 Mar 26;80(3):413-420. Epub 2018 Jan 26.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753-8515, Japan.

Owl monkeys are the only one species possessing the nocturnal lifestyles among the simian monkeys. Their eyes and retinas have been interested associating with the nocturnal adaptation. We examined the cellular specificity and electroretinogram (ERG) reactivity in the retina of the owl monkeys by comparison with the squirrel monkeys, taxonomically close-species and expressing diurnal behavior. Owl monkeys did not have clear structure of the foveal pit by the funduscope, whereas the retinal wholemount specimens indicated a small-condensed spot of the ganglion cells. There were abundant numbers of the rod photoreceptor cells in owl monkeys than those of the squirrel monkeys. However, the owl monkeys' retina did not possess superiority for rod cell-reactivity in the scotopic ERG responses. Scanning electron microscopic observation revealed that the rod cells in owl monkeys' retina had very small-sized inner and outer segments as compared with squirrel monkeys. Owl monkeys showed typical nocturnal traits such as rod-cell dominance. However, the individual photoreceptor cells seemed to be functionally weak for visual capacity, caused from the morphological immaturity at the inner and outer segments.
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http://dx.doi.org/10.1292/jvms.17-0418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880819PMC
March 2018

Improvement of implantation potential in mouse blastocysts derived from IVF by combined treatment with prolactin, epidermal growth factor and 4-hydroxyestradiol.

Mol Hum Reprod 2017 08;23(8):557-570

Laboratory of Animal Breeding and Reproduction, Division of Animal Science, Faculty of Agriculture, Utsunomiya University, 350 Mine-Machi, Utsunomiya, Tochigi 321-8505, Japan.

Study Question: Can supplementation of medium with prolactin (PRL), epidermal growth factor (EGF) and 4-hydroxyestradiol (4-OH-E2) prior to embryo transfer improve implantation potential in mouse blastocysts derived from IVF?

Summary Answer: Combined treatment with PRL, EGF and 4-OH-E2 improves mouse blastocyst implantation rates, while alone, each factor is ineffective.

What Is Known Already: Blastocyst dormancy during delayed implantation caused by ovariectomy is maintained by continued progesterone treatment in mice, and estrogen injection rapidly activates blastocysts to implantation-induced status in vivo. While the expression of many proteins is upregulated in implantation-induced blastocysts, selective proteolysis by proteasomes, such as estrogen receptor α (ESR1), occurs in implantation-induced blastocysts to achieve implantation-competent status. It is worth evaluating the proteins expressed during these periods to identify humoral factors that might improve the implantation potential of IVF-derived blastocysts because the poor quality of embryos obtained by IVF is one of the major causes of implantation failure.

Study Design, Size, Duration: Superovulated oocytes from ICR mice were fertilized with spermatozoa and then cultured in vitro in potassium simplex optimized medium (KSOM) without phenol red (KSOM-P) for 90-96 h. Blastocysts were treated with PRL (10 or 20 mIU/mL), EGF (5 or 10 ng/mL) or 4-OH-E2 (1 or 10 nM) in KSOM-P for 24 h.

Participants/materials, Setting, Methods: Levels of breast cancer 1 (BRCA1), EGF receptor (EGFR, also known as ERBB1), ERBB4, tubulointerstitial nephritis antigen-like 1 (TINAGL1) and ESR1 protein were examined with immunohistochemical analysis using immunofluorescence methods and confocal laser scanning microscopy. For embryo transfer, six blastocysts were suspended in HEPES-buffered KSOM-P medium and transferred into the uteri of recipient mice on the morning of Day 4 (0900-1000 h) of pseudopregnancy (Day 1 = vaginal plug). The number of implantation sites was then recorded on Day 6 using the blue dye method.

Main Results And The Role Of Chance: PRL, EGF and 4-OH-E2 each promoted BRCA1 protein level in the trophectoderm (TE). While PRL treatment resulted in an increase in EGFR, EGF increased both EGFR and ERBB4 in the blastocyst TE. TINAGL1 in the TE was enhanced by 4-OH-E2, which also increased localization of this protein to the basement membrane. Treatment with PRL, EGF or 4-OH-E2 alone did not improve blastocyst implantation rates. Combined treatment with PRL, EGF and 4-OH-E2 resulted in increased levels of EGFR, ERBB4, TINAGL1 and BRCA1 in the TE, whereas ESR1 was not upregulated in the treated blastocysts. Furthermore, combined treatment with PRL, EGF and 4-OH-E2 improved blastocyst implantation rates versus control (P = 0.009).

Large Scale Data: Not applicable.

Limitations, Reasons For Caution: Our studies were carried out in a mouse model, and the conclusions were drawn from limited results obtained from one species. Whether the increase in EGFR, ERBB4 and TINAGL1 protein in the TE improves implantation potential of blastocysts needs to be further studied experimentally by assessing other expressed proteins. The influence of combined supplementation in vitro of PRL, EGF and 4-OH-E2 on implantation also requires further examination and optimization in human blastocysts before it can be considered for clinical use in ART.

Wider Implications Of The Findings: Enhanced implantation potential by combined treatment with PRL, EGF and 4-OH-E2 appears to result in the upregulation of at least two distinct mechanisms, namely signaling via EGF receptors and basement membrane formation during the peri-implantation period in mice. While PRL, EGF and 4-OH-E2 each promoted BRCA1 protein level in the TE, treatment with each alone did not improve blastocyst implantation. Therefore, BRCA1 protein appears to be unnecessary for the attachment reaction in blastocysts in mice Combined supplementation of PRL, EGF and 4-OH-E2 might also be of relevance for embryo transfer of human IVF-derived blastocysts for ART.

Study Funding/competing Interest(s): This work was supported in part by the JSPS KAKENHI [Grant numbers 22580316 and 25450390 (to H.M.)] and the Joint Research Project of Japan-U.S. Cooperative Science Program (to H.M.). The authors have no conflict of interest to declare.
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http://dx.doi.org/10.1093/molehr/gax035DOI Listing
August 2017

Intracytoplasmic sperm injection into oocytes matured in vitro and early embryonic development in the owl monkey ().

Reprod Med Biol 2016 07 16;15(3):183-186. Epub 2015 Dec 16.

United Graduate School of Agricultural Science Tokyo University of Agriculture and Technology 183-8509 Fuchu-shi Japan.

Purpose: We explored the possibility of employing intracytoplasmic sperm injection (ICSI), involving oocytes and sperm of owl monkeys, to increase the availability of this species for investigations relating to malaria, etc., by increasing the number of animals in our laboratory.

Methods: Two owl monkeys (a female and a male), raised at the Amami Laboratory of the University of Tokyo, were used. Follicular oocytes surrounded with cumulus cells were cultured in vitro for approximately 25 h and cumulus cells were removed with 0.1 % hyaluronidase. Because of the poor motility of caudal epididymal sperm, sperm were injected without adding polyvinylpyrrolidone to immobilize them. The ICSI procedure was performed by an individual with considerable experience of human ICSI.

Results: We were able to produce two owl monkey embryos using ICSI of oocytes that matured to MII stage. Both embryos reached the 10-cell stage at 98 h after ICSI and showed signs of compaction, but failed to cleave further.

Conclusions: Although we successfully produced owl monkey embryos after ICSI, the embryos did not develop to the blastocyst stage. Many parameters need to be studied further, including superovulation, selection of culture media, and selection of good quality sperm in order to achieve successful ICSI in the owl monkey.
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http://dx.doi.org/10.1007/s12522-015-0229-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715853PMC
July 2016

Impaired female fertility in tubulointerstitial antigen-like 1-deficient mice.

J Reprod Dev 2016 31;62(1):43-9. Epub 2015 Oct 31.

Laboratory of Animal Breeding and Reproduction, Division of Animal Science, Faculty of Agriculture, Utsunomiya University, Tochigi 321-8505, Japan.

Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) is a matricellular protein. Previously, we demonstrated that Tinagl1 expression was restricted to extraembryonic regions during the postimplantation period and detected marked expression in mouse Reichert's membranes. In uteri, Tinagl1 is markedly expressed in the decidual endometrium during the postimplantation period, suggesting that it plays a physical and physiological role in embryo development and/or decidualization of the uterine endometrium during pregnancy. In the present study, in order to determine the role of Tinagl1 during embryonic development and pregnancy, we generated Tinagl1-deficient mice. Although Tinagl1(-/-) embryos were not lethal during development to term, homologous matings of Tinagl1(-/-) females and Tinagl1(-/-) males showed impaired fertility during pregnancy, including failure to carry pregnancy to term and perinatal lethality. To examine ovarian function, ovulation was induced with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG); the number of ovulated oocytes did not differ between Tinagl1(-/-) and Tinagl1(flox/flox). In vitro fertilization followed by embryo culture also demonstrated the normal developmental potential of Tinagl1-null embryos during the preimplantation period. Our results demonstrate that Tinagl1 deficiency affects female mice and results in subfertility phenotypes, and they suggest that although the potential of Tinagl1(-/-) oocytes is normal, Tinagl1 is related to fertility in adult females but is not essential for either fertilization or preimplantation development in vitro.
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http://dx.doi.org/10.1262/jrd.2015-109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4768111PMC
January 2017

Histocytological specificities of adrenal cortex in the New World Monkeys, Aotus lemurinus and Saimiri boliviensis.

J Vet Med Sci 2016 Jan 28;78(1):161-5. Epub 2015 Aug 28.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.

The New World monkey Aotus spp. (night monkeys) are expected for use of valuable experimental animal with the close species of Saimiri spp. (squirrel monkeys). Saimiri is known to show spontaneous hypercortisolemia, although few reports in Aotus. We compared basic states of blood steroid hormones and histological structure of the adrenal glands in two monkeys. Serum cortisol and ACTH levels were statistically lower in Aotus than Saimiri. Conversely, Aotus adrenocortical area showed significant enlargement, especially at the zona fasciculata. Electron microscopic observation at Aotus fasciculata cells revealed notable accumulation of large lipid droplets and irregular shapes of the mitochondrial cristae. These results suggest potential differences in cellular activities for steroidogenesis between Aotus and Saimiri and experimental usefulness in adrenocortical physiology and pathological models.
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http://dx.doi.org/10.1292/jvms.15-0290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751139PMC
January 2016

Molecular and cellular events involved in the completion of blastocyst implantation.

Reprod Med Biol 2016 04 15;15(2):53-58. Epub 2015 Aug 15.

Laboratory of Animal Breeding and Reproduction, Division of Animal Science, Faculty of Agriculture Utsunomiya University 321-8505 Utsunomiya Tochigi Japan.

Blastocyst implantation is an interactive process between the embryo and the uterus. The synchronization of embryonic development with uterine differentiation to a receptive state is essential for a successful pregnancy. The period of uterine receptivity for implantation is limited. Although implantation involves the interaction of numerous signaling molecules, our understanding of the hierarchical mechanisms that coordinate with the embryo-uterine dialogue is not yet sufficient to prevent infertility caused by implantation failure. This review highlights our knowledge on uterine receptivity and hormonal regulation of blastocyst implantation in mice. We also discuss the adhesion molecules, cross-linker proteins, extracellular proteins, and matricellular proteins involved in blastocyst implantation. Furthermore, our recent study reveals that selective proteolysis in an activated blastocyst is associated with the completion of blastocyst implantation after embryo transfer. A better understanding of uterine and blastocyst biology during the peri-implantation period would facilitate further development of reproductive technology.
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http://dx.doi.org/10.1007/s12522-015-0222-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715846PMC
April 2016

Degradation of estrogen receptor α in activated blastocysts is associated with implantation in the delayed implantation mouse model.

Mol Hum Reprod 2014 May 16;20(5):384-91. Epub 2014 Jan 16.

Laboratory of Animal Breeding and Reproduction, Division of Animal Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan.

Implantation of a blastocyst into a receptive uterus involves a series of highly coordinated cellular and molecular events directed by ovarian estrogen and progesterone. In particular, estrogen is essential for on-time uterine receptivity and blastocyst activation in mice. Although estrogen receptor α (ERα) is expressed in blastocysts, its targeted disruption leaves embryonic development and implantation unaffected. Therefore, the role of ERα in implanting blastocysts remains unclear. Using a delayed implantation model in mice, we showed increased expression of ERα in implantation-induced (activated) blastocysts; however, this ERα expression in activated blastocysts decreased within 6-h culture. In contrast, breast cancer 1 (Brca1) was maintained in the blastocysts during the culture. The treatment of activated blastocysts with the proteasome inhibitor MG132 demonstrated that proteolysis is associated with down-regulation of ERα expression in activated blastocysts. Embryo transfer of MG132-treated activated blastocysts into recipient mice on the morning of Day 4 of pseudopregnancy (Day 1 = vaginal plug) showed a decreased implantation rate, whereas combined treatment with MG132 and the ER antagonist, ICI 182,780, resulted in recovery of the rate of implantation. This study has revealed that down-regulation of ERα in activated blastocyst is associated with completion of blastocyst implantation after embryo transfer on the morning of Day 4 of pseudopregnancy. Our results also suggest that selective protein turnover, such as that of ERα, occurs in activated blastocysts, while expression of other proteins, including Brca1, is maintained at the same stage.
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http://dx.doi.org/10.1093/molehr/gau004DOI Listing
May 2014

Individual identification of racehorses from urine samples using a 26-plex single-nucleotide polymorphism assay.

J Forensic Sci 2013 Jan 12;58(1):21-8. Epub 2012 Oct 12.

Genetic Analysis Section, Laboratory of Racing Chemistry, 1731-2, Tsuruta-machi, Utsunomiya, Tochigi, 320-0851, Japan.

To construct a system for identifying individual horses from urine samples that are submitted for postracing doping tests, we developed a genotyping assay based on 26-plex single-nucleotide polymorphisms (SNPs). DNA was isolated from urine using a commercially available DNA/RNA extraction kit, and SNP genotyping was achieved with a SNaPshot(™) technique. DNA profiles including 26 SNPs were acquired from urine samples and blood/hair samples. Within the studied Thoroughbred population, the 26-plex assay showed a probability of identity of 5.80 × 10(-11). Compared to the conventional short tandem repeat assay, the SNP assay used less DNA, and the rate of successful genotyping was improved to 97% using aliquots of horse urine as small as 140 μL. The urinary DNA could be successfully genotyped under proper storage concerning refrigeration or freeze-thawing. This SNP assay can be used for individual identification when suspicious results are obtained from horse doping tests.
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http://dx.doi.org/10.1111/j.1556-4029.2012.02291.xDOI Listing
January 2013

Distribution of tubulointerstitial nephritis antigen-like 1 and structural matrix proteins in mouse embryos during preimplantation development in vivo and in vitro.

Zygote 2014 May 1;22(2):259-65. Epub 2012 Oct 1.

Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai, Miyagi, 981-8555, Japan.

Summary Tubulointerstitial nephritis antigen-like 1 (TINAGL1) is a novel matricellular protein that interacts with structural matrix proteins and promotes cell adhesion and spreading. We have previously reported unique localization of TINAGL1 to the trophectoderm (TE) of mouse blastocysts. TINAGL1 was found to be upregulated in implantation-competent blastocysts after estrogen treatment using progesterone-treated delayed-implantation models. Moreover, colocalization of TINAGL1 and extracellular matrix (ECM) protein laminin 1 was detected in the Reichert membrane on embryonic days 6.5 and 7.5. Although these data suggested a role for TINAGL1 in the embryo development at postimplantation, its relevance to other ECM proteins during preimplantation development is not clear. In this study, we examined the expression of TINAGL1 and its relevance to other ECM proteins fibronectin (FN) and collagen type IV (ColIV) during in vivo development of preimplantation embryos, particularly at blastocyst stage in detail. Localizations of TINAGL1, FN, and ColIV were similar. In 1-cell to 8-cell embryos, they were expressed in cytoplasm of blastomeres, and in morulae they were localized in the outer cells. FN and ColIV were expressed primarily on outer surface of the cells. In blastocysts, FN and ColIV were distributed in the cytoplasm of TE, but, just prior to implantation, they became localized uniquely to the blastocoelic surface of TE. In in vitro fertilized (IVF) blastocysts, expression levels of TINAGL1 and FN were lower than in in vivo blastocysts. These results suggest that, during preimplantation development, TINAGL1 may be involved in roles of structural matrix proteins, whose expression in blastocysts may be affected by in vitro culture.
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http://dx.doi.org/10.1017/S0967199412000469DOI Listing
May 2014

Differential expression of the motin family in the peri-implantation mouse uterus and their hormonal regulation.

J Reprod Dev 2012 20;58(6):649-53. Epub 2012 Jul 20.

Laboratory of Animal Breeding and Reproduction, Division of Animal Science, Faculty of Agriculture, Utsunomiya University, Tochigi 321-8505, Japan.

Increased vascular permeability and angiogenesis are hallmarks of the implantation process in the uterus. Angiomotin (Amot), which is a vascular angiogenesis-related protein, belongs to the motin family. There are two other members of the motin family, angiomotin-like 1 and 2 (Amotl1 and 2), which are also thought to be involved with angiogenesis. In the present study, the distribution of motin mRNAs in the mouse uterus during the peri-implantation period was investigated by in situ hybridization. Amot and Amotl1 were expressed in the stromal cells on days 3 and 4; expressions of Amotl2 during the same period were low. During the postimplantation period, Amot and Amotl1 were expressed in secondary decidual cells, while Amotl2 expression fell to an undetectable level. We also examined hormonal regulation of motin expression by steroid hormone treatment in ovariectomized mice. We found that expression of Amot was induced by P(4) in stromal cells. Additionally, Amotl1 expression was upregulated by both P(4) and estrogen (E(2)) in stromal cells, whereas E(2) increased this gene expression for only a limited time; after 12 h, expression dissipated. In contrast, P(4) regulated the expression of Amotl2 in stromal cells, while E(2) regulated its expression in luminal epithelium cells. Our results demonstrated that Amot, Amotl1, and Amotl2 were differentially expressed in uterine cells during the peri-implantation period, and that their expressions were differentially regulated by P(4) and E(2).
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http://dx.doi.org/10.1262/jrd.2012-075DOI Listing
May 2013

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

Reprod Med Biol 2012 Jul 25;11(3):123-128. Epub 2012 Jan 25.

Laboratory of Animal Breeding and Reproduction, Division of Animal Science, Faculty of Agriculture Utsunomiya University 321-8505 Utsunomiya Tochigi Japan.

Purpose: Our 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.

Methods: Blastocysts were transferred into VOAT mice on day 5 of pseudopregnancy. Immunohistochemical analysis was performed to examine the potential in VOAT ovarian tissues.

Results: The 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.

Conclusions: Uterine 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.
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http://dx.doi.org/10.1007/s12522-012-0119-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906846PMC
July 2012

Chromosome analysis by spectral karyotyping of spermatozoa from an oligoasthenozoospermic carrier of a 10; 21 reciprocal translocation.

Hum Cell 2011 Dec 12;24(4):146-9. Epub 2011 Oct 12.

Institute for Central Clinic, Yakushiji 3154, Simostuke, Tochigi 329-0431, Japan.

Cytogenetic analysis of germ-line cells prior to intracytoplasmic sperm injection (ICSI) treatment is thought to be necessary for infertile males with an identified chromosomal abnormality. We analyzed the chromosomal karyotype of human spermatozoa from an oligoasthenozoospermic carrier of a reciprocal translocation t(10; 21). Cytogenetic analysis of 39 spermatozoa was performed by spectral karyotyping (SKY) and by ICSI into mouse oocytes. The motile morphologically normal spermatozoa were injected into mouse oocytes. Of these spermatozoa, 38 (97.4%) were activated. Twenty-one (53.8%) of the activated oocytes formed two pronuclei. Metaphase chromosome spreads from 13 spermatozoa were analyzed. Only one spermatozoon was normal and 2 spermatozoa exhibited balanced translocation. Nine and one spermatozoa showed abnormalities related and unrelated to the translocation, respectively. The numbers of normal/balanced spermatozoa were lower than those in previous reports analyzing reciprocal translocations using a previously described technique involving penetrated golden hamster oocytes. After genetic counseling with the carrier and his partner, ICSI treatment was performed. Healthy female and male infants were delivered at 37 weeks gestation via a Caesarean section. The female infant was a carrier of the reciprocal translocation and the male infant was confirmed normal on prenatal diagnosis at 16 weeks gestation. For genetic counseling prior to ICSI treatment, the incidence of unbalanced type spermatozoa after swim-up or Percoll gradient treatment should be investigated and discussed with couples having fertility problems related to oligozoospermia autosomal structural abnormalities.
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http://dx.doi.org/10.1007/s13577-011-0035-yDOI Listing
December 2011

Vitrified-warmed ovarian tissue autotransplantation into ovariectomized mice restores sufficient ovarian function to support full-term pregnancy.

Reprod Med Biol 2011 Sep 24;10(3):185-191. Epub 2011 May 24.

Laboratory of Animal Breeding and Reproduction, Division of Animal Science, Faculty of Agriculture Utsunomiya University 321-8505 Utsunomiya Tochigi Japan.

Purpose: Our previous study demonstrated that heterotopic autotransplantation of fresh ovarian tissue followed by transfer of blastocysts supported full-term pregnancy in the mouse. In the present study, to address whether vitrified-warmed ovarian tissue has the potential to support uterine preparation for implantation and subsequent pregnancy to full term, we examined vitrified-warmed ovarian tissue autotransplantation (VOAT) in mice.

Methods: VOAT into kidney capsules was performed for sexual cycle-ceased mice after 7 days of ovariectomy. Uterine potential of decidualization was examined by oil infusion on day 4 of pseudopregnancy. Immunohistochemical analysis was performed to examine the potential in VOAT ovarian tissues. Blastocysts were transferred into uteri on day 4 of pseudopregnancy.

Results: In VOAT mice, uterine decidualization on day 8 of pseudopregnancy was the same as that in intact mice. Blastocyst transfer into the pseudopregnant VOAT mice showed the same rates of pregnancy and live birth pups as intact mice, while less steroidogenesis in the corpus luteum was detected in VOAT mice.

Conclusions: The autotransplantation of vitrified-warmed ovarian tissues after 7 days of ovariectomy restored their sexual cycle and then supported their pregnancy and production of offspring.
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http://dx.doi.org/10.1007/s12522-011-0090-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904650PMC
September 2011

Tubulointerstitial nephritis antigen-like 1 is expressed in the uterus and binds with integrins in decidualized endometrium during postimplantation in mice.

Biol Reprod 2010 Feb 23;82(2):263-70. Epub 2009 Sep 23.

Laboratory of Animal Breeding and Reproduction, Division of Animal Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Tochigi, Japan.

Extracellular matrix substrates contribute to both uterine and blastocyst functions during the peri-implantation period. Tubulointerstitial nephritis antigen-like 1 (TINAGL1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) is a novel matricellular protein that promotes cell adhesion and spreading. However, the physiological roles of TINAGL1 are still not clearly understood. We examined the expression and localization of TINAGL1 in peri-implantation mouse uteri. During the preimplantation period, TINAGL1 was expressed in the basement membranes of uterine luminal epithelial cells on Days 1 and 2 of pregnancy, while its expression levels declined after Day 3. In the whole uteri, the expression levels of Tinagl1 mRNA and TINAGL1 protein were similar on Days 1-4 of pregnancy. In contrast, the expression of Tinagl1 mRNA and TINAGL1 protein increased in postimplantation uteri. From Days 6 to 8, TINAGL1 was markedly expressed in the decidual endometrium. TINAGL1 is a ligand for integrins and promotes cell adhesion in cultured cells. Therefore, to address whether TINAGL1 interacts with integrins in the uterus, immunohistochemical analysis and immunoprecipitation were performed. Immunohistochemical analysis showed that ITGA2, ITGA5, and ITGB1 were expressed in stromal cells around the implanted embryos on Days 7 and 8. Biacore and immunoprecipitation analysis determined that TINAGL1 linked with ITGA5 and ITGB1 in the decidual endometrium. These results suggest that Tinagl1 functions during the postimplantation period; in particular, it associates with ITGA5B1 in the decidualized uterine endometrium.
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http://dx.doi.org/10.1095/biolreprod.109.080028DOI Listing
February 2010

Tubulointerstitial nephritis antigen-like 1 is expressed in extraembryonic tissues and interacts with laminin 1 in the Reichert membrane at postimplantation in the mouse.

Biol Reprod 2009 Nov 8;81(5):948-55. Epub 2009 Jul 8.

Laboratory of Animal Breeding and Reproduction, Division of Animal Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan.

Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) has been cloned from mouse adrenocortical cells and is known to be closely associated with zonal differentiation of adrenocortical cells. In cell culture systems, TINAGL1 is a matricellular protein that interacts with both structural matrix proteins and cell surface receptors. However, the physiological roles of TINAGL1 and regulation of its expression are still not clearly understood. In the present study, the expression and localization of TINAGL1 in peri-implantation mouse embryos was examined. During preimplantation, the expression of both Tinagl1 mRNA and TINAGL1 protein was increased just prior to implantation. In blastocysts, TINAGL1 expression was localized to the trophectoderm. Using a progesterone-treated, delayed-implantation model, TINAGL1 was found to be upregulated in implantation-competent blastocysts after estrogen treatment. During postimplantation, TINAGL1 expression was restricted to extraembryonic regions. Marked expression was detected in the Reichert membrane on Embryonic Days 6.5 (E6.5) and E7.5. Colocalization of laminin 1 and TINAGL1 was also examined. Using an anti-LAMA1 antibody, colocalization of LAMA1 and TINAGL1 was observed in postimplantation embryos. Colocalization was also detected in the Reichert membrane. Immunoprecipitation analysis determined that LAMA1 and TINAGL1 interact in embryos on E7.5. These results demonstrate that after implantation, TINAGL1 is an extraembryonic tissue-specific protein. In particular, TINAGL1 is a novel component of the Reichert membrane that interacts with laminin 1. These results suggest that TINAGL1 most likely plays a physical and physiological role in embryo development at postimplantation.
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http://dx.doi.org/10.1095/biolreprod.109.078162DOI Listing
November 2009

Expression profile and localization of mouse calcitonin (CT) sense/antisense transcripts in pre- and postnatal tissue development.

J Vet Med Sci 2009 May;71(5):561-8

National Institute of Agrobiological Science, Ibaraki, Japan.

Calcitonin (CT) has been shown to have various functions including osteoclast activity and calcium and phosphorus metabolism in mammals. In the present study, we measured the amounts of CT mRNA in the mouse brain, liver, kidney, heart and testis at various development stages, 14 days post-coitum (dpc), 17-dpc, newborn, 1 week and 8 weeks (adult), using real-time PCR. In the brain and kidney, the amount of CT mRNA decreased with development. In the testis, elevated amounts were observed at 17-dpc and 8 weeks. In the liver, the amount increased from the 14 dpc embryo to newborn stage and then decreased. In the heart, elevated amounts were observed at 17-dpc. Additionally, the CT antisense transcript was determined using a modified RT-PCR and nucleotide sequencing in the present study. Organs with high mRNA expressions were examined for localization of transcripts using in situ hybridization. The CT sense and antisense transcripts in the 14 dpc brain were mainly localized in the mesencephalon. In the pre- and postnatal stages, sense and antisense transcripts were shown to exist rather uniformly in the kidney, heart, liver and testis. In the 17-dpc rib and thyroid lobe and the adult ovary, the sense and antisense transcripts were found to be densely localized.
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http://dx.doi.org/10.1292/jvms.71.561DOI Listing
May 2009

Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers.

Zygote 2009 Aug 9;17(3):229-37. Epub 2009 Apr 9.

Laboratory of Animal Breeding and Reproduction, Division of Animal Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan.

Embryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.
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http://dx.doi.org/10.1017/S0967199409005280DOI Listing
August 2009

Improvement of embryonic development and production of offspring by transferring meiosis-II chromosomes of senescent mouse oocytes into cytoplasts of young mouse oocytes.

J Assist Reprod Genet 2009 Jan 19;26(1):35-9. Epub 2008 Dec 19.

United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo, Japan.

Purpose: The effects of reciprocal transplantation of meiosis-II chromosomes between senescent and young mouse oocytes were evaluated based on pre- and post-implantation development ability of resultant embryos.

Methods: Karyoplasts including meiosis-II chromosomes of oocytes from senescent Rockefeller mouse/Ms-Rb(6, 15) females (10 to 12 months, age-related infertile mice) were transferred into cytoplasts of oocytes from young F(1) females (3 to 5 months). Reconstructed oocytes were fertilized in vitro, and then the resultant embryos were cultured in vitro and transferred to recipient mice.

Results: The reconstructed oocytes that consisted of aged-karyoplasts and young-cytoplasts showed significantly improved embryonic development (from 23.2% to 30.0%) and development to term (from 6.3% to 27.1%, P < 0.05) as compared with the oocytes reconstructed from young-karyoplasts and aged-cytoplasts.

Conclusions: The present study showed successful rejuvenation for age-related infertility using transplantation of meiosis-II chromosomes in animal experimental models.
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http://dx.doi.org/10.1007/s10815-008-9282-6DOI Listing
January 2009

alphaCGRP and betaCGRP transcript amount in mouse tissues of various developmental stages and their tissue expression sites.

Brain Dev 2009 Oct 4;31(9):682-93. Epub 2008 Dec 4.

National Institute of Agro-biological Science, Tsukuba, Ibaraki, Japan.

The calcitonin gene-related peptides (CGRP), alphaCGRP and betaCGRP, have been implicated to play various roles in primates and rodent. However, since the expression information has been limited, in the present study, we measured the amount of gene expression in mouse brain, liver, kidney, heart, and testis at embryonic day (E) 14, E17, postnatal day (P) 1, P7, and adult using real-time PCR, and determined the precise localization of alphaCGRP and betaCGRP sense/antisense transcripts in tissues using in situ hybridization. The sense transcripts of alphaCGRP and betaCGRP were found mainly in brain, and their amount profiles were similar in the course of development: one expression peak was observed at E17 and the other at P7. The amounts of alphaCGRP transcripts were greater than those of betaCGRP transcripts in the range between 3.6 and 31 times. In the E17 and P7 brains, the localization pattern of alphaCGRP sense transcripts was similar with that of alphaCGRP antisense transcripts. Fewer transcripts were found in neuroblasts of E17 corpus callosum, and neuroblasts of P7 corpus callosum, olfactory bulb, plexus chorioideus, and ventriculus lateralis than in other brain areas. The localization pattern of betaCGRP sense and antisense transcripts was similar to that for alphaCGRP except that the betaCGRP antisense transcripts showed spot-like localizations. Additionally, the alphaCGRP sense transcript, and betaCGRP sense and antisense transcripts were found in parafollicular cells (C cells) of E17 thyroid lobe. These findings together indicate that alphaCGRP and betaCGRP have their own roles in the ontogenic process.
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http://dx.doi.org/10.1016/j.braindev.2008.10.011DOI Listing
October 2009

Blastocyst production from in vitro-produced day-2 bovine embryos classified by cleavage stage, and cytogenetical evaluation of the resultant day-8 blastocysts.

J Reprod Dev 2008 Dec 28;54(6):465-72. Epub 2008 Oct 28.

United Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan.

The present study was conducted to determine the criteria for selecting good quality embryos on Day-2 post-insemination and at the blastocyst stage. Bovine oocytes were matured, fertilized and cultured in vitro. First, Day-2 embryos were classified based on the number of blastomeres into 2-cell, 3- to 4-cell, 5- to 8-cell and >8-cell stage embryos; chromosome samples were then prepared. In the second experiment, the Day-2 embryos classified according to the number of blastomeres were cultured separately for an additional 6 days (Day 8). The resultant Day-8 blastocysts from each group of Day-2 embryos were classified into the following 3 grades based on morphology and diameter: Grade A, hatched and hatching blastocysts; Grade B, expanded blastocysts; and Grade C, unexpanded blastocysts. Chromosome samples were then prepared. The 5- to 8-cell stage Day-2 embryos had the lowest incidence of chromosomal abnormalities (13.5%, P<0.05) and the highest development rate to blastocysts (59.2%, P<0.05). Furthermore, the blastocysts derived from the 5- to 8-cell stage embryos had the largest mean number of cells (102.8+/-42.4, P<0.05), largest number of metaphases per blastocyst (9.5+/-4.8, P<0.05) and lowest incidence of chromosomal abnormalities (24.6%, P<0.05). The Grade A blastocysts had the largest mean number of cells (136.6+/-33.4, P<0.05), a large number of metaphases per blastocyst (11.9+/-5.5, P<0.05) and a low incidence of severe chromosomal abnormalities (17.3%). The results showed that, at Day 2, the 5- to 8-cell stage embryos were of better quality since they had the lowest incidence of chromosomal abnormalities and the highest blastocyst rate and the resultant blastocysts had the largest number of cells and lowest incidence of chromosomal abnormalities. In particular, selection of Grade A blastocysts can improve the development rate to term.
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http://dx.doi.org/10.1262/jrd.20036DOI Listing
December 2008

The blastocyst production rate and incidence of chromosomal abnormalities by developmental stage in in vitro produced porcine embryos.

J Reprod Dev 2008 Feb 19;54(1):22-9. Epub 2007 Nov 19.

United Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan.

The present study was conducted to determine the relationship between embryonic development speed at different stages (the cleaved stage at 52 h and the blastocyst stage at 6 days post insemination) and incidences of chromosome abnormalities in in vitro produced porcine embryos. Porcine oocytes were collected from 3-6-mm ovarian follicles obtained at a slaughterhouse and matured in modified NCSU-37 medium for 44-46 h. Following in vitro fertilization with a final concentration of 1 x 10(5) sperm/ml for 3 h, all oocytes were cultured in vitro for 52 h. Day-2 (52 h after insemination) embryos were classified according to their cleaved stages into 2-cell, 3- to 4-cell, 5- to 8-cell, and >8-cell stages; these were cultured separately for additional 4 days (Day 6). The resultant Day-6 blastocysts were classified according to the morphological diameter into 3 grades: Grade A, expanded blastocysts; Grade B, expanding blastocysts; and Grade C, early blastocysts. They were then analyzed chromosomally. The 3- to 4-cell and 5- to 8-cell embryos had significantly high blastocyst development rates (46.1 and 36.9%, respectively), and these blastocysts contained significantly more cells (40.2 and 42.4 cells, respectively) than those derived from 2-cell embryos and >8-cell embryos (28.6 and 26.5 cells, respectively). The incidence of chromosomal abnormalities was significantly higher in the blastocysts derived from 2-cell and >8-cell stage embryos than in the blastocysts derived from the other stage embryos. Furthermore, the grade A blastocysts had the lowest incidence of chromosomal abnormalities (35.3%) and contained the most cells (48.7 cells). Porcine in vitro production (IVP) yielded a high blastocyst rate and an excellent embryo quality when 3- to 4-cell and 5- to 8-cell stage embryos were selected on Day 2 after insemination. The same criteria yielded a higher quality of expanded blastocysts based on the stage of embryo development and morphology.
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http://dx.doi.org/10.1262/jrd.19102DOI Listing
February 2008

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

Reprod Med Biol 2007 Jun 14;6(2):85-90. Epub 2007 May 14.

United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo and.

The 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. Three-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. 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. Of 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. The 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 fertilization.
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http://dx.doi.org/10.1111/j.1447-0578.2007.00170.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906841PMC
June 2007

Cytogenetic analysis and developmental assessment of mouse embryos derived from in vitro fertilization of oocytes reconstructed by meiosis-II chromosome transplantation.

J Reprod Dev 2007 Apr 20;53(2):357-66. Epub 2006 Dec 20.

Laboratory of Animal Breeding and Reproduction, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan.

An electrofusion methodology for transferring meiosis-II chromosomes (M-II-t) has not been completely established. The present study compared the use of two temperatures (fusion at 37 C for Group A and 25 C for Group B) during an electrofusion procedure for mouse oocyte M-II-t and investigated the cytogenetic normality and developmental competence of embryos derived from in vitro fertilization using oocytes reconstructed by M-II-t. The M-II-t oocytes were fertilized in vitro and cultured to the blastocyst stage; the resultant embryos were analyzed cytogenetically. Subsequently, chromosomal normality of the resultant embryos at the prometaphase stage of first cleavage division and the integrity of the meiosis-II spindles of the reconstructed oocytes were analyzed. The success rate of electrofusion in Group B was 92.1%, which was significantly different from that in Group A (49.2%) (P<0.05). The fertilization rates (A, 80.7%; B, 77.2%) and development rates (A, 70.9%; B, 65.5%) in the M-II-t groups were significantly lower than those in the control group (95.0 and 92.2%, respectively) (P<0.05). The incidence of chromosomal abnormalities in the Group A embryos (20.5%) at the blastocyst stage was significantly higher than that in the control group embryos (8.5%) (P<0.05), but the incidence of chromosomal abnormalities in Group B (12.5%) was not significantly different compared with the other groups. A temperature of 25 C during the electrofusion procedure for M-II-t resulted in a good fusion rate, good development rate, and efficient production of chromosomally normal blastocysts. Furthermore, the incidence of chromosomal abnormalities in the first cleavage embryos at the prometaphase stage in Group B (9.6%) did not differ significantly from that in the control group (6.6%). The spindle morphology of the M-II-t oocytes in Group B was normal.
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http://dx.doi.org/10.1262/jrd.18114DOI Listing
April 2007

Visualization of human sperm chromosomes by using mouse oocytes cryothawed after enucleation.

Fertil Steril 2006 Aug 9;86(2):348-51. Epub 2006 Jun 9.

Science of Plant and Animal Production, United Graduated School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.

Objective: To evaluate whether previously enucleated mouse oocytes that are vitrified are usable for the analysis of human sperm chromosomes.

Design: Prospective, comparative laboratory study.

Setting: Animal breeding and reproduction department in a rural Japan university.

Patient(s): Sperm samples were obtained from a fertile donor.

Intervention(s): Human sperm were injected into previously enucleated-vitrified mouse oocytes.

Main Outcome Measure(s): The rates of appearance of the human sperm chromosomes that were injected into previously enucleated-vitrified mouse oocytes.

Result(s): The rates of appearance of human sperm chromosomes were 71%-72% in enucleated-vitrified mouse oocytes.

Conclusion(s): The use of enucleated-vitrified mouse oocytes allowed visualization of human sperm chromosomes.
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http://dx.doi.org/10.1016/j.fertnstert.2005.12.052DOI Listing
August 2006

A novel method for chromosome analysis of human sperm using enucleated mouse oocytes.

Hum Reprod 2005 May 21;20(5):1244-7. Epub 2005 Jan 21.

Science of Plant and Animal Production, United Graduated School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchuu-city, Japan.

Background: Mouse oocytes can be used in conjunction with intracytoplasmic sperm injection (ICSI) as a technique to permit chromosomal analysis of human sperm. However, chromosomes derived both from the human sperm and the mouse oocyte appear simultaneously following ICSI. The present study focused on evaluating whether or not previously enucleated mouse oocytes are usable for the analysis of human sperm chromosomes.

Methods: The metaphase chromosome-spindle complex was removed from a mouse oocyte. Human sperm from a donor with proven fertility were injected into mouse enucleated oocytes or intact oocytes. The presence of pronuclei in the oocytes was confirmed approximately 7-11 h after ICSI, and the oocytes were then fixed so that the nuclei could be observed as chromosome samples at 15-16 h after ICSI.

Results: The formation rate of one pronucleus in enucleated oocytes after ICSI was 93.9% (186/198) while that of two pronuclei in intact oocytes after ICSI was 85.4% (88/103). The appearance rate of metaphase chromosomes of human sperm in the enucleated oocytes, 89.4% (160/179), was significantly higher than that in intact oocytes, 78.7% (74/94) (P = 0.017).

Conclusions: An efficient ICSI method using enucleated mouse oocytes was established to allow the visualization of the human sperm chromosome complement without the risk of confusion with mouse oocyte chromosomes.
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http://dx.doi.org/10.1093/humrep/deh757DOI Listing
May 2005

Use of mouse oocytes to evaluate the ability of human sperm to activate oocytes after failure of activation by intracytoplasmic sperm injection.

Zygote 2004 May;12(2):111-6

United Graduated School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchuu-city, Tokyo, Japan.

The objective of the present study was to investigate the nuclei of human sperms that failed to fertilize human oocytes after intracytoplasmic sperm injection (ICSI). The sperms were injected into mouse oocytes by a piezo-micromanipulator, and some of these oocytes were artificially activated with strontium chloride (SrCl2) after ICSI. The oocytes were fixed, stained, and subjected to chromosomal analysis. The survival rate of mouse oocytes injected with infertile human sperms was 92.0% (46/50), while that of the control mouse oocytes injected with fertile human sperms was 73.6% (81/110). The rate of two pronuclei (2PN) formation was 0 (0/46) by the infertile sperms and 81.5% (66/81) by the fertile ones, a significant difference (p < 0.01). Sperm chromosomes in non-activated oocytes were present as premature chromosome condensation (PCC). Artificial activation after ICSI increased the 2PN formation rate in the infertile group to 90.3% (28/31). The results of the present study suggest that infertile sperms have a low potential to spontaneously activate oocytes and to form pronuclei. Thus, artificial activation after ICSI may rescue oocytes fertilized with infertile human sperms that do not produce 2PN. The present study proved the usefulness of mouse oocytes as specimens in evaluating the oocyte-activating capacity of objective human sperms prior to ICSI treatment.
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http://dx.doi.org/10.1017/s0967199404002606DOI Listing
May 2004

Pregnancy following chemical activation of oocytes in a couple with repeated failure of fertilization using ICSI: case report.

Hum Reprod 2004 Jul 29;19(7):1604-7. Epub 2004 Apr 29.

Kanayama Lady's Clinic, 1-201, Kanayama-cho, Atsuta-ku, Nagoya, 456-0002, Japan.

We report our attempts to achieve a successful pregnancy outcome with calcium ionophore A23187 and puromycin oocyte activation using sperm from a normozoospermic husband of a patient with previous repeated failed fertilization following ICSI. Oocytes from the female partner of a couple with a 4 year history of unexplained primary infertility with repeated failed fertilization following ICSI were used. In the latest ICSI attempt, oocytes were activated by treatment with calcium ionophore (5 min) and puromycin (5 h), then cultured. In this cycle, assisted oocyte activation with calcium ionophore and puromycin after ICSI resulted in a satisfactory fertilization rate (8/12; 66.7%); in prior cycles only one of 71 oocytes (1.4%) was fertilized. The outcome was a Caesarean section delivery of a healthy male infant without congenital abnormalities at 41 weeks, 2 days of gestation. In conclusion, the use of calcium ionophore and puromycin for oocyte activation was found to be a useful method in a case of repeated failed fertilization after ICSI.
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http://dx.doi.org/10.1093/humrep/deh294DOI Listing
July 2004

A method for genotyping Y chromosome-linked DYS385a and DYS385b loci.

Leg Med (Tokyo) 2003 Dec;5(4):228-32

Department of Legal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyzaki 889-1692, Japan.

We developed a method for genotyping Y chromosome-linked homologous DYS385 loci individually, combining locus-specific polymerase chain reaction (PCR) amplification and previously reported procedures. Duplicated DYS385a (5'-end) and DYS385b (3'-end) loci were located about 41 kb apart and inverted to each other in the Y chromosome, which data was obtained from the human genome sequence in National Center for Biotechnology Information (NCBI), and sequence differences were found at 424 bp down- and upstream of each locus. The locus-specific amplifications were performed using primers designed for this intergenic region, and fragments about 900 bp in length were produced. Polymorphic tetranucleotide arrays in the PCR products were typed according to procedures reported previously. Twenty male subjects were genotyped using this method. Alleles (GAAA)13-(GAAA)21 were observed at the DYS385a locus, and those at DYS385b included alleles (GAAA)9-(GAAA)15. DYS385a alleles, excluding those of identical arrays, were always larger than DYS385b alleles in the same subjects. These data suggest that the DYS385a and DYS385b loci can be amplified completely for discrimination, and the genotypes of the alleles provide information useful for forensic case work and population genetics.
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http://dx.doi.org/10.1016/s1344-6223(03)00082-8DOI Listing
December 2003

Effects of sera from infertile women with sperm immobilizing antibodies on fertilization and embryo development in vitro in mice.

Am J Reprod Immunol 2003 Aug;50(2):146-51

Department of Obstetrics and Gynecology, Jichi Medical School, Tochigi, Japan.

Problem: This study was performed to investigate if patients' sera with anti-human sperm antibodies show inhibitory effects on in vitro fertilization (IVF) and embryo development in mice.

Method Of Study: Patients' sera were collected from eight infertile women having sperm immobilizing antibodies and 17 infertile women without the antibodies. Male ICR mice and female F1 mice (BALB/c X C57BL/6J) were used. In mouse IVF, pre-incubated sperm were cultured in the medium containing patient's serum with or without sperm immobilizing antibodies, or bovine serum albumin (BSA) as a control. The fertilization rates and the incidences of blastocyst formation were compared.

Results: A mouse sperm immobilization test was established. Five (62.5%) of eight serum samples with sperm immobilizing antibodies and nine (52.9%) of 17 serum samples without the antibodies showed sperm immobilizing activities in mice. There was no significant difference between the two groups. Five sera with sperm immobilizing activities in human and mice, and five sera without sperm immobilizing activities in human or mice were used for the further experiments. The fertilization rates in BSA, patient's serum with sperm immobilizing antibodies, and that without the antibodies were 82.5% (746/904), 43.6% (508/1165), and 64.5% (669/1037), respectively. There were significant differences between the groups. The incidences of blastocyst formation were 59.9% (447/746), 31.7% (161/508), and 47.7% (319/669), respectively. There were also significant differences between the groups.

Conclusions: Some of the patient's serum with and without sperm immobilizing antibodies could immobilize sperm with complement. However, as compared with control, sera with sperm immobilizing activities against human and mouse sperm significantly blocked IVF and inhibited embryo development in mice. Further studies are required to investigate the mechanisms of the blocking effects of antisperm antibodies on fertilization and embryo development using the mouse model.
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http://dx.doi.org/10.1034/j.1600-0897.2003.00070.xDOI Listing
August 2003
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