Upregulation of iNOS and phosphorylated eNOS in the implantation-induced blastocysts of mice.

Purpose: This study aimed to examine expressions of iNOS and phosphorylated eNOS (p-eNOS) in implantation-induced blastocysts. We also examined the upstream of p-eNOS. Methods: To address the protein expressions in implantation-induced blastocysts, we performed immunohistochemical analysis using a delayed implantation mouse model. Immunostaining for iNOS, p-eNOS, and p-Akt was done. To address the relationship between p-eNOS and p-Akt, activated blastocysts were treated with an Akt inhibitor, MK-2206. Results: iNOS expression was at low levels in dormant blastocysts, whereas the expression was significantly increased in the activated blastocysts. Double staining of p-eNOS and p-Akt in individual blastocysts showed colocalization of p-eNOS and p-Akt of the trophectoderm. p-eNOS and p-Akt expressions were at low levels in dormant blastocysts, whereas both of them were significantly increased in the activated blastocysts. Both dormant and activated blastocysts showed significant positive correlations between p-eNOS and p-Akt. MK-2206 treatment for activated blastocysts showed that blastocysts with lower p-Akt had significantly lower p-eNOS levels. Conclusions: iNOS and p-eNOS, Ca2+ independent NOS, are upregulated by E2 in the blastocysts during implantation activation. Furthermore, p-eNOS is upregulated in implantation-induced blastocysts downstream of p-Akt.

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

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.

Tubulointerstitial nephritis antigen-like 1 deficiency alleviates age-dependent depressed ovulation associated with ovarian collagen deposition in mice.

PURPOSE: This study aimed to examine whether the Tinagl1 might be associated with ovulation in aged females and reproductive age-associated fibrosis in the stroma of the ovary. METHODS: To address the ovulatory ability and quality of ovulated oocytes, we induced ovulation by treatment with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) followed by in vitro fertilization. We also performed Picrosirius Red (PSR) staining to evaluate ovarian collagen deposition. RESULTS: As compared to ovulation in 8- to 9-month-old Tinagl1flox/flox mice, the number of ovulated oocytes from Tinagl1flox/flox mice decreased in an age-dependent manner in mice more than 10-11 months old, whereas the ovulated oocyte numbers in Tinagl1 -/- mice decreased significantly at 14-15 months. In vitro fertilization followed by embryo culture demonstrated the normal developmental potential of Tinagl1-null embryos during the preimplantation period. PSR staining indicated that collagen was found throughout the ovarian stroma in an age-dependent manner in Tinagl1flox/flox females, whereas those distributions were delayed to 14-15 months in Tinagl1 -/- females. This timing was consistent with the delayed timing of age-related decline of ovulation in Tinagl1 -/- females. CONCLUSIONS: The alleviation of age-associated depression of ovulation was caused by delayed ovarian collagen deposition in Tinagl1-null female mice.

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

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.

Molecular and cellular events during blastocyst implantation in the receptive uterus: clues from mouse models.

The success of implantation is an interactive process between the blastocyst and the uterus. Synchronized development of embryos with uterine differentiation to a receptive state is necessary to complete pregnancy. The period of uterine receptivity for implantation is limited and referred to as the "implantation window", which is regulated by ovarian steroid hormones. Implantation process is complicated due to the many signaling molecules in the hierarchical mechanisms with the embryo-uterine dialogue. The mouse is widely used in animal research, and is uniquely suited for reproductive studies, i.e., having a large litter size and brief estrous cycles. This review first describes why the mouse is the preferred model for implantation studies, focusing on uterine morphology and physiological traits, and then highlights the knowledge on uterine receptivity and the hormonal regulation of blastocyst implantation in mice. Our recent study revealed that selective proteolysis in the activated blastocyst is associated with the completion of blastocyst implantation after embryo transfer. Furthermore, in the context of blastocyst implantation in the mouse, this review discusses the window of uterine receptivity, hormonal regulation, uterine vascular permeability and angiogenesis, the delayed-implantation mouse model, morphogens, adhesion molecules, crosslinker proteins, extracellular matrix, and matricellular proteins. A better understanding of uterine and blastocyst biology during the peri-implantation period should facilitate further development of reproductive technology.

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

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.

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

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.

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

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.

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

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.

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

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.

Arginine with leucine drives reactive oxygen species-mediated integrin α5ß1 expression and promotes implantation in mouse blastocysts.

The implantation rate of in vitro fertilization (IVF)-derived blastocysts after embryo transfer remains low, suggesting that the inadequate expression of specific proteins in culture-induced IVF-derived blastocysts contributes to low implantation rates. Therefore, treatment with appropriate regulation may improve the blastocyst implantation ability. This study demonstrated that the combination of l-arginine (Arg) and l-leucine (Leu) exerts distinct effects on IVF-derived mouse blastocysts. Arg with Leu promotes blastocyst implantation, whereas Arg alone decreases the blastocyst ability. Integrin α5ß1 expression was increased in blastocysts treated with Arg and Leu. Arg with Leu also increased reactive oxygen species (ROS) levels and showed a positive correlation with integrin α5ß1. Ascorbic acid, an antioxidant, decreased ROS and integrin α5ß1 levels, which were elevated by Arg with Leu. Meanwhile, the mitochondrial membrane potential (ΔΨm) in blastocysts did not differ between treatments. Glutathione peroxidase (GPx) is involved in ROS scavenging using glutathione (GSH) as a reductant. Arg with Leu decreased GPx4 and GSH levels in blastocysts, and blastocysts with higher ROS levels had lower GPx4 and GSH levels. In contrast, Arg alone increased the percentage of caspase-positive cells, indicating that Arg alone, which attenuated implantation ability, was associated with apoptosis. This study revealed that elevated ROS levels induced by Arg with Leu stimulated integrin α5ß1 expression, thereby enhancing implantation capacity. Our results also suggest that ROS were not due to increased production by oxidative phosphorylation, but rather to a reduction in ROS degradation due to diminished GPx4 and GSH levels.

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

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).

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

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.

Functional analysis reveals that Tinagl1 is required for normal muscle development in mice through the activation of ERK signaling.

Tinagl1 (tubulointerstitial nephritis antigen-like 1) is a matricellular protein involved in female infertility and breast cancer tumorigenesis. In this study, we analyzed the function of Tinagl1 in skeletal muscle using knockout mice and cell experiments. Although primary myoblasts isolated from Tinagl1-decifient (Tinagl1-/-) mice differentiated into normal myotubes, and treatment with recombinant Tinagl1 did not affect the proliferation or differentiation of C2C12 myoblasts, Tinagl1-/- mice exhibited reduced body mass and calf muscle weights compared to the control group (Tinagl1flox/flox). Furthermore, Tinagl1-/- mice showed myofibers with centrally located nuclei, which is a morphological marker of regenerating muscle or myopathy. In addition, the capillary density in the soleus muscle of Tinagl1-/- mice showed a decreasing trend compared to that of the control group. Importantly, si-RNA-mediated knockdown of TINAGL1 resulted in reduced tube formation in human umbilical vein endothelial cells (HUVECs), whereas treatment with Tinagl1 promoted tube formation. Immunoblot analysis revealed that Tinagl1 activates ERK signaling in both HUVECs and C2C12 myoblasts and myotubes, which are involved in the regulation of myogenic differentiation, proliferation, metabolism, and angiogenesis. Our results demonstrate that Tinagl1 may be required for normal muscle and capillary development through the activation of ERK signaling.

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

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.

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

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.

Sustainable health promotion for the seniors during COVID-19 outbreak: a lesson from Tokyo.

The COVID-19 pandemic is novel corona virus infection outbreak that has gone global in 2020. Current prevention policies consist of hand hygiene and social distancing. Emergencies overloaded health services and shocked the logistics chains in many countries, especially Italy and China. Having more than a quarter of its population being elderly, Japan is at high risk for COVID-19 induced morbidity and mortality. This situation cancelled schedules of all routine group exercise activities for the seniors in Japan. While the outbreak is ongoing, staying at home is safe. However, successive days of being house-ridden and limited movement can lead to excessive physical inactivity. Some elderly who are not moving much can lose a significant amount of muscle strength, flexibility and aerobic capacity. It can accelerate the frailty and dependency of the seniors, and subsequently, claiming of care and health services. Moreover, existing and new evidences showed that physical activity can promote antiviral immunity. An alternative to usual group exercise activities is crucial to keep seniors active without affecting social distancing. While staying at home for long, functional exercises maintaining basic level of physical activity and movements are urgently required to be introduced to the seniors in Tokyo and around the world to prevent functional decline. Home exercise is a practical option. Therefore, we made a home-version of the functional training exercise video with different sets of 10-minutes exercise for 7 days a week. This breakthrough alternative may sustain health promotion for the elderly persons to preserve their active aging and maintain optimal health.

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

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.

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

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.

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

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.