Faculty Profiles - FUNAHASHI Hiroaki

Papers - FUNAHASHI Hiroaki

Division display 21 - 40 of about 105 ／ All the affair displays >>

Effect of removing cumulus cells from porcine cumulus-oocyte complexes derived from small and medium follicles during IVM on the apoptotic status and meiotic progression of the oocytes Reviewed International journal

Pilar Ferre, Tra Mi Thi Bui, Takuya Wakai, Hiroaki Funahashi

THERIOGENOLOGY 86 ( 7 ) 1705 - 1710 2016.10

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

The present study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small follicle (SF; 0.5-2 mm in diameter) and medium follicle (MF; 3-6 mm in diameter) when the oocytes were denuded before, during, and after IVM. Cumulus-oocyte complexes (COCs) were collected from SF or MF of prepubertal gilt ovaries. Before or 20 hours after the start of IVM culture, some oocytes were denuded and cultured for IVM. At the end of IVM, apoptotic status and meiotic progression of the oocytes were compared with oocytes matured in the presence of cumulus cells (CCs) by Annexin-V/PI assay and 4',6-Diamidino-2-phenylindole staining. Apoptotic status of the oocytes was only affected by time when the oocytes were denuded. In both oocytes from SF and MF, although the incidence of early and late apoptotic oocytes was significantly higher when the CCs were removed before IVM, the rate was significantly lower when CCs were removed 20 and 44 hours after the start of IVM. The incidence of mature oocytes was significantly affected by both the origin of COCs and time when oocytes were denuded from the COCs. Although the percentage of mature oocytes was higher in MF than SF, maturation rates were significantly higher when oocytes were denuded 20 hours, as compared with 0 and 44 hours after the start of IVM. However, the percentage of mature oocytes with a morphologically normal spindle was significantly higher when oocytes were denuded 44 hours, rather than 22 hours of IVM. In conclusion, removing CCs 20 hours after the start of IVM seems to promote meiotic progression of the oocytes to the metaphase-II stage even when the COCs were collected from SF, although factor(s) from or communication with CCs during IVM may need to obtain a morphologically normal spindle in mature oocytes. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2016.05.024

Application of a microfluidic sperm sorter to in vitro production of dairy cattle sex-sorted embryos Reviewed International journal

Jingchun Li, Sibing Zhu, Xianjing He, Rui Sun, Qianyu He, Yi Gan, Shengjun Liu, Hiroaki Funahashi, Yanbing Li

THERIOGENOLOGY 85 ( 7 ) 1211 - 1218 2016.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Viable sperm from sex-sorted semen without centrifugal treatment was separated by a microfluidic sperm sorter (MFSS) for IVF to improve in vitro embryo production of dairy cattle. The MFSS was originally developed to isolate motile human sperm by two laminar flows in the micro-channel (there are four chambers in an MFSS. Chamber A is the inlet for semen, chamber B is the inlet for the medium, chamber C is the exit chamber for motile sperm, and chamber D is the outlet for nonmotile sperm). Sex-sorted sperm were adjusted to 1 x 10(7) spermatozoa/mL (2 million cells/dose, sperm motility was 30% above after thawing). In a first experiment, diluted sex-sorted semen was mixed with modified Medium199(mM199) containing 5-mM caffeine for 5 minutes, resulting in variations in sperm concentration and quality parameters at chambers A, C, and D. In a second experiment, medium containing sperm from three MFSS chambers was collected and mitochondrial activity of the sperm was determined by flow cytometry, the relative activity of sperm mitochondria in chamber C (1.56 +/- 0.03) was the highest in three observation areas (P < 0.05). Thus, sperm motility and mitochondrial activity of sperm was high in chamber C. In a third experiment, different concentrations of sperm were added to chamber A and dairy cattle IVM oocytes were placed in chamber C, where motile spermatozoa will accumulate, with mM199 containing 5-mM caffeine for 5 minutes, and then cultured in caffeine-free mM199 for 8 hours. The results showed that sperm penetration rate, the monospermic penetration rate, and blastocyst rate of the 10 x 10(6) group (10 x 10(6) sperm/mL) were higher than in the 1 x 10(6) and 5 x 10(6) groups (P < 0.05). In the last experiment, we compared sperm penetration in the MFSS-IVF system with a modified standard IVF method (cocultured in droplets for 8 hours). The normal fertilization index (the ratio of monospermic oocytes to the number of oocytes examined) 8 hours after insemination was higher in the MFSS-IVF system than the modified standard IVF system (P < 0.05). Developmental competence of fertilized oocytes to the blastocyst stage was also higher in the MFSS-IVF system (40.12% +/- 2.61%) than the modified standard IVF technique (24.55% +/- 4.54%). These results demonstrate that a short coculture of dairy cattle oocytes with isolated motile sex-sorted spermatozoa gradually accumulated in the MFSS device improves the efficiencies of normally produced fertilized embryos and blastocyst formation. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2015.12.001

Milk supplements in a glycerol free trehalose freezing extender enhanced cryosurvival of boar spermatozoa Reviewed

Rukmali Athurupana, Hiroaki Funahashi

Asian pacific journal of reproduction 5 ( 1 ) 58 - 62 2016.3

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Objective: To evaluate the effect of skim milk and/or coconut milk in a glycerol-free trehalose extender to improve cryosurvival of boar spermatozoa. Methods: Sperm samples were diluted in an egg-yolk-based freezing extender containing 100 mM trehalose and 0.25% Equex STM supplemented with coconut milk or/and skim milk at 2% or 5% (w/v). Spermatozoa were cryopreserved in 0.5 mL straws and thawed by a rapid transient method (at 70 °C for 8 s) followed with a stabilizing procedure at 39 °C. Thawed samples were analyzed for motility, viability, high mitochondrial membrane potential (HMMP), and acrosome damage. Results: Even on the presence of egg yolk, motility, HMMP and viability were significantly higher in extender supplemented with 2% skim milk than controls without skim milk (. P < 0.05). Post-thaw viability significantly improved with the addition of 2% skim milk plus 2% coconut milk as well (. P < 0.05). Acrosome damage was considerably lower when the extender was supplemented with 2% coconut milk (. P < 0.05), whereas the benefit was masked in the presence of 2% skim milk. Conclusion: 2% skim milk can be used as supplements for a glycerol-free trehalose and egg yolk-based extender to improve post-thaw survival of boar spermatozoa, whereas 2% coconut milk has an effect to protect boar spermatozoa from acrosome damage.

DOI： 10.1016/j.apjr.2015.12.010

A phosphodiesterase type-5 inhibitor, sildenafil, induces sperm capacitation and penetration into porcine oocytes in a chemically defined medium Reviewed International journal

Sumire Ioki, Qing-Shan Wu, Osamu Takayama, Hideyuki H. Motohashi, Takuya Wakai, Hiroaki Funahashi

THERIOGENOLOGY 85 ( 3 ) 428 - 433 2016.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

The present study was undertaken to determine the effect of a phosphodiesterase (PDE) type-5 (cyclic guanosine monophosphate-specific) inhibitor, sildenafil, on capacitation and penetration of boar spermatozoa in a basic chemically defined medium (adenosine- and theophylline-free PGM-tac4). When ejaculated spermatozoa were cultured for 90 minutes in the absence or presence of sildenafil at 2.5 mM, the inhibitor significantly increased the percentage of capacitated/acrosome-reacted spermatozoa, as a result of the chlortetracycline assay. When fresh spermatozoa were co-cultured with oocytes in the presence of sildenafil at a different concentration (0, 2.5, 25, or 250 mu M), higher sildenafil concentrations (25 and 250 mu M) significantly resulted in higher sperm penetration rates. When oocytes matured in vitro were co-cultured with spermatozoa in the presence of 25 mu M sildenafil or 25 mM caffeine benzoate for 8 hours, the incidence of penetrated oocytes did not differ between two groups, whereas the incidence of monospermic oocytes in penetrated one was significantly higher in the presence of sildenafil. Immunocytochemical analysis reported the presence of PDE type-5 on the acrosome region of boar spermatozoa. These results report that regulation of cyclic guanosine monophosphate-specific PDE type5 by sildenafil somehow can increase the penetrability of boar spermatozoa in vitro. (c) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2015.09.013

In vitro fertilization in pigs: New molecules and protocols to consider in the forthcoming years Reviewed

Raquel Romar, Hiroaki Funahashi, Pilar Coy

THERIOGENOLOGY 85 ( 1 ) 125 - 134 2016.1

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Assisted reproduction technology (ART) protocols are used in livestock for the improvement and preservation of their genetics and to enhance reproductive efficiency. In the case of pigs, the potential use of embryos for biomedicine is being followed with great interest by the scientific community. Owing to the physiological similarities with humans, embryos produced in vitro and many of those produced in vivo are used in research laboratories for the procurement of stem cells or the production of transgenic animals, sometimes with the purpose of using their organs for xenotransplantation. Several techniques are required for the production of an in vitro-derived embryo. These include in vitro oocyte maturation, sperm preparation, IVF, and further culture of the putative zygotes. Without doubt, among these technologies, IVF is still a critical limiting factor because of the well-known, but still unsolved, question of polyspermy. Despite the improvements made in the past decade, current IVF systems hardly reach 50% to 60% efficiency and any progression in porcine ARTs requires an unavoidable improvement in the monospermy rate. It is time, then, to learn from what happens under in vivo physiological conditions and to transfer this knowledge into ART. This review describes the latest advances in porcine IVF, from sperm preparation procedures to culture media supplements with special attention paid to molecules with a known or potential role in in vivo fertilization. Oviductal fluid is the natural medium in which fertilization takes place, and, in the near future, could become the definitive supplement for culture media, where it would help to solve many of the problems inherent in ARTs in swine and improve the quality of in vitro-derived porcine embryos. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2015.07.017

Rapid thawing and stabilizing procedure improve postthaw survival and in vitro penetrability of boar spermatozoa cryopreserved with a glycerol-free trehalose-based extender Reviewed

Rukmali Athurupana, Sumire Ioki, Hiroaki Funahashi

THERIOGENOLOGY 84 ( 6 ) 940 - 947 2015.10

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Thawing process is important in semen cryopreservation as it brings back the sperm cell to physiologic temperature reactivating the metabolism. Aims of the present study were to evaluate survival rate and in vitro penetrability of boar frozen spermatozoa after rapid and transient thawing at a high temperature followed by a warming procedure at 39 degrees C. Ejaculated semen samples were diluted in an egg yolk based glycerol-free extender containing 100-mM trehalose and then cryopreserved in 0.5-mL straws according to a common protocol. In experiment 1, when temperature inside the straws was monitored after thawing at 40 degrees C, 60 degrees C, 70 degrees C, and 80 degrees C, the calculated average warming rate in the straws from -196 degrees C to 15 degrees C was much faster when thawed at 70 degrees C and 80 degrees C than at 40 degrees C (P < 0.01). The warming temperature rate inside the straw was 7 to 12 folds faster during the first 2 seconds than the second 2 seconds after immersing in high temperatures. In experiment 2, when frozen straws were thawed at 80 degrees C for 9 seconds, the viability, motility, and acrosomal integrity were significantly improved (P < 0.05), as compared with controls (at 39 degrees C). In experiment 3, frozen straws were thawed at 39 degrees C, 60 degrees C, 70 degrees C, and 80 degrees C for 60, 10, 8, and 6 seconds, respectively, and then maintained at 39 degrees C for 0, 50, 52, and 54 seconds. Higher viability, motility, mitochondria membrane potential, and acrosome integrity were observed (P < 0.05) when frozen straws were thawed at 70 degrees C for 8 seconds and then maintained at 39 degrees C for 52 seconds as compared with the control (39 degrees C for 60 seconds). In experiment 4, in vitro penetrability of frozen spermatozoa thawed at 70 degrees C for 8 seconds and maintained at 39 degrees C for 52 seconds was higher than that of controls. In conclusion, the rapid transient thawing at 70 degrees C for 8 seconds followed by stabilizing procedure at 39 degrees C for 52 seconds maintained the viability, motility, mitochondria membrane potential, acrosome integrity, and in vitro penetrability of spermatozoa frozen in a glycerol-free trehalose extender and recommended as an optimum thawing conditions. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2015.05.033

Trehalose in glycerol-free freezing extender enhances post-thaw survival of boar spermatozoa Reviewed

Rukmali Athurupana, Daisen Takahashi, Sumire Ioki, Hiroaki Funahashi

JOURNAL OF REPRODUCTION AND DEVELOPMENT 61 ( 3 ) 205 - 210 2015.6

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

Cryopreservation of boar semen is still considered suboptimal due to lower fertility as compared with fresh samples when glycerol, a permeating cryoprotectant, is used. Trehalose is a non-permeable cryoprotectant and nonreducing disaccharide known to stabilize proteins and biologic membranes. The aim of this study was to evaluate the cryosurvival and in vitro penetrability of boar spermatozoa when glycerol was replaced with trehalose in a freezing extender. Ejaculated Berkshire semen samples were diluted in egg yolk-based freezing extender containing glycerol (100 mM) or trehalose (0, 50, 100, 150, 200 and 250 mM) and cryopreserved using a straw freezing procedure. Thawed samples were analyzed for motility, viability, mitochondrial membrane potential (MMP), and acrosome integrity. In experiment 2, penetrability of spermatozoa cryopreserved with 100 mM glycerol or trehalose was examined. Replacement of cryoprotectant glycerol (100 mM) with trehalose had no effect on sperm viability, but replacing it with 100 mM trehalose improved motility, MMP and acrosome integrity significantly. Sperm motility and MMP were considerably higher in 100 mM trehalose, whereas the acrosome integrity was substantially higher in 100-250 mM trehalose. The in vitro penetration rate was also significantly higher in spermatozoa cryopreserved with trehalose (61.3%) than in those cryopreserved with glycerol (43.6%). In conclusion, 100 mM non-permeable trehalose can be used to replace glycerol, a permeating cryoprotectant, for maintenance of better post-thaw quality of boar spermatozoa.

DOI： 10.1262/jrd.2014-152

Development competence and relative transcript abundance of oocytes derived from small and medium follicles of prepubertal gilts Reviewed

Chiyuki Kohata, Maria Jose Izquierdo-Rico, Raquel Romar, Hiroaki Funahashi

THERIOGENOLOGY 80 ( 9 ) 970 - 978 2013.12

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

The objective of this study was to examine the competence of mature oocytes aspirated from small follicles (SF, <2 mm in diameter) and medium follicles (MF, 3-6 mm) of abattoir-derived prepubertal gilt ovaries. Oocytes were selected by the presence of the first polar body (1pb) after IVM in a chemically defined medium, for sperm penetration, pronuclear formation, cleavage rate, and development to the blastocyst stage. Relative transcript abundance of genes associated with regulation of oocyte maturation (AURKA, AURKB, and MOS), fertilization (ZP3 and ZP4), maternal effect (NALP9 and HSF1), and anti-apoptosis (BCL2) were also examined in oocytes at germinal vesicle (GV) and metaphase-II (MII) stages. In SF, compared with MF, the maturation rate post-IVM was lower (P < 0.05), but there were no differences in sperm penetration rate (78.2% and 68.5% at 6 hours after insemination and 90.8% and 91.9% at 9 hours after insemination, P = 0.51 and P = 0.67, respectively), the percentage of oocytes that formed both female and male pronuclei (27.9% and 25.8% at 6 hours after insemination and 79.4% and 76.1% at 9 hours after insemination), or cleavage rate at 48 hours after insemination (85.9% and 89.7%, respectively, P = 0.46), whereas blastocyst formation rate was lower (P < 0.05) in oocytes from SF versus MF (14.7% and 31.0%). Transcript abundances decreased (P < 0.05) in all genes examined between the GV and MII stages, although only transcript abundance for MOS was lower (P < 0.05) in CV oocytes from SF versus MF. In conclusion, mature oocytes from SF and MF of prepubertal gilts with a visible 1pb had similar fertilizability in vitro and relative transcript abundance of nine genes. However, follicle size affected meiotic competence, early embryonic development to the blastocyst stage, and transcript abundance of the MOS gene. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2013.07.023

A microfluidic device to reduce treatment time of intracytoplasmic sperm injection Reviewed International journal

Koji Matsuura, Takuya Uozumi, Takuya Furuichi, Ikuyo Sugimoto, Mieko Kodama, Hiroaki Funahashi

Fertility and sterility 99 ( 2 ) 400 - 407 2013.2

Authorship：Last author Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier science inc

Objective: To develop a microfluidic device that can reduce the intracytoplasmic sperm injection (ICSI) treatment time by increasing sperm concentration.Design: We compared the ICSI treatment time required for porcine sperm using a method employing the microfluidic device and one using the conventional microdroplet method.Settings: Academic research laboratories at Okayama University.Animal(s): Reproductive cells of porcine sperm, oocytes, and embryos.Intervention(s): Cell manipulations, ICSI, and embryo culture.Main Outcome Measure(s): Average ICSI treatment time and sperm concentration.Result(s): The average ICSI treatment time (mean +/- SEM) using the method with the microfluidic device for poor-quality semen (sperm concentration, 2.0 x 10(4) cells/mL) was significantly shorter than the treatment time using the conventional microdroplet method (265 +/- 15 seconds [n = 43] vs. 347 +/- 19 seconds [n = 50]). When diluted semen with a sperm concentration of 2.0 x 10(5) cells/mL was used, no significant difference was observed between the two methods (n = 50 and n = 48).Conclusion(s): The microfluidic device can reduce the time required for ICSI treatment that is used to increase sperm concentration in poor-quality semen samples. The results suggest that this device may be clinically useful for ICSI treatment in human assisted reproductive technology. (Fertil Steril (R) 2013;99:400-7. (C) 2013 by American Society for Reproductive Medicine.)

DOI： 10.1016/j.fertnstert.2012.10.022

Effects of caffeine on sperm characteristics after thawing and inflammatory response in the uterus after artificial insemination with frozen-thawed boar semen Reviewed International journal

S. Yamaguchi, C. Suzuki, M. Noguchi, S. Kasa, M. Mori, Y. Isozaki, S. Ueda, H. Funahashi, K. Kikuchi, T. Nagai, K. Yoshioka

Theriogenology 79 ( 1 ) 87 - 93 2013.1

Language：English Publishing type：Research paper (scientific journal)

We previously reported that AI with frozen-thawed boar semen supplemented with caffeine increased the number of uterine sperm by inhibiting migration of polymorphonuclear leukocytes (PMNs) into the uterine lumen, and also improved fertility of gilts and sows. The objective of the present study was to determine the effects of the addition of caffeine to a thawing solution on postthaw sperm quality and uterine inflammatory response after AI with frozen-thawed boar semen. Incubation of frozen-thawed sperm in Modena solution supplemented with 10 mM caffeine for 90 minutes improved (P < 0.05) percentages of progressive motility, straightness, and linearity of sperm movement compared with no caffeine, without causing damage to plasma or acrosomal membranes. Gilts inseminated once with 2 × 109 frozen-thawed sperm suspended in Modena solution with or without caffeine, and gilts that did not receive AI, were slaughtered 4 hours later. Uteri were recovered for analysis of number of uterine PMNs and mRNA expression (quantitative reverse transcription polymerase chain reaction) of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-8, monocyte chemoattractant protein-1, and cyclooxygenase 2 in the endometrium. Caffeine decreased (P < 0.05) both the number of total uterine PMNs and expression of IL-8 mRNA in the endometrium after AI. The amount of IL-8 and cyclooxygenase 2 mRNA after AI in the absence of caffeine were higher than samples from gilts that did not receive AI (P < 0.05), whereas there were no significant differences between treatments in expression levels of tumor necrosis factor-α, IL-1β, IL-6, or monocyte chemoattractant protein-1 mRNA. Pregnancy rate in sows inseminated with sperm supplemented with caffeine (16 of 23; 70%) tended (P < 0.1) to exceed that without caffeine (12 of 26; 46%), but litter size was not affected. In conclusion, the addition of caffeine to the thawing solution inhibited migration of uterine PMNs, probably by downregulating IL-8 mRNA expression in the endometrium. © 2013 Elsevier Inc.

DOI： 10.1016/j.theriogenology.2012.09.012

What is the optimal condition for fertilization of IVM oocytes? Invited Reviewed

Hiroaki Funahashi

Reproductive medicine and biology 12 ( 1 ) 15 - 20 2013.1

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Application of in vitro maturation (IVM) is recently increasing for human infertility, especially to rescue patients of polycystic ovarian syndrome and ovarian hyperstimulation syndrome. To increase the application of IVM oocytes for embryo production and the efficiency of successful production of babies using IVM oocytes, quality control of oocytes and achievement of fertilization in the most suitable condition may be very important. In this paper, suitable conditions for fertilization of IVM oocytes will be discussed with recent knowledge about IVM and in vitro fertilization of oocytes in domestic animals. Currently, human oocytes are collected mainly from patients' ovaries 36 h following mild gonadotropin stimulation and used for IVM for 24-26 h. However, asynchronous progression of those oocytes to reach the metaphase-II stage may have occurred during the IVM culture. In the oocytes that have already progressed to the metaphase-II stage, sudden aging such as reduction in maturation promoting factor and MAP kinases will start to occur. Application of specific inhibitors of phosphodiesterase to control intracellular cAMP (cyclic adenosine monophosphate) level may be effective to synchronize timings of the germinal vesicle breakdown and consequently the meiotic progression of oocytes, and to improve the developmental competence. Furthermore, treatment of aging oocytes with caffeine appears to rescue them from reductions in maturation promoting factor and MAP kinases and to improve the developmental competence. Assessment methods to select oocytes with good quality may also be important to improve the successful rates.

DOI： 10.1007/s12522-012-0134-9

Successful delivery derived from vitrified-warmed spermatozoa from a patient with nonobstructive azoospermia Reviewed International journal

Yuji Endo, Yoshitaka Fujii, Shozo Kurotsuchi, Hiroaki Motoyama, Hiroaki Funahashi

Fertility and Sterility 98 ( 6 ) 1423 - 1427 2012.12

Authorship：Last author Language：English Publishing type：Research paper (scientific journal)

Objective: To report the clinical outcomes following intracytoplasmic sperm injection (ICSI) with vitrified sperm from patients with severe male factor infertility. Design: Retrospective case series. Setting: IVF unit of a medical center. Patient(s): Three patients with severe oligozoospermia or nonobstructive azoospermia (NOA). Intervention(s): Cryopreservation of limited numbers of spermatozoa with the use of Cryotop and Cell Sleeper as nonbiologic containers. Main Outcome Measure(s): Four cycles underwent intracytoplasmic sperm injection (ICSI) with vitrified sperm. Result(s): A total of 148 spermatozoa in 18 containers (8.2 sperm per container) were vitrified and 36 of them (5 containers) were warmed. Thirty-three sperm (92%) were retrieved successfully and injected individually into 17 mature oocytes. Fertilization was observed in 12 oocytes (71%), and all zygotes (100%) cleaved. A couple with NOA achieved a singleton pregnancy and concluded with full-term delivery of a healthy boy (2,632 g). Conclusion(s): A successful delivery was achieved after transfer of a blastocyst derived from vitrified-warmed spermatozoa. A small number of vitrified sperm cells were used for ICSI to fertilize oocytes with predictable timing. © 2012 by American Society for Reproductive Medicine.

DOI： 10.1016/j.fertnstert.2012.07.1128

Glycosaminoglycans Improves Early Development of Zona-free 8-cell Rat Embryos to Blastocysts in a Chemically Defined Medium, but Not the Pregnancy Rate Following Transfer of the Blastocysts Reviewed

Masanobu Okuyama, Hiroaki Funahashi

JOURNAL OF REPRODUCTION AND DEVELOPMENT 58 ( 3 ) 295 - 301 2012.6

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

The objective of the present study was to clarify the possible role of the zona pellucida (ZP) in early development of rat embryos and to determine the effect of glycosaminoglycans on the development of ZP-free 8-cell embryos before or after embryo transfer at the blastocyst stage. Eight-cell embryos were divided into three groups comprised of 1) intact controls, 2) embryos with the ZP was removed with acidic solution and 3) pairs of ZP-free 8-cell embryos aggregated in a small hollow. These embryos were cultured in a chemically defined m RIECM for 24 h. Developmental ability to the blastocyst stage and mean cell number in the blastocyst was lower in ZP-free embryos than in intact controls. When these blastocysts were transferred, the farrowing rate and efficiency of embryos developed to term were also lower in ZP-free embryos, but not in the aggregated ones. Supplementation with hyaluronan (HA; 63-250 mu g/ml) or heparan sulfate proteoglycan (HS; 15 mu g/ml) significantly improved blastocyst formation of ZP-free embryos and the cell number in the blastocyst by reducing the incidence of apoptosis. However, there were no beneficial effects of HA or HS on farrowing and newborn rates after transfer of the blastocysts. In conclusion, the ZP plays roles in maintaining successful development of early rat embryos at least from the 8-cell stage not only to the blastocyst stage but also to posttransfer stages. Glycosaminoglycans, such as HA or HS, appear to contribute to successful cleavage during early development to the blastocyst stage but may be insufficient to maintain the posttransfer survival of ZP-free embryos.

DOI： 10.1262/jrd.11-092H

Effect of the addition of beta-mercaptoethanol to a thawing solution supplemented with caffeine on the function of frozen-thawed boar sperm and on the fertility of sows after artificial insemination Reviewed International journal

S. Yamaguchi, H. Funahashi

Theriogenology 77 ( 5 ) 926 - 932 2012.3

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

We have reported that artificial insemination (AI) with frozen-thawed boar semen supplemented with caffeine increased the number of uterine sperm by inhibiting the migration of polymorphonuclear leukocytes (PMNs) into the uterine lumen, thereby improving the fertility of gilts and sows. The objective of the present study was to examine the effects of the addition of the antioxidant beta-mercaptoethanol (bME) and caffeine to the thawing solution on the function of frozen-thawed sperm, on the phagocytic activity of PMNs for sperm, and on the fertility of sows after AI. When frozen-thawed sperm were cultured in the presence of 25 or 50 μm bME, sperm capacitation and spontaneous acrosome reactions were inhibited (P &lt
0.01). There was no effect of bME on phagocytic activity of PMNs for sperm in vitro. When hormonally treated (400 IU of equine chorionic gonadotropin + 200 IU of human chorionic gonadotropin) weaned sows experienced a single intrauterine insemination with frozen-thawed sperm (25 × 10 8 sperm per 50 ml dose) 40 h after subsequent hCG administration, pregnancy and farrowing rates were unaffected by the addition of 50 μm bME (pregnancy rate, 20 vs 21% in controls
farrowing rate, 20 vs 21%
n = 15 and 14, respectively). However, litter size tended to be higher than in the presence of 50 μm bME compared to its absence (10.0 ± 1.0 vs 5.7 ± 1.5, respectively
P &lt
0.07). Thus, the addition of bME to the thawing solution containing caffeine could be of benefit for improving the function of frozen-thawed sperm without influencing the phagocytic activity of PMNs for sperm. Although there were no statistically significant effects of bME on pregnancy or farrowing rates, the litter size tended to be higher in the sows subjected to a fixed-time single AI treatment with synchronized ovulation. © 2012 Elsevier Inc..

DOI： 10.1016/j.theriogenology.2011.09.023

Simple vitrification for small numbers of human spermatozoa Reviewed International journal

Yuji Endo, Yoshitaka Fujii, Kasumi Shintani, Momoyo Seo, Hiroaki Motoyama, Hiroaki Funahashi

Reproductive BioMedicine Online 24 ( 3 ) 301 - 307 2012.3

Authorship：Last author Language：English Publishing type：Research paper (scientific journal)

Conventional freezing procedures and containers are not appropriate for spermatozoa from the testis because of their low number and poor in-situ motility, and various types of container have been utilized to freeze small numbers of spermatozoa. This study tried to develop a vitrification method for small numbers of spermatozoa using the Cell Sleeper, which is a closed type of cell-cryopreservation container. The container with spermatozoa were cooled in liquid nitrogen vapour and then stored in a cryotank. Sperm motility parameters improved significantly (P &lt
0.05) by vitrification in oil-free droplets rather than in droplets covered with oil. After vitrification of five spermatozoa per container, all spermatozoa were recovered and the viable sperm rate was significantly higher when spermatozoa were vitrified in a 3.5-μl droplet rather than in 0.5 μl (72.0% versus 38.0%
P &lt
0.01). Recovery, motility and viability rates of vitrified-warmed spermatozoa were similar between the Cell Sleeper and the CryoTop groups. In conclusion, the Cell Sleeper is a highly effective tool for the cryopreservation of small numbers of spermatozoa and limited cells can be vitrified quickly and simply without significant loss. © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.rbmo.2011.11.016

Boar seminal plasma or hen's egg yolk decrease the in-vitro chemotactic and phagocytotic activities of neutrophils when co-incubated with boar or bull sperm Reviewed International journal

J. C. Li, S. Yamaguchi, H. Funahashi

Theriogenology 77 ( 1 ) 73 - 80 2012.1

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

The objective was to determine the effects of boar seminal plasma and hen's egg yolk on chemotaxis and phagocytosis of porcine and bovine polymorphonuclear neutrophils (PMNs) in vitro. Chemotactic activity of PMNs was determined following culture for 90 min in a blind well chamber. Phagocytosis was assayed after co-culture of PMNs with sperm for 60 min. In the presence of ≥ 5% boar seminal plasma, chemotactic activity of PMNs was reduced (P &lt
0.05) in both pigs (from 1126.1 to 934.2-1009.1 cells/mm 2) and in cows (from 1067.1 to 768.9-800.0 cells/mm 2). Furthermore, ≥ 5% boar seminal plasma reduced (P &lt
0.05) leukocyte phagocytosis in pigs (26.2-32.1%) and cows (27.2-30.0%) compared to controls (41.7 and 42.1%, respectively). Although 20% hen's egg yolk increased (P &lt
0.05) chemotactic activity of PMNs in pigs (from 790.4 to 1006.1 cells/mm 2) and cows (from 789.9 to 953.5 cells/mm 2), egg yolk increased (P &lt
0.05) phagocytotic activity of porcine PMNs (from 24.3 to 33.8%), but not the activity of bovine PMNs (15.1 vs 15.8% in controls). Boar seminal plasma and caffeine reduced (P &lt
0.05) the egg yolk-induced increase in chemotaxis in both species (from 988.6 to 795.2 or 813.2 cells/mm 2 in pigs and from 953.5 to 779.4 or 833.8 cells/mm 2 in cows), and phagocytotic activities of PMN (from 33.8% to 15.2 or 13.3%) only in pigs (but not in cows
11.2-15.1%). In conclusion, hen's egg yolk increased chemotactic activity of PMNs in both pigs and cows, whereas egg yolk increased only phagocytosis of PMNs in pigs, but not in cows. Even in the presence of egg yolk, boar seminal plasma and caffeine significantly reduced chemotactic activity of PMNs in pigs and cows, and phagocytotic activity of porcine PMNs. © 2012 Elsevier Inc.

DOI： 10.1016/j.theriogenology.2011.07.018

Caffeine, dibutyryl cyclic-AMP and heparin affect the chemotactic and phagocytotic activities of neutrophils for boar sperm in vitro Reviewed International journal

J. C. Li, S. Yamaguchi, Y. Kondo, H. Funahashi

Theriogenology 75 ( 7 ) 1336 - 1345 2011.4

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

The objective was to examine the effects of caffeine, dibutyryl cyclic AMP, and heparin on the chemotaxis and/or phagocytosis of PMNs for porcine sperm. The chemotactic activity of PMNs, determined in a blind well chamber, increased (P &lt
0.05) when fresh serum was added to the medium (control containing BSA, 1109.5 cells/mm2 vs serum, 1226.3 cells/mm2), regardless of the presence of sperm (control, 1121.1 cells/mm2 vs serum, 1245.8 cells/mm2), whereas heat-inactivated serum did not affect activity (without sperm, 1099.4 cells/mm2 and with sperm, 1132.6 cells/mm2). Regardless of live and dead sperm and of the origin of PMNs (boars vs sows), the phagocytotic activity of PMNs, as determined by co-culture of PMNs with sperm for 60 min, increased (P &lt
0.05) in the presence of fresh serum containing active complement (46.7 and 43.0%, respectively), but stimulation was decreased (P &lt
0.05) when 1 mM or higher concentrations of caffeine was added to the medium (from 40.7 to 20.8-31.6%). The origin of PMNs (sows vs boars) did not significantly affect phagocytotic activity. The percentage of PMNs that phagocytized polystyrene latex beads decreased when 2 mM caffeine was added to the medium containing porcine serum (from 43.7 to 21.5%). Serum-stimulated chemotactic activity of PMNs (1089.9 cells/mm2) was also reduced (P &lt
0.05) with 2 mM caffeine (942.5 cells/mm2). Furthermore, dibutyryl cAMP at ≥ 0.1 mM or heparin at ≥ 100 μg/mL decreased phagocytotic activity, in a concentration-dependent manner (P &lt
0.05). Supplementation of PMNs with heparin at 100 or 500 μg/mL decreased (P &lt
0.05) chemotactic activity in the presence of serum (from 1137.1 cells/mm2 to 1008.8-1026.3 cells/mm2). We inferred that opsonization in the presence of active complement stimulated phagocytotic and chemotactic activities of PMNs, whereas supplementation with caffeine and dibutyryl cAMP (which could be associated with the intracellular cAMP level of PMNs) or adding heparin decreased serum-stimulated phagocytotic and chemotactic activities. © 2011.

DOI： 10.1016/j.theriogenology.2010.12.005

Single spermatozoon freezing using cryotop Reviewed

Yuji Endo, Yoshitaka Fujii, Kasumi Shintani, Momoyo Seo, Hiroaki Motoyama, Hiroaki Funahashi

Journal of Mammalian Ova Research 28 ( 1 ) 47 - 52 2011

Authorship：Last author Language：English Publishing type：Research paper (scientific journal) Publisher：(一社)日本卵子学会

Conventional freezing procedures are not appropriate for surgically retrieved spermatozoa from the epididymis or testis because of their low numbers. Techniques for the cryopreservation of small numbers of spermatozoa have not been fully established. We tried to develop a cryopreservation method for a single spermatozoon using Cryotop, which has a simple structure and is easy to handle. Different parameters influencing the freezing procedure, types of container, sources of spermatozoa, and cryoprotectants were evaluated. The sperm recovery rate after thawing was similar between the sperm frozen using Cryotop or zona pellucida as containers (98.0% vs. 88.0%). Freezing of motile single spermatozoa obtained from ejaculates and testes were evaluated for recovery rate (90.0% vs. 95.0%) and motility rate (44.4% vs. 42.1%), which were not significantly different. The survival rate was significantly higher when sperm were treated with sucrose rather than with SpermFreeze (65.3% vs. 37.3%, P &lt
0.01). Cryotop was a highly effective tool for the cryopreservation of a single spermatozoon, and sucrose was determined to be an efficient cryoprotectant.

DOI： 10.1274/jmor.28.47

Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2011&ichushi_jid=J05623&link_issn=&doc_id=20110506280007&doc_link_id=10028124034&url=https%3A%2F%2Fci.nii.ac.jp%2Fnaid%2F10028124034&type=CiNii&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00003_1.gif

Hydrophobic silicone elastomer chamber for recording trajectories of motile porcine sperms without adsorption Reviewed

Koji Matsuura, Yuka Kuroda, Keisuke Yamashita, Hiroaki Funahashi

Journal of Reproduction and Development 57 ( 1 ) 163 - 167 2011

Authorship：Last author Language：English Publishing type：Research paper (scientific journal)

Motile porcine sperms adhere to hydrophilic materials such as glass and plastics. The adsorption of sperms to a hydrophobic poly(dimethylsiloxane) (PDMS) membrane is less compared with that to glass. We investigated the linear velocity (LV) and amplitude of lateral head displacement (ALHD) of motile porcine sperm on glass and PDMS preparations using computer-assisted sperm analysis (CASA). Significant decreases were observed in the 15-min LV (P&lt
0.05) and ALHD (P&lt
0.05) in motile porcine sperm on glass preparations compared with those on PDMS preparations. These differences were due to adsorption of the head and/or neck to hydrophilic substrates. Because of the elasticity of PDMS, we propose that a PDMS membrane should be used for CASA. To investigate the dynamics of motile porcine sperms with microfluidics, we do not recommend plasma treatment to bond PDMS and glass in the microchannel preparation
instead, we suggest that a PDMS molding process without plasma treatment be used for preparation of microfluidic channels. © 2011 by the Society for Reproduction and Development.

DOI： 10.1262/jrd.10-075N

Internalization of REIC/Dkk-3 protein by induced pluripotent stem cell-derived embryoid bodies and extra-embryonic tissues Reviewed International journal

Ken Kataoka, Masakiyo Sakaguchi, Kun Peng Li, Chika Taketa, Ken Ichi Yamamoto, Gang Du, Hiroaki Funahashi, Hitoshi Murata, Nam Ho Huh

International Journal of Molecular Medicine 26 ( 6 ) 853 - 859 2010.12

Language：English Publishing type：Research paper (scientific journal)

REIC/Dkk-3 was first identified as a down-regulated gene in a number of human immortalized cells and human tumor-derived cell lines. Overexpression of the REIC/Dkk-3 gene using an adenovirus vector (Ad-REIC) has showed a potent selective therapeutic effect on various human cancers through induction of ER stress. Furthermore, we recently showed that Ad-REIC has an indirect host-mediated anti-tumor activity by induction of IL-7. However, the physiological function of REIC/Dkk-3 is still unclear. As a first step to study the possible receptor(s) for secreted REIC/Dkk-3, we analyzed the internalization of Cy3-labeled recombinant REIC/Dkk-3 protein. Among the cell lines screened, mouse induced pluripotent stem (iPS) cells showed a unique pattern of internalization. The internalization was observed in peripheral cells of spherical colonies formed spontaneously, but not in undifferentiated iPS cells. When we analyzed embryoid bodies (EBs) derived from iPS cells, REIC/Dkk-3 protein was internalized specifically by differentiated cells located at the periphery of EBs. Interestingly, Dkk-1 was internalized by undifferentiated cells at the center of the EBs. When developmental tissue was analyzed, internalization of REIC/Dkk-3 protein was strictly limited to extra-embryonic tissue, such as the trophectoderm layer of 4.5 days post-coitus (dpc) blastocysts and the chorionic membrane at 16.5 dpc. The mechanism of the internalization was confirmed to be endocytosis. These findings will contribute to knowledge on the interaction of REIC/Dkk-3 with a possible receptor(s).

DOI： 10.3892/ijmm-00000534

　 PREV - NEXT 　

1
2
3
4
5
6