Biomedical subjects

Masashi Miyake

Publications and source records attributed to Masashi Miyake.

25 records · Page 2Linked to original sources

PubMed2003

Stage-specific effect of growth hormone on developmental competence of bovine embryos produced in-vitro.

Many efforts have been made to develop effective culture conditions for the production of bovine blastocysts. Growth hormone (GH) and glucose are known to affect in vitro embryo development. To improve in vitro culture conditions, the culture medium containing fetal calf serum (FCS) or bovine serum albumin (BSA) was supplemented with GH at various periods of development, and the effects of GH on the rate of development and the quality of the blastocysts were studied. Then, starting at the morula stage, the effect of glucose and GH on the rate of development was studied. In all experimental periods, FCS was more effective than BSA at improving the development rate and increasing the cell number of blastocysts. Adding GH to the culture medium between 18 and 48 h after fertilization (1-8 cell stage embryo) did not affect either the rate of blastulation or the cell number regardless of the serum protein (FCS or BSA). From 48 to 120 h after fertilization (5-cell to morula stage) GH increased the cell number of the blastocysts in the presence of BSA, but not in the presence of FCS. From 120 to 192 h after fertilization (morula to blastocyst stage), GH improved the developmental rate and cell number in the presence of FCS, although there was no significant difference when BSA was used instead of FCS as the serum protein. When cows were implanted with blastocysts developed in the presence of GH from the morula stage, their pregnancy rate did not differ from that of the control. Increasing the glucose concentration in the medium from 1.5 mM to 3 mM starting at the morula stage (120 h after fertilization) slightly decreased the rate of development, but on the other hand, decreasing the glucose concentration to 0 mM did not affect either the rate of development or the cell number. Also, then GH had no effect on the developmental rate or the cell number in the absence of glucose. In conclusion, when the medium was supplemented with serum, GH improved embryo development from the morula stage, but an increased concentration of glucose decreased embryo development. Furthermore, GH did not improve the pregnancy rate of blastocysts developed in vitro.

Animals↗

PubMed2002

Capacitation-like alterations in cooled boar spermatozoa: assessment by the chlortetracycline staining assay and immunodetection of tyrosine-phosphorylated sperm proteins.

This study was undertaken in order to characterize alterations occurring in cooled boar spermatozoa by a chlortetracycline (CTC) staining assay and immunodetection of tyrosine-phosphorylated sperm proteins. Spermatozoa were collected from 10 mature boars, washed and then resuspended in a Tris-citric acid-glucose (TCG) solution. The sperm suspensions were slowly cooled to 4 degrees C over 5 h and held for 2 days. Aliquots of the sperm suspensions were recovered before and after the cooling treatment and then used for the CTC staining assay and immunodetection of tyrosine-phosphorylated sperm proteins. Before the cooling treatment, almost all of the spermatozoa stained with CTC were characterized by uniform fluorescence over the whole head (an F pattern: uncapacitated spermatozoa). After the cooling treatment, however, significant higher percentages of spermatozoa exhibited a B pattern with a dark band of diminished fluorescence in the post acrosomal region and a relatively bright fluorescence in the acrosomal region (the pattern of capacitated spermatozoa). Coincidently, a 32 kDa tyrosine-phosphorylated protein appeared in the spermatozoa. However, these alterations occurring in the cooled spermatozoa were attenuated by the supplementation to the sperm suspensions with seminal plasma (20% (v/v)). Additionally, the same alterations were observed in the spermatozoa incubated in a capacitation-supporting medium (a modified Krebs-Ringer bicarbonate; mKRB) for 5 h. These results suggest that cooling could induce capacitation-like alterations in boar spermatozoa that were associated with the tyrosine phosphorylation of the 32 kDa sperm protein.

Animals↗

PubMed2002

Progesterone treatment of boar spermatozoa improves male pronuclear formation after intracytoplasmic sperm injection into porcine oocytes.

Boar spermatozoa were prepared for intracytoplasmic sperm injection (ICSI) by two different treatments to facilitate sperm chromatin decondensation and improve fertilisation rates after ICSI in pigs: spermatozoa were either frozen and thawed without cryoprotectants, or treated with progesterone. Morphological changes of the sperm heads after the treatments were examined and then the activation of oocytes and the transformation of the sperm nucleus following ICSI were assessed. After freezing and thawing, the plasma membrane and acrosomal contents over the apical region of sperm head were lost in all the spermatozoa. Following treatment with 1 mg/ml progesterone, the acrosome reaction was induced in 61% of spermatozoa. After injection of three types of spermatozoa, non-treated spermatozoa and progesterone-treated (i.e. acrosome-reacted) spermatozoa induced oocyte activation, but frozen-thawed spermatozoa induced oocyte activation at a significantly lower rate. Sixty-two per cent of sperm heads remained orcein-negative for 6 h, however, resulting in delayed sperm chromatin decondensation and low male pronuclear formation in the oocytes injected with a non-treated spermatazoon. Since the treatments of freezing and thawing and progesterone for spermatozoa accelerated the initial change in sperm chromatin and the latter treatment induced oocyte activation earlier, it is considered that the delay in oocyte activation and decondensation of sperm chromatin after injection of non-treated spermatozoa is caused by the existence of the sperm plasma membrane. These results show that progesterone treatment efficiently induces the acrosome reaction in boar spermatozoa without destroying their potency for oocyte activation, and the induction of the acrosome reaction results in the promotion of male pronuclear formation after ICSI.

Animals↗

PubMed2002

Fate of the acrosome in ooplasm in pigs after IVF and ICSI.

BACKGROUND: ICSI bypasses the sperm-oolemma interactions that, in normal fertilization, depend on completion of the acrosome reaction. Morphological changes in the acrosomes of sperm in the ooplasm were therefore examined following IVF and ICSI using pig gametes. METHODS: In-vitro-matured porcine oocytes were used for ICSI or IVF. Oocytes were then stained with fluorescein isothiocyanate-conjugated peanut agglutinin lectin (FITC-PNA), which specifically labels the outer acrosomal membrane of boar sperm and the cortical granules (CG) in porcine oocytes. This was followed by observation under a confocal laser scanning microscope. RESULTS: In ICSI, PNA showed the presence of disintegrated acrosomes that classified into four categories. Heterogeneous chromatin decondensation was observed in the sperm with intact/disintegrated acrosome, whereas acrosomes were barely detected in oocytes which had formed a male pronucleus. Both in ICSI and IVF, PNA-positive tails were concomitantly observed with one type of disintegrated acrosome, which was considered to be acrosome-reacted. The disappearance of CG in activated oocytes after ICSI was similar to that after IVF. CONCLUSIONS: The PNA-binding properties of sperm head components introduced into the ooplasm during ICSI are different from those after IVF. The delay of sperm chromatin decondensation is associated with that of acrosomal disassembly. Acrosomes appear to disintegrate in the ooplasm whether or not the acrosome reaction has taken place. Oocytes undergoing ICSI appear normally activated in terms of meiotic resumption and CG exocytosis.

Acrosome↗

PubMed2002

Characteristics of preimplantational development of porcine parthenogenetic diploids relative to the existence of amino acids in vitro.

The present study was designed to investigate the effects of amino acids on the in vitro development of porcine parthenogenetic diploids that were produced by electrostimulation (El-St) and cytochalasin B treatment of in vitro-matured oocytes. The culture medium for development, based on Whitten medium, contained 0.5 mg/ml of hyaluronic acid (mWM), and a two-step culture system in which 290 mOsmol before the 4-cell stage (48 or 72 h after El-St) and, subsequently, 256 mOsmol up to the blastocyst stage (mWMs) were used. In experiment 1, the diploids were cultured for 168 h in mWMs supplemented with 0.01-5 mg/ml of polyvinyl alcohol (PVA). In experiment 2, the diploids were cultured in mWMs containing 0.5 mg/ml of PVA (PVA-mWMs) for 0, 48, or 72 h and then cultured for 168 h after El-St in PVA-mWMs supplemented with essential amino acids for Eagle basal medium without glutamine (E-AA) and nonessential amino acids for minimum essential medium (NE-AA). The results showed that diploids can develop up to the blastocyst stage in mWMs including 0.05-5.0 mg/ml of PVA (49%-53% vs. 63%, P > 0.05), but the replacement of BSA with PVA alone could not support the expansion of blastocysts (11%-20% vs. 39%, P < 0.05) or their proliferation. The addition of both E-AA and NE-AA (E+NE-AA) to PVA-mWMs from the 1-cell stage resulted in severe inhibition of the development of diploids to the blastocyst stage. However, the addition of E+NE-AA to PVA-mWMs later than 48 or 72 h after El-St well supported the development of diploids to the blastocyst stage and supported the expansion of blastocysts. In experiments 3-5, which types of amino acids in E-AA inhibited the development of diploids during the first 48 h after El-St were determined. In experiment 6, the stimulatory effects of E-AA and/or NE-AA after the 4-cell stage were examined. The results of those experiments clearly showed that the presence of nonpolar E-AA, especially for valine, leucine, isoleucine, and methionine, during the first 48 h after El-St caused severe delay of the first division and inhibition of development beyond the 4-cell stage. The presence of NE-AA after the 4-cell stage produced a favorable condition for the expansion of blastocysts (33%), whereas the presence of E-AA increased the cleavage rates of the diploids after compaction and the total number of cells in the blastocysts (53.7 +/- 2.7) and inner cell mass (12 +/- 0.5). These findings indicate that the presence of nonpolar E-AA in a protein-free medium during the first 48 h causes the 4-cell block in porcine parthenogenetic diploids.

Amino Acids↗

PubMedDate not supplied

A cyclic adenosine 3',5'-monophosphate stimulates phospholipase Cgamma1-calcium signaling via the activation of tyrosine kinase in boar spermatozoa.

The aim of this study was to reveal a downstream part of the intracellular signaling that is mediated by cyclic adenosine monophosphate (cAMP)-dependent tyrosine kinases, including spleen tyrosine (Y) kinase (SYK), in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog; 0.1 mM) at 38.5 degrees C for 180 minutes and then used for Western blot and indirect immunofluorescence. Incubation of spermatozoa with cBiMPS induced tyrosine phosphorylation at the linker region of SYK (which was essential to binding to phospholipase C [PLC]gamma1) in the connecting and principal pieces, but the tyrosine phosphorylation was abolished by the addition of H-89 (a protein kinase A [PKA] inhibitor; 0.01-0.1 mM). Moreover, the cAMP-dependent tyrosine phosphorylation was also induced at the key regulatory residue of PLCgamma1 in the same segments of spermatozoa, but it was inhibited by the addition of herbimycin A (a tyrosine kinase inhibitor; 5 microM). These results suggest that the sperm cAMP-dependent tyrosine kinases, including SYK, are linked to the activation of PLCgamma1. Indirect immunofluorescence clearly detected both inositol 1,4,5-trisphosphate (IP(3)) receptor and calreticulin in the connecting piece, indicating the presence of internal calcium store. Cell imaging with fluo-3/AM (a cell-permeable Ca(2+) indicator) showed that incubation of spermatozoa with cBiMPS increased intracellular free calcium in the middle piece, but that it was reduced by the addition of U-73122 (a PLC inhibitor; 0.02 mM). Based on our findings, we conclude that the connecting piece of boar spermatozoa possesses the PLCgamma1-IP(3) receptor-calcium signaling that is triggered by cAMP and mediated by PKA and herbimycin A-sensitive tyrosine kinases, including SYK.

Animals↗

PubMedDate not supplied

Involvement of cytoplasmic free calcium in boar sperm: head-to-head agglutination induced by a cell-permeable cyclic adenosine monophosphate analog.

When boar spermatozoa are incubated in a medium designed for in vitro fertilization, many of them become agglutinated at the acrosomes. We previously reported that bicarbonate and cyclic adenosine 3',5'-monophosphate (cAMP) promote agglutination. The aim of the present study is to examine the role of cytoplasmic free Ca(2+) in boar sperm agglutination induced by a cell-permeable cAMP analogue. Spermatozoa were collected from five mature boars, washed, and resuspended in a modified Krebs-Ringer-Hepes solution lacking calcium chloride. The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 minutes and were then used to determine the percentages of head-to-head agglutinated spermatozoa. Percentages of head-to-head agglutinated spermatozoa in the samples rose significantly after incubation, from 28% to 61%-62%, after adding to the medium a cell-permeable, phosphodiesterase-resistant cAMP analogue (cBiMPS, 10 microM) or an adenylyl cyclase stimulator (sodium bicarbonate, 5 mM) plus a cell-permeable phosphodiesterase inhibitor (IBMX, 25 microM). However, the promoting effects of these reagents were blocked when spermatozoa were pretreated with a cell-permeable Ca(2+) chelator (BAPTA-AM, 25 microM), whereas the same pretreatment with a cell-impermeable Ca(2+) chelator (BAPTA, 25 microM) had almost no influence on sperm agglutination. Adding thapsigargin, a potential Ca(2+)-ATPase inhibitor, to the medium raised the percentages of agglutinated spermatozoa in a concentration-dependent manner for concentrations up to 4 microM. When 4 microM thapsigargin and 10 microM cBiMPS were examined for their effects on free Ca(2+) levels in sperm heads by using a cell-permeable Ca(2+) indicator (fluo-3/AM), the samples incubated with both or either of these reagents contained many head-to-head agglutinated cells that exhibited intense fluorescence in the heads. In control samples incubated without these reagents by contrast, most spermatozoa were free (unagglutinated) cells and characterized by almost no or only slight fluorescence in the heads. Moreover, morphological observation of Giemsa-stained preparations revealed that most agglutinated spermatozoa possessed darkly stained acrosomes, which distinguished them from acrosomereacted spermatozoa. This indicated that the sperm agglutination was not a result of the acrosome reaction. Furthermore, with indirect immunofluorescence of Ca(2+)-ATPases, the mouse monoclonal antibody to this enzyme demonstrated high affinity to the acrosomes of permeabilized spermatozoa. Based on these results, we conclude that cytoplasmic free Ca(2+) is involved in sperm head-to-head agglutination induced by a cAMP analogue.

1-Methyl-3-isobutylxanthine↗