Biomedical subjects

R Yanagimachi

Publications and source records attributed to R Yanagimachi.

At least 73 records · Page 4Linked to original sources

PubMed1998

Behavior of transgenic mouse spermatozoa with galline protamine.

General morphology, physical and chemical stability of nuclei, and postfertilization behavior of spermatozoa from transgenic mice [TgN (Prml Gal) 223 Bri] containing nuclear avian protamine (galline) were compared to those in the spermatozoa of wild-type (Wild) mice. Galline to protamine I ratios in spermatozoal nuclei of transgenic mice, strains 3175 (T75) and 3177 (T77), were 1.94 and 5.62, respectively. Live T75 and T77 spermatozoa were indistinguishable in their gross morphology from Wild spermatozoa. However, unlike Wild and T75 spermatozoa, T77 spermatozoa were vulnerable to mechanical handling, as about 40% of heads and tails were separated after gentle pipetting in suspension. Motility of T77 spermatozoa was markedly inferior to that of T75 and Wild. Chromatin heterogeneity and instability of transgenic spermatozoal nuclei were evident by transmission electron microscopy, staining reaction to Giemsa, and, as apparent by both light microscopy and flow cytometry, reaction to SDS detergent. Wild and T75 spermatozoa fertilized 90% and 60% of zona-intact oocytes in vitro, respectively. T77 spermatozoa completely failed to fertilize and bound to zona surfaces very weakly, and none of them inserted their heads into the zona. Although inefficiently, T77 spermatozoa could fertilize zona-free oocytes in vitro, indicating some ability to undergo capacitation and spontaneous acrosome reaction in vitro. After microsurgical injection into oocytes, the rate of nuclear decondensation was the greatest in rooster spermatozoa, followed by T77, T75, and Wild spermatozoa.

Animals↗

PubMed1998

Analysis of mouse oocyte activation suggests the involvement of sperm perinuclear material.

The mouse oocyte can be activated by injection of a single, intact mouse spermatozoon or its isolated head. Isolated tails are unable to activate the oocyte. Active sperm-borne oocyte-activating factor(s) (SOAF) appears during transformation of the round spermatid into the spermatozoon. The action of SOAF is not highly species-specific: mouse oocytes are activated by injection of spermatozoa from foreign species, such as the hamster, rabbit, pig, human, and even fish. Some SOAF can be extracted by simple freeze-thawing of (hamster) spermatozoa; additional SOAF is obtained by sequential treatment of spermatozoa with Triton X-100 and SDS. Electron microscopic examination of sperm heads during SOAF extraction suggests that the relatively insoluble SOAF is associated with perinuclear material. When microsurgically injected into oocytes, Triton X-100-treated sperm heads (with perinuclear material, but without any membranes) can activate the oocytes, leading to normal embryonic development. Whereas perinuclear components have been believed to play a purely structural role, these data suggest an additional function for them in oocyte activation.

Animals↗

PubMed1998

The first polar body can be used for the production of normal offspring in mice.

The purpose of this study was to determine whether chromosomes in the first polar body can participate in normal embryonic development. In the mouse the majority of first polar bodies degenerate soon after ovulation, but a few remain viable for 10 h or more. When the contents of a live polar body were injected into an enucleated mature oocyte and examined 2 h later, the polar body chromosomes were arranged on a metaphase plate as seen prior to the secondary meiotic division. Such oocytes were fertilized normally by sperm injection. When 2-cell embryos were transferred to foster females, 30-57% developed into fertile offspring. This outcome supports a longstanding belief that chromosomes ejected into the first polar body have the same genetic potential as those remaining in the oocyte after the first meiotic division. As the chromosomes in the second polar body are known to have full potential to participate in normal embryonic development, it is theoretically possible to reproduce four offspring by using chromosomes in one oocyte.

Animals↗

PubMed1998

Factors affecting meiotic and developmental competence of primary spermatocyte nuclei injected into mouse oocytes.

Mature mouse oocytes that have received the nuclei of pachytene primary spermatocytes (or metaphase I chromosomes of primary spermatocytes) can develop into fertile offspring. However, success rate in this study was low. No more than 3.8% of transferred 2-cell embryos arising from spermatocyte-injected oocytes developed to full term. Nevertheless, the birth of normal offspring seems to suggest that at least in some primary spermatocytes the functional genomic imprinting is complete before transfer and/or consolidated after the transfer. Although injected spermatocyte nuclei could undergo two successive meiotic divisions within oocytes, abnormalities of both divisions were commonly observed, and sister chromatids often separated prematurely during the second meiotic division. Chromosome breakage/rearrangements were also frequently seen before the first cleavage. Such abnormalities of chromosome behavior are probably the major causes of the poor preimplantation development of zygotes arising from primary spermatocyte-injected oocytes. Thus, clinical use of primary spermatocytes as substitutes for spermatozoa in assisted fertilization is not advisable until the causes of chromosomal abnormalities are better understood through extensive animal studies.

Animals↗

PubMed1998

Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection.

Epididymal mouse spermatozoa were suspended in various physiological solutions (CZB, PBS or isotonic saline) with or without 18% (w/v) raffinose before cooling to -20 degrees, -50 degrees or -196 degrees C and storage for 1-28 days. After thawing, a few spermatozoa frozen with raffinose were partially motile (about 2%) but in all other treatments they were immotile and diagnosed as 'dead' by staining that differentiates between live and dead spermatozoa. Almost all oocytes injected with sperm heads (nuclei) from spermatozoa frozen with and without raffinose were fertilized normally (95-100%) and developed to the two-cell stage (89-100%). No differences were found between the physiological media. The majority of oocytes fertilized with spermatozoa frozen in CZB medium developed to blastocysts (80-94%) but development was significantly reduced after fertilization with spermatozoa frozen in PBS and isotonic saline especially in the absence of raffinose (69 and 70% versus 51 and 50%). Normal fertile offspring were obtained in all treatments but there were significantly fewer offspring with spermatozoa stored at -196 degrees C in isotonic saline with or without raffinose and CZB with raffinose. Testicular spermatozoa were extremely sensitive to cryodamage: about 50% frozen to -196 degrees C in CZB with or without raffinose disintegrated after thawing. Almost 100% of oocytes injected with sperm heads from intact (at light microscope level) testicular spermatozoa developed to the two-cell stage but development to blastocysts was reduced significantly compared with that of controls especially those without raffinose. The data indicate that cryopreservation of sperm nuclei requires less stringent conditions than those for the retention of normal physiological function of intact spermatozoa. Motility and plasma membrane integrity are not essential for fertilization and the production of live offspring when nuclei of nonviable spermatozoa are injected into oocytes.

Animals↗

PubMed1997

Sperm nuclear envelope: breakdown of intrinsic envelope and de novo formation in hamster oocytes or eggs.

During fertilisation of a fully mature oocyte, the sperm intrinsic nuclear envelope (SINE) disappears soon after sperm-oocyte fusion. A new nuclear envelope appears around the decondensed sperm chromatin when the oocyte reaches telophase II. Whether the SINE persists or rapidly disappears after sperm entry into immature oocytes or fertilised eggs has been controversial. Nuclear envelopes have been demonstrated around the sperm chromatin, which cannot be decondensed within the ooplasm of these oocytes or eggs, but whether these envelopes are persisting SINEs or newly formed envelopes has been a point of dispute. To resolve this issue, the fate of the SINEs of hamster sperm nuclei was traced after incorporation into immature oocytes at the germinal vesicle stage (GV oocytes) or fertilised eggs at the pronuclear stage (PN eggs). The SINEs disappeared quickly within these oocytes or eggs, like those within maturing or mature oocytes, suggesting that the envelopes around the sperm chromatin must be newly formed after SINE breakdown. To obtain further evidence, a detergent-treated, SINE-free sperm nucleus was injected into a PN egg. A new envelope appeared around the still-condensed or partially decondensed sperm chromatin within 3 h after injection. Thus, disassembly of the SINE within ooplasm, unlike that of nuclear envelopes of other cells at prophase, is independent of the cell cycle stage of the oocyte or egg, whereas the ability of the ooplasm to assemble the new envelope is restricted to certain periods of the cycle, i.e. early prophase and telophase during meiosis and interphase, periods when active M-phase promoting factor (MPF) is absent from the ooplasm.

Animals↗

PubMed1997

The response of mouse oocytes injected with sea urchin spermatozoa.

Sea urchin spermatozoa were injected into mature mouse oocytes to determine whether they can activate mouse oocytes and, if so, how they behave within the oocyte cytoplasm of such a distant species. While injection of a single spermatozoon into each oocyte did not activate any of the oocytes, injection of 10 spermatozoa activated about 20%. Within the cytoplasm of unactivated oocytes, sperm heads commonly transformed into chromosome-like structures. When a single spermatozoon was injected, and oocytes were then activated by Sr2+, about 30% of the activated oocytes had both female (mouse) and male (sea urchin) pronuclei when examined 8 h after sperm injection. These results indicated that sperm-borne oocyte activating factor(s) and the cytoplasmic factors controlling the development of the sperm pronucleus are not strictly species-specific.

Animals↗

PubMed1997

Beneficial effect of medium with high concentration serum for direct sperm injection into mouse oocytes using a conventional pipette.

Although the piezo-electrically driven pipette is much more effective than a conventional pipette for direct sperm injection into mouse oocytes, the piezo-pipette driving unit is expensive and may not be readily accessible to most basic science laboratories. We report here that the inefficiency of the conventional pipette can be improved by performing the operation in a medium with high serum concentration.

Animals↗

PubMed1997

Fate of microinjected sperm components in the mouse oocyte and embryo.

Intact mouse sperm or mouse sperm tails alone, labelled with MitoTracker Green FM fluorochrome, were injected into mouse oocytes and the cells cultured in vitro for up to 5 days. The dye stained midpiece mitochondria, the sperm tail coarse fibres and the sperm perforatorium. Intact sperm (or tails injected with separated heads) induced normal embryonic development. The mitochondria could be identified in embryos up to the 4-cell stage, remaining associated with the sperm tail. They largely disappeared by the 8-cell stage, when only a minority of embryos (6/43) could be found with small patches of mitochondria. Axonemal elements could be identified coiled up in single external blastomeres as late as day 5 blastocysts. By contrast, mitochondria as well as tail components could be identified up to 5 days after injection of sperm tails alone into non-activated oocytes and also in embryos that arrested development before the 8-cell stage. We conclude that disappearance of the labelled sperm mitochondria in normally cleaving embryos is not due to fading or inactivation of the fluorochrome marker, but is rather an event specifically tied to cell cycle activities around the second cell division.

Animals↗

PubMed1996

Inadequate function of sterile tw5/tw32 spermatozoa overcome by intracytoplasmic sperm injection.

Mice carrying two t complementary haplotypes (tw5/tw32) are totally sterile. Their spermatozoa have poor motility and fertilize neither zona-intact nor zona-free oocytes, even though they are structurally indistinguishable from control (wild-type) spermatozoa. However, when injected directly into oocytes, these infertile spermatozoa are able to participate in normal development. This suggests that infertility of tw5/tw32 male (spermatozoa) is more likely to be due to poor sperm-oocyte interaction than to genetic incompetence of sperm nuclei.

Animals↗

PubMed1996

Sperm membrane incorporation into oolemma contributes to the oolemma block to sperm penetration: evidence based on intracytoplasmic sperm injection experiments in the mouse.

Mouse oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and reinseminated after the removal of zonae pellucidae at pronuclear stage or at the 2-cell stage. Although these oocytes were activated normally by ICSI, as evidenced by resumption of meiosis and cortical granule exocytosis, they did not develop oolemma block to sperm penetration. They could be penetrated by spermatozoa at pronuclear stage and even at the 2-cell stage. This supports the notion that incorporation of sperm plasma membrane into oolemma contributes to the changes in oolemma that block sperm penetration.

Animals↗

PubMed1996

Mouse oocytes injected with cryopreserved round spermatids can develop into normal offspring.

PURPOSE: This study was performed to determine whether frozen-thawed mouse round spermatids can fertilize oocytes and contribute to normal embryo development. METHODS: Freshly collected mouse testicular cells were frozen in PBS containing 7.5% glycerol and 7.5% fetal bovine serum. After thawing and removal of the cryoprotectants, round spermatids were selected and injected individually into mature oocytes which had been previously activated with Sr(2+)-containing Ca(2+)-free medium. RESULTS: After thawing, 75-85% of testicular cells were alive. About 90% of the oocytes were fertilized by intracytoplasmic injection of frozen-thawed round spermatids; 11% (17/150) of embryos transferred to foster mothers developed into normal offspring. CONCLUSIONS: Mouse round spermatids can be cryopreserved for production of normal offspring.

Animals↗

PubMed1996

The use of piezo micromanipulation for intracytoplasmic sperm injection of human oocytes.

INTRODUCTION: This paper describes the use of piezo actuator technology for intracytoplasmic sperm injection of human eggs. METHODS: Piezo micromanipulation involves a simple and easily made injection pipette of very thin diameter and wall thickness, and it has recently been developed using a mouse model. No bevel or spike is required on the pipettes used. The piezo actuator attaches to conventional micromanipulators and acts to mechanically advance the pipette tip through the zona pellucida using a piezoelectric effect. The oolemma could, in some cases, also be broken using piezo. This technique was applied to 39 patients presenting for in vitro fertilization due to male-factor infertility. RESULTS: The fertilization rate of eggs injected (60.5%), damage rate (7.6%), abnormality rate (3%), and ongoing pregnancy rate beyond 8 weeks (28.2%) are comparable to those for other techniques that have been successfully reported.

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PubMed1996

Comparison of methods for activating mouse oocytes for spermatid nucleus transfer.

In the mouse, mature oocytes injected with prespermatozoal cell nuclei remain unactivated. Additional stimulation is needed to trigger oocyte activation leading to embryo development. We compared various electrical stimulations, treatment with cycloheximide alone or in combination with electrical stimulation, and injection of sperm-borne oocyte-activating factor (oscillogen) in terms of their oocyte activation and embryo development rates. Of all the treatments tested, a single electrical pulse (1.0 kV/ cm, 128 microns) was the simplest, yet very effective, in allowing the development of the oocytes injected with spermatid nuclei.

Animals↗

PubMed1996

Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes.

The chromosome constitution of human spermatozoa was determined after injecting individual spermatozoa into mouse oocytes. Of a total 279 eggs arrested at first cleavage metaphase, 200 (71.7%) were suitable for the analysis of sperm chromosomes. Incidences of spermatozoa with numerical and structural chromosome aberrations were 1.3 and 6.9% respectively in spermatozoa with normal head morphology, showing values comparable with those found in previous studies using the hamster oocyte-human sperm fusion system. The ratio of X- to Y-bearing spermatozoa did not differ significantly from the expected 1:1 ratio. The incidence of structural chromosome aberrations was about four times higher in spermatozoa with amorphous, round and elongated heads (26.1%) than in those with morphologically normal heads, whereas the incidence of aneuploidy was not significantly different between the two groups. No increase in chromosome aberrations was found in spermatozoa with large heads. The same was true for spermatozoa with small heads. Although the sample size used in this study is rather small, the results nevertheless indicate that some morphological abnormalities in the sperm heads are associated with their chromosome defects.

Animals↗

PubMed1996

Normal mice develop from oocytes injected with spermatozoa with grossly misshapen heads.

Because in the mouse some structurally abnormal spermatozoa can penetrate oocytes, we decided to determine whether mouse oocytes fertilized by spermatozoa with grossly misshapen heads are able to develop normally. We injected such spermatozoa from BALB/c mice individually into mature oocytes of hybrid mice B6D2F1. Eighty to ninety percent of the oocytes were activated and developed into blastocysts in vitro and, when transferred to foster mothers, some developed into apparently normal fertile adults. This finding indicates that a proportion of abnormal spermatozoa carry all the genome and organelles necessary for normal embryonic development and growth to fertile maturity.

Animals↗

PubMed1996

Fertilization and development of mouse oocytes injected with isolated sperm heads.

To determine whether spermatozoa must be structurally intact before microsurgical injection into oocytes for normal fertilization, intact spermatozoa, as well as sperm heads separated from tails by sonication, were individually injected into oocytes. When whole spermatozoa were injected immediately after their immobilization, the majority of the oocytes were fertilized and developed normally. Sonication in the presence or absence of Triton X-100 decapitated more than 95% of spermatozoa. Although all decapitated spermatozoa were diagnosed as "dead" by live/dead sperm staining, separated sperm heads (nuclei) could participate in normal embryo development when injected into the oocytes. The ability of isolated sperm heads (nuclei) to participate in normal embryo development was maintained under cryopreservation conditions that were not suitable for the survival of plasma membrane-intact spermatozoa. These results indicate that 1) spermatozoa do not need to be structurally intact for intracytoplasmic injection, 2) the plasma and acrosomal membranes and all tail components are not essential for normal embryo development, at least in the mouse, and 3) the cryopreservation conditions required for maintenance of the genetic integrity of sperm nuclei are less stringent than those necessary for keeping plasma membrane-intact spermatozoa alive.

Animals↗

PubMed1996

Penetration by field vole spermatozoa of mouse and hamster zonae pellucidae without acrosome reaction.

Spermatozoa of the field vole (Microtus montebelli) that bound to the zona pellucida of field vole oocytes underwent the acrosome reaction before passing through it. In contrast, vole spermatozoa that bound to the zonae of mouse and hamster oocytes penetrated the zonae without any sign of the acrosome reaction. The presence or absence of proteinase/hyaluronidase inhibitors in the medium did not make any difference to zona penetration by acrosome-intact vole spermatozoa. These observations suggest that field vole spermatozoa can cross mouse and hamster zonae mechanically without assistance from zona-hydrolysing enzymes.

Acrosome↗