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Biomedical subjects

R Yanagimachi

Publications and source records attributed to R Yanagimachi.

At least 91 records · Page 5Linked to original sources
PubMed1995

Comparison of the acrosome reaction-inducing ability of the outer and inner surfaces of the zona pellucida and oolemma: a study using the golden hamster.

The outer surface of hamster zona pellucida has a strong ability to induce the sperm acrosome reaction, whereas neither the inner surface of the zona nor the oolemma has this ability. Therefore, the spermatozoa that fertilise oocytes after subzonal insemination of spermatozoa (SUZI) must have been either acrosome-reacted prior to injection or acrosome-reacted spontaneously while swimming within the perivitelline space. The outer surface of fresh mouse zona is not capable of inducing the acrosome reaction of hamster spermatozoa. When fixed with glutaraldehyde, however, it became capable of inducing the hamster sperm acrosome reaction rather efficiently. The reason for this is not clear.

Acrosome↗
PubMed1995

Spermatids as male gametes.

Intracytoplasmic sperm injection (ICSI) is becoming increasingly popular in human infertility clinics as an efficient method for the treatment of male infertility. It is proposed that spermatids can be used as substitutes for spermatozoa if men are unable to produce sperm in their testes. At least in the hamster and mouse, the nuclei of round spermatids were capable of participating in syngamy when incorporated into homologous mature oocytes either by microsurgical ICSI or electrofusion. Normal mouse offspring were born after after electrofusion of oocytes with round spermatids. When culture in vitro of spermatogonia and spermatocytes is perfected, then spermatids, transforming spermatids and spermatozoa will all be able to be used as male gametes.

Animals↗
PubMed1995

Intracytoplasmic sperm injection in the mouse.

Intracytoplasmic sperm injection (ICSI) was successful in the mouse when a piezo-driven micropipette was used instead of a mechanically driven conventional pipette. Eighty percent of sperm-injected oocytes survived, and approximately 70% of them developed into blastocysts in vitro. When 106 embryos at the 2- to 4-cell stage were transferred to eight naturally mated foster mothers, 30% of the embryos (25-43%, depending on the host) reached the full term. Except for two that were cannibalized soon after birth, all of the young (30 pups) grew into normal adults. Studies of this type on the mouse may increase understanding of the fertilization process and of how ICSI works.

Animals↗
PubMed1995

Development of normal mice from oocytes injected with secondary spermatocyte nuclei.

This study shows that the nucleus of the secondary spermatocyte can participate in syngamy and normal embryonic development. Spermatogenic cells were released from the seminiferous tubules of adult mice, and the secondary spermatocytes were selected according to the size of the whole cell and nucleus. The accuracy of this selection, evaluated by chromosome analysis, was 86%. Nuclei of presumptive secondary spermatocytes were freed from the cytoplasm and then injected individually into mature oocytes. This process itself did not activate the oocytes. The oocytes were electroactivated about 2 h after injection, at which time prematurely condensed chromosomes of the spermatocyte had become associated with the microtubules of a spindle. Following activation, the chromosomes of both the oocyte and spermatocyte completed their second meiotic division, culminating in the extrusion of two separate polar bodies and the formation of one male and one female pronucleus in about 75% of the oocytes into which spermatocyte nuclei had been injected. Two- or four-cell embryos arising from such oocytes were randomly selected and transplanted to foster mothers. Twenty-four percent developed into normal fertile offspring. The young born later to these offspring were all normal. The results of this study indicate that gametic imprinting of mouse spermatogenic cells is completed either in the testis before the second meiotic division or within the cytoplasm of a mature oocyte after artificial nuclear transfer.

Animals↗
PubMed1995

Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring.

Genomic imprinting occurs in both male and female gametes during gametogenesis, but the exact time when imprinting begins and ends is unknown. In the present study we injected nuclei of testicular spermatozoa and round spermatids into mature mouse oocytes to see whether these nuclei are able to participate in syngamy and normal embryonic development. If the injected oocytes develop into normal fertile offspring, imprinting in the male germ cells used must have been completed by the time of injection. Ninety-two percent of mouse oocytes injected with testicular spermatozoa survived and 94% of these were fertilized normally (extrusion of the second polar body and formation of male and female pronuclei). When 44 two-cell embryos so created were transferred to 5 foster mothers, 24 (54.5%) developed into normal offspring. Unlike testicular spermatozoa, round spermatids could not activate the oocytes, and therefore the oocytes had to be activated artificially either before or after spermatid injection. The highest rate (77%) of normal fertilization was obtained when the oocytes were first activated by electric current, then injected individually with a single spermatid nucleus. When 131 two-cell embryos were transferred to 15 foster mothers, 37 (28.2%) reached full term. All but two grew into healthy adults. Thus, it would appear that gametic imprinting in mouse spermatogenic cells is completed before spermiogenesis begins. Under the experimental conditions employed, spermatid nuclei were less efficient than testicular sperm nuclei in producing normal offspring, but perhaps this was due to technical rather than inherent problems.

Animals↗
PubMed1995

Activation of hamster zona-free oocytes by homologous and heterologous spermatozoa.

Spermatozoa of a wide variety of species can fuse with zona-free hamster oocytes. Zona-free hamster oocytes were inseminated with spermatozoa of homologous (hamster) and other (mouse, guinea-pig and human) species, and their responses were closely examined to determine whether such interspecific sperm-oocyte fusion always induces normal oocyte activation. While guinea-pig and human spermatozoa could activate hamster oocytes as efficiently as hamster spermatozoa, mouse spermatozoa could not. Mouse spermatozoa fused readily with hamster oocytes, yet most oocytes remained inactivated at least during the first 1.5-2 h. The amount of M-phase (metaphase) promoting factor was reduced in hamster oocytes fused with one or several mouse spermatozoa; however, repetitive Ca2+ transients failed to occur unless oocytes were inseminated with a concentrated sperm suspension and penetrated by very many spermatozoa. These observations suggest that sperm-oocyte membrane fusion per se is not sufficient to trigger oocyte activation, and that putative sperm-derived oocyte activating factors show some degree of species specificity.

Animals↗
PubMed1994

Birth of normal young after electrofusion of mouse oocytes with round spermatids.

Normally, round spermatids, the youngest male germ cells with a set of haploid chromosomes, cannot fertilize mature oocytes. However, when mouse spermatids were fused with oocytes, some of the resulting zygotes developed into normal fertile mice of either sex. This demonstrates that the nuclei of spermatids can provide the paternally imprinted chromosomes needed for full embryonic development and also that the complex postmeiotic modifications involved in sperm formation in the testis and their maturation in the epididymis merely serve to facilitate natural delivery of the paternal genome. This finding may find an application in the treatment of male infertility due to defective spermiogenesis/sperm maturation.

Animals↗
PubMed1994

Inhibition of sperm-egg fusion in the hamster and mouse by carbohydrates.

After spermatozoa bind to and penetrate the extracellular matrix of the egg, the zona pellucida, they adhere to and fuse with the plasma membrane of the egg. Since sperm-egg fusion may involve membrane glycoproteins and/or carbohydrate binding proteins, we sought to test this hypothesis by challenging sperm-egg fusion in hamster and in mouse with added carbohydrates. In this study, a number of carbohydrate and glycoconjugates were examined for their ability to inhibit sperm-egg fusion. In the hamster, D(+)-glucosamine, D(+)-galactosamine, albumin-bovine-glucosamide and -galactosamide, fucoidan and dextran sulphate inhibited the fusion of spermatozoa with zona-free eggs. The same effects were seen in the mouse, except for the toxic effects of D(+)-galactosamine. These facts suggest a role of carbohydrate binding proteins or glycoproteins in the fertilisation process at the level of binding to and fusing with the oolemma.

Acrosome↗
PubMed1994

Cryopreservation of spermatozoa from cynomolgus monkeys (Macaca fascicularis).

Fertilization of eggs from cynomolgus monkeys (Macaca fascicularis) was achieved in vitro using frozen-thawed spermatozoa. The best sperm survival rate was obtained after freezing spermatozoa in a semen-diluting medium (TTE: Tes, Tris, egg yolk base) containing 5% glycerol with 30 min equilibrium time. Although frozen-thawed spermatozoa could fertilize eggs in vitro and some of the fertilized eggs developed into blastocysts or hatched blastocysts, a sperm fertility assay using salt-stored eggs indicated that frozen-thawed spermatozoa had a shorter lifespan in capacitation medium than did fresh, unfrozen spermatozoa. This may be attributable to damage to their plasma membranes.

Animals↗
PubMed1993

Behaviour of hamster and mouse round spermatid nuclei incorporated into mature oocytes by electrofusion.

Round spermatids of the hamster and mouse were electrofused with homologous mature oocytes to examine the behaviour of their nuclei within the ooplasm. A single spermatid was inserted in the perivitelline space of a mature oocyte and an electric fusion pulse given. In the hamster, the best spermatid-oocyte fusion took place when the oocytes were pretreated with neuraminidase, subjected to 30 s AC (2 MHz, 20 V/cm) followed by a single fusion DC pulse (3000 V-cm, 10 microseconds) and another 30 s AC current. Inclusion of micromolar Ca2+ and Mg2+ in the fusion medium was essential for oocyte activation. Under these conditions all oocytes were activated and 20-40% fused with spermatids. Of these fused oocytes only 5-10% had fully developed spermatid (male) and oocyte (female) pronuclei. In the rest the spermatid-derived pronuclei remained small throughout the pronuclear stage. However, nucleolus-like structures appeared de novo and DNA synthesis occurred in these small pronuclei. Regardless of the size of male pronuclei, chromosomes from the spermatid and oocyte appeared to mingle and participate in the first cleavage. About 70% of the fused oocytes developed into the 2-cell stage. Electrofusion of mouse oocytes with spermatids was less efficient. Even under the best conditions tested, less than 10% of the oocytes fused with spermatids. Here again, most spermatid nuclei remained small throughout the pronuclear stage.

Animals↗
PubMed1993

Evidence that acrosin activity is important for the development of fusibility of mammalian spermatozoa with the oolemma: inhibitor studies using the golden hamster.

The sperm plasma membrane over the equatorial segment of the acrosome gains the ability to fuse with the oolemma some time during, or after, the acrosome reaction. Since acrosin is a major component of the acrosome matrix that dissolves during the acrosome reaction, we sought to determine the effect of acrosin inhibitors on the sperm's ability to fuse with the oolemma. Five acrosin inhibitors (soybean trypsin inhibitor (SBTI), leupeptin, benzamidine, N-p-tosyl-1-lysin-chloromethyl ketone (TLCK) and phenylmethylsulphonyl fluoride (PMSF) and one non-acrosin inhibitor (N-p-tosyl-1-phenylalanine chloromethyl ketone (TPCK) were tested at non-toxic levels (below motility-disturbing concentrations). These inhibitors were added at three different times: (1) during the acrosome reaction of spermatozoa, (2) during sperm-oocyte contact and fusion, and (3) soon after sperm-oocyte fusion was completed. TLCK prevented sperm-oocyte fusion by inhibiting the acrosome reaction. PMSF inhibited gamete fusion, without inhibiting the acrosome reaction. SBTI, leupeptin and benzamidine also inhibited gamete fusion, but they had no effect if spermatozoa were allowed to acrosome-react in inhibitor-free medium. TPCK was without any inhibitory effects, suggesting that chymotrypsin-like enzymes are not involved in gamete fusion. Although acrosin inhibitors prevented acrosome-reacted spermatozoa from becoming fusion-competent, acrosin (and trypsin) alone could not make the plasma membrane of acrosome-intact spermatozoa fusion-competent. The data suggest that (1) the plasma membrane of the acrosomal region first undergoes dramatic changes immediately before or during the acrosome reaction and (2) acrosin released from the acrosome during the acrosome reaction further alters biophysical and biochemical characteristics of the plasma membrane over the equatorial segment. Such dual changes make the plasma membrane of this specialised region of the spermatozoon competent to fuse with the oolemma. Acrosin may not be the only acrosomal enzyme to participate in these changes.

Acrosin↗
PubMed1993

Retention of hamster oolemma fusibility with spermatozoa after various enzyme treatments: a search for the molecules involved in sperm-egg fusion.

The plasma membrane (oolemma) of the hamster egg retains the ability to fuse with spermatozoa even after exhaustive treatment with proteases and glycosidases. In contrast, when mouse oolemma is treated with proteases, the ability of eggs to fuse with spermatozoa is reduced. In the present study, similar treatments effective in reducing fusibility in the mouse were reexamined in the hamster. Of the several enzymes and treatments tested, only trypsin in Ca(2+)-free medium significantly reduced the hamster oolemma's ability to fuse with spermatozoa. This is suggestive of a cadherin-like system of binding and fusion. When hamster oolemmae were treated with the same protease regimen that reduced fusibility of mouse oolemma for mouse spermatozoa, heterologous fusion of hamster oolemmae with mouse spermatozoa was reduced, without affecting the fusion of these oolemmae with hamster spermatozoa. These data suggest that a protease-sensitive oolemma molecule is of critical importance for mouse sperm-oolemma fusion but not for hamster sperm-oolemma fusion.

Animals↗
PubMed1993

Moderate heat treatment increases the penetrability of zonae pellucidae of salt-stored mammalian oocytes by spermatozoa.

The zona pellucida of mammalian oocytes stored in highly concentrated solutions of neutral salts is known to retain its biological and biochemical properties. However, the zona may become resistant to sperm penetration as the storage period is increased. In cattle and hamsters, the penetrability of zonae of salt-stored oocytes was restored or increased by treating the oocytes with moderate heat without altering the gross morphology of the zona. Although this technique did not work for salt-stored human ova, this may have been due to the use of so-called inseminated-unfertilised ova which (1) may have been fertilised but failed to activate, or (2) were not fertilised because of functionally defective zonae.

Animals↗
PubMed1993

Round spermatid nuclei injected into hamster oocytes from pronuclei and participate in syngamy.

Round spermatids are spermatogenic cells that have just completed meiosis. To discover whether nuclei of these haploid cells are genetically ready for fertilization, nuclei were individually injected into mature hamster oocytes via a microsurgical technique. In the majority of oocytes that were successfully injected and activated, spermatid nuclei transformed into pronuclei and underwent DNA synthesis. Ultimately their chromosomes mingled with ootid chromosomes immediately prior to the first cleavage. Although we could not determine the developmental potential of these zygotes, spermatid nuclei appear capable of participating in syngamy.

Animals↗