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  • Author: R. Yanagimachi x
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R. YANAGIMACHI

With some important exceptions (Chang & Hancock, 1967), fertilization of mammalian eggs is species-specific in that the eggs of a species can be penetrated only by spermatozoa of the same species. The analysis of the species-specificity of fertilization is very difficult when in-vivo systems are used. In-vitro systems, on the other hand, eliminate many factors and enable us to examine directly the interactions between the eggs and spermatozoa.

Barros (1968) inseminated rat eggs in vitro with capacitated golden hamster spermatozoa and found that only one out of 294 eggs had been penetrated. In this egg, two spermatozoa had penetrated the zona pellucida but failed to enter the vitellus. On the other hand, none of 280 mouse eggs inseminated in vitro with capacitated hamster spermatozoa were found to be penetrated.

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R. YANAGIMACHI

Summary.

Female golden hamsters were mated at various times before or after ovulation, and the time of sperm penetration into the ova was examined. When the females were mated several hours prior to ovulation, penetration of spermatozoa into the ova started about 3 hr after the commencement of ovulation or about 1·5 hr after the first ovum passed into the Fallopian tube; virtually all the ova were penetrated in the next 4 hr. In the females mated during or several hours after ovulation, sperm penetration occurred between 3 and 6 hr after coitus. The experiments in which spermatozoa were deposited artificially in the uteri of females shortly after ovulation, demonstrated that the spermatozoa recovered from the uterus of other females penetrated ova significantly faster than epididymal spermatozoa. The uterine spermatozoa deposited about the ova in vitro were also able to penetrate the ova more quickly than epididymal spermatozoa. The whole process of sperm penetration through the zona pellucida was observed on one occasion. This particular observation showed that: (1) the acrosome (at least, the outer acrosome membrane) of the fertilizing spermatozoon was absent before the spermatozoon started to penetrate the zona pellucida; (2) the direction of the passage of the spermatozoon was not vertical, but at an angle to the surface of the zona pellucida; (3) the time required for the sperm head to traverse the zona pellucida and the perivitelline space was 3 to 4 min and 1 to 2 sec, respectively; and (4) the head of the spermatozoon lay flat on the vitelline surface and sank into the vitellus without lively movements of the sperm flagellum.

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R. YANAGIMACHI

Epididymal spermatozoa of the golden hamster can be capacitated in vitro in the presence of oviduct fluid from the oestrous female hamster (Yanagimachi & Chang, 1964; Yanagimachi, 1966; Barros & Austin, 1967), follicular fluid from mature ovarian follicles of the hamster (Barros & Austin, 1967; Yanagimachi, 1969a), detoxified bovine follicular fluid (Gwatkin & Andersen, 1969; Yanagimachi, 1969b), and detoxified blood sera of the hamster and some other species (Yanagimachi, 1970). When incubated in media containing these biological fluids, the spermatozoa agglutinate head to head within ½ hr. About 2½ to 3 hr later, agglutinated spermatozoa disperse spontaneously and free spermatozoa show an extraordinarily active movement. In the majority (sometimes, 100%) of these spermatozoa, the acrosomal reaction is in progress or has been completed and the spermatozoa are ready to penetrate the

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R. YANAGIMACHI

Mammalian ovarian oocytes can mature in vitro when liberated from Graafian follicles and placed in appropriate culture media. This was first demonstrated in the rabbit (Pincus & Enzmann, 1935, 1937; Chang, 1955a, b) and has been confirmed in a variety of animals (see Donahue, 1972; Biggers, 1973; Fowler & Edwards, 1973).

According to Jagiello (1969), 80 to 100% of guinea-pig oocytes isolated from ovaries of adult females in the middle (Days 5 to 8) of their oestrous cycle resume meiosis upon culture and may reach metaphase II by 14 hr of culture. Unfortunately, Jagiello gave no detailed information of the techniques which she used for isolation and culture of the oocytes and did not determine whether the oocytes matured in vitro were fertilizable. This paper describes a method of culturing

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R. YANAGIMACHI

Summary.

When hamster epididymal spermatozoa were incubated in vitro in the presence of follicular fluid in Tyrode's solution for 3 hr or more, they became fully capacitated. The majority of the capacitated spermatozoa had lost the acrosome cap, and they displayed an extremely vigorous motility. When placed in contact with freshly ovulated eggs in vitro, the spermatozoa started to enter the zona pellucida of the eggs as early as 10 min after insemination; sperm penetration through the zona pellucida, however, occurred most often between 30 and 50 min after insemination. The follicular fluid markedly reduced its spermcapacitating potency when diluted with a large volume of Tyrode's solution. Cumulus oophorus cells, their matrix, corona radiata cells and the eggs had no potency to induce functional capacitation of the spermatozoa. Follicular fluids of four different species were tested. In respect to their potency to capacitate hamster spermatozoa, the fluids of three species could be arranged in the following order: hamster>mouse >rat. Rabbit follicular fluid was totally ineffective.

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A. D. Fleming, R. Yanagimachi and H. Yanagimachi

Summary. Semen from a male dolphin in captivity was collected by electroejaculation and frozen to −176°C. Sperm motility was excellent after thawing 10 days later. Electron microscopy showed 14–16 parallel ridges in the post-acrosomal region and two types of mitochondria in the mid-piece. The spermatozoa were capable of fusing with zona-free hamster eggs only after preincubation for 2 h, suggesting the need for sperm capacitation and acrosome reaction before fertilization in this species.

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T. OIKAWA and R. YANAGIMACHI

Summary.

Antibody produced in rabbits against hamster ovary blocked fertilization of golden hamster eggs in vitro by binding to the surface of the zona pellucida and rendering it impenetrable to spermatozoa. The antibody was also effective in blocking fertilization in vivo. An intraperitoneal injection of the antibody into females resulted in the complete inhibition of fertilization for approximately 12 days, i.e. three oestrous cycles. This temporary sterility was apparently due to the binding of the antibody to the zona pellucida of oviducal and ovarian oocytes, and not due to a failure of sperm ascent or capacitation in the female genital tract. Neither the oestrous cycle nor ovulation was affected by the antibody injection.

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T Wakayama and R Yanagimachi

Cloning methods are now well described and in almost routine use. However, the frequencies of production of live offspring from activated oocytes remain at < 3% and little is known about the factors that affect these frequencies. The effects of cytokinesis inhibitors, dimethylsulphoxide (DMSO) and the cell cycle of recipient cytoplasm on the cloning of mice were examined. Reconstructed oocytes, which were activated immediately after nucleus injection and cultured without cytochalasin B, developed into blastocysts at a frequency of 30--54% and into live cloned offspring at a frequency of 2--3%. Activated zygotes did not support development to full term after nuclear transfer. Reconstructed oocytes were activated 1--3 h after nuclear transfer and were exposed separately to three inhibitors of cytokinesis (cytochalasin B, cytochalasin D or nocodazole) to examine the toxicity of these inhibitors on cloning. All of the oocytes exposed to nocodazole-containing media formed many small pseudo-pronuclei, whereas with cytochalasin-containing media most of the activated oocytes formed only two pseudo-pronuclei. Despite such differences, 42--61% of reconstructed embryos developed to the morula-blastocyst stage and 1--3% developed to full term in all groups. Addition of 1% (v/v) DMSO to the activation medium significantly improved the frequency of development to the blastocyst stage and full term; however, this improvement did not lead to a higher success rate in the generation of live cloned offspring. These results show that activated mouse oocytes/zygotes are not effective cytoplasmic recipients with the methods described and that the lack of success of cloning is not due to inhibition of cytokinesis.

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A. SATO and R. YANAGIMACHI

Since Heape (1890) first succeeded in transplanting rabbit embryos (four-cell stage), the transplantation of fertilized eggs and preimplantation embryos of laboratory animals has been progressively more successful (for review and bibliography, see Chang & Pickworth, 1969; Adams & Abbott, 1971), though few studies seem to have been made on the transplantation of hamster embryos. Blaha (1964) reported that 49·2% of six- to eight-cell embryos from young (2½- to 6-month-old) donor hamsters could develop into term fetuses when transplanted into young recipients, but that only 8·3% of embryos of the same developmental stage developed into fetuses when transplanted into old (14- to 10-month-old) recipients. The incidence of successful fetal development was also very low (4·5%) when the embryos from old donors were transplanted into young recipients. Orsini

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T. IWAMATSU and R. YANAGIMACHI

Summary.

Oocytes of various sizes were isolated from ovaries of sexually mature and prepubertal hamsters and cultured to determine whether all the oocytes were capable of maturing in vitro. It was found that only the oocytes that had attained maximum size (about 80 μm in the vitelline diameter) were capable of undergoing maturation in vitro. Smaller oocytes (<80 μm in diameter) failed either to initiate or to complete maturation. The oocytes which were capable of maturing in vitro (competent oocytes) were present in the ovaries of adult females 136 to 138 hr before ovulation. The competent oocytes first appeared in the ovaries of prepubertal females on the 23rd day after birth, about 10 days before puberty.