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The need for spermatozoa to undergo a physiological change in the female genital tract before they can penetrate ova was described by Austin (1951, 1952) and Chang (1951, 1955).

It has been demonstrated that rabbit spermatozoa survive for as long as 48 hr in the uterus of an oestrous rat. Incubation of rabbit spermatozoa for different lengths of time in the rat uterus resulted in the fertilization of a high proportion of rabbit ova when the spermatozoa were injected into the Fallopian tube 12 to 13 hr after an ovulation-inducing injection of gonadotrophins (Bedford & Shalkovsky, 1967; Hamner & Sojka, 1967). However, if spermatozoa were injected 14 to 14½ hr after the gonadotrophins, practically no ova were fertilized (Bedford & Shalkovsky, 1967). An inference was that only partial capacitation of rabbit spermatozoa had taken place

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C. Barros and E. Herrera

Summary. Zona-free hamster oocytes inseminated in vitro with acrosome-reacted guinea-pig spermatozoa were examined with the electron microscope. Guinea-pig spermatozoa, in the vicinity of the oocytes, consistently lacked the whole acrosome including the equatorial segment region. In cross fertilization sperm–egg membrane fusion does not differ significantly from that of normal fertilization. However, it was sometimes possible to observe protrusions of oocyte cytoplasm containing sperm chromatin in the process of dispersion.

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Yanagimachi (1972a) published the first report of capacitation in vitro of guinea-pig spermatozoa. He incubated the spermatozoa in a medium developed by Biggers, Whitten & Whittingham (1971). After 12 to 18 hr incubation, the spermatozoa showed evidence of the acrosomal reaction and were able to penetrate guinea-pig eggs in vitro, with subsequent dispersion of the sperm head and pronuclear formation. So far, all the systems assayed for capacitation in vitro of mammalian spermatozoa have in their composition complex macromolecules such as those present in the body fluids or blood serum fractions.

We decided to undertake a further investigation of the conditions under which guinea-pig spermatozoa would undergo capacitation. For this purpose, the epididymal tails were removed from adult guinea-pigs and the epididymal spermatozoa were suspended in 0·9% sodium chloride. From this sperm suspension,

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Hamster spermatozoa preincubated in a human blood serum fraction for 2 to 3 hr fertilize over 90% of hamster eggs with intact zonae pellucidae. Spermatozoa preincubated for 4 to 6 hr lose the ability to cross the zona pellucida, but can enter the cytoplasm of zona-free eggs.

Electron microscopic examination of briefly incubated spermatozoa showed that the large majority had `intact' or slightly vesiculated equatorial segments, while prolonged incubation led to extensive vesiculation of the equatorial segments. It is inferred that the loss of the ability of spermatozoa to penetrate the zona pellucida is associated with excessive vesiculation of the equatorial segments.

In the light of this finding, the possible location of the sperm-borne `zona lysin' is discussed.

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Blood serum collected from female hamsters on each day of the oestrous cycle was assayed for its ability to induce the acrosome reaction. The maximum activity was found in sera obtained from females around the time of ovulation, and the minimum on Day 2. Ovariectomy did not affect the activity of the serum but oestrogen treatment significantly increased it. Blood serum from progesterone-treated and pregnant females significantly depressed the incidence of the acrosome reaction. Progesterone counteracted the effect of oestrogen with regard to the incidence of the acrosome reaction. The counteracting effect was maximal when both hormones were administered at the same time.

The possible rôle played by the hormones in this phenomenon is discussed.

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P. Quinn, C. Barros, and D. G. Whittingham

Summary. Between 70 and 80% of zona-intact hamster ova survived freezing after slow cooling (~0·3°C/min) to −80°C in Medium PB1 containing 1·5 or 2·0 m-DMSO before transfer to −196°C. After slow warming (~8°C/min), there was no difference in survival if the DMSO was diluted out by a slow stepwise or a rapid single addition of medium. When slow cooling was terminated at −40°C by direct transfer to −196°C, up to 75% of the ova survived rapid warming (~500°C/min) and rapid dilution if the medium contained 2·0 m-DMSO. The survival rates were calculated on the basis of the number of thawed ova which retained their normal morphological appearance after a 1 h incubation before removal of the zona pellucida with trypsin. All of these ova were penetrated after incubation with mouse spermatozoa, indicating that the freezing procedure per se does not adversely affect the penetration of frozen—thawed hamster ova by heterologous spermatozoa.

There was no difference in the penetration rate of human spermatozoa into frozen (34%) or fresh (42%) oocytes when a Hepes-buffered Tyrode solution containing 30 mg BSA/ml and 2·0 m-DMSO was used as the freezing medium. However, fewer ova frozen in Medium PB1 containing 4 mg BSA/ml and 2·0 m-DMSO were penetrated by human spermatozoa (18%) compared with freshly collected ova (38%). Zona-free ova did not survive the freezing procedure as well as zona-intact ova.

The survival of hamster oocytes stored at −196°C offers a convenient means of supplying and transporting these ova for the assessment of the fertilizing capacity of human and other heterologous spermatozoa.

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The mechanisms for the prevention of polyspermy in mammalian eggs appear to be associated with the development of the so-called `zona reaction' (Braden, Austin & David, 1954) and with the development of a vitelline surface block to polyspermy (Austin & Braden, 1956). The time required for the development of the zona reaction has been estimated to be not less than 10 min and not more than 1½ to 2 hr (Braden et al., 1954).

Hamster eggs recovered from mated females show a low incidence (1·6%) of polyspermy (Austin & Braden, 1956) while eggs fertilized in vitro show a high incidence, often as high as 100% (Yanagimachi & Chang, 1964; Barros & Austin, 1967; Barros, 1968b; Yanagimachi, 1969).

It has not been established whether the high incidence of polyspermy in vitro is due to the penetration of several spermatozoa at the same time or to a continuous flow of spermatozoa

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C. Barros, A. Jedlicki, I. Fuenzalida, E. Herrera, B. Arguello, P. Vigil, P. Villaseca, and E. Leontic

Summary. Samples of semen and cervical mucus were provided by 18 couples. Cervical mucus was obtained for each day possible and stored at 4°C until all the samples were collected. Flat capillary tubes were loaded with the mucous samples and spermatozoa from the husband's semen sample were allowed to migrate through the cervical mucus (3 cm column) into culture medium. The spermatozoa recovered after migration through cervical mucus were assayed in vitro with zona-free hamster oocytes. Control experiments were carried out using spermatozoa from the same semen sample but prepared by the swimming-up technique. Altogether, 557 eggs in the control group and 1236 eggs in the experimental group were analysed, and the results demonstrated that the % of sperm penetration, the mean number of sperm decondensations per penetrated egg and the mean number of spermatozoa adhering per egg all had higher values (P < 0·05) for the control samples than for the experimental samples. We suggest that cervical mucus modifies human spermatozoa, as measured by their interaction with zona-free hamster oocytes.

Keywords: human; cervical mucus; gamete membrane fusion test; spermatozoa

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J Buratini Jr, A B Teixeira, I B Costa, V F Glapinski, M G L Pinto, I C Giometti, C M Barros, M Cao, E S Nicola, and C A Price

Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.