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B. D. BAVISTER

Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, 1960 East West Road, Honolulu, Hawaii 96822, U.S.A.

(Received 8th November 1974)

The dialysable heat-stable fraction of human serum stimulates the motility of hamster epididymal spermatozoa in vitro (Yanagimachi, 1970) and produces favourable conditions for capacitation, although the presence of protein is also required to stimulate the acrosome reaction efficiently (Yanagimachi, 1970; Morton & Bavister, 1974). The active fraction from gel-filtered human serum was able to substitute for the motility-stimulating activity normally associated with the hamster cumulus mass (Bavister & Morton, 1974), and it is reasonable to assume at present that the active substances from both sources are similar. Some properties of the serum-derived substance that may assist in its identification are described in the present communication.

Freshly collected male human serum was heated to 56°C for 30 min, and then subjected to ultrafiltration (UM 20E filter, Amicon

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B. D. BAVISTER

Golden hamster spermatozoa recovered from the cauda epididymidis can penetrate eggs in vitro after a delay of 2 to 3 hr, which is attributed to the need for sperm capacitation (Barros, 1968). During an investigation into the optimal conditions for fertilization of hamster eggs in vitro (Bavister, 1971), I found that this delay could be substantially reduced by preincubating epididymal spermatozoa in a simple culture medium (a modification of Tyrode's solution, containing bovine serum albumin, designated Tyrode-B; Bavister, 1969). The preliminary findings in this work have already been reported (Austin, Bavister & Edwards, 1973); the present communication describes the techniques used and contains results of a larger series of experiments. Since there is some controversy over the meaning of the term `capacitation', it is used below to denote only those changes undergone by spermatozoa after leaving the male reproductive tract and

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B. D. BAVISTER

Summary.

The motility of hamster epididymal spermatozoa has been studied in modified Tyrode's solution; a high level of motility was maintained when bovine serum albumin, used in previous studies, was replaced by polyvinyl pyrrolidone, and when no macromolecule was included. In these culture media, it was found that sperm motility and survival were considerably enhanced if the osmotic pressure was lowered by 20% from the usual level of 290 mosmol. The reduced osmotic pressure caused leakage of a motility-stimulating substance from spermatozoa; the need to lower the osmotic pressure for optimum sperm motility could be eliminated by adding an extract of spermatozoa to the culture medium. With this culture system, spermatozoa could be maintained in a high state of motility for several hours, and the `sperm dilution effect' was substantially reduced.

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B. Fischer and B. D. Bavister

Oxygen tension was measured using flexible polarographic microelectrodes within the oviductal and uterine lumen in rhesus monkeys (n = 9), golden hamsters (n = 21) and rabbits (n = 6), during the reproductive cycle (monkey), during oestrus and pseudopregnancy (hamsters, rabbits) and during pregnancy (hamsters). In general, oxygen tensions in each species were much less than half of atmospheric O2, ranging from high values of about 60 mm Hg (8.7% O2) in the rabbit oviduct, rabbit and hamster uterus, to as low as 11 mm Hg (1.5% O2) in the monkey uterus. Oxygen tensions did not vary significantly between left and right sides of the reproductive tracts (all species), nor between pregnant and pseudopregnant states nor between oviduct and uterus (hamsters). Differences owing to reproductive stage were found in the monkey oviduct, hamster oviduct and uterus, and rabbit uterus. Oxygen tensions were consistently very low (11–14 mm Hg) in the monkey uterus throughout the menstrual cycle. In hamsters and rabbits, intrauterine O2 decreased significantly at about the normal time of blastocyst formation and implantation, to 37 mm Hg (5.3% O2) and 24 mm Hg (3.5% O2), respectively. This study indicates that embryos develop in vivo under low oxygen concentrations, especially during the peri-implantation period. The data have implications for investigations of embyro metabolism and for improving embryo development in vitro.

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S. Kito and B. D. Bavister

Male pronuclear (MPN) formation in oocytes after in vitro maturation (IVM) was compared with that of matured follicular oocytes that had matured in vivo (controls). Cumulus–oocyte complexes were matured in vitro for 13 h in modified Tyrode's solution (TLP–PVA); cumulus-free oocytes were then incubated in 20% oviductal fluid for 3 h, and washed and capacitated spermatozoa were added. MPN formation was significantly lower (P < 0.05) in IVM oocytes 3 to 12 h after insemination (0 to 34%, respectively) than in control oocytes (range, 98–100%). Female pronuclear formation was 84–100% in controls and IVM oocytes, but spermatozoa incompletely decondensed in IVM oocytes. The addition of 10 μmol l−1 during IVM, significantly increased (P < 0.05) MPN formation (from 17% in the absence of cysteine to 47% in the presence of cysteine), but was lower than that in controls (88%). During IVM, the addition of 10% serum or gonadotrophins (FSH and LH) with or without amino acids did not support MPN formation without cysteamine, whereas the treatment with gonadotrophins and 11 amino acids plus 200 μmol cysteamine l−1 (82%) equalled controls (92%). Development of oocytes after IVM (in 0, 10, 20% serum) in TLP–PVA, gonadotrophins, 11 amino acids and 200 μmol cysteamine l−1 was compared with development in controls. Of the IVM treatments, 20% serum was inferior at fertilization, but yielded the highest percentage of fertilized oocytes developing to or beyond the four-cell stage (20% serum versus controls, respectively): fertilized oocytes, 75% versus 88%; ≥four-cell embryo, 40% versus 53%; blastocyst, 8% versus 14%. It was concluded that during IVM, gonadotrophins plus 11 amino acids interacted with cysteamine, enhancing the decondensation of spermatozoa and MPN formation; oocytes matured in this medium with 20% serum were fertilized and some developed to the blastocyst stage.

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D. B. MORTON and B. D. BAVISTER

Efficient capacitation of hamster spermatozoa can be induced by follicular fluid (Barros & Austin, 1967; Yanagimachi, 1969a, b) and blood sera of several species (Barros & Garavagno, 1970; Yanagimachi, 1970a; Talbot, Franklin & Fussell, 1974). Two components are involved: one is dialysable, heat-stable and stimulates sperm motility, while the other is non-dialysable, heat-labile and induces the acrosome reaction (Yanagimachi, 1969a). Our understanding of capacitation and the acrosome reaction might be enhanced if the nature and mode of action of these factors were known. Blood serum is an easily available source of both factors and this preliminary report describes the recovery of the sperm motility-stimulating activity from human serum.

Fresh human serum was heated to 56°C for 30 min to destroy unidentified toxic factor(s) (Yanagimachi, 1970a). The serum was chromatographed on Sephadex G-25 (medium grade) and equilibrated in Tyrode's solution supplemented with 0·33 mm-sodium pyruvate, 0·01 mg phenol red/ml,

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B. D. BAVISTER and D. B. MORTON

A fraction that stimulates hamster sperm motility can be obtained from human serum by Sephadex gel filtration (Morton & Bavister, 1974). The protein-rich fractions potentiate this activity and effectively induce the acrosome reaction. Here, we describe attempts to determine the type of protein that may be involved.

The methods used to prepare the motility-stimulating fraction from human serum and for assessing its activity were as previously described (Morton & Bavister, 1974). To help identify which serum component induced the acrosome reaction, serum proteins were chromatographically separated on Sephadex G-150 (5 ml pooled human serum fractionated on 130 ml bed volume G-150 in a 1·5×90 cm column, fraction size 1·6 ml, flow rate 10 ml/hr). Protein was monitored by absorption at 280 nm with an LKB Uvicord II (see Text-fig.

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D. G. WHITTINGHAM and B. D. BAVISTER

Although many workers have fertilized hamster eggs in vitro since the technique was first described by Yanagimachi & Chang (1963), there is only one reported attempt to obtain further development of these eggs in culture (Yanagimachi & Chang, 1964). In this instance, development ceased at the two-cell stage. The present study examines the development in culture of hamster eggs fertilized in vitro and after transfer to recipient foster mothers.

Immature female hamsters (5 to 6 weeks old and 60 to 80 g in weight) were induced to superovulate with intraperitoneal injections of 25 i.u. PMSG and HCG given 48 to 56 hr apart. Following removal of the oviducts 15 to 17 hr after the injection of HCG, they were blotted on sterile filter paper to remove excess blood and immersed in liquid paraffin contained in a Petri dish (35 mm in diameter, Falcon Plastics). The eggs and surrounding cumulus cells

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D. E. Boatman and B. D. Bavister

Summary. Capacitation of rhesus monkey spermatozoa was assessed by monitoring sperm flagellar beat and trajectory changes during incubation in vitro and by determining sperm penetration into rhesus oocytes and hamster zona-free ova. Rhesus sperm capacitation in vitro depended on the addition to the culture medium of the cyclic nucleotide mediators, caffeine and dibutyryl cyclic AMP. Capacitation was correlated with the development of hyperactivated motility. Spermatozoa treated with the cyclic nucleotide mediators, and showing hyperactivated motility, penetrated 57·4% of all rhesus oocytes and fertilized 88·9% of mature rhesus oocytes that were morphologically normal. Control spermatozoa did not penetrate any of the eggs. Some sperm penetration into hamster ova occurred but was not statistically significant. These data provide a basis for achieving in-vitro fertilization in the rhesus monkey and information on specific sperm motility characteristics associated with fertilizing ability.

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M. Lorraine Leibfried and B. D. Bavister

Summary. Using an experimental design in which the addition of hypotaurine or epinephrine was staggered through time, evidence was found that suggests these two compounds are working independently and sequentially to stimulate the fertilizing capacity of hamster spermatozoa in vitro. Prior exposure of spermatozoa to hypotaurine is a prerequisite for the action of epinephrine in causing activation and penetration of hamster ova. A definite role for hypotaurine in inducing capacitation of hamster spermatozoa is also demonstrated.

The α-adrenergic antagonist, phentolamine, was more effective in blocking fertilization of hamster ova in vitro than was propranolol, a β-antagonist. This indicates that catecholamines may be working by way of α-adrenergic receptors in causing capacitation of hamster spermatozoa. The failure to block fertilization with phentolamine after epinephrine has exerted its effect implies that epinephrine acts in a hormone-like fashion.