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J. M. BEDFORD

The first barrier presented to rabbit spermatozoa at the site of fertilization is the cell mass which comprises the cumulus oophorus and, immediately surrounding the ovum, the corona radiata. Fertilization in the mouse, rat and rabbit occurs before dispersion of these granulosa cells (Lewis & Wright, 1935; Austin, 1948a, b; Chang, 1951a). In studies of the interaction of spermatozoa with the cumulus of the rat, rabbit and hamster in vitro, Austin ( 1960) observed that epididymal or ejaculate spermatozoa were apparently unable to penetrate the cumulus oophorus, whereas under similar conditions spermatozoa were seen moving freely through the cumuli obtained from mated animals. This observation led Austin to suggest that capacitation, which occurs while the spermatozoa are in the female tract (Austin, 1951; Chang, 1951b), may facilitate sperm passage through the cumulus oophorus to the surface of the zona pellucida. This suggestion has been tested in the rabbit, in vivo, in the manner described below.

Spermatozoa flushed from rabbit uteri with Hanks' solution 12 to 14 hr after natural mating were concentrated by centrifugation at 1000 rev/min for 5 min.

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J. M. BEDFORD

Summary.

The phagocytosis of rabbit spermatozoa has been studied with the electron microscope, and the process of ingestion of both intact and damaged spermatozoa by leucocytes in the oestrous uterus is described. The cell membrane and acrosome cap remained intact in many sperm heads wholly ingested by uterine leucocytes up to 12 hr after mating. However, only spermatozoa with damaged acrosomes and/ or damaged head membranes were ingested to any extent in the pseudopregnant uterus, in the pleural cavity, or following in-vitro incubation with leucocytes obtained from the oestrous uterus, intact sperm heads merely adhering to the leucocyte surface. It is suggested that some change occurs in the surface membrane of spermatozoa in the oestrous uterus which renders the intact sperm head acceptable to uterine leucocytes. Such surface changes may be associated with the process of sperm capacitation.

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J. M. BEDFORD

Summary.

A study of rabbit ejaculate and epididymal spermatozoa, and of spermatozoa recovered from various regions of the female tract, has revealed some consistent morphological differences in the state of the acrosome between these groups. Few acrosomes were missing or changed in epididymal or ejaculate spermatozoa. On the other hand, the acrosome was absent in spermatozoa released from the perivitelline space of fertilized ova. Uterine and tubal spermatozoa recovered 9 to 11 hr after insemination of ejaculates into oestrous or pseudopregnant females showed change or loss of the acrosome in a variable percentage (8 to 58%). The percentage acrosome loss from uterine samples taken after insemination of aliquots from the same ejaculate into pairs of females was closely similar within each pair, though variation occurred between pairs. Consistently higher acrosome losses (mean, 33%) were found following insemination of ejaculates collected after 1 month's sexual rest than were found after insemination of the third ejaculate from each of the same males (mean, 17·2%). Considerably fewer acrosomes were missing from uterine spermatozoa recovered after insemination of epididymal spermatozoa (mean, 5%) compared to the loss after insemination of ejaculate spermatozoa (mean, 27·4%) taken previously from the same males. Fertility trials with ejaculate and epididymal spermatozoa showed no difference in fertilization rate, or in numbers of supplementary spermatozoa, in favour of the ejaculate samples, i.e. those which showed the greater percentage acrosome changes.

In conclusion it is suggested that the quantitative variation in acrosome reaction between samples of uterine spermatozoa reflects differences in the samples, rather than differences in the female environment. The acrosome changes in the uterine and tubal spermatozoa of the domestic rabbit are probably not a morphological concomitant of capacitation, but more probably reflect impending sperm senility.

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J. M. BEDFORD

Summary.

The fertilizing ability of rabbit spermatozoa is acquired in passage through the epididymis, during which certain changes take place in the spermatozoon. It has been found here that a significant diminution (P<0·01) in acrosome width and length occurs during passage of spermatozoa from caput to cauda epididymis. Within-group analysis of the caput sperm group reveals a significant correlation (P<0·01) of acrosome width with the position of the cytoplasmic droplet on the midpiece. A large difference in the cytoplasmic droplet distribution is apparent between samples of spermatozoa from the cauda and caput epididymis, respectively. The occurrence and disposition of the droplet in either of these regions was not obviously altered following a series of successive ejaculations. The change in acrosome size during epididymal passage is discussed in relation to the development of fertilizing ability of rabbit spermatozoa.

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J. M. BEDFORD

Summary.

A potent agglutinin in the semen of a fertile male rabbit caused massive head-to-head agglutination of its own ejaculated spermatozoa and those of other rabbits, without reducing their motility, or destroying their fertility. By contrast, rabbit spermatozoa collected from the uterus 12 hr after insemination, and bull, rat, hamster, guinea-pig and human spermatozoa, were immobilized immediately when exposed to the active seminal plasma, the agglutinating and spermicidal properties of which were destroyed by heating to 55° C for 20 min. This indicates a specific change in the susceptibilities as well as in the functional competence of rabbit spermatozoa during the period of capacitation in the uterus. The agglutinin-active plasma also brought about marked clumping of leucocytes, but not of erythrocytes or granulosa cells. Brief comment is made on the nature of the sperm surface and on the significance of seminal antagglutins.

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J. M. BEDFORD

Summary.

The microstructure of the rabbit sperm head in ejaculate and in uterine spermatozoa has been examined with the electron microscope. The normal structure of the sperm head is described, and it is suggested that the properties of the plasma membrane may vary over different regions. No morphological change in the acrosome cap, which might account for the functional difference in capacitated spermatozoa, has been observed. There are indications, however, that the plasma membrane is more easily separable from the anterior and lateral edges of the underlying acrosome cap in spermatozoa recovered from the uterus.

Abnormal forms of acrosome cap development are described, and are discussed particularly in relation to similar abnormalities described previously in bull and boar spermatozoa.

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J. M. BEDFORD

Summary.

Fluids instilled into the rabbit vagina are prevented from passing further along the tract by the valve-like disposition of the uterine cervix. In the present study, vaginal spermatozoa were killed at 2 or 5 min after coitus by instillation of 12 to 16 ml of 1% lauryl sulphate (sodium dodecyl sulphate) into the anterior vagina. When the vaginal spermatozoa were killed 2 min after a single mating, only 25% of eggs were subsequently fertilized and there were essentially no accessory spermatozoa about the eggs. The fertilization rate was increased to 93%, with a mean of 6·6 perivitelline spermatozoa/egg when killing of the vaginal spermatozoa was delayed until 5 min after a single mating. Thus, in the rabbit, sufficient numbers of vaginal spermatozoa for normal fertility enter the cervix within 5 min after coitus.

Early passage of spermatozoa into the cervix was much enhanced by a second successive coital stimulus, since destruction of all vaginal spermatozoa only 2 min after double mating allowed a 90% fertilization rate and a mean of 5·5 perivitelline spermatozoa/egg. Comparable results were achieved even if the second intromission was accomplished by a vasectomized male, pointing to the importance of the second coital stimulus rather than of the greater sperm numbers provided by double mating.

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J. M. Bedford and R. Yanagimachi

Summary. The epididymis was reflected unilaterally or bilaterally to the abdomen in adult hamsters, leaving normally functioning testes in the scrotum. In unilateral cases, spermatozoa taken from the abdominal cauda, 1 month or more post-operatively, underwent a reversal of head agglutination and dispersed earlier, and underwent hyperactivation and fertilized cumulus-free eggs about 30–45 min sooner than did spermatozoa from the contralateral scrotal cauda. In addition, spermatozoa from the abdominal cauda began to undergo a spontaneous acrosome reaction 30–45 min earlier and to a greater extent than in control spermatozoa. Finally, in females mated at or soon after ovulation, spermatozoa ejaculated by bilaterally cryptepididymal males fertilized eggs 30–45 min before those from normal males. Other females mated to bilaterally cryptepididymal males gave birth to normal litters. The results are considered in terms of the possibility that temperature-sensitive sperm-binding macromolecules, which may be involved in sperm storage in the cauda epididymis, could be one determinant of the need for capacitation.

Keywords: cauda epididymidis; sperm capacitation; temperature; acrosome reaction; hamster

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P. Esponda and J. M. Bedford

Summary. Polyacrylamide gel electrophoresis under reducing and non-reducing conditions, and immunoelectrophoretic analysis, revealed that several characteristic proteins disappear from the luminal fluid of the rat cauda epididymidis when it is maintained at body temperature. On SDS-PAGE gels prepared under reducing conditions, one Coomassie-blue staining band of M r 18 000 disappeared and another of 52 000 was significantly reduced after only 6 days; bands of M r 23 000, several in the M r 34–38 000 range, one of M r 48 000, and others of M r 100–200 000 were eliminated or markedly reduced after 15 days at body temperature. Some were glycoprotein, as judged by their affinity for FITC–ConA. At 15 days after castration there was a broadly similar but rather more extensive disappearance of macromolecules, and of glycoproteins in particular, from caudal fluid. The fact that several similar proteins are diminished in or disappear from fluid or the cauda epididymidis maintained at body temperature, or after androgen withdrawal, raises the possibility that one or more such proteins play a role in sperm storage there.

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J. M. Bedford and A. Dobrenis

Summary. Groups of unfertilized and pronuclear stage rabbit oocytes were exposed to fluorescent light of 3250 lx for 20–30 min at 37°C. In 6 experiments with fertilization achieved in vivo, 54% of the zygotes exposed as secondary oocytes and 67% of light-protected controls had implanted and developed normally 16 days after transfer to the contralateral oviducts of synchronized recipients. When pronuclear oocytes were exposed similarly in 8 experiments, 63% had established a normal pregnancy at 16 days after transfer compared to 65% of the controls. In 5 of these pregnancies which were allowed to proceed to term, all the young born appeared normal.

Though similar in size, it is not clear whether the rabbit oocyte constitutes a suitable model for the human oocyte in regard to the effects of visible light. However, the level of exposure used here is 200–300 times that experienced during normal in-vitro manipulation of human eggs. The absence of significant effects should allay concerns that light is a negative factor in the normal procedure of in-vitro fertilization in man.

Keywords: oocytes; zygotes; light exposure; in-vitro fertilization; pregnancy rate; rabbit