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PubMed1977

Sperm/egg interaction: the specificity of human spermatozoa.

Human spermatozoa display unusually limited affinities in their interaction with oocytes of other species. They adhered to and, when capacitated, penetrated the vestments of the oocyte of an ape--the gibbon, Hylobates lar--both in vivo and in vitro. On the other hand, human spermatozoa would not even attach to the zona surface of sub-hominoid primate (baboon, rhesus monkey, squirrel monkey), nor to the non-primate eutherian oocytes tested. Among the apes the gibbon stands furthest from man. Thus, although the specificity of human spermatozoa is not confined to man alone, it probably is restricted to the Hominoidea. This study also suggests that the evolution of man and perhaps the other hominids has been accompanied by a restrictive change in the nature of the sperm surface which has limited and made more specific the complementary surface to which their spermatozoa may adhere. For the failure of human spermatozoa to attach to the zona surface of all non-hominoid oocytes stands in contrast to the behaviour of spermatozoa of the several other mammals studied which, in most combinations, adhered readily to foreign oocytes, including those of man. Taxonomically, the demonstration of a compatibility between the gametes of man and gibbon, not shared with cercopithecids, constitutes further evidence for inclusion of the Hylobatidae within the Hominoidea.

Animals↗
PubMed1976

Surface interactions between mammalian sperm and egg: variation of spermatozoa concentration as a probe for the study of binding in vitro.

The pre-penetration binding interactions between gametes of the golden hamster were investigated in vitro. Binding between capacitated spermatozoa and the surface of eggs, that is the zonae pellucidae with intact vitelli, as a function of the concentration of spermatozoa, followed a sigmoidal curve. This was in sharp contrast to the linear binding obtained with mechanically isolated zonae pellucidae (zonae lacking vitelli). Penetration of eggs as a function of the concentration of spermatozoa paralleled the binding curve that occurred between gametes. The binding curve obtained with uncapacitated spermatozoa and eggs was not sigmoidal but was linear after a slight lag and parallel to the curve obtained with uncapacitated spermatozoa and isolated zonae pellucidae. Taken together these results support previous work which implicated a vitelline factor in the binding reaction between the surfaces of eggs and capacitated spermatozoa. By scoring binding at one minute intervals it was possible to relate the rapid uninterrupted binding that occurs between capacitated spermatozoa and isolated zonae pellucidae with the equally rapid but transient and vitellus-influenced binding that occurs between gametes. It was concluded that the vitelline factor acts by preventing most of the early type of binding that occurs between spermatozoa and isolated zonae pellucidae and not by terminating the early, rapid, initial binding as previously postulated. Thus, this early binding never occurs between most of the gametes that finally bind 30 to 40 minutes later and, therefore, does not play a role in the establishment of the late binding step which leads to penetration.

Animals↗
PubMed1976

Sperm concentration dependency in the fertilization and zonae sperm binding properties of mouse eggs inseminated in vitro.

Sperm penetration of zonae and fertilization of mouse eggs in vitro was studied as a function of sperm concentration over the range 10(3)-10(6) cells/ml. Maximal fertilization was obtained at 10(5) sperm/ml with markedly reduced levels seen at lower concentrations. Eggs inseminated at low sperm concentrations were, however, characterized by the absence of multiple sperm penetrations of their zonae and by the retention of an intact cumulus oophorus during culture. Penetration levels were similar in eggs inseminated with and without an intact cumulus, although increases in supernumerary and supplemental sperm were observed with partly denuded ova. The distribution of sperm found within eggs inseminated in vitro was compared with a distribution predicted by chance alone. The similarity in observed and expected distributions indicated that a functional zona reaction was absent in eggs inseminated in vitro. Sperm binding to zonae of cumulus-free unfertilized mouse eggs was shown to be related to sperm concentration over the range 10(3)-10(6) cells/ml. Zonae sperm binding sites, while too numerous for accurate quantitation, exceeded 100 per zona. The interrelationships between sperm penetration of and sperm binding to the zonae of cumulus-free eggs were examined. At low sperm concentrations, the ratio of sperm/zonae with bound sperm to sperm/penetrated egg approached one, although the total number of bound sperm exceeded the total number of penetrating sperm. That proportion of cells in a preincubated epididymal sperm suspension, capable of binding to the zona was determined and employed in an estimation of the extent of sperm capacitation.

Animals↗
PubMed1976

Evidence for a polyspermy block at the level of sperm-egg plasma membrane fusion in Urechis caupo.

The results of sperm binding experiments reveal no change in the sperm binding properties of the egg surface coat at fertilization of Urechis caupo eggs. When fertilized eggs are reinseminated, sperm continue to attach to the egg surface coat. The acrosomal tubules of supernumerary sperm are observed in the perivitelline space closely apposed to the egg membrane. Thus, the polyspermy block in Urechis eggs involves neither alteration of sperm binding sites nor inhibition of the acrosome reaction. Our results suggest that the block is at the level of sperm-egg membrane fusion.

Acrosome↗
PubMed1976

Maturation and sperm penetration of canine ovarian oocytes in vitro.

Canine ovarian oocytes were cultured in a medium consisting of TC medium 199, fetal calf serum and antibiotics. Ninety-nine percent of the apparently healthy oocytes were in the germinal vesicle (dictyate) stage when recovered from the ovaries; 25% of them reached metaphase I or II by 72 hours of culture. Washed ejaculated spermatozoa were added to BWW medium containing oocytes which had either been removed directly from the follicles or which had been cultured for 24--72 hours. The earliest acrosome reaction and zona penetration by spermatozoa were seen at seven hours after insemination. Seventy-four percent of the oocytes examined between 11 and 24 hours after insemination showed evidence of zona penetration by spermatozoa. Neither the condition of the oocyte vitellus nor the stage of nuclear maturation influenced the incidence of zona penetration. Decondensing sperm nuclei were found in the vitellus of 27% of the oocytes which had not been cultured and in the vitellus of 20% of those which had been cultured for 24--72 hours and were in various stages of maturation. These results indicate that (1) canine ovarian oocytes can be matured in vitro, (2) the spermatozoa require capacitation which takes approximately seven hours in vitro and (3) maturation of the oocytes is not required for sperm passage through the zona pellucida or entry into the vitellus nor for sperm nuclear decondensation.

Animals↗
PubMed1976

Phospholipase activity of sea urchin sperm: its possible involvement in membrane fusion.

Phospholipase activity of egg-water treated Arbacia punctulata and Lytechinus variegatus sperm was shown to result from the sequential action of phospholipase A and lysophospholipase. A transient burst of phospholipase A activity followed induction of the acrosome reaction with egg water. The time of appearance suggested an acrosomal localization of the enzyme. The peak activity of phospholipase A correlated with initiation of sperm-egg fusion, suggesting a role for sea urchin sperm phospholipase A in membrane fusion and/or egg activation during fertilization.

Acrosome↗
PubMed1977

Involvement of a trypsin-like activity in sperm penetration of zona-free mouse ova.

The presence of trypsin inhibitors during insemination reduced penetration of zona-intact and zona-free mouse ova by capacitated sperm. This inhibition was dependent upon both concentration of inhibitor and sperm. Preincubation of gametes in inhibitors did not markedly influence their subsequent fertility, nor did it alter sperm motility. Trypsin inhibitors may exert an effect on penetration by interfering with the induction of the sperm acrosome reaction or with the process of sperm-egg fusion.

Amidines↗
PubMed1977

Is the activated spermatozoon really capacitated?

A culture medium (7M) consisting only of salts and devoid of any macromolecule was prepared to test its effect upon survival, activation and acrosome reaction of golden hamster spermatozoa. Incubation of spermatozoa in medium 7N allowed a temporal separation of activation and acrosome reaction. Over seventy per cent of spermatozoa were activated after four hours at 37 degrees C while the acrosome reaction remained very low (8%). Activated spermatozoa with intact acrosomes were incubated with zona-free hamster oocytes at 37 degrees C for one to two hours. When these oocytes were examined with the electron microscope it was found that the spermatozoa were closely bound to the egg plasma membrane, mainly by the plasma membrane overlying the acrosome, but in no instance had the gamete membranes fused. Prolonged incubation resulted in an engulfment of the sperm by the oocyte in a phagocytic manner. The experimental evidence would indicate that an activated spermatozoon is not capacitated since it cannot fertilize oocytes.

Acrosome↗
PubMed1977

Alteration of structure and penetrability of the vitelline envelope after passage of eggs from coelom to oviduct in Xenopus laevis.

The vitelline envelope (VE) that surrounds an egg released from the ovary into the coelom of Xenopus laevis differs markedly, in structure and penetrability, from the VE surrounding an oviposited egg. In a coelomic egg, the filaments that form the VE are arranged in distinct fascicles or bundles. The exterior surface of the VE is irregular in contour and is permeated by channels. In an oviposited egg, the filaments are evenly dispersed and lack a fasciculated arrangement; the exterior surface is smooth and no channels are present. The fascicular arrangement of fibrils in the coelomic VE is maintained only at neutral pH, and is not visibly altered by the cortical reaction. VEs from coelomic eggs retain their fasciculated morphology after isolation from the egg. In an in vitro test system, sperm penetrated VEs isolated from oviposited eggs, but failed to penetrate VEs isolated from coelomic eggs. The structural transformation of the VE from the coelomic type to the oviposited type occurs in the first 1-cm segment of the oviduct, prior to addition of jelly to the egg. Neither intact jelly, solubilized jelly, nor jelly extracts were capable of altering the structural organization of coelomic VEs, suggesting that the structural transformation of the VE is effected by some oviducal factor other than jelly.

Animals↗
PubMed1977

Timing of sperm penetration, pronuclear formation, pronuclear DNA synthesis, and first cleavage in naturally ovulated mouse eggs.

Timing of development of naturally ovulated mouse eggs from sperm penetration to first cleavage, including that of DNA synthesis, was established. In an attempt to limit variability, partial synchronization of ovulation was accomplished by shortening the length of the dark period to five hours, and partial synchronization of sperm entry was attempted by mating the females soon after the ovulated eggs reached the ampulla and by limiting the period at which mating could occur to only 20 minutes. Evidence of sperm penetration (presence of one or more sperm in perivitelline space or inside the vitellus) was found beginning 1.75 hours after the end of the mating period. Pronuclei were formed three to four hours after sperm entry. Pronuclear DNA, synthesis began about eight hours postmating, 3.25 to 4.5 hours after pronuclear formation, or about 6.25 to 8.5 hours after sperm entry; it was completed in almost all zygotes by 16 hours postmating. The first completed cleavage division was found 17 to 18 hours postmating, and almost all eggs had cleaved by 20 hours.

Animals↗
PubMed1978

Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro.

Zona-free eggs were introduced to fresh or preincubated sperm suspensions and the penetration of eggs by foreign spermatozoa was examined, as evidenced by enlargement of the sperm head and formation of the male pronucleus. It was found that zona-free hamster eggs can be penetrated by guinea-pig, deer mouse and rabbit spermatozoa but zona-free rat, mouse and rabbit eggs cannot be penetrated by guinea-pig spermatozoa. Furthermore, zona-free rat and mouse eggs cannot be penetrated by spermatozoa from two species of deer mice and the Mongolian gerbil. The zona pellucida of a few intact rat eggs can be penetrated by mouse (6%) and by P. leucopus spermatozoa (14%) but enlargement of the sperm head and formation of pronuclei were observed in the former but not in the latter. It seems that (1) sperm capacitation is required for the penetration of zona-free eggs, (2) the attachment of foreign spermatozoa to eggs may indicate their potential ability of penetration in some cases, (3) there is a certain affinity between the vitellus of one species and spermatozoa from another species, (4) the block to the entry of foreign spermatozoa is not only in the zona pellucida but also in the vitelline membrane, (5) zona-free hamster eggs can be penetrated by spermatozoa of six species, (6) mouse spermatozoa can penetrate zona-free eggs of three species, and (7) fertilization of intact P. maniculatus eggs can be achieved in vitro.

Animals↗
PubMed1978

Timing of sperm transport, sperm penetration and cleavage in the rat.

Times of sperm entry into the oviduct from the uterus, into the ampulla from the isthmus; of sperm penetration into oocytes, and of cleavage, were determined using three mating regions. Time intervals and their errors of estimation were calculated. Spermatozoa were first found in the isthmus of the oviduct no earlier than 15 minutes after coitus, but required four hours to ascend the oviduct to the ampulla. The rate of sperm arrival was equal to the rate of sperm penetration, i.e., about 3 sperms/hour. Time of cleavage in vivo was 20.6 hours after sperm penetration in ad libitum mated animals. In culture, oocytes cleaved at exactly the same time as in vivo. Delaying sperm arrival to the site of fertilization (by delaying mating) shortened the time interval between median time of sperm penetration and median time of cleavage. It was concluded that the time of cleavage of the oocyte reflects primarily the time of sperm penetration, but is also influenced by the postovulatory age of the oocyte.

Animals↗
PubMed1978

Electron microscopic observation on sperm penetration and pronuclear formation in the fish egg.

Unfertilized eggs of the medaka, Oryzias latipes were inseminated after being mechanically freed from the chorion. In the first step of penetration, as soon as it reached the vitelline surface, the whole spermatozoon was quickly enclosed by ooplasmic protrusions (fertilization cone) of the vitelline surface. In the second step, the egg plasma membrane fused with various regions of the plasma membrane of the enclosed spermatozoon; finally the sperm flagellum was also incorporated into the ooplasm. Initial disappearance of the nuclear envelope of the sperm with vesiculation at the apical region of the head is followed by dispersal of the sperm chromatin. The nuclear envelope is then reformed by fusion of elongated or flattened vesicles along the margin of dispersing nuclear chromatin. The mature male pronucleus has a large nucleolus within a wrinkled envelope. It seems that the fertilization process in this fish involves some features of that occurring in the marine invertebrate and the mammalian eggs.

Animals↗
PubMed1978

Temperature dependence of sperm-egg fusion and post-fusion events in hamster fertilization.

The effects of temperature (4--37 degrees C) on sperm-egg fusion and the post-fusion events were studied. At 4--10 degrees C, acrosome-reacted spermatozoa bound to egg plasma membranes, but could not fuse with them. At 25 degrees C or above, both binding and fusion took place. The post-fusion events could occur over a broad temperature range (4--37 degrees C) but the events progressed faster with increasing temperature. An abnormal development of egg pronucleus, possibly due to an incomplete functioning of the meiotic spindle mechanism, was observed in eggs inseminated at 37 degrees C and cultured at 25 degrees C.

Animals↗
PubMed1978

Rate of egg penetration in vitro accelerated by T/t locus in the mouse.

Spermatozoa from fertile mice heterozygous for tw32, a recessive lethal allele of the T/t locus, were compared to normal spermatozoa in a fertilization in vitro system. The rate of egg penetration following insemination in vitro was determined for epididymal spermatozoa from C57BL/6-tw32/+ mice and for epididymal spermatozoa from C57BL/6-+/+ mice. At one hour after insemination, the mean of penetration +/- standard deviation for spermatozoa from BL/6-tw32/+ mice was 20% +/- 2.1 (109 eggs observed, 5 experiments), while the mean for spermatozoa from BL/6-+/+ mice was 1% +/- 1.5 (107 eggs observed, 4 experiments). By five hours post-insemination, the levels of egg penetration were not significantly different. These results suggest that tw32 increases the initial rate of egg penetration. Preliminary observations of sperm motility and sperm-egg association at one hour post-insemination in vitro do not support the hypothesis that this earlier penetration is due to improved sperm progress to the egg. Rather, the earlier penetration may be a result of changes in the timing of capacitation, the acrosome reaction, or sperm-egg fusion. It is possible that the earlier penetration may play a role in the distortion of the transmission ratio of tw32.

Animals↗