Sperm-egg interaction is mediated by a sperm-associated body in quail

The present study describes the holes in the inner vitelline membrane of fertile eggs of the quail Coturnix japonica, which remain after the spermatozoa pass through. It was shown that the light-microscopically observable 'holes' correspond mostly to electron-microscopically defined 'disks', and, to a lesser extent (about 5%), real holes. Immunofluorescent staining of the vitelline membranes with an antiquail ZPC antiserum was used to discriminate the holes from the disks light-microscopically. Over 96% of holes were accompanied by calcium-coated sperm-associated bodies, indicating a close relationship between the two. There was no preferential localization of the disks, holes or sperm-associated bodies in the vitelline membrane around the egg. The sperm-associated bodies bound with the spermatozoa at the posterior end of sperm flagella. Incubation of the inner vitelline membranes, isolated from the largest follicles, with spermatozoa resulted in production only of the disks, whereas the holes (about 9%) were produced when the sperm-associated bodies were added to the system. It was suggested that the sperm-associated bodies assist fertile spermatozoa in binding to the inner vitelline membrane, making holes in the membrane and passing through them in fertile eggs.     

MAGNESIUM ION-REQUIRING STEP IN FERTILIZATION OF SEA URCHINS*

The magnesium ion-requiring step in fertilization of sea urchins was investigated. When eggs were inseminated in Mg-free sea water, several spermatozoa were found to bind to each egg surface with their reacted acrosomes without elevation of fertilization membrane. The number of binding jelly-treated spermatozoa to an egg did not differ regardless of the presence or virtual absence of magnesium ions. Although fertilization did not occur in Ca, Mg-deficient sea water (CM-deficient SW) even when jelly-treated spermatozoa were employed, some eggs could be fertilized by the addition of magnesium to the CM-deficient SW 60 sec after insemination, when jelly-treated spermatozoa had completely lost their fertilizing capacity in the CM-deficient SW. The acrosomal process of jelly-treated spermatozoa appeared to penetrate the vitelline layer in the CM-deficient SW. DTT- or pancreatin-treated eggs could not be fertilized in the virtual absence of magnesium. Re-fertilization using the fertilized eggs deprived of fertilization membrane did not occur under conditions of magnesium deficiency. These results suggest that external magnesium ions are indispensable at least for the fertilization process following penetration of the vitelline layer by the spermatozoa, such as fusion of the plasma membrane between an egg and a reacted spermatozoon, or the subsequent step(s) such as sperm penetration into egg interior and egg activation which precedes the cortical reaction.     

Characterization of the sperm-associated body and its role in the fertilization of the chicken Gallus domesticus

The present paper aimed to characterize the substance forming the sperm-associated body (SB), to find its producing sites, and to show its functions in the fertilization of chicken. The SB was found both in between the inner and outer layers of vitelline membranes around eggs and in the oviductal infundibulum. Material from which the SB is constructed (SB substance) was isolated from the vitelline membranes. It was a hydrophobic protein with a molecular size of 570 kDa. X-ray microanalysis detected calcium in the aggregates of the SB substance. Immunofluorescence and immunoelectron microscopy showed that the substance was produced in secretory cells in the luminal epithelium of the oviductal infundibulum and was provided to the egg on and in its vitelline membrane. During incubation, the SB substance bound with spermatozoa in the posterior portion of their flagella. Holes and disks were found in the vitelline membranes of fertile eggs at a ratio of 1: 19-24. Over 94% of the holes were accompanied by SB. The presence of SB is necessary for fertile spermatozoa to make holes in the membrane and to enter the fertile egg.     

Effect of gamma-radiation on fowl sperm function in vitro and in vivo

Doses of up to 300 Gy of ionizing radiation had little effect on fowl sperm morphology, ATP content and motility when measured in vitro. Fertility of eggs from hens inseminated with spermatozoa receiving 50 Gy, in terms of post-oviducal development, was less than 4%. However, 35% appeared 'fertile' by macroscopic examination of the germinal disc of unincubated eggs. These contained few embryonic cells, although the vitelline membrane contained many trapped spermatozoa. After doses of 100 Gy or more, inseminated spermatozoa were not found in the vitelline membrane and no fertile or apparently fertile eggs were produced; nor did such spermatozoa enter the utero-vaginal sperm-host glands. Genetic transformation using fowl spermatozoa irradiated with doses in excess of 100 Gy appears to be an unlikely prospect.     

Induction of the acrosome reaction on the surface of de-jellied sea urchin eggs

The vitelline coat of sea urchin eggs was disrupted by DTT and trypsin after removal of the jelly layer. Thereafter the percentage of acrosome reaction was determined and the fertilization rate was estimated, employing the treated eggs. Electron microscopical investigation of these eggs showed that the vitelline coat was disrupted but no morphological difference was observed between eggs treated with DTT and those treated with trypsin. However, the fertilizability of the eggs was markedly decreased by the treatment with trypsin. In contrast, DTT treatment did not affect the fertilizability of the eggs, indicating that some surface substance(s) necessary for fertilization which were not eliminated by DTT were digested by trypsin. At the same time, the percentage of acrosome reaction of supernumerary spermatozoa in the presence of variously treated eggs was estimated as an index of the acrosome reaction-inducing activity of the egg surface. The acrosome reaction of spermatozoa actually occurred at the surface of de-jellied and DTT-treated eggs. However, the eggs treated with trypsin lost the capacity to induce the acrosome reaction. The surface substance which induces the acrosome reaction and renders the eggs fertile was removed by trypsin and found in the supernatant fraction. The necessity of an acrosome reaction for fertilization was demonstrated by the fact that the low fertilizability of trypsin-treated eggs was brought back to the control level by insemination with spermatozoa previously treated with egg water to evoke the reaction of the acrosomes.     

Quantification of spermatozoa in the sperm-storage tubules of turkey hens and the relation to sperm numbers in the perivitelline layer of eggs

This study was conducted to determine the number of spermatozoa residing in the oviduct sperm-storage tubules (SST) and the relationship between these numbers and the number of spermatozoa embedded in the perivitelline layer of oviductal eggs after a single insemination of 200 x 10(6) spermatozoa. The SST of hens inseminated within one week before the expected onset of egg production were filled faster (4 h vs. 2 days) and possessed more spermatozoa (4.1 vs. 2.0 x 10(6)) than the SST of hens inseminated after the onset of egg production. Furthermore, for hens in egg production, significantly fewer spermatozoa were recovered from the SST if the hen was inseminated within 2 h before or after oviposition than if inseminated more than 2 h before or after the oviposition. There was a strong positive correlation between the number of spermatozoa in the SST and the number of spermatozoa embedded in the perivitelline layer of the oviductal eggs (r = 0.85, p less than 0.01). These data show that the population of spermatozoa actually accepted by the SST is quite small relative to the number of spermatozoa inseminated and that maximum sperm-storage is achieved when the hen is inseminated just prior to the onset of egg production. It is suggested that the sperm-storage capacity of the oviduct and the quality of the semen sample can be estimated on the basis of numbers of spermatozoa embedded in the egg perivitelline layer.     

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.     

Ultrastructural changes during fertilization envelope assembly in Lytechinus pictus eggs revealed by quick-freeze,deep-etch electron microscopy

Morphological features of fertilization envelope assembly in egges from the sea urchin Lytechinus pictus were examind in platinum replicas of samples quick-frozen, deep-etched, and rotary-shadowed at various times after insemination. Unfertilized eggs are surrounded by the vitelline layer, a glycocalyx, which faith-fully follows the contours of the microvillus-studded egg surface. The vitelline layer is secured to the plasma membrane below via a series of short projections called vitelline posts. The vitelline matrix itself is an elaborate meshwork of uniformly sized filaments, which are decorated in places with globular particles. At fertilization, the vitelline layer elevates off the egg surface and by 1 min after insemination appears as a thin, airy network of fibers. In contrast to Strongylocentrotus purpuratus, impressions of the underlying microvilli are not retained in this species. The vitelline template appears to become filled in by the deposition of amorphous secretory material between 1 and 5 min after fertilization. This smooth, amorphous layer is then coated with a thin sheet of paracrystalline material. Paracrystalline coating is incomplete at 5 min, but by 20 min after insemination the coat is complete, consisting of ordered parallel rows of roset-telike particles.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

Possible involvement of a sperm-associated body in the process of fertilization in quail

The present paper describes a novel structure, termed the sperm-associated body, which is found both in the lumen at the oviductal infundibulum and in the vitelline membrane of the ovum in the quail Coturnix japonica. The fully developed sperm-associated body, which is about 100 microm long, consisted of two parts; a core of concentric-circular appearance and a cortex of needle-like projections. The outer surface of the body was coated with CaCO3. The body was always accompanied by spermatozoa. About 70 sperm-associated bodies were observed in a single ovum. Electron-microscopically, small numbers of holes were detected in the vitelline membranes of a fertile ovum, and the sperm-associated bodies were always present in these holes. Frequently observed in the vitelline membranes was a disk speculated to be a portion of the inner layer of the membrane partially affected by spermatozoa. However, neither sperm-associated bodies nor spermatozoa were observed there. It was suggested that the sperm-associated bodies assist fertile spermatozoa in binding the inner layer of the vitelline membrane and penetrating it.     

The follicle cells of styela plicata (ascidiacea, tunicata): a sem study

The morphological aspect of the follicle cells of Styela plicata eggs is described by means of scanning electron microscope investigations. The follicular layer is made of spaced, cylindrical box-like cells which are arranged hexagonally. They adhere to the egg through a complex network of membrane extensions making an overall thin layer on the vitelline coat. The walls of the follicle cells are plentifully provided with microvilli, filopodia and lamellipodia, which allow a connection among the cells. At their apical end lies a large vacuole containing a granule, probably involved in secretion. At insemination the majority of spermatozoa is distributed on the apical membrane of the follicle cells. The membrane often breaks after sperm-egg impact and the granule is therefore displaced. By means of the present investigations it is once again suggested a role played by follicle cells in ascidian eggs at fertilization.     

Polyspermic fertilization of sea urchin eggs treated with protease inhibitors: Localization of sperm receptor sites at the egg surface

The normal elevation of the fertilization membrane and the establishment of the block to polyspermy are retarded in Arbacia punctulata eggs by specific protease inhibitors, soybean trypsin inhibitor (SBTI), leupeptin, and antipain. Ultrastructural observations show that the vitelline layer remains attached to the plasma membrane of fertilized SBTI treated eggs at numerous sites (cortical projections). Quantitive morphometric analysis indicates that the vitelline layer elevates from about 65% of the surface of SBTI treated eggs during the first 3 min post insemination. However, the vulnerability of SBTI treated eggs to refertilization (polyspermy) only declined during the subsequent gradual detachment of the vitelline layer from the cortical projections over the next 15 min. Antipain and leupeptin (10^?5 to 10^?3M) also promoted polyspermy in Arbacia eggs by a process of refertilization extending for a 10- to 15-min period after the initial monospermic insemination. Normal cleavage and development was obtained when eggs were placed in leupeptin and antipain (10^?3M) after the fertilization membrane had elevated. The data indicate that the normal secretory function (or functions) of the cortical granule protease in establishing the block to polyspermy is retarded by these protease inhibitors, and that the vitelline layer is transformed into a mechanical barrier to prevent penetration by supernumerary sperm during its detachment from the plasma membrane of the egg. Furthermore, the vitelline layer in unfertilized eggs appears to be a mosaic structure, with sperm receptor sites localized in regions of the egg's surface, which give rise to cortical projections in the presence of SBTI.     

Fertilized and unfertilized ova are transported at different rates by the hamster oviduct

To determine if the egg provides any clues for the regulation of ovum transport in the hamster, oocyte and embryo transport were compared. On the evening preceding ovulation, the animals were randomly assigned to one of five groups. They were caged overnight with a male of proven fertility (Group 1) or they were isolated (Group 2). Other females were artificially inseminated in both uterine horns at 2200 h either with fertile epididymal spermatozoa (Group 3), spermatozoa rendered infertile by freezing and thawing (Group 4), or with fertile spermatozoa in one uterine horn and infertile spermatozoa in the contralateral horn (Group 5). The number, condition, and distribution of ova in the genital tract were assessed at various intervals during the next 4 days. The rate of fertilization and normal development in females or sides inseminated with fertile or infertile spermatozoa was over 90% and 0% respectively. Embryos in Groups 1 and 3 reached the uterus 1 day earlier than unfertilized oocytes in Groups 2 and 4. In group 5, the transport of embryos resulting from insemination with fertile spermatozoa followed a pattern similar to those in Groups 1 and 3; the oocytes in the contralateral tract resembled those of Groups 2 and 4. The different transport rates of embryos and oocytes were not associated with the reproductive state of the female but with the condition of the ova. Moreover, the different transport rates were observed in animals transporting the two types of eggs simultaneously on different sides indicating that there is a local recognition of some unidentified factor unequally present in fertilized and unfertilized eggs.     

Methods for quantitating sea urchin sperm-egg binding

Two simple photometric methods are described for determining the average number of bound sea urchin spermatozoa per egg between 0 and 60 sec after insemination. One method is based on stopping gamete interaction with formaldehyde and then, after the eggs settle with sperm bound to their surfaces, measuring the turbidity of sperm remaining in suspension. An alternative method involves removal of the dead, unbound sperm from the formaldehyde fixed eggs by repeated washing in sea water. The bound sperm are then released from the egg surface by pronase digestion and the turbidity of the sperm suspension measured and related to sperm concentration by direct cell counts. Two phases of gamete interaction exist (1) the binding phase, from 0 to 20 or 25 sec, during which time sperm continuously bind to the egg surfaces; (2) the unbinding phase, from 20 or 25 to 50 sec, during which time the sperm are unbound from the eggs and return to the suspension. The results of experiments in which the method is used to assess sperm binding at different pH values and at different calcium concentrations are presented. Data are presented suggesting that a definite number of sperm binding sites may exist on the vitelline layer.     

Concanavalin A Modifies Allo-specific Sperm-Egg Interactions in the Ascidian, Ciona intestinalis

In the self sterile ascidian, Ciona intestinalis , the spermatozoa rarely bind to the vitelline coat of autologous eggs and never penetrate it. We report here that concanavalin A (ConA), a lectin recognizing mannose or glucose residues of carbohydrates, can modify these self- and nonself-specific sperm-egg interactions. When eggs were pretreated with 0.1–0.5 mg/ml of ConA, about two thousand spermatozoa became attached to the autologous vitelline coat within five minutes of insemination. The effect of ConA was not modified by the addition of D-mannose or pretreatment of spermatozoa with ConA, showing that ConA does not function merely as a ligand bridging the sperm and vitelline coat. In contrast to the marked enhancement of sperm-egg binding, ConA did not facilitate the penetration of spermatozoa through the autologous vitelline coat. Even in non-autologous insemination, it blocked the sperm penetration and, consequently, fertilization did not occur, as shown by Rosati et al. (1978). D-Mannose, when mixed with ConA in advance, completely abolished this inhibitory effect of ConA. Lotus agglutinin, a fucose-binding lectin, was less effective and wheat germ agglutinin and soy bean agglutinin had no effect on sperm entry in the perivitelline space. The results of this study are discussed in relation to the possible involvement of mannosyl and/or glucosyl glycoconjugates in allo-specific sperm-egg interactions.     

Electron microscopic observations on sperm entry and pronuclear formation in naked eggs of the rose bitterling in polyspermic fertilization

Unfertilized eggs of the rose bitterling (Rhodeus ocellatus ocellatus) were squeezed out of females that had an elongated ovipositor and were dechorionated mechanically with fine forceps in physiological saline. The dechorionated eggs were transferred into fresh water then inseminated at once by spermatozoa of the same species. A large number of spermatozoa was found on the surface of eggs that had not yet had cortical reaction following insemination. The surface of the naked eggs responded by formation of many small cytoplasmic protrusions (viz., fertilization cones) at sperm attachment sites. The formed fertilization cones were rosettelike structures formed by the aggregation of some bleblike swellings devoid of microvilli and microplicae. About 10 min after insemination, the fertilization cones retracted, but marks of their presence characterized by less microvilli and microplicae remained in the eggs 15 min after insemination. Many spermatozoa penetrated into the cytoplasm of each naked egg. The sperm nuclear envelope disappeared by means of vesiculation resulting from fusion of the inner and outer membranes. The sperm nucleus decondensed and developed into a larger male pronucleus. Smooth-surfaced vesicles surrounded the decondensing sperm nucleus and formed the new male pronuclear envelope. Sperm mitochondria and flagella were found in the egg 15 min after insemination. The response of the egg surface to sperm entry and pronucleus formation are discussed.     

Early events of in-vitro fertilization of cat eggs by epididymal spermatozoa

Newly ovulated eggs from mature queens treated with PMSG and hCG were inseminated in modified KRB solution with spermatozoa recovered from the cauda epididymidis of male cats. When 5 eggs were examined 15 min after insemination, no signs of sperm penetration into the vitellus were observed. However, in an egg examined before fixation 20 min after insemination, a spermatozoon whose head had passed through the zona pellucida was observed. Very high proportions (90-100%) of the eggs were penetrated when they were examined 0.5-5 h after insemination. Male and female pronuclei were first observed in eggs examined 4 h after insemination.     

THE SURFACE EVENTS AT FERTILIZATION OF THE SEA URCHIN EGG I. EVENTS ON THE SURFACE OF THE VITELLINE COAT1

Sperm-egg interaction during normal fertilization in the sea urchins, Strongylocentrotus intermedius and Hemicentrotus pulcherrimus, was studied by scanning and transmission electron microscopy. Several seconds after insemination, acrosome-reacted spermatozoa were found attached to the surface of the vitelline coat on each egg. Soon, several bulges of the vitelline coat appeared surrounding the fertilizing spermatozoon. These bulges then spread over the surface increasing in number, while they became fewer and disappeared around the sperm head. Thin sections of the bulging areas revealed discharging cortical granules. As the bulging vitelline coat was elevated, the sperm head was incorporated into the perivitelline space, passing through a small hole in the coat that resulted from penetration of the sperm acrosomal process immediately before fusion of the gametes. When the spermatozoon disappeared beneath the fertilization membrane, a hole was left in the membrane and the cortical reaction had finished on the other hemispheric surface. Mechanical removal of the membrane at that time exposed a spermatozoon protruding perpendicularly from the egg plasma membrane surface. The anterior tip of the sperm head was smoothly connected with the egg surface, and neither microvillous projections nor cytoplasmic covering of the egg cytoplasm could be found around the spermatozoon.     

The possibility of obtaining intergeneric hybrids via White Kołuda (Anser anser L.) goose insemination with fresh and frozen-thawed Canada goose (Branta canadensis L.) gander semen

The objective of the present experiments was to produce the intergeneric hybrids of domesticated and wild goose via artificial insemination with fresh and frozen-thawed semen. The experiments were carried out during two successive goose reproductive seasons, on eight five-year-old Canada Goose (Branta canadensis L.) males used as semen donors and 16 two-year-old White Ko?uda geese designated to fertility tests. Pooled semen was collected twice a week by the dorso-abdominal massage. In freshly collected semen, ejaculate volume, color, consistency, degree of fecal or blood contamination, spermatozoa concentration, motility, and morphology were evaluated. Part of the semen collected in the first year of the experiment (Experiment 1) was used for geese insemination with fresh semen, while the remainder was frozen. In Experiment 2 all samples were subjected exclusively to freezing procedure. Geese were inseminated once a week with fresh semen in a dose of 80 μl or 160 μl, and twice a week with frozen-thawed semen in a dose of 80 μl (160 μl per wk) or 100 μl (200 μl per wk). Eggs were set weekly and incubated up to hatching.The volume of ejaculates varied from 0.100 to 0.470 ml; spermatozoa concentration from 140 to 310 million ml⁻¹; progressive movement was observed in 40 to 60% of spermatozoa; the percentage of total live spermatozoa ranged from 69.3 to 92.0%, the highest percentage (34.0-68.3) was represented by live normal spermatozoa and those with bulb-head (13.3-41.0). Cryopreservation caused a decrease in percentage of motile cells to 30%; total live spermatozoa contribution by 27.2%p, including those live normal by 15.9%p (in relation to the fresh semen), bulb-head spermatozoa by 10.9%p, and increase (by 5.9%p) in number of spermatozoa with other deformations. Goose insemination 1×/week with fresh semen containing about 10.3 million live normal spermatozoa resulted in 66.7% of fertile eggs and with dose higher by 2.8 million spermatozoa (on average) the fertility increased by 20.9%p (up to 87.6% on average). Hatchability from set and fertile eggs was 55.9% and 83.9% vs. 66.3% and 75.6%, respectively. After twice a week insemination with frozen-thawed semen containing about 10.2 million live normal cells 58.2% eggs were fertile; hatchability from set eggs was 42.8% and from fertile eggs 71.7%, while insemination dose increase by 2.7 million spermatozoa per week caused a fertilization increase by 3.8%p (62.0% on average), this increase was not statistically significant, but hatchability from the fertile eggs (95.4%), was significantly (P < 0.05) higher.The use of AI with fresh semen in the creation of intergeneric hybrids of Canada goose males and White Ko?uda females allows a high level of egg fertility to be obtained. Furthermore, one limitation which is the short reproductive season of the Canada goose may be overcome by the use of cryopreserved semen.     

Quantitative aspects of sperm:egg interaction in chickens and turkeys

Spermatozoa embedded in the outer perivitelline layer and points of hydrolysis (holes) produced by spermatozoa in the inner perivitelline layer of chicken and turkey eggs were found to be evenly distributed and linearly correlated (r = 0.80 for both species) throughout the layers from most regions of the egg, except from those directly over the germinal disc, in which there were more holes. In turkey eggs there appeared to be relatively fewer perivitelline spermatozoa, since many had degenerated beyond recognition. In eggs from both species, there were approximately 25 times more holes mm⁻² in the inner perivitelline layer from over the germinal disc region than that from other regions of the egg. The relationship between these two frequencies could also be described as linear (r = 0.81 for chicken and 0.78 for turkey eggs), although there was some evidence for a saturation effect for holes over the germinal disc. The fertile status of eggs was shown to be a function of all of the above parameters. Eggs from both species had a 50% probability of being fertile when around 3 spermatozoa penetrated the inner perivitelline layer over the germinal disc and showed maximum fertility when more than 6 spermatozoa penetrated this region. Spermatozoa in the outer perivitelline layer and holes in the inner perivitelline layer from regions other than over the germinal disc could also be used to predict fertility, although with less certainty. Since the number of spermatozoa interacting with the egg reflects the numbers of those stored in the uterovaginal sperm storage tubules, the relationships derived in this work should be useful for understanding how fertility in chickens and turkeys is a function of oviducal sperm storage and transport.