Sperm surface heparin/heparan sulfate is responsible for sperm binding to the uterine envelope in the newt, Cynops pyrrhogaster

The sperm-binding properties of egg envelopes are investigated in the newt, Cynops pyrrhogaster. Sperm binding was only seen on the uterine envelope when acrosome-reacted sperm were inseminated. No acrosome-intact sperm bound to the envelopes. By scanning electron microscopic observation, acrosome-reacted sperm were revealed to bind to a seat-like structure present on the surface of the uterine envelope. Sperm binding to the uterine envelope was inhibited by treatment of eggs with heparin or heparan sulfate, or treatment of acrosome-reacted sperm with heparinase prior to insemination. A molecule with a molecular mass of 75 kDa was purified from the uterine envelope by affinity chromatography with heparin-Sepharose. These results indicated that sperm binding was mediated by heparin-like molecules expressed on the surface of acrosome-reacted sperm and the 75 kDa molecule was present as a constituent of uterine envelopes.     

Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O- linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and - reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.     

The role of structural integrity of the fertilising spermatozoon in early human embryogenesis.

While the fertilising spermatozoon supplies the active centre directing the human zygote's first mitotic division, the relative contributions of the sperm head and tail (as well as the importance of the sperm's general structural integrity) to subsequent developmental processes remain incompletely studied. The sperm nucleus contains paternal chromatin necessary for restoration of a diploid genome, but the functional role of the sperm tail (either attached or dissected) in early human embryonic growth is not known. In this investigation using oocytes donated by in vitro fertilisation patients, human oocytes were injected with isolated sperm heads (n = 73), isolated sperm flagella (n = 11) or both (dissected sperm heads + free sperm tails, n = 26). The formation of bipronucleate zygotes was recorded for each method. Among oocytes surviving injection with isolated sperm heads, 44 of 66 (67%) formed two pronuclei. Of oocytes receiving only sperm tails, 2 of 11 (18%) displayed two pronuclei, but a single polar body was evident in both cases. When dissected spermatozoa parts (head + tail) were jointly injected, 12 of 26 (46%) developed two pronuclei. From embryos resulting from each of these three fertilisation regimes, blastomere biopsies were obtained and subjected to multiprobe fluorescent in situ hybridisation (FISH) analysis to detect mosaicism or aneuploidy arising from these experimental treatments. Only embryos with growth sufficient to permit sampling of at least two blastomeres were evaluated, and FISH analysis was successful in 25 of 29 (86%) embryos tested. Of 12 embryos derived from injection of an isolated sperm head, only one was normal diploid; the remaining 11 were mosaic. Both embryos resulting from injection of an unattached sperm tail were mosaic. Of 11 embryos generated from oocyte injection with sperm head + tail segments, 10 (91%) were mosaic and only one was normal diploid. Results from this study show that injection of isolated sperm segments can permit oocyte activation and bipronuclear formation. However, a high rate of mosaicism in human embryos originating from disrupted sperm or sperm components suggests that more than a 'sum of parts' is needed for later development. The structural integrity of the intact fertilising spermatozoon appears to contribute to normal human early embryogenesis.     

Induction of acrosome reactions by the human zona pellucida

We have used two approaches to test the ability of the human zona pellucida to induce acrosome reactions in human sperm. First, nonviable human oocytes were incubated for 1 min in a suspension of capacitated sperm (of which fewer than 5% were acrosome-reacted) to allow binding of about 200 sperm per oocyte. Some of the oocytes were fixed immediately, and the remainder were fixed after a further 1-h incubation without free-swimming sperm. As determined by light microscopy, sperm on the zona were only 3 +/- 2% (avg. +/- SD) acrosome-reacted at 1 min, and the incidence increased to 46 +/- 15% during the next hour. Electron microscopy confirmed that most sperm on the zona at 1 min were acrosome-intact. A few sperm were in an early stage of the acrosome reaction. Acrosome reactions occurring on the zona during the subsequent hour appeared to be morphologically normal. Second, treatment of sperm in suspension with acid-disaggregated zonae (2 to 4 zonae/microliter) increased the incidence of acrosome-reacted sperm from 3 +/- 1% to 24 +/- 4%. We conclude that the human zona pellucida, or material intimately associated with it, can induce acrosome reactions in human sperm.     

Acrosomal status and motility of guinea pig spermatozoa during in vitro penetration of the cumulus oophorus

Previous studies have suggested that both acrosome-intact and acrosome-reacted guinea pig sperm are capable of binding to the zona pellucida of cumulus-free oocytes, but the acrosomal status of guinea pig sperm during penetration of the cumulus has not been reported. We made video recordings of the interaction between capacitated guinea pig sperm and cumulus-invested guinea pig oocytes. The videotapes were analysed to identify sperm with hyperactivated motility and to classify the acrosomal status of sperm during penetration of the cumulus and after binding to the zona pellucida. The resolution of the video recordings was not sufficient to recognise sperm with swollen acrosomes. However, sperm that had completed the acrosome reaction were easily identified. Acrosome-reacted sperm were found adherent to the outer boundary of the cumulus, but were never observed to penetrate the cumulus. The percentage of acrosome-intact, hyperactivated sperm was higher in the cumulus oophorus than in culture medium, suggesting that changes in motility were elicited in response to contact with the cumulus. Fully acrosome-reacted sperm were found adherent to the zona pellucida, and solubilised guinea pig zona pellucida was capable of inducing acrosome reactions in capacitated guinea pig sperm. Acrosome-intact sperm were also observed on the zona, but they were not tightly bound and did not have hyperactivated motility, suggesting that these sperm were not functionally capacitated. Our observations demonstrate that guinea pig sperm penetrate the cumulus matrix in an acrosome-intact state. Although we did not observe sperm undergoing the acrosome reaction, our observations and experimental data suggest that the acrosome reaction of guinea pig sperm is completed on or near the surface of the zona pellucida.     

REINSEMINATION OF FERTILIZED SEA URCHIN (ARBACIA PUNCTULATA) EGGS

Embryos of Arbacia punctulata , treated to remove the fertilization membrane and hyaline layer, were mixed with sperm (reinseminated) at 20–30 min (streak stage) and at 70 min (2-cell stage) postinsemination. Sperm, incorporated into embryos reinseminated at the streak stage, metamorphosed into male pronuclei which subsequently migrated to and fused with the zygote nucleus. Although blastomeres were capable of incorporating sperm and forming fertilization cones, less than 10% of the 2-cell stage embryos reinseminated. Sperm, were found entrapped within an amorphous material along the surface of 2-cell stage embryos; many had failed to undergo an acrosome reaction. These results indicate that conditions necessary for incorporation and metamorphosis of sperm nuclei into male pronuclei are present in the embryo after the normal period of fertilization.     

Can spermatozoa with abnormal heads gain access to the ovum in artificially inseminated super- and single-ovulating cattle?

The collective efficiency of barriers in the female tract against spermatozoa with abnormal heads was studied. In Experiment 1, Day 6 ova/embryos were recovered nonsurgically from superovulated (n = 24) and single-ovulating (n = 44) cows following artificial insemination with semen of bulls selected for normal spermatozoal motility (> or = 50%) and high content (> 30%) of spermatozoa with misshapen heads, random nuclear vacuoles or the diadem defect. To assess characteristics of spermatozoa capable of traversing barriers in the female tract, accessory spermatozoa were classified morphologically (x 1250) and compared with those of the inseminate. Superovulated cows proved inadequate for assessment of accessory spermatozoa due to evidence of poor sperm retention in the zona pellucida; thus, only single-ovulating cows were used. Accessory spermatozoa (n = 479) from 31 ova/embryos recovered from 44 cows were more normal in head shape than those in the inseminate (76 vs 62%; P < 0.05). Spermatozoa with normal head shape, but with nuclear vacuoles appeared as accessory spermatozoa at the same frequency as they were found in the inseminate (20 vs 17%, respectively). Only sperm cells with subtly misshapen heads appeared as accessory spermatozoa. In Experiment 2, semen pooled from 4 bulls having large numbers of spermatozoa exhibiting a gradation from severely asymmetrically misshapen heads to subtly misshapen heads was evaluated. Again, the accessory sperm population (960 sperm cells recovered from 64 ova/embryos) was enriched with spermatozoa of normal head shape relative to the inseminate (53 vs 26%, respectively; P < 0.05). Sperm cells with only nuclear vacuoles and those with subtly misshapen heads were not different between the accessory and inseminate populations (11 vs 8%, and 20 vs 25%, respectively). We conclude that morphologically abnormal spermatozoa are excluded from the accessory sperm population based upon severity of head shape distortion.     

ELECTRON MICROSCOPIC OBSERVATIONS OF GUINEA PIG SPERMATOZOA PENETRATING EGGS IN VITRO*

Guinea pig ovarian oocytes matured in vitro were inseminated in vitro with capacitated, acrosome-reacted spermatozoa and sperm penetration through the zona pellucida and into the egg cytoplasm were examined. Sperm heads passing through the zona pellucida had already lost all their acrosomal elements except for the inner acrosomal membrane and the equatorial segment. It was often observed that the texture of the zona material around the sperm head was distorted, giving the impression that the zona pellucida was parted, at least partially, by a shearing force produced by the sperm head advancing through the zona. When eggs were freed from their zonae pellucidae and inseminated, the acrosome-reacted spermatozoa immediately bound to the egg surfaces and began to fuse with the eggs; whereas the spermatozoa with intact acrosomes failed to do so. Fusion began between the egg plasma membrane and the sperm plasma membrane at the central region of the sperm head. The anterior half of the sperm head was engulfed by the egg in a phagocytic fashion, while its posterior half was incorporated into the egg by a fussion between egg and sperm plasma membranes. Incorporation of the sperm tail into the egg was achieved by fusion between the sperm and egg plasma membranes.     

Differential binding of gold-labeled zona pellucida glycoproteins mZP2 and mZP3 to mouse sperm membrane compartments.

Egg zona pellucida glycoproteins mZP3 and mZP2 serve as primary and secondary sperm receptors, respectively, during initial stages of fertilization in mice [Wassarman (1988) A. Rev. Biochem. 57, 415-442]. These receptors interact with complementary egg-binding proteins (EBPs) located on the sperm surface to support species-specific gamete adhesion. Results of whole-mount autoradiographic experiments suggest that purified egg mZP3 and mZP2 bind preferentially to acrosome-intact (AI) and acrosome-reacted (AR) sperm heads, respectively [Bleil and Wassarman (1986) J. Cell Biol. 102, 1363-1371]. Here, we used purified egg mZP2, egg mZP3 and fetuin, which were coupled directly to colloidal gold ('gold-probes'), to examine binding of these glycoproteins to membrane compartments of AI and AR sperm by transmission electron microscopy. mZP3 gold-probes were found associated primarily with plasma membrane overlying the acrosomal and post-acrosomal regions of AI sperm heads. They were also found associated with plasma membrane overlying the post-acrosomal region of AR sperm heads. mZP2 gold-probes were found associated primarily with inner acrosomal membrane of AR sperm heads, although some gold was associated with outer acrosomal membrane of AI sperm that had holes in plasma membrane overlying the acrosome. Fetuin gold-probes, used to assess background levels of binding, were bound at relatively low levels to plasma membrane and inner acrosomal membrane of AI and AR sperm, respectively. None of the gold-probes exhibited significant binding to sperm tails, or to red blood cells and residual bodies present in sperm preparations. These results provide further evidence that mZP2 and mZP3 bind preferentially to heads of AR and AI sperm, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sperm-oocyte membrane fusion in the human during monospermic fertilization

Sperm-oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro. Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH-surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy. Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome-reacted and had been partly incorporated into the ooplasm, while the anterior two-thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized. The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.     

Determining acrosomal status of the cynomolgus monkey (Macaca fascicularis) sperm by fluorescence microscopy

The acrosome of Macaca fascicularis sperm cannot be distinguished by conventional light microscopy, so determining whether sperm are acrosome-intact or-reacted is difficult. We describe methods for labeling the acrosomal region of sperm with two different probes: fluoresceinated Pisum sativum agglutinin and anti-sperm antiserum. Acrosome-intact sperm are much more heavily labeled in the acrosomal region than are acrosome-reacted sperm, providing a simple means of differentiating the two types of sperm. The two probes detect similar numbers of acrosome-reacted sperm following treatment with the divalent cation ionophore, A23187.     

An investigation of the latency period between sperm oolemmal adhesion and oocyte penetration

In clinical studies of the ability of capacitated human sperm to penetrate zona-free hamster eggs, we have previously observed that the ratio of oolemmal adherent to penetrating sperm varied between men. Sperm incorporation did not occur immediately following gamete adhesion and not all adherent sperm penetrated the egg. To further investigate this phenomenon, comparisons were made of the kinetics of gamete adhesion, membrane fusion, and sperm incorporation of capacitated mouse and human spermatozoa by zona-free hamster eggs and of mouse sperm by zona-free mouse and hamster eggs. Eggs were inseminated with either capacitated human or mouse sperm or combinations of both, washed out of sperm suspension after initial gamete adherence, and further incubated in sperm-free medium. Gamete membrane fusion was judged by dye transfer of Hoechst 33342 and sperm entry of the cortical ooplasm by observation of expanded sperm heads within acridine orange stained eggs. Oolemmal adherent mouse and human sperm fused with and penetrated zona-free hamster eggs at different times whether eggs were inseminated in parallel or with combinations of sperm of both species. Oolemmal adherent mouse sperm penetrated zona-free hamster eggs prior to their penetration of zona-free mouse eggs. Ultrastructural studies of zona-free human eggs inseminated with human sperm confirmed prior observations with hamster eggs that only acrosome-reacted human sperm adhere to the oolemma. These results have lead us to postulate that sperm entry into the egg may occur through a "zipper" mechanism involving the ligation of local gamete receptors similar to the incorporation of target particles by phagocytes and suggest that not all oolemmal adherent human sperm are capable of being incorporated although they have undergone an acrosome reaction.     

Acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida

Mammalian sperm must be acrosome reacted before penetrating the zona pellucida. In some species the sperm undergo the acrosome reaction before binding to the zona pellucida and in other species only acrosome intact sperm can initiate binding to the zona. In this study we addressed the question of acrosomal status and sperm-zona binding with human gametes. Sperm acrosome reactions were induced by treatment with human follicular fluid or N-(6-amino-hexyl)-5-chloro-naphthalene sulfonamide (W-7). The sperm suspensions, containing various percentages of acrosome-reacted sperm, were then incubated with human oocytes for 1 min. The acrosomal status of the sperm population bound to the zona was similar to the acrosomal status of the population of sperm in suspension (R2 = 0.77), regardless of the treatment to induce acrosome reactions. Our interpretation of these results is that both acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida. However, we reported earlier (N. L. Cross, P. Morales, J. W. Overstreet, and F. W. Hanson, 1988, Biol. Reprod. 38, 235-244) that the human zona pellucida is able to induce acrosome reactions. Thus, to exclude the possibility that sperm had undergone the acrosome reaction on the zona within 1 min of binding, sperm were suspended in a nominally calcium-free Tyrode's medium (0 Ca-mTyr) before incubation with oocytes (this medium was supplemented with SrCl2 and spermine to support sperm motility and zona binding). In 0 Ca-mTyr, the proportion of acrosome-reacted sperm on the zona was still highly correlated with the proportion of reacted sperm in suspension, indicating that the sperm were reacted before binding. Evidence that 0 Ca-mTyr effectively inhibited acrosome reactions induced by the zona pellucida was derived from experiments in which sperm were treated with human follicular fluid or control medium and the suspensions were diluted with either 0 Ca-mTyr or control medium.4+ Human oocytes were added for 1 min (pulse) at which time some oocytes were fixed and other oocytes were transferred to sperm-free medium and incubated for 35 min (chase) before fixation. Sperm diluted in control medium, pretreated with either human follicular fluid or control medium, showed a similar increase (40%) in the percentage of acrosome reactions among the zona-bound sperm after the chase. Sperm diluted in 0 Ca-mTyr did not show an increase in the percentage of acrosome-reacted sperm on the zona pellucida after the chase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Full-term development of golden hamster oocytes following intracytoplasmic sperm head injection

The golden hamster is the mammalian species in which intracytoplasmic sperm injection (ICSI) was first tried to produce fertilized oocytes. Thus far, however, there are no reports of full-term development of hamster oocytes fertilized by ICSI. Here we report the birth of hamster offspring following ICSI. Keys to success were 1) performing ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under a microscope with a red light source and 2) injecting sperm heads without acrosomes. All oocytes injected with acrosome-intact sperm heads died within 3 h after injection, while those oocytes injected with acrosomeless sperm heads survived injection. Under illumination with red light in a dark room, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally (77%), cleaved (91%), and developed into morulae (49%). Of the 47 morulae transferred to five recipient females, nine (19%) developed to live offspring.     

Guinea pig fertilin exhibits restricted lateral mobility in epididymal sperm and becomes freely diffusing during capacitation

The guinea pig sperm protein fertilin functions in sperm-egg plasma membrane binding. Fertilin is initially present in the plasma membrane of the whole head in testicular sperm, then becomes concentrated into the posterior head domain during epididymal passage. Fertilin remains localized to the posterior head plasma membrane following the acrosome reaction, when it functions in sperm-egg interaction. Fluorescence redistribution after photobleaching was used to examine the lateral mobility of fertilin in both acrosome-intact and acrosome-reacted sperm. Fertilin exhibited highly restricted lateral mobility in both testicular and epididymal sperm (D < 10(-10) cm(2)/s). However, fertilin in acrosome-reacted sperm was highly mobile within the membrane bilayer (D = 1.8 x 10(-9) cm(2)/s and %R = 84). Measurement of the lateral mobility of fertilin in capacitated, acrosome-intact sperm revealed two populations of cells. In approximately one-half of the cells, lateral mobility of fertilin was similar to sperm freshly isolated from the cauda epididymis; while in the other half fertilin was highly mobile. The release of fertilin from interactions that restrict its lateral mobility may regulate its function in sperm-egg interaction.     

Production of motile acrosome-reacted mouse sperm with nanomolar concentration of calcium ionophore A23187

A method to generate a population of motile, acrosome-reacted mouse sperm is described. Sperm retrieved from the cauda epididymis and vas deferens were first capacited in a 3% bovine serum albumin (BSA) containing medium. Sperm were then resuspended in medium with low BSA content (0.01%) and treated with 30 nM of the calcium ionophore, A23187, which was added as a singel dose of 30 nM for 15 min at 37°C; or three sequential 10 nM doses over three 5 min intervals. Approximately 55–60% of the treated sperm population became acrosome reacted. The motility of the treated sperm sample was 40–65%, slightly lower than that of the control sperm, following addition of medium containing 3% BSA, This is in contrast to the <10% motility observed for capacitated mouse sperm treated with 10 μM A23187, a concentration that had been used by other investigators to induce the acrosome reaction. The ultrastructure of the 30 nM A23187-induced acrosome-reacted sperm ws similar to that of the acrosome-reacted sperm induced by solubilized zonae pellucidae. These motile, acrosome-reacted sperm were able to penetrate zone-free mouse eggs at a higher rate than the control sperm. Thus this method of treatment will be useful for further physiological experimentation with acrosome-reacted sperm. © 1994 Wiley-Liss, Inc.     

Chromatin and microtubule organisation in maturing and pre-activated porcine oocytes following intracytoplasmic sperm injection

Chromatin and microtubule organisation was determined in maturing and activated porcine oocytes following intracytoplasmic sperm injection in order to obtain insights into the nature of sperm chromatin decondensation and microtubule nucleation activity. Sperm chromatin was slightly decondensed at 8 h following injection into germinal vesicle stage oocytes. Sperm-derived microtubules were not seen in these oocytes. Following injection into metaphase I (MI)-stage oocytes, sperm chromatin went to metaphase in most cases. A meiotic-like spindle was seen in the sperm metaphase chromatin. In a few MI-stage oocytes, sperm chromatin decondensed at 8 h after injection, and a small sperm aster was seen. Sperm injection into oocytes at 5 h following activation failed to yield pronuclear formation. Maternally derived microtubules were organised near the female chromatin in these oocytes, and seemed to move condensed male chromatin closer to the female pronucleus. At 18 h after sperm injection into pre-activated oocytes, a condensed sperm nucleus was located in close proximity to the female pronucleus. These results suggest that the sperm nuclear decondensing activity and microtubule nucleation abilities of the male centrosome are cell cycle dependent. In the absence of a functional male centrosome, microtubules of female origin take over the role of microtubule nucleation for nuclear movement.     

Toluidine blue staining reveals changes in chromatin stabilization of mouse spermatozoa during epididymal maturation and penetration of ova

Sperm samples, recovered from various regions of the reproductive tract of male and female mice, were air dried, fixed for 2 min in acetic alcohol and stained with toluidine blue. Testicular sperm heads were deeply stained, while in the samples from the successive regions of caput epididymidis the proportion of stained heads gradually decreased and many of them were stained in their distal part only. Spermatozoa from the corpus and cauda epididymidis, vas deferens, uterus, oviduct and from the perivitelline space were colourless (except for one type of misshapen head). Sperm heads that had penetrated the vitellus became stained distally. Vas deferens sperm heads were fully stained after treatment with dithiothreitol for 30 min. We suggest that the inability to stain with toluidine blue is characteristic of sperm chromatin stabilized by disulphide bonds.     

Soybean trypsin inhibitor as a probe for the acrosome reaction in motile cynomolgus macaque sperm

Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases, and has been shown to bind to acrosin, an acrosomal hydrolase which is not exposed on the surface of macaque sperm until after the acrosome reaction. Following activation with caffeine and dibutyryl cAMP, cynomolgus macaque sperm were induced to acrosome react with calcium ionophore A23187 in the presence of SBTI and were fixed for ultrastructural observation. Transmission electron microscopy (TEM) revealed secondary labelling of anti-SBTI-IgG with colloidal gold in association with the acrosomal matrix and fused membranes of sperm undergoing the acrosome reaction, but gold labelling was not observed on acrosome-intact sperm. When SBTI was conjugated with the fluorochrome Alexa 488, labelled (acrosome-reacted) sperm showed bright fluorescence that ranged from a patchy or punctate appearance to solid labelling over the region of the acrosomal cap. Following treatment with ionophore, the percentages of total acrosome-reacted sperm (motile and non-motile) as assessed with Alexa-SBTI, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and TEM were 54.6%, 51.6% and 61.5%, respectively. Measures of acrosomal status with FITC-PSA and Alexa-SBTI were highly correlated (r = 0.94; n = 3). Macaque zonae pellucidae were co-incubated with activated sperm for 1 min and then rinsed in medium containing Alexa-SBTI and immediately observed with epifluorescence microscopy. The mean percentage of Alexa-SBTI-labelled (acrosome-reacted) motile sperm bound to the zona was 45.7 +/- 14 (range: 22-80.4%; n = 4). Fewer than 1% of the motile sperm in suspension surrounding the zonae were acrosome-reacted. Alexa-SBTI had no effect on sperm motility, survival, or zona binding capability.     

Transformations of sperm nuclei incorporated into sea urchin (Arbacia punctulata) embryos at different stages of the cell cycle

In order to test the hypothesis that regulators of male pronuclear development may have a more general role, sharing some relation to factors involved with the cell cycle, Arbacia zygotes and 2- to 8-cell stage embryos were inseminated during different phases of the cell cycle and examined by light and electron microscopy. Differences in the development and morphology of fertilization cones and sperm asters were observed in embryos inseminated during different stages of the cell cycle. Extremely large fertilization cones, approximately four times the length of those found in fertilized eggs, formed in embryos inseminated during metaphase to telophase. Sperm asters developed only in embryos inseminated during prophase to anaphase. These variations are believed to reflect changes in the status of the cortex and cytoskeletal system of the embryo. Although sperm nuclei underwent morphological changes subsequent to incorporation, in general, they failed to develop into male pronuclei. There was a consistent correlation in sperm nuclear transformations and the cell cycle which was expressed in two patterns of morphogenesis: (1) sperm nuclei incorporated into embryos just prior to prophase and at telophase failed, for the most part, to disperse and transformed into aggregations of chromatin granules approximately 40 nm in diameter; and (2) sperm nuclei incorporated into prometaphase-anaphase embryos dispersed and then condensed into chromatin masses, morphologically similar to chromosomes of the embryo. Evidence is discussed which indicates that following the normal period of fertilization changes occur in the zygote, rendering it unable to fully support the transformation of sperm nuclei into male pronuclei.