Showing papers on "Sperm published in 1984"

Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics

Abstract: The objective of this study was to develop a relatively simple test to evaluate the functional integrity of the membranes of human spermatozoa. As in some other species, human spermatozoa 'swell' under hypo-osmotic conditions due to the influx of water and the expansion of the membranes. A mixture of equal parts of fructose and sodium citrate (150 mosmol) with calculated ionic strength of 0.15 resulted in a maximal number of clearly identifiable swollen spermatozoa. Only small variations were seen when different aliquants of the same semen samples were separately evaluated. A high correlation (r = 0.94) was obtained between expected and observed values of swollen spermatozoa when known amounts of heat-treated spermatozoa, unable to undergo swelling, were added to untreated spermatozoa. A good correlation (r = 0.90) was also observed between the % spermatozoa in a semen sample that were capable of undergoing swelling and the % of denuded hamster oocytes that were penetrated by capacitated spermatozoa from the same semen sample. By contrast, the correlations between % sperm swelling in ejaculates and % normal sperm forms, % motile spermatozoa and % spermatozoa that do not stain with eosin-Y (supravital stain) in the same ejaculates were 0.30, 0.61 and 0.52, respectively. Therefore, the hypoosmotic swelling technique to evaluate the functional integrity of the sperm membrane appears to give high repeatability and accuracy and is closely correlated to the in-vitro fertilizing ability of spermatozoa. It may be a useful addition to the standard semen analysis.

Changes in actin distribution during fertilization of the mouse egg

Abstract: Unfertilized mouse oocytes and eggs 1-8 h after fertilization in vitro were examined at the light microscope level for structural changes, distribution of actin (as assessed by both antiactin antibodies and NBD-phallicidin), surface binding of concanavalin A (Con A) and chromosomal distribution and condensation. The influence of cytochalasin D on these events was also assessed. Changes in actin distribution were associated with rotation of the second anaphase spindle, formation of the second polar body, the events following incorporation of the sperm nucleus, and formation and migration of the pronuclei. Cytochalasin D prevented spindle rotation, polar body formation, pronuclear migration and the restoration of Con A binding over the site of sperm entry and the site of polar body formation, but did not affect sperm fusion and entry, the loss of Con A binding at the site of sperm entry and pronuclear formation.

Sperm transport in the cow : peri-ovulatory redistribution of viable cells within the oviduct

Abstract: Using a surgical approach involving double ligatures and transection, together with subsequent recovery of the eggs, heifers mated at the onset of oestrus have been examined for progression of spermatozoa within the oviducts relative to the time of ovulation; the latter occurred 28-31 hours after the onset of oestrus. Evidence was obtained that spermatozoa competent to penetrate the egg(s) do not pass directly to the site of fertilisation at the isthmic-ampullary junction but instead are largely sequestered before ovulation in the caudal region of the isthmus, possibly for 18-20 hours or more. Thus, 3 of 14 eggs (21%) were fertilised when the oviduct was transected in pre-ovulatory animals 2.0 cm proximal to the utero-tubal junction 16 or more hours after mating compared with 7 of 8 eggs (88%) with a similar post-ovulatory transection 28-36 hours after mating (P less than 0.01). A redistribution of spermatozoa appears to be associated with imminent release of the egg. The distal portion of the oviduct is therefore seen as the functional sperm reservoir--that is the immediate source of viable spermatozoa at the time of ovulation. Parallels are drawn with storage of relatively quiescent sperm cells in the distal portion of the epididymal duct, and procedures of insemination are examined in the light of this storage role of the isthmus and the reported incidence of fertilisation in cattle.

Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs.

Abstract: A cell-free cytoplasmic preparation from activated Rana pipiens eggs could induce in demembranated Xenopus laevis sperm nuclei morphological changes similar to those seen during pronuclear formation in intact eggs. The condensed sperm chromatin underwent an initial rapid, but limited, dispersion. A nuclear envelope formed around the dispersed chromatin and the nuclei enlarged. The subcellular distribution of the components required for these changes was examined by separating the preparations into soluble (cytosol) and particulate fractions by centrifugation at 150,000 g for 2 h. Sperm chromatin was incubated with the cytosol or with the particulate material after it had been resuspended in either the cytosol, heat-treated (60 or 100 degrees C) cytosol or buffer. We found that the limited dispersion of chromatin occurred in each of these ooplasmic fractions, but not in the buffer alone. Nuclear envelope assembly required the presence of both untreated cytosol and particulate material. Ultrastructural examination of the sperm chromatin during incubation in the preparations showed that membrane vesicles of approximately 200 nm in diameter, found in the particulate fraction, flattened and fused together to contribute the membranous components of the nuclear envelope. The enlargement of the sperm nuclei occurred only after the nuclear envelope formed. The pronuclei formed in the cell-free preparations were able to incorporate [3H]dTTP into DNA. This incorporation was inhibited by aphidicolin, suggesting that the DNA synthesis by the pronuclei was dependent on DNA polymerase-alpha. When sperm chromatin was incubated greater than 3 h, the chromatin of the pronuclei often recondensed to form structures resembling mitotic chromosomes within the nuclear envelope. Therefore, it appeared that these ooplasmic preparations could induce, in vitro, nuclear changes resembling those seen during the first cell cycle in the zygote.

Male crickets feed females to ensure complete sperm transfer

Abstract: The spermatophore transferred by the male decorated cricket Gryllodes supplicans to the female during copulation includes a large gelatinous portion (spermatophylax), which the female removes and feeds on immediately after mating. Females usually removed and ate the smaller sperm-containing portion (ampulla) within 1 to 7 minutes after fully consuming or losing the spermatophylax. Complete sperm transfer requires that the ampulla remain attached for a minimum of 50 minutes; this corresponds to the average time at which females actually removed ampullae, 52.0 ± 2.2 minutes after mating. These results indicate that nuptial feeding of the female cricket functions to deter females from removing the sperm ampulla before sperm transfer is complete.

Eunuchs: The Role of Apyrene Sperm in Lepidoptera?

Abstract: Moths and butterflies (Lepidoptera) generally produce two types of spermatozoa: a typical nucleated (eupyrene) spermatozoon and a smaller anucleate (apyrene) spermatozoon. The apyrene sperm often predominate over the eupyrene sperm in an ejaculate and, in the female, they migrate actively to the sperm storage organ, the spermatheca. There they usually degenerate, apparently not playing any role in fertilization of the eggs. Several hypotheses for the function of the apyrene sperm have been proposed. These center around the notions that the apyrene sperm may assist the eupyrene sperm in their migration from the testes in the male to the spermatheca in the female or that they function as a nutritional supplement in the female. No experimental support for these notions has been adduced and observational evidence seems to argue against them. We propose that apyrene sperm may play, at least additionally, a role in competition between rival sperm deposited by different males. They may either eliminate, by displ...

A sulfated glycoprotein synthesized by Sertoli cells and by epididymal cells is a component of the sperm membrane.

Abstract: We report here the purification, partial characterization and immunofluorescent localization of a dimeric acidic glycoprotein (DAG-protein) secreted by cultures of Sertoli cells of rat testes. Partially purified protein was obtained after chromatography over Sepharose 4B under conditions which favored a soluble, nonaggregated form of the protein. Rechromatography over the same column under reducing conditions yielded very pure monomers of 41,000 daltons and 29,000 daltons. Antibodies were prepared against the mixed monomers and used to immunoprecipitate proteins in spent medium from cultures incubated with [35S] methionine, 35SO4 = or tunicamycin. DAG-protein and another protein (Band 4, 70,000 daltons) were coprecipitated by the antiserum and all contained 35SO4 = in their structures. It was shown by Western blotting that the antiserum cross-reacted very weakly with Band 4 protein. The DAG-protein polypeptides secreted in the presence of tunicamycin were assumed to lack N-glycosylation and exhibited apparent molecular weights of 27,000 and 21,000 daltons. Immunoprecipitations of media from organ cultures of testis and epididymis yielded DAG-protein of slightly lower molecular weight than the protein secreted in Sertoli cell cultures. Indirect immunofluorescence of DAG-protein in paraffin sections of testis and epididymis revealed that the protein was concentrated in the cytoplasm of Sertoli cells, on the stereocilia of epididymal principal cells, in the cytoplasm of epididymal halo cells, and was associated with late spermatids and mature sperm. Sperm were specifically labeled on the acrosome, at the neck, and on the endpiece of the tail. No enzymatic or structural function has been ascribed to DAG-protein as yet, but the protein must play a pivotal role in spermatogenesis because it is secreted by both the testis and epididymis and becomes an integral component of sperm.

Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization.

Abstract: We have previously defined distinct localizations of antigens on the surface of the guinea pig sperm using monoclonal antibodies. In the present study we have demonstrated that these antigen localizations are dynamic and can be altered during changes in the functional state of the sperm. Before the sperm is capable of fertilizing the egg, it must undergo capacitation and an exocytic event, the acrosome reaction. Prior to capacitation, the antigen recognized by the monoclonal antibody, PT-1, was restricted to the posterior tail region (principle piece and end piece). After incubation in capacitating media at 37 degrees C for 1 h, 100% of the sperm population showed migration of the PT-1 antigen onto the anterior tail. This redistribution of surface antigen resulted from a migration of the surface molecules originally present on the posterior tail. It did not occur in the presence of metabolic poisons or when tail-beating was prevented. It was temperature-dependent, and did not require exogenous Ca2+. Since the PT-1 antigen is freely diffusing on the posterior tail before migration, the mechanism of redistribution could involve the alteration of a presumptive membrane barrier. In addition, we observed the redistribution of a second surface antigen after the acrosome reaction. The antigen recognized by the monoclonal antibody, PH-20, was localized exclusively in the posterior head region of acrosome-intact sperm. Within 7-10 min of induction of the acrosome reaction with Ca2+ and A23187, 90-100% of the acrosome-reacted sperm population no longer demonstrated binding of the PH-20 antibody on the posterior head, but showed binding instead on the inner acrosomal membrane. This redistribution of the PH-20 antigen also resulted from the migration of pre-existing surface molecules, but did not appear to require energy. The migration of PH-20 antigen was a selective process; other antigens localized to the posterior head region did not leave the posterior head after the acrosome reaction. These rearrangements of cell surface molecules may act to regulate cell surface function during fertilization.

The Pollen Grain

Abstract: The pollen grain is the carrier of the male gametes or their progenitor cell, in higher plants In a single unit, each grain contains all the genetic information required to specify an entire haploid plant organism (for example, pollen embryoids in tissue culture), or to unite with the female gamete at fertilization and form a diploid zygote and, hence, a new sporophyte The male gametes, the sperm cells (or their progenitor, the generative cell) are housed entirely within the cytoplasm of the vegetative cell This is dehydrated like a seed at maturity, and is filled with storage reserves The vegetative cell is surrounded by a complex, intricately patterned outer wall, and its nucleus controls at least the initial growth and metabolism of the pollen tube following germination

Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligozoospermic sperm populations.

Abstract: Suspensions of capacitating human spermatozoa were analyzed for potential hyperactivated movements using videomicrographic methods. Analysis was carried out on aliquots of 22 sperm suspensions, which were proved fertile several hours later during human in vitro fertilization. After approximately 3 h of capacitation, 22.1% of the fertile spermatozoa displayed motility patterns designated as hyperactivated. Over 80% of these hyperactivated spermatozoa moved with a wide-amplitude, two-dimensional whiplash pattern, displaying marked lateral displacement of the head. Only 8.4% of capacitating spermatozoa from oligozoospermic patients showed these hyperactivated movements. The incidence of hyperactivated movements by fertile and oligozoospermic spermatozoa could be significantly increased after exposure to various motility stimulants. The clinical significance of hyperactivation as a functional assay of fertilizing capacity is discussed.

The importance of hydrolytic enzymes to an exocytotic event, the mammalian sperm acrosome reaction

Abstract: Summary The mammalian sperm acrosome reaction is a unique form of exocytosis, which includes the loss of the involved membranes. Other laboratories have suggested the involvement of hydrolytic enzymes in somatic cell exocytosis and membrane fusion, and in the invertebrate sperm acrosome reaction, but there is no general agreement on such an involvement. Although reference was made to such work in this review, the focus of the review was on the evidence (summarized below) that supports or fails to support the importance of certain hydrolytic enzymes to the mammalian sperm acrosome reaction. Because the events of capacitation, the prerequisite for the mammalian acrosome reaction, and of the acrosome reaction itself are not fully understood or identified, it is not yet always possible to determine whether the role of a particular enzyme is in a very late step of capacitation or part of the acrosome reaction. (1) The results of studies utilizing inhibitors of trypsin-like enzymes suggest that such an enzyme has a role in the membrane events of the golden hamster sperm acrosome reaction. The enzyme involved may be acrosin, but it is possible that some as yet unidentified trypsin-like enzyme on the sperm surface may play a role in addition to or instead of acrosin. Results obtained by others with guinea pig, ram and mouse spermatozoa suggest that a trypsin-like enzyme is not involved in the membrane events of the acrosome reaction, but only in the loss of acrosomal matrix. Such results, which conflict with those of the hamster study, may have been due to species differences or the presence of fusion-promoting phospholipase-A or lipids contaminating the incubation media components, and in one case to the possibly damaging effects of the high level of calcium ionophore used. The role of the trypsin-like enzyme in the membrane events of the hamster sperm acrosome reaction may be to activate a putative prophospholipase and/or to hydrolyse an outer acrosomal or plasma membrane protein, thus promoting fusion. A possible role of the enzyme in the vesiculation step rather than the fusion step of the acrosome reaction cannot be ruled out at present. (2) Experiments utilizing inhibitors of phospholipase-A2, as well as the fusogenic lysophospholipid and cis-unsaturated fatty acid hydrolysis products that would result from such enzyme activity, suggests that a sperm phospholipase-A2 is involved in the golden hamster sperm acrosome reaction. Inhibitor and LPC addition studies in guinea pig spermatozoa have led others to the same conclusion. The fact that partially purified serum albumin is important in so many capacitation media may be explained by its contamination with phospholipase-A and/or phospholipids. Serum albumin may also play a role, at least in part, by its removal of inhibitory products released by the action of phospholipase-A2 in the membrane. The demonstration of phospholipase-A2 activity associated with the acrosome reaction vesicles and/or the soluble component of the acrosome of hamster spermatozoa, and the fact that exogenous phospholipase A2 can stimulate acrosome reactions in hamster and guinea pig spermatozoa, also support a role for the sperm enzyme. The actual site or the sites of the enzyme in the sperm head are not yet known. The enzyme may be on the plasma membrane as well as, or instead of, in the acrosomal membranes or matrix. A substrate for the phospholipase may be phosphatidylcholine produced by phospholipid methylation. It is possible that more than one type of ‘fusogen’ is released by phospholipase activity (LPC and/or cis-unsaturated fatty acids, which have different roles in membrane fusion and/or vesiculation. In addition to acting as a potential ‘fusogen’, arachidonic acid released by sperm phospholipase-A2 probably serves as precursor for cyclo-oxygenase or lipoxygenase pathway metabolites, such as prostaglandins and HETES, which might also play a role in the acrosome reaction. Although much evidence points to a role for phospholipase-A2, phospholipase-C found in spermatozoa could also have a role in the acrosome reaction, perhaps by stimulating events leading to calcium gating, as suggested for this enzyme in somatic secretory cells. (3) A Mg2+-ATPase H+-pump is present in the acrosome of the golden hamster spermatozoon. Inhibition of this pump by certain inhibitors of ATPases (but not by those that only inhibit mitochondrial function) leads to an acrosome reaction only in capacitated spermatozoa and only in the presence of external K+. The enzyme is also inhibited by low levels of calcium, and such inhibition, combined with increased outer membrane permeability to H+ and K+, and possibly plasma membrane permeability to H+ (perhaps by the formation of channels), may be part of capacitation and/or the acrosome reaction. The pH of the hamster sperm acrosome has been shown to become more alkaline during capacitation, and such a change may result in the activation of hydrolytic enzymes in the acrosome or perhaps in a change in membrane permeability to Ca2+. A similar Mg2+-ATPase has not been found in isolated boar sperm head membranes. However, that conflicting result could have been due to the use of noncapacitated boar spermatozoa for the preparation of the membranes or to protease modification of the boar sperm enzyme during assay. (4) Inhibition of Na+, K+-ATPase inhibits the acrosome reaction of golden hamster spermatozoa, and the activity of this enzyme increases relatively early during capacitation. A late influx of K+ is important for the acrosome reaction. However, this late influx may not be due to Na+, K+-ATPase, but instead may be due to a K+ permeability increase (possibly via newly formed channels) in the membranes during capacitation. It is suggested in this review that Na+, K+-ATPase has a role early in capacitation rather than directly in the acrosome reaction (although such a role cannot yet be completely ruled out). One possible role for the enzyme in capacitation might be to stimulate glycolysis (which appears to be essential for capacitation and/or the acrosome reaction of hamster and mouse spermatozoa). The function of the influx of K+ just before the acrosome reaction is probably to stimulate, directly or indirectly, the H+-efflux required for the increase in intraacrosomal pH occurring during capacitation. Direct stimulation of the acrosome reaction by a change in membrane potential resulting directly from K+-influx is not a likely explanation for the hamster results. However, the importance of an earlier membrane potential change, due to increased Na+, K+-ATPase during capacitation, and/or of later membrane potential changes resulting from the pH change, cannot be ruled out. Although K+ is required for the hamster acrosome reaction, other workers have reported that K+ inhibits guinea pig sperm capacitation. However, the experimental procedures used in the guinea pig sperm studies raise some questions about the interpretation of those inhibition results. (5) Ca2+-influx is known to be required for the acrosome reaction. Others have suggested that increased Ca2+-influx due to inhibition or stimulation of sperm membrane calcium transport ATPases are involved in the acrosome reaction. There is as yet no direct or indirect biochemical evidence that inhibition or stimulation of such enzymatic activity is involved in the acrosome reaction, and further studies are needed on those questions. (6) I suggest that the hydrolytic enzymes important to the hamster sperm acrosome reaction will also prove important for the acrosome reaction of all other eutherian mammals.

Inhibition of bovine spermatozoa by caudal epididymal fluid: II. Interaction of pH and a quiescence factor.

Abstract: Previous studies (Carr and Acott , 1984) indicate that bovine sperm are maintained in a quiescent state in the caudal epididymis (CE) by a pH-dependent inhibitory factor. Here, we have determined that the pH of bovine CE fluid and of CE semen is approximately 5.8, and that the motility of CE sperm in undiluted CE fluid increases as the pH is elevated. Therefore, the acidity of CE fluid may play a physiological role in the maintenance of sperm quiescence. The changes in sperm motility, in response to changes in the pH of CE fluid, are reversible and rapid. Dilution of CE fluid with buffers at either pH 5.5 or 7.6 produces a much slower initiation of motility. In buffer a significantly lower pH is required to inhibit sperm motility than is required in CE fluid. The apparent pKs for inhibition are 5.3 in buffer and 6.6 in CE fluid. However, the motility of sperm in buffers that contain lactate, shows a pH dependence similar to sperm in CE fluid. That is, lactate inactivates sperm in buffer at pH 5.5 but not at pH 7.6. Lactate, and several other permeant weak acids, have previously been shown to reduce the intracellular pH of bovine sperm and many other types of cells. We show that these permeant weak acids, but not impermeant weak acids, reversibly reduce CE sperm motility in buffer at pH 5.5 but not at pH 7.6. Weak bases, which have previously been shown to elevate intracellular pH, initiate sperm motility in CE fluid. These results suggest that intracellular pH can regulate CE sperm motility and may be the intracellular messenger for the pH-dependent quiescence factor. Although sperm cyclic AMP levels have been previously correlated with motility stimulation, cyclic AMP levels do not change when the pH of CE fluid is elevated, even though full motility is initiated.

Sperm dependence of female receptivity to remating in drosophila melanogaster.

Abstract: As a consequence of copulation, males of Drosophila melanogaster induce a variety of physiological and behavioral effects in the female. Egg production and oviposition are stimulated by products of the male's accessory glands (Kummer, 1960; Garcia-Bellido, 1964; Merle, 1968; Bumet et al., 1973), and female attractiveness and receptivity are both reduced following copulation. Changes in female attractiveness are mediated pheromonally. A change in pheromonal profile from courtship-eliciting to courtship-discouraging pheromones is induced during the first 3 min of copulation (Tompkins et al., 1980; Tompkins and Hall, 1981; Venard and Jallon, 1980). The seminal fluid enzyme esterase-6 metabolizes the seminal fluid component cis-vaccenyl acetate to produce the antiaphrodisiac cis-vaccenyl alcohol (Mane et al., 1983). Behavioral effects of this metabolite on female attractiveness have been demonstrated and have been shown to be short-lived. There is a close correspondence between the time course of these behavioral effects and those first identified by Manning (1967) as the "copulation effect." Manning (1962, 1967) also identified a "sperm effect" on female receptivity which is longer lasting and causes females to remain unreceptive to male courtship. The strength of the sperm effect seems to be proportional to the number of sperm in storage (Gromko and Pyle, 1978; Gromko et al., 1984). The sperm effect is characterized behaviorally by the use of ovipositor extrusion to reject males, a behavior used only in very low frequency by virgin females (Connolly and Cook, 1973; Tompkins and Hall, 1981). The "sperm effect" -or the sperm dependence of the return of female receptivity-is not evident when mated females are confined with second males continuously for 24 h (Newport and Gromko, 1984). In this paper we investigate the details of the sperm dependence of female receptivity within the context of an experimental design which allows females periodic interactions with second males. We also quantify the impact of sperm-dependent female receptivity on the reproductive outcome of double matings. We show that by causing females to delay remating, first males suffer very little reduction in reproductive success due to female remating.

Monoclonal antibody to a human germ cell membrane glycoprotein that inhibits fertilization.

Abstract: A monoclonal antibody to an antigen in the human germ cell membrane did not agglutinate or immobilize sperm but inhibited binding and penetration of zona-free hamster ova by human sperm and blocked murine fertilization in vitro. The antibody, of the 2a subclass of immunoglobulin G, was germ cell-specific but not species-specific. It recognized a single antigen of 23 kilodaltons that has been isolated from human germ cells. This fertilization antigen, located on the postacrosome , midpiece, and tail of human sperm, is a glycoprotein of testicular origin associated with some types of human involuntary immunoinfertility .

Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators

Abstract: Capacitation of rhesus monkey spermatozoa was assessed by monitoring sperm flagellar beat and trajectory changes during incubation in vitro and by determining sperm penetration into rhesus oocytes and hamster zona-free ova. Rhesus sperm capacitation in vitro depended on the addition to the culture medium of the cyclic nucleotide mediators, caffeine and dibutyryl cyclic AMP. Capacitation was correlated with the development of hyperactivated motility. Spermatozoa treated with the cyclic nucleotide mediators, and showing hyperactivated motility, penetrated 57.4% of all rhesus oocytes and fertilized 88.9% of mature rhesus oocytes that were morphologically normal. Control spermatozoa did not penetrate any of the eggs. Some sperm penetration into hamster ova occurred but was not statistically significant. These data provide a basis for achieving in-vitro fertilization in the rhesus monkey and information on specific sperm motility characteristics associated with fertilizing ability.

Polyandry in honey bees (apis mellifera l.): sperm utilization and intracolony genetic relationships

Abstract: Sperm usage by queen honey bees was examined by progeny analyses using six phenotypically distinct genetic markers. No evidence was found for sperm displacement or precedence. All queens used the sperm of all males that inseminated them during all sampling periods. Sperm usage, as measured by phenotypic frequencies, did fluctuate nonrandomly but did not result in abnormally high representation of a single phenotype or the elimination of other phenotypes as has often been suggested. The genetic relationships of workers within honey bee colonies are estimated from the data presented. Average genetic relatedness is shown to be low among colony nestmates and probably approaches 0.25 in colonies with naturally mated queens. There is no evidence for elevated relatedness among colony subfamilies due to nonrandom fluctuations in sperm usage by queens or for numerical dominance of any subfamilies.

Inviability of parthenogenones is determined by pronuclei, not egg cytoplasm

Abstract: Parthenogenetic mouse embryos pose an interesting problem in the study of early mammalian development. Haploid or diploid parthenogenones, resulting from spontaneous1 or experimental2–4 activation of unfertilized eggs, will undergo apparently normal preimplantation development but die in the early post-implantation stages5–8. However, in aggregation chimaeras with fertilized embryos, parthenogenetic embryos have the ability to differentiate into many tissue types, including gametes which can give rise to normal offspring9. Furthermore, it has been reported that viable young were obtained from the transfer of inner cell-mass nuclei of parthenogenetic blastocysts to enucleated fertilized eggs10. These observations suggest that sperm have some additional role, apart from restoring a complete genome, that is necessary for normal development. To investigate whether sperm-related modifications to the egg cytoplasm are important, we have used an efficient nuclear transfer technique in which a complete karyoplast, comprised of pronuclei, surrounding cytoplasm and a portion of the egg plasma membrane, is transferred utilizing Sendai virus membrane fusion11. Embryos produced by the transfer of pronuclei from diploid parthenogenetic eggs to enucleated fertilized eggs died very soon after implantation, whereas viable young were obtained from the transfer of fertilized egg pronuclei into enucleated parthenogenetic eggs. This shows that the death of parthenogenones is not due to a lack of cytoplasmic factors from the sperm.