Ultrastructural study of polyspermy during early embryo development in pigs, observed by scanning electron microscope and transmission electron microscope

Polyspermy is generally considered a pathological phenomenon in mammals. Incidence of polyspermy in porcine eggs in vivo is extremely high (30-40%) compared with other species, and polyspermy rate in the in vitro fertilized eggs in pigs can reach 65%. It is still unknown whether polyspermy to a certain degree is a physiological condition in pigs, and whether porcine eggs have any capability with which to remove the accessory sperm in the cytoplasm. The objectives in the present study are to observe the ultrastructural changes of accessory sperm during early embryonic development in pigs. A total of 58 normal, early embryos at one-, two, three-, and four-cell and morular stages were collected from gilts and were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The surface ultrastructure showed that sperm fusion with the zona pellucida was a continuous process during one-, two-, three-, and four-cell and morular stages, as observed by the SEM. Accessory sperm were present in the cytoplasm of cleaved embryos. The sperm heads in the cytoplasm of cleaved embryos did not decondense. TEM revealed the presence of a condensed sperm head within a lysosome (or phagolysosome) in a three-cell embryo. These observations suggest that polyspermy may be a physiological condition in pigs and that early embryos may develop to term if accessory sperm do not interrupt the embryo genome. Furthermore, lysosome activity could be another physiological mechanism for removing accessory sperm in the cytoplasm of fertilized eggs and cleaved embryos after fertilization in pigs.     

Evaluation of morpholine, 3-morpholinone, and N-substituted morpholines in the rat hepatocyte primary culture/DNA repair test

Although mature mammalian sperm are incapable of DNA repair, repair of damaged sperm DNA can occur after fertilization, as the sperm head decondenses and forms the male pronucleus. To quantify the cytogenetic effects of damage to sperm DNA we adapted the sister-chromatid exchange (SCE) test for use in early mouse embryos. After ultraviolet (UV) irradiation of sperm, eggs were fertilized in vitro and cultured for 2 cell cycles in medium containing fluorodeoxyuridine and bromodeoxyuridine; chromosomes were then prepared for SCE analysis. We found that UV-induced SCEs could be detected at the second cleavage division, and that eggs of different strains showed different frequencies of SCEs when fertilized by damaged sperm of a single strain. These results may indicate strain-specific differences in DNA repair of UV-induced DNA lesions by the early mouse embryo.     

After fertilization, sperm surface components remain as a patch in sea urchin and mouse embryos

Sea urchin and mouse sperm that are labeled on their surfaces with fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TMRTC) or 125I-diiodofluorescein isothiocyanate (125IFC) remain viable and can fertilize eggs. When sea urchin eggs were fertilized with 125IFC-labeled sperm, the radioactivity from the sperm was quantitatively transferred to the egg (at a ratio of one sperm equivalent per egg) and persisted in the embryo as it developed to the pluteus larval state (5 days at 12 degrees C). The radioactivity was acid-precipitable and was associated with the particulate fraction of embryo homogenates. In addition, FITC-labeled sea urchin sperm were used to fertilize eggs, and the labeled components were followed by fluorescence microscopy. In the embryo, labeled sperm components were present as a discrete patch that was partitioned unequally during early cleavages. In experiments using mouse sperm labeled with TMRTC, the labeled sperm components were also transferred to the embryo as a discrete patch that was again distributed unequally after cleavage. This physiological cell fusion system therefore has distinctive characteristics: there is limited lateral mobility of surface components, which have a low turnover rate unlike that see in other systems. In this paper, we discussed the possible morphogenetic role of this unusual behavior.     

Detrimental effects of cryopreservation of loach (Misgurnus fossilis) sperm on subsequent embryo development are reversed by incubating fertilised eggs in caffeine

Cryopreservation can cause changes to the genetic material of cells, but the mechanism and significance of these changes are still unknown. It has been suggested that some damage to the sperm genome could be repaired by the DNA repair system of the oocyte after fertilisation. Caffeine has been reported to be an inhibitor of such repair processes. In this study the effect of caffeine on the repair system of Loach (Misgurnus fossilis) oocytes was investigated. Loach eggs were fertilised using cryopreserved sperm. Embryos derived from cryopreserved sperm were exposed to 2.6mM caffeine for 1h after fertilisation. The experiments were carried out using 32313 embryos from four females and eight males. Embryo survival was evaluated for 46 h until the hatching stage. Reduction in embryo survival after 20th stage is generally believed to result from the failure in the genome function of embryos. Cryopreservation of sperm significantly decreased embryo survival (53.4+/-2.8% compared to 68.4+/-2.8% of control) after the 20th stage. However, the addition of caffeine to the embryos derived from cryopreserved sperm, in contrast to our expectation, significantly increased survival of loach embryos (70.9+/-2.8% compared to 53.4+/-2.8% of embryos derived from cryopreserved sperm in the absence of caffeine). The effect of individual donors of sperm and eggs on overall embryo survival was also studied. Whilst no significant differences were observed between males, the effect of individual females on embryo survival was significant. The analysis of embryo survival at different developmental stages showed that embryo survival both before and after 20th stage decreased with embryo development. When fresh sperm were used the decline of embryo survival with development was more pronounced compared with those embryos derived from cryopreserved sperm. Possible explanations of these effects are presented.     

PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca(2+) oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca(2+) release. The 'sperm factor' hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca(2+) transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLC zeta, that triggers Ca(2+) oscillations in mouse eggs indistinguishable from those at fertilisation. PLC zeta removal from sperm extracts abolishes Ca(2+) release in eggs. Moreover, the PLC zeta content of a single sperm was sufficient to produce Ca(2+) oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLC zeta as the molecular trigger for development of a fertilised egg into an embryo.     

Fertilization and aqueous development of the puerto rican terrestrial-breeding frog,Eleutherodactylus coqui

The Puerto Rican tree frog, Eleutherodactylus coqui, has internal fertilization and direct development on land. In light of these reproductive adaptations, the events of fertilization and early development were studied. Cytological examination of just-fertilized eggs showed that sperm entry is restricted to about 10% of the surface of these large, yolky eggs, and all nuclear events of the first cell cycle occur near the animal pole. Although the oocytes have cortical granules, a number of polyspermic fertilizations were found. One clutch consisted of eggs with a high frequency of polyspermy and of normal development. This raises the possibility that normal development can occur despite multiple sperm entry, a situation not found in other anuran amphibians. With respect to saline requirements, the sperm and the embryo are similar to those in amphibians with external fertilization and aqueous development. Sperm motility was high in low-tonicity conditions, and the normally terrestrial embryo could develop completely from a fertilized egg to a froglet in a low-tonicity aqueous solution.     

Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis

Now that transgenic strains of Xenopus laevis and X. tropicalis can be generated efficiently and with genomic sequence resources available for X. tropicalis, early amphibian development can be studied using integrated biochemical and genetic approaches. However, housing large numbers of animals generated during genetic screens or produced as novel transgenic lines presents a considerable challenge. We describe a method for cryopreserving Xenopus sperm that should facilitate low maintenance, long-term storage of male gametes. By optimising the cryoprotectant, the rates of cooling and thawing, and conditions for fertilisation, sperm from the equivalent of one-eighth of a X. laevis testis or of two X. tropicalis testes have been cryopreserved and used to fertilise eggs of both species after thawing. Sperm undergo a substantial loss of viability during a freeze-thaw cycle, but sufficient survive to fertilise eggs. Gametes of mutagenised frogs are being stored in connection with a screen for developmental mutations.     

Mechanism of Ca2+ release at fertilization in mammals.

At fertilization in mammals the sperm triggers a series of oscillations in intracellular Ca2+ within the egg. These Ca2+ oscillations activate the development of the egg into an embryo. It is not known how the sperm triggers these Ca2+ oscillations. There are currently three different theories for Ca2+ signaling in eggs at fertilization. One idea is that the sperm acts as a conduit for Ca2+ entry into the egg after membrane fusion. Another idea is that the sperm acts upon plasma membrane receptors to stimulate a phospholipase C (PLC) within the egg which generates inositol 1,4, 5-trisphosphate (InsP(3)). We present a third idea that the sperm causes Ca2+ release by introducing a soluble protein factor into the egg after gamete membrane fusion. In mammals this sperm factor is also referred to as an oscillogen because, after microinjection, the factor causes sustained Ca2+ oscillations in eggs. Our recent data in sea urchin egg homogenates and intact eggs suggests that this sperm factor has phospholipase C activity that leads to the generation of InsP(3). We then present a new version of the soluble sperm factor theory of signaling at fertilization. J. Exp. Zool. (Mol. Dev. Evol.) 285:267-275, 1999.     

Sequential transformations of human sperm nucleus in human egg

In-vitro insemination of human zona-free oocytes prepared from oocytes that failed to fertilize in an in-vitro fertilization programme was used as an experimental model to study the time course and morphological events during the development of sperm nuclei into male pronuclei. At 30 min after insemination, 22 eggs were cultured in a CO2 incubator for further 3.5 h and 17 eggs were placed individually between a slide and coverslip for randomly repeated microscopical observations in a controlled environment for at least 3.5 h. Simultaneous arrest of maternal meiosis and sperm nuclear development occurred in 36.4% (8/22) eggs cultured in the CO2 incubator and 47.1% (8/17) of those cultured between a slide and coverslip. Sequential transformation of the human sperm nucleus in human eggs was studied in 6 eggs that showed continuous development of sperm nuclei into male pronuclei during at least 3.5 h after insemination. The early sperm nuclear development in human egg ooplasm can be divided into three phases: the sperm nucleus first decondenses (phase 1) then partly recondenses (phase 2) before expanding again to form an early male pronucleus (phase 3). The prepronuclear stages (phases 1 and 2) took about 60 min each and the pronuclear formation (phase 3) began between 120 and 170 min after insemination. Early pronuclear formation was associated with the occurrence of dense outline material, probably a precursor of the future pronuclear membrane, around the recondensed nucleus in re-expansion (phase 3). Between 30 and 60 min after the beginning of phase 3, numerous (greater than 20) dense grains, considered as nucleolar precursors, were clearly visible inside the growing male pronucleus. Moreover, we have examined sperm nuclear changes in some eggs in which the progression of late meiosis was abnormal. Meiotic arrest of maternal chromatin was always associated with arrest of sperm head development. In 75% (6/8) of the eggs arrested in the metaphase II stages and in 87.5% (7/8) of the eggs arrested in late anaphase II, sperm nuclear development was stopped at the decondensed and recondensed stages, respectively. We have always observed male pronuclei when a maternal pronucleus was present in the egg. These observations suggested that maternal chromatin and sperm nuclear development are probably regulated by common factor(s).     

Nervous necrosis virus replicates following the embryo development and dual infection with iridovirus at juvenile stage in grouper

Infection of virus (such as nodavirus and iridovirus) and bacteria (such as Vibrio anguillarum) in farmed grouper has been widely reported and caused large economic losses to Taiwanese fish aquaculture industry since 1979. The multiplex assay was used to detect dual viral infection and showed that only nervous necrosis virus (NNV) can be detected till the end of experiments (100% mortality) once it appeared. In addition, iridovirus can be detected in a certain period of rearing. The results of real-time PCR and in situ PCR indicated that NNV, in fact, was not on the surface of the eggs but present in the embryo, which can continue to replicate during the embryo development. The virus may be vertically transmitted by packing into eggs during egg development (formation) or delivering into eggs by sperm during fertilization. The ozone treatment of eggs may fail to remove the virus, so a new strategy to prevent NNV is needed.     

Causes of hatching failure in endangered birds

About 10 per cent of birds'' eggs fail to hatch, but the incidence of failure can be much higher in endangered species. Most studies fail to distinguish between infertility (due to a lack of sperm) and embryo mortality as the cause of hatching failure, yet doing so is crucial in order to understand the underlying problem. Using newly validated techniques to visualize sperm and embryonic tissue, we assessed the fertility status of unhatched eggs of five endangered species, including both wild and captive birds. All eggs were classified as ‘infertile’ when collected, but most were actually fertile with numerous sperm on the ovum. Eggs of captive birds had fewer sperm and were more likely to be infertile than those of wild birds. Our findings raise important questions regarding the management of captive breeding programmes.     

Sperm surface proteins are incorporated into the egg membrane and cytoplasm after fertilization

Using an antibody to sperm surface proteins, we have investigated the fate of the sperm membrane after fertilization. By immunofluorescence, two distinct loci of sperm surface proteins were found in the embryo: a large, restricted domain on the embryo surface and a smaller locus that apparently had moved from the point of sperm entry into the egg cytoplasm. The surface domain was initially over the fertilization cone and slowly disappeared, so that by 2 hr after fertilization it was no longer seen. The small internal locus of staining remained intact throughout the one-cell stage. When fluorescein isothiocyanate (FITC)-labeled sperm were used to fertilize eggs, the FITC-patch in the eggs was at a site distinct from either locus of sperm surface proteins. Thus, while most sperm surface proteins are incorporated into the egg surface at the site of fertilization and then slowly disappear, other sperm surface components are internalized to be retained for longer times. The differential handling of these sperm cytoplasmic components by the embryo raises the possibility that some of the sperm components may play a role in later embryonic events.     

The relative sensitivity of sperm, eggs and embryos to copper in the blue mussel (Mytilus trossulus)

Copper, an essential element, is toxic at elevated concentrations, and as a result of anthropogenic activities is becoming increasingly prevalent in marine environments. In this study, we examined the effects of copper on early life stages of the blue mussel, Mytilus trossulus. We assessed the impacts of increasing copper concentrations on embryo development, egg viability, sperm fertilization capacity and, in particular, on sperm swimming speed using computer-assisted sperm analysis. Sensitivity to copper followed the pattern: embryos > sperm > eggs. A dramatic increase in abnormal embryo development was observed following exposure to copper concentrations exceeding 10 microg/L. Sperm swimming speeds decreased significantly when exposed to 100 microg/L of copper, but lower doses did not influence sperm swimming speed. Copper exposure (at any tested concentration) did not affect sperm flagellum length, or alter egg viability. Based on our results, we suggest that exposure of sperm to copper may interfere with mitochondrial activity, which reduces sperm swimming speed during the extended duration of sperm motility in blue mussel.     

Hormonal priming, induction of ovulation and in-vitro fertilization of the endangered Wyoming toad (Bufo baxteri)

The endangered Wyoming toad (Bufo baxteri) is the subject of an extensive captive breeding and reintroduction program. Wyoming toads in captivity rarely ovulate spontaneously and hormonal induction is used to ovulate females or to stimulate spermiation in males. With hormonal induction, ovulation is unreliable and egg numbers are low. The sequential administration of anovulatory doses of hormones (priming) has increased egg numbers and quality in both anurans and fish. Consequently, we tested the efficacy of a combination of human Chorionic Gonadotrophin (hCG) and Luteinizing Hormone Releasing Hormone analogue (LHRHa) administered as one dose, or two or three sequential doses to Bufo baxteri on egg numbers, fertilization and early embryo development. Spawning toads deposited eggs into Simplified Amphibian Ringers (SAR) solution to enable controlled in-vitro fertilization (IVF) with sperm from hormonally induced male toads. Unprimed females receiving a single mixed normally ovulatory dose of 500 IU hCG plus 4 micrograms of LHRHa produced no eggs. Whereas females primed with this dose and an anovulatory dose (100 IU hCG and 0.8 micrograms of LHRHa) of the same hormones, or primed only with an anovulatory dose, spawned after then receiving an ovulatory dose. Higher total egg numbers were produced with two primings than with one priming. Moreover, two primings produced significantly more eggs from each individual female than one priming. The cleavage rate of eggs was not found to differ between one or two primings. Nevertheless, embryo development with eggs from two primings gave a significantly greater percentage neurulation and swim-up than those from one priming. Of the male toads receiving a single dose of 300 IU hCG, 80% produced spermic urine with the greatest sperm concentration 7 hours post-administration (PA). However, peak sperm motility (95%) was achieved at 5 hours PA and remained relatively constant until declining 20 hours PA. In conclusion, Bufo baxteri egg numbers and quality benefited from sequential priming with LHRHa and hCG whereas spermic urine for IVF was produced from males with a single dose of hCG. The power of assisted reproduction technology in the conservation of endangered amphibians is shown by the release of nearly 2000 tadpoles produced by IVF during this study.     

Comparison of fertility and embryo mortality following artificial insemination of common duck females (Anas Platyrhynchos) with semen from common or Muscovy (Cairina Moschata) drakes

The purpose of this study was to compare fertility and early embryo mortality rates (< or = 5 days of incubation) following artificial insemination (AI) of common duck females (Anas Platyrhynchos) with semen from either common or Muscovy (Cairina Moschata) drakes at various periods of the reproductive season (Period I, 27-35 weeks; Period II, 39-43 weeks and Period III, 49-56 weeks). Based on observations performed by stereomicroscopy on eggs laid from Days 2 to 10 after AI, we confirmed that fertility was significantly lower in the interbred compared to the purebred cross at each of the periods tested (purebred 58.1, 61.2 and 54.2 versus crossbred 31.0, 40.4 and 39.5 at Periods I, II and II, respectively; 0.01 < P < 0.001). In a complementary experiment, we demonstrated that the number of perivitelline spermatozoa (NPS) was markedly lower in mule (crossbred) eggs compared to common (purebred) eggs, a strong indication that initial sperm selection occurring in the lower oviduct is probably more intense after crossbred compared to purebred insemination. Comparison of early embryo mortality (EEM) between mule and common duck eggs indicated that increased levels of EEM in mule embryos corresponded to Stages II-IV of the Eyal-Giladi and Kochav classification (EGK). While a similar age-dependent increase in early embryo mortality was observed in eggs from both genetic origins during the latter periods of the reproductive season, it was also established that embryo mortality due to parental age was related rather to Stages X-XIV of the EGK classification in eggs from both genetic origins. It is concluded that the relative subfertility of mule compared to common duck eggs is probably the consequence of a more intense rate of selection of heterologous than homologous spermatozoa occurring in the vaginal portion of the oviduct while the causal origins of EEM in mule duck eggs can at least in part be identified on the basis of precise staging (by stereomicroscopy) of dead embryos.     

Early patterning of the mouse embryo--contributions of sperm and egg

The first cleavage of the fertilised mouse egg divides the zygote into two cells that have a tendency to follow distinguishable fates. One divides first and contributes its progeny predominantly to the embryonic part of the blastocyst, while the other, later dividing cell, contributes mainly to the abembryonic part. We have previously observed that both the plane of this first cleavage and the subsequent order of blastomere division tend to correlate with the position of the fertilisation cone that forms after sperm entry. But does sperm entry contribute to assigning the distinguishable fates to the first two blastomeres or is their fate an intrinsic property of the egg itself? To answer this question we examined the distribution of the progeny of early blastomeres in embryos never penetrated by sperm - parthenogenetic embryos. In contrast to fertilised eggs, we found there is no tendency for the first two parthenogenetic blastomeres to follow different fates. This outcome is independent of whether parthenogenetic eggs are haploid or diploid. Also unlike fertilised eggs, the first 2-cell blastomere to divide in parthenogenetic embryo does not necessarily contribute more cells to the blastocyst. However, even when descendants of the first dividing blastomere do predominate, they show no strong predisposition to occupy the embryonic part. Thus blastomere fate does not appear to be decided by differential cell division alone. Finally, when the cortical cytoplasm at the site of sperm entry is removed, the first cleavage plane no longer tends to divide the embryo into embryonic and abembryonic parts. Together these results indicate that in normal development fertilisation contributes to setting up embryonic patterning, alongside the role of the egg.     

Cytological mechanisms of gynogenesis and sperm incorporation in unreduced diploid eggs of the clonal loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae)

The loach Misgurnus anguillicaudatus comprises diploid clonal, triploid and diploid-triploid mosaic individuals in a wild population on Hokkaido island, Japan. When diploid eggs of clonal loaches are fertilized by haploid sperm of normal bisexual loaches, both diploid clonal and non-diploid aclonal individuals occur in the progeny. Flow cytometry and microsatellite analyses revealed that the occurrence of triploid, diploid-triploid and other progeny was essentially due to the genetic incorporation of sperm to diploid clonal genomes of unreduced eggs. In this study, we examined the influence of water temperature from fertilization to early embryogenesis on frequencies of diploid clonal and other progeny and observed that progeny of three out of four clonal females examined exhibited approximately constant rates of diploid clonal individuals (54.2-68.9%) at hatching stage. Thus, no drastic increase of non-diploid progeny was detected. However, the 28 degrees C group of the fourth clonal female gave significantly lower rate (28.1%) of diploid clonal progeny, suggesting that this temperature might be a critical or a borderline temperature inducing sperm incorporation. We also examined the cytological process by which diploid clonal and other aclonal progeny develop after fertilization. In some fertilized eggs, the sperm nucleus remained condensed throughout fertilization and early embryogenesis and never fused with the female pronucleus. This cytological observation concludes that clonal eggs develop by the mechanism of gynogenesis. However, some other eggs showed the cytological process of syngamy between the female pronucleus and an accidentally formed male nucleus, suggesting the formation of triploid progeny. The syngamy between an accidentally activated sperm nucleus with a male pronucleus-like structure and nucleus of a blastomere of gynogenetically developing clonal diploid embryo might produce a diploid-triploid mosaic individual.     

Spatial separation of parental genomes in preimplantation mouse embryos

We have used two different experimental approaches to demonstrate topological separation of parental genomes in preimplantation mouse embryos: mouse eggs fertilized with 5-bromodeoxyuridine (BrdU)-labeled sperm followed by detection of BrdU in early diploid embryos, and differential heterochromatin staining in mouse interspecific hybrid embryos. Separation of chromatin according to parental origin was preserved up to the four-cell embryo stage and then gradually disappeared. In F1 hybrid animals, genome separation was also observed in a proportion of somatic cells. Separate nuclear compartments during preimplantation development, when extreme chromatin remodelling occurs, and possibly in some differentiated cell types, may be associated with epigenetic reprogramming.     

A profile of fertilization in mammals

Fertilization is defined as the process of union of two gametes, eggs and sperm. When mammalian eggs and sperm come into contact in the female oviduct, a series of steps is set in motion that can lead to fertilization and ultimately to development of new individuals. The pathway begins with species-specific binding of sperm to eggs and ends a relatively short time later with fusion of a single sperm with each egg. Although this process has been investigated extensively, only recently have the molecular components of egg and sperm that participate in the mammalian fertilization pathway been identified. Some of these components may participate in gamete adhesion and exocytosis, whereas others may be involved in gamete fusion. Here we describe selected aspects of mammalian fertilization and address some of the latest experimental evidence that bears on this important area of research.