Triploid Eggs Research Articles

Simultaneous formation of haploid, diploid and triploid eggs in diploid–triploid mosaic loaches

In the loach Misgurnus anguillicaudatus, very few diploid–triploid mosaic individuals, which are generated by accidental incorporation of the sperm nucleus into diploid eggs produced by clonal diploid loach, occur in nature. Ploidy examination of gynogenetic progeny induced by activation with ultraviolet‐irradiated goldfish sperm indicated that diploid–triploid mosaic females laid haploid, diploid and triploid eggs, simultaneously. In addition, triploid eggs exhibited larger egg sizes. Microsatellite genotyping of diploid–triploid mosaics revealed that triploid genotypes of mosaic mothers possessed two alleles specific to the clonal diploid and one allele from normal diploid male. Diploid eggs from a mosaic mother had genotypes absolutely identical to the diploid clone. Most genotypes of triploid eggs were identical to the mosaic mother, and one of the three alleles of the mosaic mother was transmitted to haploid eggs. These results suggested that diploid germ cells, which had a clonal genome, were differentiated into clonal diploid eggs, and triploid and haploid eggs were produced from triploid germ cells in the same ovary of mosaic individuals.

Comparative histology of the testes of the spined loach Cobitis taenia L. and natural allotetraploids of Cobitis (Pisces, Cobitidae)

The spined loach Cobitis taenia L. creates exclusively diploid and mixed diploid–polyploid populations. Allotriploid females, which co-exist with C. taenia or C. elongatoides and a few tetraploid males and females dominate in most Cobitis mixed populations. They reproduce gynogenetically and produce triploid eggs that are stimulated to development by sperm from Cobitis males. Some of these eggs are fertilized, which leads to the production of bisexual tetraploids. Males of C. taenia (2n = 48) from a diploid population in Lake Klawoj, Northern Poland (46 individuals) and from a mixed Cobitis population in the Bug River, Eastern Poland (7 individuals), and three tetraploid males (4n = 98) from the same mixed population were examined. All the fish were analyzed karyologically and histologically. Tubules with cysts of the testes of C. taenia from both populations were filled with germ cells at various developmental stages. Among fishes from Lake Klawoj sperm maturation in batches simultaneous with the batch spawning of C. taenia females was found. The testes of the loach C. taenia, from a mixed population in the Bug River, were filled with spermatozoa over the entire reproductive season. Sperm maturation in batches was not observed. Sperm maturation in batches seems to be only connected with a few diploid males in this population. So, a continuous process of spermatogenesis in their testes is required. Only in the testes of all tetraploid Cobitis males were cells characteristic of the early stages of spermatogenesis observed, i.e. without spermatids and spermatozoa. Furthermore, the histological sections of the testis of a male captured in August, revealed fragments with connective tissue between the germ cells. However the participation of tetraploid, infertile Cobitis males in the process of reproduction in the investigated mixed population remains controversial. The results obtained so far, reveal that even the infertile sperm of tetraploid males may induce gynogenesis in Cobitis triploid females.

Allotriploidy increases sterility in the hybrid between Clarias macrocephalus and Clarias gariepinus

Hybrids between female Thai walking catfish ( Clarias macrocephalus) and male African catfish ( Clarias gariepinus) are capable of reproducing, and this has led to introgression of genes from the paternal species into natural populations of the native C. macrocephalus. Therefore this study was conducted aiming at producing sterile triploid hybrids by means of cold shock. Eggs from C. macrocephalus were fertilized with sperm from C. gariepinus and were subsequently immersed in cold water (7°C) starting 4.5 min after water activation of the eggs, and lasting for 14 min. The cold shock induced 72%, 74% and 88% triploid fish with 44–49% hatching success compared to the control value of 69–78%. At 28 weeks old body weight of the triploid fish was smaller than the diploid counterparts. The frequencies of retarded gonads and ovotestes were smaller in diploid as compared to the triploid hybrids. The average gonado-somatic index (GSI) of female diploid F 1 hybrids was significantly higher than those of the triploids whereas male GSI of the two groups were not statistically different. Fifty percents of the diploid testes produced a few spermatozoa while only 15.8% of the triploid testes did. Likewise larger percentage of the female diploid hybrids (64.3%) had well-developed ovaries marked with vitellogenic oocytes as compared to the triploids (21%). At 44-week-old, although the triploid and diploid females showed similar response to hormone injection, the triploids produced significantly less eggs than the diploids. Both diploid and triploid eggs were capable to fertilize sperm of C. macrocephalus. However, all fry from the triploid F 1 hybrids died at yolk absorption, whereas approximately 80% of the fry from diploid F 1 hybrids survived. No survived fry at yolk absorption were obtained from fertilization between C. macrocephalus eggs and sperm obtained from both triploid and diploid F 1 hybrids.

Growth and reproductive traits of diploid and triploid forms of the Squalius alburnoides cyprinid complex in a tributary of the Guadiana River, Portugal

The Squalius alburnoides hybrid complex has diploid, triploid and some rare tetraploid male and female forms, with triploid females predominating in most populations. The reproductive traits of diploids and triploids from a population in a tri- butary of the Guadiana River, Portugal, were compared, during a two-years period. Few differences were found in the growth and reproductive traits of diploid and trip- loid females. Marginal differences were found in the longevity, with a few triploid females living up to six years compared with a maximum of five years for diploid females and a maximum of four years for diploid males. Both diploid and triploid females exhibited rapid growth in the first two years of life followed by rapid drop off linked to attaining sexual maturity. Reproductive effort increased with age but was mainly linked to number of eggs produced and not to egg size, since oocyte size did not vary with age or length of fish in either ploidy forms. Both forms exhibited a pro- tracted spawning period between March and June, suggesting multiple-spawning be- haviour. Triploid females had a slightly smaller oocyte diameter that may be due to the production of reduced (haploid) oocytes while diploid females produced diploid (unre- duced) oocytes The potentially higher production of diploid oocytes (which become triploid eggs after fertilisation) by diploid females could at least partially account for the higher percentage of triploids in natural populations. In addition, the production of bigger (diploid) oocytes, with possibly higher energetic content could lead to bigger fish larvae, which tend to have a higher survival rate. The time of spawning of both diploids and triploids appears to be synchronised, although differences were found in

Optimization of tetraploid induction in Pacific oysters, Crassostrea gigas, using first polar body as a natural indicator

Tetraploid Crassostrea gigas were first successfully produced in 1993 by inhibiting the first polar body of eggs from triploids that had been fertilized with sperm from diploids (Guo and Allen method). However, attempts to repeatedly produce high yields of tetraploids were inconsistent. Because of these uncertainties, we examined some of the fundamental aspects of tetraploid production in an attempt to optimize tetraploid induction using the Guo and Allen method. Varying the duration of the treatment to inhibit polar body 1 (PB 1) of triploid eggs had clear effects on ploidy of progeny. Short treatments (15–35 min after fertilization — about half the period of meiosis 1 in triploid eggs) yielded tetraploid and heptaploid cells. Long treatments (7–43 min — about three quarters of the period of meiosis I in triploid eggs) yielded only heptaploid cells among the embryos. Tetraploid induction was most consistent when treatments were accomplished on eggs from individual triploid females rather than pooled from a number of females, and when treatments were metered according to biological landmarks. That is, eggs from individual triploids were fertilized and 0.5 mg/l cytochalasin B (CB) added after 10 min. A subsample of the fertilized eggs was kept aside untreated. When 50% of the untreated eggs showed PB 1 extrusion (as judged by microscopic examination of dividing, untreated eggs), the CB treatment was discontinued. In eight treatments based on these “biological criteria,” proportions of tetraploids ranged from 13% to 92% after 8 days for an average of 55%, and seven of eight replicates went through metamorphosis and settlement. At settlement, the percentage of tetraploids ranged from 7% to 96%, averaging 45%. Average survival in all the replicates at 8 days was 4.4%, which is acceptable considering tetraploid progeny are destined for use as brood stock.

Delayed meiosis and polar body release in eggs of triploid Pacific oysters, Crassostrea gigas, in relation to tetraploid production.

The dynamics of polar body release are important for creating polyploid shellfish. For producing triploids, these dynamics concern meiosis in diploid eggs and are well understood. For creating tetraploids, eggs from triploids are employed and the dynamics, variation, and environmental influences upon polar body release are less studied. We investigated the effects of several agents on the timing of 50% first polar body (PB1) release in eggs of triploids. PB1 release is generally slower in triploid eggs than diploid ones at 26 degrees C. Lowering the temperature (from 26 to 19 degrees C) had a marked effect on timing of 50% PB1 in both diploid and triploid eggs. While lower temperature merely slowed development in diploid eggs, it nearly halted it in triploid eggs. At any temperature, the variability in 50% PB1 release was much higher in triploid eggs than diploid ones; this variation occurred both within eggs from individual females and among eggs from different females. The amount of time eggs remain in seawater between the time they are stripped and fertilized (or time of hydration) also affected rate of meiosis. In triploid eggs, the average time necessary for the expulsion of 50% PB1 was 23 min post-fertilization (PF) for 75 min of hydration versus 29 min PF for 35 min. However, increasing the time of hydration had no effect on the variability in the timing among females. Serotonin also had no effect on the dynamics of polar body release in triploids. Variability among triploid females in timing of meiosis cannot be improved with any treatments we tried. Consequently we recommend that treatments of triploid eggs to produce tetraploids incorporate a single female at a time.

Cytogenetic mechanisms for triploid and haploid egg formation in the triploid loachMisgurnus anguillicaudatus

The triploid (diploid × tetraploid) loach Misgurnus anguillicaudatus lays large, triploid eggs and small, haploid eggs simultaneously. We conducted cytogenetic examination of gonadal cells to determine the mechanisms responsible for this unusual oogenesis. In immature ovaries of triploids, both triploid (3n = 75) and hexaploid (6n = 150) metaphases were detected. Oocytes at the pachytene stage were categorized into two types: small and large. The small oocytes gave about 50 chromosome elements, comprising 25 thick, densely stained bivalents and 25 thin, faintly stained univalents, that is, bivalent-univalent complexes. The large ones showed no fewer than 60 thick elements, considered to be 75 bivalents. Cytological examination of full-grown oocytes cultured in vitro revealed that both large and small oocytes undergo two conventional successive meiotic cycles: formation of a bipolar spindle in meiosis I and equal segregation of homologues, extrusion of the first polar body, and the appearance of a bipolar spindle in meiosis II. In the small, full-grown oocytes, some chromosomes were observed to detach from the spindle and remain in the surrounding cytoplasm, and then these chromosomes (presumably unpaired univalents) were eliminated from the meiotic event. We conclude that triploid eggs are formed by a quasinormal meiosis of the hexaploid oocytes of large size, which are generated by premeiotic endomitosis, while the haploid eggs are produced from bivalents after eliminating univalents in the meiosis of triploid oocytes of small size. J. Exp. Zool. 281:608–619, 1998. © 1998 Wiley-Liss, Inc.

Electrophoretic analysis of the diploid progenies from triploid × diploid crosses in the loachMisgurnus anguillicaudatus (Pisces: Cobitidae)

In the loach Misgurnus anguillicaudatus (Pisces: Cobitidae), triploid females derived from the hybridization of normal diploid females and natural tetraploid males generate both triploid and haploid eggs simultaneously. Diploid progenies were produced by fertilizing the haploid eggs of triploids with haploid spermatozoa of normal diploids. Ldh-1 and Mdh-2 allozyme loci were electrophoretically diagnostic, because diploid and tetraploid loach exhibited the diverged genotype aa and bbbb at each locus, respectively. Diploids from triploid (abb) × diploid (aa) crosses frequently exhibited a heterozygous ab genotype at each locus but sometimes showed genotypic frequencies that fit a random pairing model (aa:ab = 1:2) of two of three chromosomes in each set of homologues. These results show that the entire haploid genome of one parent is not completely excluded in the oogenesis of triploid loach, and the haploid eggs are not formed in a typical hybridogenetic manner. However, the two homologous chromosomes derived from the tetraploid parent are likely to pair with each other preferentially during the meiosis of triploid loach. J. Exp. Zool. 280:368–374, 1998. © 1998 Wiley-Liss, Inc.

Clonal nature of gynogenetically induced progeny of triploid (diploid × tetraploid) loach,Misgurnus anguillicaudatus (pisces: cobitididae)

In the loach Misgurnus anguillicaudatus, triploid fish, which had been produced by hybridizations between normal diploid females and natural tetraploid males, laid mature hexaploid (triploid egg nucleus + triploid polar body nucleus) eggs of large size as well as diploid (haploid egg nucleus + haploid polar body nucleus) eggs of small size. Gynogenetic triploidy was artificially induced by activating the large eggs with UV-irradiated spermatozoa. Gynogenetic hexaploidy was also generated by blocking the polar body release with pressure shock after induction of gynogenesis. Unexpected mosaics and pentaploid gynogens were infrequently observed in these crosses. Most triploid and all hexaploid gynogens from each triploid female demonstrated absolutely identical DNA fingerprints, suggesting clonal nature among the mature large eggs of each female. However, a small number of triploid gynogens were aclonal with slightly different DNA fingerprints. These results indicate that triploid loach generate viable large eggs which are genetically identical or highly similar to the somatic cells. J. Exp. Zool. 278:412–421, 1997. © 1997 Wiley-Liss, Inc.

Expression of the Giant egg gene (Ge) in triploid eggs of the silkworm, Bombyx mori

Expression of the Giant egg gene (Ge) in triploid eggs of the silkworm, Bombyx mori

Male meiosis in polyploid silkworms, Bombyx mori L. (Lepidoptera : Bombycidae)

Tetraploid and hexaploid silkworms, Bombyx mori L. (Lepidoptera Bombycidae) were induced by applying a cold shock to diploid and triploid eggs at the first cleavage stage. Male meiosis in the primary spermatocytes of these silkworms having a different ploidy was observed. In the polyploid cells, chromosome bridges, 2 for the tetraploid and 3 for the hexaploid, occurred between the newly formed daughter nuclei at telophase. Observation in the living spermatocytes showed that tetraploid cells needed a longer time than the diploid ones to complete the first meiotic division. The delay in the cell division may be responsible for the high sterility of the tetraploid males.

A Tripolar Spindle Formed at Meiosis I Assures the Retention of the Original Ploidy in the Gynogenetic Triploid Crucian Carp, Ginbuna Carassius auratus langsdorfii: (fish oocytes/gynogenesis/meiosis/spindle formation/histone H1 kinase).

A triploid crucian carp, ginbuna (Carassius auratus langsdorfii), reproduces by gynogenesis, in which sperm of diploid ginbuna or of other species triggers the development of the triploid eggs, but a male genome makes no contribution to the zygotic genome. Gynogenesis is maintained by two mechanisms: exclusion of male genome during fertilization and retention of somatic ploidy levels during oogenesis. We examined the mechanisms responsible for producing unreduced eggs. Microfluorometry with a DNA staining dye showed that DNA content in the ginbuna oocytes was not reduced in half during meiosis I. Cytological observations revealed that a tripolar spindle was formed at meiosis I and the first polar body was not extruded, whereas an ordinary bipolar spindle was formed and the second polar body was extruded at meiosis II. Activity of histone H1 kinase (as an indicator of maturation-promoting factor) decreased transiently between meiosis I and II, strongly suggesting a "normal" meiotic cycle progression in the ginbuna oocytes. These results have indicated that in the gynogenetic ginbuna the somatic ploidy levels are maintained by inhibiting the first polar body extrusion via the formation of the tripolar spindle at meiosis I.

A novel method to produce triploids in bivalve molluscs by the use of 6-dimethylaminopurine

To date, pressure shock, heat shock, and chemical treatment with cytochalasin B have been the major methods used to induce triploid bivalves. In this study, triploid bivalves were induced by a new chemical treatment using 6-dimethylaminopurine (6-DMAP). The capacity of 6-DMAP to produce triploid eggs and larvae was investigated in the Pacific oyster, Crassostrea gigas, the giant sea scallop, Placopecten magellanicus, and the blue mussel, Mytilus edulis. The triploid yields from the 6-DMAP treatments were compared with those of cytochalasin treatments. The highest percentage of triploid production was 90% in the Pacific oysters when the fertilized eggs were treated for 20 min with 300 μM 6-DMAP at 15 min after fertilization and 95% in the giant scallops when treated for 15 min with 400 μM 6-DMAP at 70 min after fertilization. Increasing durations of 6-DMAP treatments improved the efficiency of triploid induction. However, long incubations with 6-DMAP, which overlapped the period of first mitotic cleavage, led to the development of abnormal larvae. These included low percentages of normal veliger larvae in the Pacific oysters and developmental arrest at the trochophore stage in the blue mussels. The percentage of abnormalities increased with increased treatment duration. Triploid larvae of Pacific oysters produced by 6-DMAP or cytochalasin treatments had equivalent growth rates and were similar to those of control diploid larvae. However, triploid larvae showed high mortalities following these two chemical treatments. Overall, the results clearly demonstrate that 6-DMAP was an efficient and practical inducer of triploidy in bivalve molluscs. Moreover, the described procedure is the most simple and reproducible method ever reported. In addition, 6-DMAP is safer to handle than cytochalasin which is classified as a carcinogen.

Cytogenetic analysis of human oocytes after subzonal insemination.

Subzonal insemination has been proposed to achieve fertilization in cases where standard in vitro fertilization has failed. We present the results of chromosome analysis of oocytes after subzonal insemination. Our data suggest that the main cause (76 per cent) of the absence of cleavage after subzonal insemination is the total absence of sperm nucleus evolution of the injected spermatozoa. Our results also suggest that spermatozoa chromatin development is normal after subzonal insemination. Aneuploidy does not seem to be increased in zygotes after subzonal insemination. However, polyploidy was often more important than predicted by the observation of pronuclei (PN). Pronucleus development might be asynchronous and can appear earlier or later than after standard IVF. The cytogenetic risk after subzonal insemination might therefore be triploidy (if a triploid egg is transferred, because only 2 PN were seen) rather than aneuploidy or structural abnormalities.

GYNOGENETIC REPRODUCTION IN HYBRID MOLE SALAMANDERS (GENUS AMBYSTOMA).

Ambystoma platineum, a unisexual clonal triploid taxon of mole salamander, originated by hybridization between the Mendelian species A. jeffersonianum and A. laterale. Studies of lampbrush chromosomes indicated that A. platineum reproduces gynogenetically, that is, sperm from a sexual host species is required to activate egg development but makes no genetic contribution to the developing embryo. Nevertheless, electrophoretic diversity in populations of some hybrid Ambystoma suggested continual in situ recreation of unisexual hybrids and bidirectional gene exchange between the parental species and the hybrids. A. platineum usually lives with, and is sexually dependent on, one of its parental species, A. jeffersonianum. In central Indiana, however, A. platineum populations have shifted their host dependency to A. texanum. Such A. texanum-dependent populations of A. platineum provide an almost ideal system for studying reproductive mode in A. platineum, because both replacement of a jeffersonianum or laterale genome of A. platineum by a texanum genome, and movement of genes from A. platineum to the host species, A. texanum, would be readily detected by electrophoretic markers. Our samples of A. texanum provided no evidence for the transfer of jeffersonianum or laterale genes into A. texanum. Similarly, among 32 A. platineum sampled from six localities in east-central Illinois and central Indiana, we find no texanum alleles, and thus no evidence for genome replacement. The one diploid hybrid individual contained only a jeffersonianum and a laterale genome; because of the absence of either parental species from these populations, this hybrid could only have come from a diploid ovum produced by A. platineum. Both morphometric and electrophoretic results for the two tetraploid individuals indicate that they resulted from fertilization of triploid oocytes of A. platineum by sperm of A. texanum. Because genome replacement in A. texanum-dependent populations of A. platineum is irreversible, the persistence of A. platineum in A. texanum-dependent populations demonstrates conclusively that the major mode of reproduction in A. platineum populations is clonal: A. platineum produces mainly triploid eggs that develop gynogenetically.

Reconstitution of Diploid Ambystoma jeffersonianum (Amphibia: Caudata) in a Hybrid, Triploid Egg Mass with Lethal Consequences

Reconstitution of Diploid Ambystoma jeffersonianum (Amphibia: Caudata) in a Hybrid, Triploid Egg Mass with Lethal Consequences

Chromosomal Analysis of First-Cleavage Mouse Eggs Fertilized in Caffeine-Containing Medium.

Eggs derived from superovulated F1(BALB/c × C57BL/6) mice were fertilized in a modified Krebs-Ringer bicarbonate medium (TYH) containing 2-mM caffeine. Spermatozoa obtained from the cauda epididymis of ICR male mice were preincubated in the caffeine-containing for 2 h before the mixing of gametes. Gametes preincubated and inseminated in the caffeine-free medium served as controls. Chromosome analysis at the first cleavage showed a significantly (P<0.05) higher incidence of triploidy in eggs fertilized in the caffeine-containing medium (22.9%) than in the control (15.8%). The incidence of aneuploidy and the sex ratio were not significantly different between the two groups of eggs. Most of the triploid eggs observed were considered to be dispermic in origin.

Cytogenetic analysis and developmental capacity of normal and abnormal embryos after IVF.

Chromosome abnormalities represent the major cause of pre- and post-implantation embryo wastage. Indeed, 29% of embryos produced by in-vitro fertilization have an abnormal karyotype. Parthenogenesis (1.6% of the cases) and triploidy (6.4%) are the only abnormalities detectable 17 h after insemination. A total of 23% of activated oocytes with a single pronucleus (1 PN) and 18% of fertilized eggs with three pronuclei (three PN) remained uncleaved, compared with 4% for diploid eggs. The rate of cleavage for parthenogenes is not different from normal eggs. On the contrary, we observed that 29% of three PN-eggs reached the 5- to 8-cell stage 42 h after insemination when compared to 15% for diploids mainly due to a direct division of 56% of three PN-eggs in three cells. A model of development for triploid eggs is proposed, taking into account physiological and cytogenetic observations. The quality of embryos expressed in terms of morphology did not show any difference between embryos proceeding from parthenogenes or diploid eggs. On the contrary, the rate of fragmentation is decreased for triploid (3%) compared with diploid (13%) or activated (17%) embryos. Embryos resulting from one, two, three or four pronucleate ova display different developmental capacities during 5 days' culture in vitro.

Giant diploid oocytes as a cause of digynic triploidy in mammals.

Giant oocytes and zygotes twice the normal size from the Chinese hamster were studied embryologically and cytogenetically in order to clarify their significance as a cause of digynic triploidy. There were 6 (0.62%) giant eggs among 973 primary oocytes, 5 (1.37%) among 365 secondary oocytes from mature follicles missing ovulation, 3 (0.44%) among 677 secondary oocytes from oviducts, 4 (0.45%) among 886 one-cell zygotes, and 3 (0.49%) among 608 two-cell zygotes. The results indicated that follicles containing a giant oocyte might have a tendency to miss ovulation. But once ovulated, the giant eggs were fertilized normally and developed normally. Nuclear and chromosomal aspects were studied in 6 primary and 18 secondary oocytes, and 6 one-cell and 5 two-cells zygotes. There were mononuclear and binuclear giant oocytes, but the latter state was rarely maintained to metaphase II. The primary oocytes showed a diploid number of bivalents (22 tetrads) with normal chiasma configuration, and the secondary oocytes had 22 dyads with normal features, indicating normal segregation. The one-cell zygotes exhibited one set of diploid female pronuclear chromosomes and one set of haploid male pronuclear chromosomes in the late prophase. The two-cell zygotes were all digynic triploids; the tail of a fertilizing spermatozoon was attached. There were five XXX and six XXY giant triploids, giving a ratio of 1:1, as expected. It was suggested that the nuclear-cytoplasmic ratio of giant triploid eggs may be more favorable at least for preimplantation development than that of any other type of triploidy. Giant diploid oocytes may be an important source of human digynic triploids, in spite of the prevailing view from studies of marker chromosomes that suppression of first polar body extrusion is the predominant cause.

Effects of extremely low osmolarity on fertilized mouse eggs

When fertilized one-cell eggs are subjected to distilled water treatment for 2-6 min, cytoplasm bulges through the sperm-slit in the zona pellucida and forms a cytoplasmic fragment (CF). CFs were observed in 86-5% of eggs; in 20-9% of cases CFs contained a pronucleus (or pronuclei). In 53-4% of eggs permanent incorporation of the second polar body (2 P.B.) into the egg cytoplasm occurred. These phenomena occurring in different combinations produced 6-2% of haploid eggs, 10-3% of diploid eggs with a pronucleus replaced by 2 P.B. nucleus, and 43-1% of triploid eggs. 4-4% of eggs were enucleated. The remaining group comprised diploid eggs which were either not affected by the treatment (6-4%) or lost a certain amount of cytoplasm by formation of an anucleate CF (29-6%). The frequencies of the types of reaction were related to the post-fertilization stage of eggs. All eggs except the enucleated ones were able to develop to the stage of morula or blastocyst. Triploids developed until the 12th day of pregnancy and diploids that had lost up to 15% of the cytoplasm developed to term. There was a twofold reduction in the percentage of preimplantation development when treated eggs originated from induced rather than spontaneous ovulation.