Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.     

In vitro development of polyspermic porcine oocytes: Relationship between early fragmentation and excessive number of penetrating spermatozoa

Embryo development during in vitro culture of polyspermic porcine oocytes was investigated in the present study. After in vitro fertilization (IVF) of in vitro matured oocytes, putative zygotes were centrifuged to visualize pronuclei. Two pronuclear (2PN) and poly-pronuclear (PPN) zygotes were selected and cultured in vitro. Their development to the blastocyst stage and total cell numbers, dead cell rates and ploidy at the blastocyst stage and morphology of resultant embryos after first cleavage were compared. A cleavage rate of PPN embryos was lower than that of 2PN (61.3% and 82.2%, respectively), however, the ability of cleaved embryos to develop to the blastocyst stage did not differ between the PPN and the 2PN groups (22.4% and 32.9%, respectively). Also there was no difference in total cell numbers and rates of dead cells between PPN and 2PN blastocysts. The majority of blastocysts in 2PN group were found to be diploid. In contrast, blastocysts in PPN group showed heterogeneous status in their ploidy including polyploidy and mixoploidy, whereas a remarkable proportion (31.3%) of them was found to be diploid. After the first cleavage (at 36 h after IVF), there was no difference in the number of nuclei/embryo between the two groups, nevertheless embryos in PPN group had significantly higher numbers of blastomeres than that of embryos in 2PN group, mainly due to an increased frequency of anuclear blastomeres. The present results indicate that correction of embryo ploidy in polyspermic embryos can occur during IVC. Nevertheless the frequency of partial fragmentation in polyspermic embryos is increased.     

Development of polyspermic zygote and possible contribution of polyspermy to polyploid formation in angiosperms

Fertilization is a general feature of eukaryotic uni- and multicellular organisms to restore a diploid genome from female and male gamete haploid genomes. In angiosperms, polyploidization is a common phenomenon, and polyploidy would have played a major role in the long-term diversification and evolutionary success of plants. As for the mechanism of formation of autotetraploid plants, the triploid-bridge pathway, crossing between triploid and diploid plants, is considered as a major pathway. For the emergence of triploid plants, fusion of an unreduced gamete with a reduced gamete is generally accepted. In addition, the possibility of polyspermy has been proposed for maize, wheat and some orchids, although it has been regarded as an uncommon mechanism of triploid formation. One of the reasons why polyspermy is regarded as uncommon is because it is difficult to reproduce the polyspermy situation in zygotes and to analyze the developmental profiles of polyspermic triploid zygotes. Recently, polyspermic rice zygotes were successfully produced by electric fusion of an egg cell with two sperm cells, and their developmental profiles were monitored. Two sperm nuclei and an egg nucleus fused into a zygotic nucleus in the polyspermic zygote, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle. The two-celled proembryos further developed and regenerated into triploid plants. These suggest that polyspermic plant zygotes have the potential to form triploid embryos, and that polyspermy in angiosperms might be a pathway for the formation of triploid plants.     

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.     

Effect of centrifugation of mouse eggs on their development in vitro and in vivo

Fertilized mouse eggs were centrifuged at 5,000g, 10,000g, 15,000g, and 20,000g for 3 and 10 min or at 25,000g for 10 min. The pronuclei of centrifuged eggs became more distinctive than those of uncentrifuged eggs. The proportion of centrifuged and uncentrifuged eggs that developed to blastocysts was not significantly different. There was also no significant difference in the proportion of live fetuses obtained following the transfer of blastocysts developed from centrifuged and uncentrifuged eggs. This study demonstrated that there is no effect of centrifugation on the subsequent development of mouse eggs.     

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.     

Distribution and reproductive capacity of natural triploid individuals and occurrence of unreduced eggs as a cause of polyploidization in the loach,Misgurnus anguillicaudatus

Loaches (Misgurnus anguillicaudatus) were collected from 35 localities in Japan and assayed by flow cytometry to determine ploidy status. No tetraploids were found, with samples from 33 localities having no or few (1.2–3.2%) triploids. Samples collected from Ichinomiya Town, Aichi Prefecture, showed a relatively high rate of triploidy (7.7%). Samples collected from a fish farm in Hirokami Village, Niigata Prefecture, also showed high proportions of triploids (2.0–15.8%), these triploid males being sterile, but the females producing both large-sized triploid and small-sized haploid eggs. Such eggs developed bisexually rather than gynogenetically, giving rise to viable tetraploid and diploid offspring after normal fertilization. Of eight diploid females obtained from the same locality, one produced a high incidence of viable diploid gynogens (55%) after gynogenetic induction by fertilization with UV-irradiated spermatozoa. These observations indicated the presence of diploid fish which produced both diploid and haploid eggs. Thus, triploid and diploid individuals were also produced after fertilization with haploid spermatozoa. These results suggested that the occurrence of such unreduced eggs may be a cause of natural polyploidization in this species.     

Nuclear transfer of embryonic cell nuclei to non-enucleated eggs in zebrafish, Danio rerio

We previously established a novel method for nuclear transfer in medaka (Oryzias latipes) using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio), we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8%) operated eggs developed normally and reached the adult stage. Twenty-eight (15.2%) of these transplants showed wild-type phenotype and the remaining three (1.6%) were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4%) were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism). Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5) as well as the phenotype, the wild-type body color, to their F(1) and F(2) progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.     

Clonal diploid sperm of the diploid-triploid mosaic loach, Misgurnus anguillicaudatus (Teleostei:Cobitidae)

The loach Misgurnus anguillicaudatus comprises diploid, triploid and diploid-triploid mosaic individuals in a wild population of the Hokkaido island, Japan. Previous studies revealed the presence of a cryptic clonal lineage among diploid loaches, which is maintained by uniparental reproduction of genetically identical diploid eggs. In the present study, we analyzed distribution and genetic status of diploid and triploid cells in infrequent mosaic males. Flow cytometry, microsatellite genotyping and DNA fingerprinting verified that mosaic males consisted of diploid cells with genotypes identical to the natural clone and triploid cells with diploid genomes of the clonal lineage plus haploid genome from sperm nucleus of the father. Thus, the occurrence of diploid-triploid mosaicism might be caused by accidental fertilization of a diploid blastomere nucleus with haploid sperm after the initiation of clonal development of unreduced eggs. Such mosaic males produced fertile sperm with diploid DNA content. The experimental cross between normal diploid female and diploid-triploid mosaic male gave rise to the appearance of triploid progeny which exhibited two microsatellite alleles identical to the clonal genotype and one allele derived from the normal female. In DNA fingerprinting, such triploid progeny gave not only all the DNA fragments from the clone, but also other fragments from the normal female. Induced androgenesis using UV irradiated eggs and sperm of the mosaic male gave rise to the occurrence of diploid individuals with paternally derived microsatellite genotypes and DNA fingerprints, absolutely identical to the natural clonal lineage. These results conclude that the diploid-triploid mosaic male produced unreduced diploid sperm with genetically identical genotypes. The spermatogenesis in the clonal diploid cells under the mosaic condition suggests that triploid male somatic cells might transform genetically all-female germ cells to differentiate into functionally male gametes. The discovery of the mosaic male producing unreduced sperm suggests the theoretical occurrence of triploids and other polyploids by the syngamy of such paternally derived diploid gametes.     

Numerical chromosome errors in day 7 somatic nuclear transfer bovine blastocysts

Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona-free manipulation method: half-cytoplasts were made from zona-free oocytes by bisection, after which two half-oocytes and one granulosa cell (serum-starved primary culture) were fused together and activated. The NT embryos were cultured in modified synthetic oviductal fluid containing essential and nonessential amino acids, myoinositol, sodium citrate, and 5% cattle serum in microwells for 7 days, at which time nuclei from all blastocysts were extracted and chromosome aberrations were evaluated using dual-color fluorescent in situ hybridization with bovine chromosome 6- and 7-specific probes. Five embryo clone families, consisting of 112 blastocysts reconstructed from five different primary granulosa cell cultures, were examined. Overall, the mean chromosome complement within embryos was 86.9 +/- 3.7% (mean +/- SEM) diploid, 2.6 +/- 0.5% triploid, 10.0 +/- 3.1% tetraploid, and 0.5 +/- 0.2% pentaploid or greater; the vast majority (>75%) of the abnormal nuclei were tetraploid. Completely diploid and mixoploid embryos represented 22.1 +/- 4.5% and 73.7 +/- 5.5%, respectively, of all clones. Six totally polyploid blastocysts, containing or=5N chromosome complements, respectively) between two clone families were different (P < 0.01), as were blastocyst yields between other clone families (P < 0.01). Blastocyst yield was not correlated to % total ploidy error between clone families, but an inverse relationship (P < 0.01) between blastocyst total cell number and total % chromosome abnormality was observed within embryos. Categorization of the blastocysts into three quality grades (good, medium, and poor) and comparison of the distribution of ploidies when classified into 0%, 0.1-5.0%, 5.1-10.0%, 10.1-15.0%, and 15.1-100% errors within embryos indicated that medium- and poor-grade embryos were different (P < 0.05) from good-quality, in vitro-produced embryos. In a separate study, 11 different granulosa cell cultures (that did not correspond to those used for NT) were evaluated and found to possess only 0.23 +/- 0.12% ploidy errors. These results demonstrate that 1) the percentage of ploidy errors in bovine NT blastocysts is inversely related to total blastocyst cell number, 2) the mixoploid condition is representative of the majority of embryos, 3) 100% polyploid NT blastocysts can exist, and 4) the ploidy errors seem not to be derived from the donor cells.     

Action of colcemid in sea urchin eggs

The effects of Colcemid, the deacetyl-N-methyl derivative of colchicine, on the eggs of Arbacia punctulata were investigated. Colcemid in concentrations of 2.7 x 10^-5 M or greater blocks syngamy (the fusion of the pronuclei) in these eggs. Although a tenfold decrease in concentration of Colcemid usually permits the pronuclei to fuse, the subsequent division is blocked. In the sea urchin egg, the duration of presyngamy is about 15 min during which time there is no DNA synthesis. However, DNA synthesis is recorded in Colcemid-blocked cells prior to syngamy. Radioautographs of Colcemid-blocked cells which were immersed into thymidine-³H exhibited silver grains above each of the pronuclei. The action of Colcemid on Arbacia eggs is reversible. Nevertheless, exposures to 2.7 x 10^-5 M Colcemid for only 3 min, initiated 5 min after insemination, caused delays of 70 min in subsequent division. In general, cells are more sensitive to Colcemid prior to the time when the mitotic spindle is being assembled than at presyngamy stages. The results are discussed in terms of Colcemid action on pronuclear fusion and cell division.     

Polyspermy in angiosperms: Its contribution to polyploid formation and speciation

Polyploidization has played a major role in the long‐term diversification and evolutionary success of angiosperms. Triploid formation among diploid plants, which is generally considered to be achieved by fertilization of an unreduced gamete with a reduced one, has been accepted as a means of polyploid production. In addition, it has been supposed that polyspermy also contributes to the triploid formation in maize, wheat, and some orchids; however, such a mechanism has been considered uncommon because reproducing the polyspermic situation and unambiguously investigating developmental profiles of polyspermic zygotes are difficult. To overcome these problems, rice polyspermic zygotes have been successfully produced by electrofusion of an egg cell with two sperm cells, and their developmental profiles have been monitored. The triploid zygotes progress through karyogamy and divide into two‐celled embryos via a typical bipolar mitotic division; the two‐celled embryos further develop into triploid plants, indicating that polyspermic plant zygotes, unlike those of animals, can develop normally. Furthermore, progenies consisting of triparental genetic materials have been successfully obtained in Arabidopsis through the pollination of two different kinds of male parents with a female parent. These different pieces of evidence for development and emergence of polyspermic zygotes in vitro and in planta suggest that polyspermy is a key event in polyploidization and species diversification.     

Reproductive capacity of triploid loaches obtained from Hokkaido Island, Japan

Reproductive capacity was investigated in naturally occurring triploid individuals of the loach Misgurnus anguillicaudatus collected from Memanbetsu Town, Abashiri County, Hokkaido Island, Japan. These triploids have been considered to appear by accidental incorporation of the haploid sperm genome from normal diploid into unreduced diploid eggs from the clonal lineage that usually reproduces unisexually. By fertilization with sperm from the normal male, one triploid female gave many inviable aneuploid (2.1–2.7n) and very few tetraploid progeny, whereas the other produced both diploid and triploid progeny. The results suggest that at least four different types of eggs can be formed in triploid females in this locality. In contrast, no progeny hatched when eggs of the normal female were fertilized with sperm or sperm-like cells obtained from triploid males. These gametes exhibited inactive or no motility after adding ambient water. They had larger head sizes than those of normal haploid sperm and had a short or no tail. Although their ploidy was triploid or hexaploid, a small number of haploid cells were detected in the semen by flow cytometry. Thus, triploid males were generally sterile, but they have a little potential for producing very few haploid sperm.     

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.     

Improved techniques for use of the triploid cell marker in the axolotl, Ambystoma mexicanum

Techniques for using the triploid cell marker for studying cell lineage during the development and regeneration of the axolotl limb are described. Triploid animals possess cells with three nucleoli while diploid animals possess cells with two nucleoli. We have developed a technique for isolating the limb dermis as a sheet of cells for whole-mount analysis of cellular ploidy. Whole-mount tissue preparations as well as paraffin-embedded sectioned tissues were stained specifically for nucleoli with bismuth. Cell counts from a number of triploid and diploid dermal preparations show that (1) diploid dermal cells never possess three nucleoli, (2) the frequency of trinucleolate cells in whole-mount triploid dermal preparations is not 100% but varies between animals from 30 to 76%, (3) within a single triploid animal, the frequency of trinucleolate cells in different dermal preparations is constant. These data establish the usefulness of this technique and emphasize the need for appropriate control cell counts when using the triploid cell marker in the axolotl.     

Reproduction and the origin of polyploids in hybrid salamanders of the genus Ambystoma

Eggs and larvae produced by diploid, triploid, and tetraploid females collected from breeding ponds on Pelee Island in Lake Erie were studied to examine the reproductive mechanism. No instance of parthenogenesis was found as all examined females required sperm to produce viable progeny. Diploid females produced diploid and triploid larvae, triploid females produced triploid and tetraploid larvae, and tetraploid females produced triploid and tetraploid larvae. The majority of the eggs produced by hybrid females do not develop or do not complete embryogenesis. Electrophoretic examination of females and their offspring demonstrate that the male genome is being incorporated in reduced as well as unreduced eggs produced by all three ploidy classes of females. The elevation of ploidy among Pelee Island Ambystoma is attributed to sperm incorporation in unreduced eggs. Triploid as well as tetraploid individuals are constantly being produced. A critical examination of the literature on parthenogenetic or gynogenetic modes of reproduction in North America Ambystoma hybrids shows no conclusive evidence supporting these modes and it is suggested that the reproductive mechanism found among Pelee Island female hybrids may be more generally applied to other hybrid Ambystoma populations.     

Genetics of Parthenogenesis in DROSOPHILA MELANOGASTER. II. Characterization of a Gynogenetically Reproducing Strain

A strain of Drosophila melanogaster, named gyn-F9, can reproduce by gynogenesis. On mating with a male sterile mutant, ms( 3)K81, gyn-F9 females produced impaternate progeny at a rate of about 15 flies per female, which was almost 2000 times as frequent as that of the control. When the females were mated with normally fertile males, the number of offspring varied extremely from parent to parent, with average fertility being much lower than that of normal females. Nearly one-third of these bisexual progeny were either triploid females or intersexes. Among the rest of the progeny, some were diploid impaternates having developed without syngamy. The gynogenetic property of gyn-F9 is primarily governed by a few genes, most likely two recessive genes, one each located on the second and third chromosomes. The impaternates were found to restore their diploidy by the fusion of two nonsister nuclei out of the four egg pronuclei which result from the second meiotic division (central fusion). Although nondisjunction occurs frequently in the meiosis of gyn-F9, this is unlikely to bring about an appreciable number of diploid gametes developing into impaternates. Possible mechanisms of the evolutionary origin of parthenogenesis are discussed in relation to these findings.     

Host ploidy, parasitism and immune defence in a coevolutionary snail-trematode system

We studied the role of host ploidy and parasite exposure on immune defence allocation in a snail-trematode system (Potamopyrgus antipodarum-Microphallus sp.). In the field, haemocyte (the defence cell) concentration was lowest in deep-water habitats where infection is relatively low and highest in shallow-water habitats where infection is common. Because the frequency of asexual triploid snails is positively correlated with depth, we also experimentally studied the role of ploidy by exposing both diploid sexual and triploid asexual snails to Microphallus eggs. We found that triploid snails had lower haemocyte concentrations than did diploids in both parasite-addition and parasite-free treatments. We also found that both triploids and diploids increased their numbers of large granular haemocytes at similar rates after parasite exposure. Because triploid P. antipodarum have been shown to be more resistant to allopatric parasites than diploids, the current results suggest that the increased resistance of triploids is because of intrinsic genetic properties rather than to greater allocation to defence cells. This finding is consistent with recent theory on the advantages of increased ploidy for hosts combating coevolving parasites.     

Egg transfer in the Mongolian gerbil (Meriones unguiculatus) during lactational delay of implantation

Lactating pregnant gerbils in which one oviduct was ligated during early pregnancy were used on Day 6-14 p.c. as recipients for eggs (morulae and blastocysts) recovered on Day 6-12 p.c. from lactating donors. Eggs were transferred to the ligated (experimental) horn and the litter was removed. About half of the recipients had implantations in the non-ligated (control) horn 15 days after litter removal. Of these 40 females, 37 had implantations in the experimental horn and 32 had some normal fetuses present. Of 179 eggs transferred into the ligated uteri, 61% implanted and 41% developed into normal fetuses. The time after copulation of recipient and donor did not appear to influence the number of implantations or post-implantation development of the transferred eggs.     

A comparative cytological investigation of the reproductive cycle of an amphimictic diploid and a parthenogenetic triploid form of Lumbricillus lineatus (O.F.M.) (Oligochaeta,Enchytraeidae)

Summary The cytology of a diploid amphimictic (2x=26) and a triploid parthenogenetic (3x=39) cytotype of Lumbricillus lineatus has been described. The triploid type never produces mature sperm, but it must be fertilized by sperm from the diploid cytotype in order to produce viable eggs. However, the sperm does not enter the egg, but eggs which have not been activated by sperm die at an early stage. Oogenesis in the triploid cytotype, which results ultimately in the restoration of the somatic chromosome number, follows a pattern which has not been described in other parthenogenetic organisms. The first meiotic division is asynaptic and no typical metaphase plate is formed; the univalents are distributed to opposite poles without undergoing an equational division. The first division is therefore numerically approximately reductional. The nuclei are at anaphase when the eggs leave the animal, whereas the nuclei of the diploid amphimictic cytotype are in MI when the eggs are laid. Following activation by spermatozoa, the first anaphase spindle is much elongated, and becomes V-shaped. The first anaphase spindle persists and no second anaphase spindle is formed. At the second division the chromosomes divide mitotically. The resulting four chromosome groups fuse two by two in such a way that two nuclei are formed each with the full somatic (triploid) chromosome set (cf. Figs. 14–16).The different steps involved in the evolution of this mechanism are discussed in relation to the evolution of the triploid cytotype.