Gynogenetic Diploids Research Articles

Gynogenesis and sex determination in Atlantic Halibut ( Hippoglossus hippoglossus)

Gynogenesis refers to a process of uniparental inheritance whereby the resulting offspring retain only maternal DNA. It has been used to identify sex-determining mechanisms in fish and to produce all-female populations for aquaculture. The objective of this study was to develop a protocol for the production of gynogenetic Atlantic halibut for both these purposes. Various milt concentrations (1:20, 1:40 and 1:80 dilutions in seminal plasma) and UV doses (0–1382 mJ/cm 2) were tested for providing genetically inactivated, yet motile, spermatozoa for the production of gynogenetic haploids. Spermatozoon motility, assessed both as duration of swimming and percentage of motile spermatozoa, declined with increasing UV dose. Haploidy in developing embryos was determined either visually (‘haploid syndrome’) or by genetic analysis using microsatellite DNA. The optimum milt treatment for the production of haploids was 1:80 dilution followed by a UV exposure of 65 mJ/cm 2. Retention of the second polar body for the production of gynogenetic diploids was achieved by exposing newly activated eggs (15 min post-activation at 5–6 °C) to hydrostatic pressure of 8500 psi for 5 min. These treatments were combined to produce a population of gynogenetic diploids that was subsequently demonstrated to be comprised solely of females, thereby demonstrating that female is the homogametic sex in this species. It should therefore be possible to produce all-female populations of Atlantic halibut for commercial culture through gynogenesis or by crossing hormonally masculinized genotypic females to normal females.

A gynogenetic screen to isolate naturally occurring recessive mutations in Xenopus tropicalis

In the rapidly developing, diploid amphibian Xenopus tropicalis, genetics can be married to the already powerful tools of the amphibian system to overcome a disability that has hampered Xenopus laevis as a model organism: the difficulties inherent in conducting genetic analyses in a tetraploid organism with a longer generation time. We describe here a gynogenetic screen to uncover naturally occurring recessive mutations in wild X. tropicalis populations, a procedure that is both faster and easier than conventional genetic screens traditionally employed in model organisms to dissect early developmental pathways. During the first round of our screen, gynogenetic diploids from over 160 females comprising four different wild-caught populations were examined. Forty-two potential mutant phenotypes were isolated during this round of gynogenesis. From this group, we describe 10 lines that have genetically heritable recessive mutations. A wide range of developmental defects were obtained in this screen, encompassing effects limited to individual organs as well phenotypes characterized by more global changes in tadpole body morphology. The frequency of recessive mutations detected in our screen appears lower than that seen in other vertebrate genetic screens, but given constraints on the screening procedure used here, is likely to be consistent with rates seen in other animals, and clearly illustrates how wild-caught animals can be a productive source of developmental mutations for experimental study. The development of genetic strategies for the Xenopus system, together with new genomic resources, existing technologies for transgenesis, and other means for manipulating gene expression, as well as the power of performing embryonic manipulations, will provide an impressive set of tools for resolving complex cell and developmental phenomena in the future.

Sex ratios of triploids and gynogenetic diploids induced in the hybrid sturgeon, the bester ( Huso huso female× Acipenser ruthenus male)

The sex ratios of triploids and gynogenetic diploids in the hybrid sturgeon, the bester ( Huso huso female× Acipenser ruthenus male), have been investigated to confirm the possibilities of sex control by chromosome manipulation in sturgeon. Viable gynogenetic diploids were produced by activating eggs with ultraviolet-irradiated bester sperm followed by heatshock treatment. Triploids were induced by the same method after fertilization of eggs with normal bester sperm. A total of 380 progeny individuals were analyzed by flow cytometry, and classified into 217 diploids (2n) and 163 triploids (3n). The percentages of females are 40–55% in control diploid, 60.0–73.3% in triploid and 70.0–80.0% in gynogenetic groups. Since these progeny reared under same environmental conditions, the presence of males in gynogenetic progeny groups derived from five different pairs suggested the involvement of a female heterogametic {ZW (female)/ZZ (male)} sex-determination system in bester. The percentage of males in each groups most closely approximated the percentages expected when the sex-determining element on the W chromosome segregates independently of the centromere during meiosis in females. If the WW “super”-female is viable in sturgeon, such females would be produce all female progeny when crossed to normal ZZ males. Moreover, inducing gynogenetic diploid in endangered sturgeon population with genetically inactivated “donor” sperm from other sturgeon species is likely to offer a useful method for restoration, since both sexes and not just an all-female population would be predicted.

Induction of gynogenesis in the turbot ( Scophthalmus maximus): : Effects of UV irradiation on sperm motility, the Hertwig effect and viability during the first 6 months of age

Fish in which gynogenesis has been induced have all their chromosomes inherited from the mother and, if females are the homogametic sex, they usually are all females. Because turbot females grow faster than males, the production of all-female populations is highly desirable. The sperm of turbot is of poor quality and its larvae are small and fragile. These circumstances represent a challenge for the induction of gynogenesis in the turbot. As a first step towards this goal, effective conditions for the induction of gynogenesis through UV irradiation of sperm followed by a cold shock were established. When diluted 1:10 with Ringer-200 saline solution and placed in a thin layer (∼0.3 mm), a dose-dependent effect of UV light on sperm motility was found. The dose at which both the amount of motile sperm and the duration of sperm motility was reduced to 50% of the original value (ID 50) was ∼28,000 erg mm −2. A typical Hertwig effect was elicited with a dose of 30,000 erg mm −2. The resulting embryos exhibited the typical “haploid syndrome” and died shortly after hatching. Application of a cold shock (−1 to 0°C for 25 min starting at 6.5 min after fertilization) to activated eggs with UV-irradiated (30,000 erg mm −2) and diluted (1:10) sperms restored diploidy and resulted in the production of gynogenetic diploids (2 n=44 chromosomes). These conditions were used in a pilot-scale experiment and found effective in inducing gynogenesis in ∼47,000 eggs. The rate of gynogenesis induction was 100% as verified by an analysis with microsatellite DNA markers. Survival of the gynogenetics was approximately 10% of diploids at 6 months of age, although growth was similar during this period. If this species turns out to have female homogamety, as is the case in most pleuronectiformes examined so far, the method presented here is the first necessary step for the production of all-female populations of this economically important species.

Induction of diploid gynogenesis in southern flounder ( Paralichthys lethostigma) with homologous and heterologous sperm

Effective methods for induction of diploid gynogenesis in North American flounder of the genus Paralichthys are needed to initiate monosex culture, which will allow growers to take advantage of the more rapid growth and larger size attained by females. To test methods for inducing diploid gynogenesis in southern flounder ( Paralichthys lethostigma) using homologous sperm, four treatments, named for their expected outcome, were employed: haploid, diploid, triploid, and gynogenetic diploid. Diploid gynogenesis was induced by activating egg development with UV-irradiated flounder sperm (70 J/cm 2) for 3–4 min in seawater, and then subjecting the eggs to cold shock in 0–2 °C seawater for 45–50 min. Cold shock was used to prevent extrusion of the second polar body. Control treatments omitted one or more of these steps to separately assess the effectiveness of UV irradiation and cold shock. Larvae were observed for physical abnormalities and then histologically processed for ploidy determination. Haploid larvae exhibited abnormal external morphology while diploid, gynogenetic diploid, and triploid larvae showed normal morphologies. Cross-sectional areas of erythrocyte nuclei were measured for larvae in each treatment group and significant differences were found. Nuclear areas for treatment groups corresponded to predicted ploidy (triploid>diploid>haploid) and did not differ between normal diploid controls and gynogenetic diploids. These results suggest that the procedures of sperm irradiation and egg cold shock successfully generated gynogenetic diploids. Due to the low volumes of semen produced by male flounder, and to eliminate any potential genetic contribution by homologous sperm, activation of flounder eggs with heterologous sperm was also investigated. Induction of diploid gynogenesis was successful when flounder eggs were fertilized with irradiated (50 J/cm 2) sperm from striped mullet ( Mugil cephalus), and then cold shocked. This work provides procedures for induction of diploid gynogenesis in southern flounder using homologous and heterologous sperm, and validates a method for verification of ploidy in larval fish.

Induction of gynogenetic diploids and cytological studies in the zhikong scallop,Chlamys farreri

The induction of gynogenetic diploids during meiosis II in the scallop Chlamys farreri was attempted using the treatments of cytochalasin B (0.5 μg ml−1; CB) and 6-dimethylaminopurine (60 μg ml−1; 6-DMAP). The effects of CB and 6-DMAP treatments on meiosis and fertilization of eggs were also examined. Haploid gynogenesis was induced by sperm which were ultraviolet-irradiated for 30 s at an intensity of 2561 μW cm−2 s−1. CB and 6-DMAP treatments were highly effective in suppressing meiosis II, yielding 19% and 28% gynogenetic diploids, respectively. Compared with the CB treatment, the 6-DMAP treatment produced more expected D-shaped larvae. Cytological observations revealed that 6-DMAP inhibited pronuclear movements and the segregation of chromosomes, resulting in the formation of one big diploid female pronucleus with strong fluorescence, while CB inhibited cytokinesis, resulting in two female pronuclei formations. The UV-irradiated sperm nucleus developed into a male pronucleus, but did not participate in karyokinesis at mitotic anaphase, suggesting that gynogenetic diploids were induced in the scallop.

An efficient method for blocking the 1st mitotic cleavage of fish zygote using combined thermal treatment, exemplified by mud loach ( Misgurnus mizolepis)

Blocking the first mitotic cleavage of the zygote is a key tool for chromosome-set manipulations in fish. We developed an improved method for inducing tetraploidy by blocking the mitosis with a combination of heat shock at 40.5 °C for 1, 2, or 3 min followed by cold shock at 1.5 °C for 30, 45, or 60 min. When applied during the first cleavage metaphase of mud loach ( Misgurnus mizolepis) zygotes, the optimal combination was heat for 2 min followed by cold for 45 min. At 1 month, the frequency of 4N survivors and the yield from total eggs fertilized was 55.7 and 14.4%, respectively, compared to heat shock alone with 20.0% efficiency and 3.6% yield. The effectiveness of the procedure was confirmed by diploid mitotic gynogenesis using transgenic markers. The overall yield of homozygous diploids, 34.0%, was better than that for single heat shock, 17.3%. The tetraploids and homozygous diploids had higher early mortality than normal diploid controls. However, at 1 month, the viability of the tetraploids was the same as normal diploids. For gynogenetic diploids, the survival was similar to normal diploids after 3 months. The high efficiency of this new protocol extends the opportunity to study polyploidy in basic and applied research.

Production of cloned red sea bream, Pagrus major, by chromosome manipulation

Red sea bream, Pagrus major, is one of the most important fish cultured in Japan. Two clones of red sea bream were produced. Eggs from a mitotic gynogenetic diploid (mitotic-G2N) red sea bream were inseminated either with sperm from a mitotic-G2N male to produce a heterozygous clone (hetero-clone), or with UV-irradiated sperm of Japanese parrot fish ( Oplegnathus fasciatus) and the second meiotic division suppressed by cold shock to produce a homozygous clone (homo-clone). Normal diploids were also produced from one male and female as a control. The clonal status of the fish was confirmed by multilocus DNA fingerprinting. The fingerprinting patterns differed between individuals within the normal diploids. However, there was no variation between individuals within hetero- or homo-clones. The patterns of the homo-clones and the mother were identical, and all the bands of homo-clones were also observed in hetero-clones. Thus, the clonal status of homo- and hetero-clones was confirmed and the production of clones from the broodstock of mitotic-G2N was achieved. The hatching rates, survival rates and growth of the hetero- and homo-clones were recorded for a brief comparison with results of diploid controls.

Histological observations of gonadal development in gynogenetic diploids and triploids of a hybrid sturgeon, bester

Histological observations of gonadal development in gynogenetic diploids and triploids of a hybrid sturgeon, bester

Study on embryonic development and early growth of triploid and gynogenetic diploid left-eyed flounder,Paralichthys olivaceus (T. et S.)

The early effects of chromosomal manipulation of eggs and sperm on the yields of triploid and gynogenetic diploid larvae ofParalichthys olivaceus were investigated Triploidy was achieved by cold shocking fertilized eggs at 0–2°C for 45 minutes duration 5 minutes after, fertilization, and the induced triploidy rates were 31.2%–50% and the relative hatching rates were 53.3%–99%. Gynogenetic diploids were obtained when eggs were inseminated with irradiated sperm and cold shocked at 0–2°C for 45 minutes duration 5 minutes after fertilization. The induced gynogenetic diploid rates and the relative hatching rates were 94%–96% and 48.5%–68.5% respectively. The embryonic development of the triploid experimental group and of the gynogenetic diploid experimental group was delayed at first compared with the control group. But from the gastrula stage, it was not delayed anymore. There were no significant differences in the growth of the triploid experimental group larvae and the control group larvae, and in the growth of the gynogenetic diploid experimental group larvae and the control group larvae according to Student’s t-test (α=0.05). The relationship between the early growth of the triploid experimental group larvae and that of gynogenetic diploid experimental group larvae was also studied.

A Microsatellite Linkage Map of Rainbow Trout (Oncorhynchus mykiss) Characterized by Large Sex-Specific Differences in Recombination Rates

We constructed a genetic linkage map for a tetraploid derivative species, the rainbow trout (Oncorhynchus mykiss), using 191 microsatellite, 3 RAPD, 7 ESMP, and 7 allozyme markers in three backcross families. The linkage map consists of 29 linkage groups with potential arm displacements in the female map due to male-specific pseudolinkage arrangements. Synteny of duplicated microsatellite markers was used to identify and confirm some previously reported pseudolinkage arrangements based upon allozyme markers. Fifteen centromeric regions (20 chromosome arms) were identified with a half-tetrad analysis using gynogenetic diploids. Female map length is approximately 10 M, but this is a large underestimate as many genotyped segments remain unassigned at a LOD threshold of 3.0. Extreme differences in female:male map distances were observed (ratio F:M, 3.25:1). Females had much lower recombination rates (0.14:1) in telomeric regions than males, while recombination rates were much higher in females within regions proximal to the centromere (F:M, 10:1). Quadrivalent formations that appear almost exclusively in males are postulated to account for the observed differences.

The relationship between the effects of UV light and thermal shock on gametes and the viability of early developmental stages in a marine teleost fish, the sea bass (Dicentrarchus labrax L.)

To improve the efficiency of gynogenetic induction, the effects of UV light and thermal shock on gametes were investigated in the sea bass. Exposure of sperm to UV light (>/=15 000 erg mm-2) reduced the amount of motile spermatozoa, without affecting the duration of motility in the spermatozoa that remained motile. The Hertwig effect was elicited in eggs fertilized with sperm exposed to >/=35 000 erg mm-2 of UV light, indicating the inactivation of the DNA of the spermatozoa while retaining their ability to trigger development. Resulting embryos (24 chromosomes; one NOR) exhibited the haploid syndrome and died at hatch. Diploidy was restored in eggs fertilized with irradiated sperm by blocking meiosis II with a thermal shock (0 +/- 0.5 degrees C for 10 min, starting 5 min after fertilization). Resulting larvae ( approximately 35% survival at hatching) had 48 chromosomes, one or two NOR and no paternal chromosome fragments (gynogenetic diploids). In eggs fertilized with sperm not exposed to UV light, the same thermal shock induced 100% triploidy (72 chromosomes; one, two or three NOR), with 70% survival at hatching. Multifactorial ANOVA showed that, compared to external factors (sperm diluent, UV light and thermal shock), the contribution of broodstock to the viability of the early developmental stages was not significant (P > 0.05). Effects of the thermal shock were most evident after fertilization (30.7%) but disappeared (0%) at hatching, suggesting that the lower survival of triploids is a consequence of handling, not of the triploid condition per se. However, effects of UV light increased through development (42.5-69.7%), probably reflecting cumulative deficits in protein synthesis.

Studies on sex-manipulation and production of cloned populations in hirame, Paralichthys olivaceus (Temminck et Schlegel)

Hirame ( Paralichthys olivaceus) is an important cultured flatfish species in Japan, Korea and China. Because females grow faster, methods of feminized culture were developed. Sex ratios were examined in gynogenetic diploids and normal diploids and their progenies in order to clarify genetic sex-determination and enable its artificial manipulation. It was revealed that this species has male heterogametic sex-determination. However, sex-differentiation of genetic females (XX) seems to be so unstable that spontaneous sex-reversal to physiological males can be caused by exogenous factors, especially high water temperature. Manipulating techniques for physiological as well as genetic sex were established using sex-steroids and/or controlling rearing water temperature. Homozygous clones were produced by the combination of inducing mitotic gynogenetic diploids and meiotic gynogenetic diploids over two generations. More vigorous heterozygous clones were produced by hybridization between different clones. These appear to be good material for fish breeding and commercial fish culture.

Sex-determining mechanism in Buergeria buergeri (Anura, Rhacophoridae). III. Does the ZZW triploid frog become female or male?

Both triploids and gynogenetic diploids (GDs) were produced to clarify the relationship between the sex-chromosome constitution and the expression of sex in the common bell-ring frog, Buergeria buergeri. The sex differentiation of triploids in B. buergeri is quite remarkable. Triploid frogs consisted of three sex genotypes, ZZZ, ZWW and ZZW. All ZZZ triploids were males, and all ZWW triploids were females. It is very interesting that half of the ZZW triploids became female, and the other half became male. The GD frogs consisted of two sex genotypes, ZW and ZZ, which did not differ from the controls in sex differentiation. Since the ratios of ZZ and ZW eggs were significantly different among female parents, it is assumed that most (approximately 80-90%) of the eggs made pre-reductional division in some females and post-reductional division in others during meiosis. It seems that ZW eggs were produced by the occurrence of recombination between the centromere and the sex-determining genes in B. buergeri. It was also found that the number of Z chromosomes in each cell of these triploids and GDs agreed with that of the nucleoli in each cell.

Survival, growth and sex ratios of gynogenetic diploid honmoroko

Survival, growth and sex ratios of gynogenetic diploid honmoroko Gnathopogon caerulescens induced by blocking the release of the second polar body were examined. Mean survival of gynogenetic juveniles at 130 days after hatching was about 33% lower than that of the controls. No significant difference was seen in early growth between control and gynogenetic diploids. Standard length and body weight in six groups of gynogenetic progeny were significantly greater but in two groups were significantly smaller than in the controls. Although 69% of gynogenetic diploids had well-developed gonads, the remaining 30% had undeveloped gonads (small in size or thread-like), and those gonads were divided into four types. The mean proportion of females in the 10 gynogenetic groups was 87·2% which was significantly (P <0·01) higher than in the controls (44·7%). Gynogenetic diploids included 3·0–35·3% males. Most of those males produced a high proportion of female progeny, but the proportion of male offspring varied widely. From these results, the sex determining mechanism in honmoroko was presumed to be female homogamety, but other factors resulted in the production of males.

Survival, growth and sex ratios of gynogenetic diploid honmoroko

Survival, growth and sex ratios of gynogenetic diploid honmoroko Gnathopogon caerulescens induced by blocking the release of the second polar body were examined. Mean survival of gynogenetic juveniles at 130 days after hatching was about 33% lower than that of the controls. No significant difference was seen in early growth between control and gynogenetic diploids. Standard length and body weight in six groups of gynogenetic progeny were significantly greater but in two groups were significantly smaller than in the controls. Although 69% of gynogenetic diploids had well‐developed gonads, the remaining 30% had undeveloped gonads (small in size or thread‐like), and those gonads were divided into four types. The mean proportion of females in the 10 gynogenetic groups was 87·2% which was significantly (P &lt;0·01) higher than in the controls (44·7%). Gynogenetic diploids included 3·0–35·3% males. Most of those males produced a high proportion of female progeny, but the proportion of male offspring varied widely. From these results, the sex determining mechanism in honmoroko was presumed to be female homogamety, but other factors resulted in the production of males.

Complete interference and nonrandom distribution of meiotic crossover in a mollusc, Mulinia lateralis (Say).

In gene mapping, the genetic distance between two genes is measured by the frequency of meiotic crossovers occurring between them. It is generally assumed that there is more than one crossover per chromosome, and the distribution of crossovers along chromosomes is random and follows a Poisson distribution (no interference), or that interference is inversely correlated with distance. Although those assumptions may be correct for some organisms, we report here a novel exception in the marine mollusc Mulinia lateralis Say. Using segregation analysis of gynogenetic diploids, we found surprisingly high gene-centromere recombinant frequencies for most of the 13 allozyme loci studied. For at least six loci, there was always one and only one crossover occurring between the gene and its centromere, suggesting complete interference where the occurrence of one crossover completely suppressed the occurrence of another. The complete interference was confirmed by the cytogenetic observation that there was only one chiasma for all bivalents. Further, sites of the single crossover seem not to be randomly distributed along chromosomes, but preferentially located in a recombination hot-region proximal to the centromere. The restricted distribution of a single crossover per chromosome provides one explanation for the unique phenomenon of heterozygote deficiency in M. lateralis and other molluscs.

Optimization of shock parameters in inducing mitotic gynogenesis in the carp Cyprinus carpio L.

The relative effects of heat-shock parameters on the induction of first-cleavage suppression of common carp (Cyprinus carpio L.) eggs were examined. Eggs of females with scattered scales were fertilized with sperm that had been irradiated with gamma-ray cobalt-60. Fertilized eggs were exposed to heat shock at different temperatures (39, 40, 41 °C) for periods of 1–3 min at 0.5-min intervals, initiated at 41, 44, or 47 min post fertilization in a factorial design. The use of three treatment factors made it possible to analyze the main effects and all possible combinations of the three factors. The highest percentage of mitotic gynogenetic diploids (mitotic-G2n) (up to 10.6% survival at the feeding larva stage relative to the total number of inseminated eggs) was obtained from the groups treated at 40 °C for 2 min at 44 min post fertilization (ambient temperature 21 °C). Optimum duration was observed to be 2.5–3 min at 39 °C and 1–1.5 min at 41 °C. The embryos were developed under hatchery condition, while larvae were reared in a flow-through system with natural feeding. Isozyme and scale pattern analysis performed on putative mitotic-G2n individuals both proved that their genome is of maternal origin.

Suppression of Meiotic and Mitotic Cell Divisions in Nile tilapia, Oreochromis niloticus L., and Induction of Triploids and Two Types of Gynogenetic Diploids

Studies were conducted to interfere with the normal functioning of spindle apparatus during meiotic and mitotic cell divisions in Nile tilapia, Oreochromis niloticus L., eggs using both temperature and hydrostatic pressure shock treatments, therefore, leading to the induction of triploids and two types of gynogenetic (meiotic and mitotic) diploids.

Present state of common carp ( Cyprinus carpio L.) stocks in Indonesia

The common carp ( Cyprinus carpio L.) stocks in Indonesia are descendants of common carp brought from China, Europe, Taiwan and Japan. The Punten carp and Majalaya carp are the result of selection conducted in Indonesia. Based on morphological characteristics, there are at least 10 common carp stocks known to date. Some stocks can be easily distinguished by observing their morphological characteristics, e.g. Majalaya, Sinyonya, Domas, Kancra-domas, red (merah), mirror, long-fin (Kum-pay), and koi. The Punten and Taiwan carp stocks are relatively difficult to identify morphologically. In the field, uncontrolled breeding systems for common carp stocks result in mixed stocks. Purification of the stocks has been conducted since 1989. The second generation of gynogenetic diploids of some stocks has been obtained. Experiments of sex-reversal to change genotype females to functional males are being conducted.