Gynogenetic Progeny Research Articles

First successful inducing gynogenesis in Crassostrea angulata by inactivated tetraploid Crassostrea gigas sperm

Artificial induction of gynogenesis is widely practiced in aquatic animals. However, there have been few reports of obtaining adult gynogenetic marine bivalves. This study established a new and effective method for inducing gynogenesis in C. angulata using inactivated tetraploid C. gigas sperm, ultimately obtaining gynogenetic diploids. The optimal induction conditions were as follows: sperm were irradiated with ultraviolet light (254 nm) at an intensity of 1035 μW/cm2 for 60 s before fertilization with the eggs. Fifteen minutes post-fertilization, zygotes were treated with cytochalasin B (0.5 mg/L) for a duration of 20 min under a condition of 28 °C. Six different ploidy levels (2n, 3n, 4n, 5n, 6n and aneuploid) of larvae were found by karyotype analysis and flow cytometry methods, with a diploid rate of 63.57 ± 2.80 % and an induction efficiency of 7.47 ± 0.33 %. At 6 months after fertilization, induced progenies of 4 different ploidy levels (2n, 3n, 4n and aneuploid) were examined from 3 replicate groups. The proportions of diploids, triploids, tetraploids and aneuploids were 31.67 ± 3.68 %, 57 ± 3.74 %, 7.67 ± 0.94 % and 3.67 ± 0.94 %, respectively. Diploidy was determined by microsatellite analysis, which did not contain any paternal genes, suggesting them as true gynogenetic progenies. The growth traits (shell length, width, height and whole weight) of the gynogenetic diploids did not show any significant difference (P < 0.05) compared to the control diploids. Meanwhile, triploids demonstrated a significant advantage in growth. Gynogenetic diploids underwent sexual differentiation and consisted of females (31.40 ± 1.52 %), males (67.67 ± 2.75 %), and hermaphrodites (0.93 ± 1.31 %). Interestingly, the gonads of diploids developed well and produced functional gametes. This research provides robust technical support for chromosome engineering breeding and the creation of pure lines of bivalves.

The analysis of growth performance and expression of growth-related genes in natural gynogenic blunt snout bream muscle derived from the blunt snout bream (Megalobrama amblycephala, ♀) × Chinese perch (Siniperca chuatsi, ♂)

Gynogenesis is an advanced breeding technique that can quickly establish pure lines, increase progenies genetic variation, cultivate strains with higher dominant traits. As a good model for cross order species hybridization, there are few reports on the study of natural gynogenetic blunt snout bream (GBSB), even on the differences in growth between gynogenesis progenies and their parent fish (blunt snout bream♀ and Chinese perch♂). In this research we compared the growth characteristics of the natural GBSB with its parents. It was found that GBSB had a faster growth rate and higher meat content than its female parent, and the diameter and density of muscle fibers were between those of paternal and maternal fish. We performed transcriptome sequencing of muscle tissues from GBSB and BSB, it identified 1353 significant DEGS in the muscle of the GBSB compared to the BSB, with 820 up- and 533 down-regulated genes. And the relative expression of myosins was notable rise in GBSB. In addition, the MRFs of gene structure, motifs, and phylogenetic relations were characterized in BSB and CP. It showed that MRFs and mstn central domain was highly conserved, but the number of conservative motifs in MRFs of CP is generally higher than that of BSB. Further, the relative expression of myosin complex, myogenic regulatory factors (MRFs), growth hormone/insulin-like growth factor (GH/IGF)-axis and most hypothalamic-pituitary-gonadal (HPG)-axis genes in GBSB were higher than that of the female fish and some were closer to that of the paternal fish. Through the distant hybridization between BSB and CP, a gynogenetic progeny with enhanced growth rate compared to the maternal fish was successfully generated. The research has contributed significant insights into the genes and molecular properties associated with muscle development, as well as their potential functional correlations in response to different functional requirements during the growth process of hybrid progeny formed by cross breeding.

Conservation of Vulnerable Sterlet Sturgeon, Acipenser ruthenus Using Artificial Gynogenesis

Sterlet sturgeon has been identified as a vulnerable species in the IUCN criteria, which is currently lack in most propagation and rehabilitation centers of Iran. This study was conducted with the aim of creating male sterlet sturgeon using gyneogenesis induction without using semen of this sturgeon. Siberian sturgeon, Acipenser baerii sperm was used in this study. To inactivate the sperm DNA, 473 μW/cm2 UV irradiation was used for 90 seconds to maintain sperm motility for the fertility of the ovum. For diploidization of activated eggs, 34°C heat shock was used for 2 minutes, 15 minutes after activation. In this case, fertilization and hatching rate in the gynogenetic progeny were 59.5% and 41.6%, respectively. Sterlet sturgeon gynogenetic progeny was confirmed using microsatellite markers (AFU68 and AFUG9), which had only maternal heritability. Gynogenetic progeny was analyzed by histology of gonads at the age of 17 months. The results showed that both male and female sexes of sterlet sturgeon were created, but these progenies had different sex ratios.

Structural Abnormalities of Spermatozoa in Triploid Gynogenetic Crucian Carp (Carassius auratus).

The spermatozoa of triploid gynogenetic crucian carp (Carassius auratus) (3nDTCC) possess a spermatogenesis process with a normal genetic background. However, the genetic materials of these spermatozoa do not completely inherit gynogenetic progeny in general. Understanding the intrinsic mechanism may be helpful for developing breeding strategies of gynogenetic fishes. In this study, the spermatozoa ultrastructure was systematically studied in diploid red crucian carp and 3nDTCC to demonstrate their cytological structural differences. In addition, the artificial breeding tests of 3nDTCC(♀) with different ploidy spermatozoa were performed to verify the contributions of genetic materials from 3nDTCC spermatozoa to the gynogenesis progeny. Furthermore, the mRNA expression of centriole-related genes (i.e., cep57, cetn1, rootletin, and nek2) involved in spermatozoa packaging was also determined by quantitative real-time PCR (qPCR) to illustrate the molecular expression characteristics of the spermatozoa packaging process in 3nDTCC. The results reveal the adaptive features of spermatozoa in 3nDTCC, including the loose midpiece structure, abnormal head structure, and abnormal expression of centriole-related genes, which may influence the motility of spermatozoa and make it not involved normally in the genetic composition of the gynogenesis offspring.

Gonadal histology and concentration of 11-ketotesterone of meiotic gynogens confirm female heterogametic sex determination in sterlet (Acipenser ruthenus)

Gynogenetic progeny of sterlet Acipenser ruthenus were obtained by inseminating eggs with UV-inactivated sperm followed by meiotic restoration of ploidy by heat shock. Gynogenetic origin of progeny was assessed by parentage assignment using 15 microsatellite markers. Sex ratio was investigated by gonad histology in 15 3-year-old fish, eight of which were confirmed gynogenotes, with five of those containing primary oocytes (62.5%). Blood plasma concentration of 11-ketotestosterone (KT-11) was analyzed in 63 4-year-old juveniles, 46 of which were genetically confirmed gynogenotes. Measurement of KT-11 showed the proportion of female sterlet to be 65.2%. This sex ratio conformed to the current hypothesis of female heterogametic sex determination but was at the low end of the reported female ratio in sturgeon gynogenetic progeny.

Production and verification of the first Atlantic salmon (Salmo salar L.) clonal lines

BackgroundIn several fish species homozygous and heterozygous clonal lines have been produced using gynogenetic and androgenetic techniques. These lines are standardized and can be reproduced over generations. In rainbow trout such lines have existed for decades and has become important research tools in genome studies as well as in studies of commercially important traits. The Atlantic salmon is one of the best studied fish species globally, but all experiments are done on fish of wild or domesticated origin and access to standardized immortal fish lines would be of great benefit. Here, we describe the protocols developed to produce mitotic gynogenes, and from these the first clonal lines in Atlantic salmon.ResultsAtlantic salmon eggs fertilized with UV irradiated sperm combined with a pressure shock applied at 4700–4800 minC at 8 °C gave all homozygous (doubled haploid) gynogenetic progeny with high survival. From the six first maturing females, five all homozygous clonal lines were produced by meiotic gynogenesis and were verified as clonal and identical to their mother with microsatellite markers.ConclusionsWe have now produced the first documented cloned Atlantic salmon lines. This work demonstrates the potential for production of further Atlantic salmon clonal lines, potentially with distinct characteristics. Such lines will provide an important resource for further elucidation of phenotypic and genetic traits in this globally important species.

Further evidence for paternal DNA transmission in gynogenetic grass carp.

Gynogenesis is an important breeding method in aquaculture and has been widely applied to many fish species. If gynogenetic progenies are to inherit paternal partial genomic DNA, this will increase genetic variation and will provide a useful outcome for breeding. In this study, we investigated the genetic variation in homeobox (Hox) gene clusters (HoxA4a, HoxA9a, HoxA11b, HoxB1b, HoxC4a, HoxC6b, and HoxD10a) among koi carp (Cyprinus carpio haematopterus, KOC; the stimulation sperm source), grass carp (Ctenopharyngodon idellus), and gynogenetic grass carp (GGC). We found paternal DNA (a special DNA fragment and HoxC6b) derived from KOC and a recombinant gene belonging to HoxC6b in GGC. We are the first to report the recombinant HoxC6b in GGC. Our study provides further evidence for paternal DNA transmission to gynogenetic progenies, which is a finding with great significance for the genetic breeding of fish.

The first case of induced gynogenesis in Neotropical fishes using the yellowtail tetra (Astyanax altiparanae) as a model organism

Gynogenesis is a chromosome manipulation technique applicable on the production of all-female populations, generation of inbred or clonal lines and identification of genetic sex determination mechanisms in fish. Therefore, the aim of this study was induce meiotic gynogenesis in Astyanax altiparanae and determine the genetic basis of sex determination. Firstly, we evaluated the best UV-irradiation dosage in order to inactivate sperm genome, preserving the capacity of egg activation without the genetic material contribution to the offspring. After the fertilization using the inactivated sperm, diplodization was achieved by heat-shock (40 °C; 2 min) by suppression of the 2nd polar body extrusion. Diploid control and gynogenetic individuals were raised until 240 days. The gynogenetic offspring were confirmed by genotyping using microsatellite markers and the sex ratio of gynogenetic progenies suggests that most data fit to a XX/XY model of sex determination. However, unexpected sex ratios were also observed and possible reasons are discussed. This was the first protocol of induced gynogenesis in a Neotropical fish and such a procedure may be used to produce clonal linage of fish and for future mass production of monosex populations in A. altiparanae and other related species.

Meiotic gynogenesis with heterologous sperm in the mandarin fish Siniperca chuatsi and evidence for female homogamety

The mandarin fish Siniperca chuatsi is a historically important aquaculture species in China and exhibits sexually dimorphic growth. However, sex determination of this fish remains unclear so far. In this study, we induced meiotic gynogenesis in S. chuatsi using irradiated heterologous sperm from spotted mandarin fish (Siniperca scherzeri) to uncover its mechanism of sex determination. Up to 7.52% diploid progeny were obtained among three gynogenetic families in this study. Molecular analysis of female and male donors and sampled young gynogens by seven microsatellite loci further confirmed no genetic contributions from the ‘father’ S. scherzeri. After 8 months of culture, external morphology of adult fish showed that all gynogens were cloned from their mothers. Gonads of the gynogenetic progeny were examined by histological observations and the sexing results showed that they were almost 100% females, strongly supporting an assumption of female homogamety in mandarin fish. By this study, we obtained pure lines of S. chuatsi and elucidated its genetic mechanism of sex determination, providing a basis for possible sex control breeding in this species.

Origin and transition of sex determination mechanisms in a gynogenetic hexaploid fish.

Most vertebrates reproduce sexually, and plastic sex determination mechanisms including genotypic sex determination (GSD) and environmental sex determination (ESD) have been extensively revealed. However, why sex determination mechanisms evolve diversely and how they correlate with diverse reproduction strategies remain largely unclear. Here, we utilize the superiority of a hexaploid gibel carp (Carassius gibelio) that is able to reproduce by unisexual gynogenesis and contains a rare but diverse proportion of males to investigate these puzzles. A total of 2248 hexaploid specimens were collected from 34 geographic wild populations throughout mainland China, in which 24 populations were revealed to contain 186 males with various incidences ranging from 1.2 to 26.5%. Subsequently, the proportion of temperature-dependent sex determination (TSD) was revealed to be positively correlated to average annual temperature in wild populations, and male incidence in lab gynogenetic progenies was demonstrated to increase with the increasing of larval rearing temperature. Meanwhile, extra microchromosomes were confirmed to play genotypic male determination role as previously reported. Thereby, GSD and TSD were found to coexist in gibel carp, and the proportions of GSD were observed to be much higher than that of TSD in sympatric wild populations. Our findings uncover a potential new mechanism in the evolution of sex determination system in polyploid vertebrates with unisexual gynogenesis ability, and also reveal a possible association of sex determination mechanism transition between TSD and GSD and reproduction mode transition between unisexual gynogenesis and bisexual reproduction.

Presence of gynogenetic males suggests a female heterogamety in sterlet Acipenser ruthenus L.

Investigation of the heterogametic sex in sterlet Acipenser ruthenus L. was performed using meiotic gynogenesis and gonadal histology. Eggs from the albino females were activated by UV irradiated sperm of wild colored males and exposed to a heat shock. The resultant fish were all albino and exhibited exclusively maternal inheritance of the microsatellite DNA markers. Cytogenetic analysis indicated that gynogenetic progeny were diploids with 120 chromosomes. Based on the histological analysis, more than 86% of the gynogenetic individuals were found to be females. Moreover, some males (7%), sterile speciemens (3.5%) and fish with unidentified gonads (3.5%) were observed among the gynogenetic fish. Presence of both females and males in the gynogenetic offspring is indicative that the heterogametic sex in sterlets is female.

Fertility of triploid hybrids of Prussian carp (Carassius gibelio) with common carp (Cyprinus carpio L.)

Fertility of backcross triploid hybrids containing one genome of Prussian carp and two genomes of common carp is investigated. The females of hybrids of Prussian carp and common carp (Prussian × common carp) are prolific and produce diploid gametes. Since males of such hybrids are sterile, their reproduction is realized by means of induced gynogenesis. Triploid progeny is obtained by backcrossing female Prussian × common carp with carp males. Among triploids obtained from hybrids F1 and among hybrids of the first gynogenetic generation, there were no prolific specimens. However, in reproduction of diploid hybrids by means of gynogenesis during six generations, the female fertility in the backcross progeny is restored. From backcross triploid females (daughters of Prussian × common carp of the sixth gynogenetic generation), a viable triploid gynogenetic progeny and a tetraploid backcross (by carp) progeny are obtained. The obtained data may be considered as the experimental proof of the hypothesis of reticular speciation.

Production of Tetraploid Gynogenetic Loach Using Diploid Eggs of Natural Tetraploid Loach, Misgurnus anguillicaudatus, Fertilized with UV-Irradiated Sperm of Megalobrama amblycephala without Treatments for Chromosome Doubling

The gynogenesis phenomenon in nature mainly appears in the reproduction of fish and invertebrates. So far, gynogenesis has been successfully induced in many fish species with the aid of some physical or chemical methods for chromosome doubling. However, few fish can produce gynogenetic progenies, genetically identical or similar to the somatic cells of the mothers, without a treatment for the doubling of chromosomes, which may be related to apomixis, premeiotic endoreduplication, or premeiotic endomitosis. At present, no studies are available about fish with normal ovarian structures producing gynogenetic progenies that could spontaneously double their chromosomes. According to the analyses of flow cytometry, chromosome number, and microsatellites, we found that, with the use of UV-irradiated sperm of blunt snout bream Megalobrama amblycephala, tetraploid loach Misgurnus anguillicaudatus produced tetraploid gynogenetic progenies without any treatments for the doubling of chromosomes. To determine the genetic relationships of gynogenetic progenies and their maternal parent, microsatellite genotyping was conducted. The results indicated that the reason for spontaneous chromosome duplication in gynogenetic progenies may be cytokinesis or inhibition of the extrusion of the second polar body. This is the first report on fish with normal ovarian structures that can produce gynogenetic progenies which spontaneously double their chromosomes and which are genetically identical or similar to the somatic cells of the mothers.

Application of The UV-Irradiated Homologous and Heterologous Sperm for Activa Tion of the Rainbow Trout (Oncorhynchus Mykiss) Eggs and Production of the Gynogenetic Stocks

Abstract Meiotic gynogenetic development of the rainbow trout (Oncorhynchus mykiss, Walbaum 1792) was induced in the course of egg activation performed by the UV-irradiated homologous and heterologous (European grayling Thymallus thymallus Linnaeus, 1758) spermatozoa. To recover diploidy in the gynogenetic zygotes, activated eggs were subjected to the high pressure shock in order to inhibit extrusion of the second polar body. Gynogenetic rainbow trout progeny hatched from the eggs activated by the irradiated rainbow trout and grayling milt with similar hatching rates of 28.19% and 29.22%, respectively. However, gynogenetic rainbow trout produced with grayling semen had shown lower survival than gynogenotes provided with the homologous spermatozoa during two years of rearing. Viable hybrids are not produced between rainbow trout and grayling which ensured that fish obtained in this experiment were true gynogenetic progenies. A Robertsonian polymorphism characteristic for the rainbow trout from the studied strain was also observed among the gynogenetic specimens that exhibited diploid chromosome number ranging from 58 to 62 and stable chromosome arm number (FN= 104). No radiation-induced fragments of the paternal chromosomes were observed in the gynogenetic individuals. Fish produced in both experimental variants were genotypic (XX) and phenotypic (gonads) females. The results confirmed that the gynogenetic protocol used in the present research is an efficient means of producing all-female gynogenetic rainbow trout stocks.

Activation of the Albino Sterlet Acipenser ruthenus Eggs by UV-Irradiated Bester Hybrid Spermatozoa to Provide Gynogenetic Progeny.

Meiotic gynogenesis was induced in the albino form of sterlet Acipenser ruthenus by activation of eggs with UV-irradiated bester (Huso huso x Acipenser ruthenus) spermatozoa followed by inhibition of the second meiotic division performed by a heat shock. Obtained putative gynogenetic progeny were all albinos. The genetic verification based on three microsatellite DNA markers confirmed the only maternal inheritance of the progeny from the gynogenetic experimental groups. Cytogenetic analysis proved the gynogenetic sterlets were diploids. Application of the albino phenotype together with the molecular and the cytogenetic diagnostic approaches enabled to evaluate the efficiency of the spermatozoa irradiation and application of the heat shock to restore diploid state in the gynogenetic zygotes.

Genetic variability in meiotic gynogenetic muskellunge,Esox masquinongy(Mitchell), estimated from segregation of microsatellite alleles

Muskellunge, Esox masquinongy, is an important recreational freshwater fish native to North America. Since muskellunge populations are often maintained through stocking efforts, advances in muskellunge reproductive technologies are of direct relevance to fishery enhancement. We evaluated the efficiency of inbreeding through induced meiotic diploid gynogenesis. Eggs from six female muskellunge were manually stripped and activated using ultra violet-irradiated yellow perch, Perca flavescens, sperm. Hydrostatic pressure shocking regimes (48 263 kPa) were then applied to the eggs to prevent second polar body expulsion producing unambiguous meiotic gynogens. Six female dams and samples of 12–20 of their gynogenetic progeny were genotyped at seven polymorphic microsatellite loci. Chromosomal recombination frequencies of microsatellite loci based on retention of heterozygosity among gynogens ranged from 0.043 to 0.839 (0.576 ± 0.237). There were no statistical differences in recombination frequency among females at any of the loci. The average inbreeding coefficient (F-value) ranged from 0.581 to 0.979, equivalent to three to fourteen generations of full-sibling crosses respectively. The average F-value overall was 0.712, equivalent to between five and six generations of full-sibling crosses. Centromere map distances of the seven microsatellite loci ranged from 2.15 to 41.95 cM and meiotic gynogenesis was useful in eliminating heterozygosity at loci proximal to the centromere, but not distal. Since the age at maturity of female muskellunge is approximately 5 years, gynogenesis may pose an expeditious alternative to traditional breeding strategies for creation of homozygous pedigrees for some loci that may be outcrossed to introduce positive heterozygosity effects in the offspring.

Variability in microsatellite DNA markers in gynogenetic and backcross progenies obtained from ornamental (koi) carp (Cyprinus carpioL.) × goldfish (Carassius auratusL.) hybrid females

Inheritance and segregation at five microsatellite loci were studied in diploid gynogenetic and triploid backcross progenies obtained from koi × goldfish hybrid females, which produce diploid eggs. Gynogenetic and backcross progenies were obtained from three individual hybrid females by inseminating eggs with genetically inactivated and intact sperm of parental species respectively; no shock treatments were applied to the early embryos. Complete absence of paternally specific alleles at all investigated microsatellite loci has proven successful genetic inactivation of spermatozoa by irradiation and confirmed gynogenetic origin of progenies. Genotypic segregations at microsatellite loci showed almost complete homogeneity of gynogenetic progenies and their identity to female parents. These results correspond with previous cytogenetic data on the occurrence of premeiotic endomitosis in hybrid females producing diploid eggs. Fish from triploid backcross progenies had genotypes resulting from combination of entire diploid female genome and haploid genome from male.

Microsatellite-centromere mapping in common carp through half-tetrad analysis in diploid meiogynogenetic families.

Gene-centromere (G-C) mapping provides insights into the understanding of the composition, structure, and evolution of vertebrate genomes. Common carp (Cyprinus carpio) is an important aquaculture fish and has been proposed to undertake tetraploidization. In this study, we selected 214 informative microsatellite markers across 50 linkage groups of a common carp genetic map to perform gene-centromere mapping using half-tetrad analysis. A total of199 microsatellites were segregated under the Mendelian expectations in at least one of the three gynogenetic families and were used for G-C distance estimation. The G-C recombination frequency (y) ranged from 0 to 0.99 (0.43 on average), corresponding to a fixation index (F) of 0.57 after one generation of gynogenesis. Large y values for some loci together with significant correlation between G-C distances and genetic linkage map distances suggested the presence of high interference in common carp. Under the assumption of complete interference, 50 centromeres were localized onto corresponding linkage groups (LGs) of common carp, with G-C distances of centromere-linked markers per LG ranging from 0 to 10.3cM (2.9cM on average). Based on the information for centromere positions, we proposed a chromosome formula of 2n = 100 = 58m/sm + 42t/st with 158 chromosome arms for common carp, which was similar to a study observed by cytogenetic method. The examination of crossover distributions along 10 LGs revealed that the proportion of crossover chromatids was overall higher than that of non-crossover chromatids in gynogenetic progenies, indicating high recombination levels across most LGs. Comparative genomics analyses suggested that the chromosomes of common carp have undergone extensive rearrangement after genome duplication. This study would be valuable to elucidate the mechanism of genome evolution and integrate physical and genetic maps in common carp.

Chromosomal manipulation in Senegalese sole (Solea senegalensis Kaup, 1858): induction of triploidy and gynogenesis.

In this study we have developed protocols for induced triploidy and gynogenesis of Senegalese sole (Solea senegalensis), a promising flatfish species for marine aquaculture, in order to: 1) identify the sex-determination mechanism; and 2) to improve its production by generating a) sterile fish, avoiding problems related with sexual maturation, and b) all-female stocks, of higher growth rate. Triploidy was induced by means of a cold shock. Gynogenesis was induced by activating eggs with UV-irradiated sperm, and to prompt diploid gynogenesis, a cold-shock step was also used. Ploidy of putative triploid larvae and gynogenetic embryos were determined by means of karyotyping and microsatellite analysis. Haploid gynogenetic embryos showed the typical "haploid syndrome". As expected, triploid and gynogenetic groups showed lower fertilization, hatching, and survival rates than in the diploid control group. Survival rate, calculated 49 days after hatching, for haploid and diploid gynogenetic groups was similar to those observed in other fish species (0% and 62.5%, respectively), whereas triploids showed worse values (45%). Sex was determined macroscopically and by histological procedures, revealing that all the diploid gynogenetic individuals were females. In conclusion, we have successfully applied chromosomal-manipulation techniques in the flatfish species Senegalese sole in order to produce triploid, haploid, and diploid gynogenetic progenies.

Evidence of Female Heterogamety in Largemouth Bass, Based on Sex Ratio of Gynogenetic Progeny

AbstractMeiotic gynogenetic progeny in largemouth bass Micropterus salmoides have been obtained by inseminating largemouth bass eggs with UV‐irradiated sperm from white bass Morone chrysops or striped bass Morone saxatilis and suppressing the second meiotic division by hydrostatic pressure. The sex composition of gynogenetic progeny was determined by dissection or ultrasound investigation of 1‐year‐old fish. Among the 21 fish analyzed, 7 fish (33.3%) were male and 14 fish (66.7%) were female. The presence of males in meiotic gynogenetic progeny suggests the existence of female heterogamety (WZ females, ZZ males) in largemouth bass.