Dispermic androgenesis in sturgeons with the help of cryopreserved sperm: production of androgenetic hybrids between Siberian and Russian sturgeons

Dispermic androgenesis was used to produce, for the first time, an androgenetic progeny of the Siberian sturgeon (Acipenser baerii) and the androgenetic nuclear cytoplasmic hybrids (Siberian sturgeon, A. baerii x Russian sturgeon, A. gueldenstaedtii) using cryopreserved sperm. Microsatellite DNA analysis confirmed exclusively paternal inheritance in the androgenetic progeny of Siberian sturgeon. Heterozygotes for certain microsatellite loci were detected among the androgenetic hybrids, thereby confirming a dispermic nature of androgenesis. According to the data of comparative morphological analysis, the obtained androgenetic hybrid, by the age of 15 months old, was completely identical to the paternal species. Both a female and a male were detected in the androgenetic sturgeon progenies, which is of interest for producing bisexual progenies via androgenesis. The data of this study confirm the feasibility of dispermic androgenesis using cryopreserved sperm to preserve and recover the gene pools of endangered sturgeon species.     

Failure of interspecies androgenesis in salmonids

Androgenetic development of salmonid embryos was induced in recipient oocytes from the same or other species (intra- or interspecies androgenesis). Parameters for induced androgenesis were investigated in brown trout Salmo trutta and brook trout Salvelinus fontinalis . Reciprocal androgenetic and control crosses were conducted among fishes from three genera: Oncorhynchus (rainbow trout, O. mykiss ), Salmo (brown trout) and Salvelinus (brook trout), and within two genera: Salmo (brown trout and Atlantic salmon, S. salar ) and Salvelinus (brook trout and Arctic charr, S. alpinus ). Live hatched androgenetic progenies were obtained in all intraspecies variants, where oocytes and sperm originated from the same species. Interspecies androgenesis resulted in no viable larvae, despite the fact that most hybrid controls and intraspecies androgenetic individuals were viable. When recipient oocytes originated from other genera (interspecific intergeneric androgenesis), embryonic development ceased in early developmental stages, except for haploid controls of brook trout produced in eggs of brown trout. Survival of embryos to the eyed-egg stage was relatively high in the intrageneric androgenesis experiment. Nevertheless, none of these embryos survived to hatching. Some of the presumed Atlantic salmon individuals developing in brown trout eggs contained maternal DNA, questioning the accuracy of enucleation using irradiation. The inability to induce interspecific androgenesis among the examined salmonid species may have been the result of substantial kariotypical and developmental differences between spermatozoal donors and oocyte recipients, causing an incompatibility between maternal cytoplasmic regulatory factors and the paternal nuclear genome.     

Dispermic androgenesis in sturgeons with the use of cryopreserved sperm: Production of androgenetic siberian sturgeon and androgenetic hybrids between Siberian and Russian sturgeons

Dispermic androgenesis was used to produce, for the first time, an androgenetic progeny of the Siberian sturgeon (Acipenser baerii) and the androgenetic nucleo-cytoplasmic hybrids (Siberian sturgeon, A. baerii × Russian sturgeon, A. gueldenstaedtii) using cryopreserved sperm. Microsatellite DNA analysis confirmed exclusively paternal inheritance in the androgenetic progeny of Siberian sturgeon. Heterozygotes for certain microsatellite loci were detected among the androgenetic hybrids, thereby confirming a dispermic nature of androgenesis. According to the data of comparative morphological analysis, the obtained androgenetic hybrid, by the age of 15 months old, was completely identical to the paternal species. Both a female and a male were detected in the androgenetic sturgeon progenies, which is of interest for producing bisexual progenies via androgenesis. The data of this study confirm the feasibility of dispermic androgenesis using cryopreserved sperm to preserve and recover the gene pools of endangered sturgeon species.     

All-male asexuality: origin and maintenance of androgenesis in the Asian clam Corbicula

Androgenesis is a rare form of asexual male reproduction found in disparate taxa across the Tree of Life. Phylogenetic analyses of mitochondrial genes suggest that androgenesis has arisen repeatedly in the Asian clam genus Corbicula. Two of these androgenetic species have been introduced to North America. Multiple lines of genetic evidence suggest that although nuclear recombination between these two species is rare, mitochondrial genome capture is a frequent consequence of androgenetic parasitism of heterospecific eggs. Egg parasitism may also rarely result in partial nuclear genome capture between closely related species of Corbicula, which provides a mechanism for the otherwise clonal species to avoid the deleterious effects of asexuality. Egg parasitism among congeners may explain why androgenesis has been maintained in Corbicula after fixation and has not yet led to population extinction. This mechanism also provides an explanation for the apparent multiple origins of androgenesis in Corbicula as seen on the mitochondrial DNA phylogeny. We suggest that a single androgenetic lineage may have repeatedly captured mitochondrial genomes (as well as portions of nuclear genomes) from various sexual species, resulting in several distinct androgenetic species with distantly related mtDNA genomes and divergent morphologies.     

Cryopreservation of rhesus macaque (Macaca mulatta) spermatozoa and their functional assessment by in vitro fertilization

Although spermatozoa from several species of nonhuman primates have been cryopreserved, there has been no report of success with rhesus macaque spermatozoa as judged by functional assays. Two Tris--egg yolk freezing media, TEST and TTE, which have been successfully used for cynomolgus macaque (Macaca fascicularis) spermatozoa, were compared for cryopreservation of spermatozoa from four rhesus macaques (Macaca mulatta). The postthaw motility (percentage and duration) of spermatozoa cryopreserved in TTE was much higher than that for spermatozoa cryopreserved in TEST. The function of sperm cryopreserved in TTE was evaluated by in vitro fertilization of oocytes collected from gonadotropin-stimulated prepubertal rhesus macaques. Of the inseminated oocytes, 82 +/- 13% were fertilized and 63 +/- 22 and 39 +/- 21% of the resulting zygotes developed into morulae and blastocysts, respectively. These results indicate that rhesus macaque spermatozoa can be effectively cryopreserved in TTE medium. This finding will facilitate the application of in vivo and in vitro assisted reproductive technologies in this species.     

Induced androgenesis in fish: obtaining viable nucleocytoplasmic hybrids

A self-review of long-standing studies of induced androgenesis in fish. The data are provided on the induction of diploid androgenesis in teleost fish via blocking the first cleavage division and the development of the method of dispermic androgenesis in sturgeon fish. Androgenetic nucleocytoplasmic hybrids were obtained using the method of dispermic androgenesis and eggs of previously produced true interspecific hybrids. Specific morphological features of androgenetic hybrids were described and dispermic androgenesis was realized using cryopreserved sperm. The mechanism of sex determination in sturgeon fish is discussed with reference to possible us of androgenesis for restoration of rare and disappearing species.     

Induction of Haploid Androgenesis in Pacific Oyster by UV Irradiation

Androgenesis, development from paternal but not maternal chromosomes, can be induced in some organisms including fish, but has not been induced previously in mollusk. In this study we investigated the induction of haploid androgenesis in the Pacific oyster by ultraviolet irradiation and observed nuclear behavior in the androgenetic eggs. Irradiation for 90 seconds at a UV intensity of 72 erg/mm² per second (6480 erg/mm²) was the optimal dose to achieve haploid androgenesis. The fertilization and development rates of D-shaped larvae decreased with increasing exposure time, and the development of the genetically inactivated eggs terminated before reaching the D-shaped stage. Cytologic observations showed that UV irradiation did not affect germinal vesicle breakdown or chromosomal condensation but caused various nuclear behavioral patterns during meiosis and first mitosis: 21.7% of eggs extruded all maternal chromosomes as 2 or 3 polar bodies, and 59.1% of eggs formed one female pronucleus. The maternally derived nucleus did not participate, or partially participated, in the first karyokinesis. The cytologic evidence demonstrates that the male genome is directing development in haploids produced by UV irradiation.     

Androgenesis is a maternal trait in the invasive ant Wasmannia auropunctata

Androgenesis is the production of an offspring containing exclusively the nuclear genome of the fathering male via the maternal eggs. This unusual mating system is generally considered a male trait, giving to androgenetic males a substantial fitness advantage over their sexually reproducing relatives. We here provide the first empirical study of the evolutionary outcomes of androgenesis in a haplo-diploid organism: the invasive ant Wasmannia auropunctata. Some of the populations of this species have a classical haplo-diploid sexual mating system. In other populations, females and males are produced through parthenogenesis and androgenesis, respectively, whereas workers are produced sexually. We conducted laboratory reciprocal-cross experiments with reproductive individuals from both types of populations and analysed their progenies with genetic markers, to determine the respective contribution of males and females to the production of androgenetic males. We found that androgenesis was a parthenogenetic female trait. A population genetic study conducted in natura confirmed the parthenogenetic female origin of androgenesis, with the identification of introgression events of sexual male genotypes into androgenetic/parthenogenetic lineages. We argue that by producing males via androgenesis, parthenogenetic queen lineages may increase and/or maintain their adaptive potential, while maintaining the integrity of their own genome, by occasionally acquiring new male genetic material and avoiding inbreeding depression within the sexually produced worker cast.     

Androgenesis: a review through the study of the selfish shellfish Corbicula spp

Among the asexual reproductive modes, androgenesis is probably one of the most astonishing and least studied mechanisms. In this 'paternal monopolization', the maternal nuclear genome fails to participate in zygote development and offspring are paternal nuclear clones. Obligate androgenesis is known in only a few organisms, including multiple species of clam in the genus Corbicula. Corbicula is a good system to review the evolutionary consequences of this 'all-male asexuality' because the cytological mechanisms of androgenetic reproduction have been described. In Corbicula, sperm are unreduced and, after fertilization, the maternal nuclear chromosomes are extruded as two polar bodies. Hermaphroditic lineages of Corbicula have a worldwide distribution and seem to reproduce through androgenesis, whereas their sexual relatives have restricted ranges. The invasive success of these androgenetic Corbicula lineages may be linked to their asexual mode of reproduction. We review the phenomenon of androgenesis, focusing on evolutionary perspectives, using the genus Corbicula as an exemplar system.     

Induced Androgenesis in Fish: Obtaining Viable Nucleocytoplasmic Hybrids

A self-review of long-standing studies of induced androgenesis in fish. The data are provided on the induction of diploid androgenesis in teleost fish via blocking the first cleavage division and the development of the method of dispermic androgenesis in sturgeon fishes. Androgenetic nucleocytoplasmic hybrids were obtained using the method of dispermic androgenesis and eggs of previously produced true interspecific hybrids. Specific morphological features of androgenetic hybrids were described and dispermic androgenesis was realized using cryopreserved sperm. The mechanism of sex determination in sturgeon fishes is discussed with reference to possible use of androgenesis for restoration of rare and disappearing species.__________Translated from Ontogenez, Vol. 36, No. 4, 2005, pp. 254–264.Original Russian Text Copyright © 2005 by Grunina, Recoubratsky.     

Production of androgenetic diploid rainbow trout

Haploid androgenesis was induced in rainbow trout (Salmo gairdneri) when eggs were irradiated with 60Co gamma radiation prior to fertilization. Diploidy was restored to the androgenetic haploid zygotes by suppression of first cleavage division using hydrostatic pressure. Peak survival in the androgenetic diploid lots (32.5-38.9 percent of control) occurred when a pressure shock of 9000 pounds per square inch lasting from one to three minutes was applied to the eggs 345 minutes post-fertilization. Chromosomal analysis confirmed diploidy in the androgenetic individuals and suggested that YY rainbow trout are viable to at least the "eyed stage" of development. Inheritance patterns at two loci confirmed all-paternal inheritance. The relatively high yields of completely homozygous androgenetic rainbow trout and the potential for the use of androgenesis in the production of inbred lines and in genetic studies indicate that androgenesis may become a valuable tool in fish research and breeding.     

Nucleocytoplasmic Incompatibility in Androgenetic Fish Hybrids Can Be Overcome

Many androgenetic fish hybrids are nonviable due to the disturbed interaction between the foreign nucleus and the cytoplasm. It has been proposed that this incompatibility can be overcome if eggs from interspecific hybrids and sperm from one of the parental species are used for diploid androgenesis. Indeed, the androgenetic progeny obtained as a result of the insemination of the enucleated eggs of goldfish and carp hybrids by the carp spermatozoa proved to be viable and fertile. This approach may be used for reconstitution of the genotypes of rare or disappearing fish species with the help of induced androgenesis, if their native or cryoconserved sperm is available.     

Nucleocytoplasmic incompatibility in androgenetic fish hybrids can be overcome

Many androgenetic fish hybrids are nonviable due to the disturbed interaction between the foreign nucleus and the cytoplasm. It has been proposed that this incompatibility can be overcome if eggs from interspecific hybrids and sperm from one of the parental species are used for diploid androgenesis. Indeed, the androgenetic progeny obtained as a result of the insemination of the enucleated eggs of goldfish and carp hybrids by the carp spermatozoa proved to be viable and fertile. This approach may be used for reconstitution of the genotypes of rare or disappearing fish species with the help of induced androgenesis, if their native or cryoconserved sperm is available.     

In vivo and in vitro fertilizing capacity of cryopreserved European mouflon [Ovis gmelini musimon] spermatozoa used to restore genetically rare and isolated populations

European mouflon sheep are an endangered species of ovidae residing primarily in the mountenous habitat of the islands of Sardinia and Corsica. The purpose of this study was to assess the fertilizing capacity of cryopreserved European mouflon spermatozoa after AI in synchronized mouflon and domestic ewes and after IVF in in vitro matured mouflon and domestic ewe oocytes collected by OPU technique. Domestic ram (Ovis aries) spermatozoa served as control. Semen was collected by artificial vagina from three mouflons and three domestic rams during the breeding season and was cryopreserved. At thawing, no significant differences in sperm viability were found between the wild and the domestic species (53.1 +/- 4.6% versus 56.0 +/- 4.7%) whereas the percentage of acrosome-intact sperm was lower in mouflon (55.5 +/- 4.6%) than in ram semen (62.7 +/- 3.1%; P < 0.05). Lambing rate did not differ between synchronized mouflon and domestic ewes (5/11 versus 8/12) after 150 and 156 days of pregnancy, respectively. After two OPU sessions, 87 and 132 oocytes were collected from three hyperstimulated mouflon and three domestic ewes. Cryopreserved/thawed semen was inseminated with an endoscope into the uterus of corresponding species during the non-breeding season. The oocytes were matured and fertilized in vitro; 61/73 mouflon and 81/101 domestic ewe oocytes were found to be fertilized. From these, we obtained 6/61 and 17/81 blastocysts. After vitrification and thawing, the hatching rate showed no significant difference between mouflon and sheep blastocysts (4/6 versus 14/17). In conclusion, our data showed that cryopreserved mouflon spermatozoa can be successfully used to carry out a genuine and complete program of genetic restoration in small and isolated groups of European mouflons.     

人工诱导泥鳅与大鳞副泥鳅雄核发育单倍体子代的RAPD分析

用３０个经过筛选的随机引物对３组泥鳅雄核发育单倍体、２组大鳞副尼鳅雄核发育单倍体及其相应亲本进行了ＲＡＰＤ分析。结果表明,雄核发育倍体子代与其父本的ＲＡＰＤ谱带相似率为９７．０％－９７．８％,与母本相似率为３０．３％－５９．５％,子代中的非亲本谱眩为０－０．０２９,极少母本特异谱带。这一结果说明雄性发育鱼类单倍体的遗传信息主要来自父本,且存在着个体差异,雄核发育子代存在ＤＮＡ变异和母本ＤＮＡ非特异带,但并非其必要条件。     

Inheritance of resistance of bovine preimplantation embryos to heat shock: relative importance of the maternal versus paternal contribution

Brahman preimplantation embryos are less affected by exposure to heat shock than Holstein embryos. Two experiments were conducted to test whether the ability of Brahman embryos to resist the deleterious effects of heat shock was a result of the genetic and cellular contributions from the oocyte, spermatozoa, or a combination of both. In the first experiment, Brahman and Holstein oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from an Angus bull. A different bull was used for each replicate to eliminate bull effects. On day 4 after fertilization, embryos >or= 9 cells were collected and randomly assigned to control (38.5 degrees C) or heat shock (41 degrees C for 6 hr) treatments. The proportion of embryos developing to the blastocyst (BL) and advanced blastocyst (ABL; expanded and hatched) stages was recorded on day 8. Heat shock reduced the number of embryos produced from Holstein oocytes that developed to BL (P < 0.001, 55.6 +/- 4.2% vs. 29.8 +/- 4.2%) and ABL (P < 0.01, 37.7 +/- 3.6% vs. 12.2 +/- 3.6%) on day 8 as compared to controls. In contrast, heat shock did not reduce development of embryos produced from Brahman oocytes (BL = 42.1 +/- 4.8% vs. 55.6 +/- 4.8% for 38.5 and 41 degrees C, respectively; ABL = 17.6 +/- 4.2% vs. 32.4 +/- 4.2%). In the second experiment, oocytes from Holstein cows were fertilized with semen from bulls of either Brahman or Angus breeds. Heat shock of embryos >or= 9 cells reduced development to BL (P < 0.002) and ABL (P < 0.005) for embryos sired by both Brahman (BL = 54.3 +/- 7.7% vs. 23.4 +/- 7.7%; ABL = 43. +/- 7.4% vs. 7.9 +/- 7.4%, for 38.5 and 41 degrees C, respectively) and Angus bulls (BL = 57.9 +/- 7.7% vs. 31.0 +/- 7.7%; ABL = 33.6 +/- 7.4% vs. 18.4 +/- 7.4%, for 38.5 and 41 degrees C, respectively). There were no breed x temperature interactions. Results suggest that the oocyte plays a more significant role in the resistance of Brahman embryos to the deleterious effects of heat shock than the spermatozoa.     

Induction of androgenetic development of the brook charr (Salvelinus fontinalis) × Arctic charr (Salvelinus alpinus) hybrids in eggs derived from the parental species

Failure of interspecific androgenesis between brook charr (Salvelinus fontinalis, Mitchill 1814) and Arctic charr (Salvelinus alpinus, L.) has been attributed to the conflict between the egg cytoplasm of one species and the sperm nucleus of the other species. To overcome this incompatibility, sperm derived from the brook charr × Arctic charr hybrid male was used to induce androgenetic development in eggs originating from the parental species as well as their hybrids. The eggs were subjected to 420 Gy of X-radiation to damage the maternal nuclear DNA and inseminated with untreated sperm. Haploid zygotes were exposed to high hydrostatic pressure shock (7000 psi for 4 min), which was applied 420 min after insemination to inhibit the first cell cleavage and recover the diploid state of the zygote. The androgenetic diploid offspring that hatched from the brook charr, the Arctic charr and the hybrids eggs had survival rates of 4.7 ± 0.6%, 1.2 ± 0.4% and 16.8 ± 0.5%, respectively. Drastic mortality among the hatched androgenetic individuals was observed within the first five months of rearing. Cytogenetic analysis of the androgenetic progenies exhibited residues of the irradiated maternal nuclear genome in the form of radiation-induced chromosome fragments in 47% of the specimens that were examined. Interactions between the egg cytoplasm and the sperm nucleus, the low quality of the gametes, the expression of homozygous paternal lethal alleles and the incomplete inactivation of the maternal chromosomes were identified as factors responsible for the large mortality among androgenetic embryos and hatchlings.     

Effect of the volume of medium and number of oocytes during in vitro fertilization on embryo development in pigs

The present study was designed to determine the effect of the volume of medium (VM) and the number of oocytes (NOOC) during in vitro fertilization (IVF) on embryo development in pigs. Groups of 15, 30 and 50 in vitro matured oocytes were transferred to 2, 1 and 0.1 ml of modified Tris-buffered medium (mTBM) and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) in a 3 x 3 factorial experiment. A total of 2739 oocytes from four replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The efficiency of fertilization (EF: number of monospermic oocytes/total number of inseminated oocytes) and BF decreased (P<0.03) as the VM increased (EF: 45.9+/-2.2, 43.8+/-2.6 and 36.9+/-1.6% and BF: 29.4+/-2.7, 23.2+/-1.8 and 19.9+/-2.1% for VM 0.1, 1 and 2 ml, respectively). The BF, but not EF, was also affected (P<0.04) by NOOC (19.8+/-1.6, 28.1+/-2.3 and 24.6+/-2.9% for groups of 15, 30 and 50 oocytes, respectively). The effect of the interaction VM x NOOC on EF and BF was not significant. These results indicate that when 2000 spermatozoa/oocyte were used, a low volume of IVF medium (0.1 ml) and the number of oocytes during IVF (30-50) can improve the in vitro embryo production in pigs.     

Ratio of the zygote cytoplasm to the paternal genome affects the reprogramming and developmental efficiency of androgenetic embryos

Uniparental embryos have uniparental genomes and are very useful models for studying the specific gene expression of parents or for exploring the biological significance of genomic imprinting in mammals. However, the early developmental efficiency of androgenetic embryos is significantly lower than that of parthenogenetic embryos. In addition, oocytes are able to reprogram sperm nuclei after fertilization to guarantee embryonic development by maternally derived reprogramming factors, which accumulate during oogenesis. However, the importance of maternal material in the efficiency of reprogramming the pronucleus of androgenetic embryos is not known. In this study, androgenetic embryos were constructed artificially by pronucleus transfer (PT) or double sperm injection (DS). Compared with DS embryos, PT embryos that were derived from two zygotes contained more maternal material, like 10–11 translocation methylcytosine deoxygenase 3 (Tet3) and histone variant 3.3 (H3.3). Our experiments confirmed the better developmental potential of PT embryos, which had higher blastocyst rates, a stronger expression of pluripotent genes, a lower expression of apoptotic genes, and superior blastocyst quality. Our findings indicate that the aggregation of more maternal materials in the paternal pronucleus facilitate the reprogramming of the paternal genome, improving embryonic development in PT androgenesis.     

Successful cryopreservation of Pacific oyster (Crassostrea gigas) oocytes

Protocols for cryopreservation of sperm and oocytes would provide the ultimate control over parental crosses in selective breeding programmes. Sperm freezing is routine for many species, but oocyte freezing remains problematic, with virtually zero success in aquatic species to date. This paper describes the development of a successful protocol for cryopreserving high concentrations of Pacific oyster (Crassostrea gigas) oocytes. Ethylene glycol (10%) and dimethyl sulfoxide (15%) were found to be the most effective cryoprotectants resulting in post-thaw fertilization rates of 51.0+/-8.0 and 45.1+/-8.3%, respectively. Propylene glycol was less effective and methanol resulted in zero fertilization post-thaw. The use of Milli-Q water rather than seawater as a base medium significantly improved fertilization (20.4+/-3.0 and 8.7+/-2.2%, respectively) as did the inclusion of a 5 min isothermal hold at -10 or -12 degrees C (35.9+/-5.0 and 31.9+/-4.6%, respectively). The optimal cooling rate post-hold was 0.3 degrees C min(-1), with virtually zero post-thaw fertilization with cooling rates of 3 and 6 degrees C min(-1). Using an optimized protocol, post-thaw fertilization rates for oocytes from eight individual females ranged from 0.8 to 74.5% and D-larval yields from 0.1 to 30.1%. For three individuals, larvae were reared through to spat. Development of D-larvae to eyed larvae and spat was similar for larvae produced from unfrozen (24.8+/-4.1% developed to eyed larvae and 16.5+/-3.2% to spat) and cryopreserved (28.4+/-0.6 and 18.7+/-0.5%, respectively) oocytes. The ability to cryopreserve large quantities of oyster oocytes represents a major advance in cryobiology and selective breeding.