Analysis of the chromosome complement in outbred mouse sperm fertilizing in vitro

The chromosome complements in a population of mouse sperm from random-bred ICR donors were analyzed at first-cleavage metaphase after in vitro fertilization (IVF) of oocytes from females of the same strain. The sperm were aged as donations occurred within an average of 31 days, either since last mating or at arrival at the animal facility in the case of virgin males. Of a total of 598 sperm complements studied from 22 sexually mature males aged 10–26 weeks old, there was one diploid complement (0.17%). The frequencies of hyperhaploidy and structural aberrations that were studied in 338 complements were 4.4% and 3.6%, respectively, giving an overall frequency of 8.0%. The hyperhaploid complements consisted of n + 1, n + 2, n + 3, and n + 7 counts, while the structural abnormalities were of the chromosome type and included large and small fragments and a possible translocation. This is the highest frequency of sperm chromosome abnormalities reported for mouse sperm obtained from males under physiological conditions and fertilized in vitro or in vivo. Sperm aging, strain, and/or technique differences are among the factors that may be responsible for this high frequency. Since the 8.0% frequency of hyperhaploidy and structural abnormalities is similar to the frequency reported for human sperm after IVF, the outbred murine in vitro fertilization system may be a useful model to study the origin of human sperm chromosome abnormalities.     

Life‐history traits of the Whiting polyploid line of the parasitoid Nasonia vitripennis

In hymenopterans, males are normally haploid (1n) and females diploid (2n), but individuals with divergent ploidy levels are frequently found. In species with ‘complementary sex determination’ (CSD), increasing numbers of diploid males that are often infertile or unviable arise from inbreeding, presenting a major impediment to biocontrol breeding. Non‐CSD species, which are common in some parasitoid wasp taxa, do not produce polyploids through inbreeding. Nevertheless, polyploidy also occurs in non‐CSD Hymenoptera. As a first survey on the impacts of inbreeding and polyploidy of non‐CSD species, we investigate life‐history traits of a long‐term laboratory line of the parasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) (‘Whiting polyploid line’) in which polyploids of both sexes (diploid males, triploid females) are viable and fertile. Diploid males produce diploid sperm and virgin triploid females produce haploid and diploid eggs. We found that diploid males did not differ from haploid males with respect to body size, progeny size, mate competition, or lifespan. When diploid males were mated to many females (without accounting for mating order), the females produced a relatively high proportion of male offspring, possibly indicating that these males produce less sperm and/or have reduced sperm functionality. In triploid females, parasitization rate and fecundity were reduced and body size was slightly increased, but there was no effect on lifespan. After one generation of outbreeding, lifespan as well as parasitization rate were increased, and a body size difference was no longer apparent. This suggests that outbreeding has an effect on traits observed in an inbred polyploidy background. Overall, these results indicate some phenotypic detriments of non‐CSD polyploids that must be taken into account in breeding.     

Unexpectedly low frequencies of diploid males in an inbreeding parasitoid with complementary sex determination

In hymenopterans with single locus complementary sex determination, sex depends on the genotype at one polymorphic locus. Haploids are always male, while diploids are female when heterozygous and male when homozygous at the sex‐determining locus. Brothers and sisters have a 50% chance of sharing a sex allele (i.e. of being ‘matched’), and hence half of all sibling matings are expected to produce diploid males at the expense of females. Nevertheless, observed frequencies of diploid males are often lower than predicted, as diploid males may succumb to pre‐imaginal mortality, or because unmatched mates or sperm enjoy a competitive advantage. We counted diploid males in broods of the parasitoid wasp Cotesia glomerata sampled in the field, and in broods produced through controlled laboratory crosses. Consistently, the frequency of diploid males fell below expectations based upon the estimated occurrence of sibling mating. In the staged broods with diploid males, females made up a disproportionately large share of the diploids. Broods with and without diploid males were of similar size. Hence, the shortage of diploid males cannot be accounted for by differential pre‐imaginal mortality alone. Instead, we postulate the existence of a mechanism that leads to preferential fertilization of eggs by sperm bearing unmatched alleles. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ●● , ●●–●●.     

Diploid sperm produced by artificially sex-reversed clone loaches

Clone loaches reproduce unisexually in a wild population of Hokkaido Island, Japan. These clone loaches produce genetically identical unreduced eggs which develop to diploid individuals without any genetic contribution of sperm donors. In the present study, sex reversal of clone loaches was attempted and the reproductive potential of resultant clone males was examined. Clone loaches administered 0.5 ppm of 17-alpha methyltestosterone (MT) for 30 days from 1 month after hatching differentiated into physiological males. These sex-reversed clone males produced fertile spermatozoa with a diploid DNA content. Diploid spermatozoa had significantly larger heads than normal haploid sperm, but had a normal shape showing a head, mid-piece, and tail. The motility of diploid spermatozoa was low after ambient water was added. Concentration of diploid spermatozoa per unit of sperm was lower than that of control haploid spermatozoa. Microsatellite genotyping revealed that triploid progeny from the cross between a normal diploid female and a sex-reversed clone male had two alleles specific to the diploid clone male and one allele of the mother loach. These results indicated that the sex-reversed clone males produced fertile diploid spermatozoa genetically identical to the clone lineage.     

Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid

Our objective was to examine the developmental fate of sperm nuclei in oocytes fertilized under conditions of meiotic arrest. Therefore zona-free metaphase II oocytes and oocyte fragments (nucleate and anucleate) were fertilized in the presence of colcemid. In anucleate oocyte fragments, normal male pronuclei develop. In contrast, in intact oocytes and nucleate fragments sperm nuclei after initial decondensation undergo secondary condensation. This state is maintained as long as the oocytes are treated with colcemid. When the drug is removed 3 h after insemination, the meiotic spindle(s) is reconstructed, the second polar body(ies) is extruded, and a female pronucleus (or micronuclei) forms. At the same time the sperm nucleus decondenses again and transforms into a male pronucleus. In addition oocytes fertilized in the presence of colcemid could not be refertilized. These observations suggest that oocytes and oocyte fragments fertilized in the presence of colcemid undergo activation despite the failure of pronucleus formation. The inhibitory effect of colcemid on the formation of pronuclei is expressed only in the presence of oocyte chromosomes. We suggest that colcemid stabilizes factors responsible for chromosome condensation that are associated with oocyte chromosomes but not factors (whether the same or different) present in the cytoplasm.     

Diploid male production in a leaf‐cutting ant

1. In haplodiploid social insects where males are haploid and females are diploid, inbreeding depression is expressed as the production of diploid males when homozygosity at the sex‐determining locus results in the production of diploid individuals with a male phenotype. Diploid males are often assumed to have reduced fitness compared with their haploid brothers. 2. While studying the reproductive biology of a leaf‐cutting ant, Atta sexdens, in Gamboa, Republic of Panama, we detected the presence of a larger male morph. Using microsatellite markers we were able to confirm that the large male morph was diploid in 87% of cases. 3. We infer that the Gamboa population of A. sexdens experiences inbreeding depression because diploid males were found in three out of five mature colonies. However, their frequencies were relatively low because queens were multiply mated and our estimates suggest that many diploid male larvae may not survive to adulthood. 4. We measured two traits potentially linked to male reproductive success: sperm length and sperm number, and showed that diploid males produced fewer but longer sperm. These results provide indirect evidence that diploid male reproductive success would be reduced compared with haploid males if they were able to copulate. 5. We conclude that diploid male production is likely to affect the fitness of A. sexdens queens with a matched mating, as these males are produced at the cost of workers and, if the colony survives to reach mature size, also gynes.     

Changes in motility, ATP content, morphology and fertilisation capacity during the movement phase of tetraploid Pacific oyster (Crassostrea gigas) sperm

Changes in sperm features during the movement phase are especially interesting to study in external fertilization species whose sperm duration movement is long because this implies a significant adaptation of moving cells to the external medium. This study describes the changes in tetraploid Pacific oyster sperm characteristics in relation to time post activation.Sperm individually collected on three tetraploid males were activated in seawater. Their features were analysed over a 24 h period and compared to a sperm pool collected on three diploid males as a reference. The percentage of motile spermatozoa, the intracellular ATP content, and the fine structure of spermatozoa were studied in relation to time post activation. Furthermore, the fertilisation capacity of sperm individually collected on five diploid males was assessed after 1 and 24 h post activation.A forward progressive movement was maintained for at least a 20 h duration. Compared to diploid males, the percentage of motile spermatozoa was lower in tetraploid males. The intracellular ATP concentration was higher in spermatozoa from tetraploid males than in spermatozoa from diploid males. A decrease in ATP content was observed in the first 6 h post activation and severe alterations were observed in sperm morphology after 24 h. Then, a lower fertilisation capacity of sperm from diploid males was observed at the end of the movement phase.The cessation of Pacific oyster sperm motility was unlikely caused by ATP consumption as ATP concentration was still high at the end of sperm movement but rather caused by drastic changes in sperm morphology. Compared to sperm collected on diploid males, the lower quality of sperm from tetraploid males was emphasized by a shorter movement duration and deeper morphological alterations at the end of the movement phase.     

Production of fertile unreduced sperm by hybrid males of the Rutilus alburnoides complex (Teleostei,cyprinidae). An alternative route to genome tetraploidization in unisexuals.

The hybrid minnow Rutilus alburnoides comprises diploid and polyploid females and males. Previous studies revealed that diploid and triploid females exhibit altered oogenesis that does not involve random segregation and recombination of the genomes of the two ancestors, constituting unisexual lineages. In the present study, we investigated the reproductive mode of hybrid males from the Tejo basin, using experimental crosses and flow cytometric analysis of blood and sperm. The results suggest that diploid hybrids produced fertile unreduced sperm, transmitting their hybrid genome intact to offspring. Triploid hybrids also produced unreduced sperm, but it was not possible to obtain data concerning their fertility. Finally, tetraploid hybrids produced fertile diploid sperm, which exhibited Mendelian segregation. Tetraploid R. alburnoides may reestablish biparental reproduction, as individuals of both sexes with the appropriate constitution for normal meiosis (two haploid genomes from each parental species) are likely to occur in natural populations. Tetraploids probably have arisen from syngamy of diploid eggs and diploid sperm produced by diploid hybrid males. Diploid hybrid males may therefore play a significant role in the dynamics of the complex, starting the evolutionary process that may ultimately lead to a new sexually reproducing species.     

Spermatozoa production by triploid males in the New Zealand freshwater snail Potamopyrgus antipodarum

Asexual lineages derived from dioecious taxa are typically assumed to be all female. Even so, asexual females from a variety of animal taxa occasionally produce males. The existence of these males sets the stage for potential gene flow across asexual lineages as well as between sexual and asexual lineages. A recent study showed that asexual triploid female Potamopyrgus antipodarum, a New Zealand freshwater snail often used as a model to study sexual reproduction, occasionally produce triploid male offspring. Here, we show that these triploid male P. antipodarum (1) have testes that produce morphologically normal sperm, (2) make larger sperm cells that contain more nuclear DNA than the sperm produced by diploid sexual males, and (3) produce sperm that range in DNA content from haploid to diploid, and are often aneuploid. Analysis of meiotic chromosomes of triploid males showed that aberrant pairing during prophase I probably accounts for the high variation in DNA content among sperm. These results indicate that triploid male P. antipodarum produce sperm, but the extent to which these sperm are able to fertilize female ova remains unclear. Our results also suggest that the general assumption of sterility in triploid males should be more closely examined in other species in which such males are occasionally produced. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 227–234.     

Double copulation of a female with sterile diploid and polyploid males recovers fertility in Bombyx mori

Silkworm males produce dimorphic sperm, nucleate eupyrene sperm and anucleate apyrene sperm. Apyrene sperm have been speculated to have an assisting role in fertilisation. However, the coexistence of eupyrene and apyrene sperm in the testis and female reproductive organs has made it difficult to define the role of apyrene sperm. Polyploid males are highly sterile. Microscopic observation revealed that the elimination of eupyrene nuclei by peristaltic squeezing caused the sterility of polyploids. Heat-shock applied to pupae of Daizo males (DH) also induced high sterility due to the lack of normal apyrene sperm. When eupyrene sperm of sterile DH males and apyrene sperm of sterile polyploid males were mixed by double copulation, a remarkable increase in fertility of the double-mated females was observed. This finding strongly suggests that the apyrene sperm are indispensable in fertilisation of the silkworm and that polyploid apyrene sperm function as a substitute for diploid sperm. We established an experimental system in which we can separate the two types of sperm for further studies on their functions without chemical and/or mechanical treatments.     

Sexual conflict and protein polymorphism

Sexual conflict, where male and female reproductive interests differ, is probably widespread and often mediated by male or sperm proteins and female or egg proteins that bind to each other during mating or fertilization. One potential consequence is maintenance of polymorphism in these proteins, which might result in reproductive isolation between sympatric subpopulations. I investigate the conditions for polymorphism maintenance in a series of mathematical models of sexual conflict over mating or fertilization frequency. The models represent a male or sperm ligand and a female or egg receptor, and they differ in whether expression of either protein is haploid or diploid. For diploid expression, the conditions imply that patterns of dominance, which involve neither overdominance nor underdominance, can determine whether polymorphism is maintained. For example, suppose ligand expression is diploid, and consider ligand alleles L1 and L2 in interactions with a given receptor genotype; if L1/L1 males are fitter than L2/L2 males in these interactions, then polymorphism is more likely to be maintained when L1/L2 males more closely resemble L1/L1 males in these interactions. Such fitter-allele dominance might be typical of a ligand or its receptor due to their biochemistry, in which case polymorphism might be typical of the pair.     

Low incidence of chromosome aberrations in spermatozoa of fertile boars

Chromosomal imbalance in gametes and embryos is one of the factors contributing to early embryonic mortality. Although the rate of chromosomally abnormal sperm cells is low and usually does not exceed 1%, there is no clear indication of fertilizing potential of such gametes. The aim of the experiment was to investigate the type and incidence of numerical chromosomal aberrations in spermatozoa produced by fertile boars used in artificial insemination (AI). We used the protocol of fluorescent in situ hybridization (FISH) on sperm interphase nuclei with molecular probes for porcine chromosome pairs 1 and 10. Altogether 12?348 sperm cells were examined. Disomy was observed in spermatozoa of all seven AI boars whereas only one diploid cell was identified in all screened sperm cells. The average rate of chromosomally unbalanced sperm was 0.105% (13/12 348) with an inter-individual variation from 0.048% to 0.194%. Among abnormal sperm cells, both disomy (0.097%) and diploidy (0.008%) were detected. Nullisomy was not included into calculations. The estimated aneuploidy rate calculated by doubling the number of disomic cells was 0.194%. Chromosome pair 10 was significantly more often involved in non-disjunction (75%, 9/12 aneuploid sperm cells) than chromosome pair 1 (25%, 3/12). We have shown for the pig that the rate of disomic cells falls into a range presented by other authors, whereas that of diploid spermatozoa appeared to be lower in the present study. In conclusion, numerical chromosome aberrations were present in spermatozoa of all AI boars analyzed in this study. Therefore, it can be assumed that the presence of unbalanced spermatozoa at the level observed in fertile males does not significantly affect their reproductive potential.     

Offsetting effects of Wolbachia infection and heat shock on sperm production in Drosophila simulans: analyses of fecundity, fertility and accessory gland proteins

Infection in Drosophila simulans with the endocellular symbiont Wolbachia pipientis results in egg lethality caused by failure to properly initiate diploid development (cytoplasmic incompatibility, CI). The relationship between Wolbachia infection and reproductive factors influencing male fitness has not been well examined. Here we compare infected and uninfected strains of D. simulans for (1) sperm production, (2) male fertility, and (3) the transfer and processing of two accessory gland proteins, Acp26Aa or Acp36De. Infected males produced significantly fewer sperm cysts than uninfected males over the first 10 days of adult life, and infected males, under varied mating conditions, had lower fertility compared to uninfected males. This fertility effect was due to neither differences between infected and uninfected males in the transfer and subsequent processing of accessory gland proteins by females nor to the presence of Wolbachia in mature sperm. We found that heat shock, which is known to decrease CI expression, increases sperm production to a greater extent in infected compared to uninfected males, suggesting a possible link between sperm production and heat shock. Given these results, the roles Wolbachia and heat shock play in mediating male gamete production may be important parameters for understanding the dynamics of infection in natural populations.     

Chromosome studies of progenies of tetraploid female rainbow trout

Summary Nine induced tetraploid females were artificially inseminated by UV-irradiated sperm collected from diploid males, in order to induce the gynogenetic development of their ova. Most of the resulting embryos were diploid (or minor aneuploids). Several gynogenetic tetraploids, likely to issue from unreduced ova, were also detected in these progenies. The same females fertilized by normal sperm of diploid males gave a majority of triploids and several pentaploids, while the fertilization by normal sperm of tetraploid males gave rise to a majority of tetraploids and one hexaploid. The same crosses, after the eggs had been heat-shocked to double the maternal genetic contribution, yielded about three-quarters pentaploids and one quarter haploids (normal sperm of diploids), or three-quarters hexaploids and one quarter diploids (normal sperm of tetraploids). These haploids and diploids are likely to result from androgenesis.     

Microtubule assembly is required for the formation of the pronuclei,nuclear lamin acquisition,and DNA synthesis during mouse,but not sea urchin,fertillization

Microtubule assembly is required for the formation of the male and female pronuclei during mouse, but not sea urchin, fertilization. In mouse oocytes, 50 μM colcemid prevents the decondensation of the maternal meiotic chromosomes and of the incorporated sperm nucleus during in vitro fertilization. Nuclear lamins do not associate with either of the parental chromatin sets although peripherin, the PI nuclear peripheral antigen, appears on both. DN A synthesis docs not occur in these fertilized, colcemid-arrested oocytes. This effect is limited to the first hours after ovulation, since colcemid added 4–6 hours later no longer prevents pronuclear development, lamin acquisition, or DNA synthesis. Neither microtubule stabilization with 10 μM taxol nor microfilament inhibition with 10 μM cytochalasin D or 2.2 μg/ml lalrunculin A prevent these pronuclear events; these drugs will inhibit the apposition of the pronuclei at the egg center. In sea urchin eggs, colcemid or griseofulvin treatment doe? not result in the same effect and the male pronucleus forms with the attendant accumulation of the nuclear lamins. The differences in the requirement for microtubule assembly during pronucleus formation may be related to the cell cycle: In mice the sperm enters a meiotic cytoplasm, whereas in sea urchin eggs it enters an interphase cytoplasm. Refertilization of mitotic sea urchin eggs was performed to test the possibility that this phenomenon is related to whether the sperm enters a meiotic/mitotic cytoplasm or one at interphase; during refertilization at first mitosis, the incorporated sperm nucleus is unable to decondense and acquire lamins. These results indicate a requirement for microtubule assembly for the progression from meiosis to first interphase during mouse fertilization and suggest that the cytoskeleton is required for changes in nuclear architecture necessary during fertilization and the cell cycle.     

The chemotherapeutic agents nocodazole and amsacrine cause meiotic delay and non-disjunction in spermatocytes of mice

Aneuploidy of germ cells contributes to reduced fertility, foetal wastage and genetic defects. The possible risk of aneuploidy induction by the cancer chemotherapeutic drugs amsacrine (AMSA) and nocodazole (NOC) was investigated in male mice. Two molecular cytogenetic approaches were used: (1) the BrdU-incorporation assay to test the altered duration of meiotic divisions and (2) the sperm-FISH assay to determine aneuploidy induction during meiosis by observing hyperhaploid and diploid sperm. Sperm were sampled from the Caudae epididymes of treated and solvent control males. Single intraperitoneal injections with NOC (35 mg/kg) and AMSA (15 mg/kg) caused a meiotic delay of 24h. The timing of sperm sampling for the sperm-FISH assay was adjusted accordingly, i.e. 23 days after treatment. Mice were treated with 18, 35 and 50 mg/kg of NOC, or 5, 10, 15 and 20 mg/kg of AMSA. Significant dose-dependent increases above the concurrent controls in the frequencies of hyperhaploid sperm were found with both agents. Significant increases in the frequencies of diploid sperm were found only with AMSA. These results provide a basis for genetic counselling of patients under AMSA or NOC chemotherapy. During a period of 3-4 months after the end of chemotherapy, they may stand a higher risk of siring chromosomally abnormal offspring.     

Male-Sex-Ratio Trait in Drosophila Pseudoobscura: Frequency of Autosomal Aneuploid Sperm

Males with the SR X chromosome show the "sex-ratio" (sr) phenotype in which they produce almost entirely daughters. The few sons (about 1%) are invariably sterile X/O males and result entirely from nullo-XY sperm. The "male-sex-ratio" (msr) phenotype is a modified form of sr in which SR/Y males produce a higher frequency of sterile X/O sons. The msr trait is due to the presence of the SR X-chromosome in males which are also homozygous for one or more autosomes from the L116 strain. Here the frequency of nullo-3 and diplo-3 sperm from msr males was measured by crossing to a compound-3 strain and found to be 13.8% and 3.2%, respectively, of the total viable sperm. The sr males produced very low levels of nullo-3 sperm at a frequency not different from control X/Y males and a slightly elevated frequency of diplo-3 sperm over X/Y males. The msr males were found to have only 12% the fecundity of sr males and in matings to cause a high frequency of brown inviable eggs. These results indicate that high rates of autosomal aneuploidy are not restricted to chromosome 3 but also occur for chromosomes 2, 4 and 5. The overall frequency of autosomal aneuploid sperm is estimated to be approximately 50%. Microscopic studies of meiosis in testes from msr males indicates meiotic nondisjunction and meiotic chromosome loss are responsible for the msr phenotype. Last, microscopic studies of sperm cysts from msr males reveal high levels of spermiogenic failure.     

Dual effect of protein kinase C on the induction of DNA synthesis by colcemid in G0-arrested human diploid fibroblasts

G0-arrested human diploid fibroblasts, TIG-1, was stimulated to induce DNA synthesis by serum, epidermal growth factor (EGF), colchicine, colcemid, or 12-O-tetradecanoylphorbol-13-acetate (TPA). The induction of DNA synthesis was mediated by protein kinase C (PKC) when stimulated with TPA but not when stimulated with other agents. When TPA-stimulated cells were immediately treated with colcemid, induction of DNA synthesis was reduced. This reduction diminished when colcemid was added more than 6 h after TPA treatment. Conversely, when colcemid-stimulated cells were treated with TPA, induction of DNA synthesis was also reduced. This reduction was enhanced when the interval between the addition of two stimulants was extended. PKC-deprivation abolished both stimulatory and inhibitory effects of TPA on DNA synthesis. Staurosporine blocked an induction of DNA synthesis by TPA but appeared to be ineffective on the inhibitory action of TPA on DNA synthesis by colcemid. These results suggest that the inhibitory effect of TPA on the induction of DNA synthesis by colcemid is mediated by down regulation-sensitive and staurosporine-insensitive PKC.     

Male body size and condition affects sperm number and production rates in mosquitofish,Gambusia holbrooki

Sperm number is an important predictor of paternity when there is sperm competition. Sperm number is often measured as maximum sperm reserves, but in species where mating is frequent, males will often be replenishing their reserves. Thus, variation in how quickly males can produce sperm is likely to be important in determining male success in sperm competition. Despite this, little is known about how male size, body condition or diet affects sperm production rates. We counted sperm number in large and small Gambusia holbrooki (eastern mosquitofish) after 3 weeks on either a high or low food diet. Sperm number was significantly higher in both larger males and in well‐fed males. We then stripped ejaculates again either 1, 2, 3, 4 or 5 days later to investigate subsequent sperm production. The rate of sperm replenishment was influenced by an interaction between size and diet. Large, well‐fed males had consistently high levels of sperm available over the 5 days (i.e. rapid replenishment), whereas small poorly fed males showed consistently low levels of sperm availability over the 5 days (i.e. slow replenishment). In contrast, large, poorly fed and small, well‐fed males increased their sperm numbers over the first 3 days (i.e. intermediate replenishment). Our study highlights that when mating is frequent and sperm competition is high, size and condition dependence of maximal sperm number and of sperm production rate might both contribute to variation in male reproductive success.     

Ploidy manipulation using diploid sperm in the loach, Misgurnus anguillicaudatus: a review

This paper assesses the present state of the art of ploidy manipulation in the loach, Misgurnus anguillicaudatus (Teleoste: Cobitidae). Diploid sperm can be obtained from natural tetraploid individuals with four sets of homologous chromosomes. Using diploid sperm, various polyploids and androgenetic diploids have been produced. Cryptic clonal lineages are also recognized in wild populations of the loach. They produce unreduced diploid eggs genetically identical to somatic cells of the mother fish and most diploid eggs develop gynogenetically as a member of the clone. However, some eggs develop to triploid and/or diploid-triploid mosaic individuals by incorporation of sperm nucleus. Diploid-triploid mosaic males exclusively generate fertile diploid sperm with clonal genotypes. Such diploid sperm can also be obtained from artificially sex-reversed clonal individuals. Recent population studies suggested that Japanese M. anguillicaudatus might not be a single species, but a complex involving cryptic species, because wild populations exhibited genetic differentiation at interspecific level. This implies possible relationship between atypical reproduction and natural hybridization in the loach.