Why are triploid zebrafish all male?

Adult triploid zebrafish Danio rerio has previously been reported to be all male. This phenomenon has only been reported in one other gonochoristic fish species, the rosy bitterling Rhodeus ocellatus, despite the fact that triploidy is induced in numerous species. To investigate the mechanism responsible, we first produced triploid zebrafish and observed gonad development. Histological sections of juvenile triploid gonads showed that primary growth oocytes were able to develop in the juvenile ovary, but no cortical alveolus or more advanced oocytes were found. All adult triploids examined were male (n = 160). Male triploids were able to induce oviposition by diploid females during natural spawning trials, but fertilization rates were low (1.0 ± 3.1%) compared with diploid male siblings (67.4 ± 16.6%). The embryos produced by triploid sires were aneuploid with a mean ploidy of 2.4 ± 0.1n, demonstrating that triploid males produce aneuploid spermatozoa. After confirming that adult triploids are all male, we produced an additional batch of triploid zebrafish and exposed them (and a group of diploid siblings) to 100 ng/L estradiol (E2) from 5 to 28 dpf. The E2 treated triploids and nontreated triploids were all male. The nontreated diploids were also all male, but the E2 treated diploids were 89% female. This demonstrates that triploidy acts downstream of estrogen synthesis in the sex differentiation pathway to induce male development. Based on this and the observations of juvenile gonad development in triploids, we suggest that triploidy inhibits development of oocytes past the primary growth stage, and this causes female to male sex reversal.     

Light and electron microscopic studies on the seminal secretions and the vas deferens of the penaeiodean shrimp,Sicyonia ingentis

Unlike the other penaeiodean shrimp, the ridge back shrimp, Sicyoniaingentis does not produce a spermatophore, but transfers sperm suspended in seminal plasm. This paper reports on the histomorphology and ultrastructure of the vas deferens with reference to its functional role in secreting the sperm bearing materials. The vas deferens is divisible into proximal secretory, mid storage and distal ejaculatory regions. The epithelial cells lining the proximal vas deferens are comprised of secretory and absorptive cell types. The loose sperm cells found in the lumen of this region are in an immature condition, and are agglutinated into a compact mass with signs of spermiogenesis in the mid vas deferens. The epithelial cells lining the mid vas deferens are short flattened cells. The distal vas deferens is ensheathed by muscular fibres. The inner epithelial cells are highly secretory and contain numerous microvilli at the luminal end. The sperm cord gets liquefied in this region facilitating the transfer of sperm in liquid form to the female during mating.     

Microsatellite-centromere distances and microsatellite diversity in different ploidy classes of Chinese shrimp (Fenneropenaeus Chinensis)

This is the first report of microsatellite-centromere mapping in this commercial species Fenneropenaeus Chinensis, and will be important for providing fixed points in the linkage groups of genetic maps. Triploid Chinese shrimp was induced by heat shock. The fertilized eggs were treated either by retention of the first polar body or the second polar body to produce Meiosis I (MI) or Meiosis II (MII) triploid. The triploidy status in each Chinese shrimp could be confirmed by nine polymorphic microsatellite loci, in which the parents with different alleles and the female parents were each heterozygous. The nine loci were mapped in relation to their centromeres in three MII triploid families, which were induced by retention of the second polar bodies after fertilization with sperm. Microsatellite-centromere (M-C) distances ranged from 9.6 cM to 37 cM under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in F. chinensis. Twelve polymorphic microsatellites were used to assess the heterozygosity and allelic diversity in different ploidy classes. As expected, triploids were significantly more polymorphic than diploids. The diploids had an average heterozygosity and allelic diversity value of 0.86, whereas the triploids heterozygosity averaged 0.93 and had allelic diversity value of 1.29. However, MI triploids were not significantly more polymorphic than MII in the microsatellite loci.     

Gonad morphology and sex succession in the protogynous hermaphrodite Hemanthias vivanus (Jordan and Swain)

Hemanthias vivanus , an anthiine serranid common on deep (70–80 m) reefs off eastcentral Florida, is shown to be a protogynous hermaphrodite with observed size ranges as follows: female, 49–77 mm S.L.; transitional 65–74; male, 65–96. Sexes are distinguishable externally as the dorsal spine filaments elongate and colour pattern changes with sex succession. In females, lamellae extend from the lateral and dorsal walls of the bilobed ovary into the lumen. Ova mature within the lamellae and at ovulation pass centrally into the lumen, posteriorly into a common post-ovarian sinus, and exit through the oviduct. Females possess a discrete tissue band (testicular islets) at the border of the alamellar region, from which testicular tissue proliferates during sex succession and replaces the regressing ovarian tissue. In males, spermatozoa are formed in crypts which drain peripherally into a sperm sinus which forms by rupture of muscular and connective tissue layers surrounding the gonad. The sperm sinus drains posteriorly into the vas deferens which is formed by rupture of collagenous tissue surrounding and posterior to the now vestigial post-ovarian sinus. The vas deferens and mesonephric duct join within the urogenital papilla. Two smooth muscle bands associated with the infracarinalis medius muscle bands, the collagenous tissue surrounding the gonoduct, and the intestinal lining are implicated in the extrusion of gametes, urine and faeces. At sex succession, ova are resorbed in one of at least three pathways depending upon their stage of development at the onset of sex transformation: unovulated mature ova become encysted with fibroblasts and invaded by cells of an unknown origin; non-mature yolky ova are invaded and phagocytized by granulosa cells; non-yolky ova are not invaded but fragment.     

Daily rhythm in myogenic contractions of vas deferens associated with sperm release cycle in a moth

In the gypsy moth, Lymantria dispar, release of sperm bundles from the testis into the upper vas deferens (UVD) and subsequent transfer of sperm bundles into the seminal vesicles (SV) occurs in a daily rhythm. The UVD undergoes different types of contractions despite the fact that its musculature appears to receive no innervation. Patterns of the UVD movements were recorded throughout the daily sperm release and transfer cycle. In males kept in light-dark cycles, transfer of sperm from the UVD to the SV was accompanied by a characteristic pattern of UVD contractions of high frequency and amplitude. In males kept in constant light, which fail to transfer sperm, this contraction pattern was absent. It is concluded that the vas deferens muscles undergo daily changes in contraction pattern in phase with the light-dark cycle. The increased muscular contractions appear to be a causal factor in the gated sperm transfer from the UVD to the SV.Abbreviations LD light-dark - LL constant light - SV seminal vesicle - UVD upper vas deferens     

The effect of inhibitors upon intracellular cyclic AMP levels and chick myoblast differentiation.

The small free-living nematode Caenorhabditis elegans is usually found as a hermaphrodite, but occasionally true males appear in the population. This study provides an account of gonadogenesis in the normal male and in a mutant that is a temperature-sensitive sex transformer.Male and hermaphrodite gonads develop from morphologically identical primordia. The small primordial gonad lies on the ventral side of the worm in the coelomic cavity. The gonadial primordium contains four nuclei at parturition. As this primordium develops in a hermaphrodite, it produces a double-armed, mirror symmetrical gonad that produces first sperm and then eggs. In the male, however, this primordium develops into an asymmetrical structure composed of a ventrally located testis, a loop region, a seminal vesicle, and a vas deferens. The male gonad presents a linear sequence of nuclei in successive stages of spermatogenesis beginning with a mitotic region in the testis, followed by clearly distinguishable stages of meiosis throughout the loop region to the seminal vesicle.A temperature-sensitive sex transformer mutant, tsB202, has been isolated. tsB202 carries an autosomal recessive mutation in linkage group II that at restrictive temperature transforms an XX hermaphrodite into a phenotypic male, complete with a normal male gonad and vestigial external genitalia. These transformed males are classified as pseudomales because they do not exhibit mating behavior. Temperature shift experiments have determined the specific temporal sequences of gonadogenesis, oogenesis, and spermatogenesis. Proper manipulation of the temperature regimen causes the production of intersexes. In one intersex, a male gonad complete with sperm, seminal vesicle, and vas deferens also contains oocytes. In another intersex produced by the complementary temperature shift, a hermaphrodite-shaped gonad develops that produces only sperm and no oocytes.     

Difference of fertilizing capacity between testicular sperm and vas deferens sperm in Cynops pyrrhogaster

The fertilizing capacity was compared between testicular and vas deferens sperm in Cynops pyrrhogaster. The testicular sperm was not capable of fertilizing jelly eggs. In contrast, the vas deferens sperm was already capable of fertilizing the newt jelly eggs. There was no inhibitory factor for fertilizing jelly eggs in the testis. These results suggest that the testicular sperm is immature as to the fertilizing capacity. The testicular sperm gained the fertilizing capacity for the jelly eggs by treatment with Holtfreter's solution or 1/20 strength Holtfreter's solution. The treatment may promote the step of maturation to achieve the fertilizing capacity. The treated testicular sperm did not fertilize dejellied eggs, although vas deferens sperm fertilized dejellied eggs. Therefore, the maturation state of the treated testicular sperm is different from that of vas deferens sperm. Newt sperm may be matured within the vas deferens, as the newt does not have an organ like the mammalian epididymis.     

Development of spermiduct and seminal vesicle during pharate adult of the cabbage armyworm,Mamestra brassicae, (Lepidoptera; Noctuidae)

Summary

Development of the upper vas deferens and seminal vesicle, and the ecdysteroid titers of the cabbage armyworm (Mamestra brassicae) during pharate adult stage were investigated to provide evidence of endogenous control of the sperm movement through the reproductive tract. Apparently, development of the upper vas deferens is initiated when ecdysteroid titers increase after pupation and continues until eclosion. Sperm movement correlates with the decrease of ecdysteroid titers. When ecdysteroid titers remained at low levels, sperm release from the testis was observed one day before eclosion.     

Reproductive capacity of triploid loaches obtained from Hokkaido Island, Japan

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

Erratum to Production of diploid and triploid offspring by inbreeding of the triploid planarian <Emphasis Type="Italic">Dugesia ryukyuensis</Emphasis>

Triploidy has generally been considered to be an evolutionary dead end due to problems of chromosomal pairing and segregation during meiosis. Thus, the formation of tetraploids and diploids from triploid types is a rare phenomenon. In the present study, we demonstrated that inbreeding of the triploid planarian Dugesia ryukyuensis resulted in both diploid and triploid offspring in nature. In the triploids of D. ryukyuensis, chiasmata between homologous chromosomes were observed in both female and male germ lines. This result suggests that both diploid and triploid offspring of this species are produced bisexually by zygotic fusion between sperm and eggs. Hence, this phenomenon may be a novel mechanism in planarian for escaping the triploid state.     

Mechanisms of parasite-induced sex reversal in Gammarus duebeni

The amphipod Gammarus duebeni is host to the feminising microsporidian parasite Nosema granulosis that converts males into functional females. To test the hypothesis that the parasite acts through endocrine disruption we compared the morphology of the gonad and activity of the androgenic gland, which coordinates male sexual differentiation, in infected and uninfected animals. Male gonad consisted of testis, seminal vesicle and vas deferens that was anchored to the genital papilla on segment 7. The androgenic gland was associated with the distal end of the vas deferens. In female and intersex animals the bi-lobed ovary opened into the oviduct at segment 5, vestigial vas deferens and vestigial androgenic gland were retained. The majority of parasitised individuals (38/39) were either phenotypic females or intersexes with fully developed ovaries and an undifferentiated androgenic gland. Our data suggest that the parasite prevents differentiation of the androgenic gland. In further support of this hypothesis, mass spectrometry of a single androgenic gland from males revealed a dominant molecular ion with a mass/charge ratio of 4818.4+H, corresponding to a peptide of androgenic gland hormone from Armadillidium vulgare. In contrast the vestigial androgenic gland from parasitised and unparasitised females showed only low intensity peaks. Our observations demonstrate that the parasite manipulates host sex by preventing androgenic gland differentiation, androgenic gland hormone production and consequently male differentiation. This is in agreement with observations of A. vulgare with inherited Wolbachia infection, suggesting that phylogenetically distant feminisers manipulate hosts through a common mechanism. The high frequency of infection in intersexes (89.3%) suggests that this phenotype results from incomplete feminisation by the parasite.     

黄姑鱼三倍体的诱导、鉴定及其性腺发育特征

研究采用冷休克方法诱导黄姑鱼(Nibea albiflora)三倍体并进行鉴定,进一步采用组织学和生殖相关基因表达分析比较了三倍体黄姑鱼的性腺发育特征。研究结果表明:(1)在受精后2.5min以3℃进行10min的冷休克处理,冷休克处理组的受精率和孵化率分别为(70.31±4.49)%和(21.5±6.63)%,其受精率和孵化率显著低于二倍体对照组;(2)经流式细胞仪倍性检测和染色体核型分析发现,三倍体的DNA含量为二倍体的1.5倍,染色体数目为72条,而二倍体的染色体数目为48条,三倍体的比例为100%;(3)三倍体的生殖腺指数显著低于二倍体,进一步通过组织切片观察发现三倍体的精巢和卵巢发育较二倍体滞后,在12月龄时,二倍体精巢和卵巢处于Ⅴ期,而三倍体精巢和卵巢分别处于Ⅲ期和Ⅰ期;(4)三倍体精巢的dmrt1和vasa基因及三倍体卵巢的cyp19a基因表达量均显著低于二倍体(P<0.05);三倍体卵巢的vasa基因表达量也比二倍体低(P>0.05)。综上结果表明:研究通过冷休克处理成功诱导了黄姑鱼三倍体;三倍体的性腺发育较二倍体滞后,育性明显降低。     

Functional morphology of the male reproductive system in Callichirus major (Crustacea: Decapoda: Axiidea): Evidence of oocytes in the gonad

Callichirus major inhabits the intertidal region of marine ecosystems and it is frequently used as live bait for fishing. This study aimed to describe the functional anatomy of the male reproductive system by microscopic techniques. The animals were collected along the Corujão beach, Piuma—ES, Brazil, and, in laboratory, the males were classified into two phases: immature (IM) and developed (DE) based on the macroscopic characteristics of the gonads. The gonad and vas deferens were dissected for histological routine and histochemical tests. Histologically, it was noted that in both phases, the more distal region of gonads has ovarian characteristics, showing developing oocytes. Also, different male germ cells were identified: spermatogonium (SPG), spermatocytes I and II (SPTCI, SPTCII), initial and final spermatid (IS, FS) and sperm (SPZ). Accessory cells with spherical or pyramidal nuclei were also present inside the testicular lobules. According to the vas deferens structure, three regions can be characterized: proximal (PVD), middle (MVD) and distal (DVD). In the lumen of the vas deferens, a spermatophoric matrix highly reactive for histochemical tests was observed. The presence of female germ cells in males suggests the occurrence of intersexuality or hermaphroditism in this species.     

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp.     

Cloning and expression analysis on a homolog of spermatogonial stem-cell renewal factor in Fenneropenaeus chinensis

The spermatogonial stem-cell renewal factor (SSRF) was named since its function in spermatogonial mitosis was reported in Japanese eel. Our previous study showed that a homolog of SSRF was highly expressed in the ovary of triploid shrimp, but not expressed in the ovary of diploid shrimp. To understand the function of SSRF in shrimp, the full-length cDNA of ssrf gene was cloned from Chinese shrimp Fenneropenaeus chinensis (Fcssrf) and its expression was analyzed. The full length of Fcssrf cDNA was 2588?bp and it contained an open reading frame encoding 450 amino acids. The predicted tertiary structure of FcSSRF was very similar to that of SSRF/eSRS34 from Anguilla japonica and TP/PD-ECGF from Homo sapiens. RT-PCR analysis showed that the Fcssrf was highly expressed in nerve, testis, hepatopancreas, gill, and stomach rather than in ovary. Expression of Fcssrf mRNA was not detected during embryonic stages and larval stages, from the nauplii to the post-larvae stage, in diploid, and triploid shrimp. However, it began to be expressed in juvenile stages (June–September) in diploid and triploid shrimp. Immunohistochemical analyses showed that FcSSRF was identified in both the diploid testis and triploid ovary. We inferred that the Fcssrf might be related to testis development.     

Influence of environmental parameters on growth pattern and population structure of Carassius auratus gibelio in Eastern Ukraine

Population structure and growth parameters of Prussian carp (Carassius auratus gibelio) were studied in 12 freshwater ecosystems of the Donbass region (Eastern Ukraine). These ecosystems differed significantly with respect to their surface area, water transparency and annual concentrations of phosphorus and nitrogen. Amongst the studied ecosystems, diploid males and females as well as a smaller percentage of triploid females were found. The population structure of C. a. gibelio varied significantly in terms of the percentage of triploids and sex ratio amongst diploids. A considerable proportion of triploid females (>10%) was found in four ecosystems with intermediate surface area (38–50 ha) and relatively high growth rate of specimens. The sex ratio amongst diploids was significantly female-biased in seven of 12 ecosystems, including those where triploids were present in considerable numbers. The growth parameters of Prussian carp were significantly correlated with ecosystem characteristics, but the growth patterns of diploids and triploids were not significantly different from each other. The restricted distribution of triploid specimens suggests that the triploid form might counter diverse environmental challenges, whereas the diploid form of Prussian carp seems to be efficiently adapted to a wide range of ecosystem conditions.     

Histological and immunocytochemical study of deferens ducts in the Chinese rat snake (Zaocys dhumnades)

To investigate the relationship between structure and function of the deferens ducts in the Chinese rat snake (Zaocys dhumnades), morphological changes within an annual cycle were observed by routine histological techniques. Also, the correlation of androgen receptor (AR), estrogen receptor (ER), progesterone receptor (PR) and aromatase (Ar) expressions in the vas deferens and testis were studied immunohistochemically. To confirm that the sperm and the spherical structure existed in deferens ducts, we also used routine histological technique observed deferens ducts in the Striped-tailed rat-snake (Elaphe taeniura), Red-banded snake (Dinodon rufozonatum), and Tiger-spotted neck-troughed snake (Rhabdophis tigrina lateralis). The results showed that the deferens ducts of the Chinese Rat Snake were composed of efferent duct, epididymal duct and vas deferens. Efferent duct contained sperm from August-October, and the sperm were observed in the epididymal duct from August-the following January. Throughout the year (except July) a large number of sperm were present in the vas deferens where a previously unreported spherical structure formed by spermatids was observed, which showed no significant differences in the IOD values of AR-, ER-, PR- and Ar-immunoreactivities. Since the spermatids in the spherical structure were undergoing spermatogenesis and this phenomenon also existed in the Striped-tailed rat-snake and Red-banded snake, the term, seminiferous spherule, was named for this spherical structure This study demonstrated that the testis was the main site for snake spermiogenesis, and the seminiferous spherule in vas deferens was the other Both the epididymis and vas deferens stored sperm; however, the vas deferens was the main organ for sperm storage.     

Comparative biochemical composition in gonad and adductor muscle of triploid and diploid catarina scallop (Argopecten ventricosus Sowerby II, 1842)

Biochemical components of gonad and adductor muscle for diploid and triploid catarina scallop, Argopecten ventricosus, were evaluated and compared at four periods in 1 year (January, April, June, and October). Two comparisons were done. The first one compared an untreated control (diploid) vs. a triploidy-treated group for which the percentage of triploids was 57%. The second comparison was done on a group derived from within the triploidy-treated group, separating diploids (internal control) from triploids ('putative triploids'). Regardless of which comparison, in the gonad diploid scallops had larger concentrations of proteins, carbohydrates, lipids, and acylglycerols than triploid scallops. This reflects the maturation processes in diploid scallops vs. the sterility seen in most triploid scallops, and it is particularly supported by the consistently larger concentration of acylglycerols in gonads of diploids than in triploids. The gonad index of the internal control (diploid) group was significantly larger than that seen in the putative triploid group at all sampling periods but October, when none of the gonad biochemical components were different between ploidy groups.Triploid scallops had a significantly larger muscle index than diploids from April to October. This can be caused by a larger gain in muscle tissue in triploids than diploids from January to June. However, there were no consistent differences in any of the biochemical components evaluated in adductor muscle of diploids and triploids. The use of freshly ingested food rather than reserve mobilization from muscle in diploids is suggested by these results. Nutrients derived from ingested food are apparently used for muscle growth in triploids, whereas in diploids those nutrients serve primarily for gonad development. The importance of freshly ingested food for maintenance and growth is suggested because the decline in biochemical components seen in October in both muscle and gonad was paired with a decline in weight of those two organs, especially when the control groups are considered, but a decrease was also evident for the triploid groups. This may have been caused by the presence of El Ni?o, with its characteristic high water temperatures and low productivity.     

Ultrastructure of the sperm of the deep-sea decapod Aristeus antennatus

The sperm of Aristeus antennatus presents notable differences in relation to the two basic models of decapod crustaceans considered to date. Basically, it does not present a single appendage, or spike, characteristic of the so-called unistellate sperm of the suborder Dendrobranchiata and the infraorder Caridea of the suborder Pleocyemata. Nor does it have arms or spikes characteristic of the multistellate sperm that all belong to the Pleocyemata group. The spermatozoa of A. antennatus are composed of a nucleus and an electron-dense acrosome, which have the polarity of multistellate sperm. A number of mitochondria and vesicles are present in the cytoplasm, located between the acrosome and the nucleus. In accordance with the fine structural details, the morphology of the sperm has been described at two different levels of the male gonad, the vas deferens and terminal ampulla, and in the spermatophore placed in the thelycum of the female. Three ultrastructural changes in the acrosome (unorganized structures, tubular organization, disintegration process) and the nucleus (uncondensed, condensed, and compact) are present along the male reproductive apparatus. They first appear in a non-organized manner at the level of the vas deferens, subsequently undergo a process of structural configuration in the ampulla, and finally show disorganized structures in the spermatophore. J. Morphol. 234:79–87, 1997. © 1997 Wiley-Liss, Inc.