Wnt4 expression in the differentiating gonad of the frog Rana rugosa

Wnt4, a member of the Wnt family, is known to influence the sex-determination cascade. In mice having a targeted deletion of Wnt4, masculinization occurs in XX pups. Therefore, in addition to Sry, Wnt4 is also involved in sex determination in mice. In humans, a chromosomal duplication of the WNT4 causes feminization of XY-individuals. Thus, for better understanding of the mechanism of sex determination in vertebrates, it is necessary to examine the expression of Wnt4 at early gonadal development stages in non-mammalians. We first isolated the Wnt4 cDNA from the tetsis of the frog Rana rugosa. R. rugosa Wnt4 had a high similarity (>86%) at the amino acid level with zebra fish, chicken, mouse, and human Wnt4s. We next employed RT-PCR analysis to examine whether Wnt4 was expressed in a sexually dimorphic fashion at early stages of gonadal development in R. rugosa. Wnt4 was transcribed first in the embryos at the late gastrula stage, and its expression was maintained until the indifferent gonad differentiated into a testis or an ovary. Wnt4 expression in the differentiating gonad appeared in a non-sexually dimorphic pattern. The results, taken together, suggest that Wnt4 is highly conserved through evolution, and that its expression in the indifferent gonad takes place with no sexual dimorphism. Thus, Wnt4 is not a key factor to initiate the development of a testis or an ovary from the indifferent gonads in R. rugosa. However, this gene probably forms part of a gonadal-development pathway in this species.     

Evidence for two successive pericentric inversions in sex lampbrush chromosomes of Rana rugosa (Anura: Ranidae)

The objective of this study was to clarify the course of inversions by which a ZW sex chromosome dimorphism has become established in Rana rugosa. Fortunately, R. rugosa preserves three different forms of sex chromosomes in the several isolated populations. In both males and females, the homomorphic sex chromosomes from Hiroshima were closely similar to Z, while those from Isehara were slightly different from the Z. Females from Hirosaki demonstrated heteromorphic sex chromosomes. In this study, the configuration and pairing behavior of sex lampbrush chromosomes were examined in the female offspring produced from a cross between a female from Hiroshima and a male from Isehara, as well as the female offspring of a female from Hirosaki and the male from Isehara. For the sex lampbrush chromosomes from Hiroshima and Isehara, chiasmata were exclusively formed between the distal regions of the long arms of one sex chromosome and the terminal regions of the short arms of the other. As a result, landmarks arranged in reverse order were observed in the achiasmatic regions of these chromosomes. For the sex lampbrush chromosomes from Isehara and Hirosaki, on the other hand, chiasma formation was mainly confined to the lower half of the chromosomes corresponding to the long arms, and the landmarks in the achiasmatic regions of these chromosomes were disposed in the opposite direction to each other. These results seem to indicate that in the primitive sex chromosomes of the Hiroshima type two pericentric inversions occurred, leading to the differentiation of the W chromosomes. This is the first report to substantiate the process of sex chromosome differentiation experimentally. Received: 10 November 1996; in revised form: 22 April 1997 / Accepted: 24 April 1997     

Fine structure of the dorsal lingual epithelium of the frog, Rana rugosa

Scanning and transmission electron microscopy was employed to investigate the ultrastructure of the lingual dorsal epithelial cells of the frog, Rana rugosa. The specimens for scanning electron microscopy were prepared by a method that involved osmium postfixation and treatment with acid to remove extracellular material that adhered to the surface of the tongue. Over almost the entire dorsal surface, filiform papillae, consisting of a large number of non-ciliated cells with microridges and a very small number of ciliated cells, were compactly distributed. Fungiform papillae were scattered among these filiform papillae. A round sensory disk was located on the top of each fungiform papilla. Each sensory disk was encircled by a band of ciliated cells. Transmission electron microscopy revealed that a large part of the filiform papillar epithelium was composed of cells that contained numerous electron-dense granules. These cells were coincident with the non-ciliated cells observed by scanning electron microscopy. In these cells, the nucleus was located on the basal side, and the ergastoplasm was well-developed on the basal side of the nucleus.     

辽宁产粗皮蛙的染色体组型研究(图版Ⅳ,下)

用骨髓细胞制片法分析了粗皮蛙的染色体组型 ,结果表明其二倍体染色体数为 2 6 ,可配成 13对 ,有5对大型染色体 (相对长度 >9)和 8对小型染色体 (相对长度 <6 5 ) ,其中 ,第 1、 5、 6、 7、 8对为中部着丝点染色体 ,第 12对为端部着丝点染色体 ,第 2、 3、 4、 9、 10、 11、 13对为亚中部着丝点染色体 ,第 4对为性染色体 ,属XY型 ,X染色体为亚中部着丝点 (相对长度为 10 70 ,臂比指数为 1 72 ) ,Y染色体为亚中部着丝点(相对长度为 12 83,臂比指数为 2 0 2 )。     

The origin and differentiation of the heteromorphic sex chromosomes Z, W, X, and Y in the frog Rana rugosa, inferred from the sequences of a sex-linked gene, ADP/ATP translocase

Sex chromosomes of the Japanese frog Rana rugosa are heteromorphic in the male (XX/XY) or in the female (ZZ/ZW) in two geographic forms, whereas they are still homomorphic in both sexes in two other forms (Hiroshima and Isehara types). To make clear the origin and differentiation mechanisms of the heteromorphic sex chromosomes, we isolated a sex-linked gene, ADP/ATP translocase, and constructed a phylogenetic tree of the genes derived from the sex chromosomes. The tree shows that the Hiroshima gene diverges first, and the rest form two clusters: one includes the Y and Z genes and the other includes the X, W, and Isehara genes. The Hiroshima gene shares more sequence similarity with the Y and Z genes than with the X, W, and Isehara genes. This suggests that the Y and Z sex chromosomes originate from the Hiroshima type, whereas the X and W chromosomes originate from the Isehara-type sex chromosome. Thus, we infer that hybridization between two ancestral forms, with the Hiroshima-type sex chromosome in one and the Isehara-type sex chromosome in the other, was the primary event causing differentiation of the heteromorphic sex chromosomes.      

Sex from W to Z: evolution of vertebrate sex chromosomes and sex determining genes

Sex determination in major vertebrate groups appears to be very variable, including systems of male heterogamety, female heterogamety and a variety of genetic and environmental sex determining systems. Yet comparative studies of sex chromosomes and sex determining genes now suggest that these differences are more apparent than real. The sex chromosomes of even widely divergent groups now appear to have changed very little over the last 300+ million years, and even independently derived sex chromosomes seem to have followed the same set of evolutionary rules. The sex determining pathway seems to be extremely conserved, although the control of the genes in this pathway is vested in different elements. We present a scenario for the independent evolution of XY male heterogamety in mammals and ZW female heterogamety in birds and some reptiles. We suggest that sex determining genes can be made redundant, and replaced by control at another step of a conserved sex determining pathway, and how choice of a gene as a sex switch has led to the evolution of new sex chromosome systems. J. Exp. Zool. 290:449-462, 2001.     

The hypothalamo-hypophysial system of the frog Rana temporaria

Summary The median eminence (ME) of the adult frog, Rana temporaria, was studied by means of electron microscopy including quantitative electron-microscopic autoradiography. In frogs captured in May and June numerous peptidergic neurosecretory fibres extending via the internal zone to the pars nervosa display large swellings containing few granules, mitochondria, neurotubules and cisternae of the smooth endoplasmic reticulum. In addition, few secretory globules up to 1.5 m in diameter occur in these varicosities. In animals collected during the autumn period many of these neurosecretory swellings filled with neurosecretory granules and polymorphic inclusions resemble Herring bodies. Three types of granule-containing neurosecretory fibres were observed in the external zone (EZ) of the ME of adult R. temporaria. Peptidergic A₁- and A₂-type fibres are characterized by granules 150–220 nm and 100–160 nm in diameter, respectively. Monoaminergic fibres of type B with granules approximately 100 nm in diameter represent 50% of all neurosecretory elements in the EZ of the frog ME; 12% of the total number of granule-bearing axons in the EZ actively taking up radiolabelled 5-hydroxytryptophan are thought to be serotoninergic terminals. Neurosecretory terminals of all types and glial vascular endfeet establish direct contacts with the perivascular space of the primary portal capillaries. Some neurosecretory terminals are separated from the lumen of the third ventricle by a thin cytoplasmic lamella of tanycytes. The possible physiological significance of this structural pattern is discussed.     

The yellow mutation in the frog Rana rugosa: pigment organelle deformities in the three types of chromatophore

Crossing experiments revealed that a single recessive gene mutation (yellow) gives rise to the yellow phenotype of Rana rugosa in Japan. Ultrastructural observation of dermal chromatophores showed that the pigment organelles; melanosomes, pterinosomes, and reflecting platelets, all had structural deformities. This suggests that the yellow gene acts at the level of a primordial pigment organelle common to the three types of chromatophore.     

Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica

Japonicin-1 (FFPIGVFCKIFKTC) and japonicin-2 (FGLPMLSILPKALCILLKRKC), two peptides with differential growth-inhibitory activity against the Gram-negative bacterium, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, were isolated from an extract of the skin of the Japanese brown frog Rana japonica. Both peptides show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from the skins of Ranid frogs. Circular dichroism studies, however, demonstrate that japonicin-2 adopts an alpha-helical conformation in 50% trifluoroethanol in common with many other cationic antimicrobial peptides synthesized in amphibian skin. Peptides belonging to the brevinin-1, brevinin-2, and tigerinin families, previously identified in the skins of Asian Ranid frogs, were not detected but a temporin-related peptide (ILPLVGNLLNDLL.NH(2); temporin-1Ja), that atypically bears no net positive charge, was isolated from the extract. The minimum inhibitory concentrations (MICs) of the peptides against E. coli were japonicin-1, 30 microM; japonicin-2, 12 microM; and temporin-1Ja > 100 microM. The MICs against S. aureus were japonicin-1, > 100 microM; japonicin-2, 20 microM; and temporin-1Ja, > 100 microM.     

Sex reversal and primary sex ratios in the common frog (Rana temporaria)

Sex reversal has been suggested to have profound implications for the evolution of sex chromosomes and population dynamics in ectotherms. Occasional sex reversal of genetic males has been hypothesized to prevent the evolutionary decay of nonrecombining Y chromosomes caused by the accumulation of deleterious mutations. At the same time, sex reversals can have a negative effect on population growth rate. Here, we studied phenotypic and genotypic sex in the common frog (Rana temporaria) in a subarctic environment, where strongly female‐biased sex ratios have raised the possibility of frequent sex reversals. We developed two novel sex‐linked microsatellite markers for the species and used them with a third, existing marker and a Bayesian modelling approach to study the occurrence of sex reversal and to determine primary sex ratios in egg clutches. Our results show that a significant proportion (0.09, 95% credible interval: 0.04–0.18) of adults that were genetically female expressed the male phenotype, but there was no evidence of sex reversal of genetic males that is required for counteracting the degeneration of Y chromosome. The primary sex ratios were mostly equal, but three clutches consisted only of genetic females and three others had a significant female bias. Reproduction of the sex‐reversed genetic females appears to create all‐female clutches potentially skewing the population level adult sex‐ratio consistent with field observations. However, based on a simulation model, such a bias is expected to be small and transient and thus does not fully explain the observed female‐bias in the field.     

Efferent system in the retina of the frog, Rana catesbiana.

Single units were recorded through glass microelectrodes placed on the optic disk or on the retina of the opened eye of the frog (Rana catesbiana). Units were classified as A-, B-, and C-fibers according to conduction velocities. By the method of collision between naturally elicited and electrically elicited impulses, many of the B-fibers and some A- and C-fibers, which showed unusual behavior to photic stimulation, were found to be efferent fibers. Retinal effects of the efferent nerves were studied by repetitive stimulation and cooling of the optic nerve. The effects were found to be both inhibitory and excitatory.     

The ZZ/ZW sex-determining mechanism originated twice and independently during evolution of the frog, Rana rugosa

The Japanese frog, Rana rugosa, has two distinct sex chromosome types, XX/XY and ZZ/ZW. These two types are found in localized groups, separated geographically by a boundary area predicted to lie somewhere around Lake Biwa in central Japan. To determine this precise boundary, the heterogametic sex of 18 populations around Lake Biwa was examined by genotyping sex-linked genes. Phylogenetic relationships between the populations were also analyzed using mitochondrial 12S rRNA gene. Results showed that the Suzuka-Kii mountain range located east of Lake Biwa separated the XX/XY populations from the ZZ/ZW populations. Unexpectedly, from a phylogenetic perspective, the ZZ/ZW populations around Lake Biwa belonged not to the main ZW group but to the XY group. The authors propose that the ZZ/ZW populations around Lake Biwa diverged secondarily from the XX/XY group through a change of heterogametic sex, eventually forming a new group. This group was thus named the 'Neo-ZW group'. As the main ZW group inhabiting northwestern Japan is known to have a different male heterogametic origin, this finding shows that change of heterogametic sex from male to female may have occurred twice, and independently, during the frog speciation.     

泽蛙的性腺分化及温度对性别决定的影响

通过组织学方法观察了泽蛙(Rana limnocharis)原始生殖细胞(PGCs)的迁移、原始性腺的形成和性腺分化,并且探讨在不同的培育温度条件下性腺分化的差异。泽蛙的性腺分化有其特殊性:生殖嵴形成时,其中既有体细胞,又有原生殖细胞;无论原始性腺是分化成为精巢还是卵巢,其中都出现一个初生性腔。蝌蚪孵化后的17-34d(Gosner 26-38期)为性腺分化的敏感时期。在蝌蚪孵化后的第2d(Gosner 25期),分别用不同水温18℃±1℃、30℃±1℃、32℃±1℃、34℃±1℃培育蝌蚪,直至完成变态幼蛙(Gosner 46期)形成。自然水温23℃-25℃为对照。对照组的雌、雄性比接近1∶1(1∶1.06);18℃±1℃实验组的雌、雄比例为1.83∶1,雄性率仅35.1%(P<0.01);从30℃±1℃实验组起,雄性率提高,34℃±1℃实验组的雄性率达74.0%(P<0.01)。较高的培育温度可使泽蛙蝌蚪性别分化趋向雄性,而较低的培育温度则使蝌蚪雌性化。泽蛙的性别分化属于温度依赖型性决定(TSD)。当前全球性气候变暖对两栖类性比的稳定存在着威胁。     

Isolation and structural characterization of novel Rugosin A-like insulinotropic peptide from the skin secretions of Rana saharica frog

Skin secretions of Rana saharica were evaluated for the isolation and characterization of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase HPLC yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11cells, fractions 36-43, 46-54 and 57-63 showed the significant 2-8-fold increase in insulin-releasing activity (P<0.001) compared with 5.6mM of glucose alone. A pool of fractions 36-43 was subsequently rechromatographed to 28 homogenous peaks out of which 7 were capable of subsequent 1.5-3-fold increase in insulin release (P<0.001). Structural analysis of the non-toxic peptides with greatest insulin-releasing activity was performed by mass spectrometry and Edman degradation. Mass spectrometry analysis of two peaks indicated the molecular masses of 1892.6 and 2930.8Da. The sequence of the 1892.6-Da peptide was determined as KGAAKGLLEVASCKLSKSC, which has 68% homology with Rugosin A originally isolated from the skin secretion of Rana rugosa. A partial N-terminal sequence was determined for the 2930.8-Da peptide as AVITGACERDVQCGGGTCCAVSLI.... These data indicate that the skin secretions of Rana saharica frogs contain novel peptides with insulin-releasing activity.     

Lens structural disorders in the leopard frog (Rana pipiens) supplied to the laboratory from wild populations.

Examination of lenses of 48 leopard frogs, Rana pipiens, from three commercial suppliers disclosed a high incidence of histologic aberrations. Only four of 17 lenses that appeared totally clear under the dissecting microscope were found to be free of discernible abnormalities on histologic examination. Deviations were most common in the area of elongation. Because of these findings, the applicability of frog lenses for cellular investigations was questioned.     

DNA replication and inactivation patterns in structural abnormality of sex chromosomes

Summary High resolution chromosome analysis and bromodeoxyuridine (BrdUrd) incorporation have been applied to study patterns of chromosomal replication (inactivation) in two cases of unbalanced X-autosome translocations, seven cases of X and Y chromosome rings or fragments, and five cases of dicentric isochromosomes (Xq). Our results indicate the following: (1) In (X-A) translocations, detailed replicational analysis of the translocated autosomal segment is informative. Absence of spreading effect and partial-incomplete spreading effect are the most common observations. (2) Sex chromosome derived fragments and rings can be differentiated based on their replicational features. (3) Dicentric isochromosomes (Xq) can be classified based on intercentromeric distances, replicational asynchrony, and centromere inactivation. (4) A correlation between intercentromeric distance and degree of 45,X mosaicism was observed in dicentric i(Xq) chromosomes.Evidence for spreading effect based on our results and on the review of the literature has been critically analyzed and general rules in evaluating spreading effects (SE) proposed. The cytologic detection of active regions on the late replicating X chromosome and the inactivation capacity of the juxtacentromeric region of Xp is evaluated. It is proposed that centromere suppression and underreplication are related phenomena. Finally, the analysis of informative replicational stages is emphasized and the application of their analysis in basic and clinical cytogenetics demonstrated.