Leaf Frog Research Articles

Reproduction in the Mexican Leaf Frog,Pachymedusa dacnicolor: VI. Presence and Distribution of Multiple GnRH Forms in the Brain

The presence and distribution of gonadotropin-releasing hormone (GnRH) has been investigated in the Mexican leaf frog,Pachymedusa dacnicolor,brain during development and in the adult. The ontogenetic pattern of GnRH neurons illustrates their extracranial as well as intracranial sites. Immunohistochemical analysis indicates that GnRH-immunoreactive neurons appear during the metamorphic climax. They are located in the mesencephalon and subsequently other GnRH neurons appear in the peripheral terminal nerve and anterior preoptic area of the brain. Use of specific antisera and homologous combined with heterologous preabsorption tests indicate that mammalian and chicken GnRH-II-like peptide-containing neurons are differentially located within the brain, the former in the anterior preoptic area and peripheral terminal nerve and the latter in the mesencephalon. HPLC and RIA data suggest the presence of three forms of immunoreactive GnRH in theP. dacnicolorbrain. A mammalian GnRH-like molecule and a chicken GnRH-II-like molecule are present. A third form, suspected to be [hydroxyproline9]mGnRH elutes before the mammalian GnRH.

Reproduction in the Mexican leaf frog, Pachymedusa dacnicolor: V. Immunohistochemical localization of gonadotropin-releasing hormone in the brain

The presence and distribution of immunoreactive gonadotropin-releasing hormone (GnRH) in brains of adult male and female Pachymedusa dacnicolor has been studied immunohistochemically using antisera against mammalian, chicken-II, and salmon GnRHs. The distribution map of the immunoreactive-GnRH elements in the brain of P. dacnicolor is extremely simple, being limited to the anterior preoptic area-infundibulum-median eminence circuit. No sex- or reproductive status-related difference in either the distribution pattern or intensity of immunoreaction was revealed in this study. This is also the first immunohistochemical evidence of the presence of different structural forms of GnRH in the brain of an amphibian.

Mexican leaf frog (Pachymedusa dacnicolor) as a model in endocrine research

Mexican leaf frog (Pachymedusa dacnicolor) as a model in endocrine research

Nuclear shaping in spermatids of the Thai leaf frog Megophrys montana.

Transmission electron microscopy of Thai leaf frog testis revealed a unique pattern of spermatid nuclear morphogenesis. Chromatin condenses into a continuous cylindrical coil within a roughly spherical nucleus. Later the nuclear membrane conforms to the contours of the uncoiling nuclear contents. In the mature sperm, the long, tapering nucleus is helically shaped. This developmental sequence occurs in the absence of a microtubular manchette, raising questions about the role of this structure in nuclear shaping in spermatozoa of other species.

Reproduction in the Mexican leaf frog, Pachymedusa dacnicolor: III. The female

The female of the Mexican leaf frog, Pachymedusa dacnicolor, displays a marked annual ovarian cycle. This consists of a long period of vitellogenic stasis, starting in early fall, soon after the breeding season, and ending in early spring. Oogonial proliferation continues throughout the year and new previtellogenic follicles are formed continuously. During the period of vitellogenesis, from spring to early summer, early, advanced, and postvitellogenic follicles are all found together, a situation that continues through the breeding season. This is correlated with the fact that a breeding female can lay three or more clutches per season. Breeding can begin as early as June and end as late as early September, with peak spawning activity taking place during July and August. An examination of the ovarian hormone secretion pattern in P. dacnicolor during the year revealed that plasma levels of testosterone and estradiol correlated with ovarian growth and attained highest levels in amplectant and ovulating females. Both hormones showed quite similar plasma levels and patterns of change during the annual reproductive cycle. Lowest plasma levels of testosterone and estradiol were found during fall and winter, in females possessing exclusively previtellogenic ovarian follicles. Plasma progesterone levels were maintained at a very low level throughout the year, except for the ovulatory surge, when amplectant and ovulating females may show a three- to sixfold increase. Plasma androstenedione showed a low peak during this phase of the reproductive cycle. Plasma levels of 5α-dihydrotestosterone were 13 to 30 times lower than plasma testosterone levels. The potential roles of these gonadal steroids in controlling ovarian activity and reproduction are discussed briefly.

Reproduction in the mexican leaf frog, Pachymedusa dacnicolor: II. The male

The Mexican leaf frog, Pachymedusa dacnicolor, an inhabitant of the semiarid, subtropical Mexican lowlands, displays a well-defined seasonal testicular cycle. Testis weight seems to be a reliable index of the reproductive status of the animal and plasma levels of androgens (testosterone, T; 5α-dihydrotestosterone, DHT; androstenedione, A) correlate not only with testicular growth, but with callosity development, reproductive behavior, and breeding. During the fall and winter, testis weight reaches its minimum as do plasma concentrations of T, DHT, A, and estradiol-17β (E). Plasma levels of progesterone (P) are maintained at a very low level throughout the year. During the fall and winter, spermatogenesis is almost entirely absent and the callosities are white and smooth. No signs of reproductive behavior (calling and amplexus) are evident. Late spring marks the initiation of spermatogenesis, testis weight increase, darkening of callosities, and increase in plasma levels of androgens. The magnitude of callosity development and the onset of calling and amplectant behavior are correlated with a great rise in plasma androgen levels. Although plasma T concentrations were higher than plasma levels of DHT and A, this androgen failed to stimulate the development of callosities and calling behavior in successfully castrated males.

Characterization of blue and yellow pigments in eggs of the Mexican leaf frog

Characterization of blue and yellow pigments in eggs of the Mexican leaf frog

Yolk pigments of the Mexican leaf frog.

Eggs of the Mexican leaf frog contain blue and yellow pigments identified as biliverdin and lutein, respectively. Both pigments are bound to proteins that occur in crystalline form in the yolk platelet. The major blue pigment is biliverdin IX alpha. The eggs vary in color from brilliant blue to pale yellow-green depending on the amount of each pigment. These pigments may provide protective coloration to the eggs.

Electron microscopic study of leaf frog melanophore differentiation in culture, with special reference to allopurinol effect on pterorhodin formation

Melanophores in the skin from metamorphic climax tadpoles can produce pterorhodin autonomously in culture. Thyroxine neither accelerated synthesis nor increased the amount of pterorhodin deposited. Cytoplasmic events attendant to the transformation of the larval melanosome in vitro were like those that occur in vivo. These include the uplifting of the melanosomal limiting membrane, the presence of many small cytoplasmic vesicles that can fuse with the limiting membrane, and the gradual deposition of pterorhodin flocculations on the melanin surface starting at stage 25, the completion of metamorphic climax. Allopurinol inhibits pterorhodin synthesis in melanophores in skin culture. It is proposed that the autonomous transformation of larval melanosomes to the pterorhodin-containing adult type is based upon a preprogramming that occurs near the onset of metamorphic climax. This preprogramming involves the uptake of elements necessary for pterorhodin biosynthesis and implicates current theory concerning the origin of pigment cells.

The differentiation of leaf frog melanophores in culture

During early larval development of mexican leaf frog, Pachymedusa dacnicolor, dermal melanophores are typically black, but a few brown melanophores appear about stage 18. During metamorphic climax brown melanophores begin to dominate and by stage 23 they are the exclusive type. The synthesis of the pigment, a red pteridine-dimer, occurred in the melanophores of organ-cultured back skin isolated from tadpoles. The development of this pigmentation in organ culture was independent of thyroxine at least after the onset of metamorphic climax (stage 19). Isolated melanophores in cell culture conditions proliferated only in the presence of MSH, but even these proliferating melanophores, if derived from larvae of stage 21 or younger, showed no indication of pterorhodin synthesis. Melanophores derived from a stage 22 larva and cultured for 18 days synthesized and deposited pterorhodin.

Hormone‐induced pigment translocations in amphibian dermal iridophores, in vitro: Changes in cell shape

Hormone-induced pigment translocation studies were conducted at both the light and electron microscopic levels on cultured dermal iridophores from the Mexican leaf frog, Pachymedusa dacnicolor. Two distinct types of dermal iridophores were characterized which differed in (1) their in vivo locations, (2) their overall morphologies in vitro, (3) their responses to alpha-MSH, ACTH, c-AMP or theophylline, (4) their physical alterations of light, and (5) certain ultrastructural features. One iridophore (Type I) was found to be physiologically responsive to the above hormones or agents by a reversible retraction of cellular processes and a thickening of the cell body, an event which is inhibited by cytochalasin B. The other iridophore (Type II) appeared to be unresponsive. Type I iridophores contain cube-like pigmentary organelles, refractosomes, while Type II iridophores contain larger, bar-shaped refractosomes. In addition, both iridophore types contain 60 and 100 A microfilaments as well as microtubules. By in large, micorfilaments were found within microvilli, beneath and parallel to the plasma membrane and in the perinuclear region. Occasionally, bundles of 100 A microfilaments were found between layers of refractosomes in Type I iridophores. These results are discussed in relation to hormone-induced changes in cell shape.

Melanophore differentiation in leaf frogs

In melanosomes of Pachymedusa (Agalychnis) dacnicolor and other leaf frogs, the pteridine dimer, pterorhodin, is found in fibers concentric to a kernel of eumelanin. The kernel is a remnant mature larval melanosome that is renovated at metamorphic climax and on which pterorhodin is deposited at the completion of metamorphosis. When pterorhodin is initially detected by chemical means in the skin of stage 25 individuals, flocculations of fibers are first seen in melanosomes. At stage 25+ a more intense chemical demonstration is accompanied by larger flocculations. These larval melanosomes are smaller than those of other vertebrates, but are formed from classical premelanosomes. At metamorphosis, the melanosome's limiting membrane is elevated from the surface of the eumelanin, and small spheroids are seen in the space and on the pigment surface. The Golgi complex is extremely active, numerous small vesicles are seen in the cytoplasm, and blebbing of the outer membrane of the nuclear envelope occurs. At stage 25 small thick-walled vesicles appear in the cytoplasm in contact with or within the melanosome; they may represent the transport of pterorhodin or elements necessary for its formation.

Heterochromatin dispersion and blebbing of the nuclear membrane induced by melanophore stimulating hormone.

Stimulation (in vivo and in vitro) of dermal melanophores of the leaf frog, Agalychnis dacnicolor, by melanophore stimulating hormone (MSH) elicits two responses in addition to the dispersion of melanosomes: (1) dispersion of heterochromatin; and (2) blebbing of the outer membrane of the nuclear envelope. The latter leads to the formation of dilated rough endoplasmic reticulum with the involvement of 80–100 A microfilaments. N6,O2-dibutyryl adenosine 3′,5′-monophosphate (db-cAMP) elicits a similar response. Actinomycin D prevents both heterochromatin dispersion and membrane blebbing while cytochalasin B (CB) prevents only the latter.

An analysis of color change phenomena in the leaf frog, Agalychnis dacnicolor

AbstractIn vivo and in vitro studies of integumental color change in the leaf frog, Agalychnis dacnicolor, are reported. A color index is established for the assessment of color change in the absence of melanophore visibility. Green leaf frogs adapt rapidly to background by controlling MSH release. Some members of the population are always brown regardless of background. Naturally brown specimens respond to localized reductions in illumination by becoming green in the affected area. Either skin or chromatophores in these areas are photosensitive. Isolated skins darkened with theophylline are not photosensitive; however, masking of areas of isolated skins prevented darkening of the masked area. Contact between skin and mask may inhibit darkening by physically preventing melanosome dispersion. Neither melanosome aggregation nor dispersion occur in isolated skins incubated in oxygen‐free media. Experiments involving denervation and electrical stimulation both in vivo and in vitro fail to implicate a chromatophore control by innervation.

Localization of rhodomelanochrome in melanosomes of leaf frogs

AbstractRhodomelanochrome, a new pigment extracted from the skin of leaf frogs is localized in the fibrous portion of the large melanosome typical of these frogs. Skin extracted with NaOH contains melanosomes possessing a diminished fibrous component and melanophores in thin sections of skin treated with NaOH are filled with melanosomes that lack large areas of the fibrous zone. The appearance of the electron‐dense core of the melanosome of untreated skin is unchanged and it is suggested that the leaf frog melanosome is composed of an inner kernel of eumelanin and an outer fibrous component of rhodomelanochrome.

Color changes, unusual melanosomes, and a new pigment from leaf frogs.

Melanosomes of phyllomedusid frogs are unusually large and are composed of an amorphous matrix of thick fibers. Their hitherto undescribed dark red pigment is neither phaeomelanin nor eumelanin, but seems to be related to melanins. Melanophores of at least one of these species, Agalychnis dacnicolor, exhibit color change in direct response to illumination, and it is suggested that these chromatophores are innervated.