Histology and histochemistry of androgen-stimulated nuptial pads in the leopard frog, Rana pipiens, with notes on nuptial gland evolution

M.S. Epstein and D.G. Blackburn

Abstract: Nuptial pads are digital specializations of male frogs that cycle with the reproductive season and are considered to function in mating. Glandular secretions of the nuptial pads were analyzed histochemically in androgen-stimulated overwintering leopard frogs, Rana pipiens, to provide information on gland function and physiological control. In castrated and sham-operated male frogs treated with testosterone cypionate, the secretory product of the nuptial-gland epithelium stained positive for carbohydrates and proteins, yet negative for lipids and glycogen. Secretions also stained positive for tyrosine residues and negative for acidic mucosubstances, sulphated mucosubstances, tryptophan, and cystine. Castration prior to hormone treatment had no effect on gland staining properties, and glands of cholesterol-treated castrates and intact controls appeared to be inactive cytochemically. Nuptial glands of frogs treated with 5-a-dihydrotestosterone were histologically similar to those of frogs treated with testosterone cypionate. Nuptial glands share structural and functional characteristics with integumentary mucous glands, and may have been modified evolutionarily from that parent gland population.

Resume : Les coussinets nuptiaux representent des modifications specialis6es des doigts chez les grenouilles miles, structures dont l'apparition cyclique suit la saison de reproduction et qui jouent probablement un r61e dans I'accouplement. Les secretions glandulaires des coussinets ont ete soumises a des analyses histochimiques chez des Grenouilles-leopards, Rana pipirns, stimul6es aux androgknes pendant l'hiver, ce qui a permis d'obtenir des informations sur le fonctionnement des glandes et le contr6le physiologique. Des miles castres et des miles soumis a une operation simulee ont ete traites au cypionate de testosterone; la coloration du produit de secretion de l'epithelium des glandes nuptiales a revel6 la presence d'hydrates de carbone et de proteines, mais pas celle de lipides ou de glycogkne. Les colorants ont kgalement montre l'existence de residus de tyrosine, mais pas de mucosubstances acides, des mucosubstances sulphatees, de tryptophane ou de cystine. La castration avant le traitement aux hormones n'a pas eu d'effet sur les proprietes d'absorption de colorants des glandes et les glandes des castrats traites au cholesterol et celles des miles temoins sains semblaient inactives cytochimiquement. Les glandes nuptiales de grenouilles traitees li la 5-a-dihydrotestosterone etaient histologiquement semblables h celles de grenouilles traitees au cypionate de testosterone. Les glandes nuptiales ont donc des proprietks structurales et fonctionnelles semblables celles de glandes muqueuses tegumentaires et peuvent avoir evolue a partir de cette population ancestrale de glandes. [Traduit par la Redaction]

Introduction et al. 1984). The pads are postulated to assist the male in clasping the female during mating (Parrakal and Ellis 1963;

Nuptial pads are sexually dimorphic integumental specializa- Kurabuchi 1993), but the functions of the nuptial glands tions of the digits and forelimbs in male anurans (frogs and themselves are uncertain ( ~ ~ j i k ~ ~ ~ et al. 1988; ~h~~~~ et toads). The pads are often concentrated on the "thumb" 19933. Moreover, although anuran nuptial pads are widely (actually digit 11) as prominent, thickened regions of skin used in experimental studies as indicators of testis function (Duellman and Trueb l986), and are with large and hormone levels (e.g., Kanamadi and Saidapur 1982; androgen-sensitive glands that growth and Blackburn et al. 1995), the mechanisms of hormone action regression (Saidapur and Nadkarni 1975; Polzonetti-Magni the glands are unclear.

Received July 25, 1996. Accepted October 30, 1996.

M.S. Epstein' and D.G. B l a c k b ~ r n . ~ Department of Biology, Life Sciences Center, Trinity College, Hartford, CT 06106, U.S.A.

' Present address: Department of Molecular and Cellular Life Sciences, University of California at Los Angeles, Los Angeles, CA 90024, U.S. A. Author to whom all correspondence should be addressed (e-mail: daniel.blackburn@trincoll .edu).

Can. J . Zool. 74: 472-477 (1997)

~istochemistr~ has proved to be a valuable tool for revealing the biochemical nature and functions of secretions of the granular (e.g., poison) glands and mucous glands of the anuran integument (e.g., Dapson 1970; Hostetler and Cannon 1974; Le Quang Trong 1976; Barni et al. 1987; Pederzoli et al. 1990). However, the histochemistry of the nuptial glands has received little attention (Parrakal and Ellis 1963; Thomas et al. 1993), and the precise identity and func- tional attributes of the nuptial gland secretions are not known for any species. In addition, despite numerous studies

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indicating that some skin glands are androgen responsive (e.g., d'Istria et al. 197 1 ; Rastogi and Chieffi 197 1 ; Thomas and Licht 1993; Lynch and Blackburn 1995), the means by which hormones affect gland function are poorly understood. Hormones theoretically could exert their effects directly on the glands or via the nervous system, and the capacity for interconversion of gonadal steroids by target tissues (Callard et al. 1990) potentially complicates the mechanism of control. At present, a single hormone cannot be presumed to control both nuptial gland growth and synthesis of the glandular secretions, nor can homogeneity of the secretory product be assumed (see Dapson et al. 1973; Mills and Prum 1984; Delfino et al. 1990). However, neither possibility has been explicitly addressed in the literature. In some vertebrate exo- crine glands, a complex battery of hormones controls gland function, and the glandular secretion is heterogeneous (Tucker 1974; Forsyth and Hayden 1977).

The goal of this study was to investigate the effects of androgen administration and castration on nuptial gland histol- ogy and histochemistry in the leopard frog, Rana pipiens, with the goal of providing information relevant to nuptial gland function and evolution. A recent survey (Thomas et al. 1993) has provided useful information on nuptial gland histochemistry under natural conditions. The present study allows comparison between experimentally stimulated and naturally cycling frogs, and provides a basis for ongoing work on the neuroendocrinologica1 control of sexually dimorphic features of anurans.

Materials and methods In the first experiment, adult male R. pipiens (sensu Hillis et al. 1983) that had been collected in Wisconsin, U.S.A., were obtained from commercial suppliers in January. The frogs were not in reproductive state upon arrival, as indicated by their regressed thumb pads. Frogs were maintained in aluminum cages provided with running water, at a temperature of 18°C and an increasing light cycle of 12 h light (L) : 12 h dark (D) to 14 h L: 10 h D. Animals were fed earthworms and crickets weekly. All animals were cared for in accordance with the guidelines of the Canadian Council on Animal Care.

Frogs were anesthetized with urethane and the testes were removed through a lateral abdominal incision. In sham-operated controls, the testes were manipulated surgically but not removed. Half of the frogs in each group then were treated with 5-mg pellets of testosterone cypionate (TC) (Innovative Research, Toledo, Ohio, U.S.A.) embedded subcutaneously in the dorsal lymph sac. The remaining frogs were treated with control pellets composed of cholesterol and inert carriers. Two successive 3-week doses of hormone and placebo were used. After 6 weeks of treatment, the animals were sacrificed with a urethane overdose and the first digit (digit 11) or, in some cases, the manus were harvested and fixed in Bouin's solution or neutral-buffered formalin. Tissues were dehydrated in ethanol, cleared in toluene or Hemo-De (Fisher Scientific, Pittsburgh, Pennsylvania, U .S. A.), embedded in Paraplast plus (Fisher Scientific), and sectioned at 6 - 8 pm.

Sections mounted on albumen-coated slides were treated with a battery of histological stains. Eosin and iron hematoxylin were used to demonstrate general morphology. To reveal carbohydrates, the following protocols were used: periodic acid - Schiff's reagent (PAS) (Humason 1962), the PASIsalivary amylase test (Humason 1962), and Alcian Blue tests at pH 1.0 and 2.5 (Kiernan 1990). Stains employed for protein analysis included brilliant indocyanine blue (Kiernan 1990), the Millon reaction (Humason 1962), the cysteic acid reaction (Kiernan 1990), and the dimethylaminobenzaldehyde

(DMAB) reaction (James and Tas 1984). For positive controls, sec- tions of rat intestine and liver were stained in parallel with the experimental tissues.

In the second experiment, male R. pipiens from Lake Champlain, Canada, were obtained commercially in early February and housed as in the previous experiment. Frogs were castrated and treated with 5-mg pellets of 5-a-dihydrotestosterone (DHT) or a cholesterol placebo (Innovative Research) as described above; untouched con- trols were used for comparison. At the termination of the experi- ment, some nuptial pad samples were harvested and processed for histology; others were frozen at -30°C, embedded in Tissue-Tek O.C.T. compound (Miles, Inc., Elkhart, Indiana, U.S.A), and sec- tioned in a cryostat at 15 pm. Frozen sections were stained with Sudan Black (Kiernan 1990) for lipids, using rat adipose tissue as a positive control. All histological samples were photographed with an Olympus BH-2 compound microscope fitted with apochromatic lenses and a Nikon camera.

Results The nuptial pads of male frogs treated with DHT, like those of frogs treated with TC, showed strong morphological evi- dence of activity. The dermal and epidermal layers were thick and keratinized epidermal papillae were prominent. The glands exhibited large acini lined with columnar epithelial cells with well-defined cellular boundaries (Fig. I). Cells of the gland epithelium were filled with rounded eosinophilic secretory granules. The granules were distributed unevenly throughout the cytoplasm, being somewhat more concentrated towards the cell apices. Secretory material was also visible in the lumen of some glands. Castration prior to treatment had no effect on the responses to androgen.

The nuptial glands of castrated and sham-operated frogs that had been treated with placebo appeared to be inactive (Fig. 2). Gland lumina were constricted and the epithelial cells were squamous or cuboidal, with indistinct cell bound- aries. Secretion was not evident in the gland lumina and cytoplasm, and the gland epithelia stained weakly or not at all during each of the histochemical procedures outlined below. In the untouched controls for the DHT experiment, although inactive glands predominated, some glands showed evidence of activity, probably reflecting the onset of recrudescence.

In testosterone-treated frogs, the cytoplasmic secretory granules stained bright red with the PAS reaction (Fig. 3). Amylase pretreatment caused no diminution of stain, in con- trast to its effect on positive control tissues (rat liver). The secretory material stained bright blue using the indocyanine protocol, with a particular concentration of stain at the epi- thelial cell apices (Fig. 4). When processed for the Millon reaction, the secretory granules and surrounding cytoplasm showed a weakly positive reaction (Fig. 5). However, the glands reacted negatively when treated with Sudan black (Fig. 6). The secretory product did not stain with either of the Alcian Blue protocols (pH 1.0 and 2.5) or with the cysteic acid test, although in each case the reaction of adjacent hyaline cartilage demonstrated strong activity of the stain (Figs. 7 and 8). The glandular epithelium also showed no reaction to DMAB.

Discussion Histochemistry Histochemical analysis of the tissues from the testosterone- stimulated R. pipiens allows characterization of the nuptial

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Fig. 1. Transverse section of male nuptial pad in overwintering male Runu pipiens treated with 5-a-dihydrotestosterone. The nuptial glands (N) are enlarged and epidermal papillae (P) are prominent. Hematoxylin and eosin. Fig. 2. Transverse section of a placebo-treated, castrated control male. Note the sparse, inactive nuptial glands ( N ) and the absence of papillae. Hematoxylin and eosin. Fig. 3. Nuptial pad of TC-treated, sham-operated male, showing the positive reaction of secretory granules to PAS. Fig. 4. Nuptial pad of TC-treated, sham-operated male, showing the positive reaction to the indocyanine protocol. Fig. 5. Nuptial pad of TC-treated, castrated male treated using the Millon reaction. The glands and the surrounding connective tissue show weakly positive and moderate responses, respectively. The black elements lying superficially in the dermis are melanocytes. Lightly counterstained with hematoxylin. Fig. 6. Frozen section of nuptial pad of TC-treated, sham-operated male, showing the negative reaction of the nuptial glands (N) to Sudan black. Note the subepithelial melanocytes. The remaining coloration is due to the hematoxylin counterstain. Fig. 7. Nuptial pad of TC-treated, sham-operated male, stained with Alcian Blue (pH 2.5). Note the positive reaction of the digital cartilage (C) and the lack of response by the glands. Lightly counterstained with hematoxylin. Fig. 8. Nuptial pad of TC-treated, sham-operated male, processed for the cysteic acid reaction. Lightly counterstained with hematoxylin. Scale bar = 250 pm in Figs. 1 - 8.

gland secretions. That the secretions contain carbohydrate is evident from the positive reaction to PAS, which can reveal the presence of neutral hexose sugars or sialic acids, and which occurs in the presence of glycolipids, polysaccharides (e.g . , glycogen), glycoproteins, and proteoglycans (Humason 1962; Kiernan 1990). The negative response to Sudan black indicates that lipid (including glycolipid) was not present. The amylase test indicates that glycogen did not contribute significantly to stainability, and the negative reaction to Alcian Blue at the pH values tested suggests that acidic and sulphated mucosubstances were not the carbohydrate-containing moiety.

Tissue stainability with brilliant indocyanine blue is propor- tional to the protein concentration (Kiernan 1990); thus, the intense reaction of the nuptial gland secretions indicates that proteins represent a major component. The presence of tyro- sine was indicated by the Millon reaction; although the reaction was not strong, stain intensity with this protocol is not known to correlate with tyrosine concentration (James and Tas 1984). Stains for cystine and tryptophan yielded negative results; however, the protocols used reveal only high concentrations of these amino acids. For all of the stains used, intracellular and luminal secretory products showed no difference in stain- ing characteristics, thus offering no evidence that the nuptial gland products are modified during or following secretion.

From these results, we infer that the synthetic and secre- tory product of the androgen-stimulated nuptial glands of male R. pipiens is a neutral mucosubstance with carbohydrate and proteinaceous moieties, and is most likely a glycoprotein (for a summary of carbohydrate-containing macromolecules and their stainability see Kiernan 1990). A complementary battery of tests on nuptial glands of environmentally stimulated frogs (Thomas et al. 1993) has yielded consistent results. Further characterization of the secretory product will require identifi- cation of the monosaccharide residues and amino acid composi- tion, perhaps through the use of lectins and immunochemistry .

Previous work has shown that the nuptial pads of over- wintering male R. pipiens can respond to endogenous TC, becoming indistinguishable histologically from the nuptial pads of leopard frogs sampled during the breeding season (Lynch and Blackburn 1995). The present study indicates that the secretions of the nuptial glands of the testosterone- treated overwintering frogs are histochemically similar or identical with those from leopard frogs housed under condi- tions that simulated spring or summer. Therefore, testosterone treatment appears to be an effective experimental means of stimulating normal synthetic activity in the nuptial glands of reproductively inactive frogs.

Mechanism of hormone action Many studies have reported that exogenous testosterone stimu- lates undeveloped nuptial pads in anurans (e .g . , Obert 1975; Tojio and Iwasawa 1977; Kanamadi and Saidapur 1982). However, surprisingly few studies (d'Istria et al. 197 1 ; Botte et al. 1972; Iwasawa and Kobayashi 1974) describe the effects of such treatment on nuptial gland activity, perhaps on the assumption that secretory activity invariably correlates with modification of the nuptial pad epidermis. This assumption may be premature. In sexually dimorphic "breeding glands" identified in the dorsum of male R. pipiens, changes in morphology are not accompanied by modifications in the overlying epidermis (Thomas and Licht 1993).

In our studies, exogenous TC and DHT each stimulated gland hypertrophy and synthetic activity, responses that were unaffected by castration. Whether the testosterone is aroma- tized to estrogen or converted to DHT before affecting the genome (Knudsen and Max 1980; Callard et al. 1990) is unknown. In the bullfrog, Rana cutesbiana, the major metabo- lite of testosterone in the testis is DHT (Kime and Hews 1978), although aromatase activity occurs in the forebrain (Callard et al. 1978). Our studies indicate that DHT mimics the stimulatory effects of testosterone on the nuptial glands, whereas pharmacological doses of estradiol- 176 do not affect the glands (D.G. Blackburn and L. Lynch, unpublished data). Estradiol also has been shown to lack stimulatory effects on nuptial pads in other anurans (Botte and Delrio 1967; Iwasawa and Kobayashi 1974; Kanamadi and Saidapur 1982).

From these findings, we postulate that testosterone action is likely to be mediated by 5-a-reduction rather than aromati- zation. Although both TC and DHT occur in the plasma in R. pipiens, the situation in vivo is complicated by indirect evidence of an unknown testicular factor (Wada et al. 1976). Whether androgens directly stimulate the nuptial glands or act via nerves of the thumb pad (Fox 1986; Fujikura et al. 1988) is unknown. However, the presence of androgen recep- tor in the nuptial pad (Delrio and d'Istria 1973) is consistent with a mechanism involving direct hormone action.

Nuptial gland function and evolution Anuran skin glands include the mucous glands, which keep the skin moist and aid in respiration, and granular (poison) glands, which help protect against predators (Lillywhite and Licht 1975; Fox 1986). The glands of the nuptial pads, along with sexually dimorphic glands of the male forelimbs and abdomen, constitute a third population, the "breeding glands'' (Conaway and Metter 1967; Thomas and Licht 1993). Thomas

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et al. (1993) have provided strong evidence of histochemical similarities between the dimorphic glands in representatives of six families of anurans, and have postulated a common origin for breeding glands that could transcend differences in location and perhaps function. Nuptial glands often are pre- sumed to assist the male in grasping the female (Duellman and Trueb 1986; Lynch and Blackburn 1995), but also have been postulated to release pheromones (Thomas et al. 1993).

Several features of anuran nuptial glands suggest some role in mating, notably their sexual dimorphism, androgen dependence, seasonal cylicity, and location in nuptial pads that are thought to help males grasp females during amplexus. That androgen treatment of reproductively inactive males stimulates synthesis of secretions which are cytochemically like those of reproducing males, as shown herein, offers further evidence of a reproductive function. In addition, cytochemical homogeneity of the secretions and stimulation of their synthesis by a single hormone may be evidence of a unitary function of the glands in R. pipiens. However, the presence of breeding glands on the dorsum of male R. pipiens and elsewhere on other anurans (Thomas et al. 1993) raises difficulties in making facile assumptions of a single non- pheromonal function. The histochemical findings offer evi- dence that should prove useful in studies of the properties of nuptial secretions, notably their adhesive qualities and pheromonal potential under aquatic conditions.

Theoretically, anuran breeding glands (including the nuptial glands) could have originated from either granular glands or mucous glands (Metter and Conaway 1969; Kanamadi and Saidapur 1982). Alternatively, the glands could have evolved as a neomorphic population that evolutionarily combined fea- tures of pre-existing glands, as has been proposed for the mammary gland (Blackburn 199 1, 1993). Assuming their evolutionary origins to be reflected in morphology and histo- chemistry, nuptial glands probably represent modified mucous glands. Like the nuptial glands, mucous glands produce a mucosubstance with protein and carbohydrate moieties (Dapson 1970; Park 1974) that lacks the bound lipids of granular glands (Dapson et al. 1973). Mucous glands also exhibit other features found among nuptial glands, such as androgen sensitivity (Thomas and Licht 1993) and a cuboidal epithelium that surrounds a patent, central lumen (Mills and Prum 1984). If the seromucous glands of R. pipiens are considered to be breeding glands (Thomas and Licht 1993), other similarities could include myoepithelial cells, specialized "mitochondria- rich cells," indirect innervation, and differential responses to a-adrenergic and 0-adrenergic stimulation (Mills and Prum 1984). In contrast to mucous glands and nuptial glands, granular glands are surrounded by smooth muscle (Sjoberg and Flock 1976; Neuwirth et al. 1979; Mills and Prum 1984; cf. Fox 1986), exhibit syncytial epithelium, are innervated directly (Sjoberg and Flock 1976), and produce lipid-containing secretions with biologically active properties (Fox 1986; Delfino 1991).

Their widespread occurrence (Thomas et al. 1993) proba- bly indicates that breeding glands are primitive for anurans. The possibility that the sexually dimorphic glands of urodeles share a common origin with those of anurans is less probable. Given their restricted taxonomic distribution (Duellman and Trueb 1986), sexually dimorphic glands may not be ancestral for urodeles, and in at least some species (Sever 1985),

produce secretions that are histochemically unlike those of the anuran nuptial glands studied herein. At present, nuptial glands, like the glands of the digital pads of hylid frogs (Ernst 1973), seem likely to represent mucous glands that have been modified for specialized functions following diver- gence of the anuran clade. However, if the production of skin secretions with adhesive properties is shown to be taxonomi- cally widespread among lissamphibians (Evans and Brodie 1994; Williams and Anthony 1994), this assessment may need to be revisited.

Acknowledgments

For technical assistance we thank L.E. Miller, J.E. Simmons, and C.A. Sidor. We thank the anonymous reviewers for their comments on a previous draft of the paper.

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