Annual Morphological Cycles of Testis and Thumb Pad of the Male Frog (Rana ridibunda)
Article first published online: 28 MAY 2008
Copyright © 2008 Wiley-Liss, Inc.
The Anatomical Record
Volume 291, Issue 9, pages 1106–1114, September 2008
How to Cite
Kaptan, E. and Murathanoğlu, O. (2008), Annual Morphological Cycles of Testis and Thumb Pad of the Male Frog (Rana ridibunda). Anat Rec, 291: 1106–1114. doi: 10.1002/ar.20723
- Issue published online: 25 AUG 2008
- Article first published online: 28 MAY 2008
- Manuscript Accepted: 2 APR 2008
- Manuscript Received: 1 NOV 2006
- Scientific Research Department of Istanbul University. Grant Number: T103/11112002
- Rana ridibunda;
- thumb pad;
- spermatogenetic activity;
- mixed gland;
- breeding glands
In this study, Rana ridibunda was used as samples because of their wide use in Turkey. Male frogs were collected in the East Marmara region each month throughout 1 year. Frogs from every monthly collection were used to analyze structural components of the thumb pads and testes. Spermatogenetic activity of Rana ridibunda living in the East Marmara region was determined to be “potentially continuous” type. Generally, the increase in the number and the size of nuclei of Leydig cells was inversely proportional to the fluctuation of spermatogenetic activity. The lumen of the seminiferous tubules in testes contained, in addition to the spermatogenic cells, a Periodic-acid Schiff-positive granular material. The amount of this material varied throughout the year, and that finding suggested a function related to spermiation. The components of thumb pads exhibited structural changes with respect to the activities of Leydig cells. During the periods where the Leydig cells were active, mucus glands (also called breeding glands) of thumb pads were also developed. On the other hand, we observed mixed glands with unknown function, which as first reported by us, and were poison glands in the thumb pads. The results suggest structural changes in the thumb pads are linked to changes in the testes. Anat Rec, 291:1106-1114, 2008. © 2008 Wiley-Liss, Inc.
In amphibians, male sex hormones are the most important factors among the ones determining the sexual functions and behavior of the individual (Wilson et al., 1981; Harvey and Propper, 1997). In the amphibians such as Rana pipiens, Rana esculenta, Pachymedusa dacnicolor, Rana perezi, and Rana catesbeiana plasma testosterone levels vary according to the seasonal changes (Wada et al., 1976; Pierantoni et al., 1984; Rastogi et al., 1986; Delgado et al., 1989; Sasso-Cerri et al., 2005). Most amphibians usually exhibit the highest plasma testosterone levels in winter and drops significantly in the following months when active spermatogenesis begins. However, in the Mexican tree frog, Pachymedusa dacnicolor, this level is the highest during active spermatogenesis. Rastogi et al. (1986) and Ko et al. (1998) have also demonstrated that seasonal changes in plasma testosterone levels are related to the numbers of Leydig cells and their nuclear size in some amphibian species such as Rana nigromaculata, Rana rugosa, and Rana dybowskii.
Spermatogenesis of amphibians exhibits a cystic form as in other anamniotic vertebrates such as Pisces. In this type of spermatogenesis, proliferation of the germ cells takes place in cysts composed of clusters. Frog germ cells undergoing mitotic and reductional divisions in the cyst are synchronized. Also this type of spermatogenesis is highly adequate for species showing aquatic reproduction. The spermatogenesis of amniotic vertebrates, including reptiles, aves, and mammalian, never show such a cystic form (Lofts, 1974).
In addition to the internal factors such as androgens, the habitat and the climate are external factors affecting the reproductive activity of amphibians. It has been known that, despite some exceptions, amphibians exhibit three types of spermatogenetic activities determined by external and internal factors. These are continuous, discontinuous, and potentially continuous types (Rastogi, 1976). Generally, animals living in the tropical and subtropical regions exhibit continuous type, while those living in temperate zones exhibit discontinuous or potentially continuous types of spermatogenetic activity (Lofts, 1974; Rastogi, 1976). Spermatogenesis of temperate zone anurans reveals a seasonal cycle. For example, spermatogenesis of Rana temporaria, which lives in temperate zones, is paused during mid-summer because its primary spermatogonia gains resistance to gonadotropins. The cycle of Rana temporaria is an example of discontinuous cycling. However, spermatogenesis of Rana esculenta, also living in temperate zones, is decelerated during cold months, but its primary spermatogonia does not gain resistance to gonadotropins. Decelerating of its cycle is exclusively caused by decreasing environmental temperature. Therefore, this spermatogenetic cycle is called potentially continuous (Lofts, 1974).
In most male amphibians, not only primary sex organs exhibit seasonal changes during the reproductive cycle but also some other structures such as thumb pads display different morphological or physiological properties in this period (Lofts et al., 1972; Kao et al., 1994). It has been shown in various amphibians that the activity of thumb pads is related to plasma androgen level (Lofts, 1964; Lofts et al., 1972; Zamachowski and Zysk, 1978; Izzo et al., 1982; Polzonetti-Magni et al., 1984; Thomas et al., 1993; Epstein and Blackbrun, 1997; Emerson et al., 1999).
Although thumb pads of anuran amphibia have been subject of extensive investigations, because they are the indicators of androgenic hormone levels and testis function, no detailed information is available on chemical composition of their breeding gland. Some researchers have stated that androgen-dependent glands in thumb pads or in other parts of the skin might be capable of secreting chemical signals related to reproduction. Yet, the functional properties of the secretions are not well known (Thomas and Licht, 1993; Thomas et al., 1993; Pearl et al., 2000).
Rana ridibunda is the most common frog species in Turkey. It is also exported at high percentages out of Turkey because of its nutritional and commercial values. On the other hand, any kind of information regarding its reproduction is of importance for our country. Moreover, the features associated with its reproduction strategy are important to compare with other anuran species and vertebrates. Numerous studies on various amphibia have been published to show the relationship between thumb pads, which are secondary sexual characteristics, and testes. However, this relationship has not been histologically evaluated in Rana ridibunda. Up to now, no reports have been found on histological features of the thumb pads related to this species. In our study, histological structure of the thumb pads is described in detail at the light microscopic level, and the functional relationship between thumb pads and testes is investigated.
MATERIALS AND METHODS
The adult male frogs (Rana ridibunda) were collected from the plains of Geyve (East Marmara). Four individuals were collected each month and anesthetized by submerging into 0.1% MS-222 (Tricaine methane sulfonate, Sigma A5040); testes and thumb pads were quickly removed and fixed in Bouin's fluid. Following routine processing procedures, paraffin sections of 5–6 μm thick were cut and stained with the following techniques; hematoxylin–eosin (HE), periodic acid Schiff (PAS), Masson's triple staining, bromphenol blue (Humason, 1972), and Alcian blue (AB) pH 2.5 (Bancroft and Cook, 1984).
The number of Leydig cells in the testes of each frog was determined in randomly selected interstitial area. The size of Leydig cell nuclei was measured with micrometer under ×1,000 magnification (4 cross-sections/animal; four animals). The formula of “0.5 longitudinal axis length × 0.5 transversal axis length × 3.14” was used to calculate the area of a single nucleus (Ko et al., 1998). The spermatogenetic cells in seminiferous tubule selected randomly from each cross-section were counted. The thickness of epidermal and dermal layers in thumb pads were also measured by an ocular micrometer (4 cross-sections/animal; four animals).
SPSS computer software was used in statistical evaluations. Data are shown as the mean ± standard error (SE). One-way analysis of variance was used to determine the variance in all the parameters measured or calculated and monthly means were compared with Scheffe multiple test. For the monthly comparison of the area of Leydig cell nuclei, Tukey multiple test was used. We accepted P < 0.05 as indicating statistical significance.
General Structural and Cyclic Variations in Testes
Primary spermatogonium (Spg I) are the first stage of germ cells in seminiferous tubules. These cells have spherical or oval nuclei that had variable basophilic staining properties. These cells are located in seminiferous tubule's wall together with follicle cells; the follicle cells have crescent-shaped nuclei and appear similar to mammalian Sertoli cells in their later stages (Fig. 1a). Secondary spermatogonium (Spg II) have spherical nuclei. When Spg II is compared with Spg I, their nuclei are smaller, with more intense and basophilic appearance than Spg I (Fig. 1b). Primary spermatocytes (Spc I) have spherical nuclei as well, but in comparison to Spg II, they are much larger cells. Spc I can be easily differentiated from any other cells in seminiferous tubules by their chromatin material and meiotic nuclear features (Fig. 1d). Secondary spermatocytes (Spc II) have spherical nuclei and they are smaller than Spc II. Their chromatin material is more dense and basophilic than the other germ cells (Fig. 1c). According to light microscopic appearance, spermatids (Spd) are classified as first, second, and third type spermatids. First type spermatids are spherical and smaller than the other germ cells and have coarse granular chromatin material in their nuclei (Fig. 1e). The nuclei of second type spermatids reveal higher extend of chromatin condensation than in earlier spermatid stages (Fig. 1f). The nuclei of third type spermatids are ovoid in shape and have dense chromatin material (Fig. 1g). Sperm bands (Sb) are formed by packaging many spermatozoa having long heads. These light microscopic properties of the frog germ cells are partly similar to those of mammalian germ cells, except for Spg I and Spg II.
To determine the type of spermatogenetic activity in Rana ridibunda testes, the number of various germ cell types in the lumen of seminiferous tubules was counted monthly for 1 year (Figs. 2, 3). The number of Spg I decreased during late spring and summer, while in fall, it started increasing and remained constant during the winter (Fig. 3a,d). Months with the highest Spg I counts were January to April, while in May and June, the number decreased (P < 0.05, when compared with January–April; Fig. 2a). In seasonal distribution of Spg II, a significant difference was not observed statistically (data not shown). On the other hand, Spc I and Spc II were observed more widely in late spring and they were more abundant in summer in comparison to that in winter (Fig. 3a–f). Their number was the lowest in January–April, while there was a great increase in May and June (P < 0.05, when compared with January–April; Fig. 2b,c). Spds were abundant in summer, while in winter and spring, their distribution was limited (Fig. 3a–f). There was a large increase in the number of Spds in June (P < 0.05, when compared with other months except July and October; Fig. 2d). Sb distribution was noticeably decreased in summer, while in winter, it increased significantly (Fig. 3a–f). The Sb number was low in June through October, yet the number reached the peak value after October (P < 0.05, when compared with other months; Fig. 2e).
The number of Leydig cells and sizes of their nuclei also varied seasonally (Fig. 3g–i). In May, a sharp increase was detected in its number and size (P < 0.05, when compared with other months except November and December). After May, the number started decreasing (P < 0.05, when compared with other months, except September and October), and was the lowest during July–October (P < 0.05, when compared with other months except September). In late fall and winter, the numbers were high and March was the month during which the size of nuclei was the smallest (P < 0.05, when compared with other months) (Fig. 2f,g).
PAS-positive material contained by seminiferous tubules
In addition to the germinal cells, the lumina of the seminiferous tubules contained some PAS-positive granular material. The appearance and distribution of this material in tubular lumina displayed a seasonal cycle. The material was located in the basal part of the seminiferous tubules throughout December–February (Fig. 4a). In March, this material observed in the luminal centers and the appearance increased in comparison to that in previous months. In April, the appearance and location of material was similar to those observed in January and February. In May, it was also observed that PAS-positive material was in the center of tubules but its appearance was increased (Fig. 4b). The material was located usually in the basal portion of the tubules, and was low in appearance in the individuals collected in June through August (Fig. 4c). In September through November, the material was again located in basal part of tubules, yet its appearance was notably higher.
General Structural and Cyclic Variations in Thumb Pads
The epidermal and dermal layers of thumb pads were generally thicker in comparison to other parts of the skin and its epidermal surface contained conical protrusions that were more evident in some months, such as April, and least in March and August, leaving the skin smoother (Fig. 4d,h). In November through January, the protrusions were somewhat visible as well (Fig. 4f).
The observations showed that epidermal thickness was low in summer and high in late fall and winter (Fig. 4d–f). The thickness was the lowest in August while it was the thickest in February (P < 0.05, when compared with other months). A decrease in thickness of thumb pad epidermis was observed in March (P < 0.05). The epidermal thickness was noticeably elevated in April in comparison to that in March (P < 0.05). However, it decreased again in May (P < 0.05, when compared with other months; Fig. 5a).
The sublayer of to the dermis, the stratum spongiosum, contains mucus glands. These glands are also called breeding glands by some investigators. Among them, occasionally distributed are the poison glands as well as mixed glands that possess the properties of both mucus and poison glands (Fig. 6a). In this study, the thickness of the dermis and size of the breeding glands it contains were in agreement with the monthly changes in epidermis (P < 0.05, when compared to other months; Fig. 5b). These fluctuations in dermis thickness were statistically significant (Fig. 5). In January and February, when epidermal thickness was greatest dermal glands were well-developed (Fig. 4f,g). In March and August, while epidermal thickness decreased, glands also began regressing (Fig. 4d).
The breeding glands can be distinguished from the mucus glands of the skin by certain structural differences. In our sample, Rana ridibunda, these glands may be tall, elliptical, or spherical, according to their functional states. The secretory portion of the gland contained cylindrical secretion cells with basally located nuclei (Fig. 6a,d). Their secretory material gave positive reactions to PAS (Fig. 6b), while negative to bromphenol blue and AB. Moreover, nongranular Alcian blue-positive areas with patchy appearance were also observed in secretory portions of some glands (Fig. 6c). In the periods when the glands were small and regressed, the patch-like regions that displayed increased AB positivity were amplified (Fig. 4d,e).
The poison glands had a similar structure with those in skin that covers all the body. The particles in the secretory cells of the gland were stained pink and red with eosin and with Masson's triple stain, respectively, and these particles gave negative reactions to PAS and AB (Fig. 6e) and positive reactions to bromphenol blue (Fig. 6f).
The mixed glands that possess the physical characteristics of breeding glands in one half and of poison glands in the other half display the structural properties of both glands as we described above. The zones of mixed glands that exhibited the characteristics of breeding andpoison glands were separated by a definitive border (Fig. 6g).
According to Lofts's criteria (1974), annual distribution of germ cells in Rana ridibunda has spermatogenetic activity that appears to be the continuous type. In this type of activity, the testes bear all stages of germinal cysts throughout the year. However, with respect to the spermatogenetic activity, the number and the stages of these cysts vary. In the cold months, when the spermatogenic activity decelerates, Spg I and Sbs dominate, while in warm months, all stages of the spermatogenic cells are visible (Lofts, 1974). In the region where the frogs were collected, the increase in the number of Spg I and Sbs in the cold January–April period demonstrated that spermatogenetic activity is decelerated. Yet, during May, when the temperature increased, the increase in the number of Spcs and the decrease in that of Spg I and sperm bands demonstrated the acceleration in spermatogenetic activity. Similar results have been reported by Loumbourdis and Kyriakopoulou-Sklavonou (1991) on the same species that live in the northern part of Greece, that the spermatogenetic activity is potentially continuous.
It was clear in our measurements that the number of Leydig cells and the size of their nuclei varied in a seasonal cycle. Ko et al. (1998) determined that the changes in cell counts and nuclear size are related to the amount of testosterone produced by testes; thus, they have argued that this is an indication of the plasma testosterone level. In the present study, biochemical assaying of the plasma testosterone level of Rana ridibunda was not measured. However, in consideration of the Leydig cell number and their nuclear size, presence of a seasonal fluctuation in plasma level of androgens derived from testis could be proposed. It was observed that the number of Leydig cells and their nuclear sizes were low in summer when spermatogenetic activity was high, or vice versa in fall and winter months. The contrast between plasma testosterone levels and spermatogenetic activity has also been pointed out by several other groups (Rastogi et al., 1978; Pierantoni et al., 1984; Rastogi and Iela, 1992).
Pierantoni et al. (1984), Rastogi et al. (1986), and Delgado et al. (1989) have shown that plasma testosterone levels in various amphibians are high when spermatogenetic activity is low, or it is low during high spermatogenetic activity. However, in this present study, a significant increase in Leydig cells number and their nuclear size were determined in May (that decelerated in the following months) as opposed to that claimed by the investigators mentioned above. These observations resemble the fluctuation profile of plasma testosterone level in the periods of before and after reproduction quite a lot, shown by Raucci et al. (2004) in Rana esculenta. The results obtained here point out that the unexpected increase of Leydig cells number and their nuclear size could be related to the increase of testosterone levels in the periods of before and after reproduction in Rana ridibunda. It is well known that testosterone triggers the formation of sexual behavior (Wetzel and Kelley, 1983). This finding might indicate that the peak level of testosterone in the plasma in late spring might lead the formation of sexual behavior or the initiation of new spermatogenetic wave, as stated by Basu (1968) and Lofts et al. (1972).
The results of the studies (Basu, 1968; Pierantoni et al., 1984; Rastogi et al., 1986) have shown that spermatogenetic activity is considerably inhibited by external testosterone application in castrated animals. This finding supports our results that spermatogenetic activity is increased despite the drop in Leydig cell number in Rana ridibunda.
In the present study, PAS-positive granular material was observed in seminiferous tubules and this material also exhibited a seasonal cycle. Similar results have also previously been reported in some other anuran species (Lofts et al., 1972; Sasso-Cerri et al., 2004). It has been claimed in Rana temporaria by Lofts et al. (1972) that the PAS-positive granular material observed in basal portion of seminiferous tubules is glycogen granules accumulated in cytoplasm of Sertoli cells. According to these investigators, in winter, Sertoli cells are rich of glycogen; however, following the spermiation when Sertoli cells' task is completed, they are disposed of into the lumen, thus glycogen granules also accumulate in the lumen of seminiferous tubules. After the initiation of active spermatogenesis following the reproduction period, new generation of Sertoli cells begins accumulating glycogen again, this leads to building up high glycogen levels. Sasso-Cerri at al. (2004) have postulated that the PAS-positive material in seminiferous tubules of Rana catesbeiana might be originated from Sertoli cells and related to spermiation and spermiogenesis. We observed that the PAS-positive material accumulated exclusively in the tubule lumen in May. Considering other parameters in addition to statements of Lofts et al. (1972) and Sasso-Cerri et al. (2004), it can be claimed that spermiation takes place in May in Rana ridibunda.
In some anurans, structural components of the thumb pads were shown to exhibit a seasonal cycle in terms of morphological properties (Lofts, 1964; Lofts et al., 1972; Zamachowski and Zysk, 1978). Although there are several reports related to thumb pad annual cycle of various other anurans in the literature, no study on Rana ridibunda was found. Our results show that a cycle also exists in structural components of the thumb pads in Rana ridibunda.
The annual cycle observed in the components of thumb pads of Rana ridibunda exhibits an opposite development with spermatogenetic activity. On the other hand, in June and July when the number of Leydig cells and their nuclear size began decreasing as an indication of secretory activity, all components of the thumbs pads also regressed. In August, when Leydig cell secretory activity was low, the epidermal thickness was the lowest and the breeding glands of thumb pads regressed. Subsequently, as the secretory activity of the Leydig cells increased thumb pads began developing. In January and February, when spermatogenetic activity was the slowest and the secretory activity of the Leydig cells increased, epidermis thickness peaked and the breeding glands of thumb pads also enlarged and developed. As previously reported by Lofts (1964) and Lofts et al. (1972), these findings have indicated that thumb pad development is dependent on testosterone. In the present case, unexpected regression of thumb pads in March is related to the sudden decline in secretion activity of the Leydig cells. In parallel to this decrease in the secretion activity, epidermal thickness of thumb pads and the size of the breeding glands also decreased in March. The drop in the Leydig cell number might be related to the unusual environmental temperature; according to the data obtained from the Geyve Station of the National Meteorological Institute, the maximum temperature in March reached up to 28°C (Table 1). Thus, various parameters such as epidermal and dermal thicknesses, morphological structure, and some histochemical properties of the breeding glands in thumb pads observed in March exhibited similarity to the results obtained in summer. Although a decrease in Leydig cell number was observed in April, there was a significant increase in their nuclear size in comparison to that in March. As a result, a well developed thumb pad structure was observed in April.
These data also indicate that testosterone has a significant role in the development of thumb pads in our specimen, Rana ridibunda. Additionally, Rastogi et al. (1978) propose that androgen receptors in thumb pads are activated in a critical low temperature. Therefore, it might be postulated that the environmental temperatures affect thumb pad development. This assumption may explain why the frogs have less developed thumb pads in May in comparison to those in April, although the secretory activity of the Leydig cells increase in May.
The effect of testosterone on thumb pads was earlier shown using histological methods in Rana esculenta and Rana pipiens by introducing external testosterone into individuals without testes (Izzo et al., 1982; Thomas et al., 1993; Thomas and Licht, 1993; Epstein and Blackburn, 1997). In addition, some investigators clarified the dependence of the breeding glands of thumb pads on androgens by demonstrating the androgen receptors in these glands using biochemical and immunohistochemical methods in Rana esculenta (Delrio and D'Istria, 1973; D'Istria et al., 1975, 1979; Delrio et al., 1980; Emerson et al., 1999). Although the thickness of thumb pad epidermis increased when the secretion activities of the Leydig cells increased to a certain level in November, December, and January, the conical protrusions of epidermis were less evident. This result suggested that, in addition to the male sex hormones, some other hormones or factors might be effective on thumb pad structure.
In our observations, it was determined that thumb pads also contained poison glands and mixed glands in addition to breeding glands. The poison glands were not different than those described structurally in the other parts of the skin. These glands in anuran skin are distributed throughout the body (Delfino et al., 1999). A third group of glands observed in the thumb pads were mixed glands that had the appearance of both breeding and poison glands. The presence of those glands was reported in dorsal skin of various species of urodele amphibia (Delfino et al., 1986; Tsuruda et al., 2002) and two anura species (Thomas et al., 1993). However, there are no reports on the presence of mixed glands in dorsal skin or the thumb pads in Rana ridibunda. No investigators have presented an opinion about the functions of the mixed glands in the amphibian skin. We do not have a study with these glands, either. However, these glands might be an intermediate form in the stage of conversion from one type into another.
While the secretory granules occupying the lumen of poison glands in thumb pads were PAS and AB-negative, they gave a positive reaction to bromphenol blue. These results supports the findings that these glands contain proteinaceous secretion and the secretion of poison glands are the source of some biologically active protein and peptide materials (Dapson et al., 1973; Perry, 2000). On the other hand, the secretion granules in the cytoplasm of secretory cells belonging to mucus glands were bromphenol blue-negative and PAS-positive. These results indicated that the secretion granules contained neutral mucopolysaccharides. Besides, the secretion granules gave negative reaction with AB which is an indication of acidic mucopolysaccharides. However, patch-like AB-positive locations were also evident in the cells of breeding glands. Thomas et al. (1993), have shown in Xenopus laevis that a small amount of AB-positive material exists in regressed thumb pads of testes-removed individuals. Except for this investigation, the presence of AB-positive regions in other amphibia was not reported. The distribution of AB-positive regions in Rana ridibunda breeding glands was inversely proportional to secretory activity of the Leydig cells. Therefore, in the summer when the glands were small and regressed, AB-positive regions were widespread and the staining reaction with AB was more intense. It might be proposed that the secretion content of the glands exhibits variations in terms of acid mucopolysaccharide content during certain periods of the year. This annual cycle in gland content is most likely influenced by testosterone that affects the glands morphologically. This claim is supported by those of Thomas et al. (1993). Thus, the increased AB-positive material might be an indication of the decrease in plasma testosterone levels. At the same time it might be also a determinant of functional activity of breeding glands.
A limited number of reports exists on the function of breeding glands. In Microhylid frogs a sticky mucus material is secreted from androgen-dependent abdominal glands that are equivalent of breeding glands in thumb pads. This mucus material was shown to function in tight attachment of male to the female frog during amplexus (Conaway and Metter, 1967). Moreover, it was proposed by Thomas et al. (1993) that the sex hormone-dependent skin glands might secrete some chemicals to attract the females and stimulate ovulation. It has been shown that glands, which they develop depending on androgen, secrete material to attract female individuals (Malacarne and Vellano, 1987). Some investigators identified these substances (chemically and functionally) in cloacal glands of urodele amphibians (Kikuyama et al., 1995; Rollmann et al., 1999; Yamamoto et al., 2000). One of these substances called splendiferin was also identified in anura amphibian Litoria splendid (Wabnitz et al., 1995). Another anura amphibian, Hymonochirus sp, possesses breeding glands containing a female-attracting substance in its skin (Pearl et al., 2000). These results support the previous findings on functional properties of these glands. We also believe that studies related to understand on the contents of glands have a great impact on comprehending the reproduction strategies of these animals.
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