In R. cyanophlyctis intratesticular efferent ductules arise from seminiferous tubules and join vasa efferentia. The latter, whose number varies from 7–9 are connected to a lateral kidney canal (LKC). From the LKC sperm traverse to cranial nephrons through Bowman's capsules. The urinogenital (Wolffian) duct transports both urine and sperm to cloaca. In R. limnocharis the sperm from seminiferous tubules traverse to the cranial nephrons through intratesticular efferent ductules followed by 6–8 branched vasa efferentia. The nephrons release sperm into the Wolffian duct. The LKC is absent. By contrast, in P. maculatus the testes are composed of seminiferous lobules that radiate from a longitudinal collecting ducts (LCD) situated along the inner aspect of the testes. The LCD is drained by 8–12 vasa efferentia that are connected to nephrons directly; intratesticular efferent ductules and LKC are absent. In M. rubra the intratesticular efferent ductules arise from the seminiferous tubules. From testis 3–4 branched vasa efferentia traverse through the mesorchium to join the ipsilateral kidney. The vasa efferentia transport sperm directly to the cranial uriniferous tubules. From there the sperm are released in the Wolffian duct. Both LCD and LKC are absent. In B. melanostictus 25–30 branched vasa efferentia arise from each testis and are connected directly to nephrons. In B. fergusonii 14–18 branched vasa efferentia connect testis and the ipsilateral kidney. The sperm from vasa efferentia directly pass to the nephrons and then into the Wolffian duct. Intratesticular efferent ductules, LCD and LKC are absent in both B. melanostictus and B. fergusonii. The Wolffian duct is remarkably glandular all along its length in P. maculatus unlike in other species studied presently in which only seminal vesicle (ampulla) is glandular. The present study shows a diversity in the organization of urinogenital system in anurans and lack of a phylogenetic trend.
Close anatomical relationship between the gonadal ridge and anlage of kidney is seen during embryogenesis in vertebrates (Blum, 1986). The association between urinary and genital systems is of great importance in males of most vertebrates since the former contributes to the formation of ducts that transport sperm from testes. In the course of vertebrate evolution, urinogenital organisation with respect to sperm transport has undergone diverse changes not only between different classes and orders but also within a group of closely related species (Blum, 1986). There are numerous studies describing the pattern of urinogenital connections in elasmobranchs and bony fishes. In anuran amphibians the description of sperm transport system is limited to Rhacophorus maximus (Bhaduri, 1932), Rana pipiens, Hyla crucifer (Rugh, 1939, 1941), Bombinator sp., Discoglossus sp. and Alytes sp. (references in van Tienhoven, 1983; Blum, 1986). The present comparative study elucidates the excurrent duct system of sperm transport in six south Indian anurans, Rana cyanophlyctis, Rana limnocharis, Polypedates maculatus, Microhyla rubra, Bufo melanostictus and Bufo fergusonii.
MATERIALS AND METHODS
Adult male R. cyanophlyctis (N=10), P. maculatus (N=10) and B. melanostictus (N=10) were collected in May 1996 and, R. limnocharis (N=10), M. rubra (N=10) and B. fergusonii (N=10) were collected between June and August 1999 around Dharwad city (15°17″N, 75°3″ E). In south India the main breeding season of anurans lies between June–October (Saidapur, 1989). However, R. cyanophlyctis and B. melanostictus exhibit continuous type spermatogenesis and as such sperm are found in the testes year round (Saidapur, 1989). The animals were sacrificed under anesthesia and the entire urinogenital system was dissected out and fixed in Bouin's fluid. Using a magnifying lens the number of vasa efferentia were counted by gently pulling the testis away from the ipsilateral kidney. The Bouin's fixed material was then embedded in paraffin, sectioned at 5–10 μm thickness, and stained with haematoxylin - eosin. Serial sections were examined under the light microscope to trace the excurrent duct system of sperm transport.
Testes of the adult frog are ovoidal, about 4 mm long and 2 mm wide. The kidneys are dark red, elongated, flattened and compact structures measuring about 10 mm in length and 4 mm in width. Around 7–9 vasa efferentia (Fig. 1A) traverse through the mesorchium to join the ipsilateral kidney. The number of vasa efferentia varies slightly between right and left side of an individual and also between different individuals. In cross sections of the testis, intratesticular efferent ductules filled with sperms are seen arising from the seminiferous tubules. A continuity between intratesticular efferent ductules and the vasa efferentia is clearly seen at least in some sections. On the inner aspect of each kidney, a lateral kidney canal (LKC) is seen (Fig. 2). In length, the canal is almost equal to that of the testis. The vasa efferentia carrying spermatozoa join LKC. A few vasa efferentia directly join the cranial nephrons. Spermatozoa from LKC are released into the Bowman's capsule (Fig. 3). From there, sperm traverse through cranial nephrons (Fig. 4) to the urinogenital (Wolffian) duct. The caudal nephrons are generally devoid of sperms. The Wolffian duct originates near the antero-dorsal region of the kidney and runs posteriorly. It then takes a lateral position and leaves the kidney from postero-ventral tip. The duct enlarges in size to form an ampulla (seminal vesicle) before joining the cloaca (Fig. 5). Wolffian duct is lined by a pseudostratified epithelium whose height is greatest in seminal vesicle compared to that in other regions of the duct.
Testes of the adult frog are ovoid, elongated, about 2.8 mm long and 1.2 mm wide. The kidneys are compact, flat, elongated and reddish brown measuring 6.5 mm in length and 1.8 mm in width. The intratesticular efferent ductules that arise from seminiferous tubules showed abundant sperm. The sperm from the testis are carried by 6–8 branched vasa effer entia (Fig. 1B). The number of vasa efferentia is slightly variable (2–3). The sperm from the vasa efferentia are directly transported to the cranial nephrons since LKC is absent. A few caudal nephrons also exhibit sperm. The sperm traverse through elongated, coiled nephrons and are released into the Wolffian duct. The latter slightly enlarges in size before joining cloaca to form seminal vesicle. The Wolffian duct is lined by a stratified epithelium. The epithelial height is greatest in seminal vesicle compared to other parts of urinogenital duct.
The testes of the tree frog are ovoid organs, about 6 mm long and 4 mm wide. Kidneys are dark red, flat and compact structures measuring about 14 mm in length and 4.5 mm in width. In cross sections of the testis seminiferous lobules are seen radiating (Figs. 1C, 6) from a longitudinal collecting duct (LCD) running all along the inner aspect of the testis from anterior to the posterior end (Fig. 7). It is drained by vasa efferentia (Fig. 6) whose number varies between 8–12. The vasa efferentia are connected directly to kidney (Fig. 6). Intratesticular efferent ductules as well as LKC are absent. The sperm from the seminiferous lobules are released into intratesticular LCD from where they are transported to cranial nephrons through vasa efferentia. The sperm from the cranial nephrons are released into the Wolffian duct at the anterior part of the kidney. The nephrons in the posterior region of the kidney are devoid of sperm. The Wolffian duct is lined by a columnar epithelium and it is glandular all along its length (Fig. 8). The duct enlarges slightly at its posterior end to form a seminal vesicle before opening into cloaca.
Testes of M. rubra are ovoid, about 2.9 mm long and 1.7 mm wide. Anterior part of the testis is slightly broader than the posterior part. The kidneys are flat, compact, elongated and reddish brown measuring about 7.1 mm in length and 2.1 mm in width. Posterior part of the kidney is broader than the anterior part (Fig. 1D). Testis and ipsilateral kidney are connected by 3–4 branched vasa efferentia (Fig. 1D). The number of vasa efferentia varies slightly between two sides and also between the individuals. Intratesticular efferent ductules arising from the seminiferous tubules exhibit many sperms. The intratesticular LCD and LKC are absent. The sperm from the seminiferous tubules traverse to the cranial nephrons through the intratesticular efferent ductules and from there to vasa efferentia. A few caudal nephrons also exhibit sperm. The nephric tubules release sperms into the Wolffian duct that is enlarged at its posterior end to form seminal vesicle. The Wolffian duct is lined by a columnar epithelium and its height is greatest in seminal vesicle compared to other regions of the duct.
Testes of the toad are elongated and are about 15 mm long and 4 mm wide. The kidneys are elongated, flattened and compact measuring about 23 mm in length and 7 mm in width. As many as 25–30 vasa efferentia arise from each testis and branch before joining the kidney (Fig. 1E).
The intratesticular efferent ductules, LCD and LKC are absent. Sperm from the testis reach directly to nephric tubules through vasa efferentia (Fig. 9). Most part of the inner edge of the kidney is connected to testis by the vasa efferentia (Fig. 1E). The nephric tubules from cranial to caudal region of the kidney exhibit sperm. These release sperm into the Wolffian duct (Fig. 10) that is slightly enlarged near the cloaca to form seminal vesicle or sperm receptacle. The duct is lined by a ciliated columnar epithelium. The epithelial height is greatest in the seminal vesicle compared to other regions of the Wolffian duct.
Testes of B. fergusonii are elongated about 4.9 mm long and 1.5 mm wide. The kidneys are flat, compact, elongated and reddish brown, 7.5 mm in length and 2 mm in width. Around 14–18 branched vasa efferentia traverse through the mesorchium to join the ipsilateral kidney (Fig. 1F). The intratesticular efferent ductules, LCD and LKC are absent. Most part of inner edge of the kidney is connected to the testis by branched vasa efferentia. Sperm are seen in nephric tubules throughout the kidney. The sperm from the seminiferous tubules, through vasa efferentia are directly transported to nephric tubules. From there the sperm released into the Wolffian duct that is lined by cuboidal epithelium. The latter at its posterior end enlarges in size to form seminal vesicle where height of the epithelium is greatest compared to the other parts of the Wolffian duct.
Among the extant vertebrates, only cyclostomes and teleosts have no urinogenital connections. In cyclostomes, the mature testis tubules burst releasing sperm into the body cavity from where they pass via genital funnels of coelomic origin out of the body into the surrounding water (Norris, 1987). In teleosts, the testes usually extend throughout the entire length of the body cavity and form their own secondary spermiducts (not Wolffian ducts) which unite prior to opening to the exterior. In all other vertebrates, the mesonephric kidney and/or its duct plays an important role in transporting sperm to exterior. However, the degree of association between testis and mesonephros varies greatly among anamniote vertebrates. In Chondrostei the testes contain central testicular canal through which ductuli efferentes arise and connect the outer LKC. The latter is joined by connecting ducts with an internal LKC in the mesonephros. In elasmobranchs LKC is restricted to anterior mesonephros. In some anurans ductuli efferentes lead directly into the LKC that is restricted to cranial mesonephros while in others LKC is absent. However, in all anurans sperm are eventually transported to exterior via Wolffian ducts. Interesting point here is that among anurans cranial part of mesonephros transports exclusively sperm while its caudal part transports mainly urine. Hence, the cranial part of mesonephros in anurans is comparable to epididymis of amniotes. In fact, in amniotes anterior mesonephric kidney gives rise to epididymis and, the Wolffian duct forms the vas deferens.
In anurans belonging to different genera, R. cyanophlyctis, R. limnocharis, P. maculatus, M. rubra, B. melanostictus, and B. fergusonii studied presently, there is a close association between the testis and the ipsilateral kidney. In these species, nephrons of the mesonephric kidney transport sperm to Wolffian duct as in R. maximus (Bhaduri, 1932), R. pipiens and H. crucifer (Rugh, 1939; 1941). The present as well as earlier studies show that there exists a considerable variation in the organization of excurrent duct system of sperm transport within the order Anura. Blum (1986) has summarized such variations into 3 types thereby depicting a developmental series in the organization of urinogenital system in anurans. Accordingly, the first step in this series is encountered in the genus Bombinator in which the ductuli efferentes join LKC and from the latter transverse canals make contact with the nephrons of cranial mesonephric kidney that in turn join the Wolffian duct. Interestingly, in this genus, there is an additional connection i.e. a direct path, bypassing the kidney between cranial end of LKC and Wolffian duct. The second developmental stage is seen in the genus Discoglossus in which ductuli efferentes, LKC and transverse canals linking testis and mesonephric kidneys are lost. Therefore, in Discoglossid anurans studied so far mesonephros is not involved in the transport of sperm as they traverse through a ductus deferens directly into the Wolffian duct. However, the latter also drains the cranial mesonephros. In contrast, a separate secondary urinary duct drains the caudal mesonephros. Both these ducts empty their contents into the urinary bladder. Finally, in the members of the genus Alytes, the ductus deferens arising from the testis joins the Wolffian duct directly without any connection being made to the nephrons. Hence, drainage of urine is taken over by a secondary urinary duct to which all the nephrons of the mesonephros are connected. The primary urinary (Wolffian) duct therefore becomes simply a spermiduct.
The patterns of excurrent duct system of presently studied six species does not resemble either Bombinator or Discoglossus or Alytes. In these species, testicular sperm are invariably transported to Bowman's capsule and from there to Wolffian duct that serves as an urospermiduct. There are no secondary urinary ducts comparable to that reported for other anuran families. Interestingly, LKC is present in R. cyanophlyctis. The presence of LKC vis'-a- vis' an indirect method of transporting sperm to Wolffian duct may be a primitive feature. In R. limnocharis, P. maculatus, M. rubra, B. melanostictus and B. fergusonii LKC is absent and therefore urinogenital connection is direct. The occurrence of an intratesticular longitudinal collecting duct in the testis of P. maculatus (Kanamadi and Jirankali, 1992) may have evolved in conjunction with the radial organization of the seminiferous loblules as in the urodelan amphibians (Lofts, 1984). Such an intratesticular collecting duct found in the tree frog possibly represents a primitive rete system. In the four genera studied presently, the cranial mesonephros is chiefly involved in the transport of sperm from the testis to Wolffian duct as in R. maximus (Bhaduri, 1932), R. pipiens and H. crucifer (Rugh, 1939; 1941). However, in the toads (B. melanostictus and B. fergusonii), perhaps due to a longer testis size, even the nephrons of caudal mesonephros are involved in the transport of sperm. The caudal nephrons exhibiting sperm may also indicate a temporary storage of sperm in this part of mesonephros. Generally, the caudal nephrons are involved exclusively in the drainage of urine. In all the six species the posterior part of Wolffian duct is enlarged and glandular giving rise to a seminal vesicle. The seminal vesicles of anurans are believed to store sperm (Iwasawa and Michibata, 1972). Presence of prominent seminal vesicles filled with sperm in the presently studied six species supports the view that they may indeed help in sperm storage. The present study shows a diversity in the organization of urinogenital system in anurans and a lack of phylogenetic trend.