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JOURNAL OF MORPHOLOGY 275:914–922 (2014)

Modifications of the Genital Kidney Proximal and Distal Tubules for Sperm Transport in Notophthalmus viridescens (Amphibia, Urodela, Salamandridae) Abbigail E. Nicholson* and Dustin S. Siegel Department of Biology, Southeast Missouri State University, Cape Girardeau, Missouri 63701 ABSTRACT Male salamanders use nephrons from the genital kidney to transport sperm from the testicular lobules to the Wolffian duct. The microstructure of the epithelia of the genital kidney proximal tubule and distal tubule was studied over 1 year in a population of Notophthalmus viridescens from Crawford and Pike counties in central Missouri. Through ultrastructural analysis, we were able to support the hypothesis that the genital kidney nephrons are modified to aid in the transportation of sperm. A lack of folding of the basal plasma membrane, in both the genital kidney proximal and distal tubules when compared to the pelvic kidney proximal and distal tubules, reduces the surface area and thus likely decreases the efficiency of reabsorption in these nephron regions of the genital kidney. Ciliated epithelial cells are also present along the entire length of the genital kidney proximal tubule, but are lacking in the epithelium of the pelvic kidney proximal tubule. The exact function of these cilia remains unknown, but they may aid in mixing of seminal fluids or the transportation of immature sperm through the genital kidney nephrons. Ultrastructural analysis of proximal and distal tubules of the genital kidney revealed no seasonal variation in cellular activity and no mass production of seminal fluids throughout the reproductive cycle. Thus, we failed to support the hypothesis that the cellular activity of the epithelia lining the genital kidney nephrons is correlated to specific events in the reproductive cycle. The cytoplasmic contents and overall structure of the genital and pelvic kidney epithelial cells were similar to recent observations in Ambystoma maculatum, with the absence of abundant dense bodies apically in the epithelial cells lining the genital kidney distal tubule. J. Morphol. 275:914–922, 2014. VC 2014 Wiley Periodicals, Inc.

KEY WORDS: urogenital; nephron; anatomy; histology; ultrastructure

(Spengel, 1876; Siegel et al., 2012a). The genital kidney nephrons culminate proximally when the vasa efferentia abut a genital kidney renal corpuscle. Distal to the renal corpuscle are the remaining genital kidney nephron regions in the following sequential order: 1) neck segment, 2) proximal tubule, 3) intermediate segment, 4) distal tubule, and 5) collecting tubule. The genital kidney collecting tubule empties directly into the Wolffian duct (Siegel et al., 2013). The caudally positioned pelvic kidneys have no communication with the testes, and are composed of nephrons that function primarily in filtration and urine formation (Siegel et al., 2010, 2013). Although some studies (e.g., Williams et al., 1984; Siegel et al., 2012a) have assessed variation among genital kidney nephrons, historical information is lacking in terms of how these structures are modified from the pelvic kidney nephrons to support sperm transport, and how such modifications vary within the annual sperm production and transportation cycle. Currently, Siegel et al. (2010, 2013) are the only studies to use ultrastructural techniques to assess characteristics of tissue and cell composition between the genital and pelvic kidney nephrons in Ambystoma maculatum (Ambystomatidae), but no data exist on how these structures vary within other salamander families. Siegel et al. (2010, 2013) found that the epithelial cells lining the genital kidney proximal and distal tubules were ultrastructurally different than those that line the pelvic kidney proximal and distal tubules. The genital kidney proximal tubules possessed epithelial cells that were highly ciliated and lacked complex folding of the basal plasma membrane. The genital kidney distal

INTRODUCTION Male salamanders possess dualistic kidneys that contain regions that differ primarily by function (Siegel et al., 2010, 2013). Cranially, kidney nephrons communicate with testicular ducts through vasa efferentia to transport sperm from the testes to the Wolffian ducts. This communication forges what is known as the genital kidney, a condition that is found in all families of salamanders C 2014 WILEY PERIODICALS, INC. V

*Correspondence to: A. E. Nicholson; Department of Biology, Southeast Missouri State University, Cape Girardeau, MO 63701. E-mail: [email protected] Received 12 January 2014; Revised 13 February 2014; Accepted 22 February 2014. Published online 19 March 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jmor.20268

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Fig. 1. Notophthalmus viridescens, overview of the male genital and pelvic kidney proximal and distal tubule epithelia. (A) The genital kidney proximal tubule from September, depicting ciliated columnar epithelial cells (dark cells) and nonciliated columnar epithelial cells (light cells) with microvillus brush borders (toluidine blue). (B) The genital kidney distal tubule from September, depicting nonciliated low cuboidal epithelial cells and genital kidney collecting tubule depicting a slightly higher epithelium composed of alternating light and dark cells without apical cilia or microvilli (toluidine blue). (C) The pelvic kidney proximal tubule from November, depicting microvillus columnar epithelial cells (toluidine blue). (D) The pelvic kidney distal tubule from November, depicting a simple cuboidal epithelium devoid of ciliation or microvilli (toluidine blue). Ci, Cilia; DC, dark cell; gCT, genital collecting tubule; gDT, genital distal tubule; LC, light cell; Lu, Lumen; Mv, microvilli; pDT, pelvic distal tubule; pPT, pelvic proximal tubule.

tubules also lacked basal membrane folding to the degree observed in the pelvic kidney distal tubules and also possessed an abundance of apical dense bodies of unknown function. However, those studies only used males sampled during the breeding season, and thus those studies did not provide data on potential seasonal variation in epithelial cell activity of the genital kidney nephrons. This study is the first to investigate and describe the seasonal variation of the genital kidney nephrons at the ultrastructural level in any salamander family, as well as the first to assess the modifications of the genital kidney nephrons compared to the pelvic kidney nephrons in male Central Newts (Notophthalmus viridescens), representing members of Salamandridae. Notophthalmus viridescens was selected as the model salamander for this study as it has been the focal point of many reproductive studies (for review see Sever, 2006), resulting in an extensive amount of historical information on the primary and secondary sexual organs (e.g., Siegel et al., 2012b). Thus, in this study, the hypothesis was tested that the

genital kidney proximal and distal tubules were structurally different than those of the pelvic kidney (as in Ambystoma maculatum; Siegel et al., 2010, 2013) and that the activity of the epithelial lining of those regions of the genital kidney nephrons would vary seasonally (in terms of cellular processes) in direct correlation with the reproductive cycle. Some controversial variation exists in the life history among different populations of N. viridescens (for review see Sever, 2006), and thus, a population of newts was used for this study that has received recent attention toward outlining of their reproductive cycle (i.e., sperm production and transportation; Siegel et al., 2012b). MATERIALS AND METHODS Two to three adult male Notophthalmus viridescens (Rafinesque, 1820) were collected every month. Collections took place in Crawford and Pike counties in Missouri under the Missouri Department of Conservation permit numbers 14357 and 14641. Specimens were sacrificed by submergence in MS-222 (approved by the Institutional Animal Care and Use Committee of Saint Louis University) within 24 h of collection. After

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Fig. 2. Notophthalmus viridescens, ultrastructure of the genital kidney proximal tubule. (A) Overview of the genital kidney proximal tubule (September) depicting the alternating ciliated and nonciliated cells of this region. (B) High magnification of the apical region of a ciliated epithelial cell (January) depicting the junctional complexes adhering adjacent epithelial cells, the nonlabyrinthine intercellular canaliculi between epithelial cells, and apical cytoplasmic contents. (C) High magnification of the basal region of a nonciliated epithelial cell (March) depicting the basal membrane flat against the basal lamina with areas of folding where nonciliated cells abut, and the basal cytoplasmic contents. (D) High magnification of the apical region of a nonciliated epithelial cell (February) depicting the microvillus apical membrane and apical cytoplasmic contents. Bb, basal bodies; Bl, basal lamina; Ci, cilia; Ds, desmosomes; End, endosomes; Ic, intercellular canaliculi; Lu, lumen; Mi, mitochondria; Mv, microvilli; Nc, nucleolus; Nu, nuclei; Rer, rough endoplasmic reticulum; Tj, tight junction; Va, vacuole.

euthanasia the abdominal cavities were opened ventrally, and the salamanders were submerged inverted in McDowell’sTrump solution (4% formaldehyde, 1% glutaraldehyde; Electron Microscopy Sciences, Hatfield, PA) to allow this solution to easily enter the pleuroperitoneal cavity. Fixation lasted for 24 h. After initial fixation, the urogenital tracts were removed and submerged in a second solution of McDowell’s-Trump fixative for at least 24 h. Urogenital tracts of Notophthalmus viridescens were rinsed in phosphate buffered saline (pH 7.4), dehydrated via a graded series of ethanol (35, 50, 70, 85, 95, and 100%), cleared in propylene oxide, immersed in a 1:1 mixture of propylene oxide and Embed 812 (Electron Microscopy Sciences, Hatfield Pennsylvania), and subsequently transferred to 100% Embed 812. Tissues were then oriented for transverse sectioning in flat molds in 100% Embed 812 and allowed to cure for 2 days in an oven at 70 C. Semithin sections were obtained with a Leica EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany), heat affixed to glass slides and stained with toluidine blue for 30 s to pinpoint nephron regions of interest for transmission electron microscopy. Ultrathin sections at 70 nm were obtained with a Leica EM UC6 ultramicrotome, placed on copper grids, and stained with uranyl acetate (15 min) and lead citrate (5 min). Grids were viewed with a JEOL JEM 100S transmission electron microscope (JEOL USA, Peabody, MA) and photographed with a L3C CCD digital camera (Scientific Instruments

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and Applications, Duluth, GA). Images were subsequently uploaded into Adobe Creative Suite 6 (Adobe Systems, San Jose, CA) for labeling.

RESULTS Through investigation via light microscopy, the genital kidney nephrons arise when the vas efferens abuts renal corpuscles. The renal corpuscle is easily identifiable by the presence of a capillary network surrounded by and closely associated with a squamous visceral and parietal epithelium of a Bowman’s capsule. Immediately following the renal corpuscle is a ciliated neck segment with cuboidal epithelium, which opens into the proximal tubule of the genital kidney. The columnar epithelial cells of the genital kidney proximal tubule alternate between ciliated cells (dark cells) and nonciliated cells (light cells). The nonciliated cells are covered apically by a dense microvillus brush border (Fig. 1A). Nuclei of all epithelial cells of this region are located in a basal position

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Fig. 3. Notophthalmus viridescens, ultrastructure of the genital kidney distal tubule. (A) Overview of the genital kidney distal tubule (May) depicting the homogenous epithelial lining of this region. (B) High magnification the apical region of an epithelial cell (May) depicting the lack of apical modification (i.e., cilia or microvilli), junctional complexes, and apical cytoplasmic contents. (C) High magnification of the lateral regions of two epithelial cells (May) depicting the nondistended labyrinthine intercellular canaliculi, junctional complexes adhering adjacent epithelial cells, and vacuolated regions of the cell cytoplasm. (D) High magnification of the basal region of two epithelial cells (May) depicting areas of nonfolded and folded regions of the basal plasma membrane. Bl, basal lamina; Ds, desmosome; Fp, foot process of the basal membrane; Ic, intercellular canaliculi; Inc, electron dense inclusions; Lu, lumen; Mi, mitochondria; Mv, microvilli; Nu, nucleus; Rer, rough endoplasmic reticulum; Tj, tight junction; Va, vacuole.

(Fig. 1A). After a brief ciliated intermediate segment, the transition into the genital kidney distal tubule is visible by the presence of low cuboidal epithelial cells with centrally located nuclei that have lost cilia and microvilli along the apical borders of the epithelium (Fig. 1B). The genital kidney distal tubule epithelium transitions to a slightly higher epithelium of the genital kidney collecting tubule composed of alternating light and dark cells without apical cilia or microvilli (Fig. 1B). This genital kidney collecting tubule ultimately opens into a Wolffian duct. The nephrons of the pelvic kidney contain all of the same regions that are present in the genital kidney with slight variation to the cellular composition of the pelvic kidney proximal tubule observed at the light microscopy level (Fig. 1C). The columnar epithelium of the pelvic kidney proximal tubule does not contain ciliated cells (Fig. 1C). However, a dense microvillus brush border covers the apical membranes, and filtrate is visible in the lumen (Fig. 1C). The cuboidal epithelium of

the distal tubule appears similar to that of the epithelium of the genital kidney distal tubule (Fig. 1D), with slightly taller epithelial cells. No cytological differences were observed when comparing the genital kidney proximal and distal tubules and pelvic kidney proximal and distal tubules between months of collection. The genital kidney proximal tubule is lined by a columnar epithelium that alternates between ciliated and nonciliated cells (Fig. 2A), confirming the observations from light microscopy. The nonciliated epithelial cells of the genital kidney proximal tubule have a dense microvillus brush border apically (Fig. 2A). The lateral membranes are distinctly nonlabyrinthine and sealed apically by tight junctions, followed by desmosomes located regularly along the entire length of the lateral membrane (Fig. 2B). The intercellular canaliculi were not noticeably distended along their entire lengths. The basal plasma membrane of the genital kidney proximal tubule nonciliated and ciliated cells is contiguous with the basal lamina along the entire length and Journal of Morphology

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Fig. 4. Notophthalmus viridescens, ultrastructure of the pelvic kidney proximal tubule. (A) Overview of the epithelium from the pelvic kidney proximal tubule (June) depicting the two types of principle cells found in this region. (B) High magnification of the apical region of the epithelial cells (July) depicting the microvillus brush border and cytoplasmic contents (uranyl acetate and lead citrate). (C) High magnification of the lateral region of the epithelial cells (March) depicting the junctional complexes and cytoplasmic contents. (D) High magnification of the basal region of the epithelial cells (March) depicting the basal folding of the plasma membrane and cytoplasmic contents. Ab, apical bleb; Bb, brush border; Bl, basal lamina; Ev, endocytic vesicles; Fp, foot process of basal membrane; Hd, hemidesmosome; Lu(Ft) lumen with filtrate; Ly, lysosome; Mi, mitochondria; Mv, microvilli; Nu, nuclei; Nu(Dc) nucleus of dark cell; Nu(Lc) nucleus of light cell; Tj, tight junction.

lacks complex folding except at random small regions along the basal membrane nonciliated cells (Fig. 2C). This folding creates a tight labyrinthine intercellular canaliculi involution of the basal membrane at the junction between nonciliated epithelial cells (Fig. 2C). Apically, cilia are embedded through the apical membranes of ciliated cells and anchor at discrete basal bodies (Fig. 2B). Microvilli line the entire apical membrane of nonciliated cells through membrane and cytoplasmic projections into the genital kidney proximal tubule lumen (Fig. 2D). The cytoplasm distributed throughout the epithelial cells of nonciliated cells is light and surrounds the basally oriented nuclei, which contain diffuse chromatin, scattered regions of dense heterochromatin (Fig. 2A), and discrete nucleoli (Fig. 2C). Apically, endocytic vesicles are common at the base of microvilli and appear to form via endocytosis of the apical membrane between microvilli (Fig. 2D). Electron dense lysosomes are common in the supranuclear region of nonciliated cells Journal of Morphology

scattered amongst small mitochondria (Fig. 2D). A diffuse luminal material is associated with microvilli, and may represent products filtered into the lumen through the genital kidney renal corpuscles (Fig. 2D). This material was not observable at the light microscopy level. Basal and lateral to the nucleus, the cytoplasm of nonciliated cells is filled with small lucent vesicles and dark elongated mitochondria (Fig. 2C). Small aggregations of rough endoplasmic reticulum are also common immediately basal to the nucleus (Fig. 2C). The apical cytoplasm of the ciliated cells of the genital kidney proximal tubule lack endocytic inclusions as observed in the nonciliated cells (Fig. 2B). Small dark mitochondria are abundant throughout the cytoplasm, but reach their highest density in the supranuclear region of ciliated epithelial cells (Fig. 2B). Besides the random occurrence of dark inclusions around the nucleus, the cytoplasm of ciliated cells is unremarkable in terms of its contents. The nuclei of ciliated cells possess more heterochromatin than those of

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Fig. 5. Notophthalmus viridescens, ultrastructure of the pelvic kidney distal tubule. (A) Overview of the distal tubule epithelial lining (June) depicting the homogenous epithelial cell composition. (B) High magnification of the apical and lateral regions of the epithelial cells (June) depicting junctional complexes adhering adjacent epithelial cells and apical and lateral cytoplasmic contents. (C) High magnification of the lateral and basal regions of the epithelial cells (June) depicting the lateral cell membranes, highly folded nature of the basal membrane, and cytoplasmic contents of this cellular region. (D) High magnification of the epithelial cell basal membrane (February) depicting the foot processes contacting the basal lamina and the cytoplasmic contents of this cellular region. Bb, brush border; Bl, basal lamina; Ds, desmosome; Fp, foot process of basal membrane; Ft, filtrate; Ic, intercellular canaliculi; Hd, hemidesmosomes; Lu, lumen; Mi, mitochondria; Nu, nuclei; Tj, tight junction; Va, vacuolated region.

nonciliated cells, but still possess areas of diffuse chromatin (Fig. 2A). The epithelial lining of the distal tubule in the genital kidney is low cuboidal with large, centrally located heterochromatic nuclei (Fig. 3A). The epithelial lining is homogenous along the entire length of the genital kidney distal tubule, and the apical membranes of individual epithelial cells are devoid of modification in the form of a brush border or cilia (Fig. 3A,B); however, an occasional microvillus can be observed (Fig. 3A). Folding of the lateral membranes between cells is extensive, producing labyrinthine intercellular canaliculi (Fig. 3C). The intercellular canaliculi are not distended (Fig. 3B,C), and sealed apically by tight junctions (Fig. 3B) followed by regularly aligned desmosomes more basally (Fig. 3B). The majority of the basal plasma membranes lies flat against the basal lamina; however, some folding of the basal plasma membrane exists causing involutions of the basal plasma membrane toward the interior

of individual epithelial cells (Fig. 3D). Apical, lateral, and basal to the nucleus the cytoplasm of the epithelial cells of the genital kidney distal tubule is unremarkable, and only possesses large mitochondria (Fig. 3B-D), the occasional electron dense inclusion (Fig. 3B), and random areas of vacuolation (Fig. 3A,C). The pelvic kidney proximal tubule is lined by a columnar epithelium with basal nuclei that are slightly heterochromatic and possess discrete nucleoli (Fig. 4A). The apical portion of each epithelial cell is slightly domed and covered with an extensive microvillus brush border (Fig. 4A). The majority of the epithelial cells lining the pelvic kidney proximal tubule have a light cytoplasm; however, epithelial cells with a dark cytoplasm are scattered amongst the light cells (Fig. 4A), and their concentration is higher more distally along the length of the proximal tubule. As noted previously, the apical membrane possesses an extensive microvillus brush border with Journal of Morphology

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high concentrations of filtrate dispersed in the lumen of the proximal tubule and amongst microvilli (Fig. 4A,B). Occasionally, the apical membranes of the light proximal tubule epithelial cells are distended and project into the lumen of the proximal tubule forming what appear to be blebs from the apical membrane (Fig. 4A). The intercellular canaliculi of the proximal tubule epithelia are slightly distended and sealed apically by tight junctions with desmosomes regularly found more basally (Fig. 4C). The lateral membrane of some epithelial cells forms small lateral projections, but for the most part, the intercellular canaliculi are nonlabyrinthine (Fig. 4C). Basally, the plasma membrane of the epithelial cells of the proximal tubule is folded in appearance due to projections (foot processes) of the basal membrane (Fig. 4D). At the most proximal portion of the pelvic kidney proximal tubule, the basal plasma membrane lacks folding. Apically, the cytoplasm of light cells is filled with endocytic vesicles immediately basal to the microvillus brush border (Fig. 4B). Lysosomes at varying stages of development are highly concentrated basal to the endocytic vesicles (Fig. 4B,C). Large mitochondria are found intermixed with the lysosomes (Fig. 4B,C). Mitochondria are also observed lateral to the cell nuclei and in high concentrations basal to the cell nuclei (Fig. 4C,D). Basally, hemidesmosomes are found at the most basal portions of the foot processes (Fig. 4D). Small cisternae of rough endoplasmic reticulum are also found scattered perinuclearly in the light cells of the pelvic kidney proximal tubule epithelium. The cytology of dark cells is similar to that of light cells; however, endosomes are prominent basal to apical endocytic vesicles (Fig. 4A), and the entire cell cytoplasm is filled with smooth endoplasmic reticulum, Golgi vesicles, and rough endoplasmic reticulum (Fig. 4A). The epithelial lining of the pelvic kidney distal tubule is homogenous along its entire length (Fig. 5A). The lining is cuboidal with centrally to apically position nuclei containing heterochromatin aggregated at the periphery of each epithelial cell nuclei (Fig. 5A). Filtrate is abundant in the lumen of this pelvic kidney nephron region (Fig. 5A). The apical membrane is devoid of a microvillus brush border or cilia, although small microvillus projections are occasionally observed as protrusion from the apical membrane (Fig. 5B). The lateral membranes of adjacent cells lie flat against each other forming nonlabyrinthine intercellular canaliculi (Fig. 5B,C) that are sealed apically with tight junctions (Fig. 5B) and desmosomes more basally (not pictured). Basally, the membrane is highly folded. The folds are extensive and result in invaginations of the basal membrane deep into the interior of every cell (Fig. 5D). The basal membrane makes contact with the basal lamina as Journal of Morphology

multiple foot processes with hemidesmosomes present at the terminus of each foot (Fig. 5D). Apically, the cytoplasm of the pelvic kidney distal tubule epithelial cells is devoid of organelles (Fig. 5A,B); however, large mitochondria are located immediately perinuclear (Fig. 5A,B). Vacuolated spaces around the cell nuclei are also prominent (Fig. 5A–C). Lateral to the cell nucleus, regular folds of the basal membrane break the cytoplasm (Fig. 5A–C). Large and elongated mitochondria fill the scant areas of cytoplasmic space between the basal membrane folds with their long axis perpendicular to the basal lamina (Fig. 5A–D). DISCUSSION Through ultrastructural analysis of the genital and pelvic kidney nephrons of Notophthalmus viridescens, we could not reject the hypothesis that the genital kidney nephrons of N. viridescens are modified for the purpose of sperm transportation when compared to those nephrons of the pelvic kidney. This support is strictly based on epithelial structure, as the physiology of these ducts has yet to be assessed in any of the 10 salamander families. Similar results were reported for a close relative, Ambystoma maculatum. Ambystomatidae and Salamandridae share a common ancestor (Pyron et al., 2011; Siegel et al., 2013). In both families, the lumina of the genital kidney nephrons lack dense aggregations of filtrate and epithelial cells of the genital kidney proximal tubules possess microvilli or elongated cilia. The proximal tubules of the pelvic kidney lack these cilia and lumina of the proximal tubule nephrons is almost always filled with dense filtrate (Siegel et al., 2010, 2013). The function of cilia lining the surface of the genital kidney proximal tubule epithelium has yet to be tested, but cilia may aid in the movement of immature sperm through the length of the genital kidney nephron through cilliary action, as sperm do not mature until reaching the Wolffian ducts (McLaughlin and Humphries, 1978; Russell et al., 1981; Matsuda, 1986), or the mixing of seminal fluids (Hess, 2001). Interestingly, distal to the testicular rete (amniotes) or vas efferens (anamniotes), all vertebrates examined thus far possess a sperm transport duct lined apically with cilia (reviewed by Sever, 2010), highlighting the evolutionary importance of ciliation of the proximal sperm transport ducts. Historically, it was thought that the genital kidney nephrons could have the dual functionality of modifying urine as well as transporting sperm (Spengel, 1876). Even though the genital kidney nephrons possess all the functional structures found in the pelvic kidney for urine formation, their modification is supportive of sperm transport and not that of urinary function. The lack of basal

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plasma membrane folding in the epithelia of the proximal tubule and distal tubule in the genital kidney infer a decrease in the ability for reabsorption of water and solutes (Maunsbach and Boulpaep, 1984). Nephrons that are responsible for urine formation differ from this morphology by having a highly folded basal plasma membrane (especially the distal tubule) and overall increase in surface area in both the proximal and distal tubules in not only Notophthalmus viridescens, but in every salamander studied (Clothier et al., 1978; Sakai and Kawahara, 1983; Maunsbach and Boulpaep, 1984; Siegel et al., 2010); although this folding decreases in aquatic salamanders (Maunsbach and Boulpaep, 1984), it was not as extensive in Notophthalmus viridescens when compared to terrestrial salamanders (see Maunsbach and Boulpaep, 1984; Siegel et al., 2013). Furthermore, as Siegel et al. (2013) noted, the renal corpuscles of the genital kidney in Ambystoma maculatum are ultrastructurally different than those of the pelvic kidney, indicating potential differences in primary urine filtration into the nephron; however, this hypothesis needs testing. As observed in Ambystoma maculatum (Siegel et al., 2013), the brush border of the genital kidney proximal tubule nonciliated cells increases their apical surface area and is indicative of apical transport processes. Numerous endocytic vesicles and supranuclear lysosomes support this hypothesis. Interestingly, endocytic activity through the proximal sperm transport ducts (i.e., ductuli efferentes) was also identified in numerous amniote taxa and undoubtedly is an important feature of the proximal sperm transport ducts in terrestrial vertebrates (for review see Sever, 2010). Even though the genital kidney nephrons are modified for sperm transport, we were unable to find any support for the hypothesis that these ducts vary in epithelial activity throughout the reproductive cycle. In other vertebrates (reviewed by Sever, 2010), the ductuli efferentes produce copious apical blebs. We found no evidence of complex secretory processes in the testicular ducts of Notophthalmus viridescens throughout the reproductive and nonreproductive seasons as outlined by Siegel et al. (2012b). Furthermore, dense bodies of unknown function in the apical cytoplasm of the epithelia of the genital kidney distal tubules and Wolffian ducts of Ambystoma (Siegel et al., 2013) and Rhyacotriton (Zalisko and Larsen, 1988), respectively, were not observed in the genital kidney distal tubules of N. viridescens. Thus, if these dense bodies represented some sort of addition to the seminal fluid during sperm transport, this fluid is lacking in the sperm transport ducts of some salamanders; that is, N. viridescens. The lack of secretory function along the length of the genital kidney nephrons was surprising, as salamanders lack definitive seminal fluid producing glands.

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Structure of the pelvic kidney proximal and distal tubules in Notophthalmus viridescens was identical to that of another member of Salamandridae, Cynops pyrrhogaster (Sakai and Kawahara, 1983). In both species, the primary cell type of the proximal tubule was that of a microvillus columnar cell with endocytotic and lysosomic activity. Although this was the primary cell type in N. viridescens, this taxon also possessed scattered dark cells (especially more distally) with abundant smooth endoplasmic reticulum. This epithelial structure was similar to what was also observed in the proximal tubule of other salamander families (Clothier et al., 1978; Hinton et al., 1982; Maunsbach and Boulpaep, 1984; Stanton et al., 1984; Siegel et al., 2010), although multiple regions of the proximal tubule were delineated in Ambysotma maculatum (vauolated and lysosomic regions; Siegel et al., 2010) and Amphiuma means (regions 1 through 3; Clothier et al., 1978). The distal tubule of both taxa was composed solely of cuboidal cells with no apical modifications and extensive folding of the basal plasma membrane. This epithelial structure was identical to what was also observed in the distal tubule of other salamander families (Clothier et al., 1978; Hinton et al., 1982; Maunsbach and Boulpaep, 1984; Stanton et al., 1984; Siegel et al., 2010), although an early distal tubule and late distal tubule were delineated in A. means (Stanton et al., 1984) and Hinton et al. (1982) also described two distal tubule segments in A. tigrinum; however, the two segments in A. tigrinum appear to be the delineation of the distal and collecting tubules (Siegel et al., 2010). In conclusion, we provide the first ultrastructural examination of the genital kidney nephrons in a salamandrid and the first seasonal comparison of the genital kidney nephrons to that of the pelvic kidney nephrons. With this ultrastructural investigation, we demonstrate that the genital kidney nephrons are similar in structure to those of Ambystoma maculatum (Siegel et al., 2013) and different in terms of epithelial cell composition and structure when compared to the pelvic kidney nephrons. Although the genital kidney nephrons are modified from their pelvic kidney counterparts for the action of transporting sperm, they do not express seasonal variation at the ultrastructural level when directly compared to the outlined reproductive cycle of Notophthalmus viridescens (Siegel et al., 2012b).

ACKNOWLEDGMENTS We thank Robert Aldridge and Saint Louis University for early support of this project, David Sever and Southeastern Louisiana State University for continuous use of microscopy facilities, and Journal of Morphology

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Southeast Missouri State University for continual support of our research. LITERATURE CITED Clothier R, Worley R, Balls M. 1978. The structure and ultrastructure of the renal tubule of the urodele amphibian, Amphiuma means. J Anat 127:491–504. Hess RA. 2001. The efferent ductules: structure and function. In: Robaire B, Hinter BT, editors. The Epididymis: From Molecules to Clinical Practice. New York: Kluwer Academic Press, Plenum Press. pp 49–80. Hinton DE, Stoner LC, Burg M, Trump BF. 1982. Heterogeneity in the distal nephron of the salamander (Ambystoma tigrinum): A correlated structure function study of isolated tubule segments. Anat Rec 204:21–32. Matsuda M. 1986. Difference of fertilizing capacity between testicular sperm and vas deferens sperm in Cynops pyrroghaster. Gamete Res 14:209–214. Maunsbach AB, Boulpaep EL. 1984. Quantitative ultrastructure and functional correlates in proximal tubule of Ambystoma and Necturus. Am J Physiol 246:710–724. McLaughlin EW, Humphries AA. 1978. The jelly envelopes and fertilization of eggs of the newt, Notophthalmus viridescens. J Morphol 158:73–90. Pyron RA, Burbrink FT, Colli GR, Motes de Oca AN, Vitt LJ, Kuczynski CA, Weins JJ. 2011. The physiology of advanced snakes (Colubroidea), with discovery of new subfamily and comparison of support methods for likelihood trees. Mol Phylogenet Evol 58:329–342. Rafinesque CS. 1820. Annals of nature or annual synopsis of new genera and species of animals, plants, etc. discovered in North America. Lexington: Thomas Smith. 16 p. Russell LD, Brandon RA, Zalisko EJ, Martan J. 1981. Spermatophores of the salamander Ambystoma texanum. Tissue Cell 13:609–621.

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