Histological and quantitative changes in the annual

FRANCISCO JOSE SAEZ,BENITO FRAILE, AND RICARDO PANIAGUA

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Department of Cell Biology and Genetics, University of Alcalci de Henares, Alcalci de Henares, Madrid, Spain Received March 29, 1989

Can. J. Zool. Downloaded from www.nrcresearchpress.com by China University of Petroleum on 06/05/13 For personal use only.

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Histological and quantitative changes in the annual testicular cycle of Triturus marmoratus marmoratus

SAEZ,F. J., FRAILE, B., and PANIAGUA, R. 1990. Histological and quantitative changes in the annual testicular cycle of Triturus marmoratus marmoratus. Can. J. Zool. 68: 63-72. Eight male marbled newts (Triturus marmoratus marmoratus) were collected on the 15th of each month in 1987 and their testes were studied by light and electron microscopy. Quantitative studies also were performed to establish the annual testicular cycle and the total volume per testis occupied by each germ cell type throughout the year. Characteristicultrastructural features of germ cells are the occurrence of a well-developed Golgi complex in primary spermatogonia; multiple small dictyosomes and nuclear blebs in primary spermatocytes; peripherally situated mitochondria; long strands of endoplasmic reticulum and subsurface cisternae in round spermatids; and abundant rough endoplasmic reticulum in follicular cells. Secondary spermatocytes have a short or absent interphase and are observed in the prophase. The annual testicular cycle comprises three periods: (i) germ cell proliferation (May-June), characterized by the formation of primary spermatocytesthat undergo meiosis, giving rise to round spermatids; (ii) spermiogenesis (July-September), during which round spermatids develop into spermatozoa and the interstitial boundary cells are transformed into glandular tissue cells; and (iii) testicular quiescence (October-April) in which the testis contains only spermatozoa, glandular tissue, and a few primordial germ cells and spermatogonia. In the second phase of testicular quiescence (February-April) spermatozoa are released from the testis and proliferation of secondary spermatogonia occurs. SAEZ,F. J., FRAILE, B. et PANIAGUA, R. 1990. Histological and quantitative changes in the annual testicular cycle of Triturus marmoratus marmoratus. Can. J. Zool. 68 : 63-72. En 1987, huit tritons (Triturus marmoratus marmoratus) mlles ont t t t rkcoltts le 15 de chaque mois et leurs testicules ont t t t soumis tt des examens au microscope tlectronique et au microscope photonique. Des dosages quantitatifs ont permis d'ttablir le cycle testiculaire annuel et d'tvaluer le volume total occupk par chaque type de cellule germinale dans le testicule durant toute l'annte. Parmi les caractkristiques microscopiques des cellules germinales, mentionnons la prtsence d'un complexe de Golgi bien dtvelopp6 dans les spermatogonies primaires, de nombreux petits dictyosomes et d'ampoules nuclkaires dans les spermatocytesprimaires, de mitochondries en position @riphtrique, de longs cordons de rtticulum endoplasmiqueet de citernes presque superficielles dans les spermatides rondes, enfin d'un ergastoplasme abondant dans les cellules folliculaires. Les spermatocytes secondaires ont une interphase courte ou absente et s'observent durant la prophase. Le cycle annuel des testicules comprend trois Ctapes : (i) la prolifkration des cellules germinales (mai-juin) au cours de laquelle se foment les spermatocytes primaires qui subissent une meiose, donnant naissance aux spermatides rondes, (ii) la spermiogenkse (juillet-septembre) durant laquelle les spermatides rondes se transforment en spermatozoi'deset les cellules intersticielles ptriphtriques se transforment en cellules de tissue glandulaire, (iii) la quiescence des testicules (octobre-avril), p6riode pendant laquelle le testicule ne contient que des spermatozoi'des, du tissu glandulaire, quelques cellules germinales primordiales et quelques spermatogonies. Durant la seconde phase de la quiescence (ftvrier-avril), les spermatozoi'dessont liMrts et les spermatogonies secondaires prolifkrent. [Traduit par la revue]

Introduction The morphology of the testis of urodele amphibians has been studied in many species by light (Humphrey 1921;Adams 1940; Ifft 1942; Miller and Robbins 1954; Joly 197 1 ; Rouy 1972; Tso and Lofts 1977a; Tanaka and Iwasawa 1979) and electron microscopy (Picheral 1968; Barker and Biesele 1967; Sentein and Temple 197 1; Rouy 1974; Tso and Lofts 1977b; Imai and Tanaka 1978; Hamasima and Kotani 1979; Callard et al. 1980; Ucci 1982; Bergmann et al. 1982, 1983; Pudney et al. 1983; Schindelmeiser et al. 1983, 1985). These studies describe the cell types involved in the spermatogenetic process and, in some cases, the period of the year in which the most representative of these cells are present. The most complete descriptions of the annual testicular cycle, with percentages of cell types present in each period, are for Euproctus asper (Rouy 1972), Trituroides hongkongensis (Tso and Lofts 1977a), and Cynops pyrrhogaster (Tanaka and Iwasawa 1979). There are no previous reports on spermatogenesisand the annual testicular cycle of the marbled newt, Triturus marmoratus, or other urodele amphibians inhabiting Spain. This paper offers a light and electron microscopic description of spermatogenesis in T. marmoratus and ' ~ u t h o to r whom correspondence and requests for reprints should be addressed. Printed in Canada I Imprimt au Canada

studies the changes in the total volume per testis occupied by each germ cell type and the glandular tissue cells during the different months of the year.

Materials and methods Eight male marbled newts (Triturus marmoratus marmoratus Latreille) were collected from forested areas of the Province of Leon in Spain (latitude 42"301N,longitude 5"30-45'W; altitude 800 m) on the 15th of each month in 1987. The average day length and temperature for each month were as follows: January, 9.5 h, 5°C; February, 10.5 h, 6°C; March, 11.25 h, 9°C; April, 13.5 h, 12°C; May, 14.5 h, 16°C; June, 15 h, 20°C; July, 14.75 h, 23"C, August, 13.5 h, 23°C; September, 12.5 h, 21°C; October, 11 h, 14°C; November, 10 h, 9°C; and December, 9.25 h, 5°C. To eliminate the influence of body weight, only newts weighing between 9 and 9.5 g were selected. Newt ages, determined by counting growth rings in the femur (Dolmen 1982), ranged from 6 to 9 years. The day after their capture the newts were weighed, anaesthetized with methanesulphonate (MS-222, Sandoz, Barcelona, Spain), and perfused through the aortic cone for 30min with 3% phosphatebuffered glutaraldehyde. After this, both testes were removed and weighed, and the testicular volumes were calculated by water displacement. The left testis was fixed for an additional 6 h in the same fixative, dehydrated, and embedded in paraffin and used for quantitativestudies. Because germ cell differentiation in the newt testis progresses from


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the anterior to the posterior testicular pole, only sagittal sections of the whole testis were suitable for quantitative studies. For this purpose, five 5 p,m thick sagittal sections of each left testis, taken at 116, 113, 112, 213, and 516 of the transverse testicular diameter, were selected and stained with haematoxylin and eosin. In each testis, the areas occupied in the five sections by each germ cell type (including their accompanying follicular cells and interstitial cells) and the glandular tissue cells (developed Leydig cells with their accompanying connective tissue) were measured with a semiautomatic image analyzer (Kontron, Zeiss, Oberkochen, Federal Republic of Germany). The resulting values were divided by the total surface area of the five sections, thus providing the volume densities of each cell type. The absolute volume per testis for each cell type was obtained by multiplying the volume density by the testicular volume and applying a correction factor (0.76). Because the testicular volume was calculated before fixation and the surface area occupied by each germ cell type was estimated after fixation and embedding, this correction factor is necessary to convert testicular volume before fixation to testicular volume after fixation and embedding. The factor was calculated beforehand using 50 newt testes. The means and standard deviations for each group (month) of newts were calculated from the average values for each testis. Comparison of the means between groups was carried out using the one-way ANOVA test and Sheffk's pairwise comparison test. The right testes were sliced into 1-rnm3 blocks, fixed for an additional 6 h in the same fixative, postfixed in 1 % phosphate-buffereti osmium tetroxide (4 h), dehydrated in ethanol, and embedded in Epon. Semithin sections were stained with toluidine blue. Ultrathin sections were double-stained with uranyl acetate and lead citrate.

Results The marbled newt testis is formed by two to four lobes with identical organization and synchronic maturation. Each lobe consists of multiple spherical lobules connected to a branched system of ductuli efferentes. Each lobule comprises a single or, at most, two consecutive germ cell types. Germ cell development in the lobules progresses from the anterior to the posterior testicular poles (Fig. 1). However, not all germ cell types are present throughout the year. According to the histological pattern of the testis, the cycle is discontinuous and can be divided into three periods. First period (May-June): germ cell proliferation In May a new spermatogenetic wave begins with proliferation of germ cells which leads to a remarkable enlargement of the testis (Fig. I). The following germ cells types are present. Primordial germ cells are the stem cells in spermatogenesis. They have a homogeneously pale, multilobed nucleus containing one or two small nucleoli (Fig. 2). Their cytoplasm exhibits scanty rough endoplasmic reticulum cisternae, a few polysomes, and polymorphic mitochondria that form a crown around the nucleus. Peculiar features are electron-dense vesicles and nucleolus-like structures that appear isolated (nuages) or joining mitochondria (intermitochondrial bars) (Fig. 3). The primary spermatogonia that appear intermingled with the primordial germ cells originate from mitoses in these cells. Primary spermatogonia possess a round to ovoid nucleus with

homogeneously distributed dense chromatin masses (Fig. 2). Their cytoplasm is similar to that of primordial germ cells, including the arrangement of,mitochondria around the nucleus and the presence of nuages and intermitochondrial bars. A distinctive feature is the occurrence of a developed Golgi complex (Fig. 4). Each single germ cell (primordial germ cell or primary spermatogonium) is surrounded by several follicular cells, forming a follicle or cyst (Figs. 2, 3,5,6). Follicular cells have an elongated, somewhat triangular, indented nucleus with abundant clusters of dense chromatin (Figs. 2, 4). Their cytoplasm contains abundant rough endoplasmic reticulum (Fig. 6) and displays projections which isolate each germ cell. Secondary spermatogonia originate from mitosis of primary spermatogonia and differ from these in the somewhat smaller nucleus with more abundant and irregular dense chromatin (Fig. 5), as well as in the absence of nuages and electron-dense bars between mitochondria, which are not arranged around the nucleus (Fig. 6). All the secondary spermatogonia derived from the same primary spermatogonium form a single cyst. Within each cyst, the secondary spermatogonia are not separated by follicular cells (Fig. 5). These only separate each cyst from other cysts, each derived from a different primary spermatogonium. Primary spermatocytes originate from mitoses of secondary spermatogonia. Primary spermatccytes are larger than the preceding germ cell types and their nuclei show the different stages of the first meiotic division (Fig. 7). In zygotene and pachytene the nuclear envelope displays blebs that are not found in the other germ cell types. The cytoplasm is characterized by the occurrence of multiple dictyosomes and whorls of smooth endoplasmic reticulum (Fig. 8). In June, in the lobules of primary spermatocytes there are many cells in the meiotic stages between metaphase I and anaphase I1 (Fig. 7). Secondary spermatocytes are difficult to identify because interphase cells showing morphological characteristics other than those of either primary spermatocytes or round spermatids are not seen. Nuclei intermediate in size between primary spermatocytes and round spermatids show well-defined chromosomes attached to the nuclear envelope, as in advanced prophase (Figs. 7, 9). The cytoplasm is like that of primary spermatocytes except for a peripheral arrangement of mitochondria. Lobules of round spermatids are also present in June. These cells have a round nucleus, smaller than that of primary spermatocytes, with dense chromatin strands (Figs. 7, 10). The cytoplasm exhibits small, peripherally placed mitochondria, a few very long, smooth endoplasmic reticulum cisternae, some located immediately beneath the plasma membrane (subsurface cisternae), and multiple dictyosomes that are closer to each other than in the spermatocytes (Fig. 10). Small vesicles arise from the Golgi complex and join to form the acrosomic granule which afterwards makes contact with the nucleus (Fig. 11). Near the acrosomic granule, ciliary structures suggesting the initial steps of axoneme formation can be seen (Figs. 10, 12).

FIG. 1 . Longitudinally sectioned testicular lobule of a marbled newt killed in June. pg, primordial germ cells and primary spermatogonia; sg, secondary spermatogonia; pc, primary spermatocytes; rs, round spermatids. Haematoxylin and eosin. x 25. FIG. 2. Anterior testicular

pole of the same newt, showing intermingled primordial germ cells (PGC) and primary spermatogonia (PG). Each germ cell is completely surrounded by follicular cells (FC). IC, interstitial cell. Toluidine blue. x970. FIG. 3. Primordial germ cell of the same newt, showing a pale multilobed nucleus, mitochondriajoined by electron-dense material (arrows), and electron-dense vesicles (arrowhead). The large arrow points to interdigitations with the adjacent follicular cell (FC). x5800. FIG. 4. Primary spermatogonium of the same newt, showing a developed Golgi complex (star, electron-dense material joining mitochondria (arrows), and electron-dense vesicles (arrowhead). x 12 000. FIG. 5. Lobules of secondary spermatogonia (SG) in the same newt, surrounded by follicular cells (FC). Toluidine blue. x970.


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Each lobule of developing germ cells is surrounded by interstitial cells, similar to fibroblasts (Fig. 6). A few spermatozoa formed in the preceding cycle and remnants of the glandular tissue can also be observed.

lipid droplets (Fig. 15). When spermiogenesis is completed and spermatozoa are released, the cystic pattern of these lobules disappears and the lobules are occupied by the transformed interstitial cells that form the glandular tissue (Figs. 13, 18).

Second period (July-September): spermiogenesis The testis enlarges further, reaching its largest volume in September (Fig. 13). In July, besides the preceding germ cell types, elongated spermatids are observed. These cells have an electron-dense nucleus which gradually becomes elongated, up to 80 x 3 pm (Fig. 14). During spermiogenesis elongated spermatids are grouped, forming cysts, each supported by a follicular cell (Fig. 15). These cells also undergo morphological changes; their nuclei become more irregularly outlined and paler than in the preceding stages of spermatogenesis. Their cytoplasm exhibits abundant smooth endoplasmic reticulum, residual bodies, and lipid droplets (Fig. 15). At the periphery of the follicular cells are bands of microtubules surrounding the differentiating spermatids . At the end of spermiogenesis the spermatid cysts are released from the follicular cells and form spermatozoon bundles. The first spermatozoa appear in August and their formation continues until September, when more than 60% of testicular volume is occupied by spermatozoon bundles. The spermatozoa of the marbled newt are similar to those of other urodele amphibians. They have a very elongated, somewhat bent head, covered on its tip by a small thin acrosome. Opposite to the acrosome is the neck piece, an electron-dense structure about 5 pm in length, which surrounds the initium of the axoneme (Fig. 16). The tail forms an undulating membrane which is bordered along its length by the axoneme and the axoneme cup on one side and the axial filament (axial rod) on the other side. In the middle piece, the axial filament has a horseshoe-shaped profile and is surrounded by mitochondria. In the principal piece the axial filament displays a trifoliate outline and does not show mitochondria (Fig. 17). When the spermatozoa are released from their follicular cells, the surrounding interstitial cells increase in size and become less elongated. The cytoplasm contains abundant smooth endoplasmic reticulum, mitochondria with tubular cristae, and some

Third period (October-April) First phase (October-December) In October all primary spermatocytes, round spermatids, and elongated spermatids have developed into spermatozoon bundles which occupy about 80% of the testicular volume. Only in the anterior testicular pole is there a group of primordial germ cells, primary spermatogonia, and secondary spermatogonia that will be the source of spermatogenesis in the following cycle. In December the glandular tissue reaches its greatest volume (Fig. 18). Second phase (January-April) During this period there is a slow proliferation of primary spermatogonia and secondary spermatogonia which anticipates the pronounced germ cell proliferation occurring in May, when the new cycle begins. Spermatozoon release from the testis occurs in February. Mating and egg laying occur in MarchApril. Changes in testicular volume and in the total volume per testis occupied by each germ cell type and glandular tissue cells in the different periods are shown in Table 1 and Fig. 19.

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Discussion A multilobate pattern of the testis has been reported in many urodele species (Humphrey 1921; Miller and Robbins 1954; Joly 1971; Rouy 1972). The number of lobes varies among species. About 80% of the marbled newts studied had two or three lobes; the remaining 20% had one or four lobes. Each testicular lobe comprises many lobules which, as occurs in most Salamandridae, are spherical and appear connected to a branched system of ductuli efferentes (Lofts 1974). Other urodeles possess tubular lobules that are radially arranged, as in Plethodon (Lofts 1974), or distributed like the ribs of a fan, as in Necturus (Pudney et al. 1983). Cephalocaudal germ cell development is common in urodeles (Rouy 1972; Lofts 1974; Tanaka and Iwasawa 1979).

FIG. 6. Part of the lobule from Fig. 5, showing secondary spermatogonia (SG), follicular cells (FC) with developed rough endoplasmic reticulum (rer), and a fibroblast-like interstitial cell (IC). x8800. FIG. 7. Testicular lobules of the same newt, showing different stages of the meiotic divisions. pc, primary spermatocytesat pachytene;the star indicates stages from metaphase I to anaphase 11; rs, round spermatids. The cells labelled sc are probably secondary spermatocytes with a nuclear size intermediate between that of primary spermatocytesand round spermatids. Toluidine blue. x500. FIG. 8. Primary spermatocyte at zygotene, showing nuclear blebs (stars), multiple small dictyosomes (arrows), and a whorl of smooth endoplasmic reticulum (arrowhead). x 9000. Inset: Synaptonemal complex. X 37 500. FIG. 9. Cell labelled sc in Fig. 7. Chromosomes (ch) attached to the nuclear membrane can be seen. Most mitochondria are close to the plasma membrane (arrows). X 7000. FIG. 10. Round spermatids from Fig. 7, showing peripherally placed mitochondria (m), a cross-sectioned cilium (large arrow), a long endoplasmic reticulum cisterna parallel to the nuclear surface (small arrows), and two subsurface cisternae forming a "triad" between two adjacent cells (arrowheads). X 12 000. FIG. 1 1 , Acrosomic granule (ag) near the nucleus (N) in a round spermatid from the lobule shown in Fig. 10. X 21 000. FIG. 12. Cilium (C) near the Golgi complex (stars) and the nucleus (N) in a round spermatid. x 20 000. FIG. 13. Longitudinally sectioned testicular lobule of a newt killed in September. pg, primary spermatogonia; sg, secondary spermatogonia; pc, primary spermatocytes; rs; round spermatids; es, elongated spermatids; spz, spermatozoon bundles; gt, glandular tissue. Haematoxylin and eosin. x 14. FIG. 14. Elongated spermatids (es) beside a lobule of round spermatids. The nuclei of the follicular cells (arrows) are paler and more irregularly outlined than in the preceding stages of spermatogenesis. Toluidine blue. X 675. FIG. 15. Follicular cell (FC) and interstitial cell (IC) after the end of spermatogenesis. Both cell types are larger than during spermatogenesis and exhibit abundant smooth endoplasmic reticulum (ser), lipid droplets (li), and residual bodies (arrows). x 5400. FIG. 16. Oblique section of spermatozoa, showing the head (h) and the neck piece (np). X 10000. FIG. 17. Cross-sectioned spermatozooa in a spermatozoon bundle. um, undulating membrane; arrows indicate axoneme and axoneme cup; star indicates horseshoe-shaped profile of the axial filamentin the middle piece; asterisks indicate the trifoliate profile of the axial filament in the principal piece. X 10000. FIG. 18. Longitudinally sectioned testicular lobule of a newt killed in December. sg , secondary spermatogonia; spz, spermatozoon bundles; gt, glandular tissue. x 28.


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SAEZ ET AL.

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Ultrastructurally, the cell types in the testis of the marbled newt that are described in this paper are similar to those of Salamandra salamandra (Bergmann et al. 1982; Schindelmeiser et al. 1983, 1985). The nuages are cytoplasmic ribonucleoproteins which appear isolated or closely attached to mitochondria, forming intermitochondrial bars. These ribonucleoproteins probably have a nucleolar origin and have been reported in primordial germ cells and primary spermatogonia of many urodele species including Cynops pyrrhogaster (Tanaka et al. 1980), Ambystoma mexicanum (Ikenishi and Nieuwkoop 1978), and Pleurodeles waltl (Picheral1971). The spermiogenesis is like that reported for Amphiuma tridactylum (Barker and Biesele 1967). However, we have observed the following peculiar ultrastructural features which have not been reported in other urodeles. The follicular cells show a well-developed rough endoplasmic reticulum during spermatogonial proliferation and meiosis, as well as development of abundant smooth endoplasmic reticulum during spermiogenesis; this contrasts with the small amounts of smooth endoplasmic reticulum reported for S. salamandra (Schindelmeiser et al. 1983, 1985). The characteristic central arrays of parallel tubules observed in the mitochondria of mature interstitial cells in S. salamandra (Bergmann et al. 1982) and other urodeles (Picheral 1968; Imai and Tanaka 1978; Callard et al. 1980) are lacking in the marbled newt. Primary spermatogonia show a well-developed Golgi complex which is not present in other germ cell types. Primary spermatocytes exhibit blebs in the nuclear membrane and multiple small dictyosomes in the cytoplasm. Round spermatids in the marbled newt are characterized by the occurrence of multiple small dictyosomes, long cisternae of smooth endoplasmic reticulum, subsurface cisternae, and peripherally placed mitochondria. The cells identified as secondary spermatocytes in S. snlamandra (Schindelmeiser et al. 1985) are also present in the marbled newt, but these cells seem to be round spermatids because both acrosomic granules and cilia are observed. Interphase secondary spermatocytes in the marbled newt were not identified. Probable secondary spermatocytes, that is, cells with a nuclear diameter intermediate between those of primary spermatocytes and round spermatids, showed chromosomes attached to the nuclear membrane. The cytoplasm of these cells differs from that of primary spermatocytes in the peripheral distribution of mitochondria, and from that of round spermatids in the absence of long cisternae of smooth endoplasmic reticulum, subsurface cisternae, ciliary structures, and acrosomic granules. It is probable that in the marbled newt, secondary spermatocytes have a short or absent interphase. The annual testicular cycle of the marbled newt is similar to that of the urodeles Taricha granulosa (Specker and Moore 1980), E. asper (Rouy 1972), and C . pyrrhogaster (Tanaka and Iwasawa 1979), which also live in cold-temperate areas. The longer spermiogenesis period (until October) in the latter two species might be due to the somewhat warmer temperature. In urodeles of warmer areas, such as Trituroides hongkongensis (Tso and Lofts 1977a), Ambystoma tigrinum (Norris et al. 1985), and P . waltl (Garnier 1985), the cycle is longer, from March to November. Although no complete data have been reported for Necturus maculosus (Humphrey 1921; Pudney et al. 1983) and Notophthalmus viridiscens (Adams 1940; Ifft 1942), the cycle of these species seems to be very short, because germ cell proliferation begins in summer and spermiogenesis is complete in September. Quantification of germ cell types throughout the year in the marbled newt reveals that proliferation of secondary spermatog-


SAEZ ET AL.

Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. primary spermatogonia

0 ....... elongated spermatids

secondary spermatogonia

spermatozoa

primary spermatocytes

glandular tissue

round spermatids

FIG.19. Changes in testicular volume and total volume per testis occupied by each germ cell type during the annual testicular cycle in Triturus marmoratus murmoratus. The volume occupied by each germ cell type includes that of the accompanying follicular cells and interstitial cells. Standard deviations and statistical analysis of differences between means are given in Table 1. onia begins in February, 3 months before the beginning of the cycle. Although in urodeles with a cycle similar to that of the marbled newt the highest numbers of spermatogonia have been described in April-June (Humphrey 1921; Tso and Lofts 1977a; Specker and Moore 1980; Norris et al. 1985), precise quantitative data are not provided and it is possible that early spermatogonial proliferation has not been detected. In the marbled newt and E. asper (Rouy 1972), primary spermatocytes are only observed from May to September. In other urodeles such as C. pyrrhogaster (Tanaka and Iwasawa 1979), T. hongkongensis (Tos and Lofts 1977a), and P. waltl (Gamier 1985), primary spermatocytes are present throughout the year. This finding might be related to temperature; low temperatures inhibit spermatocyte proliferation in urodeles even during the spermatogenetic period (Ifft 1942; Werner 1969; Steinborn 1984). It is possible that if winter temperatures are not very low, a certain degree of spermatocyte proliferation might occur in this season. Most studies of the testicular cycle in urodeles only report on the months at the beginning and end of spermatogenesis. This quantification study of the marbled newt permitted us to distinguish between the periods of cell proliferation (MayJune) and spermiogenesis (July-September) . Such a distinction is important because the two processes occur under different natural environmental conditions. Germ cell proliferation takes place when the photoperiod lengthens and the ambient temperature increases, whereas during spermiogenesis both the photo-

period and temperature gradually decline. Experimental studies on the effect of these two environmental factors should take into account the time of the experiment. In a previous study we showed that a short photoperiod has negative effects on spermiogenesis during the phase of germ cell proliferation but not during spermiogenesis, and elevated temperatures favor germ cell proliferation and hinder spermiogenesis (Fraile et al. 1989).

Acknowledgements This work was supported by grants from the Junta de Castilla y Le6n, Fondo de Investigaciones Sanitarias de la Seguridad Social, and Universidad de Alcalh de Henares, Spain. We are indebted to Mr. Antonio Priego for technical assistance. ADAMS,A. E. 1940. Sexual conditions in Triturus viridiscens. 111. The reproductive cycle of the adult aquatic form of both sexes. Am. J. Anat. 66: 253-37 1. BARKER, K. R., and BIESELE,J. J. 1967. Spermateleosis of a salamander Amphiuma tridactylum Cuvier. A correlated light and electron microscopic study. Cellule, 67: 9 1- 129. BERGMANN, M., SCHINDELMEISER, J., and GREVEN, H. 1982. The zone of mature spermatozoa in the testis of Salamandra salamundra (L.) (Amphibia-Urodela). Z . Mikrosk. Anat. Forsch. 96: 221234. 1983. The glandular tissue in the testis of Salamandra salamundra (L.) (Amphibia-Urodela). Acta Zool. (Stockh.), 64: 123-130. CALLARD, G. V., CANIK,J. A., and PUDNEY, J. 1980. Estrogen


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synthesis in Leydig cells: structural-functional correlations in Necturus testis. Biol. Reprod. 23: 461 -479. DOLMEN,D. 1982. Skeletal growth marks and testis lobulation as criteria for age in Triturus spp. (Amphibia) in central Norway. Acta Zool. (Stockh.), 63: 73-80. FRAILE, B., PANIAGUA, R., RODRIGUEZ, M. C., and SAEZ,F. J. 1989. Effects of photoperiod and temperature on spermiogenesis in marbled newts (Triturus marmoratus marmoratus). Copeia, 1989: 357-363. GARNIER, D. H. 1985. Androgen and estrogen levels in the plasma of Pleurodeles waltl Michah during the annual cycle. I. Male cycle. Gen. Comp. Endocrinol. 58: 376-385. HAMASIMA, N., and KOTANI, M. 1979. Nuage-chromosome association in metaphase spermatogonia of the newt Cynops pyrrhogaster. Dev. Growth Differ. 3: 221-227. HUMPHREY, R. R. 1921. The interstitial cells of the urodele testis. Am. J. Anat. 29: 213-237. IFFT,J. D. 1942. The effect of environmental factors on the sperm cycle of Triturus viridiscens. Biol. Bull. (Woods Hole, Mass.), 83: 111-128. IKENISHI, K., and NIEUWKOOP, P. D. 1978. Location and ultrastructure of primordial germ cells (PGCs) in Ambystoma mexicanum. Dev. Growth Differ. 20: 1-9. IMAI,K., and TANAKA, S. 1978. Histochemical and electron microscopic observations on the steroid hormone-secreting cells in the testis of the Japanese red-bellied newt Cynops pyrrhogaster pyrrhogaster. Dev. Growth Differ. 20: 151- 167. JOLY,J. 1971. Les cycles sexuels de Salamandra (L.). I. Cycles sexuels des mAles. Ann. Sci. Nat. Zool. Biol. Anim. 13: 452-499. LOFTS,B . 1974. Reproduction. In Physiology of the Amphibia. Vol. 2. Edited by B. Loft. Academic Press, New York. pp. 197-218. MILLER,M. R., and ROBBINS, M. E. 1954. The reproductive cycle in Taricha torosa (Triturus torosus). J. Exp. Zool. 125: 415-445. NORRIS,D. O., NORMAN, M. F., PANCAK, M. K., and DUVAL,D. 1985. Seasonal variations in spermatogenesis, testicular weights, vasa deferentia, and androgens levels in neotenic male tiger salamanders, Ambystoma tigrinum. Gen. Comp. Endocrinol. 60: 51-57. PICHERAL, M. B . 1968. Les tissus klaborateurs d'hormones stkroi'des chez les amphibiens urodkles. I. Ultrastructure des cellules du tissue glandulaire du testicule de Pleurodeles waltlii Michah. J. Microsc. 7: 115-134. 1971. Ultrastructure du noyau en rapport avec l'kvolution des protCines basiques nuclCaires au cours de la spermiogknksedu Triton Pleurodeles waltlii Michah. 11. J. Microsc. 12: 107- 132. G. V. 1983. The PUDNEY, J., CANICK, J. A., MAK,P., and CALLARD, differentiation of Leydig cells, steroidogenesis, and the spermato-

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