Myoid cells in the peritubular tissue (lamina propria) of the reptilian testis

Cell Tiss. Res. 163, 545-560 (1975) - �9 by Springer-Verlag 1975

Myoid Cells in the Peritulmlar Tissue (Lamina propria) of the Reptilian Testis *

K. Unsicker

Department of Zoology, University of Melbourne, Parkville, Victoria, Australia

G. Burnstock

Department of Anatomy and Embryology, University College, London

Summary. The arrangement and fine structure of peritubular myoid cells was studied in the testes of three species of reptiles (Lacerta dugesi, Testudo graeca and Natrix natrix) during two short periods of the seasonal cycle (European spring and autumn) and correlated with some ultrastructural properties of Leydig cells. The lamina propria consists of myoid cells, fibroblasts and non-cellular components comprising collageneous and non-striated microfibrils. Both components are arranged in alternating layers surrounding seminiferous tubules. In spring the lamina propria of lacertilian testis shows 1-5 layers of myoid cells which are rich in 50-70 A filaments and exhibit plasmalemmal and intracellular dense patches, smooth vesicles along the cell membrane and a concentration of organelles in a juxtanuclear position. Leydig cells are rich in smooth ER profiles and have few lipid droplets. In autumn most myoid cells are replaced by fibroblast-like elements. Leydig cells display large numbers of lipid droplets and dense bodies, but only small amounts of agranular ER. Similar changes are noted in Leydig cells of Testudo and Natrix. However, in these species the boundary tissue of seminiferous tubules fails to show significant alterations comparing spring and autumn animals. In both species the lamina propria exhibits a few fibroblast-like cells interspersed among myoid cells.

Key words: Myoid cells - Interstitial (Leydig) cells - Testis - Reptiles - Seasonal cycle.

Introduction

Since Roosen-Runge (1951) recorded contractile activity cinematographically in seminiferous tubules of rat and dog, and Clermont (1958, 1960) observed in

Send offprint requests to : Dr. K. Unsicker, Department of Anatomy, University of Kiel, Federal Republic of Germany

* Supported by a grant from Deutsche Forschungsgemeinschaft (Un 34/3) and a Research Fellow- ship of the University of Melbourne to K. Unsicker. We are particularly grateful to Dr. O'Shea, Veterinary School, University of Melbourne, and Dr. Gabella, University College, London, for their criticisms of the manuscript.

546 K. Unsicker and G. Burnstock

the electron microscope cells similar to smooth muscle cells in the connective tissue underlying the germinal epithelium of the rat, a considerable volume of work has been carried out on the arrangement and ultrastructure of these smooth muscle-like cells (synonyms: myofibroblasts, contractile fibroblasts, peritubular cells, myoid cells) particularly in mammals (mouse: Gardner and Holyoke, 1964; Ross, 1967; Bressler and Ross, 1972; Chung, 1974; rat: Clermont, 1958, 1960; Br6kelmann, 1960; Lacy and Rotblat , 1960; Leeson and Leeson, 1963 ; Kormano , 1970; D y m and Fawcett, 1970; Korm ano and Hovatta , 1972; Hovatta, 1972a, b; hamster: McCord, 1970; guinea-pig: Fawcett et al., 1969; pig: Dierichs and Wrobel, 1973; sheep: Bustos-Obreg6n and Courot, 1974; monkey (Macaca): Dym, 1972; man: Ross and Long, 1966; Strauss and Kao, 1968; B6ck et al., 1972; Bustos-Obreg6n and Holstein, 1973). These papers have provided evidence for differences in the arrangement and ultrastructure of testicular myoid cells (see also review by Burgos et al., 1970). For several species (Bressler and Ross, 1972; Hovat ta , 1972 b; Dierichs and Wrobel, 1973; Bustos-Obreg6n and Courot, 1974) it seems well-established that the myoid cells respond to androgenic stimulation.

Only one electron microscopic study has been devoted to the boundary tissue of seminiferous tubules in non-mammal ian vertebrates (domestic fowl: Rothwell and Tingari, 1973), al though lower vertebrates are particularly suitable for studies in this field since they show cyclical variations of reproductive activity and hor- monal levels. This is one reason why reptiles were chosen for the present study. Another one is their special phylogenetic position and the fact that in a previous investigation, three species of anuran amphibia (Xenopus, Rana, Bufo) have been shown to lack a peritubular layer of contractile cells comparable to that in the mammal ian testis (Unsicker, 1975).

Materials and Methods

Three species of reptiles were chosen for the present study: Lacerta dugesi, Testudo graeca and Natrix natrix. The animals (6 mature males of each species) were obtained from dealers in West Germany, kept under conditions similar to their particular habitat as far as temperature, environment and day/night cycles are concerned and sacrificed during the following months:

La~erta : May and August ; Testudo: May and October; Nutrix: May and October.

Fixation occurred either by perfusion via the heart (phosphate buffered 2.5% glutaraldehyde, pH 7.4, for 15 minutes and phosphate buffer, 0.1 in pH 7.4 for 10 minutes) or immersion of testis slices 1 mm thick into the fixation medium and subsequent rinsing in phosphate buffer for at least 2 hours. All specimens were postfixed in 2% aqueous OsO4, dehydrated in ethanol and embedded in Araldite. Sectioning was carried out with a Reichert-Sitte Om U2. The tissue was stained with uranyl acetate in 70% methanol, and lead citrate for 5 minutes each. The electron microscopes used were Zeiss EM 9A, Siemens 101 and Joel JEM 100B.

Results

Lacerta (May) : The lamina propria surrounding the seminiferous tubules consists of cellular and non-cellular components arranged in alternating but not distinctly

Myoid Cells in the Reptilian Testis 547

Fig. 1. Lacerta dugesi, May. Three layers of attenuated processes of myoid cells adjacent to a seminiferous tubule (ST). Myoid cells display thin filaments (F), plasmalemmal attachment sites for filaments (arrows) and glycogen particles (GP). Basal lamina of germinal epithelium (BL). Note collagen fibres (C) and microfibrils (M). Inset: Cell interdigitations and short desmosome-like junctions (D) provide contacts between adjacent myoid cells. Discontinuous basal lamina (DB) covering myoid cells. Germinal epithelium (G). x 18000

separated layers (Fig. 1). Parallel to the basal surface of the germinal epithelium a basal lamina is observed which is about 500 A thick. On the outer (non-epithelial) side of the basal lamina there is a space 0.1-0.5 la wide, relatively electron-lucent, containing collagen fibres and non-striated microfibrils. The fibres are loosely arranged, run in various directions, and may form bundles. The next layer is the first cellular one of up to five. These cells are elongated with respect to the transverse axis of the tubule, widest in the nuclear region and with long tapering cytoplasmic processes, which may be as thin as 0.3 l.t. Occasionally


they are found to be covered by an incomplete basal lamina (Fig. 1, inset). The cytoplasmic processes of neighbouring cellular layers may overlap for dis- tances up to 4 ~ and, in a few instances, exhibit short desmosome-like junctions. In some regions cells are observed in close contact, narrowing the intercellular space to 150-200 A (Fig. 2). The most conspicuous feature of peritubular cells is the presence of cytoplasmic filaments, 50-70 A thick (Figs. 1 and 2). They are both more numerous and densely packed in peripherally situated cells giving these elements a darker appearance particularly at low magnification. The filaments are commonly arranged in bundles with a parallel array; however, single or loosely arranged filaments can also be observed. Bundles of filaments do not only run parallel to the long axis of the cells; frequently they are found running in a variety of directions. Filaments are seen to merge with hemidesmo- some-like dense areas along the cell membrane and intracellular dense patches. Another ultrastructural detail which makes the peritubular cells resemble smooth muscle cells is the existence of plasmalemmal vesicular in-pocketings. However, they do not cover more than 5-10% of the cell surface. Peritubular cells exhibit slender, centrally placed nuclei often with deep indentations (Fig. 2). Golgi appara- tuses and membrane-bounded dense bodies are concentrated at the nuclear poles. Mitochondria, rough endoplasmic reticulum (ER), free ribosomes and glycogen particles are widely distributed throughout the cells.

At this stage of the seasonal cycle Leydig cells are rich in smooth ER and display only few lipid inclusions. Mitochondria show predominantly tubular cristae, and there are large amounts of free ribosomes.

In August, the appearance of the peritubular cells is as follows. Most of the previously dark cells have gained a light cytoplasm mostly due to a loss of thin filaments (Fig. 3). The rough ER is prominent with dilated cisternae being filled with floccular material of medium electron density. Lipid droplets are constantly to be seen. As a whole the peritubular cells more closely resemble fibroblasts than smooth muscle cells. Specialised cell junctions were not seen. Leydig cells show very low amounts of agranular ER and numerous liposomes and dense bodies. Mitochondria exhibit lamellar cristae.

Testudo (May): Very similar to Lacerta, the lamina propria of seminiferous tubules is composed of 2-7 layers of flattened cells with collagen fibrils and microfibrils interposed (Fig. 4 a). Most of the cells show structural features charac- teristic of smooth muscle cells: an abundance of thin filaments (50-70 ~ in diameter), plasmalemmal attachment sites and intracellular dark patches, but only few membrane inpocketings. Occurrence and distribution of cell organelles are comparable to the situation seen in Lacerta. Cells which according to the predominance of granular ER and free ribosomes and low amounts of filaments can be described as fibroblasts (Fig. 4 a) may form up to two of the seven cellular layers; however, their location is not constant. Cells showing features of both smooth muscle and fibroblasts are sometimes observed. Myoid and fibroblast-like cells and pairs of myoid cells frequently form contacts by bulbous protrusions which reach into similarly shaped invaginations of the adjacent cell (Fig. 4a). In sections of the lamina propria tangential to the tubular wall it appears as if both intra- and extra-cellular filaments are oriented in the same directions (Fig. 5). A striking feature of Testudo testes, which has been described in a previous


Fig. 2. Lacerta dugesi, May. Nuclei of peritubular myoid cells with scalloped outlines. The space between the cells has the size of a normal intercellular cleft (arrows). Juxtanuclear area (J) with mitocbondria. • 36000


Fig. 3. Lacerta dugesi, August. Myoid cells in the peritubular boundary tissue are widely replaced by fibroblast-like cells (FI and F2) showing dilated cisternae of granular ER (GE), lipid droplets (L) and free ribosomes, but only few thin filaments (Ft. x 48000

Fig. 4. (a) Testudo graeca, October. Lamina propria showing dark myoid cells (M) and a fibroblast-like cell (F) connected with a myoid cell by a bulbous protrusion (B) and a corresponding invagination. Note basal foot-like processes of a Sertoli cell (P) containing prominent bundles of thin filaments. x 24000. (b) Part of a Leydig cell (Testudo) in May with prominent profiles of smooth ER (ER),

tubular mitochondria (M) and lipid droplets (L). x 18000. (c) Leydig cell (Testudo) in October containing large numbers of lipid droplets (L) and lipofuscin inclusion bodies (B). x 7200



paper (Unsicker, 1974), is the occurrence of prominent bundles of thin filaments in basal processes of Sertoli cells (Fig. 4a),

Leydig cells display a fine structure comparable to that seen in Lacerta indicat- ing a high degree of secretory activity (Fig. 4b).

In specimens fixed in October, the structure of peritubular cells is not notice- ably different. Myoid cells remain highly differentiated. On the other hand, Leydig cells have accumulated large numbers of lipid droplets and lipofuscin particles (Fig. 4c), and both the agranular ER and the tubular form of mitochondria are less pronounced than in animals sacrificed in May.

Natri?c (May): The peritubular tissue is made of 1-5 cellular layers (Fig. 6) with no more than one representing a fibroblast-like type of cell if two or more layers are present. A darker and a lighter type of cell are discernible among myoid cells. Dark cells contain abundant thin filaments which almost completely fill the cell body. Mitochondria and scanty profiles of granular ER are mostly restricted to a juxtanuclear position. The attenuated cell processses display, apart from filaments, a few mitochondria and small islets of free ribosomes. Inpocket- ings at the cell surface are scarce. In electron-light cells which are still clearly identifiable as contractile on the basis of the presence of filaments, granular ER and ribosomes appear more prominent.

Dark myoid cells are connected by desmosome-like junctions. Fibroblasts have a well-developed rough ER. Their non-dilated cisternae show a content of medium electron density. Thin filaments either in bundles or single are ubiqui- tously present.

Leydig cells (Fig. 6) of snakes killed in May are devoid of lipid droplets, but possess a well-developed smooth ER with a few inter-mingled ribosomes and mitochondria with tubular cristae.

In October essentially the same morphological aspect of the lamina propria is found (Fig. 7). Myoid cells contain numerous, densely-packed filaments. The number of layers and the proportions of both the cellular and non-cellular components are the same as in May. On the other hand there are heavy structural alterations in Leydig cell which can be seen in Fig. 7. There are numerous large lipid droplets, few tubular mitochondria and only very scanty profiles of agranular ER.

Discussion

The present study gives evidence for the existence of myoid cells in the peritubular tissue of the testes in three species of reptiles belonging to three different orders or sub-orders, respectively. This is of particular significance in view of the recent finding that anuran amphibians lack such a set of contractile peritubular cells (Unsicker, 1975).

Two of the amphibian species studied (Rana and Bufo) showed contractile cells as vascular smooth musculature only. The intertubular spaces in Xenopus, on the other hand, were rich in non-vascular smooth muscle cells, which formed thick bundles, however, without being constituents of a lamina propria. Leydig cells which bordered the basal lamina of the germinal epithelium were quite


Fig. 5. Testudo graeca, May. Intra- and extra-cellular filaments running in the same direction suggest a functional integration of cellular and non-cellular compartments, in the lamina propria. Attachment sites (A). Germinal epithelium (G) and its basal lamina (B). • 60000


Fig. 6. Natrix natrix, May. Lamina propria with three layers of myoid cells (1-3) the parts of which are shown are completely filled with thin filaments. Plasmalemmal attachement sites (arrows). An adjacent Leydig cell (L) shows a well developed smooth ER, glycogen particles and with mitochondria tubular cristae. Endothelium (E). Germinal epithelium (G). x 18 000

common in these species thus establishing an arrangement which resembles the teleost pattern and differs fundamentally from that seen in electron microscopic studies of amniote testes hitherto.

In spite of species differences, arrangement and ultrastructure of the lamina propria elements in the reptiles used for this study show a basic similarity with both the avian and mammalian pattern. This includes the existence of smooth muscle-like cells, fibroblasts, and collagen and microfibrillar fibres, and the arrangement of cells and fibres in layers surrounding the seminiferous tubules.

In mammals, Burgos et al. (1970) have distinguished three types of arrangement in the peritubular tissue. Type a is characterised by a single layer of smooth muscle-like cells bordering an inner and an outer non-cellular layer and is found


Fig. 7. Natrix natrix, October. Myoid cells are highly developed (M), while the adjacent Leydig cell is filled with large lipid droplets (L). Major amounts of smooth ER do not exist. Germinal epithelium (G). x 7200

in the rat, mouse and hamster. In type b inter-lamellar cells are present in two to four layers, intermingled with a peripheral network of collagenous fibres and fibroblast-like cells. Type b is present in the guinea-pig. In type c which is found in primates, cat, and slightly modified, in the ram (Bustos-Obreg6n and Courot,


1974), the peritubular contractile component is multilayered, and the inner, non- cellular lamella has an inner stratified layer formed by microfibrils and an outer layer with collagen fibres. Fibroblasts are associated with the latter. For the domestic fowl, Rothwell and Tingari (1973) have described a type of arrangement which most closely resembles that of type c. However, in deviation from any mammalian species, a typical fibroblast lamella forms the innermost of the cellular layers.

The reptilian pattern as found in this study is not identical with either of these types. In lizards sacrificed in May there are up to five lamellae of myoid cells. Filaments are more abundant in cells farther from the germinal epithelium. However, neither are they absent in the innermost cells nor do these cells display clear fibroblast features. Certainly, fibroblast-like cells are interspersed among myoid cells in the lamina propria of tortoises, but they do not occupy a constant position in close proximity to the tubular wall. The same situation holds true for the snakes studied.

Myoid cells in the reptilian testis share several ultrastructural attributes with smooth muscle cells, as do testicular myoid cells of mammals. They contain intracytoplasmic filaments, 50-70 A thick, i.e. approximately the same size as actin filaments. The variations in size from 40 A in the mouse (Ross, 1967) up to 80 A in man (B6ck et al., 1972) are paralleled by variations in diameter of thin filaments reported in smooth muscle cells of different origin (Burnstock, 1970). Thick filaments which can be visualised in smooth muscle cells under various conditions (Campbell and Chamley, 1975), and 100 A filaments, have never been reported to occur in peritubular myoid cells nor have they been found in the course of this study. Both plasmalemmal and intracellular dense attachment sites for thin filaments exist in myoid cells of reptiles, and the occurrence of smooth membrane inpocketings which are typical of mammalian myoid cells as well (Clermont, 1958; Lacy and Rotblat, 1966; Leeson and Leeson, 1963; Ross and Long, 1966; B6ck e ta l . , 1972; Kormano and Hovatta, 1972; Dierichs and Wrobel, 1973) provides an additional analogy between myoid and smooth muscle cells. Myoid cells in all reptiles studied make contacts by short desmosome-like junctions and cell membrane interdigitations as has been seen in rat (Leeson and Leeson, 1963), pig (Dierichs and Wrobel, 1973) and man (Bustos-Obreg6n and Holstein, 1973). Tight junctions or nexuses which may play an important role in intermuscle fibre conduction and have been reported to occur in various smooth muscle tissues (cf. Lane and Rhodin, 1964; Bennett and Rogers, 1967; Yamauchi and Burnstock, 1969) have not been found to occur between reptilian myoid cells. Chung (1974) mentions the presence of nexuses in peritubular contractile cells in mice. However, tight junctions have been demonstrated between myoid cells of guinea-pigs and rats (Fawcett et al., 1970; Dym and Fawcett, 1970).

Intrusions like that of reptilian myoid cells processes into neighbouring cells are common in smooth muscle cells. In our material they do contain organelles (cf. Merrillees et al., 1963) and the distance between the adjacent cell membranes is not decreased.

In a few instances reptilian peritubular cells are covered by basal lamina material, which, as in mouse (Ross, 1967), pig (Dierichs and Wrobel, 1973)


and man (B6ck et al., 1972; Bustos-Obreg6n and Holstein, 1973) does not build up a continuous sheet. Together with the homogeneous ground substance, collagen fibres and microfibrils this material is probably predominantly produced by the fibroblasts which are interspersed among myoid cells. Moreover, it seems likely that myoid cells, with their considerable proportions of rough ER, take part in the formation of intercellular fibrillar proteins. Some of our pictures suggest that the functional significance of the non-cellular layers is not to allow some degree of mobility between cellular layers, but also to establish contacts between adjacent layers thus making the peritubular boundary tissue a functional unit. No evidence comes from this investigation regarding peritubular cells to form part of the blood-testis barrier (if such a barrier exists in reptiles).

At present, evidence for a possible contractility of peritubular myoid cells in reptiles is only indirect coming mainly from two sources: first, there is a resemblance with smooth muscle cells and, second, there is a close analogy in location with mammalian peritubular cells which have been shown to exhibit spontaneous contractions in isolated tubules (Roosen-Runge, 1951), under oxytocin stimulation (Niemi and Kormano, 1965) and in tissue culture (Hovatta, 1972a). As in mammals (Baumgarten and Holstein, 1971) these cells receive no innervation, despite the heavy supply of adrenergic nerves to the reptilian testis (Unsicker, 1973), favouring the view of these cells as an autocontractile aid in sperm transport. Application of an immunofluorescence method with an actomyosin-antibody would be an important step in elucidating the contractile character of reptilian myoid cells.

For a variety of mammals it is well established that myoid cells in the testis are sensitive to pituitary and testicular hormones. Bressler and Ross (1969) have shown in mice that differentiation of myoid cells is dependent on normal pituitary function. From a series of experiments carried out in tissue culture it is obvious that testosterone and HCG increase both the number of filaments in myoid cells and the percentage of tubules showing contractile activity, whereas the antiandro- genic cyproterone acetate inhibits the development of both structure and function (Hovatta, 1972). Chung (1974) has reported on immature myoid cells in mice with testicular feminization.

In Lacerta dugesi a high degree of secretory activity of Leydig cells in May is accompanied by a maximum development of contractile properties of myoid cells as far as it can be assessed by ultrastructural features. It is widely accepted that large amounts of agranular ER in combination with mitochondrial vesicles or tubules and few or no lipid inclusions may reflect a high levels of androgen hormone synthesis and secretion (see Christensen and Gillim, 1969).

Studies correlating the fine structure of Leydig cells with androgen levels in lower vertebrates are still lacking. However, it can be cautiously extrapolated from investigations which correlate, for example, the development of secondary sex characteristics with ultrastructure (cf. Br6kelmann, 1964), that in non-mammalian species, too, production of testicular interstitial hormones depends on the development of smooth ER and to a lesser extent special mitochondrial membranes which are known to bind the essential enzymes for steroid hormone synthesis.

Well-developed smooth membranes and mitochondria with tubular cristae


have been found also by Dufaure (1970) in interstitial cells of the testis of Lacerta vivipara in spring, i.e. at the same time when spermiation takes place. In early au tumn the morphology of both myoid and Leydig cells in Lacerta testis has deeply changed, Leydig cells are rich in dense bodies and liposomes with low amounts of smooth ER. Peritubular cells exhibit fibroblast features and contain lipid droplets which also occur in rat contractile cells following administration ofcyproterone acetate (Hovatta, 1972 b) and in lamb myoid cells after hypophysec- tomy (Bustos-Obreg6n and Courot, 1974). Since changes like those observed in Leydig cells presumably reflect a less active state, the conclusion is not unlikely that lacertilian myoid cells, as their mammal ian counterparts, are androgen- dependent.

A more complicated relation between Leydig and myoid cells seem to exist in Testudo and Natrix where low interstitial cell activities in autumn are not paralleled by significant changes in peritubular contractile cells compared to their morphology in spring. Considering the fact that during the prepuberal regression of Leydig cells in the pig myoid cells undergo further differentiation, Dierichs and Wrobel (1973) have suggested that other factors than sexual steroid hormones could be involved in the differentiation processes of myoid cells. A similar suggestion has been made by Bustos-Obreg6n and Courot (1974) based on their observation on the differentiation of the lamina propria around the time of birth in lamb. A final answer with regard to the androgen dependency of myoid cells in Testudo and Natrix cannot be given here. It could perhaps be assumed that relatively low androgen levels which do not require exceptional amounts of smooth ER and tubular mitochondria in Leydig cells could maintain the differentiation of contractile cells. Nevertheless, it should be noted that a high degree of differentiation of Testudo myoid cells in autumn seems reasonable since in chelonian reptiles spermatozoa pass into the epididymides in autumn where they are stored for several months (Lofts, 1968). Although in Natrix sperm product ion and sperimation commonly occur in spring (Herlant, 1933), it has been reported that copulation may also occur in autumn producing sperm which is stored in the genital tract of the female for a successful fertilisation of eggs in spring (Sosnovsky, 1940).

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Unsicker, K. : lnnervation of the testicular interstitial tissue in reptiles. Z. Zellforsch. 146, 123 138 (1973)

Unsicker, K. : Contractile filamentous structures in Sertoli cells of the Greek tortoise (Testudo graeca) ? Experientia (Basel) 30, 272-273 (1974)

Unsicker, K. : Fine structure of the male genital tract and kidney in the anura Xenopus laevis Daudin, Rana temporaria L. and Bufo bufo L. under normal and experimental conditions. I. Testicular interstitial tissue and seminal efferent ducts. Cell Tiss. Res. 158, 215-240 (1975)

Yamauchi, A., Burnstock, G.: Postnatal development of smooth muscle cells in the mouse vas deferens. A fine structural study. J. Anat. (Lond.) 104, 1-15 (1969)

Received July 22, 1975

Documents

Myoid cells in the peritubular tissue (lamina propria) of the reptilian testis