The postnatal maturation of efferent tubules in the rat: a light and electron microscopy study

The postnatal maturation of the genital duct in the rat seems to precede the first appearance of sperms (Sun & Flickinger, 1979; Francavilla et al. 1986), and it is claimed to be influenced by testicular factors like androgens (Orgebin-Crist, Danzo & Davies, 1975; Sun & Flickinger, 1979). The information so far available on the maturation of the genital duct is, however, restricted to few ultrastructural investigations in this species, dealing with epididymis (Leeson & Leeson, 1964; Flickinger, 1969; Sun & Flickinger, 1979).

The postnatal differentiation of efferent tubules was never analysed in detail in the rat, although efferent tubules and epididymis are embryologically (Bovy, 1929; Brambell, 1927; Flickinger, 1969; Gier & Marion, 1970), structurally (Hamilton, 1975), and functionally (Levine & Marsh, 1971) distinct. We therefore decided to undertake a detailed investigation, by light and transmission electron microscopy, of the maturation of efferent tubules in the rat from birth to the time of sperm appearance in the genital duct.

The aim of this study was to follow the development of those specializations of the epithelium and tubule wall of efferent tubules that are related to absorption and to the progression of luminal content.

Efferent tubules in adult animals transport fluids across the epithelium from the lumen (Levine & Marsh, 1971), and take up endocytic markers (Hermo & Morales, 1984). Moreover, they facilitate sperm progression through the beating of epithelial cilia and contractility of the tubule wall (Hamilton, 1975).

Thirty-six Long-Evans rats (Angelini breeding colony) were utilized in this investigation. Three animals were studied on each of the postnatal days 1, 3, 5, 8,10,15, 20, 25, 30, 35, 40, 50; the rats were anaesthetized with ether and killed by decapitation.

Gonads were carefully exposed and efferent tubules were dissected, along with the initial segment of the caput epididymis and rete testis with the most dorsal region of the attached testis.

Tissue samples were rapidly fixed by immersion in picric acid-formaldehyde (Zamboni & De Martino, 1967) containing 2·5% glutaraldehyde in Sorenson buffer, pH7·2, for 12 h. The material was stored at 4°C and postfixed in 1·5% unbuffered OsO₄ solution for 1·5 h, and embedded in Epon 812. Thick sections for light microscopy were stained in buffered toluidine blue (pH 8·0), and thin sections were stained with uranyl acetate and lead hydroxide and examined by a Siemens Elmiskop 101 electron microscope.

At birth, the efferent tubules appeared very coiled and easily distinct from the epididymis because of their small cross-sectional size and thin peritubular wall (Fig. 1). The epithelium was formed by cuboidal cylindrical cells with a basal located nucleus and small scattered microvillar specializations on the luminal surface (Fig. 2). Stromal cells surrounded the tubules (Fig. 2).

3-day-old animals did not show any remarkable change.

5 days after birth, the epithelial cells showed an increased number and size of microvilli, and scattered apical invaginations of the plasma membrane seemed to engulf an electron-dense fuzzy material (Fig. 3). In the same animals, a lumen appeared in the neighbouring rete testis, while seminiferous cords did not show any sign of canalization.

In 8-day-old animals, a complex endocytotic apparatus was built up in the epithelial cells. Numerous caveolae and coated pits of the plasma membrane were interspersed among the bases of long, packed and regularly arranged microvilli. Hundreds of coated and uncoated vesicles crowded the apical cytoplasm, along with a network of dense tubules and multivesicular bodies (Fig. 4). The functional relationship of all these specializations of the plasma membrane, during a fluid phase, and receptor-mediated endocytosis is well documented (Ericsson & Trump, 1969; Djakiew, Byers & Dym, 1984).

One cilium was sometimes visible along the apical border of epithelial cells (Fig. 4). The lateral plasma membranes showed numerous interdigitations along both the apical (Fig. 5) and basal cell region (Fig. 6), as described in epithelial cells specialized for transport of water and salts (Ericsson & Trump, 1969).

In 8-day-old animals, a wide lumen was visible in the tubules of the rete testis, but not yet in the seminiferous cords (Fig. 7). The epithelium of tubules in the rete was formed by cuboidal cells with a smooth luminal surface. The transition with efferent tubules was abrupt, and featured a cylindrical epithelium provided with packed microvilli resembling a brush border (Fig. 8).

The epithelium of efferent tubules was seen to be formed at both the light and electron microscope level by principal and scattered dense-stained cells. The latter ones (Fig. 9) showed an electron-dense cytoplasm, round- and rod-shaped mitochondria, like those of principal cells, and packed microvilli. The apical cytoplasm contained numerous vesicles and scattered multivesicular bodies, but not the network of dense tubules visible in principal cells. The nuclei of dark cells had a coarser chromatin than that of neighbouring principal cells. A third cell type, resembling the wandering halo cells of adults (Hamilton, 1975) and developing epididymis (Sun & Flickinger, 1979), was seldom present in the epithelium. The tubules were surrounded by loosely arranged, slender stromal cells; those next to the tubule basal lamina showed a decreased amount of rough endoplasmic reticulum and a more electron-dense cytoplasm (Fig. 10)

No changes were recorded in 10-day-old animals.

In 15-day-old rats, efferent tubules underwent changes involving the tubule wall and the epithelium. The stromal cells surrounding the tubules were regularly arranged in two to four circular strips, and acquired features of myoid elements. Numerous bundles of thin (5–6nm) filaments were visible in the cytoplasm, anchored to dense patches along the inner plasma membrane. The cisternae of rough endoplasmic reticulum were not so numerous as those of younger animals, and the cells appeared surrounded by a discontinuous basement membrane. These changes appeared first in the cells next to the epithelial basal lamina, and progressively involved outer cells (Fig. 11).

The differentiation of a contractile wall was associated with an active ciliogenesis, involving scattered epithelial cells. Cilia appeared interspersed among microvilli, while components of the endocytotic apparatus in the apical cytoplasm were greatly reduced as compared with neighbouring principal cells (Fig. 12).

In 20- and30-day-old rats, the efferent tubules did not show new changes, except for a light growth of the Golgi complex in epithelial principal cells (Fig. 13). Microvilli and components of the endocytotic apparatus had almost disappeared in the ciliated elements.

In 35-day-old rats, the efferent tubules appeared mature. The epithelium was formed by a palisade of tall cylindrical principal cells and scattered ciliated elements (Fig. 14). The latter had nuclei with coarse chromatin, often located in an apical position, and a fairly dense-stained cytoplasm. Their apical border had packed cilia, while microvilli and specializations related to endocytosis were absent (Fig. 15). The wall was formed by three to four layers of circularly arranged myoid cells.

The appearance of sperms in the duct of 50-day-old rats was not associated with any remarkable structural change, as also reported for the epididymis maturation in the same species (Sun & Flickinger, 1979; Francavilla et al. 1986).

This study shows that the postnatal maturation of efferent tubules in the rat occurs in two sequential steps. In the first one, an endocytotic apparatus is differentiated in the epithelial cells. During the second one, the apparatus for transport of sperms and fluids is built up, and comprises the differentiation of ciliated epithelial cells among the principal cell type, and peritubular myoid elements.

The differentiation of an endocytotic apparatus in the epithelial cells largely precedes the initial postnatal maturation of seminiferous cords and secretion of testicular fluid. This, in fact, appears in the genital duct at the end of the second week of life (Tindall, Vitale & Means, 1975), and parallels the differentiation of the ciliated epithelium and peritubular contractile wall of efferent tubules.

The two steps of morphological maturation of this segment of the genital duct do not overlap. The endocytotic apparatus of epithelial principal cells is fully developed in 8-day-old animals, while ciliated cells are still absent, and the wall is formed by poorly differentiated stromal cells. This maturation pattern of efferent tubules is quite unique. In other regions of the genital duct, like epididymis and intragonadal ductus deferens, the epithelium and tubule wall undergo a simultaneous differentiation (Francavilla et al. 1986).

The peculiar sequence of changes during the postnatal life seems to be related to the embryonal ontogenesis of efferent tubules, which are the only component of the genital excurrent pathway originated in mammals from mesonephric tubules (Bovy, 1929; Gier & Marion, 1970; Upadhyay, Luciani & Zamboni 1981; Zamboni & Upadhyay, 1981, 1982). In embryos, the mesonephron fulfils an excretory function, and the tubules transiently develop a phenotype that is strictly related to absorption, when they represent the uriniferous ducts of a functioning, though ephemeral, nephron (Leeson, 1959; De Martino & Zamboni, 1966; Krause, Cutts & Leeson, 1979; Wettstein & Tiedeman, 1981; Zamboni & Upadhyay, 1981).

The endocytotic apparatus of the epithelium in efferent tubules, during the early postnatal life, could be but a remnant of a phenotype this epithelium had in embryonal life, and which is resumed as soon as a fluid appears in the tubules of 1-week-old animals. A direct demonstration of the passage of fluids in the genital duct, at this age, is not available. The appearance of a lumen in the cords of rete testis in 5-day-old animals could be indirect evidence of the initial transport of fluids in the duct. A coincidence between the appearance of a lumen and the occurrence of fluid secretion has, in fact, been demonstrated in developing seminiferous tubules (Tindall et al. 1975). Hence the lack of a lumen in seminiferous cords of 5- and 8-day-old rats suggests that the canalization of rete testis cords could be related to the initial transport of fluids across the epithelium of the rete, as demonstrated in adult animals (Setchell & Waites, 1975).

In 8-day-old rats, the endocytotic apparatus of epithelial cells in efferent tubules has reached the degree of development described in the epithelium of adult rats (Hamilton, 1975) and other mammals (opossum, Ladman, 1967; monkey, Ramos & Dym, 1977; bull, Goy al & Hrudka, 1980). It is worth mentioning that at this early postnatal age (Fig. 8), the epithelium of efferent tubules shows a degree of morphological specialization as an absorbing organ which is comparable to that of functioning mesonephric tubules of different mammal embryos (man, De Martino & Zamboni, 1966; opossum, Krause et al. 1979; mouse, Zamboni & Upadhyay, 1981).

During the third and following weeks of life, the efferent tubules undergo changes which represent an adaptation of already differentiated ‘uriniferous-like’ tubules, to promote transport of sperms. These are, in fact, poorly motile in the initial segment of the adult genital duct (Bedford, 1975), and their progression in the efferent tubules is accomplished by beating of epithelial cilia and contractility of the tubule wall.

Stromal cells that surround the tubules of neonatal rats progressively acquire features of myoid elements, like those that form the wall of seminiferous tubules (Leeson & Leeson, 1963) and the initial segment of the epididymis (Francavilla et al. 1983).

Of particular interest is the differentiation of ciliated cells. The epithelium of efferent tubules up to 15 days after birth is formed by a single cell type provided with packed microvilli and, rarely, one cilium, as frequently reported in developing epithelial cells not involved in the differentiation of ciliated elements (De Martino & Zamboni, 1966). Scattered dark cells observed in the epithelium of 8-day-old rats could be precursors of ciliated cells. Both share a dense-stained cytoplasm and nuclei with coarse chromatin. Principal and dark cells are, however, provided with microvilli and a developed endocytotic apparatus, even though the latter does not show the apical dense tubules but numerous vesicles only. Hence, whichever is the precursor, ciliated cells arise from fully differentiated elements, which progressively lose the features of absorbing cells and switch their phenotype toward specializations related to movement of luminal content.

The second step in the maturation of efferent tubules largely coincides with the initial differentiation of the epithelium and tubule wall of the neighbouring caput epididymis (Francavilla et al. 1986). Both segments of the genital duct reach a full differentiation 35 days after birth, i.e. before the arrival of sperms which appear in the duct of 50-day-old rats. The coincidence of developing changes of efferent tubules and caput epididymis suggests that factors like androgens, which are claimed to induce the foetal development (Jost, 1947; Price & Pannabecker, 1956; Alexander, 1972) and postnatal maturation of the epididymis in mammals (Orgebin-Crist et al. 1975), could also influence the maturation of efferent tubules or, more precisely, the adaptation of survived ‘mesonephric tubules’ to promote sperm progression. The occurrence during the third week of life in the rat of increased plasma testosterone levels (Yukitaka, Nieschlag & Lipsett, 1973) and the first appearance of the androgen-rich testicular fluid in the genital duct (Tindall et al. 1975) seem to be more than accidental with respect to maturation changes involving, at the same time, the epididymis and efferent tubules.

The authors gratefully acknowledge the excellent collaboration of Mrs Ingrid Adam for typing and editing the manuscript.