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Revista chilena de anatomía

versión impresa ISSN 0716-9868

Rev. chil. anat. v.18 n.1 Temuco  2000

http://dx.doi.org/10.4067/S0716-98682000000100009 

ISTOCHEMICAL STUDY IN THE EPIDIDYMIS OF THE DOG (Canis familiaris, L.)

ESTUDIO HISTOQUÍMICO DEL EPIDÍDIMO EN EL PERRO (Canis familiaris, L.)

* Schimming, B. C.
** Vicentini, C. A.

* Laboratory of Anatomy, Faculty of Health Sciences, University of Marília, UNIMAR, São Paulo, Brazil
** Department of Biological Sciences, Faculty of Sciences of Bauru, UNESP, São Paulo, Brazil

SUMMARY: This paper deals some characteristics of mucosubstances reactivities in the epididymis of the dog. The PAS reactivity of the epithelium lining was moderate in all the segments of the duct, although strongly PAS positive granules were observed in the cytoplasm of the principal cells. These granules are probably formed by glycoproteins. Comparisons were made with data in the previous observations.

KEY WORDS: 1. Epididymis; 2. Dog; 3. Histochemistry.

INTRODUCTION

The epididymis is an important segment of the excurrent ducts of mammals. It is evident that spermatozoa undergo maturational changes concerning their fertility and motility as they proceed along the epididymis (GREENBERG & FORSSMANN, 1983). BRETON et al. (1998) reported that the lumen of the epididymis is the site where spermatozoa undergo their final maturation and acquire the capacity to become motile.

In accordance with this, the epididymis secrete compounds into the luminal fluid as mucosubstances and glycogen (ALSUM & HUNTER, 1978; RAMOS JR., 1980; VICENTINI, 1984 and VIOTTO et al., 1988). VOGLMAYR et al. (1985) have suggested that glycoproteins are a major factor in surface transformations of ram spermatozoa in the epididymis, especially during the initial stages of maturation.

Some of these glycosylated surface components may be intrinsic to the sperm cell from the outset (VOGLMAYR et al., 1980, 1982 and JONES et al., 1981), others may be synthesized and secreted by the epididymis (KOHANE et al., 1979 and BROOKS, 1981, 1983) and subsequently adsorbed by the sperm cell (LEA et al., 1978; BROOKS & HIGGINS, 1980; KOHANE et al., 1980 and GONZALES ECHEVERRÍA et al., 1982). Surface glycoproteins are known to control several critical cellular functions (e.g., differentiation, recognition, adhesion, motility) (VOGLMAYR et al., 1985).

Histological and histochemical studies on the epididymidis in various laboratory and domestic animals have been reported (NICANDER, 1957; HAMILTON, 1975; VICENTINI, 1984; VIOTTO et al.; MARTINELLI & NOGUEIRA, 1992; ORSI et al., 1993 and SANCHEZ et al., 1998).

A review of the literature on the subject indicates that histochemical studies in the dogs are rare. NICANDER & MALMQVIST (1977) suggested merocrine secretion in the initial segment, SINOWATZ et al. (1979 a, b) located the site of the glycosidases and hydrolases in the epididymis of the dog. In the present report, an histochemical study of mucosubstances was undertaken in order to obtain supplementary evidence for glycoprotein secreted by the epididymis.

MATERIAL AND METHOD

Epididymis were removed from ten adult, mongrel, sexually mature dogs (Canis familiaris, L.). The tissues were collected from the morphophysiological segments of the epididymis (see GLOVER & NICANDER, 1971, for review) and fixed in Bouin's and Gendre's fluids. The samples were dehydrated, embedded in paraffin, and sectioned at 7 µm.

The material was submitted to the following histochemical reactions: periodic acid-Schiff (PAS) (McMANNUS, 1946, modified by PEARSE, 1972), PAS after salivary amylase (LISON, 1960), alcian blue (AB) (1%) at pH 0.5 (LEV & SPICER, 1964) and pH 2.5 (LISON), combined alcian blue (pH 2.5) and periodic acid-Schiff method (AB-PAS) (VIALLI, 1955 and PEARSE, 1972), metachromasia (at pH 1.7, 2.5, 5.6) (LANDSMEER, 1951), blockage by acetylation plus PAS (McMANNUS & CASON, 1950), blockage by acetylation and by methylation plus AB pH 2.5 (FISHER & LILLIE, 1954).

The histochemical reactions were analyzed on the basis of tissue staining under a light microscope. So, the material was photographed. Each slide was graded subjectively for intensity of reactions as follows: negative reaction (-), trace (±), weak (+), moderate (++), strong (+++). The epithelial lining, stroma periductal and luminal contents were observed in all sections.

RESULTS

The epididymis of the dog was divided in initial, middle and terminal segments, according to GLOVER & NICANDER. The results of the histochemical tests of neutral and acid mucosubstances in the epithelial lining, stroma periductal and luminal contents of the epididymis of the dog are summarized in Table I.

Table I. Mucosubstances histochemistry in the epididymis of the dog

Reactions Initial segment   Middle segment   Terminal segment
  E L S   E L S   E L S

PAS ++ ++ +++   ++ ++ +++   ++ ++ +++
Amylase + PAS ++ ++ +++   ++ ++ +++   ++ ++ +++
Acetylation + PAS - - -   - - -   - - -
AB pH 0.5 - - -   - - -   - - -
AB pH 2.5 - - +   - - +   - - +
AB pH 2.5 + PAS ++ ++ +++   ++ ++ +++   ++ ++ +++
Acetylation + AB - - -   - - -   - - -
Methylation + AB - - -   - - -   - - -
Metachromasia pH 1.7 - - -   - - -   - - -
Metachromasia pH 2.5 - - -   - - -   - - -
Metachromasia pH 5.6 ± ± ±   ± ± ±   ± ± ±

E, epithelial lining; L, luminal contents; S, stroma periductal.
Reaction intensity: (-) no reaction, (±)trace reaction, (+) weak reaction, (++) moderate reaction, (+++) strong reaction.

The epithelial lining of three segments of epididymis reveals moderate PAS-positivity (Figs. 1, 2 and 3). This reaction was the same after salivary amylase. The principal cells of initial segment and terminal segment showed intensely PAS-positive granules in the cytoplasm (Figs. 1 and 3). The granules were more observed in the terminal segment. The PAS staining reaction was stronger in the apical region than the basal cytoplasm of the principal cells. This form of distribution and staining intensity was similar along the length of the duct.


Fig. 1- Initial segment. Note the PAS-positive granules (arrowhead), stereocilia (rose), basal lamina (arrows) and stroma periductal (stars). PAS. x200.


Fig. 2- Middle segment. The stereocilia (rose), basal lamina (arrows) and stroma periductal (stars) are indicated. PAS. x200.


Fig. 3- Terminal segment. The PAS-positive granules (arrowhead) and stereocilia (rose) in the epithelium. PAS. x400.

In general, the stereocilia were moderately to strongly PAS positive throughout the epididymis. The collagenous fibers of the basal lamina surrounding the epididymal duct, were strongly PAS positive (Fig. 1). However, the epithelial lining exhibited negative reactions for alcian blue. For the AB pH 2.5 method, the reaction is only positive in the apical border of the principal cells.

Moderated activity for periodic acid-Schiff was noted in the luminal contents of three segments, while a strong activity was observed in the acrosoms of spermatozoa. The stroma periductal was characterized by its strong PAS-positivity and weak alcianophily.

DISCUSSION

The PAS-positive material detected in the epithelial lining of epididymis of mammals has been demonstraded may be involved in secretion of glycoproteins (KOPECNY & PECH, 1977; FLICKINGER, 1983, 1985; VIOTTO et al., 1988 and VICENTINI & ORSI, 1989). Glycoprotein dynamism in the mouse epididymal epithelium using histoautoradiography after injection of L-fucose-3H has also been demonstraded (KOPECNY & PECH).

The epithelial lining of initial, middle and terminal segments in the epididymis of the dog reveals moderate PAS-positivity, suggesting a secretion of neutral mucosubstances. This finding is in agreement with studies by VIOTTO et al., and MARTINELLI & NOGUEIRA. MANEELY (1955) noted considerable PAS reactive material in the epithelia throughout the rat epididymis. ALSUM & HUNTER proposed a secretory cycle which was most active in the proximal segments in the Rhesus monkey epididymis, however, VICENTINI demonstraded a weak PAS-positivity in the initial segment of bull epididymis.

The principal cells of initial and terminal segments showed intensely PAS-positive granules in the cytoplasm, which were similarly reported for the hamster (VICENTINI et al., 1992). These granules has been showed in the terminal segment of bull epididymis (VICENTINI). VIOTTO et al. has been observed these granules in the initial segment of cat ductus epididymidis.

These granules variated in size, and intensity of PAS staining, and may also indicate a degree of accumulation or transformation of products associated with biosynthetic activity of the cells. On the basis of these preliminary histochemical tests alone, it would appear that these granules do contain abundant carbohydrates, especially glycoproteins and mucopolysaccharides (RAMOS JR.).

These granules are also positive at salivary amylase plus PAS reaction but negative at AB pH 2.5, suggesting are probably formed by glycoproteins, similarly to VIOTTO et al. The activity of secretion of glycoproteins by epididymis epithelium confirm the ultrastructural findings (NICANDER & MALMQVIST and ORSI et al.).

No glycogen has been detected in the dog epididymis, is basically similar to that described in the camel (TINGARI & MONIEM, 1979), and cat (VIOTTO et al.). This observation contrasts with other findings of glycogen in the epithelial lining. In the rat epididymis, for example, MANEELY detected a PAS-positive reaction in the epithelium, which was then found to be glycogen.

Glycogen has been reported also in the middle segment of several other species, including rabbit (NICANDER, 1957), stallion, bull and ram (NICANDER, 1958), and south american marsupial (MARTINELLI & NOGUEIRA. Furthermore, MONIEM (1972) demonstrated the presence of glycogen in epididymidis of rat, rabbit, ram and hamster, but noted that even in animals of the same species and in different parts of the same duct, the presence of glycogen was not constant.

According to VIOTTO et al., and ORSI et al. descriptions, the stroma periductal was characterized by its strong PAS-positivity. This PAS-positivity gives evidence for the presence of collagen fibers and smooth muscle fibers in the stroma. This stroma compounds were observed in ultrastructural studies (LOPES & BREUCKER, 1986 and VIOTTO & ORSI, 1989).

The apical border of the principal cells and stroma periductal were weakly stained by AB pH 2.5. The reaction for metachromasia was weak at pH 5.6 and negative at pH 1.7 and pH 2.5, detected more nonsulfated mucosubstances than sulfated mucosubstances. Thus, in the dog epididymis a acid mucosubstances was generally seen, although more less than neutral mucosubstances. HOLT et al. (1980) reported the cells of elephant epididymis secreted acid mucosubstances, probably sialic acid. VOGLMAYR et al. (1985) suggested that sialic acid is adsorbed by the spermatozoa after be synthesized and secreted by the cells of the ductus epididymidis.

Sialic acid is known to be an immunoprotectant and it may protect spermatozoa from phagocytosis by leucocytes or epithelial cells in the female reproductive tract by masking specific antigenic groups (HOLT, 1980). UEDA et al. (1998) has been reported sialic acids could participate in different physiological functions characteristic in the epididymis.

The moderate staining of stroma periductal for AB-PAS suggesting that stroma was constitued by neutral and acid mucosubstances. The dog's epididymis seems contain of an unbalanced condition between neutral and acid mucosubstances, with predominance of the neutral mucosubstances.

The present histochemical observations is according to previous report about structural and histochemical studies of epididymis of mammals (KOPECNY & PECH; RAMOS JR.; VICENTINI & ORSI and ORSI et al.), which correpond to histophysiological features, may be interpreted as evidence for secretion of glycoproteins by epididymal epithelium.

RESUMEN: Para la determinación de glucosaminoglicanos en el epidídimo del perro fueron usadas reacciones histoquímicas. En los tres segmentos del epitelio ductal la determinación de mucosustancias neutras reveló reacciones medias. No obstante, la presencia de gránulos PAS positivos en el epitelio, indicarían una probable secreción glucoproteica de las células principales. Las determinaciones histoquímicas confirman las interpretaciones previas.

PALABRAS CLAVE: 1. Epidídimo; 2. Perro; 3. Histoquímica.

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Correspondence to:
Prof. Dr. Bruno Cesar Schimming
Laboratory of Anatomy
Faculty of Health Sciences
University of Marília _ UNIMAR
Av. Hygino Muzzi Filho, 1001
Marília _ 17525-902
São Paulo
BRAZIL
Email:schimmin-fcs@unimar.br

Recibido : 29-03-2000
Aceptado: 01-06-2000

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