THE ANATOMICAL RECORD 209:209-218 (1984)

Microvasculature of the Mouse Epididymis, With Special Reference to Fenestrated Capillaries Localized in the Initial Segment

KAZUHIRO ABE, HIROKO TAKANO, AND TAKASHI IT0 Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060, Japan

ABSTRACT The blood supply, microvasculature, and ultrastructure of the capillaries in the epididymis in adult mice were regionally examined. The epididymal duct of the initial segment is surrounded with a dense network of fenestrated capillaries running just under the epithelium. The other segments have loose networks of nonfenestrated capillaries running in the interductal connective tissue. The fenestration of capillaries in the initial segment was markedly reduced in frequency immediately after cutting the efferent duct. In adult mice which were subjected to cutting of the efferent duct neonatally, the dense capillary network did not develop, and fenestrated capillaries were absent in the initial segment. We interpret our results to indicate that the fenestrated capillaries in the initial segment provide for absorption of the testicular fluid and that their development is dependent upon the testicular fluid entering the epididymal duct.

The epididymis consists of an extremely convoluted duct which is morphologically di- vided into several segments that seem to vary functionally (see reviews by Glover and Ni- cander, 1971; Bedford, 1975; Hamilton, 1975). In the mouse, we distinguish five segments of the epididymal duct: segments I, 11, and 111 constitute the head, segment lV the body, and segment V the tail (Takano, 1980; Abe et al., 1982, 1983a,b). Segment I corresponds to the initial segment which has the highest epithelium; functionally, it is considered to absorb the testicular fluid (Levine and Marsch, 1971). Ligation of the efferent duct results in marked regression of this segment (Fawcett and Hoffer, 1979).

Blood flow to the initial segment is greater than to any other segment (Setchell et al., 1964; Brown and Waites, 1972). After vascu- lar perfusion with India ink in the mouse, we observed that the initial segment is sur- rounded by a very dense network of capillar- ies (Takano, 1980). Such a capillary network has also been demonstrated by plastic casts of the blood vessels when observed by the scanning electron microscope (Suzuki, 1982).

During examination of the ultrastructure

of the mouse epididymis (Abe et al., 1983a), we found that the capillaries forming the dense network in the initial segment were of the fenestrated type whereas capillaries in the other segment were nonfenestrated. De- spite extensive electron microscopic studies of the epididymis, regional differences in ul- trastructure of the epididymal capillaries have not been reported (review by Hamilton, 1972; Reijonen et al., 1975; Nicander, 1979; Suzuki, 1982). The specialized microvascula- ture in the initial segment appeared closely related to the function of this segment. Thus, this study is concerned with the microvascu- lature of the mouse epididymis in particular relation to regional differences. To under- stand the functional meaning of the micro- vasculature, we examined capillary changes after interruption of the testicular fluid flow entering the epididymal duct by cutting the efferent duct in neonatal and adult mice.

Received July 11, 1983; accepted January 3, 1984.

0 1984 ALAN R. LISS, INC

2 10 K. ABE, H. TAKANO, AND T. IT0

MATERIALS AND METHODS Animals

In this study, 77 male dd-mice were used. They were killed at 2-3 months of age.

Examination of Vascular Supply Ten mice were anesthetized with Nembu-

tal (pentobarbital sodium) injected intraperi- toneally. The abdomen was opened and the vasculature of the epididymis was examined by means of the dissecting microscope.

India Ink Perfusion Study Ten mice received a vascular perfusion of

India ink following saline. Then the epidi- dymis was removed, fixed in 5% formalin-0.1 M phosphate buffer (pH 7.41, dehydrated, and embedded in Coulter’s Epon mixture (Coul- ter, 1967). The blocks were warmed and cut into 0.2-0.5-mm-thick sections with a razor blade. The sections were mounted on slide glasses and observed by light microscopy.

Light Microscopy The epididymis was fixed with 5% for-

malin-0.1M phosphate buffer (pH 7.4) for 20 hours, embedded in methacrylate (Bennett et al., 1976) and cut into 2-pm-thick sections using glass knives. The sections were stained with hematoxylin and eosin and observed with the light microscope.

Electron Microscopy The epididymides from ten mice were fixed

with 5% formalin-1.5% sucrose in 0.05 M phosphate buffer (pH 7.4) or 3% paraformal- dehyde-1% glutaraldehyde-0.1 M cacodylate buffer (pH 7.4) for 2 hours, postfixed with 2% OsO4 for 2 hours, dehydrated, and embedded in Luft’s Epon mixture (Luft, 1961). The ul- trathin sections were stained with uranyl acetate and lead citrate, and observed by electron microscopy.

Cutting of the Efferent Duct 1) Twenty-one mice at 60 days of age were

anesthetized with Nembutal, and the epidi- dymis and testis were exposed through a me- dian incision of the lower abdomen. Under the dissecting light microscope, the efferent duct was cut off avoiding damage to the blood vessels of the epididymis and testis. The mice served for India ink perfusion or electron mi- croscopy 3, 6, 12, and 24 hours, 3 and 7 days, and 4 weeks after the operation.

2) Ten mice at the day of birth were anes- thetized by hypothermia in a freezer and the

efferent duct was cut as described above. They served €or India ink perfusion study or electron microscopy at 60 days of age.

Number of Pores in the Capillary Endothelium

In normal mice and in those at various intervals after cutting the efferent duct, the capillaries in each segment of the epididymal duct were photographed at ~ 5 , 0 0 0 and en- larged to x 10,000. The capillary pores were counted along the lateral aspect of the endo- thelial cells, and the circumferential length of the cells was measured. From this, the number of pores per circumferential length was obtained. The perinuclear cytoplasm of the endothelial cells was omitted from mea- surement. For each epididymis at each post- operative time, 20-40 capillaries were randomly selected for measurement.

Vascular Supply (Figs. 1,2) The testis and epididymis are located with

fat tissue in a peritoneal fold arising from the inferior posterior abdominal wall. The testicular artery runs in the peritoneal fold to reach the testis. Along the long margin of the testis, the epididymis lies lateral to the artery. In the peritoneal fold, the testicular artery gives off a n inferior epididymal ar- tery, two arteries to the efferent duct, and an artery to the fat tissue, respectively, and fi- nally becomes a superior epididymal artery- that is, the main channel.

The superior epididymal artery reaches the border of segments I and I1 and supplies branches to segments I, 11, and 111 in the epididymal head. The inferior epididymal ar- tery reaches the proximal region of the body and is divided into an ascending branch to- ward the head and a descending branch to segment IV. The descending branch anasto- moses with the deferential artery ascending from the epididymal tail.

The deferential artery is a branch of the internal iliac artery, runs along the deferent duct, reaches the epididymal tail, and as- cends along the epididymal body. The artery supplies blood not only to the deferent duct but also to segments V and IV. At the tip of the epididymal tail, the deferential artery anastomoses with a small artery from the external iliac artery, which enters the epidi- dymal tail from the scrotum.

The epididymis usually shows the foremen- tioned arterial supply, but some variations occur, as shown in Figure 2. For example, in

RESULTS

FENESTRATED CAPILLARIES IN THE EPIDIDYMIS 211

Fig. 1. Diagram showing blood supply to the right testis and excurrent duct system. The fat tissue of the peritoneal fold is dotted. TA, testicular artery; EA, effer- ent duct arteries; SEA, superior epididymal artery; IEA, inferior epididymal artery; CIA, common iliac artery; EIA, external iliac artery; IIA, internal iliac artery; DA,

deferential artery. The superior and inferior epididymal arteries (SEA, IEA) are branches of the testicular artery (TA). The deferential artery (DA) arises from the internal iliac artery (IIA). The branch from the external iliac artery (EIA) enters the epididymal tail from the scrotum (see the lowest artery in the figure).

some cases, the epididymis may receive two arteries: an epididymal artery to the head and a deferential artery to the tail and body (see figure legends for more detail).

The efferent duct receives two arteries from the superior epididymal artery. These arter- ies reach the middle portion of the duct on the medial side of the epididymal head. The fine arteries running on the efferent duct anastomose with the superior epididymal ar- tery at the junction between the efferent duct and the epididymal head.

India Ink Perfusion Study Immediately after India ink perfusions,

segment I appears black, but the other seg- ments are gray. In living animals, segment I appears reddish because of its dense micro- vasculature. Light microscopy of thick sec- tions reveals that the epididymal duct in segment I is surrounded by a dense capillary network (Figs. 3, 4). The network forms meshes with narrow spaces which are elon- gated in the transverse direction of the duct (Fig. 4). However, capillaries in segments II-

V form a coarse network with large meshes elongated in the longitudinal direction of the duct (Fig. 4). Thus, while the capillary net- work differs in pattern between segment I and the remaining segments, it exhibits no regional difference between segments 11-V.

Light and Electron Microscopy (Figs. 5-10) The epithelium of the epididymal duct is

closely surrounded with two to six layers of smooth muscle cells, among which elongated fibrocytes are occasionally seen. Outside the smooth muscle layer is stromal areolar con- nective tissue. Capillaries are found between the epithelium and smooth muscles and in the stroma (Figs. 5-7,9). Thus the capillaries in the epididymis can be divided into subepi- thelial and stromal capillaries according to their location (Figs. 5-7, 9).

Segment I has subepithelial capillaries just beneath the epithelium (Figs. 5, 7). The epi- thelial basal surface facing the capillaries shows depressions of grooves along the cap- illaries, and the smooth muscle cells have their flat surface toward the capillaries (Fig.

212 K. ABE, H. TAKANO, AND T. IT0

7). The subepithelial capillaries are 2-5 pm in diameter and the endothelial cells are at- tenuated and fenestrated (Fig. 7). The pores of the endothelial cells are about 60 nm in diameter and have a diaphragm with a cen- tral knob (Fig. 8). They are distributed throughout the cytoplasmic sheet of the en- dothelial cells but tend to be more frequent on the side toward the epithelium of the epi- didymal duct (Fig. 7).

The capillary endothelium facing the epi- thelium lacks pericytes and adventitial cells, which, however, are found around the endo- thelium facing the smooth muscles. The space between the endothelial cells and the epithelial cells is 0.2-0.5 pm wide and con- tains two basal laminae which relate to both the endothelial and epithelial cells (Fig. 8).

In segment I, the stromal capillaries are less frequent than subepithelial ones (Fig. 5).

Fig. 2. Diagram showing variations of the arterial supply to the epididymis. Dotted lines indicate the bor- der between the head and body of the epididymis. The upper four epididymides show no anastomosis between the artery of the head and the artery ascending from the tail. The double asterisk indicates the most frequent type of epididymal arterial supply. The single asterisk indicates the type which lacks the inferior epididymal artery and this type appears to represent the basic pat- tern of the vascular supply to the epididymis. The epidi- dymis between the marked two shows the intermediate form. The rightmost represents the inferior epididymal artery reaching the border between the epididymal head and body. The lower three epididymides show anasto- mosis of the ascending artery from the tail with the artery of the head. The right two lack the inferior epidi- dymal artery.

Fig. 3. Thick section of the epididymal head after India ink perfusion. Segment I appears black due to much denser microvasculature than that in segments I1 and I11 (I1 and 111). DD, deferent duct. Epon embedded. x 20. Epon embedded. ~ 7 0 .

Fig. 4. Thick section of the epididymal head after India ink perfusion. Epididymal duct in segment I is surrounded with a dense capillary network. Capillaries in segments I1 and I11 (I1 and 111) form coarse networks.

FENESTRATED CAPILLARIES IN THE EPIDIDYMIS 213

They are 5-10 ,um in diameter, being of the nonfenestrated type (Fig. 9).

In segments 11-V, the subepithelial capil- laries are of rare occurrence (Fig. 6). The capillaries are usually found in the smooth muscle layer, and one or two layers of smooth muscle cells andor fibrocytes intervene be- tween the endothelium and epithelium. The subepithelial capillaries, if present are of the nonfenestrated type (Fig. 10). The cyto- plasmic processes of pericytes and adventi- tial cells are found all around the endo- thelium.

The stromal capillaries in segments 11-V are similar in appearance to those in seg- ment I. Effect of Efferent Duct Cutting (Figs. 11, 12) In the epididymis subjected to efferent duct

cutting at 60 days of age, the capillary net-

work appears unchanged even 4 weeks after operation. In living animals, segment I ap- pears less reddish. By India ink perfusion, segment I macroscopically is paler than the normal, though it microscopically reveals a dense capillary network.

On the other hand, electron microscopy shows that the pores in the subepithelial cap- illaries in segment I decrease in number im- mediately after efferent duct cutting (Fig. 12). After the cutting, the number of pores is slightly decreased at 3 hours and then rap- idly reduces to half at 12 hours, to one-third at 24 hours, and to very few at 3 days. Thus, after 3 days the subepithelial capillaries in segment I become nonfenestrated, as seen in segments 11-V (Figs. 11, 12).

During the decrease, the pores tend to dis- appear a t first on the side facing the smooth muscle. In addition, when the pores have

Fig. 5. Segment I. Many subepithelial capillaries Fig. 6. Segment 11. No subepithelial capillaries are seen. SC, stromal capillaries. Methacrylate embedded. Hematoxylin and eosin. X450.

(SEC) containing red blood cells run between the basal surface of the duct epithelium and the thin cellular layer surrounding the duct. SC, stromal capillary. Methacry- late embedded. Hematoxylin and eosin. X450.

2 14 K. ABE, H. TAKANO, AND T. IT0

Fig. 7. Segment I. Subepithelial capillary (SEC) lo- cated between the duct epithelium (Ep) and the sur- rounding smooth muscle (SM) and fibrocyte (F) layer. The capillary endothelium, especially that facing the epithelium, is thinly attenuated and fenestrated (ar- rows). P, pericyte. ~11,000.

epithelial cell; BL, basal laminae in the space between the endothelial cell and the epithelial cell. X27,OOO.

Fig. 9. Segment I. The stromal capillary (SC) is non- fenestrated. P, pericyte; SM, smooth muscles surround- ing the epididymal duct. X 7 , 2 0 0 .

Fig. 8. Segment I. Pores of the endothelium of the subepithelial capillary have diaphragms (arrows). Ep,

FENESTRATED CAPILLARIES IN THE EPIDIDYMIS 215

Fig. 10. Segment 11. Subepithelial capillary (SEC) seen between the epithelial cell (Ep), and smooth muscles (SM) surrounding the duct is nonfenestrated. x 12,000.

Fig. 11. Segment I, 4 weeks after efferent duct cut- ting. The subepithelial capillary (SEC) shows disappear- ance of the fenestrations. P, pericyte; Ep, epithelial cell; SM, smooth muscles. x 12,000.

T

Fig. 12. Number of pores of the subepithelial capil- lary endothelium. I, segment I; 11-V, segments 11-V; N, normal. The arrow indicates the time of efferent duct

cutting. The number of pores (mean k SE) decreases significantly with time (h, hour; d, day; w, week) after operation.

2 16 K. ABE, H. TAKANO, AND T. IT0

disappeared in the endothelium facing the epithelium, the cytoplasmic processes of per- icytes are observed between the endothelium and epithelium.

The subepithelial capillaries in segment I did not change in frequency after cutting the efferent duct.

The stromal capillaries in segment I and the capillaries in segments 11-V show no change in ultrastructure and frequency.

Effect of Neonatal Efferent Duct Cutting In adult mice neonatally subjected to effer-

ent duct cutting, India ink perfusion reveals that the epididymis has no dense network of capillaries in the head.. The capillaries in the region corresponding to segment I are the same in nature as those in the other regions. The capillaries show no regional difference in ultrastructure. They are also similar in structure to those in segments 11-V of the normal epididymis.

DISCUSSION

The epididymal duct is considered to be involved in functional maturation and stor- age of spermatozoa (see reviews by Bedford, 1975; Hamilton, 1975). It has been reported for many species that the maturation of sper- matozoa occurs in the head of the epididymis and that maturated spermatozoa are stored in the body and tail (see reviews by Bedford, 1975; Hamilton, 1975). The vascular supply to the head of the epididymis is different from that to the body and tail; the former is from the testicular artery and the latter from the deferential artery (Chubb and Desjar- dins, 1982; Suzuki, 1982). The epididymal head and the testis are considered to cooper- ate in production and maturation of sperma- tozoa, and the remaining portion of the epididymis and the deferential duct to form a unit for discharge of mature spermatozoa. In this relation, it is of interest that such functionally combined structures have their vascular supply from the same origin.

The dense vascularization of the initial segment has been observed by microangiog- raphy in the rat and human epididymides (Kormano, 1968; Kormano and Reijonen, 1976). In the mouse, scanning electron mi- croscopy of vascular casts (Suzuki, 1982) and light microscopy of blood vessels perfused with India ink (Takano, 1980) revealed that a dense, basketlike capillary network sur- rounds the duct in the initial segment. The high blood flow to this segment has also been demonstrated by physiological methods

(Setchell et al., 1964; Brown and Waites, 1972).

The dense vascularization and high blood flow in the initial segment suggest that this segment is metabolically very active. In the epididymal duct, this segment has been known most actively to secrete protein or glycoprotein and absorb the testicular fluid (Levine and Marsch, 1971; Kafika and Ko- peCny, 1977; KopeEny and Pech, 1977). Of the testicular fluid entering the epididymis, the largest volume is thought to be absorbed in the initial segment (Levine and Marsch, 1971).

The secretory and absorptive functions in the initial segment appear to be dependent upon the testicular fluid (Hoffer et al., 1973a; Fawcett and Hoffer, 1979). Efferent duct lig- ation or cutting, which excludes the testicu- lar fluid from the epididymal duct, causes marked decrease in height of the epididymal epithelium, and the epithelial cells of the initial segment cytologically show dediffer- entiation that appears to impair the secre- tory and absorptive functions (Smith, 1962; Fawcett and Hoffer, 1979). With reduction in the functional activity after ligation or cut- ting of the efferent duct, the blood flow to this segment also shows significant decrease after 7 days (Brown and Waites, 1972). The reduction in blood flow caused the initial seg- ment to become paler in appearance after operation in living animals or after India ink perfusion, though the capillary network did not change in density. The decrease in blood flow may be attributed to a functional con- striction of the capillaries.

When adult mice are subjected to neonatal efferent duct cutting, the dense capillary net- work is not observed in the head of the epi- didymis. We interpret this finding to indicate that testicular fluid may be necessary for the development of the dense capillary network in the initial segment.

The development and functional differen- tiation of the epididymal duct are thought to be dependent upon androgen, and after cas- tration the epididymal duct loses the seg- mental differentiation due to depletion of the circulating androgen (Cavazos, 1958). How- ever, since i t is known that the testicular fluid entering the epididymal duct contains a high concentration of androgen which is associated with the androgen-binding pro- tein in the testis (Hansson and Djgseland, 1972), intraluminal androgen appears neces- sary for maintenance of the differentiation of the initial segment. Fawcett and Hoffer

FENESTRATED CAPILLARIES IN THE EPIDIDYMIS 217

(1979) also reported that the initial segment is responsive only to the luminal androgen, because regression of this segment after ef- ferent duct cutting is not prevented by a high concentration of exogenous testosterone. The functional relationship between the initial segment and the testicular fluid may become clearer when the nature of the fine structure of the capillaries surrounding the duct of this segment is known, as described in our results.

It has been briefly reported that the epidi- dymis possesses both fenestrated and non- fenestrated capillaries, but no regional dif- ferences have been described (Hamilton, 1972; Reijonen et al., 1975; Nicander, 1979; Suzuki, 1982). The present findings establish that the epididymal capillaries show re- gional differences in fine structure: The cap- illaries form a dense network around the duct in the initial segment just under the epithe- lium, and are fenestrated; in the other seg- ments, less-dense networks of nonfenestrated capillaries are seen in the interductal con- nective tissue. In addition, pores of the fenes- trated capillaries in the initial segment show dramatic changes in number after efferent duct cutting.

Fenestrated capillaries are usually distrib- uted in organs which show a very active fluid exchange with the blood (Karnovsky, 1967). The pores of capillaries are inferred to vary in number according to functional activity of the organ (Carsten and Merker, 1965), though additional evidence is needed. The subepithe- lial capillaries in the initial segment of the epididymis may serve as a dynamic model for suggesting a functional significance of the fenestrated capillaries.

In the mouse epididymis, fenestration of the capillaries as well as remarkable devel- opment of the capillary network is consid- ered to be related to high functional activity in the initial segment (Levine and Marsch, 1971; Hoffer et al., 197313; Kafika and Ko- peEny, 1977; KopeEny and Pech, 1977). After efferent duct cutting, loss of capillary fenes- tration occurs within 1 day prior to the epi- thelial dedifferentiation and blood flow- decrease that were confirmed a few weeks later (Brown and Waites, 1972; Hoffer et al., 197313). Therefore, the response of the capil- laries is not a secondary reaction due to the epithelial dedifferentiation but a primary re- sponse related directly to possible absorption of the luminal testicular fluid. Since the pores of the capillaries in the initial segment facil- itate the absorption of the testicular fluid, their decrease in number immediately after

efferent duct cutting suggests that the reduc- tion of fenestration accompanies the dimin- ished availability of testicular fluid for absorption. After operation, the pores de- crease in number slowly for the first 3 hours. The initial slow decrease may be due to the time lag in the removal of the luminal testic- ular fluid and the decrease of fluid to elicit the morphological reaction of the capillary wall.

The distribution of pores in the epididymal capillaries is similar to that in the intestinal capillaries; these also show fenestrations in the endothelium oriented toward the absorp- tive epithelium of the intestinal villi (Cle- menti and Palade, 1969). The polarization in the pore distribution appears more obvious after efferent duct cutting. The findings also suggest that the epididymal capillaries are functionally involved in absorption of the lu- minal material.

The initial segment shows epithelial dam- age by alpha-chlorhydrin and cadmium and high stainability to dyes administered into the blood vessels; these responses become weak and disappear after efferent duct liga- tion (Gunn et al., 1970; Hoffer et al., 1973b; Reijonen et al., 1975). Our results suggest that such characteristic features of the ini- tial segment can be explained by the pres- ence of fenestrated capillaries, which allow great permeation of drugs, and by reduced fenestration, which limits the permeability of the capillaries after efferent duct ligation. The failure of testosterone administration to prevent regression of the initial segment after efferent duct cutting, mentioned above, may also be primarily related to the reduc- tion of the capillary fenestration (Fawcett and Hoffer, 1979). After efferent duct cutting the capillaries probably do not have available concentrations of testosterone high enough to sustain the normal epithelial functions in the initial segment.

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