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Keywords:

  • microvasculature buffalo epididymis;
  • endothelium fenestrations;
  • peripheral lymphatic vascular system;
  • lymphatic capillary network;
  • cylindrical forms

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The microvasculature of the water buffalo (Bubalus bubalis) epididymis was investigated using light (LM), scanning electron (SEM), and transmission electron (TEM) microscopy techniques. SEM analysis of the buffalo epididymis showed fenestrations that occupied ovoid inside the endothelium of the postcapillary venules located in the caput,corpus, and cauda. They varied in shape and dimension, but more importantly, they connected the venules of the blood vascular system to the capillaries of the peripheral lymphatic vascular system. Morphofunctional analysis of these connections suggests that the microvasculature of the buffalo epididymis plays a role in facilitating the circulation of biologically active substances, and the absorption and secretion processes necessary for the survival and maturation of spermatozoa. The lymphatic capillaries at the connection points formed a network of variously sized polygonal links. These capillaries then converged to form the precollector lymphatic vessels, which in turn converged with the larger vessels originating from the testis. It was further noted that in the capillary endothelium there were no fenestrations, and in the large veins there were many diverticula. These diverticula appear to play a role in the regulation of the seasonal variations of the blood reflux. In general, the microvascular architecture of the buffalo epididymis, particularly its connection to the lymphatic vascular system, appears to play an important role in the absorption and secretion processes of the epididymal epithelium. Anat Rec 266:58–68, 2002. © 2002 Wiley-Liss, Inc.

The vasculature of the epididymis–testis complex plays an important role in the regulation of the reproductive activity of many mammals. In particular, the microvasculature of the epididymis, and the absorptive and secretive processes of the epididymis epithelium are morphofunctional prerequisites for the capacity of sperm to fertilize. Past studies of the epididymis microvasculature have mostly focused on laboratory mammals (Clavert et al., 1980; Chubb and Desjardins, 1982; Suzuki, 1982; Abe et al., 1984; Markey and Meyer, 1992), large breeding mammals (Hees et al., 1989; Stoffel et al., 1990), and man (Kormano and Reijonen, 1976). However, few of these studies dealt with species exhibiting seasonal sexual activity, and they revealed very little regarding the role that vascular structures play in the regulation of nutrition and hormone exchanges. It has been shown for the fox, whose sexual activity is seasonal, that the male's testis undergoes morphological changes due to an abrupt increase in the capillary blood flow during the sexual cycle (Joffre and Kormano, 1975). The regulation of this capillary blood flow occurs primarily at the microvascular level (Setchell, 1970). There has been only one study of the vasculature of the epididymis in large breeding animals which have seasonal sexual cycles. Paino et al. (1983) studied the water buffalo (Bubalus bubalis) epididymis, and suggested that the vascular structure connecting the testis to the epididymis plays an important role in the transfer of the substances involved in sperm maturation. The present study investigates the microvasculature of the water buffalo epididymis using light (LM), scanning electron (SEM), and transmission electron (TEM) microscopy in order to better describe its structure, and to shed light on its functions.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Twenty-four epididymis–testis complexes from adult water buffalos (B. bubalis) were collected over the periods of November–December and March–April, which correspond to the most active and least active periods of reproduction, respectively. The complexes were divided into four groups, each of which was studied using different microscopy techniques.

SEM—Vascular Corrosion Cast Technique

Six complexes were each perfused through the testicular artery (A. testicularis) with a physiological solution to wash the blood vessels. They were then injected with a low-viscosity, colored methylmethacrylate mixture (Gannon, 1981) to obtain a cast, and were corroded by immersion in KOH solution (30%) for 1–2 weeks; the solution was changed every 4–5 days. Upon complete corrosion, the casts were rinsed with tap water, rinsed with bidistilled water, dried in a desiccator, and separated into epididymis and testis parts. The epididymis casts were photographed using a digital macrophotographic camera (Nikon Coolpix 990, Tokyo, Japan). Each epididymis cast was separated into caput,corpus, and cauda parts; each part was then cut into numerous 1-cm3 samples that were mounted on stubs (25 mm diameter) and coated with gold using a sputter coater (SC500, BIORAD, Hemel Hempstead, UK). All gold-coated samples were examined and photographed under a scanning electron microscope (LEO 435 VP, Cambridge, UK) at 10 kV.

SEM—Intact Tissue Technique

Six complexes were each perfused through the testicular artery with phosphate buffer 0.1 M, pH 7.3, to wash the blood vessels, and then fixed with Karnovsky's solution (4% paraformaldehyde, 2.5% glutaraldehyde). After 12 hr, the epididymes were separated from the complexes and divided into caput,corpus, and cauda parts. Each part was cut into numerous pieces (0.5 cm long) which were immersed in a glucose phosphate buffer for 24–48 hr, and dehydrated in ethyl alcohol and critical point dryer (CPD 030, BALZERS, Liechtenstein). The piece specimens were mounted on stubs (12.5 mm diameter), examined under SEM (LEO 435 VP) at 17 kV, and photographed.

TEM

Six complexes were each perfused through the testicular artery with a cacodilate buffer 0.1 M, pH 7.2, to wash the blood vessels, and then fixed with a mixture of this buffer and glutaraldehyde 2%. After 1 hr, the epididymes were separated from the complexes and divided into caput,corpus, and cauda parts. Each part was cut into minute pieces that were immersed in glutaraldehyde for 1 hr, rinsed in cacodilate buffer, postfixed with 2% OsO4 for 2 hr, dehydrated, and embedded in an EM bed of EMbed-812 (EMS, Fort Washington, PA, USA). All embedded specimens were sliced into ultrathin sections using an ultramicrotome (Ultratome IV-LKB, Bromma, Sweden), stained with uranyl acetate and lead citrate (Ultrastain-LKB), examined under a transmission electron microscope (Philips EM 201, Eindhoven, The Netherlands) at 40 kV, and photographed.

LM

Six complexes were each perfused with bidistilled water to wash the blood vessels, and colored with China ink (Pelikan, Milano, Italy) through the testicular arteries. The epididymes were separated from the complexes and divided into caput,corpus, and cauda parts. Each part was cut into numerous pieces (0.5 cm long) which were then fixed in paraformaldehyde and picric acid (PAF) for 12–18 hr, rinsed in a glucosate phosphate buffer for 24–36 hr, dehydrated, and pre-embedded twice in 12 hr in a solution of 2-hydroxyethyl metacrylate (85 ml), 2-butoxyethanol (15 ml), and benzoyl peroxide (1 g). Specimens were embedded using a mixture of the pre-embedding solution and N,N-dimethylaniline (ratio 30:1). After polymerization, all specimens were sectioned using a microtome (Historange-LKB), stained with toluidine blue, and examined under a light microscope (Leitz Orthoplan, Wetzlar, Germany) to study blood vessel topography.

All nomenclature in this work was adopted from the Nomina Anatomica Veterinaria, Nomina Histologica and Nomina Embryologica Veterinaria (World Association of Veterinary Anatomists, 1994).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

General Vascular Arrangement of the Epididymis

Macroscopic examination of the buffalo epididymis showed a very well developed vascular arrangement. The integral casts of the epididymis had a half-moon shape, opened cranially, and could be divided into three parts: the caput,corpus, and cauda. The cranio-lateral or external side of the caput was convex, while the caudo-medial or internal side of the caput was concave and embraced the dorso-cranial pole (Extremitas capitata) of the testis. The corpus was shaped like a thin plate with a convex external side caudally, and a concave internal side cranially. The cauda was ladle-shaped, and embraced the ventro-caudal pole (Extremitas caudate) of the testis (Fig. 1).

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Figure 1. A microvascular corrosion cast of the buffalo epididymis. a: Lateral view. b: Medial view. H, caput of the epididymis. B, corpus of the epididymis. C, cauda of the epididymis. D, branches of the deferent duct. Scale bar = 1 cm.

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Blood Vessel Architecture of the Epididymis

The proximal portion of the testicular artery gave rise to many branches (Rami epididymales) that extended through the tunica albuginea (Tunica fibromuscularis) into the caput and corpus of the epididymis. The distal portion of the testicular artery, before going on to the deferent, gave rise to branches (Ramus ductus deferentis) that extended through the tunica into the cauda epididymis. All branches had a spiral-shaped appearance, which remained constant regardless of the season in which the specimen was collected. In addition, all branches subdivided immediately outside the tunica in order to irrigate the tunica, and subdivided immediately inside the epididymis (100 μm internal diameter) to form the interlobular arteries, which continued tortuously towards the lateral and medial margins of the epididymis (Fig. 2).

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Figure 2. Caput of the buffalo epididymis. SEM of the microvascular corrosion cast of the caput efferent ducts. A, interlobular artery. N, capillary network surrounding the epithelium of the mucosa. V, interlobular venule.

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The interlobular arteries gave rise to further branches (20–30 μm internal diameter), viz., the arterioles of the caput efferent ducts (Ductuli efferentes testis) and the arterioles of the epididymis ducts (Ductus epididymidis) in the corpus and cauda. The architecture formed by the branches of these arterioles was markedly different in the caput compared with the corpus-cauda segments. The arterioles of the caput efferent ducts put out numerous winding branches that entered into the fibromuscular layer (Stratum fibromusculare) of the efferent ducts, forming a capillary network made up of elongated and polygonal links. The network surrounds the epithelium of the duct mucous membrane (Fig. 3). The arterioles of the corpus-cauda segments continued to the anses (hairpin bends) of the epididymis ducts, fanned out into numerous branches between the interductal connective tissue, and entered into the fibromuscular tunica (Tunica fibromuscularis) (Fig. 4). In the superficial layers of the fibromuscular tunica, the arterioles narrowed (15–20 μm internal diameter) to form a precapillary arteriole covering, and in the deeper layers to form a network of elongated, polygonal, capillary links surrounding the epithelium of the duct mucous membrane (Tunica mucosa) (Fig. 5). Spiral anses that anastomosed between adjacent precapillary arterioles were very often observed (Fig. 6).

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Figure 3. Caput of the buffalo epididymis. SEM of the microvascular corrosion cast of the efferent duct. Polygonal links of the network surrounding the epithelium of the mucosa. N, capillary network.

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Figure 4. Corpus of the buffalo epididymis. SEM of the microvascular corrosion cast of the anses (hairpin bends) of the epididymis duct. A, interlobular artery. R, branches of the superficial layer of the fibromuscular tunica. V, interlobular venule.

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Figure 5. Cauda of the buffalo epididymis. SEM of the microvascular corrosion cast of the anses (hairpin bends) of the epididymis duct. A, interlobulary artery. R, branches of the superficial layer of the fibromuscular tunica.

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Figure 6. Cauda of the buffalo epididymis. SEM of the microvascular corrosion cast of the anastomosis spiral anses between precapillary arterioles. R, branches of the superficial layer of the fibromuscular tunica. S, anastomosis spiral anse.

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The capillary network gave rise to the postcapillary venules, which converged in the lobular venule of the interlobular septa and terminated in the thick superficial veins of the caput,corpus, and cauda. These veins, located in the tunica albuginea, traveled along the length of the epididymis where they frequently anastomosed with adjacent venules, and terminated in the plexus pampiniformis. In addition, these veins frequently showed diverticula (20–25 μm long) with irregular margins (Fig. 7).

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Figure 7. SEM of the superficial vein of the buffalo epididymis. Diverticula showed the superficial veins. T, diverticula with irregular margins.

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Blood Vessel Microstructure of the Epididymis

The microvasculature structure of the blood vessels was similar in the caput,corpus, and cauda of the epididymis.

The capillary vessels were covered by a continuous basal membrane which either split into pockets that contained thin cytoplasmic extensions of the pericytes, or blended into the fibrous muscular membrane.

The endothelial cells of the capillary vessels had fairly thick walls and were either superimposed on or interdigitated between each other. Their nuclei were elongated and irregular due to the presence of invaginations, which were sometimes very deep. They had abundant chromatin, which was often irregularly thickened along the inner side of the nuclear membrane, and nucleoplasm, which was very dense and granular. The cytoplasm had numerous micropinocytotic vesicles, which varied in size from 30–66 nm, and numerous mitochondria which also varied in size. In addition, many granular endoplasmic reticulum cisternae, numerous free ribosomes, numerous glycogen granules, and very few Golgi apparatus were observed. No fenestrate structures were present (Figs. 8 and 9).

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Figure 8. TEM of the capillary vessels of the buffalo epididymis. No fenestrations were present. Z, nucleus of the endothelial cell. Scale bar = 1.5 μm.

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Figure 9. TEM of the capillary vessel of the buffalo epididymis. The cytoplasm has numerous micropinocytotic vesicles. P, cytoplasm of the endothelial cell. Scale bar = 1 μm.

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Lymphatic Vasculature of the Epididymis

The SEM examination of the fixed, dehydrated specimens (intact tissue technique) showed fenestrations (0.5–1.5 μm diameter) that occupied ovoid zones (7–8 μm diameter) inside the endothelium of the postcapillary venules (Fig. 10). These fenestrations were present in venules throughout the epididymis, and varied in shape and dimension. The SEM examination of the casts (vascular corrosion cast technique) clearly showed that the endothelium fenestrations connected the venules of the blood vascular system to the capillaries of the peripheral lymphatic vascular system (Fig. 11). These lymphatic capillaries (1.5–2.0 μm wide) formed a network of variously sized polygonal links that connected to the precollector lymphatic vessels, which in turn connected to larger vessels (2.5–3.0 μm internal diameter) that led to the testis (Fig. 12).

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Figure 10. SEM of the ovoid zone showing the fenestrations inside the endothelium of the postcapillary venules. F, ovoid zone of the fenestrations.

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Figure 11. SEM of the vascular corrosion cast of the buffalo epididymis showing the connections of the blood vascular system to the capillaries of the peripheral lymphatic vascular system. L, lymphatic capillary. V, postcapillary venule.

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Figure 12. SEM of the network of the peripheral lymphatic vascular system. M, precollector lymphatic vessel.

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Along the external surface of the lymphatic capillaries and precollector lymphatic vessels, cylindrical forms (7–10 μm long) were frequently present either singly or in groups of various sizes (Fig. 13).

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Figure 13. SEM of the cylindrical forms along the peripheral lymphatic vessels. E, cylindrical form. L, lymphatic capillary.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The results of the present study show that, in buffalo, the spiral form of the epididymis microvasculature did not change during the seasons observed. However, the epididymis microvasculature of the fox does exhibit seasonal changes. In fact, the adult fox exhibits a notable increase in the spiralization of the testis veins and arteries during the nonbreeding season (Joffre and Kormano, 1975). Furthermore, the present results show that in buffalo there are neither qualitative nor quantitative seasonal changes in the vascular network of the epididymis microvasculature. This is not the case for camel testes, which during winter and spring exhibit a substantial increase in the number of blood and lymphatic vessels (Abdel-Raouf et al., 1975; Zayed et al., 1995). Thus, in buffalo seasonal reproduction, activity does not affect the macrovasculature of the epididymis–testis complex, whereas in fox and camel it does.

As noted in Results (Blood Vessel Architecture of the Epididymis section), the blood vessel architecture of the epididymis caput, and the corpus-cauda segments showed marked differences. Epididymis segment differences have also been noted in the bull (Hees et al., 1989), boar (Stoffel et al., 1990), mouse (Suzuki, 1982; Abe et al., 1984), rat, rabbit (Chubb and Desjardins, 1982), and man (Kormano and Reijonen, 1976). Stoffel et al. (1990), in a study of the boar, observed differences in the organization of the epididymis capillary networks of the caput, as compared to those of the corpus-cauda segments. Dacheux and Dacheux (1989) provided a morphofunctional explanation of these differences based on their observations that, in boar, there are more synthesis and secretion sites in the caput than in the other two segments. However, the results of the present study show that the epididymis microvascularization in buffalo is organized differently from that in boar. In buffalo, the microvascularization in the epididymis is much more dense in the corpus and cauda than in the caput. This particular organization may be explained on the basis of the following considerations. In buffalo, the spermatozoa spend an extended period of time in the ducts of the epididymis corpus and cauda, and hence, need a relatively efficient system of thermoregulation in order to provide adequate conditions for the survival and maturation of the spermatozoa. In fact, it is well known that male buffalos continually emerge their testes in water and mud to facilitate the thermoregulation of this zone.

It was further noted in Results (Blood Vessel Architecture of the Epididymis section) that the surface veins of the epididymis showed numerous diverticula. These diverticula are not spurious, i.e., are not due to any inherent error in the casting technique, since the casts clearly showed all the surfaces of the blood vessels and exhibited the precise structural characteristics of the vascular walls. It may be hypothesized that these diverticula play a role in the regulation of the blood reflux to the large veins of the epididymis, in accord with the seasonal variations of the blood supply requirements of the reproductive system.

The most surprising of our results regards the absence of fenestrated capillaries along the entire buffalo epididymis (see the Lymphatic Vasculature of the Epididymis section). This absence is in sharp contrast with the results of previous studies (Abe et al., 1984) in the mouse, where fenestrations of varying diameters were always present along the capillary walls of the epididymis. However, the most interesting of our results concerns the presence of large fenestrations in the postcapillary venule endothelium of the buffalo epididymis, which connects the blood vascular system to the lymphatic vascular system. A previous study by Pressman and Simon (1961) noted direct connections between the blood vascular system and the lymphatic system using lymphographic techniques to investigate pathologic conditions in man and various laboratory animals. Even though the presence of a well developed lymphatic system has been greatly documented in man (Orlandini et al., 1979; Holstein et al., 1979; Moller, 1980; Aleksieiev, 2000), mouse (Itoh et al., 1998), rat (Pérez-Clavier et al., 1982), and bull (Zhang et al., 1996), a direct morphostructural connection between the blood vascular system and the lymphatic system is still uncertain.

It can be hypothesized that, in buffalo, the direct connections between the venules of the epididymis and lymphatic periphery are involved in the reabsorption of substances secreted from the testis and the epididymis.

The cylindrical formations along the lymphatic capillaries are most probably the casts of the lacunae located in the extracellular matrix. Miserocchi (1993) and Miserocchi et al. (1984, 2001) described in detail the presence of lacunae which collect the fluid coming from the three or four pleuric stoma in various mammalian species. They hypothesized that these lacunae are the beginning of the lymphatic periphery, based on the fact that the interstitial fluids drain through the stoma via the lymphatic capillaries. Moreover, Castenholz (1998) emphasized the role of the extracellular matrix as a supporting element and prefilter for the lymphatic endothelium that constitutes the lymphatic periphery in rat tongue.

In conclusion, the blood vessel architecture of the buffalo epididymis connects the blood vessel system to the lymphatic vascular system in order to facilitate the circulation of biologically active substances. It also facilitates the absorption and secretion processes which take place in the epididymal epithelium, and which are necessary for the survival and maturation of spermatozoa.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED
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