Differentiation of Lymphatics from Blood Vessels in the Broad Ligament of the Swine Using S-Ioo Protein Immunohistochemical Localization in the Vascular Endothelium

95 Lymphology 28 (1995) 95-104 DIFFERENTIATION OF LYMPHATICS FROM BLOOD VESSELS IN THE BROAD LIGAMENT OF THE SWINE USING S-IOO PROTEIN IMMUNOHISTOCHEMICAL LOCALIZATION IN THE VASCULAR ENDOTHELIUM B. Gawronska, T. Doboszynska, A. Zezula-Szpyra Department of Reproductive Histophysiology, Division of Reproductive Endocrinology & Pathophysiology, Centre for Agrotechnology & Veterinary Sciences, Polish Academy of Sciences, Olsztyn, Poland ABSTRACT properties of lymphatic vessels in the broad ligament of the swine (10-12). From studies of We examined the immunohistochemical other species it is known that the lymphatics staining characteristics of S-100 protein in the of the reproductive tract vary in size and vessels of the broad ligament of the swine shape depending on the stage of the sexual uterus. The endothelial cells of arterial vessels, cycle (13,14). In sheep, for example, lymphatics and blood capillaries as well as lymphatics vary from large and filled with nerve fiber bundles showed S-lOO protein concentrated lymph during the luteal phase to positivity. In contrast, the endothelial cells of narrow and barely detectable during the veins did not react for the S-lOO antiserum. follicular phase (14). Immunoreactivity for S-100 protein in the Morphological studies on lymph vessels endothelial cells of lymphatics did not are severely hampered by difficulties in proper consistently demonstrate strong staining identification including distinction from blood intensity. Accordingly, we filled lymphatics vessels. Accordingly, we pursued immuno with colored gelatin before immunohisto histochemical localization using S-100 protein chemical staining to facilitate identification of in the vascular endothelium of conducting lymphatics under light microscopy. Numerous lymphatics and blood vessels in the broad arterioles and capillaries (of which the ligament of the uterus in swine. endothelial cells were immunopositive for s- S-100 protein, so named for its solubility 100 protein) in the lymphatic walls, especially in saturated ammonium sulfate solution, was those in the paraovarian vascular plexus, first isolated from the bovine brain (15). For a support the existence of a microvascular long time S-100 protein was regarded as arterio-arterial rete mirabile network. specific for the nervous system (16). Later, biochemical and immunohistochemical studies Endocrinological studies suggest revealed that S-100 protein was a dimer with participation of lymph and blood vessels in the subunits aa, afi and fifi, and that it was transfer of hormones between the uterus and common among various cell types of non the ovary within the broad ligament in various neural tissues (17-19). Although the biological animals (1,2) including the swine (3-9). More role of S-1 00 has not as yet been precisely investigation of morphology is needed, recognized, its presence has been shown in the however, with the paucity of reports on the endothelial cells of lymphatics (20-23), arteries Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 96 (22-25) and capillaries of the continuous type sections were treated with 0.3% HzOz in (20,24) but not in the venous vessels (22,24). methanol for 30 min to block endogenous However, Amselgruber et al (20) who studied peroxidase activity and then immersed in non the cattle testis found immunoreactivity for immune goat serum (diluted 1:10) and bovine S-100 protein in the endothelial cells of venous serum albumin (diluted 1:100) to inhibit vessels and some immunoreactivity within the nonspecific binding of immunoglobulins. The endothelium of arterioles, although the sections were incubated with anti-S-100 reaction was usually faint. protein serum (diluted 1:700) for 24 hours and Because no other work describes locali then processed with the ABC method. Finally, zation of S-lOO protein in the vasculature of the sections were treated with 3.3' diamino the broad ligament of the swine, we examined benzidine. Some sections were counterstained the specificity of S-100 protein localization in with hematoxylin for better visualization of various vessels of the mesometrium, meso cell nuclei. All reactions were performed at varium, and mesosalpinx and whether lymph room temperature. Normal goat serum and vessels could be identified and differentiated bovine serum albumin were used instead of from blood vessels. the primary antibody as a control-the results were negative also using nerve fiber bundles. MATERIALS AND METHODS Observations and photographs were made with a light microscope (Nikon FXA, Japan). Ten mature pigs (about 1 year old, 90-120 kg body weight) from local industrial RESULTS farms were investigated. The reproductive organs including the broad ligament of the Immunoreactivity for S-100 protein in the uterus were removed immediately after broad ligament of the uterus of the swine was slaughter. In five, the lymphatics were injected seen in the endothelium of both lymph and with carmine-gelatin mass (32-40°C) (26); arterial vessels as well as nerve fiber bundles. interstitially into the ovary and subserously The endothelium of venous vessels was into the mesometrial margin of the uterine immunonegative for S-lOO protein (Figs. lA horn at 1-2 cm intervals. Both tissues with and B). Colored deposits of S-100 protein in lymphatics filled with colored gelatin, and the endothelial cells of lymphatics, visible also those not filled, were fixed in 10% buffered where lymphatics had a collapsed lumen (Fig. neutral formalin. Blocks of tissue (lOx10 mm) IB), permitted their positive identification. cut out from various parts of the meso Additional counterstaining of slices with metrium, mesovarium and mesosalpinx were hematoxylin for better visualization of the cell dehydrated and embedded in paraffin in nuclei corroborated a negative reaction to the conventional fashion. Paraffin sections were presence of S-100 protein in the endothelium serially sectioned with a Reirchert-Jung 2040 of veins and a positive reaction in the microtome into 3-6 JIm slices. Some slices were endothelium of lymph vessels (Fig. IB) stained with hematoxylin and eosin (HE); A highly positive reaction for S-lOO others were immunohistochemically treated protein in the form of condensed brown preci for localization with S-100 protein. pitate was uniformly found in the endothelium Deparaffinized sections were submitted for of arteries in the broad ligament especially immunoperoxidase technique-avid in-biotin uterine and ovarian arteries and their large peroxidase complex (ABC method branches (Figs. 1-3). There was a clear Vectastain® ABC kit Vector Laboratories). difference in the immunoreactivity of the The antiserum used was a polyclonal rabbit endothelia of arteries and veins comparing anti-S-lOO protein antibody obtained adjacent slides containing the tissues of commercially (Sigma Chemical Co.). The ovarian artery and branches of the utero- Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 97 Fig 1. Overview of various vessels of the mesometrium of the swine: positive reaction for S-100 protein in the endo thelium of artery (A), arteriole (a), lymphatics (L) including those with narrow lumen (arrowheads) and nerve fiber bundles (N), and negative reaction in the endothelium of venous vessels (V). Conden sation of reaction products around the cell nuclei counterstained with hema toxylin. lA x250, IB x500 L ovarian vein, stained either with hematoxylin tiny adventitial nerves. Similar pheno-mena eosin (Fig. 3A) or immunostained (Fig. 3B). were observed in other parts of the swine Venous endothelial cells did not demonstrate broad ligament. colored products of immunohistochemical Determining the paths of lymphatic reaction to S-lOO protein whereas in the vessels filled with carmine-gelatin mass adventitia such reaction was seen only in tiny (Fig. 4A) further confirmed localization of vessels (probably arterioles) and in nerve fiber S-lOO protein in the lymphatic endothelium bundles. In the artery, however, a positive (Fig. 4B). Interstitial injection with carmine reaction was evident in the endothelium and gelatin mass did not consistently fill all the Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 98 Fig 2. Cross-section of the uterine artery (2A) and ovarian artery (2B). 2A x250; 2B x500. lymph vessels in a relevant area and dash-like appearance for cross-section consequently in identifying all the lymphatics. endothelium, a positive reaction for S-lOO Here, however, the brown deposits ofS-lOO protein was clearly marked especially in protein localized in the endothelial cells of the slightly protruding nuclear areas of cells. vessel served as the indicator for a lymphatic Thus, the endothelium of lymphatics typically (Fig. 4B). By these criteria, thin-walled, size looked like a thin broken line which made it diversified lymphatic segments of various readily distinguishable from arterial and parts of the broad ligament of the swine were venous vessels (see Figs. SA-C and 6A-C). It demonstrated (Fig. SA-C). Despite a delicate should be emphasized, however, that the Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 99 Fig 3. Sequential sections of the ovarian artery and branches of the utero-ovarian vein: (3A) - staining with hematoxylin-eosin; (3B) - immunohistochemical

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