Localized increases in ovarian vascular permeability and leucocyte accumulation after induced ovulation in rabbits U. Gerdes1, M. G\l=a%o\fvels2, A. Bergh1,3 and S. Cajander1 Departments of ' Pathology, 2Physiology and 3Anatomy, University ofUmeà, Sweden Summary. Colloidal carbon was injected i.v. in mature virgin rabbits at different times after induction of ovulation by human chorionic gonadotrophin (hCG, 100 iu) or mating. Before induction of ovulation, slight carbon leakage was observed in the inner vascular ring of the theca interna of antral follicles, but blood vessels in the other ovarian compartments were unstained. Between 4 and 10\m=.\5h after hCG-treatment or mating, vascular leakage was most marked in the blood vessels of the interstitial gland and in the theca interna of antral follicles. Just before ovulation, carbon particles were observed between granulosa cells and some carbon was seeping into the follicular fluid of preruptured follicles. Vascular leakage was also observed over the follicle dome before rupture as well as at the dorsomedial junction between the mesovarium and the ovary. The blood vessels stained with carbon were 7\p=n-\70\g=m\mdiameter, representing capillaries and postcapillary venules. About 6 h after hCG injection, an increased number of polymorphonuclear leucocytes migrated from the vessels of these ovarian compartments into the surrounding interstitial tissue. The number of leucocytes seen in the follicular wall and ovarian medulla increased markedly towards ovulation. During early corpus luteum formation, the number of leucocytes decreased markedly. The localized vascular changes seen after mating and hCG stimulation were similar to an inflammatory reaction and could form the basis for the formation of peritoneal exudate after ovulation in rabbits and periovulatory ascitic accumulation seen in the peritoneal cavity of women during the menstrual cycle. Keywords: ovary; hCG; mating; vascular changes; leucocyte; ovulation; rabbit Introduction Ovulation in mammals is preceded by rapid follicle expansion under constant hydrostatic pressure (Blandau & Rumery, 1963; Espey & Lipner, 1963). This can occur because of increases in proteo- lytic enzyme activities in ovulatory follicles (Beers, 1975; Hsueh et ai, 1988; Cajander et ai, 1989) and an increase in vascular permeability (Bjersing & Cajander, 1974a, b, c; Okuda et ai, 1980, 1983; Damber et ai, 1987; Cajander, 1989). Hyperaemia, oedema and accumulation of leucocytes are noted around ovulatory follicles in rabbit ovaries (Burr & Davies, 1951; Morris & Sass, 1966; Zachariae et ai, 1958; Bjersing & Cajander, 1974a, b, c; Cavender & Murdoch, 1988) when ovulation is induced by mating or by treatment with human chorionic gonadotrophin (hCG). Treatment with luteinizing hormone (LH) and hCG increases release of ovarian histamine and concentration of eicosanoid, leukotriene, platelet-activating factor and bradykinin (Murdoch et al., 1986; Munalulu et ai, 1987; Espey et ai, 1989a, b,c; Krishna et ai, 1989). It has, thus, been suggested that these inflammatory mediators are involved in increases in ovarian blood flow and vascular permeability induced by LH and hCG (Espey, 1980; Cavender & Murdoch, 1988; Wallach "Present address: University of Pennsylvania, Department of Obstetrics and Gynecology, Division of Reproductive Biology, 7th Floor East-Clinical Research Building, 422 Curie Boulevard, Philadelphia, PA 19104-6147, USA. Downloaded from Bioscientifica.com at 10/09/2021 04:04:47PM via free access et ai, 1989). These observations have further substantiated the hypothesis that ovulation is related to an inflammatory reaction in the ovulatory follicle (Espey, 1980), particularly as several studies have reported that anti-inflammatory drugs, such as antihistamine and inhibitors of prostaglandin synthesis, lipoxygenase and platelet activators, blocked ovulation (Espey, 1980, 1986; Reich et ai, 1983; Abisogun et ai, 1988, 1989; Lipner, 1988). Other morphological studies have demonstrated the appearance of fenestrations and interendothelial cell gaps in perifollicular capillaries after hCG treatment (Bjersing & Cajander, 1974c; Okuda et ai, 1983; Otsuki et ai, 1986), but vascular changes in other regions of such ovaries have not been well characterized. The volume and protein composition of peritoneal fluid sampled during the menstrual cycle varies in relation to cycle day. Studies indicate that this fluid could emerge from the ovaries as a result of transudation from leaking blood vessels (Bouckaert et ai, 1986). The present study characterized the localization of leaking blood vessels and their relation to leucocyte accumulation in the ovary. The colloidal car¬ bon labelling technique (Majno et ai, 1961) was used for morphological investigation of changes in vascular permeability in rabbit ovaries after ovulation induced by mating or hCG treatment. Materials and Methods Animals White Swedish landrace rabbits, 6 months old and weighing 3-4 kg, were purchased from a local breeder. They were kept in separate cages and fed a standard rabbit chow and water adlibitum for 2-3 weeks before the experiments. Rabbits were used because they ovulate 10-12 h after ovulation induced by hCG or mating (Harper, 1961; Cajander, 1976). The total number of rabbits in the permeability studies was 26. The animals were divided into three groups: one received 100 iu hCG (Gonadex: Leo Co., Helsingborg, Sweden or Pregnyl: Organon, Netherlands) i.v. in the lateral ear vein; the second group was mated; and the third group received no treatment and served as controls. In the experiments where leucocyte accumulation was tested, only hCG-treated rabbits (n = 16) were used. Protocols for these studies were reviewed and approved by the Regional Ethical Committee for Animal experiments in Umeá. Assessment of vascular permeability Colloidal carbon (Pelikan Drawing Ink: Pelikan Werke Hannover, Germany) in a dose of 1-4 ml kg1 body weight was given i.v. 1 h before the rabbits were killed. Intravascularly injected carbon particles (average diameter 20-30 nm) are too large to penetrate an intact endothelium, but they may escape from the blood via open inter¬ endothelial cell junctions. Their further passage is, however, partly restricted by the basement membrane and thus sites of vascular leakage (open gaps or gaps that have been open and later closed) are thus labelled black and can later be localized in tissue sections. This technique has been used extensively to localize vascular leakage in various models of experimental inflammation (Cotran & Majno, 1964; O'Donnell et al, 1987) and in rat testis after hCG treatment (Bergh et al, 1987). The animals were anaesthetized with sodium pentobarbital (30mg kg"' i.v. of Mebumal vet.: ACO, Sweden) 1-5,4, 6, 8, 9-5 or 10-5 h after induction of ovulation and the ovaries and parts of the mesovarium were quickly removed and fixed for at least 24 h in 4% (v/v) formaldehyde, 3% (w/v) glutaraldehyde and 005% (w/v) picric acid in 01 mol Na-cacodylate buffer l"1. Animals were killed according to procedures described by Adams (1987), by overdose of barbiturates. Ovarian slices about 1 mm thick were dehydrated and embedded in glucol- methacrylate plastic (Histo-Resin, LKB, Sweden) and sections 1-2 pm thick were stained with haematoxylin and eosin. Other parts of the ovaries were fixed in 1 % (w/v) Os04, dehydrated and embedded in Epon. Thin sections were counterstained with lead citrate and uranyl acetate before observation in a Zeiss EM9 electron microscope. For morphological assessment of the distribution of carbon, the ovaries were categorized into cortex, interstitial gland and mesovarium. The follicles were divided into preantral, secondary antral and tertiary or ovulatory. Atresia was defined according to the criteria of Peters & McNatty (1980). Vessels were classified as capillaries when the wall was composed of an endothelial cell surrounded by occasional pericytes and the diameter was < 10 pm (Ham, 1971). Vessels with a larger diameter, but of the same appearance, were classified as large sinusoidal capillaries. Vascular spaces without erythrocytes were classified as lymphatics. The amount of carbon visible was classified from + to ++++ (+ indicated carbon particles just visible with the light microscope at 1000 magnification). Light micrographs were taken in a Zeiss microscope. Assessment of leucocyte accumulation All rabbits in this part of the study were injected intravenously with lOOiu hCG At 0, 1,6, 10, 14-5 or 26 h after injection, the rabbits were oophorectomized. Three animals were operated at each time except at 1 h after hCG injection (n 1). The ovaries and about 1 cm of mesovarian tissue, including the ovarian vessels, were immersed in — Downloaded from Bioscientifica.com at 10/09/2021 04:04:47PM via free access sodium-phosphate-buffered, neutral 10% (v/v) formaldehyde. After 1-2 days of fixation, the ovaries were cut into slices about 1 mm thick. These specimens were dehydrated and embedded in glycol-metacrylate (Histo-Resin: LKB, Stockholm, Sweden). Sections 1-2 pm thick were cut on a Sorval, Porter-Blum microtome, JB-4, and stained with haematoxylin and eosin. Quantitative analysis of number of leucocytes in different ovarian regions was performed at a magnification of 500 with the aid of frames inserted in the eyepiece. The frames enclosed areas measuring 120 120 or 120 24 pm. The square frame was used for morphometry of leucocytes in mesovarian tissue and ovarian medulla, while the rectangular one was used for tunica albugínea