THE ANATOMICAL RECORD 197: 489-493 (1980) Rat Ovarian Mast Cells: Distribution and Cyclic Changes RICHARD E.JONES, DAVID DWALL, AND LOUIS J. GUILLElTE, JR. Laboratory of Compamtiue Reprcductwn, Department of Environmental, Population, and Organismic Biology, University of Colomdo, Boulder, Colomdo 80309 ABSTRACT Numerous tissue mast cells are present in the ovarian medulla and hilus and in the oviduct of rats. In the medulla, most of these mast cells are in the connective tissue of the stroma near blood and lymphatic vessels. During proestrus, many of the medullary mast cells totally degranulate and thus are not visible histochemically; they then regranulate during estrus. In contrast, the number of stainable mast cells in the ovarian hilus and oviduct does not change during the estrous cycle. Histofluorometric methods demonstrate that mast cells in the ovarian medulla and hilus, as well as the oviduct, contain histamine. In addition, the lining of small blood vessels in the ovarian medulla contains histamine. Thus, mast cell and blood vessel histamine secretion may play a role i n ovarian function. In most mammals studied, ovarian blood plays a vital role in follicular growth and flow markedly increases shortly before ovula- ovulation (Ellinwood e t al., 1978), may be tion (see Ellinwood et al., 1978). More specif- mediated by release of ovarian histamine. ically, ovarian blood flow in the rat is low That histamine plays a n essential role in during diestrus and early proestrus, but in- ovarian follicular growth is supported by the creases t o a peak at 2000 hr of proestrus. The inhibitory effects of exogenous antihistamines flow rate remains high until early estrus, and on this phenomenon (Lipner, 1971; Knox, then decreases to a low level at 1500 hr of 1974; Gergely et al., 19761, as well a s by the estrus (Wurtman, 1964; Yoshinaga e t al., finding that cystic follicle formation is related 1969). Large, preovulatory mammalian folli- to hypersecretion of histamine (Hunter and cles receive most of this increase in blood Leathem, 1968). In mammals, histamine exists in bound delivery (Ellinwood et al., 1978). The blood vessels of rat preovulatory follicles become form in granules of tissue mast cells as well hyperemic, and their permeability greatly in- as in circulating basophils and blood platelets creases (Basset, 1943; Parr, 1974). In the rat, (see Beaven, 1978). Also, the endothelium of as in other mammals (Ellinwood et al., 1978), small blood vessels synthesizes and secretes the preovulatory surge of luteinizing hormone “inducible” histamine i n response to local, (LH) appears to cause the proestrous increase intravascular conditions (Schayer, 1962). The in ovarian blood flow (Ellis, 1961; Parlow and source of histamine within the ovary, howReichert, 1963; Szego and Gitin, 1964; Wurt- ever, is not known. In this paper, we report man, 1964; Piacsek and Huth, 1971). that the ovarian hilus and medulla of the Histamine is a potent, vasoactive biogenic diestrous, estrous, and metestrous rat contain amine, and the above-described effect of LH a large number of mast cells. In addition, on ovarian blood flow in the rat is mimicked degranulation of medullary mast cells during by histamine but not by follicle-stimulating proestrus results in a significant reduction in hormone, prolactin, serotonin, epinephrine, or the number of these cells visible under the norepinephrine (Wurtman, 1964). Exogenous light microscope. We also report that ovarian LH causes depletion of ovarian histamine in mast cells contain histamine, as does the linthe rat (Szego and Gitin, 19641, and the anti- ing of small blood vessels present in the ovarhistamine promethazine hydrochloride blocks ian medulla. the hyperemic effect of LH on rat ovaries (Piacsek and Huth, 1971). Therefore, the LHReceived December 17. 1980. accepted March 5. 1980 induced increase in ovarian blood flow, which 0003-276x180/1974-0489$01.40 @ 1980 ALAN R. LISS, INC. 489 490 R.E. JONES, D. DUVALL, AND L.J. GTJILLETTE, JR. MATERIALS AND METHODS We removed one ovary from each of 18 adult, cycling rats (200 gm, Sprague-Dawley). Sample-size for each stage of the estrous cycle (as determined by vaginal smears) was: proestrus, 6; estrus, 4; metestrus, 3; diestrus, 5. Rats were killed in the late afternoon each day of the estrous cycle. Each ovary was fixed in 10% neutral-buffered formalin, embedded in Paraplast, and serially sectioned a t 6 pm. The sections were stained with toluidine blue, which stains granules of mast cells (Humason, 1972). For each ovary, a total of 10-20 individual sections, each a t least 36-pm apart, were scored to produce a mast cell index, or MCI. A 0.083 mm2 square grid (400 X ) was used. Each section was viewed and the grid fixed over a n area of maximal mast cell concentration. All mast cells within the grid were counted, and the average score from the 10-20 sections produced the MCI for an individual ovary. An MCI for the ovarian medulla, the ovarian hilus, and the oviduct was obtained in this manner. We also scored each mast cell as being either partially degranulated or granulated. Our "partially degranulated" stage was equal to stages 1 and 2 of Gibbons and Chang (1972), and our "granulated" stage was their stage 3. Finally, a n ovary from two rats in each of the four stages of the estrous cycle was examined for the presence of histamine using the histofluorometric method of Enerback (1969). D P E M D P E M E M x 8 O r w OVIDUCT " D P Fig. 1. Index of total mast cells (granulated plus partially degranulated) in the ovarian medulla, hilus, and oviduct throughout the rat estrous cycle. Means S.E. D, diestrus; P, proestrus; E, estrus; M, metestrus. For ovarian medulla, analysis of variance detected significant variation (F [3,141= 14.84,p < 0.001).Duncan's Multiple Range Group Comparison Test indicated that E > D = M > P a t p < 0.05. Numbers of mast cells in hilus and oviduct were statistically similar across the estrous cycle. RESULTS Mast cells are present in medullary tissue of the rat ovary. These cells also are abundant in the connective tissue of the hilus and in the TABLE 1. Distribution and condition of mast cells in the rat ovarian medulla, hilus, and ouiduct durinrr the estrous cycle. Stage Diestrus Proestrus Estrus Metestrus Arteriole Gran. Degran. 0 0 .01 ? .01 .02 ? .02 .08 4 .04 0 .13 2 .05 0 Venule Gran. Degran. .07 2 .04 0 .03? .03 .03 ? .02 .13 ? .04 .08 ? .03 .40 ? .13 .12? .04 Ovarian Medulla Lymphatic Gran. Degran. .O1 2 .O1 0 0 0 .06? .03 0 .03 ? .01 0 "Means * S.E. bConsidering all ovarian compartments, analyses of variance detected significant main-effects for granulation conditions IF (1.101 = 79.19, p < 0.001) and cycle stagp IF 13.14) = 14.48. p < 0.001) for ovarian medulla There was also a significant interaction between cycle stage and granulation condition of mast cells in the medulla (1.'[3.10] = 290.49, p < 0.001). Duncan's Multiple Range Test detected significant differences lp < 0.051 among degranulated cells in the ovary a t each stage of the estrous cycle. 49 1 OVARIAN MAST CELLS interfascicular connective tissue of the oviduct. In contrast, no mast cells were seen in the theca of ovarian follicles or corpora lutea. The MCI for the ovarian medulla was significantly higher at estrus, moderate a t diestrus and metestrus, and extremely low a t proestrus (Fig. 1). In contrast, the MCI for the hilus and oviduct was relatively high and similar through the estrous cycle (Fig. 1).In all tissues (ovarian medulla, ovarian hilus, and oviduct), a great majority of the mast cells were partially degranulated a t all stages of the estrous cycle (Table 1). In the ovarian medulla, most of the mast cells were in the connective tissue of the medullary stroma, many were in the connective tissue surrounding venules, and a few were adjacent to arterioles, lymphatic vessels, and interstitial tissue (Table 1; Fig. 2). The changes in the total MCI for the ovary during the estrous cycle were reflected in each medullary compartment, and these changes were mainly accounted for by differences in the index for partially degranulated cells during the cycle (Table 1). Histofluorometric methods demonstrated that histamine was present (yellow fluorescence) in the mast cells of the ovarian medulla and hilus as well as the oviduct. Also, the lining of arterioles and venules in the ovarian medulla and hilus exhibited yellow fluorescence, indicating that these vessels contain histamine. Our sample was not large enough t o determine if the content of histamine in the mast cells and vessels changed during the estrous cycle. Gran. Stroma Degran. .18 t .07 .01 t .01 .05 -c .02 .10 f .06 .78 f .23 .15 f .04 1.05 f .ll .58 t .07 Interstitial Gran. Degran. .06 t .03 0 0 0 .10 + .04 0 .ll t .07 0 DISCUSSION It is clear that tissue mast cells are abundant in the ovarian medulla and hilus of the rat. This is the first report of mast cells in the rat ovary. In cow ovaries, a few mast cells along with basophilic leucocytes and plasma cells congregate in the thecal interstitial spaces of preovulatory follicles (Cupps et al., 1959). A similar event occurs in the rabbit ovary (Zachariae et al., 1958; Bjersing and Cajander, 1974).Our observation that no mast cells occur around growing or preovulatory rat follicles, the finding of Peppler and Greenwald (1970) that ovaries of diestrous rats contain no preovulatory follicles, and the report of Szego and Gitin (1964) that diestrous rat ovaries contain high amounts of histamine, all suggest that mast cells in the ovarian medulla or hilus and not in preovulatory follicles are a source of ovarian histamine. The blood vessels in the medulla and hilus also contain “inducible” histamine, and the relative roles of mast cell histamine and inducible histamine in influencing ovarian blood flow are not known. Nevertheless, the presence of variation in mast cell number during the estrous cycle suggests that the mast cells degranulate during proestrus and are involved in the increase in ovarian blood flow during this stage of the cycle. Mast cell degranulation is a normal stage in the secretion of histamine (Goth and Johnson, 1975). We detected a decrease in mast cell number during proestrus, and an increase during estrus. These changes in number could reflect changes in synthesis and release of granules, with cells completely Hilus Gran. Degran. .26 + .09 .03 t .03 .03f .03 .05 .05 5.38 t -99 5.85 f .46 6.55 f .77 5.29 + .65 * Oviduct Gran. Degran. .77 t .26 .03 t .03 .12 t .12 .63 f .16 5.22 f .64 6.40 t .28 5.88 + .64 5.75 + .61 ‘Considering a l l mast cells. analyses of variance detecLed significanL main effects far ovarian compartment [F14.70) = 27.15. p < 0.0011 and stageof theestrouscyclelF[3.14] = 14 R4.p < 0 OOl).Theinteraction hetweenstageotcycleandovariancompartment was not significant. Among the ovarian compartment, Duncan’s Multiple Range Test detected t h a t the mast cells in stromal Connective tissue > venules = Interstitial = arterioles = lymphatics ( p < 0.05). 492 R.E. JONES, D. DUVALL, AND L.J. GUILLETE, JR. Fig. 2. Mast cells (-+) in the ovarian medulla of an estrous rat. Note the arteriole (A) and interstitial tissue (IT). Toluidine blue, x 400. discharging granules being invisible using toluidine dye. Even though this dye stains heparin (and not histamine) in the granules (Beaven, 1978), histamine is released with heparin as granules are discharged. The LH surge during the late afternoon or evening of proestrus in the rat (Daane and Parlow, 1971; Butcher et al., 1974) may cause the lowering of mast cell number during proestrus, because this hormone may stimulate mast cell degranulation. The mast cells then regranulate during estrus. Whatever factor causes ovarian mast cell degranulation, its effects may be limited to the medulla, since mast cells in the ovarian hilus and oviduct do not change in number during the estrous cycle. Whether LH influences the secretion of inducible histamine in ovarian blood vessels is not known. However, both compound 48/80 (which degranulates mast cells) and an antihistamine are needed to inhibit ovarian follicular growth in the rat (Lipner, 1971), suggesting that inducible histamine as well as mast cell histamine play a role in ovarian function in this species. Our discovery that the rat ovary contains mast cells that vary in number during the estrous cycle poses more questions. For example, what are the changes in mast cell condition throughout the day of proestrus? Does exogenous LH induce ovarian mast cell degranulation followed by regranulation? What are the roles of mast cell and inducible histamine in ovarian function? Further research should be conducted to answer these questions. ACKNOWLEDGMENTS We thank David 0. Norris and Kevin Fitzgerald for criticizing this manuscript. Supported by a research grant (HD 11482-02) and a Research Career Development Award (HD701650 5 ) from the National Institute of Child Health and Human Development. LITERATURE CITED Bassett, W.S. 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