J. Anat. (1983), 137, 3, pp. 457-466 457 With 7 figures Printed in Great Britain The vascular anatomy of the and the relative contribution of the ovarian and uterine to the blood supply of the ovary in the guinea-pig M. I. HOSSAIN* AND J. D. O'SHEAt * Department ofAnatomy and Histology, Bangladesh Agricultural University, Mymensingh, Bangladesh and t Department of Veterinary Preclinical Sciences, University ofMelbourne, Parkville, Victoria, Australia 3052 (Accepted 13 January 1983)

INTRODUCTION The mammalian ovary derives its main arterial supply from the ovarian which arises from the abdominal aorta or from the renal artery (Mossman & Duke, 1973; Reynolds, 1973). The blood vessels supplying and draining the of the guinea-pig have been described briefly (Potter, Jones & Hermann, 1958; Shively & Stump, 1975; Cooper & Schiller, 1975), although DelCampo & Ginther (1972) have given a detailed account of the anatomy of the extrinsic ovarian vasculature and have related it to the presence of a unilateral luteolytic mechanism in this species. There are no published data on the intrinsic vasculature of the ovary of the guinea-pig. However, it has been reported that the intrinsic vasculature of the ovary differs widely both between species and during different functional states (Clark, 1900; Burr & Davies, 1951; Delson, Lubin & Reynolds, 1948; Delson, Lubin, Brooklyn & Reynolds, 1949; Reynolds, 1973). In most mammals, there are anastomoses between branches of the ovarian and uterine arteries (Mossman & Duke, 1973; Reynolds, 1973). In relation to their potential contribution to ovarian blood supply, it is clearly important to know the direction of blood flow in these anastomoses. The simplest approach is to compare the diameters of the various vessels. On this basis, it has been concluded that flow in these anastomoses is probably towards the uterus in the horse, sheep and pig (DelCampo & Ginther, 1973), and towards the ovary in the rat, hamster, guinea-pig (DelCampo & Ginther, 1972), rhesus monkey (Ginther, Dierschke, Walsh & Del- Campo, 1974) and, on the side ipsilateral to the corpus luteum, in the ox (Ginther & DelCampo, 1974). However, in a later study in which microspheres labelled with different isotopes were injected into the left ventricle, and into the aorta between the origins of the ovarian and iliac arteries, Chaichareon, Rankin & Ginther (1976) have concluded that the flow in this anastomosis is towards the uterus in the guinea-pig. In view of such contradictory results, further investigation, using different methodology, has been considered worthwhile. This paper reports a study of the extrinsic and intrinsic vasculature of the ovary in the guinea-pig, and of the direction of blood flow along the arterial anastomosis between the uterine and ovarian arteries. 458 M. 1. HOSSAIN AND J. D. O'SHEA

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Fig. 1. Diagrammatic representation of the ventral view of the arteries supplying, and the major draining, the ovary of the guinea-pig. AO, aorta; VC, vena cava; AD RA, additional renal artery; 0 V B A D RA, ovarian branch of additional renal artery; R 0 V, right ovary; CL, corpus luteum; E ARC, extra-ovarian arterial arcade; R 0 V V, right ovarian ; L 0 V V, left ovarian vein; L O VA, left ovarian artery; L R V, left ; RK, right kidney. Arrow indicates the region where direction of blood flow was observed.

MATERIALS AND METHODS Animals Adult female Dunkin-Hartley guinea-pigs were used in these studies. Timing of the stages of the oestrous cycle was based on daily observation for vaginal opening; vaginal smears were taken on days when the vagina was open. The first day of com- plete vaginal opening when the smear contained abundant comified epithelial cells, with very few nucleated epithelial cells or leucocytes, was designated Day 1 of the cycle. In all the animals, at least two consecutive oestrous cycles of 15-19 days had been observed immediately preceding the cycle in which the experiment was conducted. Ovarian vasculature Latex studies were carried out on fifteen animals between Days 6 and 12 of the cycle. Latex was injected either into the arterial system or venous system or both. Latex of two contrasting colours was used when both systems were injected. Specimens were fixed by immersion in a solution containing 30 % of 95 % ethanol, 10 % formalin, 10 % glacial acetic acid and 50 % distilled water (DelCampo & Ginther, 1972) for 48-72 hours. They were then studied with a dissecting microscope after either dissection, clearing in benzene and benz> benzoate (Orsini, 1962; DelCampo & Ginther, 1972) or maceration for 48 ho rs in 20% sodium hydroxide at 50 'C. Two other methods were each used on a single animal on Day 10 of the oestrous Vascular anatomy of ovary in guinea-pig 459

OVA Fig. 2. Diagrammatic representation of the arteries to the ovary and corpora lutea of the guinea-pig. OV, ovary; OVA, ovarian artery (here ovarian branches of the ovarian artery); LA, luteal artery; CL, corpus luteum; I ARC, intra-ovarian arterial arcade. cycle. A silicon rubber cast was formed by injecting silicon rubber (Microfil, MV-1 12, Canton Biochemical Products, Inc., Boulder Co.) via the thoracic aorta. The injected specimen was fixed in 10 % formalin, dehydrated in alcohol and cleared using methyl salicylate. In the other method, a 2 % Berlin blue solution was injected via the aortic cannula. The reproductive tract, together with its attached ligaments, the contiguous peritoneal tissues, kidneys and aorta was dissected out as a single unit, pinned flat on a cardboard sheet, and fixed in 10 % formalin. The specimen was then cleared in dimethyl sulphoxide (Analar, BDH Chemical Ltd., Poole, England) for a week before examination. Direction ofbloodflow in utero-ovarian arterial anastomosis Six female guinea-pigs weighing 500-550 g, two animals each on Days 10, 12, and 16 of the cycle, were included in this experiment. The method was based on that described by O'Shea & Lee (1974) in the rat. The animals were fed on a restricted diet, with water ad libitum, so that weight remained almost constant over a period of 35 days, thereby reducing the amount of fat present in the broad ligament and around the ovarian and uterine vessels. Under either urethane or rentobarbitone anaesthesia (three animals, one each on Days 10, 12, and 16 of the cycle, were anaesthetised with each anaesthetic), the right common carotid artery was cannula- ted and the cannula was advanced to the left ventricle. The abdominal cavity was then opened with a mid-ventral incision, and the viscera carefully retracted to one side to expose the region of anastomosis between the ovarian and uterine arteries. The region was then -viewed under a dissecting microscope, care being taken to avoid touching the vessels. Since only small amounts of fat were present, the anastomotic vessels were clearly visible. Approximately 0-15 ml of a 2-5 % solution of Lissamine green, adjusted to pH 7*4 with 0 1 M sodium hydroxide solution, was 460 M. I. HOSSAIN AND J. D. O'SHEA

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ov V Fig. 3. Diagrammatic representation of the veins to the ovary and corpora lutea of the guinea- pig. CV, ovary; OV V, ovarian vein (here ovarian tributary of ovarian vein); LV, luteal vein; CL, corpus luteum. then injected via the carotid catheter from a 1 ml tuberculin syringe. The artery in the region of anastomosis was viewed simultaneously under a dissecting microscope to observe the direction of flow of the injected dye. Several repeated injections were made in all animals. The above procedure was performed on both left and right sides of all the animals studied.

RESULTS Extrinsic ovarian blood vessels Arterial supply The ovaries were supplied by the paired ovarian arteries (Figs. 1, 4, 5). These arteries originated either from the aorta at about the level of origin of the renal arteries, or as branches of renal arteries, or by common trunks with renal arteries (Fig. 1). From their origins, the ovarian arteries passed caudally and laterally around the caudal poles of the kidneys (Figs. 1, 4, 5). These arteries were in close apposition to the corresponding veins on each side (Figs. 1, 6), supplied small branches to the tissues of the mesovarium and to the perirenal fat, and in some cases formed anastomoses with branches of the renal arteries. Each ovarian artery then gave rise to two or three ovarian and tubal branches, after which the main trunk ran more caudally to form a major end-to-end anasto- mosis with the cranial end of the uterine artery, so completing a closed loop with this vessel (Fig. 4). The ovarian and tubal branches formed spiralling coils around their longitudinal axis as they approached the ovary. Before reaching the hilus of the ovary, further branching took place from these ovarian and tubal branches, pro- ducing a total of 5- or 6 small, tightly coiled branches. Anastomoses between these Vascular anatomy ofovary in guinea-pig 461

Fig. 4. Ventral view of partially cleared Berlin blue injected arteries to the female genital tract ofthe guinea-pig. 0 V, ovary; O V A, ovarian artery; UA, uterine artery; K, kidney; 0 VB O V A, ovarian branches of ovarian artery. Arrow indicates the regions where direction of blood flow was observed. x 1-4. arteries were observed in the pedicle. The most caudal of the arteries supplied the uterine tube, infundibulum and mesosalpinx, and formed anastomoses (a) caudally with a branch or branches of the uterine artery on the tubal wall and (b) cranially with a branch or branches of one of the ovarian arteries coming around the cranial pole of the ovary, to form an extra-ovarian arterial arcade (Fig. 1). Three or four of the coiled arterial branches entered the hilus of the ovary (Figs. 1, 2, 5). Venous drainage Several small veins drained the ovary. At the hilus they usually joined to form a single ovarian vessel which then fused with a large tributary from the uterus to form a single ovarian vein, corresponding to the ovarian artery. These veins were in close contact with the corresponding arteries. In the ovarian pedicle, the ovarian arterial branches commonly spiralled around the ovarian venous tributaries. The right ovarian vein finally joined the vena cava and the left ovarian vein opened into the left renal vein (Fig. 1). Intrinsic ovarian blood vessels Arterial supply As stated above, three or four ovarian arterial branches entered separately into the hilus of the ovary (Fig. 2). They followed tortuous, helical courses within the medulla, subsequently dividing into numerous, spirally coiled smaller branches 462 M. 1. HOSSAIN AND J. D. O SHEA'

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Ak:..~~*r _ .. w . . 5 Fig. 5. Higher magnification of parts of Fig. 4. 0 V, ovary; O VA, ovarian artery; OV B O V A, ovarian branches of ovarian artery. x 2-4. which supplied the medullary region, and into larger, less convoluted branches which passed towards the cortex. These larger arteries commonly ran parallel to the ovarian surface, giving off smaller branches which supplied the cortical tissue and often formed anastomotic intra-ovarian arterial arcades with each other (Figs. 1, 2). Most of the arterial branches supplying the cortex were spirally coiled vessels of gradually diminishing diameter. The majority were given off radially from the arterial arcades described, or were the terminal branches of cortical arteries which did not form arcades. Graafian follicles and corpora lutea were generally supplied by single branches originating directly from the cortical arteries (Fig. 2). In the case of the corpus luteum, the branch at first became coiled. As it approached the luteal surface, it divided into two or more major vessels which ramified over the surface of the corpus luteum, producing a wreath of circumferential yessels encircling the entire surface of the corpus luteum (Figs. 2, 6). From various points on the surface these arteries sent smaller, radially oriented branches into the luteal tissue. Venous drainage Veins were less coiled than the corresponding arteries and showed less tendency to form anastomotic arcades (Fig. 7). A corpus luteum was generally drained by one or two major veins (Fig. 3); these issued from, and the supplying artery entered the same side of the corpus luteum. The veins then ran a relatively straight course to the medulla, where they fused with other veins, ultimately forming a single large channel running parallel to the hilar surface of the ovary, from which the main ovarian tributary of the ovarian vein arose (Fig. 3). Within the cortex and medulla, veins tended to form anastomoses, but arcade formations were less prominent than with the corresponding arteries. Vascular anatomy of ovary in guinea-pig 463

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6 ; E.sJ § ; 6> Fig. 6. Photograph of the Latex-injected specimen showing the relationship of the ovarian arteries and ovarian veins. RO VA, right ovarian artery; 0 V V, ovarian vein; D CL, dissected corpora lutea. x 2. Direction of bloodflow in utero-ovarian arterial anastomosis In all guinea-pigs receiving injections of Lissamine green, the direction of flow of dye-coloured blood was clearly visible under the dissecting microscope. The direction of flow was consistently towards the uterus in all animals at all stages of the cycle observed. Furthermore, flow was invariably in this caudal direction even after multiple small injections of dye, and was the same on both left and right sides of the body. DISCUSSION This study has demonstrated in the guinea-pig that the ovarian artery of each side, throughout its course, is in close apposition to, and sometimes coiled around, the ovarian vein which drains blood from both the uterus and ovary. This close anatomical relationship has been described previously in this species and several others (reviewed by Ginther, 1974). A correlation with a local uterine luteolytic mechanism has been reported by Ginther (1974), who points out that the artery and vein are less closely apposed in species lacking this local mechanism, for example the rabbit. However, the correlation may not be absolute, for a very similar relationship between ovarian artery and vein exists in several marsupials in which no uterine luteolytic mechanism is believed to occur (Lee & O'Shea, 1977). The prominent anastomosis between ovarian and uterine arteries in the guinea- pig, rat, hamster and other species has been reported previously (DelCampo & Ginther, 1972). This anastomosis provides the ovary with an alternative source of blood supply in the event of obstruction of the ovarian artery, and similarly to the uterus if instead the uterine artery should become blocked. 464 M. I. HOSSAIN AND J. D. O'SHEA

7 _ Ni t Fig. 7. Photograph of the Latex-injected specimen showing the origin and course of the ovarian vein. 0 V, ovary; OV B 0 V V, ovarian branches of ovarian vein; R CV V, right ovarian vein. x3.

No previous description of the anatomy of the intrinsic blood supply to the ovary of the guinea-pig is available. However, the results of the present study conform closely with earlier observations on human (Clark, 1900) and rat ovaries (Bassett, 1943). The single arterial supply to the corpus luteum indicates that the luteal arteries are commonly end arteries, and that their obstruction would lead to ischaemia of the entire corpus luteum. However, more proximally, the arteries form extensive anastomoses with one another, and it is only the distal part of the luteal artery close to the corpus luteum which is an end artery. Anastomosis is, of course, a characteristic feature of the ovarian artery itself and of its ovarian branches both within and without the ovary. The presence of anastomoses and of multiple branches supplying the ovary should ensure that the ovary as a whole is well protected against the occlusion of any single artery. The significance of coiling of the ovarian artery, which is a prominent feature in the guinea-pig both outside and inside the ovary, has been discussed in some detail by Reynolds (1973) who suggests several possible functional applications. These include (a) adaptability to changes in size of the ovary associated with growth of the Graafian follicle, ovulation and development of the corpus luteum, (b) regulation of the equalization of blood flow throughout the ovarian stroma, responding to localized changes in growth or function, such as ovulation and luteinization, (c) con- trol of ovarian blood pressure and stream lining of flow in the ovarian artery and its branches. By extrapolation from the situation in the tightly coiled spermatic arteries of mammals (Setchell, 1977), it might also be postulated that the marked coiling could produce a reduction in pulse pressure in the ovarian arterial blood. The direction of blood flow in the utero-ovarian arterial anastomosis in the guinea-pig was towards the uterus in all the animals studied. This is consistent with the findings of the microsphere study of Chaichareon et al. (1976), and uniform results have thus been obtained with two different methods. It is clear that under the conditions of these experiments involving cycling animals with or without functional corpora lutea, the entire ovarian blood supply is derived from the ovarian artery, with no uterine arterial contribution. The only features common to both these studies have been the use of general anaesthesia and some form of prior Vascular anatomy of ovary in guinea-pig 465 surgical intervention during the period of the experiment. In view of these factors, some caution is required concerning firm conclusions as to the direction of blood flow in the conscious animal. Nevertheless this functional study provides no support for the suggestion of DelCampo & Ginther (1972), based on anatomical considera- tions, that there is a uterine contribution to the ovarian blood supply.

SUMMARY Guinea-pig ovaries were supplied by paired ovarian arteries which on both sides were in close apposition to the corresponding ovarian veins along their course, and which formed end-to-end anastomoses with the cranial ends of the uterine arteries. A total of three or four spirally coiled branches of the ovarian artery entered the ovarian hilus and branched within the ovary to supply both cortex and medulla. Usually, each Graafian follicle and corpus luteum was supplied by a single arterial branch from one of the cortical vessels. The veins were less coiled than the arteries. One or occasionally two veins drained each corpus luteum. Several large veins draining each ovary joined to form a single ovarian vein, which finally joined the caudal vena cava on the right side and the left renal vein on the left side. The direc- tion of blood flow in the utero-ovarian arterial anastomosis was studied by direct visual observation in anaesthetised guinea-pigs; blood flowed towards the uterus in all observations on all guinea-pigs studied. The functional significance of the above observations is discussed. This work is part of a Ph.D. thesis submitted to the University of Melbourne by M. I. Hossain.

REFERENCES BASSETT, D. L. (1943). The changes in the vascular pattern of the ovary of the albino rat during the oestrous cycle. American Journal ofAnatomy 73, 251-291. BURR, J. H. & DAVIES, J. 1. (1951). The vascular system of the rabbit ovary and its relationship to ovulation. Anatomical Record 111, 273-294. CHAICHAREON, D. P., RANKIN, J. H. & GINTHER, 0. J. (1976). Factors which affect the relative contribu- tions of ovarian and uterine arteries to the blood supply of reproductive organs in guinea-pigs. Biology ofReproduction 15, 281-290. CLARK, J. G. (1900). The origin, development and degeneration of the blood vessels of the human ovary. Johns Hopkins Hospital Reports 9, 593-676. COOPER, G. & SCHILLER, A. L. (1975). Anatomy ofthe Guinea-pig. Cambridge, Mass.: Harvard University Press. DELCAMPO, C. H. & GINTHER, 0. J. (1972). Vascular anatomy of the uterus and ovaries and the unilateral luteolytic effect of the uterus: Guinea-pigs, rats, hamsters and rabbits. American Journal of Veterinary Research 33, 2561-2578. DELCAMPO, C. H. & GINTHER, 0. J. (1973). Vascular anatomy of the uterus and ovaries and unilateral luteolytic effect of the uterus: horses, sheep and swine. American Journal of Veterinary Research 34, 305-316. DELSON, B., LUBIN, S., BROOKLYN, N. Y. & REYNOLDS, S. R. M. (1949). Vascular patterns in the human ovary. American Journal of Obstetrics and Gynecology 57, 842-853. DELSON, B., LUBIN, S. & REYNOLDS, S. R. M. (1948). Spiral arteries in the human ovary. Endocrinology 42, 124-128. GINTHER, 0. J. (1974). Internal regulation of physiological processes through local venoarterial pathways. A view. Journal ofAnimal Science 39, 550-564. GINTHER, 0. J. & DELCAMPO, C. H. (1974). Vascular anatomy of the uterus and ovaries and the unilateral luteolytic effect of the uterus: Cattle. American Journal of Veterinary Research 35, 193-203. GINTHER, 0. J., DIERSCHKE, D. J., WALSH, S. W. & DELCAMPO, C. H. (1974). Anatomy of arteries and veins of uterus and ovaries in rhesus monkeys. Biology of Reproduction 11, 205-219. LEE, C. S. & O'SHEA, J. D. (1977). Observations on the vasculature of the reproductive tract in some Australian marsupials. Journal ofMorphology 154, 95-114. 466 M. I. HOSSAIN AND J. D. O'SHEA MossMAN, H. W. & DUKE, K. L. (1973). Comparative Morphology of the Mammalian Ovary. Madison, Wisconsin: University of Wisconsin Press. ORSINI, M. W. (1962). Technique of preparation, study and photography of benzyl-benzoate cleared material for embryological studies. Journal ofReproduction and Fertility 3, 283-287. O'SHEA, J. D. & LEE, C. S. (1974). Studies on the mechanism of prolongation of pseudo-pregnancy following section of utero-ovarian vascular connections in the rat. Australian Journal ofExperimental Biology and Medical Science 52, 265-270. POTrrER, G. E., JomEs, W. D. C. & HERMANN, C. L. (1958). The of the guinea-pig. Bioscience 29, 3-13. REYNOLDS, S. R. M. (1973). Blood and lymph vascular systems of the ovary. In Handbook ofPhysiology (ed. R. 0. Greep & E. B. Astwood), sect. 7, vol. II, part 1. Washington, D.C : American Physiological Society. SETCHELL, B. P. (1977). Male reproductive organs and semen. In Reproduction in Domestic Animals (ed. H. H. Cole & P. T. Cupps), 3rd ed. New York, San Francisco, London: Academic Press. SHIVELY, M. J. & STUMP, J. E. (1975). The systemic arterial pattern of the guinea-pig: The . Anatomical Record 182, 355-366.