Institute of Biological Science, Mitsui Pharmaceuticals Inc., , Nihonbashi, Chuo-ku, Tokyo 103, Japan

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1 Journal of Reproduction and Development, Vol. 43, No. 2, 1997 Enhancement of Number of Ovulations by Administration of a Pyrimidine Compound with Potentiating Activity of Angiogenesis in Mice Superovulated by PMSG and hcg Kazuya MATSUMOTO, Seiki HARAGUCHI, Kazuchika MIYOSHI, Akira AWAYA 1) and Eimei SATO Department of Reproductive and Developmental Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, and 1) Institute of Biological Science, Mitsui Pharmaceuticals Inc., , Nihonbashi, Chuo-ku, Tokyo 103, Japan Abstract. We evaluated the influence of the administration of a synthesized heterocyclic pyrimidine compound, 2-piperadino-6-methyl-5-oxo-5,6-dihydro(7H) pyrrolo-[3,4-d] pyrimidine (MS-818), which has promoting activity of basic fibroblast growth factor(bfgf)-induced angiogenesis, on the number of ovulations in mice (Jcl:ICR strain) superovulated with pregnant mare s serum gonadotropin (PMSG) and human chorionic gonadotropin (hcg). When injected with PMSG and hcg, ovulation failed to be induced in 13.5% of mice, but all of those injected with MS-818 in addition to PMSG and hcg, responded to the stimulation and superovulated. There was also a significant increase in superovulatory response in the mice injected with MS-818 compared with those injected with saline. The mean actual number of ovulations for mice injected with 0 (saline) or 50 µg of MS- 818 for 3 days was 13.1 ± 2.1 and 26.9 ± 4.2, respectively, indicating that MS-818 affected the actual number of ovulations. Subcutaneous injection of MS-818 resulted in more actual ovulations than intraperitoneal injection. There was no difference between the control (saline) and MS-818-injected groups in the proportion of ova fertilizing and cleaving to the blastocyst stage in vitro. Key words: Superovulation, Pyrimidine compound, MS-818, Angiogenesis, Mouse (J. Reprod. Dev. 43: , 1997) G onadotropins are administered to females to increase the number of eggs that are ovulated, i.e., to induce superovulation [1]. Pregnant mare s serum gonadotropin (PMSG) and human chorionic gonadotropin (hcg) are usually used for many species of animals, but differences among individuals in the number of ovulations are observed for example in superovulated mice, requiring the development of chemicals to induce a highly stable number of ovulations by potentiating the activity of the gonadotropins. Accepted for publication: February 4, 1997 Correspondence: E. Sato Development of the microvasculature in the mouse ovary has been studied by histological methods including vascular cast methods [2 6]. These studies show that there is a striking difference in the distribution of blood vessels to individual follicles. Smaller follicles mainly have vessels of large circumference with few branches. As the follicles grow, the number of follicular vessels increases remarkably and these vessels form multilayered, complex vascular networks surrounding follicles at ovulation [4, 7]. These morphological observations suggest that vascularization around the follicles and ovulation are linked and that angio-

2 138 MATSUMOTO et al. genic factors may potentiate the activity of gonadotropins via the proliferation of capillaries carrying hormones to target tissues. A newly synthesized pyrimidine compound has recently been found to promote angiogenesis induced by basic fibroblast growth factor (bfgf) in chorioallantoic membrane mode [8, 9]. In this study, we then investigated the effect of a synthesized heterocyclic pyrimidine compound, 2-piperadio-6-methyl-5-oxo-5,6-dihydro(7H) pyrrolo-[3,4- d] pyrimidine (MS-818) (Fig.1), on ovulation in mice after the administration of pregnant mare s serum gonadotropin (PMSG) and human chorionic gonadotropin (hcg). Materials and Methods Animals Mice were given a commercial diet (Oriental Kobo MF, Oriental Co., Japan) ad libitum and maintained at C in a 13(L):11(D) photoperiod, the middle of the dark period being set at midnight. Induction of ovulation Female mice (Jcl:ICR), days of age, were injected subcutaneously with 5 IU of PMSG (Sigma, St. Louis, MO) followed by an intraperitoneal injection of 5 IU of hcg (Sigma) at 48 h after the first injection. Thirty, 50 or 100 µg of MS-818 synthesized as described before [8] dissolved in saline or saline alone was also administered to the mice injected with PMSG and hcg for 3 or 8 consecutive days according to the timetable shown in Figs.2A and 2B. They were killed by cervical dislocation under ether anesthesia at hr after the injection of hcg to remove the oviducts, because ovulation occurred at 10 h after the hcg injection in our experiments [7]. The extramural portion of the oviduct was then blotted on filter paper to remove any blood. The distended portion identified in the oviduct, where the ovulated oocyte mass is located, was torn off in HTF medium by the means of 26-gauge needles, and the number of oocytes was counted. Fig. 1. Chemical structure of the pyrimidine compound (MS-818) having a promoting activity of angiogenesis induced by bfgf. In vitro fertilization Female mice(jcl:icr), days of age, were superovulated by subcutaneous injections of 5 IU PMSG, followed after 48 h with 5 IU hcg. MS-818 dissolved in saline, or saline alone was administered to mice injected with PMSG and hcg for 3 consecutive days (50 µg/day/mouse). Fifteen to 16 h after the hcg injection, the animals were killed by cervical dislocation. Cumulus-oocyte complexes were collected by tearing the ampulla wall of the oviducts and were transferred to a 200 µl drop of HTF medium [10]. Spermatozoa were obtained from 13- to 22-weekold males (Jcl:ICR) by extrusion from the slitted portion of the epididymal duct of the excised cauda epididymides and were directly introduced into 400 µl drops of HTF medium ( sperm/ µl). Small aliquots of sperm suspension, preincubated for 60 min at the original concentration, were transferred to 400 µl drops of HTF medium. Sperm were introduced into the fertilization medium at a sperm concentration of 250 sperm/µl after 2-h preincubation and were kept in a CO 2 incubator for min. Preloaded and preincubated eggs were then transferred to 200 µl drops of sperm suspension adjusted to 250 sperm/µl. Coincubation of eggs with sperm for 60 min was stopped by transferring to another HTF medium and culturing for 96 h. Statistical analysis Significant differences between values were determined by chi-square analysis.

3 A PYRIMIDINE COMPOUND AFFECTING OVULATION 139 Results Effect of MS-818 on the number of ovulations induced by gonadotropins When injected with PMSG (5 IU) and hcg (5 IU), ovulation failed to be induced in 13.5% of mice, all of those injected with MS-818 (30 µg/day/ mouse) in addition to PMSG and hcg, responded to stimulation and superovulated. In the first experiment, one hundred and twenty mice were placed at random into one of six treatment groups. In all groups, virgin female mice were injected subcutaneously with 5 IU of PMSG, followed 48 h later intraperitoneally by 5 IU of hcg, and given 0, 30, 50 or 100 µg of MS-818 per a day for 3 consecutive days subcutaneously or intraperitoneally, after which they were killed and the number of oocytes in the oviducts was recorded. There was a significant increase in superovulatory response in the mice injected with MS-818 compared with the mice injected with saline (control) (Fig. 2A). The mean actual number of ovulations for mice injected with 0 (saline) or 50 µg of MS-818 for 3 consecutive days was 13.1 ± 2.1 and 26.9 ± 4.2, respectively, indicating that MS-818 affected the actual number of ovulations. Subcutaneous injection of MS-818 resulted in a greater number of ovulations than intraperitoneal injection (Fig. 2A). In the second experiment, 30 µg of MS-818 was administered for 8 consecutive days according to the timetable shown in Fig. 2B. In this case also, the number of ovulations increased, but there was no significant difference between the number of ova ovulated by the mice injected with MS-818 for 3 and 8 consecutive days. Discussion The efficient induction of superovulation in mice depends on several variables [1]. The sexual maturity of the female is a major factor affecting the Fertilizability and early embryogenesis of ova superovulated by the administration of MS-818 As shown in Table 1, there was no difference between the groups in the proportion of ova fertilizing and cleaving to the morula/blastocyst stage in vitro, with 71.4% (saline) and 75.2% (MS-818; 50 µg/day for 3 days) of ova fertilizing, and on development, with 85.2% and 86.4% of the ova cleaving, and 41.7 and 38.6% of the ova developing to blastocysts, indicating that MS-818 has no deteriorative effect on eggs in fertilization and early embryogenesis. Fig. 2. Effect of MS-818 on the ovulation number in mice induced by PMSG and hcg. MS-818 was administered for 3 (A) or 8 (B) days according to the schedule shown underneath the figure. n=20. Each point is mean + S.D.

4 140 MATSUMOTO et al. Table 1. Fertilizability and early embryogenesis of ova superovulated following the administration of PMSG, hcg and MS-818 Dose No. of eggs No. of eggs No. (%) 2) of eggs developed to (µg/day) 1) examined fertilized (%) 2-cell 4-cell blastocysts (control) (71.4) (85.2) (59.1) (41.7) (75.2) (86.4) (55.7) (38.6) 1)Mice were injected subcutaneously with MS-818 according to the schedule shown in Fig. 2A. 2)No. of eggs developed to 2-cell, 4-cell and blastocyst stage/ No. of eggs fertilized 100. number of eggs that are superovulated. The nutritional status and health of a female can also affect follicular maturation: underweight and/or sick animals tend to be retarded in development and yield a reduced number of eggs after superovulation. The best age for superovulation of mice varies from strain to strain, but is usually between 3 and 5 weeks of age, during the prepubescent stage of development. The optimal age for the ICR female is 4 weeks [11]. We therefore used day-old healthy mice for the induction of ovulation. The recommended dose of PMSG for most strains is 5 IU injected intraperitoneally or subcutaneously. hcg is then administered to induce superovulation. Injections of 5IU are generally administered. The time that the PMSG and hcg are administered relative to each other and to the light-dark cycle of the mouse room will affect the number of eggs in superovulated mice. For most strains, a 42- to 48-h interval between the PMSG injection and the hcg injection has been found to be best in terms of egg yield. In this study hcg was therefore injected at 48 h after PMSG injection. Even in the supposed best condition to induce superovulation, however, a certain percentage of mice did not respond to the gonadotropin to induce ovulation in this study. Even when there was a response, the number of ovulation is quite small in some of the mice. It was therefore thought that it is important to find and develop some chemicals capable of potentiating the activity of gonadotropin. One strategy is to find chemicals which affect the proliferation of the microvascular network. Hormones secreted from the cells circulate via capillaries and reach the cells of the target organs. When capillaries around the cells of target organs are well vascularized, it is supposed that hormones easily reach the target cells. In the ovary, there is a striking difference in the distribution of blood vessels to individual follicles. As the follicles grow, the number of follicular vessels increases remarkably and these vessels form multilayered, complex vascular networks surrounding the follicles [7]. These morphological features suggest that microvascular networks and angiogenic factors affect oocyte and follicular growth and ovulation. In this study, we have shown that the supplementation of MS-818, one of the chemical enhancers of growth factor-induced angiogesis, for 3 consecutive days prior to ovulation increased the number of ovulations in the mice superovulated by PMSG and hcg. The mean number of ovulated oocytes in the groups injected with MS-818 was significantly higher than that in control group. The number in the group injected with MS-818 was almost double that in the control group. This study has also shown that ability in fertilization and early embryogenesis of eggs obtained from mice injected with MS-818 did not change, indicating that MS-818 has activities which enhance the number of ovulation in mice superovulated by PMSG and hcg. To our knowledge, this is the first report on the potentiation of the activity of gonadotropins by the chemical with potentiating the activity of angiogenesis. EGF and bfgf are produced by thecal and interstitial cells in the ovary [12], and in cell culture systems EGF and bfgf were shown to enhance the proliferation of vascular endothelial cells [13]. EGF and bfgf also effect neovascularization in vivo [14, 15]. When EGF and bfgf were tested in vivo, with a slow release form polymer composed of Elvax 40 implanted in the rabbit cornea, capillary proliferation was induced. EGF and bfgf are therefore considered to be those of the major an-

5 A PYRIMIDINE COMPOUND AFFECTING OVULATION 141 giogenic factors in the ovary. There is an example that exogenous compounds potentiate the angiogenic activity of EGF [15] indicating the possibility that MS-818 potentiates endogenous bfgf in inducing angiogenesis at the thecal layers and stimulating the growth of follicles and ovulation. Indeed, MS-818 potentiates the angiogenesis induced by bfgf with angiogenic activity in a dose-dependent manner [9] and activates macrophages which secrete angiogenic factors [9]. How MS-818 potentiates the activity of gonadotropins to stimulate follicular growth and/or to induce ovulation is therefore now being investigated. Acknowledgments This work was supported in part by grant Nos and from the Ministry of Education, Science and Culture, and a grant for a pioneering research project in biotechnology from the Ministry of Agriculture, Forestry and Fisheries, Japan. References 1. Hogan B, Beddington R, Costantini F, Lacy E. Manipulating the Mouse Embryo, A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press 1994; Kanzaki H, Okamura Y, Okuda Y, Takenaka A, Morimoto K, Nishimura T. Scanning electron microscopic study of rabbit ovarian follicle microvasculature using resin injection-corrosion casts. J Anat 1982; 134: Kitai H, Yoshimura Y, Wright KH, Santulli R, Wallach EE. Microvasculature of preovulatory follicles: comparison of in situ and in vitro perfused rat ovaries following stimulation of ovulation. Am J Obstet Gynecol 1985; 152: Takada S, Shimada T, Nakamura M, Mori H, Kigawa T. Vascular pattern of the mammalian ovary with special reference to the three-dimensional architecture of the spiral artery. Arch Histol Jpn 1987; 50: Murakami T, Ikebuchi Y, Ohtsuka A, Kikuta A, Taguchi T, Ohtani O. The blood vascular wreath of rat ovarian follicle, with special reference to its changes in ovulation and luteinization: a scanning electron microscopic study of corrosion casts. Arch Histol Cytol 1988; 51: Cavender JL, Murdoch WJ. Morphological studies of the microcirculatory system of periovulatory ovine follicles. Biol Reprod 1988; 39: Shimoda K, Sato E, Tanaka T, Takeya T, Toyoda Y. Morphological differentiation of the microvasculature during follicular development, ovulation and luteinization of mouse ovaries. Develop Growth Differ 1993; 35: Awaya A, Kobayashi H, Horikomi K, Tanaka S, Kabir AM, Yokoyama K, Ohno H, Kato K, Kitahara T, Tomino I, Isayama S, Nakamura S. Neurotropic pyrimidine heterocyclic compounds. I. The newly synthesized pyrimidine compounds promote neurite outgrowth of GOTO and neuro 2a neuroblastoma cell lines, and potentiate nerve growth factor (NGF)-induced neurite sprouting of PC 12 cells. Biol Pharm Bull 1993; 16: Yasuhara S, Kashiwagi S, Ito H, Awaya A. Neurotropic pyrimidine heterocyclic compounds promote the angiogenesis induced by b asic FGF. Clin Pharm Res 1996 (In press). 10. Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985; 44: Sato E, Ishibashi T. Relationship between fetal body weight and number of fetuses in Jcl:ICR mice following gonadotropin treatment. Jpn J Anim Reprod 1979; 25: Carson RS, Zhang Z, Hutchinson LA, Herington AC, Findlay JK. Growth factors in ovarian function. J Reprod Fert 1989; 86: Carpenter G. Epidermal growth factor. Annu Rev Biochem 1979; 48: Gospodarowicz D, Bialecki H, Thakral TK. The angiogenic activity of the fibroblast and epidermal growth factor. Exp Eye Res 1979; 28: Sato E, Tanaka T, Takeya T, Miyamoto H, Koide SS. Ovarian glycosaminoglycans potentiate angiogenic activity of epidermal growth factor in mice. Endocrinology 1991; 128: