Variations in Uterine Kallikrein during Cycle and Early Pregnancy in ...

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The authors are indebted to Maria Elena Ortiz for valuable advice and the ob- servation of the oocytes, to Rubia .... McRae AC, Heap RB. Uterine vascular ...
BIOLOGY OF REPRODUCTION 50, 1261-1264 (1994)

Variations in Uterine Kallikrein during Cycle and Early Pregnancy in the Rat' JENNY CORTHORN 3 and GLORIA VALDES' 2'3 4 Centro de Investigaciones Mddicas,3 Departamentosde Nefrologia y Obstetriciay Ginecologia4 Facultadde Medicina, Pontificia Universidad Cat6lica, Santiago, Chile ABSTRACT We have previously demonstrated the presence of tissue kallikrein and its mRNA in rat uterus, and an increase of the immunoreactive enzyme on Day 7 of gestation, which suggests a hormonal regulation and a role in implantation. This study pursued the sequential variations during the cycle and early pregnancy. During the estrous cycle, immunoreactive uterine kallikrein levels showed a recurrent pattern, with the highest value on proestrus (12.9 + 1.5 ng/uterus or 0.49 ± 0.03 ng/mg protein), and the lowest on metestrus (4.1 ng 0.5 ng or 0.30 ± 0.03 ng/mg protein);p < 0.05. During gestation, values on Day 1 (6.1 ± 0.4 ng/uterus or 0.30 ± 0.01 ng/mg protein) and Day 3 (4.9 ± 0.3 ng or 0.35 ± 0.01 ng/mg protein) were similar to levels during estrus and diestrus; a progressive rise, observed from Day 5 (8.2 ± 1.1 ng or 0.43 0.02 ng/ mg protein), attained the highest value on Day 7 (15.8 ± 1.7 ng or 0.78 ± 0.05 ng/mg protein); p < 0.05. The variations observed during the cycle and early gestation coincide with those described for ovarian steroids and uterine vasoactive changes, suggest the hormonal regulation of uterine kallikrein levels, and support its role in implantation.

INTRODUCTION

Tissue kallikreins are serine proteases that participate in blood flow regulation and through the generation of kinins in the processing of enzymes, hormones, and growth factors [1]. They belong to a multigene family, consisting in the rat of about 20 members, of which 13 have been identified; only in 6 have the corresponding proteins been described [2, 3]. Their regulation is tissue- and protease-specific, and has been related to estrogens in the pituitary gland, to androgens in the salivary glands, and to steroids and potassium in the kidney [2-8]. We have recently reported in rat uterus the presence of two kallikrein-like kinin-generating enzymes, with immunological reactivity and inhibition profiles identical to rat urinary kallikrein; and by finding mRNA for tissue kallikrein, we have confirmed the local synthesis. The increase of immunoreactive (IR) kallikrein on Day 7 of gestation suggests a hormonal dependence and a role in implantation [9]. In this study, we have extended our evaluation of kallikrein content to the whole estrous cycle and to early gestation, with the aim of associating any variations in uterine kallikrein levels with previously described changes in the pattern of hormonal regulation and in local blood flow and vascular permeability in the uterus during implantation. MATERIALS AND METHODS

Materials The following materials were obtained from Sigma Chemical Co. (St. Louis, MO): soybean trypsin inhibitor Accepted January 24, 1994. Received September 13, 1993. 'Financial support: Grant 749/92 from Fondo Nacional de Ciencia y Tecnologia (Fondecyt) and donation from Ricardo Claro. ZCorrespondence: Gloria Valdes, M.D., Centro de Investigaciones Medicas, Marcoleta 391, Santiago, Chile. FAX: (562) 6321924.

(SBTI), leupeptin, PMSF, gammaglobulin, polyethylene glycol (PEG), BSA, and Triton-X-100. Rabbit kallikrein antiserum was prepared by us as described [9]. Methods Female Sprague-Dawley rats, weighing 200-250 g, were housed in a light- and temperature-controlled room. Vaginal smears were taken daily, and after two consecutive 4day cycles, animals were assigned randomly to two experimental groups: cycling and pregnant. On the night of proestrus, the rats assigned to the second group were caged with fertile males. Spermatozoa found in the vaginal smear the following morning defined Day 1 of pregnancy. Tissue collection. Rats in the four days of the estrous cycle and in Days 1, 3, 5, 6, and 7 of pregnancy were killed, and uteri were collected between 1100 and 1300 h. The animals were anesthetized with nembutal (40 mg/kg i.p.), given injections of heparin (1000 U in 0.2 ml) into the femoral vein, and perfused via the abdominal aorta with cold saline until the uterine horns were completely free of visible blood. The uteri were removed, trimmed of adherent fat and mesometrium, blotted, and weighed; in Day 1 animals uteri were gently slit open and flushed with saline to remove blood and seminal fluid. The specimens were snapfrozen in liquid nitrogen and stored at -20 0C. In Day 3 uteri, pregnancy was confirmed by the observation, under low-power light microscopy, of ova obtained by flushing of the oviducts with saline; in Day 5, 6, and 7 uteri, pregnancy was confirmed at the time of dissection by the presence of implantation sites. Preparationof rat uterine homogenate. Tissues were homogenized as previously described [9], with some modifications. Briefly, tissues were quickly minced and homogenized in 8 ml/g of 20 mM TRIS/HCl buffer [10], pH 8.2, containing 25 mM EDTA, 5 mM PMSF, 2 M leupeptin, and 50 ig/ml SBTI with 3 pulses of 15 sec each with a Tis-

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suemizer homogenizer (Tekmar Co., Cincinnati, OH) in an ice bath. After centrifugation at 4500 x g for 5 min, the supernatant was treated with 0.05% (v/v) Triton X-100 at 4°C for 1 h and then centrifuged at 12 350 x g for 10 min; the supernatant was stored at -20 0C until assayed. Only blood-free homogenates were used. Recovery of added purified rat urinary kallikrein to minced uteri before homogenization was 104% (n = 6), as determined by RIA for tissue kallikrein. The final supernatant contained 96% (n = 8) of the total kallikrein extracted. Determination of IR kallikrein. Determination of IR kallikrein was performed by RIA with use of an antiserum raised in rabbits against purified female rat urinary kallikrein, as described previously for rat uterine homogenates [9], except that the standard rat urinary kallikrein curve was run in the presence of inhibitors and detergent, in amounts equal to those contained in the uterine samples. Cross-reactivity of the kallikrein antiserum was analyzed by RIA towards plasma kallikrein (60-480 Rg), partially purified rat urinary esterase A (12.5-500 jLg of protein), epidermal growth factor (0.1 ng-1 RIg), porcine pancreatic kallikrein (0.25-100 ILg), and partially purified rat kininogen (50-500 jIg); none of them showed significant cross-reaction. Analytical methods. Protein concentration was measured by the method of Lowry et al.[11], with BSA used as standard. All data are expressed as mean - SEM. Significance of differences between different days of cycle and gestation was determined with a one-way Student-NeumanKeuls analysis of variance, verified by the nonparametric Wilcoxon rank-sum test. Statistical significance was considered to bep - 0.05. RESULTS

During the estrous cycle, uterine IR kallikrein content expressed both as kallikrein per uterus and kallikrein per milligram protein was significantly increased in proestrus (12.9 ± 1.5 ng or 0.49 + 0.03 ng/mg protein); the lowest values were found for metestrus and diestrus (4.1 ng + 0.5 ng or 0.30 ± 0.03 ng/mg protein and 5.1 ± 0.5 ng or 0.30

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FIG. 1. Uterine IR kallikrein content expressed as ng/uterus (white bars) and ng/mg protein (black bars) during the estrous cycle. D, diestrus; P, proestrus; E, estrus; M, metestrus. *p < 0.05 for P vs. all other groups; #p < 0.05 for E vs. M; ##p < 0.05 for P vs. all other groups.

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FIG. 2. Uterine and ng/mg protein and 7 of pregnancy groups; *p < 0.05 groups.

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IR kallikrein content expressed as ng/uterus (white bars) (black bars) during proestrus (P) and Days 1, 3, 5, 6, (P1, P3, P5, P6, P7). #p < 0.05 for P and P7 vs. all other for P vs. P1 and P3; **p < 0.05 for P7 vs. all other

+ 0.03 ng/mg protein, respectively). Estrus showed intermediate values of 7.7 ± 0.7 ng or 0.39 ± 0.02 ng/mg protein (Fig. 1). Uterine weight was highest during proestrus (629 ± 80 mg) and lowest during metestrus and diestrus (356

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20 and 403 ± 20 mg); (p < 0.05).

On Days 1 and 3 of gestation, uterine IRkallikrein values resembled those for estrus and diestrus (6.1 + 0.4 and 4.9 + 0.3 ng or 0.30 + 0.01 and 0.35 ± 0.01 ng/mg protein, respectively). A progressive rise was observed on Days 5 (8.2 ± 1.1 ng or 0.43 ± 0.02), 6 (9.2 ± 0.8 ng or 0.43 ± 0.04 ng/mg protein), and 7 (15.8 ± 1.7 ng or 0.78 ± 0.05 ng/mg protein) (Fig. 2). The lowest uterine weight was found in Day 3 uteri (396 + 20 mg); from Days 5 to 7, weight tended to increase (467 ± 30 to 511 + 60 mg, n.s.). DISCUSSION

Tissue kallikreins are serine proteases that participate in blood flow regulation and through the generation of kinins in the processing of enzymes, hormones, and growth factors [1]. They are encoded by a multigene family composed of a series of closely related members, about 20 in the rat, of which 13 have been identified; only in 6 have the corresponding proteinases been characterized (tissue kallikrein [rK1], tonin [rK2], rK7, rK8, rK9, and rK10) [2,3]. These kallikrein proteinases are very similar; and since they share common epitopes, they are difficult to differentiate by polyclonal antisera, but they can be separated through their different electric charges by anion exchange chromatography. True tissue kallikrein is classically defined by its ability to generate kinins from kininogen, and by its resistance to SBTI and susceptibility to aprotinin inhibition [12]. In rat uterine homogenates, we have recently isolated and characterized two kinin-generating enzymes, with the immunological properties and the characteristic inhibition profile of purified rat urinary kallikrein [9]. One of them has a molecular mass (38-43 kDa) similar to that of true tissue kallikrein (rK1, 41 kDa), while the second has a higher molecular mass (120-125 kDa) and may represent another

RAT UTERINE KALLIKREIN IN CYCLE AND EARLY GESTATION

form of rK1, or a different enzyme of the kallikrein family very closely related to it. In rat uterus, J. Clements (unpublished data) has recently found the mRNA encoding rK1, rK3, rK7, and rK9. Of these four, the resistance to SBTI inhibition shown by the fractions we isolated permits us to rule out rK7 and rK9; the same characteristic also rules out rK2 and rK10 [13]. In this study, in which we quantified total uterine IR kallikrein content (the sum of both forms), we demonstrated that its levels show a recurrent pattern during the normal estrous cycle, and a rise in early pregnancy that attains its maximum at Day 7, the last day studied. During the estrous cycle, the changes in uterine IR kallikrein were similar to the variations described for uterine weight, DNA, and RNA, which are maximum during proestrus and minimum during diestrus [14]. All these changes could be expressions of estrogen regulation, since the two extreme values coincide with the high and low levels of estrogen attained in ovarian vein and systemic circulation [15,16]. Further support for an association between estrogens and kallikrein is provided by the vasoactive effects of estrogens [17-21] and by the fact that estrogen-induced vasodilation can be diminished by interfering with the generation of kinins [22]. Progesterone levels during the cycle show a peak on the evening of proestrus and another during metestrus [23], and are thus not connected with those here reported for IR kallikrein. During gestation, the rise observed from Day 5 onwards occurs after the estrogen peak [20] and coincides with the maintained early rise of progesterone from Days 5 to 9 [16, 24]. The associations of IR kallikrein levels found in this study with the reported levels of estrogen and progesterone during the cycle and pregnancy differ, suggesting a bimodal regulation. During gestation, the rise of IR kallikrein starting on Day 5 concurs with the first contact of the embryo with the uterine wall [16, 25]. The interaction of the embryo and the uterine epithelium seems to be necessary for implantation to proceed [26], and could also participate in the regulation of kallikrein levels. This study demonstrates the similarity between the variations described for blood flow and those of uterine kallikrein levels during cycle [27]. During early gestation, the IR kallikrein content rises steadily from Day 5 to Day 7, in a pattern that resembles the variations described for vascular permeability in this condition [28]. The possibility that the kallikrein-kinin system is involved in these changes is also supported by our previous finding of a preferential localization of kallikrein in the implantation nodes [9], coinciding with the zone where increased vascular permeability and blood flow has been described on Days 5 and 6 [29]. Exogenous bradykinin has a potent vasodilator effect on uterine blood flow [30,31]. Moreover, bradykinin induces gaps in capillary endothelium [32], such as those described in the implantation site, related to the increase in vascular

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permeability, the first sign of decidualization [26, 33, 34]. Apart from its direct effects on vasodilation and permeability, the kallikrein-kinin system increases the synthesis of prostaglandins by stimulating phosphoinositide hydrolysis and arachidonic acid release in human chorion, amnion, and decidual and stromal cells in vitro [35-37]. Kallikrein can also stimulate histamine release [38-40], such as has been described in decidualization [41]. Up to now the implantation reaction had been mainly attributed to histamine and prostaglandins [26, 41-47]. Our findings, added to the relationships among the kallikrein-kinin system, prostaglandin, and histamine, point to a cascade of these three interrelated vasodilator agents acting in the development of endometrial vascular permeability and decidual transformation, as has been described for inflammation [48]. Besides intervening in this vasoactive cascade, bradykinin may be one more of the growth factors involved in the hormone-dependent cell proliferation of the endometrium [49], since it is able to stimulate DNA synthesis in human endometrial stromal cells [50]. In conclusion, we believe that the changes in uterine IR kallikrein levels during the cycle and early gestation described here, and their coincidence with changes in uterine blood flow and vascular permeability, constitute a strong argument for relating the kallikrein-kinin system to the vasoactive changes of early gestation. ACKNOWLEDGMENTS The authors are indebted to Maria Elena Ortiz for valuable advice and the ob25 servation of the oocytes, to Rubia Pellegrini for the labeling of I-kallikrein, to Luis Villarroel for the statistical analysis, and to Maricarmen Berrios for excellent technical assistance.

REFERENCES 1. Bhoola KD, Figueroa CD, Worth K. Bioregulation of kinins, kallikreins, kininogens and kininases. Pharmacol Rev 1992; 44:1-80. 2. Clements JA The glandular kallikrein family of enzymes:tissue-specific expression and hormonal regulation. Endocr Rev 1989; 10:393-419. 3. Scicli GA, Carbini LA,Carretero OA The molecular biology of the kallikrein-kinin system: II. The rat gene family. J Hypertens 1993; 11:775-780. 4. Clements JA, Fuller P, MacNally M, Nikolaidis I, Funder J. Estrogen regulation of kallikrein gene expression in the rat anterior pituitary. Endocrinology 1986; 119:286-273. 5. Powers CA. Elevated glandular kallikrein in estrogen-induced pituitary tumors. Endocrinology 1987; 120:429-431. 6. Margolius HS, Chao J, Kaizu T. The effects of aldosterone and spironolactone on renal kallikrein in the rat. Clin Sci Mol Med 1976; 51:S279-S282. 7. Vio C, Figueroa CD. Evidence for a stimulatory effect of high potassium diet in renal kallikrein. Kidney Int 1987; 32:26-30. 8. Vald6s G, Vio CP, Montero J, Avendafio R. Potassium supplementation lowers blood pressure and increases urinary kallikrein in essential hypertensives. J Hum Hypertens 1991; 5:91-96. 9. Vald6s G, Corthorn J, Scicli GA, Gaete V, Soto J, Ortiz ME, Foradori A, Saed G. Uterine kallikrein in the early pregnant rat. Biol Reprod 1993; 49:802-808. 10. Colowick SP, Kaplan NO. Preparation of buffers for use in enzyme studies. Methods Enzymol 1955; 1:138-146. 11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reaction. J Biol Chem 1951; 193:265-275. 12. Vogel R. Kallikrein inhibitors. In: Erdos EG (ed.), Handbook of Experimental Pharmacology, Vol. 25 supplement. New York: Springer Verlag; 1979: 163-225. 13. Moreau T, Brillard-Bourdet M, BouhnikJ, Gauthier F. Protein products of the rat kallikrein gene family. J Biol Chem 1992; 267:10045-10051.

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14. Shelesniak ML, Tic L. Studies on the mechanism of decidualization. IV. Synthetic processes in the decidualizing uterus. Acta Endocrinol 1963; 42:465-472. 15. Shaikh AA Estrone and estradiol levels in the ovarian venous blood from rats during the estrous cycle and pregnancy. Biol Reprod 1971; 5:297-307. 16. Forcelledo ML, Vera R, Croxatto HB. Ovum transport in pregnant, pseudopregnant, and cycling rats and its relationship to estradiol and progesterone blood levels. Biol Reprod 1981; 24:760-765. 17. Szego C, Roberts S. Steroid action and interaction in uterine metabolism. Recent Prog Horm Res 1953; 8:419-469. 18. Kalman MS. The effect of estrogens on uterine blood flow in the rat. J Pharmacol Exp Ther 1958; 124:179-181. 19. Hechter O, Krohn L, Harris J. The effect of estrogens on the permeability of the uterine capillaries. Endocrinology 1968; 82:333-341. 20. Shelesniak MC, Kraicer PF, Zeilmaker GH. Studies on the mechanism of decidualization. I. The estrogen surge of pseudopregnancy and progravidity and its role in the process of decidualization. Acta Endocrinol 1963; 42:225-232. 21. Ham K, HurleyJV, Lopata A, Ryan GB. A combined isotopic and electron microscopic study of the response of the rat to exogenous estradiol. J Endocrinol 1970; 46:71-81. 22. Phaily S, Senior J. Inhibition of estrogen-induced increase in uterine blood flow in the rat. J Pharm Pharmacol 1978; 30:734-735. 23. Hashimoto I, Henricks DM, Anderson LL, Melampy RM. Progesterone and preg4-en-20a-ol-3-one in ovarian venous blood during various reproductive states in the rat. Endocrinology 1968; 82:333-341. 24. Pepe GP, Rothchild I. A comparative study of serum progesterone levels in pregnancy and various types of pseudopregnancy. Endocrinology 1974; 95:275-279. 25. Enders AC, Schlafke S. A morphological analysis of the early implantation stages in the rat. Am J Anat 1967; 120:185-226. 26. Abrahamson PA, Zom TMT. Implantation and decidualization in rodents. J Exp Zool 1993; 266:603-628. 27. Harvey C, Owen DAA. Changes in uterine and ovarian blood flow during the estrous cycle in rats. J Endocrinol 1976; 71:367-369. 28. Takemori K, Okamura H, Kanzaki H, Yoshida M, Konishi I. Scanning electron microscope study on corrosion cast of uterine vasculature during the first half of pregnancy. J Anat 1984; 138:163-173. 29. McRae AC, Heap RB. Uterine vascular permeability, blood flow and extracellular fluid space during implantation in rats. J Reprod Fertil 1988; 82:617-625. 30. Resnik R, Killam AP, Barton MD, Battaglia FC, Makowski EL, Meschia G. The effect of various vasoactive compounds upon the uterine vascular bed. Am J Obstet Gynecol 1976; 125:201-206. 31. Clark KE, Mills EG, Stys SJ, Seeds AE. Effects of vasoactive polypeptides on the uterine vasculature. Am J Obstet Gynecol 1981; 139:182-188. 32. Gabbiani G, Baddonel MC, Majno G. Intraarterial injections of histamine, serotonin, or bradykinin: a topographic study of vascular leakage. Proc Soc Exp Biol Med 1970; 135:447-452.

33. Psychoyos A. La reaction d6ciduale est precede de modifications prcoces de la perm6abilite capillaire de l'ut6rus. C R Soc Biol (Paris) 1960; 154:1384-1387. 34. Abrahamson P, Lundkvist O, Nilsson O. Ultrastructure of the endometrial blood vessels during implantation of the rat blastocyst. Tissue Res 1983; 229:269-280. 35. Acker GM, Pesty A Effects of fetal urinary corticosteroids, catecholamines and kallikrein in PGE2 synthesis in monolayer cultures of human amnion and chorionic cells. Prostaglandins Leukotrienes Essent Fatty Acids 1988; 34:135-140. 36. Bonney RC, BeesleyJC, Rahman C, Franks S. Arachidonic acid release and inositol lipid metabolism in response to bradykinin and related peptides in human decidual cells in vitro. Prostaglandins Leukotrienes Essent Fatty Acids. 1993; 48:253260. 37. Schrey MP, Holt J, Cornford P, Monaghan H, Al-Ubaidi F. Human decidua as a target for bradykinin and kallikrein. Phosphoinositide hydrolysis accompanies arachidonic acid release in uterine decidua cells in vitro. J Clin Endocrinol & Metab 1992; 74:426-435. 38. Amundsen E, Haugen PO. Synergistic action of kallikrein and phospholipase A on histamine release. Scand J Clin Lab Invest Suppl 1969; 107:95-96. 39. Johnson AR, Erdos EG. Release of histamine from mast cells by vasoactive peptides. Proc Soc Exp Biol Med 1973; 142:1252-1256. 40. Devillier P, Renoux M, Giroud GP, Regoli D. Peptides and histamine release from rat peritoneal mast cells. Eur J Pharmacol 1985; 117:89-96. 41. Shelesniak MC. A history of research in nidation. Ann NY Acad Sci 1986; 476:524. 42. Kennedy JG. Evidence for a role for prostaglandins in the initiation of blastocyst implantation in the rat. Biol Reprod 1977; 16:286-291. 43. Finn CA. Implantation, menstruation and inflammation. Biol Rev 1986; 61:313328. 44. Kennedy TG. Prostaglandins and uterine sensitization for the decidual reaction. Ann NY Acad Sci 1986; 476:43-48. 45. Parr M, Parr EL, Munaretto K, Martin C, Dey SK Immunohistological localization of prostaglandin synthase in the rat uterus and embryo during the peri-implantation period. Biol Reprod 1988; 38:333-343. 46. Kennedy TG, Martel D, Psychoyos A. Endometrial prostaglandin E2 binding: characterization in rats sensitized for decidual cell reaction and changes during pseudopregnancy. Biol Reprod 1983; 29:556-564. 47. Weitlauf HM. Biology of implantation. In Knobil E, Neill J (eds.), The Physiology of Reproduction. New York: Raven Press; 1988: 231-262. 48. Proud D, Kaplan AP. Kinin formation: mechanisms and role in inflammatory disorders. Annu Rev Immunol 1988; 6:49-83. 49. FindlayJK, Salamonsen lA Paracrine regulation of implantation and uterine function. In: Seppala M (ed.), Factors of Importance for Implantation. Bailliere's Clinical Obstetrics and Gynaecology. London: Bailliere Tindall; 1991: 117-131. 50. Endo T, Fulane H, Kanaya M, Mizunuma M, Fujii M, Yamamoto H, Tanaka S, Hashimoto M. Bombesin and bradykinin increase inositol phosphates and cytosolic free Ca+ + and stimulate DNA synthesis in human endometrial stromal cells. J Endocrinol 1991; 131:313-318.