00l3-7227/90/1261-0631$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 1 Printed in U.S.A.
Prolactin Inhibits Plasminogen Activator Activity in the Preovulatory Follicles YASUNORI YOSHIMURA, KUNIYUKI MARUYAMA, MAKOTO SHIRAKI, SEIJI KAWAKAMI, MINORU FUKUSHIMA, AND YUKIO NAKAMURA Department of Obsterics and Gynecology, Fujita-Gakuen Health University School of Medicine, Aichi, Japan 470-11; and the Department of Obstetrics and Gynecology, Kyorin University School of Medicine (Y.N.), Tokyo, Japan 181
ABSTRACT. The present study was designed to determine the effects of PRL on changes in morphology and plasminogen activator (PA) activity in the preovulatory follicles. Rabbit ovaries were perfused with hCG alone or with hCG plus at 10, 102, or 103 ng/ml. PRL at 103 ng/ml directly inhibited the degeneration and decomposition of surface epithelial cells induced by hCG exposure. The subsurface connective tissue was visualized by treatment with sodium dodecyl sulfate, which removed surface epithelial cells from the ovary, thereby exposing collagen fibrils and the basal lamina. Sodium dodecyl sulfate treatment revealed inhibition of connective tissue disruption at the apex of the follicle wall in PRL-treated ovaries. PA activity in mature
H
YPERPROLACTINEMIA can perturb human ovarian physiology at several levels, including follicular maturation and steroidogenesis, ovulation, the process of luteinization, and corpus luteum formation (1, 2). PRL may act on the hypothalamic-pituitary axis to suppress gonadotropin secretion or may act directly on the ovary to suppress follicular development (3-5). Several observations derived from the in vitro experiments have demonstrated that at the ovarian level, PRL interferes with follicle rupture induced by gonadotropin (6, 7). Furthermore, PRL has been shown to inhibit ovulation in the isolated in vitro perfused rabbit ovary by mechanisms independent of ovarian progesterone synthesis (7). These findings suggest that PRL may act by interfering with mechanical events within the ovary that are required for the rupture of mature Graafian follicles. Rat ovarian granulosa cell production of plasminogen activator (PA) correlates with ovulation (8, 9). Plasminogen is present in follicular fluid, and plasmin, the product of the interaction between PA and plasminogen, has been shown to weaken the wall of the follicle in vitro (10). Reich et at. (11) measured follicular levels of PA at
follicles in perfused rabbit ovaries exposed to hCG increased from 1.40 ± 0.08 to 28.4 ± 4.25 IU/g tissue after 4 h of perfusion. The addition of PRL to the perfusate inhibited the hCG-stimulated increase in intrafollicular PA activity in a dose-dependent fashion. Although at 7 h mature follicles treated by hCG alone showed greater intrafollicular PA activity than those treated with hCG plus PRL, this difference was not significant. These results suggest that PRL may act directly by interfering with mechanical events within the ovary that are required for the rupture of mature Graafian follicles, probably via the inhibition of intrafollicular tissue PA activity. (Endocrinology 126: 631-636, 1990)
the different stages of ovulation and confirmed that the preovulatory LH surge stimulated PA activity. We also have found a preovulatory increase in intrafollicular PA activity in mature ovulatory follicles. A failure to increase PA activity in immature follicles leads to impaired follicular development, resulting in reduced ovulatory efficiency (our unpublished observation). These results suggest that specific proteolytic events, including increased PA activity in mature follicles, allow folliculogenesis to proceed appropriately. The in vitro perfusion of viable rabbit ovaries makes it possible to monitor mammalian ovulation in a controlled environment (12-14). This ovarian perfusion can serve as an effective model for exploring the direct effect of various substances in the process of ovulation while eliminating the possibility of systemic influences. Experimetal conditions in which PRL inhibits hCG-induced ovulation in the rabbit ovary in vitro may provide important clues concerning the local intrafollicular cellular mechanism regulating the normal ovulatory processes. The present study was designed to determine the effects of PRL on changes in morphology and PA activity in preovulatory follicles.
Materials and Methods
Received July 26,1989. Address requests for reprints to: Yasunori Yoshimura, M.D., Department of Obstetrics and Gynecology, Fujita-Gakuen Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake, Toyoake City, Aichi, Japan 470-11.
Animals Thirty-nine sexually mature female Japanese White rabbits, weighing 3.0-3.5 kg, were isolated for a minimum of 3 weeks 631
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632
INHIBITORY EFFECTS OF PRL IN INTRAFOLLICULAR PA ACTIVITY
and used in all experiments. The rabbits were housed individually under controlled lighting (14 h of light, 10 h of darkness) and temperature and fed water and Purina rabbit chow (Clea Japan Inc., Tokyo, Japan) ad libitum. Rabbits were anesthetized with iv sodium pentobarbital (32 mg/kg), given heparin sulfate (120 U/kg) for anticoagulation, and then subjected to laparotomy. Ovaries were excluded from further study if they appeared immature or if 50% or more of the surface follicles appeared hemorrhagic. Ovarian perfusion Each ovarian artery was cannulated in situ after all major vascular connections had been ligated. The ovary, with its artery, vein, and supporting adipose tissue still attached, was removed from the rabbit and immediately placed in a perfusion chamber. The perfusion fluid consisted of a total of 150 ml medium 199 (Gibco, Grand Island, NY) containing 1% BSA (Sigma Chemical Co., St. Louis, MO), which was supplemented with heparin sulfate, insulin, streptomycin, and penicillin G, and adjusted to a pH of 7.4. The technique for cannulation and perfusion has been described in detail previously (15). In a modification of this technique, 1% BSA was added to the basic perfusion fluid to increase oncotic pressure and decrease edema formation (16). Experimental design Porcine PRL (USDA pPRL B-l, NIDDK; biological activity, 111 IU/mg) at a concentration of 10, 102, or 103 ng/ml was added to the perfusate of one ovary. The contralateral ovary of each rabbit was perfused simultaneously in a separate chamber with medium alone and served as a control. Thirty minutes after the onset of perfusion, 50 IU hCG (CH-446, Organon, Oss, The Netherlands; biological activity, 3830 IU/mg) were added to the perfusate of both ovaries. The first ovulation in this sytem occurred after approximately 7 h of exposure to hCG. The mean time interval from ovulatory stimulus to ovulation has been shown to be 8.34 ± 0.48 h after exposure to hCG (7). Ovulatory efficiency, defined as the percentage of mature follicles that proceeded to rupture, was about 80% in hCG-treated ovaries during 12 h of perfusion (7,12,14). Therefore, perfusion was terminated after 4 or 7 h of exposure to hCG in order to determine changes in the morphology and intrafollicular PA activity in the process of ovulation. Six rabbits were used for each time point of perfusion. Twelve unstimulated ovaries were obtained from six rabbits not treated with hCG. Perfusion medium was injected into the cannulated ovarian artery at a rate approximating normal blood flow to the ovary (1.5 ml/min). Perfusion was maintained for 10 min until blood had been flushed from the ovary. These ovaries served as 0 h controls for the study of follicular PA activity. Surface morphology of ovarian follicles Twelve rabbits were used to study the surface morpology of ovarian follicles. Perfused rabbit ovaries were removed immediately from the perfusion chamber and perfusion-fixed with 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4). After 10 min, the fixed ovaries were
Endo • 1990 Vol 126 • No 1
excised and immersed in fixative to await further processing for scanning electron microscopy (SEM). Ovarian subsurface morphology was examined in 12 ovaries treated with the detergent sodium dodecyl sulfate (SDS), according to the method described by Martin et al. (17,18). Perfused rabbit ovaries were immersed in 5% SDS in distilled water for 6-7 min and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 3 h. The fixed ovaries were treated with 0.02% deoxyribonuclease for 1 h at 20° C and then processed for examination by SEM. Fixed ovaries with or without SDS were dehydrated in a graded series of ethanol for 20 min each at 50%, 70%, 90%, 95%, and 100%. After dehydration, ovaries were placed in acetone for 30 min and transferred to amyl-acetate for an additional 30 min. All specimens were mounted on SEM stubs using carbon paint, dried using a critical point drying apparatus, and sputter-coated with a 100 A coating of gold palladium. These specimens were examined by SEM (JSM-840, Nippondenshi, Tokyo, Japan) operated at 5-15 kV. Preparation of the follicular extract Mature follicles were dissected from 12 unstimulated ovaries and 42 perfused rabbit ovaries 4 or 7 h after hCG administration. Tissue samples were treated in a homogenizer (Bio-Mixer, Kenis Scientific Inst., Tokyo, Japan) cooled in an ice water with 2 M KSCN solution, freshly adjusted to pH 7.75 with solid NaHCO3 using 1.0 ml KSCN solution/30 mg tissue, as previously described (19). After homogenization, the suspension was slowly shaken mechanically for 1 h at room temperature. The tissue suspension was centrifuged for 10 min at 2900 X g at 4 C. The supernatant was diluted with 7 vol 1 M hydrochloric acid to pH 1. After 30 min at room temperature the mixture was centrifuged, and the sediment was redissolved in 1 ml 1 M KSCN solution neutralized with solid NaHCO3. This solution was referred as the stock solution of the sample. PA acitivity assay Intrafollicular PA activity was determined using the Spectrolyse tissue PA (t-PA) activity assay kit produced by American Diagnostica Inc. (New York, NY). This assay is based on the functional parabolic rate assay described by Ranby et al. (20). The two-chain form of t-PA was prepared from Bowes' melanoma cells. Fibrinolytic activity, expressed in international units, was determined using a human melanoma t-PA reference standard (lot 831517 of the NIBSC, London, England). The specific activity of the t-PA standard in the present study was 74,600 IU/mg t-PA in the standard clotlysis assay. Glu plasminogen was prepared from fresh frozen human plasma by affinity chromatography on lysine-Sepharose, gel filtration, and ion exchange chromatography (21). To minimize plasmin and PA contamination, the preparation was treated with matrix-bound aprotinin and matrix-bound antibodies against human tissue PA. H-D-Norleucyl-hexahydro-tyrosyl-lysine-P-nitroanilide diacetate salt (H-D-Nle-HHT-Lys-pNA 2AcOH) was used as the chromogenic substrate for the amidolytic assay of plasmin and reactions in which plasmin was generated or consumed. The samples and the t-PA standard were added to a solution of the plasminogen. Soluble fibrin was added to the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 15:01 For personal use only. No other uses without permission. . All rights reserved.
INHIBITORY EFFECTS OF PRL IN INTRAFOLLICULAR PA ACTIVITY tissue activator reagent, followed immediately by mixing. After incubation for 20 min, the substrate was added to the reaction solution at 37 C in cuvettes. PA activity was determined by measuring the initial rate of the cleavage of the substrate spectrophotometrically at 405 nm (COBAS FARA, Roche, Basel, Switzerland). The standard curve for t-PA activity ranged from 0.2-3.0 IU/ml. Data regarding PA activity are presented as the mean ± SEM in at least 24 follicles derived from 6 ovaries. Statistical analysis was determined by Student's t test. The level of significance was established with P < 0.05.
Results Surface morphology of ovarian follicles
After 4 h of hCG exposure in vitro, the surface epithelial cells had become detached from the follicular apex, and the length and number of the microvilli on the remaining epithelial cells had decreased (Fig. la). In PRL-treated ovaries, most surface epithelial cells were spherical or polyhedral, with numerous tall microvilli (Fig. lc). However, the microvilli of these cells appeared thinner and less numerous than in in situ ovaries before hCG stimulation. After 7 h of exposure to hCG, the surface epithelial cells showed progressive degeneration (Fig. lb). At the apex, the cells had decomposed and had been sloughed off, thereby exposing the connective tissue of the basal lamina. In PRL-treated ovaries, apical surface epithelial cells exhibited a prominent loss of microvilli at their upper aspect (Fig. Id). The cells surrounding the apical region had fairly well preserved microvilli extending into the intercellular spaces. The apical epithelial cells demonstrated a flattened characteristics and appeared to adhere to each other, yielding a cobblestone
633
appearance. The detergent SDS is effective in removing surface epithelial cells in the rabbit ovary, allowing examination of the integrity and distribution of subepithelial tissues in the preovulatory follicle (Fig. 2). After 4 h of hCG exposure in vitro, SDS treatment revealed that the honeycomb-like architecture characteristics in the subsurface connective tissue had become flattened and resembled the basal lamina (Fig. 2a). Collagen fibrils of the tunica albuginea could be observed beneath the degenerated basal lamina at the apex of mature follicles. In PRL-treated ovaries, the honeycomb-like architecture remained thick, without any defects in any region of the follicle (Fig. 2c). After 7 h of hCG exposure, SDS treatment revealed prominent degeneration of the basal lamina, with large defects (Fig. 2b). The apical region was covered by bundles or wide bands of decomposed collagen fibrils in the tunica albuginea which could be seen beneath the degenerated basal lamina. In PRL-treated ovaries, however, the honeycomb-like architecture was preserved at the apex (Fig. 2d). Intrafollicular PA activity with or without PRL Exposure of the perfused rabbit ovaries to hCG increased PA activity in mature follicles from 1.40 ± 0.08 IU/g tissue in unstimulated ovaries to 28.4 ± 4.2 IU/g tissue after 4 h of perfusion (Fig. 3). Intrafollicular PA activity declined by 7 h after hCG administration, when the first in vitro ovulation occurred. PRL at 103 ng/ml inhibited hCG-stimulated PA activity significantly (P < 0.01) in mature follicles after 4 h of exposure to hCG.
FlG. 1. Surface morphology of ovarian follicles treated with or without PRL in the perfused rabbit ovary model, a, After 4 h of hCG exposure (original magnification, X2000); b, after 7 h of hCG exposure (X2000); c, after 4 h of hCG plus PRL exposure (X5000); d, after 7 h of hCG plus PRL exposure (X2000). Porcine PRL at 103 ng/ml was added to the perfusate of one ovary. The contralateral ovary of each rabbit was simultaneously perfused in a separate chamber with medium alone and served as a control. Thirty minutes after the onset of perfusion, 50 IU hCG were added to the perfusate of both ovaries. The perfusion was terminated 4 or 7 h after exposure to hCG to observe changes in the morphology.
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634
INHIBITORY EFFECTS OF PRL IN INTRAFOLLICULAR PA ACTIVITY
Endo • 1990 Voll26«Nol
FIG. 2. Changes in the morphology of SDS-treated ovaries with or without PRL in the perfused rabbit ovary model. Surface morphology was examined in the perfused ovaries after 4 or 7 h of hCG exposure, a, After 4 h of hCG exposure (original magnificaton, X1000); b, after 7 h of hCG exposure (X1500); c, after 4 h of hCG plus PRL exposure (X1000); d, after 7 h of hCG plus PRL exposure (X2000).
40
40i
Lt)hCG(50IU) ( I P C G (50IU) plus PRL(IOVml)
30-
CO
+1
30-
20-
o 10-
Ohr
4hr
7hr
•p
Vol. 126, No. 1 Printed in U.S.A.
Prolactin Inhibits Plasminogen Activator Activity in the Preovulatory Follicles YASUNORI YOSHIMURA, KUNIYUKI MARUYAMA, MAKOTO SHIRAKI, SEIJI KAWAKAMI, MINORU FUKUSHIMA, AND YUKIO NAKAMURA Department of Obsterics and Gynecology, Fujita-Gakuen Health University School of Medicine, Aichi, Japan 470-11; and the Department of Obstetrics and Gynecology, Kyorin University School of Medicine (Y.N.), Tokyo, Japan 181
ABSTRACT. The present study was designed to determine the effects of PRL on changes in morphology and plasminogen activator (PA) activity in the preovulatory follicles. Rabbit ovaries were perfused with hCG alone or with hCG plus at 10, 102, or 103 ng/ml. PRL at 103 ng/ml directly inhibited the degeneration and decomposition of surface epithelial cells induced by hCG exposure. The subsurface connective tissue was visualized by treatment with sodium dodecyl sulfate, which removed surface epithelial cells from the ovary, thereby exposing collagen fibrils and the basal lamina. Sodium dodecyl sulfate treatment revealed inhibition of connective tissue disruption at the apex of the follicle wall in PRL-treated ovaries. PA activity in mature
H
YPERPROLACTINEMIA can perturb human ovarian physiology at several levels, including follicular maturation and steroidogenesis, ovulation, the process of luteinization, and corpus luteum formation (1, 2). PRL may act on the hypothalamic-pituitary axis to suppress gonadotropin secretion or may act directly on the ovary to suppress follicular development (3-5). Several observations derived from the in vitro experiments have demonstrated that at the ovarian level, PRL interferes with follicle rupture induced by gonadotropin (6, 7). Furthermore, PRL has been shown to inhibit ovulation in the isolated in vitro perfused rabbit ovary by mechanisms independent of ovarian progesterone synthesis (7). These findings suggest that PRL may act by interfering with mechanical events within the ovary that are required for the rupture of mature Graafian follicles. Rat ovarian granulosa cell production of plasminogen activator (PA) correlates with ovulation (8, 9). Plasminogen is present in follicular fluid, and plasmin, the product of the interaction between PA and plasminogen, has been shown to weaken the wall of the follicle in vitro (10). Reich et at. (11) measured follicular levels of PA at
follicles in perfused rabbit ovaries exposed to hCG increased from 1.40 ± 0.08 to 28.4 ± 4.25 IU/g tissue after 4 h of perfusion. The addition of PRL to the perfusate inhibited the hCG-stimulated increase in intrafollicular PA activity in a dose-dependent fashion. Although at 7 h mature follicles treated by hCG alone showed greater intrafollicular PA activity than those treated with hCG plus PRL, this difference was not significant. These results suggest that PRL may act directly by interfering with mechanical events within the ovary that are required for the rupture of mature Graafian follicles, probably via the inhibition of intrafollicular tissue PA activity. (Endocrinology 126: 631-636, 1990)
the different stages of ovulation and confirmed that the preovulatory LH surge stimulated PA activity. We also have found a preovulatory increase in intrafollicular PA activity in mature ovulatory follicles. A failure to increase PA activity in immature follicles leads to impaired follicular development, resulting in reduced ovulatory efficiency (our unpublished observation). These results suggest that specific proteolytic events, including increased PA activity in mature follicles, allow folliculogenesis to proceed appropriately. The in vitro perfusion of viable rabbit ovaries makes it possible to monitor mammalian ovulation in a controlled environment (12-14). This ovarian perfusion can serve as an effective model for exploring the direct effect of various substances in the process of ovulation while eliminating the possibility of systemic influences. Experimetal conditions in which PRL inhibits hCG-induced ovulation in the rabbit ovary in vitro may provide important clues concerning the local intrafollicular cellular mechanism regulating the normal ovulatory processes. The present study was designed to determine the effects of PRL on changes in morphology and PA activity in preovulatory follicles.
Materials and Methods
Received July 26,1989. Address requests for reprints to: Yasunori Yoshimura, M.D., Department of Obstetrics and Gynecology, Fujita-Gakuen Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake, Toyoake City, Aichi, Japan 470-11.
Animals Thirty-nine sexually mature female Japanese White rabbits, weighing 3.0-3.5 kg, were isolated for a minimum of 3 weeks 631
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 15:01 For personal use only. No other uses without permission. . All rights reserved.
632
INHIBITORY EFFECTS OF PRL IN INTRAFOLLICULAR PA ACTIVITY
and used in all experiments. The rabbits were housed individually under controlled lighting (14 h of light, 10 h of darkness) and temperature and fed water and Purina rabbit chow (Clea Japan Inc., Tokyo, Japan) ad libitum. Rabbits were anesthetized with iv sodium pentobarbital (32 mg/kg), given heparin sulfate (120 U/kg) for anticoagulation, and then subjected to laparotomy. Ovaries were excluded from further study if they appeared immature or if 50% or more of the surface follicles appeared hemorrhagic. Ovarian perfusion Each ovarian artery was cannulated in situ after all major vascular connections had been ligated. The ovary, with its artery, vein, and supporting adipose tissue still attached, was removed from the rabbit and immediately placed in a perfusion chamber. The perfusion fluid consisted of a total of 150 ml medium 199 (Gibco, Grand Island, NY) containing 1% BSA (Sigma Chemical Co., St. Louis, MO), which was supplemented with heparin sulfate, insulin, streptomycin, and penicillin G, and adjusted to a pH of 7.4. The technique for cannulation and perfusion has been described in detail previously (15). In a modification of this technique, 1% BSA was added to the basic perfusion fluid to increase oncotic pressure and decrease edema formation (16). Experimental design Porcine PRL (USDA pPRL B-l, NIDDK; biological activity, 111 IU/mg) at a concentration of 10, 102, or 103 ng/ml was added to the perfusate of one ovary. The contralateral ovary of each rabbit was perfused simultaneously in a separate chamber with medium alone and served as a control. Thirty minutes after the onset of perfusion, 50 IU hCG (CH-446, Organon, Oss, The Netherlands; biological activity, 3830 IU/mg) were added to the perfusate of both ovaries. The first ovulation in this sytem occurred after approximately 7 h of exposure to hCG. The mean time interval from ovulatory stimulus to ovulation has been shown to be 8.34 ± 0.48 h after exposure to hCG (7). Ovulatory efficiency, defined as the percentage of mature follicles that proceeded to rupture, was about 80% in hCG-treated ovaries during 12 h of perfusion (7,12,14). Therefore, perfusion was terminated after 4 or 7 h of exposure to hCG in order to determine changes in the morphology and intrafollicular PA activity in the process of ovulation. Six rabbits were used for each time point of perfusion. Twelve unstimulated ovaries were obtained from six rabbits not treated with hCG. Perfusion medium was injected into the cannulated ovarian artery at a rate approximating normal blood flow to the ovary (1.5 ml/min). Perfusion was maintained for 10 min until blood had been flushed from the ovary. These ovaries served as 0 h controls for the study of follicular PA activity. Surface morphology of ovarian follicles Twelve rabbits were used to study the surface morpology of ovarian follicles. Perfused rabbit ovaries were removed immediately from the perfusion chamber and perfusion-fixed with 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4). After 10 min, the fixed ovaries were
Endo • 1990 Vol 126 • No 1
excised and immersed in fixative to await further processing for scanning electron microscopy (SEM). Ovarian subsurface morphology was examined in 12 ovaries treated with the detergent sodium dodecyl sulfate (SDS), according to the method described by Martin et al. (17,18). Perfused rabbit ovaries were immersed in 5% SDS in distilled water for 6-7 min and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 3 h. The fixed ovaries were treated with 0.02% deoxyribonuclease for 1 h at 20° C and then processed for examination by SEM. Fixed ovaries with or without SDS were dehydrated in a graded series of ethanol for 20 min each at 50%, 70%, 90%, 95%, and 100%. After dehydration, ovaries were placed in acetone for 30 min and transferred to amyl-acetate for an additional 30 min. All specimens were mounted on SEM stubs using carbon paint, dried using a critical point drying apparatus, and sputter-coated with a 100 A coating of gold palladium. These specimens were examined by SEM (JSM-840, Nippondenshi, Tokyo, Japan) operated at 5-15 kV. Preparation of the follicular extract Mature follicles were dissected from 12 unstimulated ovaries and 42 perfused rabbit ovaries 4 or 7 h after hCG administration. Tissue samples were treated in a homogenizer (Bio-Mixer, Kenis Scientific Inst., Tokyo, Japan) cooled in an ice water with 2 M KSCN solution, freshly adjusted to pH 7.75 with solid NaHCO3 using 1.0 ml KSCN solution/30 mg tissue, as previously described (19). After homogenization, the suspension was slowly shaken mechanically for 1 h at room temperature. The tissue suspension was centrifuged for 10 min at 2900 X g at 4 C. The supernatant was diluted with 7 vol 1 M hydrochloric acid to pH 1. After 30 min at room temperature the mixture was centrifuged, and the sediment was redissolved in 1 ml 1 M KSCN solution neutralized with solid NaHCO3. This solution was referred as the stock solution of the sample. PA acitivity assay Intrafollicular PA activity was determined using the Spectrolyse tissue PA (t-PA) activity assay kit produced by American Diagnostica Inc. (New York, NY). This assay is based on the functional parabolic rate assay described by Ranby et al. (20). The two-chain form of t-PA was prepared from Bowes' melanoma cells. Fibrinolytic activity, expressed in international units, was determined using a human melanoma t-PA reference standard (lot 831517 of the NIBSC, London, England). The specific activity of the t-PA standard in the present study was 74,600 IU/mg t-PA in the standard clotlysis assay. Glu plasminogen was prepared from fresh frozen human plasma by affinity chromatography on lysine-Sepharose, gel filtration, and ion exchange chromatography (21). To minimize plasmin and PA contamination, the preparation was treated with matrix-bound aprotinin and matrix-bound antibodies against human tissue PA. H-D-Norleucyl-hexahydro-tyrosyl-lysine-P-nitroanilide diacetate salt (H-D-Nle-HHT-Lys-pNA 2AcOH) was used as the chromogenic substrate for the amidolytic assay of plasmin and reactions in which plasmin was generated or consumed. The samples and the t-PA standard were added to a solution of the plasminogen. Soluble fibrin was added to the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 15:01 For personal use only. No other uses without permission. . All rights reserved.
INHIBITORY EFFECTS OF PRL IN INTRAFOLLICULAR PA ACTIVITY tissue activator reagent, followed immediately by mixing. After incubation for 20 min, the substrate was added to the reaction solution at 37 C in cuvettes. PA activity was determined by measuring the initial rate of the cleavage of the substrate spectrophotometrically at 405 nm (COBAS FARA, Roche, Basel, Switzerland). The standard curve for t-PA activity ranged from 0.2-3.0 IU/ml. Data regarding PA activity are presented as the mean ± SEM in at least 24 follicles derived from 6 ovaries. Statistical analysis was determined by Student's t test. The level of significance was established with P < 0.05.
Results Surface morphology of ovarian follicles
After 4 h of hCG exposure in vitro, the surface epithelial cells had become detached from the follicular apex, and the length and number of the microvilli on the remaining epithelial cells had decreased (Fig. la). In PRL-treated ovaries, most surface epithelial cells were spherical or polyhedral, with numerous tall microvilli (Fig. lc). However, the microvilli of these cells appeared thinner and less numerous than in in situ ovaries before hCG stimulation. After 7 h of exposure to hCG, the surface epithelial cells showed progressive degeneration (Fig. lb). At the apex, the cells had decomposed and had been sloughed off, thereby exposing the connective tissue of the basal lamina. In PRL-treated ovaries, apical surface epithelial cells exhibited a prominent loss of microvilli at their upper aspect (Fig. Id). The cells surrounding the apical region had fairly well preserved microvilli extending into the intercellular spaces. The apical epithelial cells demonstrated a flattened characteristics and appeared to adhere to each other, yielding a cobblestone
633
appearance. The detergent SDS is effective in removing surface epithelial cells in the rabbit ovary, allowing examination of the integrity and distribution of subepithelial tissues in the preovulatory follicle (Fig. 2). After 4 h of hCG exposure in vitro, SDS treatment revealed that the honeycomb-like architecture characteristics in the subsurface connective tissue had become flattened and resembled the basal lamina (Fig. 2a). Collagen fibrils of the tunica albuginea could be observed beneath the degenerated basal lamina at the apex of mature follicles. In PRL-treated ovaries, the honeycomb-like architecture remained thick, without any defects in any region of the follicle (Fig. 2c). After 7 h of hCG exposure, SDS treatment revealed prominent degeneration of the basal lamina, with large defects (Fig. 2b). The apical region was covered by bundles or wide bands of decomposed collagen fibrils in the tunica albuginea which could be seen beneath the degenerated basal lamina. In PRL-treated ovaries, however, the honeycomb-like architecture was preserved at the apex (Fig. 2d). Intrafollicular PA activity with or without PRL Exposure of the perfused rabbit ovaries to hCG increased PA activity in mature follicles from 1.40 ± 0.08 IU/g tissue in unstimulated ovaries to 28.4 ± 4.2 IU/g tissue after 4 h of perfusion (Fig. 3). Intrafollicular PA activity declined by 7 h after hCG administration, when the first in vitro ovulation occurred. PRL at 103 ng/ml inhibited hCG-stimulated PA activity significantly (P < 0.01) in mature follicles after 4 h of exposure to hCG.
FlG. 1. Surface morphology of ovarian follicles treated with or without PRL in the perfused rabbit ovary model, a, After 4 h of hCG exposure (original magnification, X2000); b, after 7 h of hCG exposure (X2000); c, after 4 h of hCG plus PRL exposure (X5000); d, after 7 h of hCG plus PRL exposure (X2000). Porcine PRL at 103 ng/ml was added to the perfusate of one ovary. The contralateral ovary of each rabbit was simultaneously perfused in a separate chamber with medium alone and served as a control. Thirty minutes after the onset of perfusion, 50 IU hCG were added to the perfusate of both ovaries. The perfusion was terminated 4 or 7 h after exposure to hCG to observe changes in the morphology.
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634
INHIBITORY EFFECTS OF PRL IN INTRAFOLLICULAR PA ACTIVITY
Endo • 1990 Voll26«Nol
FIG. 2. Changes in the morphology of SDS-treated ovaries with or without PRL in the perfused rabbit ovary model. Surface morphology was examined in the perfused ovaries after 4 or 7 h of hCG exposure, a, After 4 h of hCG exposure (original magnificaton, X1000); b, after 7 h of hCG exposure (X1500); c, after 4 h of hCG plus PRL exposure (X1000); d, after 7 h of hCG plus PRL exposure (X2000).
40
40i
Lt)hCG(50IU) ( I P C G (50IU) plus PRL(IOVml)
30-
CO
+1
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20-
o 10-
Ohr
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7hr
•p
