Neutral endopeptidase is expressed on the follicular granulosa cells of rabbit ovaries

Comparative Biochemistry and Physiology Part B 129 Ž2001. 863᎐870

Neutral endopeptidase is expressed on the follicular granulosa cells of rabbit ovaries Jacques Philippe Zappulla, Luc DesGroseillersU Department of Biochemistry, Uni¨ ersity of Montreal, P.O. Box 6128, Station Centre-¨ ille, Montreal Quebec, ´ Canada H3C 3J7 Received 8 January 2001; received in revised form 16 March 2001; accepted 19 March 2001

Abstract Neutral endopeptidase ŽNEP. is a zinc metallopeptidase ubiquitously distributed in various tissues in mammals. This peptidase is involved in the post-secretory metabolism of various neuropeptides and peptide hormones in vivo, such as enkephalins, bradykinin, atrial natriuretic peptide, substance P and endothelins. In this paper we show that NEP is expressed in ovaries as a 110-kDa glycosylated integral membrane protein with enzymatic properties similar to those of the kidney protein. Using immunohistochemistry, we localize the peptidase in the granulosa cells of follicles at all stages of maturation, with the exception of atretic follicles. We also observe immunoreactive staining in the epithelia that lines the blood vessels in the medulla and the surface of the ovary. The co-localization of NEP and bioactive peptides known to be physiological substrates of NEP in other tissues suggests an important role for this protein in processes such as follicle maturation, ovulation, andror regulation of ovarian blood flow, by modulating the physiological function of these peptides. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Neutral endopeptidase; Neprilysin; Ovary, Granulosa cells; Peptide degradation; Immunofluorescence; Enzyme kinetics; Localization

1. Introduction Although the central roles of gonadotrophins and of gonadal steroids in ovarian folliculogenesis are well accepted, additional ovarian systems are required for its modulation Žsee review by Roy, 1994.. Among the new ovarian regulators, neuropeptides have been the subject of increasingly intense investigations. Numerous observations inU

Corresponding author. Tel.: q1-514-343-5802; fax: q1514-343-2210. E-mail address: [email protected] ŽL. DesGroseillers ..

dicate that mammalian ovaries receive not only adrenergic but also peptidergic innervations. Neuropeptide Y ŽNPY. ŽPapka et al., 1985; McDonald et al., 1987., substance P ŽSP. ŽDees et al., 1985; Papka et al., 1985; Klein and Burden, 1988; Majewski et al., 1995., vasoactive intestinal polypeptide ŽVIP. ŽPapka et al., 1985; Dees et al., 1986; Klein and Burden, 1988; Majewski et al., 1995., enkephalins ŽKilpatrick et al., 1985., bradykinin ŽSmith and Perks, 1979., and angiotensin II ŽThomas and Sernia, 1990. have been located in immunoreactive fibres in ovaries. The fate of endogenous neuromodulatory peptides is commonly coupled with metabolic pepti-

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dases that ensure their rapid degradation and thus the termination of the signal they carry. In ovaries, membrane bound peptidases are thought to play important regulatory functions regarding follicular growth andror ovulation. Aminopeptidases have been localized in ovaries and implicated in gonadotrophin-stimulated ovulation in mice ŽFujiwara et al., 1992; Nakamura et al., 1996., in restoring follicular growth and ovulation suppressed by stress in mice ŽNakamura et al., 1998., and in enhancing progesterone and oestradiol secretion by porcine granulosa cells in certain conditions ŽTachibana et al., 1996.. The presence of endothelin-converting enzyme-1 ŽYoshioka et al., 1998; Korth et al., 1999; Karam et al., 1999., endopeptidase-24.15 ŽPierotti et al., 1991., dipeptidyl aminopeptidaseX-S ŽWada et al., 1992., dynorphin-processing enzyme ŽDevi, 1993., aminopeptidase P ŽHoltzman et al., 1987., and a soluble cysteine endopeptidase ŽSatoh et al., 1990. were also reported in mammalian ovaries. Recently, the presence of neutral endopeptidase ŽNEP, CALLA, EC 3.4.24.11, enkephalinase . in ovaries has been suggested by radioimmunoassay ŽRIA. ŽHowell et al., 1991.. NEP is a cell surface metalloendopeptidase ubiquitously distributed in mammals, that plays critical roles in various peptide-mediated physiological responses in vivo Žsee reviews by Roques et al., 1993 and by Kenny, 1993.. NEP was in particular shown to be implicated in the degradation of inflammatory peptides ŽShipp et al., 1990, 1991; Connelly et al., 1985; Martins et al., 1990; Kenny, 1993., endothelin ŽVijayaraghavan et al., 1990., LHRH ŽYang et al., 1998., enkephalins ŽRoques et al., 1980. and atrial natriuretic peptide ŽGros et al., 1989, 1990; Seymour et al., 1995; Thompson and Morice, 1996.. Although results of RIA suggested that NEP is present in ovaries, previous immunological experiments failed to detect this enzyme in this tissue ŽFujiwara et al., 1992.. It is thus important to document the presence of NEP in this tissue, considering the fact that many neuropeptides present in ovaries were shown to be natural endogenous substrates for NEP. In this study, we provide clear biochemical, enzymatic and immunological evidences for the presence of NEP in ovaries. Using the technique of immunofluorescence, we localized rabbit NEP at the surface of the follicular cells at all stages of folliculogenesis, and in endothelial cells of blood vessels. The localization of NEP at sites where peptides play

several functions on the ovarian cycle suggests that NEP may be a pivotal regulator of follicle maturation, ovulation andror vascular transport.

2. Materials and methods 2.1. Animals Sexually mature New Zealand White female rabbits weighing 3.5᎐4.5 kg were caged individually under controlled light and temperature and provided with water and a diet of Purina rabbit chow ŽRalston-Purina, St Louis, MO. ad libitum. Rabbits were anesthesized with i.v. sodium pentobarbital Ž32 mgrml., given heparin sulfate Ž120 unitsrkg. for anticoagulation, and then subjected to laparotomy. 2.2. Chemicals, reagents and antibodies 1-O-n-octyl ␤-D-glucopyranoside Žoctyl glucoside., phosphoramidon and amastatin came from Sigma Chemical Co. ŽSt. Louis, MO, USA.. Captopril was obtained from Squibb ŽPrinceton, NJ, USA.. Thiorphan was obtained from Dr Bernard P. Roques ŽUniversite ´ Rene´ Descartes, France.. The labelled substrate wTyr-3,5- 3 H xwD-Ala 2 , Leu 5 xenkephalin Ž50 Cirmmol. was obtained from Le Commissariat ` a l’Energie Atomique ŽGif-SurYvette, France.. Guinea-pig polyclonal antibodies ŽLemay et al., 1989. and mouse monoclonal 18B5 antibody ŽJalal et al., 1992., both raised against purified rabbit kidney NEP were a generous gift from Dr Philippe Crine ŽUniversite ´ de Montreal, ´ Canada.. Both antibodies are specific for NEP and do not recognize other members of the NEP family Žunpublished data and Ruchon et al., 2000.. Peroxidase-conjugated anti-Žguinea-pig immunoglobulins., anti-Žmouse immunoglobulins. were purchased from Dako Diagnostics Canada Inc. ŽMississauga, ON.. 2.3. Plasma membranes preparations and assay of enzymatic acti¨ ity Tissues from rabbits were dissected out, homogenized at 4⬚C with a Polytron homogenizer and centrifuged at 95 000 = g for 1 h at 4⬚C through a 41% Žwrv. sucrose cushion ŽMaeda et al., 1983.. The white interfacial band of plasma membranes was collected and pelleted by centrifugation at

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95 000 = g for 30 min at 4⬚C. The membrane pellet was solubilized for 1 h at 4⬚C in phosphate-buffered saline ŽPBS., pH 7.4, containing 1% Žwrv. octyl glucoside. The unsolubilized proteins were eliminated by centrifugation at 13 000 = g for 15 min at 4⬚C. The enzyme activity of solubilized NEP was measured as described previously ŽDion et al., 1993. using 50 nM of the tritiated substrate, in the presence of the aminopeptidase inhibitor amastatin at a concentration of 10 ␮M, and the angiotensin-converting enzyme inhibitor captopril at a concentration of 1 ␮M. Enzymatic assays were repeated four times in duplicates, with membrane preparations prepared from ovaries of four different rabbits. IC 50 were done twice in duplicates.

bodies were applied to the section at a 1:100 dilution in TPBSr1% NGSr0.2% saponin for 12᎐16 h in a humid chamber at 4⬚C. Slides were then washed six times in TPBS, and incubated with biotin conjugated anti-guinea pig IgG ŽDako, Carpinteria, CA. 1:100 in TPBSr1% NGSr0.2% saponin for a further 12᎐16 h at 4⬚C. Slides were washed six times in TPBS, revealed using the TSATM -Direct kit as recommended by the manufacturer ŽNEN TM Life Science Products, Boston, MA., and mounted in Immuno Floure Mounting Medium ŽICN Biomedicals Inc., Montreal, QC. for microscopic examination. Immunofluorescence experiments were performed on serial sections of ovaries isolated from two different rabbits.

2.4. Endoglycosidase digestions and immunoblotting

3. Results

Endoglycosidase digestions were performed by adding PNGase F directly to aliquots of solubilized membrane proteins according to the manufacturer’s instructions ŽNew England Biolabs Ltd., Missaussaga, ON.. The treated proteins were separated by electrophoresis on a 6% SDS-polyacrylamide gel under reducing conditions, and electrophoretically transferred to nitrocellulose filters. The blots were incubated with a monoclonal antibody against rabbit kidney NEP Ž18B5. that recognizes a conformational epitope on the extracellular domain of rabbit NEP, and developed using horseradish peroxidase-conjugated anti-mouse IgG antibodies ŽDako, Mississauga, ON. and the SuperSignal substrate ŽPierce, Rockford, IL. as recommended by the manufacturer. Western blotting has been performed three times, using ovaries from different rabbits.

3.1. Neutral endopeptidase is a membrane protein of rabbit o¨ aries

2.5. Immunocytochemistry Ovaries from two rabbits were dissected out and fixed for 2 h at room temperature in 2% paraformaldehyde in TPS Ž40 mM Tris᎐HCl, 8 mM Na 2 HPO4 ⭈ 7H 2 O, 3 mM KH 2 PO4 . before being frozen. Frozen sections were brought to room temperature and post-fixed in 4% paraformaldehyde in TPBS Ž40 mM Tris᎐HCl, 120 mM NaCl, 8 mM Na 2 HPO4 ⭈ 7H 2 O, 3 mM KH 2 PO4 . for 1 h. Slides were washed three times in TPBS and blocked 1 h in 10% normal goat serum ŽNGS. in TPBS containing 0.2% saponin for permeabilization. Anti-NEP polyclonal anti-

To determine whether NEP is expressed in ovaries, we first performed a Western blot with protein extracts isolated from rabbit ovary plasma membranes ŽFig. 1.. Using a monoclonal antibody Ž18B5. directed against rabbit NEP, we detected a protein band of approximately 110 kDa. Since the size of the immunoreactive component was higher than the 94 kDa of the NEP isolated from kidneys, we tested whether NEP is differentially glycosylated in ovaries, as compared to kidneys. Removal of N-linked sugar moieties with PNGase F produced a polypeptide with an apparent molecular mass of 85 kDa, which is the size that is also observed for the deglycosylated kidney enzyme ŽFig. 1.. This demonstrates that the NEP gene is expressed in both tissues and that NEP is differentially glycosylated in kidneys and ovaries. The amount of NEP protein is much lower in the ovaries compared to that found in the kidneys, because for our Western analysis, 10-fold more protein was used Ž5 vs. 50 ␮g for kidney vs. ovary protein. and the exposure time was 15 times longer for the ovary compared with the kidney blot. 3.2. Enzymatic acti¨ ity of NEP expressed in rabbit o¨ aries To determine whether the ovary NEP has the same enzymatic activity as the kidney NEP, we

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J.P. Zappulla, L. DesGroseillers r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 863᎐870

Fig. 1. Western blot analysis of the expression of NEP in rabbit ovaries. Proteins Ž50 ␮g of ovarian proteins, and 5 ␮g of kidney cortex proteins. from octyl glucoside-solubilized membrane preparations were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane and analysed by Western blotting with monoclonal anti-NEP antibodies. ŽF., protein extracts digested with PNGase F; Žy. untreated preparations. The blot with ovary proteins was exposed 15 times longer than the one with kidney proteins.

prepared plasma membranes from ovaries and used them to determine the NEP kinetic parameters. Using wTyr-3,5- 3 HxwD-Ala2 ,Leu 5 xenkephalin as a substrate, we showed that ovary membranes cleave the substrate at a rate of 0.0078 pmolrŽmin mg. of protein, which is weak as compared to the 1.6 pmolrŽmin mg. previously reported for kidney cortex membranes ŽAubry et al., 1987.. However, this is consistent with the low level of expression of NEP in ovaries ŽFig. 1.. This activity was sensitive to two NEP inhibitors: phosphoramidon and thiorphan. The respective IC 50 for phosphoramidon and thiorphan were 18.63" 2.24 nM and 2.97" 0.12 nM, which concur with the values of 8 " 2 nM and 2.1" 1.2 nM described for the enzyme purified from rabbit kidneys ŽLemay et al., 1990; Fossiez et al., 1992.. Altogether our results strongly support the fact that rabbit ovary NEP has catalytic properties similar to those of the kidney enzyme. 3.3. Immunolocalization of NEP in rabbit o¨ aries To pinpoint the location of NEP in ovaries more precisely, we performed immunofluorescence experiments using polyclonal anti-NEP antibodies. As control, serial sections were stained by pre-immune serum. Rabbits are reflex ovulators and the state of their ovaries is uncontrolled.

In mature rabbit ovaries, positive staining was clearly observed in follicles at all stages of folliculogenesis, from the primary to the Graafian follicles. In the follicles, the signal was observed on the granulosa cells and on the cells of the corona radiata, but not on the thecal cells ŽFigs. 2 and 3c,d.. A line of staining was visible approximately where the oocyte membrane is expected ŽFig. 3c.. Interestingly, several atretic follicles found in the stroma were clearly negative for NEP, suggesting that the expression of NEP is repressed in these cells once the follicles enter this phase of regression ŽFig. 2.. The primordial follicles located near the outer surface Žcortex. of the organ, were

Fig. 2. Immunolocalization of neutral endopeptidase in rabbit ovaries. The montage of this picture is from 8 adjacent areas, fixed with paraformaldehyde and treated with polyclonal antiNEP antibodies. Ža. Rabbit ovary sections stained with hematoxylin and eosin. Žb. Same sections labeled by polyclonal anti-NEP antibodies. Abbreviations: Gc, granulosa cells of follicles at all stages of maturation; Th, thecal cells; Oo, oocyte; Žv, e. endothelium of blood vessels in the medulla and of ovarian cell surface, respectively; atf, atretic follicles; pf, primary follicles; Gf, Graffian follicle. Arrow heads indicate the position of atretic follicles, which are not stained. Magnification: 100 = .

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Fig. 3. NEP is a peptidase expressed in the granulosa cells in the rabbit ovaries. Ža. Rabbit ovary section labeled by polyclonal anti-NEP antibodies. A secondary follicle, and the ovarian cell surface epithelium are clearly stained. Magnification: 100 = . Žb. Serial section stained by pre-immune serum. Magnification: 100 = . Žc. Graffian follicle labeled by polyclonal anti-NEP antibodies. Magnification: 200 = . Žd. Higher magnification showing the granulosa cells of a Graffian follicle. Magnification: 400 = . Abbreviations: Gc, granulosa cells; Th, thecal cells; Oo, oocyte; An, antrum. The arrows indicate the position of secondary follicle, the arrowheads the position of primary follicles, and square brackets the ovarian cell surface.

stained in the absence ŽFig. 3b. or presence ŽFig. 3a. of the primary anti-NEP antibodies, precluding any conclusion as to whether they express NEP or not. The mesenchyme is not stained ŽFig. 2.. However, the vasculature in the medulla shows clear staining, suggesting that the endothelial cells composing the ovarian vasculature express NEP ŽFig. 2.. This staining was not seen with the pre-immune serum. Finally the surface epithelium of the ovary is also highly positive by immunostaining suggesting that the epithelial cells surrounding the ovary express the neutral endopeptidase ŽFigs. 2 and 3a,b..

4. Discussion In this paper, we provide biochemical, enzymatic and immunocytochemical evidence that NEP is expressed in ovaries. We showed that the protein is present in plasma membranes, and degrades enkephalin with the same inhibitor pro-

file as the kidney NEP. However, the protein is differentially glycosylated as compared to kidney NEP. Differential glycosylation of NEPs in various human tissues was described before, and this phenomenon does not change the enzymatic parameters of the proteins ŽRoques et al., 1993.. The rates of degradation of enkephalin by plasma membranes, and the strength of the signals observed on Western blot, are both consistent with the fact that NEP is approximately 200 times less abundant in ovaries than in the kidney. This may reflect a low transcription rate of the NEP gene in ovaries or the fact that NEP is mainly found on the granulosa cells of healthy follicles, and they make up a relatively small fraction of the ovary. In contrast, NEP is widely distributed in the brush border epithelium of the kidney proximal tubules ŽRoques et al., 1993.. At the cellular level, we localized NEP on the granulosa cells ŽGCs. of the follicles, the blood vessels and the external epithelium. The ovarian compartments where NEP is expressed are known to be critical targets for mitogenic and differentiation-induction peptides. Interestingly most of

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these peptides are in vivo substrates for NEP in other mammalian tissues ŽRoques et al., 1993.. The presence of NEP in a peptidergic pathway that controls some of the ovarian functions is likely to modulate the level of the colocalized peptideŽs. and consequently itsŽtheir. physiological roleŽs.. The expression of NEP on the GCs is likely to modulate the levels of endothelin 1 ŽET1., VIP, angiotensin II andror BK, which all play significant roles in GCs. ET-1 regulates the proliferation and differentiation of GCs ŽTedeschi et al., 1992; Kamada et al., 1995., while VIP modulates many aspects of granulosa cell function, and prevents GC apoptosis and follicular atresia in the absence of gonadotropin influence ŽFlaws et al., 1995.. For its part, angiotensin II induces ovulation and oocyte maturation via the AT2 receptor subtype present on GCs ŽPellicer et al., 1988; Yoshimura et al., 1996.. Finally, BK is involved in the process of follicle rupture ŽYoshimura et al., 1988., a phenomenon similar to plasma extravasation that occurs on the earliest steps of inflammation ŽEspey, 1994.. This similarity is interesting since studies of NEP knockout mice clearly established that NEP is involved in the regulation of basal microvascular permeability mediated by SP and BK ŽLu et al., 1997.. Therefore, NEP is probably one of the numerous regulators of follicle development, oocyte selection for fertilization, ovulation, angiogenesis andror prostaglandin production. The inhibition of NEP in ovaries with specific inhibitors will be necessary to determine its exact site of action, as reported recently for an aminopeptidase activity ŽNakamura et al., 1996, 1998.. Neuropeptides are critical modulators of ovarian physiology. A better knowledge of their expression, the dynamics of their receptors and of their degradative enzymes is crucial for a better understanding of folliculogenesis and ovulation. The fact that a large number of these molecules which were shown to be in vivo substrates for NEP, were also shown to be expressed in the same cells as this enzyme in the ovaries, strongly suggests that NEP could be an important regulator of their actions. However, NEPyry mice are fertile ŽLu et al., 1995.. A detailed examination of the ovarian function and studies of other peptidases whose overexpression in ovaries could have compensated the absence of NEP in NEPyry mice, will be critical to understand the relative importance of this enzyme in vivo.

Acknowledgements The authors wish to thank Dr Philippe Crine and Dr Guy Boileau for the generous gifts of anti-NEP antibodies and Louise Wickham for critical reading of the manuscript. We would also like to acknowledge the skilled technical assistance of Marie-Eve Lane, Mireille Fyfe, Nadia Bonvouloir and Jeanne Lavoie. This research was supported by FCAR ŽFonds pour la Formation des Chercheurs et l’Aide ` a la Recherche. grant to L.D.G. References Aubry, M., Berteloot, A., Beaumont, A., Roques, B.P., Crine, P., 1987. The use of monoclonal antibody for the rapid purification of kidney neutral endopeptidase Ž‘enkephalinase’. solubilized in octyl glucoside. Biochem. Cell Biol. 65, 398᎐404. Connelly, J.C., Skidgel, R.A., Schulz, W.W., Johnson, A.R., Erdos, E.G., 1985. Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide. Proc. Natl. Acad. Sci. USA 82, 8737᎐8741. Dees, W.L., Kozlowski, G.P., Dey, R., Ojeda, S.R., 1985. Evidence for the existence of substance P in the prepubertal rat ovary. II. Immunocytochemical localization. Biol. Reprod. 33, 471᎐476. Dees, W.L., Ahmed, C.E., Ojeda, S.R., 1986. Substance P- and vasoactive intestinal peptide-containing fibers reach the ovary by independent routes. Endocrinology 119, 638᎐641. Devi, L., 1993. Tissue distribution of a dynorphinprocessing endopeptidase. Endocrinology 132, 1139᎐1144. Dion, N., Le Moual, H., Crine, P., Boileau, G., 1993. Kinetic evidence that His-711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state. FEBS Lett. 318, 301᎐304. Espey, L.L., 1994. Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction. Biol. Reprod. 50, 233᎐238. Flaws, J.A., DeSanti, A., Tilly, K.I. et al., 1995. Vasoactive intestinal peptide-mediated suppression of apoptosis in the ovary: potential mechanisms of action and evidence of a conserved antiatretogenic role through evolution. Endocrinology 136, 4351᎐4359. Fossiez, F., Lemay, G., Labonte, ´ N., Parmentier-Lesage, F., Boileau, G., Crine, P., 1992. Secretion of a functional soluble form of neutral endopeptidase-24.11 from baculovirus-infected insect cell line. Biochem. J. 284, 53᎐59. Fujiwara, H., Maeda, M., Imai, K. et al., 1992. Differential expression of aminopeptidase-N on human ovarian granulosa and theca cells. J. Clin. Endocrinol. Metab. 74, 91᎐95.

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