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The effects of gonadotropins on metabolism of isolated rat granulosa cells
THE EFFECTS OF GONADOTROPINS ON METABOLISM OF ISOLATED RAT GRANULOSA CELLS Darhl Foreman, Florence Dusek and Beth Overmoyer Department of Biology, Case Western Reserve University, Cleveland, Ohio (Received in final form July 9, 1984)
ry FSH in v i t r o , but not LH, increased the 02 uptake of isolated granulosa cells f r o m ' - 2 ~ old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the c e l l u l a r binding of FSH was highest. LH increased the ~ uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise~in in 02 O?uptake when the rats were implanted with DES capsules unless FSH was injected-to induce LH ;. Addition of dbcAMP in v i t r o Inm 30 day old rats at concentrations creased 02 uptake of granulosa IOX lower than those required t< e 02 uptake in cells from 23 day old rats treated with DES alone. FSH in v i t r o increased lactate formation in the absence of added substrates but did id not do so when glucose was added to th, the media. In contrast, LH greatly increased ncreased lactate formation with added gllucose. Dose response studies showed d that less than 0.6 ug/ml LH $21 was effective e in increasing control levels. These data suggest that th~ FSH affects aerobic )rocess of the differene LH affects anaerobic pathways in the pr anulosa cells toward luteal cells. I t is well known that FSH and LH interact with their target cells in the ovary by binding to specific receptors and that FSH stimulates LH-receptor production (1). Receptor binding by either hormone activates adenylate cyclase (2) raising cyclic adenosine monosphosphate (cAMP) levels (3) and increasing protein kinase a c t i v i t y (4). Such changes probably trigger changes in the major metabolic pathways that support f o l l i c u l a r development because cells of corpora lutea have glycogen (5) which is not present in f o l l i c u l a r granulosa cells (6-9). Several studies suggest that FSH and LH may regulate cesses in the ovary. LH increases lacta 1,12) and also increases the uptake of g n uptake in chick ovaries (14), rat ovari~ However, only one study has been done rger (17) has reported that FSH increase( of immature rats while LH increased the Since granulosa cells from immature rat~ while theca cells have LH receptors the mones appear unclear unclear. The present studies were undertaken to more accurately characterize the actions of FSH, LH, and dibutyryl cAMP (dbcAMP) on the oxygen uptake of isolated granulosa cells and remaining tissues of immature ovaries and to determine the effects of FSH and LH on the production of lactate by granulosa cells.
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press I
Methods Reagents and hormones. NIH FSH B1,$1,$4,$12 and S14 were i n j e c t e d in doses e q u i v a l e n t to FSH $1 (1 mg = 1U). NIH FSH $4 and $12 were equivalent to $I , NIH FSH BI was 0.4U/mg and $14 was 9U/mg. The doses given were 100 ug $1, $4 and $12 and 11.11ug $14. The hormones used i n v i t r o were p u r i f i e d prepara t i o n s of ovine FSH (FSH G4-150C, 50U/mg) and o~ne--L-IT-(LH G3-222B, 2.75x NIH LH SI) both prepared by Dr. Harold Papkoff. NIH LH 21 (2.5x NIH LH SI) was used f o r a l l l a c t a t e s t u d i e s , l o d i n a t e d oFSH (B900) and u n l a b e l l e d oFSH (B882), generously supplied by Dr. A. R. M i d g l e y , J r . were used i n the b i n d i n g DES, dbcAMP and t h e o p h i l l i n e were purchased from the Sigma studies. Chemical Co Medical grade Si bing was purchased from Dow Corning, Midland, MI. Silicone coating Electric Drifilm) for glassware was purchased from Pierce Chemical Ill
E2'
Animals and treatments. Immature r a t s of 3 cage-bred s t r a i n s were used in these experiments. Twenty-three or 28 day old r a t s were implanted w i t h i0 mm S i l a s t i c capsules c o n t a i n i n g DES or Ep and e i t h e r autopsied a t 48 hours or at v a r i o u s i n t e r v a l s f o l l o w i n g FSH t r e a t m e n t . Thus, 23 day old r a t s received DES capsules w i t h autopsy at 48 hours (Group I ) , DES capsules and FSH at 36 hours with autopsy at 48 hours , DES capsules and FSH at 36, 48 and 60 hours with autopsy at 72 hou I l l ) , DES capsules and FSH at 36, 48, 60 and 72 hours with autopsy a~ (Group IV). Sixteen rats implanted with DES capsules UI~ W were ~r'~ injected i r l j ~ u w1 U I d L otal ULdl of 600 (8 rats) or 700 (8 rats) ug FSH (Groups V and Vl). Thirty day old rats were used to determine the effects of FSH and LH on oxygen uptake and lactate production. They were implanted with 10 mm capsules containing E2 alone or al so injected with FSH at 36, 48 and 60 hours with autopsy at 72 hours.
t of cells and tissues. Isolated granulo~ ranulosa cells were i n i t i a l l y echanically expressing the cells d i r e c t l y into Krebs buffer with 8) but a greater y i e l d was obtained by the method of Campbell (19) of putting the ovaries f i r s t into 6.8M EGTA in 0.1M phosphate buffered saline pH 7.2 (PBS) for 15 min. then in O.5M sucrose in PBS for 15 min. before ~xpressing the cells into Krebs buffer with substrates (0.015M glucose, 0.036M pyruvate, O.07M fumarate, 0.039M glutamate). The substrate used for lactate studies was 0.27M glucose to prevent glucose from l i m i t i n g the reaction (18). Twelve to 15 pairs of ovaries were used in Group I but fewer animals were used in groups in which FSH was injected because more cells could be harvested from the ovaries. All the glassware used was silicone-treated (20) to prevent adhesion of the c e l l s . The granulosa cells and remaining tissues were divided )urg (7ml) flasks with one flask containi dbcAMP, while the other flask served as ured by manometric methods (21) for 2 h( sues were removed for determination of DNA for the percentage viable cells as det( ). Remaining tissues could not be correct recepto~ binding of 125I-FSH. Whole gra Lion of For Qe~ermlnd~ion OT - 251-FSH - 1 - r 3 m binding DinGing while wnlle a a homogeniz nomogenlzea membrane p r e p a r a t l o n . ' o l l e c t e d from remaining t i s s u e s (x 20,000 g) was used f o r comparison. Two ".oncentrations of c e l l s or membranes (I00 and 50 u l ) were used i n each r e p l i "ate to o b t a i n s a t u r a t i o n . Both c o n c e n t r a t i o n s were run in t r i p l i c a t e with Jnlabeled FSH added to determine n o n s p e c i f i c b i n d i n g . A l l tubes were pretreated w i t h bovine serum albumin to decrease n o n - s p e c i f i c b i n d i n g . The b i n d ing was done at room temperature, on a shaker f o r 4 hours and the i n c u b a t i o n d by the addition of 2 ml PBS (4°C). Ce 20,000 x g for i hour and the supernatant rice and the tubes were counted for 2 min
)del 4230 gamlna well counter. The specific binding of 1251-FSH was 7.8% in ~oup I with 10,000 counts added to each tube. DNA was determined on each ~be as above. Lactate determination. Samples were prepared for gas chromatography by addin( iding 2ml methanol, O.4ml 50% H~SO4and O.Iml benzoic acid (100 meq/lO0 ml) to) 1.0ml of granulosa cells and ~Ted]~a removed from each of the paired Warburg flasks. After standing overnight, lml water and O.5ml chloroform was added to extract the methylated derivative of lactate into the chloroform layer. Extraction was monitored with benzoic acid. A dual flame ionization detector gas chromatograph (F and M 810-19 Analytical Gas Chromatograph) with a Supelco 15% SP 1220 (1% H~PO~, H~PO.. 100, 100/200 Chromabsorb, WAW) column (0.1 mu x 6 f t long) Methylated pyruvic, l a c t i c , oxalic was used. The c~rrler gas wa! r i c , and succinic acids were prepared oxaloacetic, malonic, methylmal, for each determination and were used to measure the resolution of the column, Lactic acid and benzoic acid standards were used to measure variation between assays. The mean coefficient of variation for the area under the curve for the 0.05 uM l a c t i c standard was 3.19% (N=8) and is the intra-assay variation. The coefficient of variation for ratios of the l a c t i c standard to the internal benzoic acid standard was 9.05% and is the inter-assay variation. ts Ovarian weight increased w of treatment with DES capsules in 23 day old rats (25 day control weight m + SE = 10.29 + _ 0.I55 mg; DES capsules m + SE = 12.74 + 0.65 mg; t = 2.28; P
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FIGURE I. The e f f e c t s of d i f f e r e n t flask concentrations of FSH in v i t r o on 02 uptake of isolated granulosa c e l l s from 23 day rats treated with DES alone or with DES and FSH. Group I is DES alone, Group II is DES and lOOug FSH and Group I I I is DES and 300ug FSH. Data are reported as the mean + SE of the d i f ference in 02 uptake between flasks (ul 02/mg DNA/h). Substrates were present in a l l flasks.
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FIGURE 2. Specific binding of z251-FSH by isol ated granulosa c e l l s and remair Ining tissue membranes isolated from ovaries of 23 day old rats treated tre with DES and injected for lon longer times with I00 ug doses of FSH. The mean + SE of the counts s p e c i f i c i a l l y bou-nd are presented.
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days DES 2 2 1injected - .1 ~mg)
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FIGURE 3. The el in v i t r o concent~ the formation of ed granulosa cell im~anted with DES 300 ug FSH. The + SE of the d i f f e formed between f l and control flasks. The media has 0.27M glucose as a substrate.
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1.0 2.0 3.0 LH Flask Concentration(ug/ml)
LH in vitro failed to increase O^ uptake by either granulosa cells or ~maining-'-tissdes of any treatment grou~ (data not presented). Granulosa cells from 30 day old rats showed increased O~ uptake of 30% above ~ove control levels when O.9~g/ml LH G3-222B was added in v~tro the flasks DES treatment prevented the LH stimulated in,ease in 07 uptake (Table I ) . (Table I). Three concentrations of dbcAMP added in v i t r o were tried but only 0.018 mM was effective in increasing O~ uptake. T~s concentration was IOX lower than that effective on granulosa ~ells of Group I. TABLE I Effects of in vitro of LH G3-222B and dbcAMP on Oxygen Uptake of Isolat~ sa Cells from 30 Day Old Rats. Differences are between Control and Experimental Flasks with Substrates in Both.
Animal Treatment None None DES capsules
Flask Concentration LH or dbcAMP LH dbcAMP dbcAMP dbcAMP LH
Difference in 02 Uptake (mean ~ SE)#I/O2/mg DNA(h) No__~.
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16.25 8* 19.94 5 16.56 8* 17.63 7 28.83 7 *p< 0.05 (two tailed)
Granulosaa cells of untreated 30 day old rats incuibated with 0.27 M glu2.292 ~g/ml LH 21 added in vitro did not increase oxygen uptake ~ase lactate production ~ a ~ f f e r e n c e in lactate = 0.071 + ate/mg DNA, N = 7, P <0.01). When 27 day old rats were implanteB with E~ capsules, LH in vitro neither increased the O~ uptake nor stimulated lactate formation b y ~ o ~ granulosa c e l l s . Granu~osa cells from animals implanted with E~capsules and injected with 300 ug FSH showed greater lactate production than ~id those from untreated rats and these cells were used for dose response studies. Dose-response studies of the difference in ~M lactate formed with in vitro LH showed that flask concentrations of LH 21 as low as 0.572 ~g/ml -s~gnl--f1~'cantly stimulated lactate production (Fig. 3). In contrast, FSH G4-150C (0.9~g/ml) added in vitro to granulosa cells of untreated rats without added substrates increased ~ O~ uptake and lactate production (m~an difference ~_ uptake = 6 ~3.q2 rice 0 7uptake 3 . 9 2+ ll.3? l l . 3 2 ~uIVma m g DNA. DNA, N = 5. P <0.0]: mean c t a t e ~ 0.1204 + .0382 #-M/mg DNA, N = 5, P incubated with -higher glucose concentrat in vitro FSH to increase either Oe uptak rice O? uptake = 1.34 + 7.81Ml Op/n/g DNA, d = -0.1036 + .0272 ~M lactate/m~DNA, N = Discussion Granulosa cells are apparently the only ovarian compartment that can respond to in vitro FSH by increasing their O~ uptake. The concentrations of FSH which st-~mu~d O~ uptake in the 23 day'model are similar to those reported to increase cAMP ~in granulosa cells (24). The concentrations of dbcAMP required to stimulate De uptake of these cells was much higher than needed to stimulate granulosa celts from 30 day old rats not treated with DES capsules. e could be due to the different permeabili MP or to different levels of phosphodiest(
r results with theophilline do not suggest that there was appreciable phosodiesterase a c t i v i t y in granulosa cells of Group I . I t has been reported that untreated ovaries from immature rats have FSH rece ceptors on granulosa cells and LH receptors on theca cells ( i ) . Th6refore, thee report of Hamberger (12) that LH acts to increase oxygen uptake of granulosa cells and FSH acts to increase oxygen uptake of theca cells of untreated rats suggests a lack of correlation between receptor content and the in v i t r o response of the cells to these gonadotropins. Our data using the 23 day treatment model do not support the results of Hamberger (12). The most sensitive cells to FSH were those isolated from Group I and these cells had the most FSH receptors. The decre ing present in Groups I I and I l l is probably not due to the occup~ SH receptors by FSH given 12 hours e a r l i e r (26), Ubut Ub there bll~l E is 13 a ~[ U ~ I U I 0 I I ~ y that ~ll~b bile b UUpI IIl~ U I the coupling of the occupied receptors with i n t r a c e l l u l a r enzyme systems (adenylate cyclase and protein kinases) is changed. Similarly, the absence of LH receptors in the granulosa cells of 30 day rats treated only with DES or estradiol capsules prevented an increase in either oxygen uptake or lactate formation when LH was added in v i t r o . Estrogen capsules i n h i b i t the small endogenous secretion of FSH (2/]w--BXc-Ecould have caused the development of LH receptors. The production of lactate losa cells from 30 day rats treated with FSH and estradiol was depe :he presence of s u f f i c i e n t glucose in the flasks to support )ort metabol" me~aDollsm over tne z 2 hour nour perloo )eriod. Our experiments used 4 mg/ml glucose ;e because concentrations less than 2 mg/~ /ml can be rate l i m i t i n g to cells in v i t r o (18). When FSH was added to granulos ranulosa cells in the absence of glucose, a small but significant lactate productior on was found. However, only LH was abl )le to increase lactate formation in the pl )resence of 4 mg/ml glucose and i t was is more effective in cells from rats treat treated with both estradiol n cells from untreated 30 day old rats. Therefore, the response ~lated to the increased LH receptor prese )resent after FSH treatment periments are the f i r s t reporting an LH induced stimulation of lactate production by isolated granulosa cells from rat ovaries. They are ; i m i l a r to those reported by Zakar and Hertelendy (29) using isolated avian ]ranulosa cells and to the results of others on isolated Graafian f o l l i c l e s , )r slices of whole ovaries (30, 7, 9). Thus, i t appears that the granulosa : e l l s are a major source of lactate production in ovaries. The overall in v i t r o effects of FSH and LH on the metabolism of isolated )ranulosa cells suggest that FSH acts on metabolic processes within the mito:hondria while LH acts on metabolic processes within the cytoplasm. The steps ~tion of adenylate cyclase and these met~ ~nt as are the enzyme pathways affected b ;abolic pathways. I t is probable that pro for steroid synthesis and synthesis of ne metabolic changes. Acknowledgements we WOUlG llike i k e to CO thank ~nanK John Jonn and Mar Mary Bates ~ates for partlal )~ support o? tn~s )roject. Dr. Harold Papkoff kindly provided FSH G4-150C and LH G3-222B. The ~ational P i t u i t a r y Agency of NIAMKD provided NIH FSH and LH preparations. Dr. k. R. Midgley, Jr. provided iodinated FSH for the binding studies. References ~RDS and A.R. MIDGLEY, J r . , Biol. Reprod. I MER, Receptors and Hormone Action, ed. B.~ v. 1 485-547, Academic Press, N.Y. (1977)
•
J.M. MARSH, Advances in Cyclic Nucleotide Research, ed. P. Greengard and G.A. Robinson, V. 6 137-199, Raven Press, N.Y. (1975). • M. HUNZICKER-D~.A. JUNGMANand L. BIRNBAUMER, Ovarian F o l l i c u l a r Development and Function, ed. A.R. Midgley, Jr. and W.A. Sadler, 267--304, Raven Press, N.Y. T19-97"9-)'. 5. D.T. ARMSTRONG, R. KILPATRICK and R.O. GREEP, Endocrin. 73 165-169 (1965). 6. K. AHREN, L. HAMBERGERand L. RUBENSTEIN, Acta physiol. ~and. 74 79-90 (1968). 7. S.W. FARMER, M.K. SAIRAMand H. PAPKOFF, Endocrin. 92 1022-1027 (1973). 8. K. AHRENand L. HAMBERGER,Acta physiol. Scand. 77 ~J'7-260 (1969). 9. D.T. ARMSTRONG, Endocrin. 72 908-913 (1963). -Ckl ~D 1 ~mk~,,~l vn'-'--Mmm.h 1 1 1 1 1 [ 1 Q~q I0• CS. ~BEN-OR, J. Embryol. mexp-: • I - I I (1963). 11. G. EJSMONT, Folia Histochem T. 3 291-300 (1965)• 12. J.E. JIRASEK J PRESLand A~ta Histochem. 37 162-169 (1970) 13. E.J• BLANCHETTE,J. Cell Biol. 31 501-516 (1966). -14. O. NALBANDOVand A.V. NALBANDO~/~-Endocrin. 45 195-203 (1949). 15, D. FOREMAN,Physiol. Zool. 42 76-84 (1969) - 16. D. FOREMAN,Gen. Comp. Endo~inol. 8 66-71 (1967). 17. L. HAMBERGER,Acta physiol. Scand. T4 410-425 (1968). 18, H.A. KREBS, Biochim. Biophys. Acta 4---249-268 (1950). 19. K.L. CAMPBELL, Biol. Reprod '86 (1979). 20. G. SAYERS, Ann. N.Y. Acad. !20-241 (1976)• 21. W.W. UMBREIT, R.H. BURRIS a "AUFFER, Manometric and Biochemical Techniques 5th ed. Burgess Ig Co Minneapolis (1972). 22. K. BURTON, Biochem J. 62 315-323 (1956). ERGER, Method-{-in Enzymology, ed. J.G. Hardman Har and B.W. 23. A. STEINBERGER O'Malley, v. 39 p. 139, Acade6ih]~-P'~s, N.Y. (1975). (1975) 24. A.K. GOFF~ . T . ARMSTRONG, Endocrin. 101 1461-1467 1461-14 (1977). 25. J.J. IRELAND AND and J.S. RICHARDS, Endocrin.' 102 876-883 876-8 (1978}. ARDS, J.J. IRELAND, M.C. RAO, G.A. BERNATH, BERNATH A.R. MIDGLEY, JR. and HERT, JR., Endocrin. 99 1562-1570 (1976). ER and W. WUTTE Endoc~in. 97 898-907 (1974). (197 !8• A.D. ZELESNIK, A.R. MIDGLEY, JR. a n ~ . E . REICHERT, JR., Endocrin• 95 818 (1974). !9. T. ZAKARand F. HERTELENDY, Biol. Reprod. 22 810-816 (1980). ~0. M.E. LIEBERMAN, K. AHREN, A. TSAFRIRI, S. ~UMINGER and H. R. LINDNER, J. Steroid Biochem. 6 1445-1449 (1975). .i
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ry FSH in v i t r o , but not LH, increased the 02 uptake of isolated granulosa cells f r o m ' - 2 ~ old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the c e l l u l a r binding of FSH was highest. LH increased the ~ uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise~in in 02 O?uptake when the rats were implanted with DES capsules unless FSH was injected-to induce LH ;. Addition of dbcAMP in v i t r o Inm 30 day old rats at concentrations creased 02 uptake of granulosa IOX lower than those required t< e 02 uptake in cells from 23 day old rats treated with DES alone. FSH in v i t r o increased lactate formation in the absence of added substrates but did id not do so when glucose was added to th, the media. In contrast, LH greatly increased ncreased lactate formation with added gllucose. Dose response studies showed d that less than 0.6 ug/ml LH $21 was effective e in increasing control levels. These data suggest that th~ FSH affects aerobic )rocess of the differene LH affects anaerobic pathways in the pr anulosa cells toward luteal cells. I t is well known that FSH and LH interact with their target cells in the ovary by binding to specific receptors and that FSH stimulates LH-receptor production (1). Receptor binding by either hormone activates adenylate cyclase (2) raising cyclic adenosine monosphosphate (cAMP) levels (3) and increasing protein kinase a c t i v i t y (4). Such changes probably trigger changes in the major metabolic pathways that support f o l l i c u l a r development because cells of corpora lutea have glycogen (5) which is not present in f o l l i c u l a r granulosa cells (6-9). Several studies suggest that FSH and LH may regulate cesses in the ovary. LH increases lacta 1,12) and also increases the uptake of g n uptake in chick ovaries (14), rat ovari~ However, only one study has been done rger (17) has reported that FSH increase( of immature rats while LH increased the Since granulosa cells from immature rat~ while theca cells have LH receptors the mones appear unclear unclear. The present studies were undertaken to more accurately characterize the actions of FSH, LH, and dibutyryl cAMP (dbcAMP) on the oxygen uptake of isolated granulosa cells and remaining tissues of immature ovaries and to determine the effects of FSH and LH on the production of lactate by granulosa cells.
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press I
Methods Reagents and hormones. NIH FSH B1,$1,$4,$12 and S14 were i n j e c t e d in doses e q u i v a l e n t to FSH $1 (1 mg = 1U). NIH FSH $4 and $12 were equivalent to $I , NIH FSH BI was 0.4U/mg and $14 was 9U/mg. The doses given were 100 ug $1, $4 and $12 and 11.11ug $14. The hormones used i n v i t r o were p u r i f i e d prepara t i o n s of ovine FSH (FSH G4-150C, 50U/mg) and o~ne--L-IT-(LH G3-222B, 2.75x NIH LH SI) both prepared by Dr. Harold Papkoff. NIH LH 21 (2.5x NIH LH SI) was used f o r a l l l a c t a t e s t u d i e s , l o d i n a t e d oFSH (B900) and u n l a b e l l e d oFSH (B882), generously supplied by Dr. A. R. M i d g l e y , J r . were used i n the b i n d i n g DES, dbcAMP and t h e o p h i l l i n e were purchased from the Sigma studies. Chemical Co Medical grade Si bing was purchased from Dow Corning, Midland, MI. Silicone coating Electric Drifilm) for glassware was purchased from Pierce Chemical Ill
E2'
Animals and treatments. Immature r a t s of 3 cage-bred s t r a i n s were used in these experiments. Twenty-three or 28 day old r a t s were implanted w i t h i0 mm S i l a s t i c capsules c o n t a i n i n g DES or Ep and e i t h e r autopsied a t 48 hours or at v a r i o u s i n t e r v a l s f o l l o w i n g FSH t r e a t m e n t . Thus, 23 day old r a t s received DES capsules w i t h autopsy at 48 hours (Group I ) , DES capsules and FSH at 36 hours with autopsy at 48 hours , DES capsules and FSH at 36, 48 and 60 hours with autopsy at 72 hou I l l ) , DES capsules and FSH at 36, 48, 60 and 72 hours with autopsy a~ (Group IV). Sixteen rats implanted with DES capsules UI~ W were ~r'~ injected i r l j ~ u w1 U I d L otal ULdl of 600 (8 rats) or 700 (8 rats) ug FSH (Groups V and Vl). Thirty day old rats were used to determine the effects of FSH and LH on oxygen uptake and lactate production. They were implanted with 10 mm capsules containing E2 alone or al so injected with FSH at 36, 48 and 60 hours with autopsy at 72 hours.
t of cells and tissues. Isolated granulo~ ranulosa cells were i n i t i a l l y echanically expressing the cells d i r e c t l y into Krebs buffer with 8) but a greater y i e l d was obtained by the method of Campbell (19) of putting the ovaries f i r s t into 6.8M EGTA in 0.1M phosphate buffered saline pH 7.2 (PBS) for 15 min. then in O.5M sucrose in PBS for 15 min. before ~xpressing the cells into Krebs buffer with substrates (0.015M glucose, 0.036M pyruvate, O.07M fumarate, 0.039M glutamate). The substrate used for lactate studies was 0.27M glucose to prevent glucose from l i m i t i n g the reaction (18). Twelve to 15 pairs of ovaries were used in Group I but fewer animals were used in groups in which FSH was injected because more cells could be harvested from the ovaries. All the glassware used was silicone-treated (20) to prevent adhesion of the c e l l s . The granulosa cells and remaining tissues were divided )urg (7ml) flasks with one flask containi dbcAMP, while the other flask served as ured by manometric methods (21) for 2 h( sues were removed for determination of DNA for the percentage viable cells as det( ). Remaining tissues could not be correct recepto~ binding of 125I-FSH. Whole gra Lion of For Qe~ermlnd~ion OT - 251-FSH - 1 - r 3 m binding DinGing while wnlle a a homogeniz nomogenlzea membrane p r e p a r a t l o n . ' o l l e c t e d from remaining t i s s u e s (x 20,000 g) was used f o r comparison. Two ".oncentrations of c e l l s or membranes (I00 and 50 u l ) were used i n each r e p l i "ate to o b t a i n s a t u r a t i o n . Both c o n c e n t r a t i o n s were run in t r i p l i c a t e with Jnlabeled FSH added to determine n o n s p e c i f i c b i n d i n g . A l l tubes were pretreated w i t h bovine serum albumin to decrease n o n - s p e c i f i c b i n d i n g . The b i n d ing was done at room temperature, on a shaker f o r 4 hours and the i n c u b a t i o n d by the addition of 2 ml PBS (4°C). Ce 20,000 x g for i hour and the supernatant rice and the tubes were counted for 2 min
)del 4230 gamlna well counter. The specific binding of 1251-FSH was 7.8% in ~oup I with 10,000 counts added to each tube. DNA was determined on each ~be as above. Lactate determination. Samples were prepared for gas chromatography by addin( iding 2ml methanol, O.4ml 50% H~SO4and O.Iml benzoic acid (100 meq/lO0 ml) to) 1.0ml of granulosa cells and ~Ted]~a removed from each of the paired Warburg flasks. After standing overnight, lml water and O.5ml chloroform was added to extract the methylated derivative of lactate into the chloroform layer. Extraction was monitored with benzoic acid. A dual flame ionization detector gas chromatograph (F and M 810-19 Analytical Gas Chromatograph) with a Supelco 15% SP 1220 (1% H~PO~, H~PO.. 100, 100/200 Chromabsorb, WAW) column (0.1 mu x 6 f t long) Methylated pyruvic, l a c t i c , oxalic was used. The c~rrler gas wa! r i c , and succinic acids were prepared oxaloacetic, malonic, methylmal, for each determination and were used to measure the resolution of the column, Lactic acid and benzoic acid standards were used to measure variation between assays. The mean coefficient of variation for the area under the curve for the 0.05 uM l a c t i c standard was 3.19% (N=8) and is the intra-assay variation. The coefficient of variation for ratios of the l a c t i c standard to the internal benzoic acid standard was 9.05% and is the inter-assay variation. ts Ovarian weight increased w of treatment with DES capsules in 23 day old rats (25 day control weight m + SE = 10.29 + _ 0.I55 mg; DES capsules m + SE = 12.74 + 0.65 mg; t = 2.28; P
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FIGURE I. The e f f e c t s of d i f f e r e n t flask concentrations of FSH in v i t r o on 02 uptake of isolated granulosa c e l l s from 23 day rats treated with DES alone or with DES and FSH. Group I is DES alone, Group II is DES and lOOug FSH and Group I I I is DES and 300ug FSH. Data are reported as the mean + SE of the d i f ference in 02 uptake between flasks (ul 02/mg DNA/h). Substrates were present in a l l flasks.
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FIGURE 2. Specific binding of z251-FSH by isol ated granulosa c e l l s and remair Ining tissue membranes isolated from ovaries of 23 day old rats treated tre with DES and injected for lon longer times with I00 ug doses of FSH. The mean + SE of the counts s p e c i f i c i a l l y bou-nd are presented.
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days DES 2 2 1injected - .1 ~mg)
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FIGURE 3. The el in v i t r o concent~ the formation of ed granulosa cell im~anted with DES 300 ug FSH. The + SE of the d i f f e formed between f l and control flasks. The media has 0.27M glucose as a substrate.
~,02 u
1.0 2.0 3.0 LH Flask Concentration(ug/ml)
LH in vitro failed to increase O^ uptake by either granulosa cells or ~maining-'-tissdes of any treatment grou~ (data not presented). Granulosa cells from 30 day old rats showed increased O~ uptake of 30% above ~ove control levels when O.9~g/ml LH G3-222B was added in v~tro the flasks DES treatment prevented the LH stimulated in,ease in 07 uptake (Table I ) . (Table I). Three concentrations of dbcAMP added in v i t r o were tried but only 0.018 mM was effective in increasing O~ uptake. T~s concentration was IOX lower than that effective on granulosa ~ells of Group I. TABLE I Effects of in vitro of LH G3-222B and dbcAMP on Oxygen Uptake of Isolat~ sa Cells from 30 Day Old Rats. Differences are between Control and Experimental Flasks with Substrates in Both.
Animal Treatment None None DES capsules
Flask Concentration LH or dbcAMP LH dbcAMP dbcAMP dbcAMP LH
Difference in 02 Uptake (mean ~ SE)#I/O2/mg DNA(h) No__~.
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Granulosaa cells of untreated 30 day old rats incuibated with 0.27 M glu2.292 ~g/ml LH 21 added in vitro did not increase oxygen uptake ~ase lactate production ~ a ~ f f e r e n c e in lactate = 0.071 + ate/mg DNA, N = 7, P <0.01). When 27 day old rats were implanteB with E~ capsules, LH in vitro neither increased the O~ uptake nor stimulated lactate formation b y ~ o ~ granulosa c e l l s . Granu~osa cells from animals implanted with E~capsules and injected with 300 ug FSH showed greater lactate production than ~id those from untreated rats and these cells were used for dose response studies. Dose-response studies of the difference in ~M lactate formed with in vitro LH showed that flask concentrations of LH 21 as low as 0.572 ~g/ml -s~gnl--f1~'cantly stimulated lactate production (Fig. 3). In contrast, FSH G4-150C (0.9~g/ml) added in vitro to granulosa cells of untreated rats without added substrates increased ~ O~ uptake and lactate production (m~an difference ~_ uptake = 6 ~3.q2 rice 0 7uptake 3 . 9 2+ ll.3? l l . 3 2 ~uIVma m g DNA. DNA, N = 5. P <0.0]: mean c t a t e ~ 0.1204 + .0382 #-M/mg DNA, N = 5, P incubated with -higher glucose concentrat in vitro FSH to increase either Oe uptak rice O? uptake = 1.34 + 7.81Ml Op/n/g DNA, d = -0.1036 + .0272 ~M lactate/m~DNA, N = Discussion Granulosa cells are apparently the only ovarian compartment that can respond to in vitro FSH by increasing their O~ uptake. The concentrations of FSH which st-~mu~d O~ uptake in the 23 day'model are similar to those reported to increase cAMP ~in granulosa cells (24). The concentrations of dbcAMP required to stimulate De uptake of these cells was much higher than needed to stimulate granulosa celts from 30 day old rats not treated with DES capsules. e could be due to the different permeabili MP or to different levels of phosphodiest(
r results with theophilline do not suggest that there was appreciable phosodiesterase a c t i v i t y in granulosa cells of Group I . I t has been reported that untreated ovaries from immature rats have FSH rece ceptors on granulosa cells and LH receptors on theca cells ( i ) . Th6refore, thee report of Hamberger (12) that LH acts to increase oxygen uptake of granulosa cells and FSH acts to increase oxygen uptake of theca cells of untreated rats suggests a lack of correlation between receptor content and the in v i t r o response of the cells to these gonadotropins. Our data using the 23 day treatment model do not support the results of Hamberger (12). The most sensitive cells to FSH were those isolated from Group I and these cells had the most FSH receptors. The decre ing present in Groups I I and I l l is probably not due to the occup~ SH receptors by FSH given 12 hours e a r l i e r (26), Ubut Ub there bll~l E is 13 a ~[ U ~ I U I 0 I I ~ y that ~ll~b bile b UUpI IIl~ U I the coupling of the occupied receptors with i n t r a c e l l u l a r enzyme systems (adenylate cyclase and protein kinases) is changed. Similarly, the absence of LH receptors in the granulosa cells of 30 day rats treated only with DES or estradiol capsules prevented an increase in either oxygen uptake or lactate formation when LH was added in v i t r o . Estrogen capsules i n h i b i t the small endogenous secretion of FSH (2/]w--BXc-Ecould have caused the development of LH receptors. The production of lactate losa cells from 30 day rats treated with FSH and estradiol was depe :he presence of s u f f i c i e n t glucose in the flasks to support )ort metabol" me~aDollsm over tne z 2 hour nour perloo )eriod. Our experiments used 4 mg/ml glucose ;e because concentrations less than 2 mg/~ /ml can be rate l i m i t i n g to cells in v i t r o (18). When FSH was added to granulos ranulosa cells in the absence of glucose, a small but significant lactate productior on was found. However, only LH was abl )le to increase lactate formation in the pl )resence of 4 mg/ml glucose and i t was is more effective in cells from rats treat treated with both estradiol n cells from untreated 30 day old rats. Therefore, the response ~lated to the increased LH receptor prese )resent after FSH treatment periments are the f i r s t reporting an LH induced stimulation of lactate production by isolated granulosa cells from rat ovaries. They are ; i m i l a r to those reported by Zakar and Hertelendy (29) using isolated avian ]ranulosa cells and to the results of others on isolated Graafian f o l l i c l e s , )r slices of whole ovaries (30, 7, 9). Thus, i t appears that the granulosa : e l l s are a major source of lactate production in ovaries. The overall in v i t r o effects of FSH and LH on the metabolism of isolated )ranulosa cells suggest that FSH acts on metabolic processes within the mito:hondria while LH acts on metabolic processes within the cytoplasm. The steps ~tion of adenylate cyclase and these met~ ~nt as are the enzyme pathways affected b ;abolic pathways. I t is probable that pro for steroid synthesis and synthesis of ne metabolic changes. Acknowledgements we WOUlG llike i k e to CO thank ~nanK John Jonn and Mar Mary Bates ~ates for partlal )~ support o? tn~s )roject. Dr. Harold Papkoff kindly provided FSH G4-150C and LH G3-222B. The ~ational P i t u i t a r y Agency of NIAMKD provided NIH FSH and LH preparations. Dr. k. R. Midgley, Jr. provided iodinated FSH for the binding studies. References ~RDS and A.R. MIDGLEY, J r . , Biol. Reprod. I MER, Receptors and Hormone Action, ed. B.~ v. 1 485-547, Academic Press, N.Y. (1977)
•
J.M. MARSH, Advances in Cyclic Nucleotide Research, ed. P. Greengard and G.A. Robinson, V. 6 137-199, Raven Press, N.Y. (1975). • M. HUNZICKER-D~.A. JUNGMANand L. BIRNBAUMER, Ovarian F o l l i c u l a r Development and Function, ed. A.R. Midgley, Jr. and W.A. Sadler, 267--304, Raven Press, N.Y. T19-97"9-)'. 5. D.T. ARMSTRONG, R. KILPATRICK and R.O. GREEP, Endocrin. 73 165-169 (1965). 6. K. AHREN, L. HAMBERGERand L. RUBENSTEIN, Acta physiol. ~and. 74 79-90 (1968). 7. S.W. FARMER, M.K. SAIRAMand H. PAPKOFF, Endocrin. 92 1022-1027 (1973). 8. K. AHRENand L. HAMBERGER,Acta physiol. Scand. 77 ~J'7-260 (1969). 9. D.T. ARMSTRONG, Endocrin. 72 908-913 (1963). -Ckl ~D 1 ~mk~,,~l vn'-'--Mmm.h 1 1 1 1 1 [ 1 Q~q I0• CS. ~BEN-OR, J. Embryol. mexp-: • I - I I (1963). 11. G. EJSMONT, Folia Histochem T. 3 291-300 (1965)• 12. J.E. JIRASEK J PRESLand A~ta Histochem. 37 162-169 (1970) 13. E.J• BLANCHETTE,J. Cell Biol. 31 501-516 (1966). -14. O. NALBANDOVand A.V. NALBANDO~/~-Endocrin. 45 195-203 (1949). 15, D. FOREMAN,Physiol. Zool. 42 76-84 (1969) - 16. D. FOREMAN,Gen. Comp. Endo~inol. 8 66-71 (1967). 17. L. HAMBERGER,Acta physiol. Scand. T4 410-425 (1968). 18, H.A. KREBS, Biochim. Biophys. Acta 4---249-268 (1950). 19. K.L. CAMPBELL, Biol. Reprod '86 (1979). 20. G. SAYERS, Ann. N.Y. Acad. !20-241 (1976)• 21. W.W. UMBREIT, R.H. BURRIS a "AUFFER, Manometric and Biochemical Techniques 5th ed. Burgess Ig Co Minneapolis (1972). 22. K. BURTON, Biochem J. 62 315-323 (1956). ERGER, Method-{-in Enzymology, ed. J.G. Hardman Har and B.W. 23. A. STEINBERGER O'Malley, v. 39 p. 139, Acade6ih]~-P'~s, N.Y. (1975). (1975) 24. A.K. GOFF~ . T . ARMSTRONG, Endocrin. 101 1461-1467 1461-14 (1977). 25. J.J. IRELAND AND and J.S. RICHARDS, Endocrin.' 102 876-883 876-8 (1978}. ARDS, J.J. IRELAND, M.C. RAO, G.A. BERNATH, BERNATH A.R. MIDGLEY, JR. and HERT, JR., Endocrin. 99 1562-1570 (1976). ER and W. WUTTE Endoc~in. 97 898-907 (1974). (197 !8• A.D. ZELESNIK, A.R. MIDGLEY, JR. a n ~ . E . REICHERT, JR., Endocrin• 95 818 (1974). !9. T. ZAKARand F. HERTELENDY, Biol. Reprod. 22 810-816 (1980). ~0. M.E. LIEBERMAN, K. AHREN, A. TSAFRIRI, S. ~UMINGER and H. R. LINDNER, J. Steroid Biochem. 6 1445-1449 (1975). .i
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