molecular and Cellular Endocrinology, a 1992 Elsevier Scientific Publishers

MOLCEL

115

87 (1992) 115-123 Ireland. Ltd. 0303.72~7/92/$05.00

02812

Isolation of luteinising hormone receptor binding inhibitor from bovine corpus luteum S. Chari, W. Kail, E. Daume and G. Sturm

Kq wordc Corpus

luteum;

Luteinizing

hormone;

Granulosa

cell; Binding

inhibitor;

Ovulation;

(Bovine

ovary)

Summary A luteinising hormone receptor binding inhibitor (LHRBI) has been purified from bovine corpus luteum (CL). Steroid-free extract of the CL was subjected to successive chromatographies on Sephadex G-SO, Q-Sepharose, Orange A dye and metal chelate affinity columns followed by high performance-reverse phase and gel filtration columns. Purification was monitored by the ability of the fractions to inhibit the binding of ‘2”I-human chorionic gonadotropin (hCG) to porcine granulosa cells in vitro. The final isolate showed an SOOO-foldenrichment of activity. It was also capable of inhibiting porcine granulosa cell secretion of estradiol and progesterone (PI in vitro. Administration of LHRBI into follicle-stimulating hormone (FS~)-stimulated, immature rats strongly inhibited the ovarian ovulatory response to hCG as revealed by decreased P levels and the number of ova released. The M, of LHRBI as assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was ca. 15 kDa and the pl was between 5.0 and 5.5.

Introduction A variety of nonsteroidal factors which appear to function as paracrine/ autocrine regulators has been identified in the ovary of diverse species (see Tsafriri, 1988 for a review). The presence of a substance that inhibits the binding of luteinising hormone (LH) to ovarian receptors (LHRBI) has been demonstrated in the luteal extracts of the rat (Yang et al., 1976), cat, dog, sheep, goat (Yang et al., 19781, pig (Sakai et al., 1977) and human (Kumari et al., 1980). Evidence for the

Correspondence ogy, Endocrinology kiinik, Pilgrimstein

to: Dr. S. Chari, Department of Gynecoland Reproduction, Universitats Frauen3, DW-3550 Marburg, Germany.

existence of both high and low molecular weight inhibitors has been presented by Yang et al, (1978). LHRBI from pig (Ward et al., 1982; Kwok et al., 1986) and sheep (Kumar et al., 1989) corpora lutea have been partially purified. However, to date no reports on bovine LHRBI are available. It was, therefore, of interest to purify LHRBI activity from bovine corpus luteum and examine its biological effects in vivo and in vitro. Materials and methods Unless otherwise stated, all reagents were purchased from Sigma (Diesenhofen, Germany). Bovine ovaries were collected from the slaughter-house in Wittlich/ Eifel. The follicles were punctured and the fluid thus collected was kept

aside were tissue before

frozen for other purposes. Corpora lutea separated, cleaned from the surrounding and kept frozen at -80°C for 4-12 weeks extraction.

Extraction The tissue was thawed slowly at 4°C and homogenised in 4 volumes of chilied physiological saline containing 0.001% phenylmethylsulfony~ fluoride (PMSF). The homogenate was stirred overnight at 4°C subsequently centrifuged at 30,000 x g for 30 min at 4°C. The supernatant was recovered. To ensure complete recovery of the activity from the tissue, the residue was reextracted as above. The supernatants from the two extractions were pooled and mixed with an equal volume of chilled (-20°C) 1-propanol (Merck, Darmstadt, Germany). The proteins thus precipitated were recovered by centrifugation and dried by Iyophilisation (crude extract). Purification Unless otherwise stated all operations were done at 4°C. 5 g of crude extract were processed each time. The material was dissoIved in 0.05 M ammonium acetate buffer pH 7.6. The insoluble material was separated by centrifugation and the clear supernatant was loaded on Sephadex G-50 (50 X 5.0 cm column; Pharmacia/LKB, Uppsala, Sweden) equiiibrated with the above buffer. Fractions of 10 ml were collected, the flow rate being 100 ml/h. The active fractions were pooled, concentrated by ultrafiltration using Amicon UM 5 filter (M, cut-off limit 5000 Da; Amicon, Lexington, MA, USA) and loaded on a 25 x 5 cm coIumn filled with an anion-exchange gel Q-Sepharose (Pharmacia/LKB) equilibrated with the starting buffer 0.05 M ammonium acetate, pH 7.6. The column was washed with several volumes of the starting buffer. The adsorbed proteins were eluted by increasing NaCI concentrations in the starting buffer in a step-wise manner from 0.1 M to 1.0 M. The 1.0 M fraction containing LHRBI activity was then subjected to Orange A dye (Amicon) affinity chromatography. After allowing 60 min for the binding of the proteins to the dye gel, the unbound material (OAUB) was eluted with 0.05 M ammonium acetate buffer pH 7.6, whereas the

bound proteins were eluted with the above buffer containing 1.5 M KCI. The active fraction from this step was subjected to metal chelate affinity chromatography (MCAC). For this purpose, a chelating Sepharose column (Pharmacia/LKB) was charged with the copper ion according to the manufacturer’s instructions. The unbound (CuUB) and the bound proteins from this column were eluted with 0.1 M ammonium acetate pH 7.6 and 0.1 M sodium acetate buffer pH 4.0 respectively. Final purification of the active fraction was done by high performance liquid chromatography (HPLC) at room temperature using Beckman System GoId apparatus (Beckman, CA, USA). Reverse-phase (RP) HPLC was carried out on an analytical C,, column (250 X 4.5 mm. pore size 5 pm; Vydac, CA, USA). After loading the sample, the column was eluted isocratically for 5 min with 0.1% trifluoroacetic acid (TFA; Pierce, Cologne, Germany) in HPLC grade distilled water (FSA, ~ughborough, UK) followed by a 20 min linear gradient from 0 to 100% B buffer (80% acetonitrile; FSA, UK) containing 0.1% TFA. Fractions of 0.8 ml/min were collected. The final run was done on an analytical HPLC gel fiItration column (TX; Beckman, CA, USA), the column dimensions being 7.5 mm X 30 cm. The column was eluted with 0.1 M ammonium acetate buffer, pH 7.6, the flow rate being 0.8 ml/min. Analyticai Protein was determined by measuring the samples at 280 nm assuming 1 OD unit (1 cm light path) = 1 mg/ml. Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulphate (SDS) was done on a 12% gel according to the method of Laemlh (1970) using BioRad mini vertical slab gel. Before loading, the samples dissolved in 0.05 M Tris-HCl buffer, pH 6.8, containing 5% pmcrcaptoethanol and 2% SDS, were reduced by heating at 95°C for 5 min. The gel was electrophoresed at 200 V for 50 min at a constant current of 20 mA. The protein bands were stained with silver stain (BioRad, CA, USA>. Analytical isoelectric focusing (IEF) was performed using ultrathin acrylamide geIs, pH range 3.5-9.5, supplied by LKB, in a horizontal Multi-

117

phor system (Pharmacia/LKB, Bromma, Sweden). Bovine serum albumin (Sigma) was used as marker. The gel was stained with Coomassie blue.

Biological tests In rlitro: radioreceptor binding assay.

LHRBI activity of the samples was assessed by their ability to inhibit the binding of ‘251-human chorionic gonadotropin (hCG) to porcine granulosa cells in vitro. Commercial, iodinated hCG (specific activity > 50 pCi/pg) was purchased from NEN, Du Pont (Dreieich, Germany). According to the manufacturer’s report, highly purified hCG was iodinated using carrier-free 12’1 by the Hunter and Greenwood (1962) method and purified by ion exchange column chromatography. Fresh porcine ovaries were obtained from the local slaughterhouse. Follicular fluid was aspirated and the granulosa cells were harvested from the fluid. The cells were washed 3 times with 0.05 M TrisHCl buffer pH 7.0 containing 0.1 M sucrose, 5 mM magnesium chloride and 0.1% egg albumin. The washed cells were resuspended in the above buffer to give a final concentration of approximately 10” cells (counted by Coulter counter; Krefeld, Germany) in 100 ~1 buffer. For the assay 100 ~1 of the cell suspension together with 20 ~1 of 12”1-hCG (ca. 12,000 cpm per tube) and 100 ~1 of buffer or test samples were incubated for 1 h at 37°C in a shaking water bath. Following this 1 ml of cold Tris buffer was added to the incubation tubes, centrifuged at 1500 x g for 15 min at 5°C. The supernatant was decanted and the radioactivity bound to the pellet was counted in a gamma counter (Berthold, Germany). This was defined as the total binding. Nonspecific binding of the labelled hCG was calculated by adding IOOO-fold excess unlabelled hCG to some tubes. The specific binding was obtained by deducting the nonspecific binding (NSB) of “‘1-hCG from the total binding to the cells.

Effect on steroid secretion: short-term incubation. The effect of the test samples on estradiol (E,) and progesterone (P> secretion by porcine granulosa cells in vitro was tested as follows. The granulosa cells from 5-12 mm follicles were harvested as described above. They were washed and resuspended in Dulbecco’s phosphate buffered saline (DPBS; Sigma) containing 0.3% bovine

serum albumin. Nearly 90% of the cells thus prepared were found viable as checked by trypan blue dye exclusion. l-2 X 10h cells/tube in the presence or the absence of the test samples with or without hCG (10 IU/tube: Pregnesin, Serono; 5000 IU = 500 pug hCG) were incubated in a total volume of 500 ~1 for 3 h at 37°C in a shaking water bath set at 120 oscillations per minute. All treatments were performed in triplicates. Following incubation, the tubes were centrifuged at 1500 x g for 15 min at 5°C the supernatant recovered and kept frozen until assayed for P and E, by radioimmunoassays. Cell viability following incubation was checked again by the trypan blue exclusion method. Long-term incubation. The purified sample was also examined for its ability to inhibit hCGstimulated P and E, secretion by bovine granulosa cells in vitro. Granulosa cells from fresh bovine ovaries obtained from the local slaughterhouse were processed as described above for porcine ovaries. The washed cell pellet was resuspended in serum-free Dulbecco’s minimum essential medium (DMEM; Sigma) supplemented with 50 pg/ml penicillin, 50 pg/ml streptomycin and 0.3% bovine serum albumin (bSA). The cells were incubated in 24-well tissue culture plates (Nunc, Wiesbaden, Germany) at 37°C in a humidified chamber under an atmosphere of 95% air and 5% CO,. Each well contained approximately 4000 viable cells in a final volume of 0.6 ml. The assay samples were dissolved in the culture medium and added to the culture wells. The cells were incubated in the presence of hCG (10 II-l/well) for 24 h after which the medium was withdrawn and the amount of P and E, accumulated in the medium estimated by radioimmunoassays. In ciao: effect on oculation. Using the experimental design described earlier (Chari et al., 1982) the effect of OAUB on hCG-induced P and ovulation in FSH-primed, immature, Wistar-derived rats was tested. A priming dose of 7 IU of human follicle-stimulating hormone (FSH; Urofollitropin, Serono) was administered S.C. into rats on day 26 of birth. Starting immediately thereafter, the test sample or equivalent amounts of bovine serum albumin (bSA)/saline (control group was administered i.p. in a total of five or

I IX

seven injections at 12 h intervals. One hour after the last injection of the sample or bSA, 10 IU of hCG (Pregnesin, Serono) was administered i.p. to all the rats. In one series of experiments, the total dose of the sample or bSA was given in a single injection 1 h prior to hCG. Nine hours after hCG the rats were bled. The fallopian tubes were flushed and the number of ova released was counted. The serum was analysed for P and E, by radioimmunoassay. Radioimmunoassays of the steroids Radioimmunoassays of P and E, were performed using commercial kits (ICN Biomedicals; CA, USA). Both steroids were estimated in the samples without extraction according to the manufacturer’s instructions. Sensitivity of the assay for E, was 10 pg/ml and for P 0.2 ng/ml. The inter- and intra-assay variations were 4% and 6% for Ez, and 7% and 9% for P respectively. Statistical analysis Values are presented as means and their associated standard errors. Statistical significance was evaluated by Student’s t-test, the level of significance being set at p < 0.05. Results The major part of the proteins was eluted in the void volume of the G-50 column followed by two small broad peaks (Fig. 1). LHRBI activity was found in the first (V,,) and the third peak ( < 5 kDa). For further purification, however, only the macromolecular proteins were processed. By anion-exchange chromatography the biological activity could be recovered in the fraction eluted with 1 M NaCl in the buffer (DE-1M). Orange A dye chromatography of DE-IM resulted in two protein peaks, one being unbound (= 70%) and the other bound to the dye. The activity was found in the unbound fraction (OAUB). Metal chelate affinity chromatography of OAUB resulted in nearly 60% of the material being bound to the gel. The biological activity was again associated with the unbound proteins (CuUB). Following RP-HPLC of CuUB on a C,, column, the activity was recovered in the major peak eluting at the 13th minute. A typical elution profile of RP chromatography is shown in Fig. 2. Rechro-

160

100

SO

0

Vol (ml x 10)

Elullor

Fig. 1. A typical elution diagram of bovine corpus luteal crude extract (= 5 g) on Sephadex G-SO. Elution with 0.05 M ammonium acetate buffer pH 7.6. Flow rate 100 ml/h, 10 ml/fraction. V,, = 398 ml and V, = 1200 ml. Shaded area (fraction Nos. 25-50 and 103-l 12) showed biological activity.

matography of this peak on an HPLC-TSK column yielded a homogeneous peak eluting at 16.3 min (Fig. 3). Isoelectric focusing of CuUB along with marker proteins (FMC/Biozyme Diagnostic, Hameln, Germany) showed the pI of CuUB in the range of 5.0-5.5 (data not shown). Therefore, for the final isolate (TSK), bSA was used as a marker. The pl of TSK was found to be around 5.2 (Fig. 4). Following SDS-PAGE under reduced conditions, the final isolate appeared as a single band migrating close to the dye front. The estimated molecular weight (M,) of TSK was about 15 kDa (Fig. 5).

0.3

L-+J! I

,/”

0.2

/1/111-1/

::: i. ,

:w

.:.:,

0.1

*;::

/”

/

*ii;;

:i:i:;‘\>

0 0

10

6

20

16

Retention -

El”. ,,rollle

the -

gradlent

Fig. 2. RP-HPLC on CuUB. Vydac TP C,, (250x4.6 mm). Buffer A: 0.1% TFA in water. Buffer B: 0.1% TFA in acetonitrile/water 80:X). Gradient: O-100% B in 20 min. Flow rate: 0.8 ml/min. X-axis: retention time in minutes; Y-axis: absorption at 280 nm. Biological activity found in the shaded area (fraction Nos. 12 and 13).

1lY

Biological activity in vitro: binding inhibition tests The specific binding of labelled hCG to lo6 granulosa cells under the conditions described was 35 f 2% (n = 20). A dose-related inhibition of the specific binding of hCG to the cells by fractions at various stages of purification is shown in Fig. 6. The yield and the specific activity of the purified fractions are shown in Table 1. An 8000-fold increase in LHRBI activity was found in the TSK sample. Employing the method described by Yang et al. (1976), the possibility of the inhibitory activity being due to damage of the hCG molecule by LHRBI, or due to soluble hCG receptors, was examined. Porcine granulosa cells were incubated with ““I-hCG in the presence or absence (controls) of CuUB (50 pg) as described above. The free or unbound radioactivity following incubation was chromatographed on Sephadex G-100 (0.6 X 32 cm column) equilibrated with 0.05 M Tris-HCl buffer pH 7.5. The unbound radioactivity in the control (receptors + ‘251-hCG only) eluted in one major peak with a V,/V, ratio of 1.64 (intact ‘251-hCG) followed by a very small peak (less than 2% of the total unbound radioactivity loaded on the column) with a V,/V, ratio of 2.3. The elution profile of the unbound radioactivity in the treated group (receptors + 50 pg CuUB + ‘2sI-hCG) was not different from that of the control. Further, there was no additional radioactive peak eluting before the intact ‘251-hCG

m-m

gin

+l ‘SK

+

Fig. 4. Horizontal isoelectric focusing of TSK using 0.5 mm acrylamide gel, pH range 3.5-9.5. Running time 2 h at 750 V and 20 mA. Lane 1: bovine serum albumin ( = 10 pg loaded); lane 2: TSK ( = 50 ng loaded). TSK sample was restained with silver stain.

peak as would be expected for a hCG-receptor complex.

01

0

5

10

15

20

25

Retsntlon time 0.4ln.l

Fig. 3. HP-molecular sieve chromatography of the active peak from Fig. 2. Column: TSK-Ultraspherogel SEC 3000, 300 x 7.5 mm. Eluent: 0.1 M ammonium acetate buffer pH 7.6. Flow rate: 0.8 ml/min. Fraction No. 16 showed activity.

Effect on E, and P secretion in clitro At doses between 6 and 60 pg of protein, OAUB effected a significant decrease in the basal levels of E, secreted by porcine granulosa cells in vitro. The highest dose effected a 64% reduction in E, secretion (p < 0.001). A significant inhibition of P was effected only by 60 pg of OAUB (p < 0.005; Fig. 7). CuUB at 5 and 20 pg doses highly significantly suppressed basal as well as hCG-induced E, and P secretion by porcine



60 I

i

116 K 97.4K 66.2

K

0

III~IU

10

UT,,

100

I,,,,,,

1000 Proleln

45.OK

TSK

+

C”“B

--c

OAUB

I

ioooo cont. -s

I

,m~Tr,-mnT,

1000000

100000

(ng)

Q-Seph

*

t-50

-g

Extr*Ct

Fig. 6. Inhibition of the specific binding of ‘251-hCG to porcine granulosa cells in vitro by various samples. Each point is the mean of triplicate determinations and the variation was less than 3% of the mean. Under the conditions described in the text, the specific binding of ‘*‘I-hCG to the receptors (IO” cells) was 35 f 3% and the NSB was 4 k 1% 01 = 10).

31.OK 21.5K

* -TSK

144K

Fig. 5. Vertical SDS-PAGE of the TSK fraction (Fig. 3) on a 12% gel. Run time: 50 min at 200 V and 20 mA. After fixing, the gels were silver stained. Lane 1: marker proteins ( = 200 ng loaded); lane 2: TSK sample t = 50 ng loaded).

TABLE

1

RELATIVE

ACTIVITY

Sample

OF BOVINE Yield ” (mg/lOO

Extract Sephadex DE-I M OAUB CuUB TSK

Fig. 7. Effect of OAUB on porcine granulosa cell (2x IOh) production of E, and P in vitro. Vertical lines in the bar indicate i SEM.

666.0 G-50

283.0 34.0 19.0 2.1 0.4

CORPUS

g CL)

LUTEAL

(CL) LHRBI Relative (U/mg)

activity

h

Purification (fold)

0.30 (ED,,, 3,300) 0.58 2.50 20.00 50.00 2,500.OO

(ED,,, (EDS,, (ED,,, (ED,, (ED,,,

” Yield in mg protein per 100 g wet weight of corpus luteum. h Relative activity is the amount of protein needed to effect 50% inhibition in vitro. The effective dose in pg protein is given in parentheses.

1,700) 400) 50) 20) 0.4)

(ED,,,)

of “SI-hCG

_ 1.94 8.25 66.00 165.00 8,250.OO

binding

to porcine

granulosa

cells

TABLE

2

EFFECT PORCINE

OF CuUB ON STEROID SECRETION GRANULOSA CELLS IN VITRO

All treatments were incubated

were done as described

in triplicates. in the text.

Ix 10h cells/tube

Treatment

E, (pg/mll

P (ng/mll

CuUB

75 * 2.0 42+ 1.2 27+ I.15 12Sk3.5 9lk I.2 21 i0.6

4.0+0.17 2.8 * 0.06 1.3k0.2 12.0 * 0.87 7.3 f 0.29 4.2i0.21

O@cg 5fig 20 CLg 10 IU

hCG hCG + CuUB hCG + CuUB

5 fig 20 CLg

BY



Protein I

Eslradio,

Ea

looonp

ng (ngl Progesterone

Fig. 8. Effect of TSK on Ez and P secreted by bovine granulosa cells (= 4000 cells) incubated for 24 h. All treatments were done in the presence of hCG (IO lU/well) in triplicates and the variation was less than 2? of the mean.

granulosa cells (p < 0.001: Table 2). Cell viability following incubation in the control as well as the treated groups was found to be greater than 65%. The effect of the final isolate (TSK) on steroid secretion by bovine granulosa cells incubated for 24 h is shown in Fig. 8. A dose-related inhibition of hCG-induced P and E2 production was effected by 500 and 1000 ng of TSK (p < 0.0005). In rir?o: effects on P secretion and ovulation In vivo, hCG given at 48 (series a and b) or 72 h (series c, Table 3 and Fig. 9) after FSH resulted in an increased production of P. The total dose of 250 pg of the sample (OAUB/CuUB) administered in five or seven injections at 12 h intervals prior to hCG effected a highly significant (p < 0.0005) inhibition in serum P levels (Table 3; Fig. 9). Estradiol levels in the control as well as in the TABLE

500

0

Control

I

(01

OAUE (60 111)

Progelterone m

Nr.Oocytes

Fig. Y. Effect of OAUB on the serum levels of P and the number of ova shed into the oviduct in immature, gonadotropin-stimulated rats. Vertical bars indicate k SEM of three experiments, n = IO/group per experiment,

3

EFFECTS

OF LHRBI

All groups

received

Series

ON P SECRETION

AND OVULATION

FSH on day 26. bSA-injected Treatment

groups

served

IN FSH-PRIMED

P

$ i: 01 = 7)

c ** (n = 12)

* hCG injected

SO +g bSAx5 at I2 h interval 50 pg CUUBXS at I2 h interval 250 FL: bSA x I 250 wg CuUB x I

69.5 + 4.5

250pgbSAxl

88.5 & 4.6

250 wg CuUB x 1

79.4 * 3.4

48 h after FSH: * * hCG injected

RATS

No. rats ovulated (oocyte No.)

(ng/ml) a ‘: (n = 9)

IMMATURE

as controls.

43.2i4.2

***

78.2k3.5 69.4k5.8

72 h after FSH: * * * p < 0.0007.

2 (X.5 * 3.5) I (9) 0 1 (13) 10 (44.8k8.1) 9 (32.9 i 8.6)

122

treated groups in all experiments were in the range of 65-80 pg/ml. Ovulation occurred only in 24% of the rats (control group) in response to hCG given 48 h after FSH. On the other hand, in three different experiments conducted (n = 10,’ group per experiment), hCG injected 72 h after FSH induced ovulation in more than 90% of the rats. In the group treated with OAUB in seven injections prior to hCG only 20 + 10% of the rats were ovulatory. Furthermore, the number of ova released in these rats was also highly significantly ( p < 0.001) reduced (Fig. 9). A single administration of the sample prior to hCG did not significantly alter the ovarian responses to hCG (Table 3). Discussion Using a procedure modified from the one described for porcine LHRBI (Kwok et al., 1986), it has been possible to obtain an 8000-fold purified LHRBI activity from bovine corpus luteum. Although the activity was found in more than one protein peak (Sephadex G-50 filtration, Fig. I), in view of the meagrc yield of the third peak, we have processed only the macromolecular proteins for further purification. However, the existence of different molecular species of LHRBI activity needs further examination. In the ion-exchange chromatography, the 0.5 and 1.0 M NaCl fractions were pooled as they were equally potent, and in later experiments, the 0.5 M step was deleted. The metal chelate affinity chromatography (MCAC) step employed in this study exploits the affinity of the proteins for metal ions immobilised by complex formation with a chelating ligand, such as iminodiacetic acid. This method has been successfully used for the purification of membrane proteins, interferons, nucleotides, serum and plasma proteins (see review by Arnold, 199 I). MCAC has proved to be advantageous for LHRBI purification, in that the activity recovered in the unbound (CuUB) fraction revealed only one major and two minor components when tested by SDS-PAGE (data not shown). Further purification of the substance could be achieved by two steps of HPLC. The IEF of the final isolate indicated LHRBI to be an acidic protein. An enrichment of biological activity after each step

of purification is evident in Table 1 and Fig. 6. The tissue extracts of bovine lung and kidney prepared in a similar manner did not show any LHRBI activity indicating that the activity is unique to the corpus luteum. The identical elution profiles revealed by Sephadex G-100 chromatography of the unbound radioactivity in the control and the treated groups exclude the possibility of the LHRBI activity being due to the degraded hCG molecule or due to the soluble receptors. In addition to direct inhibition of LH binding to granulosa cells, murine, porcine (for a review see Tsafriri, 1988) and ovine (Dhir et al., 1989) LHRBI has been shown to inhibit the LH-stimulated, but not the basal production of progesterone/CAMP by rat ovarian slices/ granulosa cells in vitro. Interestingly, in the present study the bovine LHRBI was capable of inhibiting both LH-induced and the basal secretion of Ez and P by porcine and bovine granulosa cells in vitro. Several factors, such as species differences in the source and the target cells as well as the stage of purification of the test sample may be responsible for this discrepancy in the biological charactcristics. Furthermore, it is also likely that the shortterm steroid production by pooled granulosa cells from medium and preovulatory follicles is influenced by previous exposure to endogenous LH and that this event is also being inhibited by LHRBI. The results, however, further rule out the possibility that the LHRBI activity may be due to internalised hCG-receptor. It was also seen that the inhibitory effects on both E, and P were more pronounced with increasing purification of the inhibitor (Table 2 and Fig. 8). This cannot be attributed to a possible toxic effect of the test sample since the cell viability following incubation in the treated groups was not different from that of the controls. It is likely that other contaminants in the less purified substance (OAUB) interfere with LHRBI activity. We reported earlier that inhibin-like material from human (Chari et al., 1985) and bovine (unpublished data) follicular fluid was also capable of suppressing E, secretion by porcine granulosa cells in vitro. That the corpus luteum is also a major source of inhibin has been observed by Davis et al. (19871, Tsonis et al. (1987) and

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McLachlan et al. (1989). These observations point to the need for checking LHRBI for inhibin-like activity. Of interest is a recent report by Aten and Behrman (1989). They observed that a gonadotropin-releasing hormone binding inhibitor (GnRH-BI; 16 kDa) purified from bovine ovaries also inhibited gonadotropin-stimulated function of rat ovarian cells. On the basis of their amino acid composition and the partial sequence, they identified the inhibitor to be histone H,A. The identity of the bovine ovarian inhibitor reported by the above authors and the one in the present study, is questionable in view of the acidic nature of the corpus luteum LHRBI. Any conclusion, however, regarding the homology of the bovine LHRBI with the other peptides discussed above should await information on the amino acid composition and sequence of the inhibitor. In immature rats, treatment with pregnant mare’s serum gonadotropin (PMSG) induces the development of preovulatory follicles which respond to an ovulatory dose of hCG (Peluso et al., 1980; Chari et al., 1982). It is demonstrated here that pretreatment with LHRBI suppressed the hCG-stimulated P secretion and ovulation in the FSH-primed, immature rats. It was also observed that frequent, but not single, administration of the sample was essential to block the ovarian responses to hCG stimulation. At this stage it is difficult to explain this observation due to lack of information on the mechanism of action of bovine LHRBI. Work is underway to examine the timecourse effects of CuUB/TSK in vivo. Channing et al. (1981) demonstrated that administration of crude extracts of porcine corpus luteum could inhibit coitus-induced ovulation in the rabbit. Dhir et al. (1989) observed that i.p. administration of partially purified ovine LHRBI to adult cycling rats on the day of proestrus partially blocked ovulation and the number of ova shed. These in vivo studies suggest a significant role for LHRBI in the regulation of ovarian functions. It ‘is possible that the inhibitor, by intraovarian diffusion into granulosa cells, influences the follicular maturation processes. It is also likely that the luteal inhibitor plays a role in determining the responsiveness of the corpus luteum to LH during the luteal phase. Future studies with purified material should clarify some of the questions regarding the mechanism of its actions.

Acknowledgements We gratefully acknowledge the financial support of Dr. C.E. Geacintov, Marburg. We thank Miss B. Hartmann and Mrs. T. Plaum for performing radioimmunoassays of the steroids, and Mrs. D. Wenz for help with photography. References Arnold. F.H. (19911 Bio/Technology 9, 151-156. Aten, R.F. and Behrman. H.R. (1989) .I. Biol. Chem. 264. 11065-l 1075. Charming, C.P., Batta, SK. and Bae, I.H. (19811 Proc. Sot. Exp. Biol. Med. 166, 479-483. Chari, S., Duraiswami, S., Daume, E. and Sturm, G. (19821 in Nonsteroidal Regulators in Reproductive Biology and Medicine (Fujii. T. and Channing, C.P., eds.), pp. 61-72, Pergamon Press. Oxford. Chari. S., Daume, E., Sturm. G., Vaupel, H. and Schiiler, I. (19851 Mol. Cell. Endocrinol. 41. 137-145. Davis, S.R., Krozowski, Z., McLachlan, R.I. and Burger. H.G. (19871 J. Endocrinol. 115. R21-R23. Dhir, R.N.. Kumar. N., Dhingra. S.R., Kumari, G.L. and Duraiswami, S. (1989) Endocr. Res. 14, 3455364. Hunter, W.M. and Greenwood, F.C. (19621 Nature 194. 495. Kumar. N., Kumari, G.L., Dhir, R.N. and Duraiswami. S. (19891 Endocr. Res. 14, 319-343. Kumari, G.L.. Vohra. S., Joshi, L. and Roy, S. (19801 Horm. Res. 13, 57-67. Kwok, P., Chari. S., Daume, E. and Sturm, G. (19861 IRCS Med. Sci. 14, 1023-1024. Laemlli. U.K. (1970) Nature 227, 680-685. McLachlan, R.I., Cohen, N.L., Vale. W.W., Rivier. J.E., Burger, H.G.. Bremner. W.J. and Soules, M.R. (19891 J. Clin. Endocrinol. Metab. 68. 1078-1085. Peluso, J.J.. Stude. D. and Steger. R.W. (1980) Acta Endocrinol. 93, 505-512. Sakai, C.N., Engel, B. and Channing, C.P. (1977) Proc. Sot. Exp. Biol. Med. 155, 373-376. Tsafriri, A. (19881 in The Physiology of Reproduction, Vol. 1 (Knobil, E. and Neill, J.D., eds.1, pp. 527-567, Raven Press, New York. Tsonis, C.G., Hillier, S.G. and Baird, D.T. (19871 J. Endocrinol. 112. RI 1-R14. Ward, D.N., Liu. W.-K., Glenn, S.D., Channing, C.P. and Sugino, H. (19821 in Nonsteroidal Regulators in Reproductive Biology and Medicine (Fujii, T. and Channing, C.P., eds.1, pp. 227-228, Pergamon Press, Oxford. Yang, K.P., Samaan. N.A. and Ward, D.N. (1976) Endocrinology 98. 233-241. Yang, K.P., Gray, K.N., Jardine, J.H.. Nancy, H.L., Samaan, N.A. and Ward, D.N. (1978) in Novel Aspects of Reproductive Physiology (Spilman, C.H. and Wilks, J.W., eds.). pp. 61-80. Spectrum Publications, New York - London.

Isolation of luteinising hormone receptor binding inhibitor from bovine corpus luteum.

A luteinising hormone receptor binding inhibitor (LHRBI) has been purified from bovine corpus luteum (CL). Steroid-free extract of the CL was subjecte...
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