GENERAL AND COMPARATIVE ENDOCRINOLOGY 41, l-7 (1980)

Effects of Mammalian and Avian Gonadotropins on in Vitro Progesterone Production by Avian Ovarian Granulosa Cells

COLING.SCANES AND JOHN H. FAGIOLI

Department of Physiology, Cook College, Rutgers, The State University. New Brunswick, New Jersey 08903

Accepted October 1, 1979

Chicken ovarian granulosa cells were incubated in vitro following their preparation by collagenase treatment. The secretion of progesterone by the cell suspension was increased (between 2.3- and 3.8-fold) by the addition of either avian or mammalian luteinizing hormone (LH). The effectiveness of chicken gonadotropin fractions in stimulating progesterone re- lease agrees well with their known LH contents. The addition of an antiserum against avian LH, to the incubation, blocked the effect of chicken LH on progesterone release. Mamma- lian FSH induced small (20 to 70%) increases in progesterone secretion.

Recently, the role of gonadotropins in the control of gonadal functioning has been subject to detailed investigation in a number of lower vertebrate species (re- viewed Licht et al., 1977). However, there is not yet complete agreement over the rel- ative importance of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the control of steroidogenesis. In males, there is strong evidence that LH is the more important gonadotropin stimulat- ing androgen production in birds (Maung and Follett, 1977). However in male rep- tiles, FSH appears to be the principal steroidogenic gonadotropin (e.g., Callard and Ryan, 1977; Lance er al., 1977; Tsui and Licht, 1977). In male amphibians LH is the major stimulator of steroidogenesis (Light et al., 1977). The situation in the female appears to be similar: with LH being the principal gonadotropin-promoting pro- gesterone secretion in amphibians, and it being probable that FSH promotes proges- terone and estrogen production in reptiles (Licht and Crews, 1976; Lance and Callard, 1978).

The relative importance of LH and FSH in promoting steroidogenesis by the avian ovary is unknown. In the adult female domestic fowl, the administration of mam- malian LH has been found to increase the

levels of progesterone and testosterone in both plasma and the follicular wall (Shahabi ef al., 1975). While in the laying turkey, cir- culating concentration of estradiol and progesterone are elevated by the injection of either mammalian LH or FSH (Camper and Burke, 1977). Very recently mamma- lian LH has been found to increase proges- terone production by chicken granulosa cells incubated in vitro (Hammond et al., 1978; Huang et al., 1979). The present study describes the effect of preparations of mammalian and chicken gonadotropins on chicken ovarian granulosa cells in vitro.

MATERIALS AND METHODS Preparation of granulosa cells. Ovarian tissue was

obtained from laying hens (White Leghorn strain) at approximately 4 and 8 hr following the last ovi- position. Granulosa tissue was obtained by the method of Gilbert et al. (1977) from the three largest follicles. The method, briefly, entailed the rupturing the follicle with a scapel blade and subsequently removing the granulosa layer from the yolk, under 0.9% saline. The cells were dispersed by collagenase treatment (Maung and Follett, 1977; Lance and Callard, 1977). Tissue from two birds (i.e., six follicles) was incubated for 60 min in 3.4 ml incubation buffer [Krebs-Ringer bicar- bonate, pH 7.4, containing (2.0 mg/ml) glucose, (1 mg/mI) bovine serum albumin, and (120 IU/ml, Sigma Type IV) collagenase]. The enzyme treatment was performed in polystyrene tubes at 37”, under 95% O,, 5% CO,, with shaking (100 cycleslmin). After filtering the mixture through gauze (ca. 80 pm mesh) to remove

1 0016~6480/80/050001-07$01.00/O Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.

2 WANES AND FAGIOLI

tissue pieces, the cells were collected by centrifugation (15 min x 6OOg). The cells were then washed in fresh incubation buffer (4 ml), recentrifuged (15 x 6OOg), and resuspended in 12 ml incubation buffer.

Incubation. The suspensions of granulosa cells were incubated in polystyrene tubes in a shaking water bath (30 cycleslmin). Unless otherwise stated, all incuba- tions were performed in a volume of 200 ~1, at 39.5”, under 95% 0,, 5% CO, for periods of 3 hr. Hormone preparations were added to the incubation in 10 ~1 0.9% saline. At the end of the incubation, the granulosa cell suspensions were centrifuged (10 min x 9OOg) at 4”. After separation, the media were stored at -20” prior to assay for progesterone. The concentra- tion of cells in the suspension was estimated in each incubation run using a hemocytometer. This was con- sistent; the mean being 2.27 5 SEM 0.24 (N = 9) x lo5 live cells per ml. At this time the viability of the cells was also assessed by their ability to exclude trypan blue. In all cases this was in excess of 85%.

Hormones. The avian hormones employed were obtained by previously described methods (Stockell Hartree and Cunningham, 1969; Scanes and Follett, 1972; Godden and Scanes, 1975). The potencies of these are given in Table 1. Fraction GTN contained the extracted glycoprotein adenohypophyseal hormones (LH, FSH, and thyroid-stimulating hormone, TSH). Fraction CM1 (a crude FSH fraction) was separated from fraction CM2 on DEAE-Cellulose. The mam- malian pituitary hormones (NIH-LH-S19 and NIH- FSH-Sl 1) were provided by the National Institute of Health.

Progesferone Assa.v. Progesterone concentrations in

TABLE 1 POTENCIES OF AVIAN GONADOTROPINS EMPLOYED IN

THIS STUDY

Fraction

LH Activity FSH Activity

BY BY BY radioligand radioimmuno- radioimmuno-

aSSay aSSap assay’

GTN 18.5d CM1 0.7’ CM2 39 1’ AEI 1316

2090 12.1’

432 568 c

Not tested* 42’

Not tested <30’

O Potencies in microgram equivalents of ovine LH (NIH- LH-Sl7) per milligram in a radioreceptor assay using rat testes receotors and human LH as tracer.

* Potencies in microgram equivalents fraction IRCZ per mil- ligram by radioimmunoassay (Follett ef al., 1972).

’ Potencies in microgram equivalents fraction DC3 per mil- ligram by homologous radioimmunoassay @canes er al.. 1977).

d Calculated from Follett et al. (1978). ’ Kindly performed by Dr. R. Gibson (see Godden, 1976). ’ From Godden (1976). 0 From Jenkins et al. (1978). * A similarly obtained fraction had a potency of 46 &mg

(Scanes er al.. 1976).

the incubation media were determined by radioim- munoassay. Medium (100 ~1) was extracted with pe- troleum ether (1 ml; 40-60” bp) and the extract dried by flow of nitrogen [mean recovery of progesterone extraction 84.3 2 (N = 5) 2.6%]. The progesterone was taken up in 3 or 5 ml assay buffer. The assay sys- tem employed was that suggested by New England Nuclear for use with their supplied antibody (proges- terone [3H] radioimmunoassay pak). The assay had a quoted cross-reactivity with pregnenolone O.l%, tes- tosterone O.l%, and 17cu-hydroxyprogesterone 4.3%. Briefly, the assay method involved the use of standard or unknown in 100 ~1 assay buffer, 10,000 cpm [ 1,2- 3H]progesterone (New England Nuclear; Specific Ac- tivity 40-60 Ci/mmol) and dextran-coated charcoal for the separation of bound and free.

Statistics. Differences between treatments were determined by Student’s r test or by analysis of vari- ance where appropriate.

RESULTS Effect of Mammalian Gonadotropins on

Progesterone Production Granulosa cells, in the absence of gonad-

otropins, produced progesterone. This was largely similar in the separate incubations, with tissue from different individual hens [mean progesterone released 49.5 + (AI = 6) 7.5 ng/mU105 cells in 3 hr] . The production in three incubation runs is summarized in Table 2. Mammalian LH increased proges- terone secretion by a maximum of between 2.3- and 3.8-fold over basal release. Doses of 100 ng, 1 pg, and 10 pg LH were accom- panied by similar plateau levels of pro- gesterone release. Mammalian FSH was much less effective in stimulating ste- roidogenesis. However, the various doses of FSH consistently and significantly elevated progesterone release by between 20 and 70%, these effects being observed by relatively low doses of FSH.

Effect of Zncubation Time The effect of varying incubation time on

progesterone release was examined in the presence or absence of mammalian LH (100 ng) (Table 3). Progesterone levels in the medium increased with time in an approxi- mately linear manner in the absence of go- nadotropin (Table 3). The secretion of progesterone was significantly (P < 0.001)

AVIAN OVARIAN STEROIDOGENESIS 3

TABLE 2 EFFECT OF MAMMALIAN LH (NIH-LH-S19) AND FSH (NIH-FSH-Sll) ON PROGESTERONE PRODUCTION BY

CHICKEN OVARIAN GRANULOSA CELLS

Treatment (dose/tube)

hz) Run 1

Progesterone released as a percentage of the mean control level + (N) SEM

Run 2 Run 3 Run 4

Control 100 k (3) 1.2” 100 f (4) 4.1” 100 k (3) 1.6’ 100 c (10) 1.7 LH

0.01 - 119 2 (4) 4.6 353 + (3) 15.7*** 219 + (7) 44.4 0.1 - 226 zk (4) 16.1*** 355 + (3) 10.7*** 281 + (7) 26.2*** 1 - 230 k (4) 17.3*** 383 + (3) 11.3*** 295 + (7) 30.7***

10 354 ” (3) 20.7*** 225 2 (4) 7.9*** - 280 t (7) 26.2*** FSH

0.01 - 127 + (4) 21.4 119 2 (3) 0.5*** 123 + (7) 12.3* 0.1 119 + (4) 27.4 123 + (3) 6.1* 121 + (7) 15.9 1 - 171 + (4) 19.0 150 + (3) 1.0*** 166 + (7) 11.1***

10 129 + (3) 6.0*** 138 ? (4) 1.2*** - 134 k (7) 3.3***

fl Control progesterone release 35 k (3) 1.1 ng/ml/105 cells. b Control progesterone 44 2 (4) 3.5 ng/ml/105 cells. c Control progesterone 89 + (3) 1.5 ng/ml/105 cells. * P < 0.05, **P < 0.01, ***P < 0.001 compared to control (by Student’s t test).

increased by LH within 0.5 hr and with as long as 3 hr of incubation. It may be noted that LH appeared to produce a greater stimulation of progesterone release the longer the incubation progressed. (Proges- terone produced first hour: control 24.9, LH 44.4; in second hour: control 9.6, LH 55.0; in third hour: control 17.1, LH 194.8 ng progesterone/ml/IO5 cells/hi-; the in- crease due to LH being 19.5, 45.4, and 177.7 ng progesterone/ml/105 cells/hr for the first, second, and third hours of the incuba- tion, respectively.)

Effects of Mammalian and Avian LH and FSH Figure 1 summarizes the effects of differ-

ent doses of avian and mammalian gonado- tropin fractions (details see Table 1). Chicken LH (fraction AEl) and ovine LH were the most potent preparations for stimulating progesterone production by the granulosa cells in vitro. A less pure avian LH preparation (CM2) was less potent in stimulating steroidogenesis. The crude gly- coprotein adenohypophyseal extract (GTN containing LH and FSH-Stockell Hat-tree

TABLE 3 THE EFFECT OF INCUBATION TIME AND MAMMALIAN (NIH-LH-Sl9) LUTEINIZING HORMONE

(100 ng PER TUBE) ON PROGESTERONE SECRETION BY CHICKEN GRANULOSA CELLS

Time (hr) Treatment

Progesterone released (nglml/lW cells i (N) SEM)

0.5 0.5 1

Control Luteinizing hormone Control Luteinizing hormone Control Luteinizing hormone Control Luteinizing hormone

5.2 5 (5) 24.9

0.2*** + (5) 0.6

24.9 + (5) 1.3 44.4 f (5) 0.9*** 34.5 + (5) 2.3***

101.4 + (5) 6.5 5i.6 -t (5) 3.0***

295.2 t (5) 48.3

*** P < 0.001 compared with control (by Student’s 1 test).

SCANES AND FAGlOLl

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Hormone (ng 1 FIG. 1. The effect of different doses of avian and mammalian gonadotropins on the release of

progesterone from chicken granulosa cells. Mammalian LH (NIH-LH-S19) x-‘-‘-x, mammalian FSH (NIH-FSH-S17) +-.-.-+, chicken LH preparations: -AEl O---O; CM2 A----A, chicken LH and FSH (GTN) O---O and chicken FSH (CMl) O----O were added to the incubations. Vertical bars signify SEM (JV = 3).

and Cunningham, 1969) had lower progesterone-stimulating activity, while the impure avian FSH (CMl) had even lower activity. Mammalian FSH had the lowest ability to increase progesterone production. Estimates were made of the doses of hor- mone fractions which induced a stimulation of progesterone secretion 50% that of the maximum (206 ng/mY105 cells) (NIH-LH 5.0 ng; AEl 5.6 ng; CM2 36.3 ng; GTN 299 ng; CM1 818 ng). Using these data it was estimated that the relative potencies equiv- alents of the gonadotropic fractions were: AEl-892; CM2-138; GTN-16.8; CM1 6.1 pg NIH-LH per mg.

Effect of an Antiserum to Avian LH In this study, chicken LH (AEl) again

stimulated progesterone production by granulosa cells (Table 4). The addition of an antiserum to avian LH tended to depress basal progesterone release. However, the addition of as little as 10 ~1 of the antiserum completely blocked the effects of avian LH on granulosa progesterone secretion.

Effects of Glucose Concentration The ability of the system to withstand re-

ductions in the glucose concentration in the incubation was tested, in the presence or absence of LH. Reduction of glucose con-

TABLE 4 EFFECT OF AVIAN LH (AEl) AND ANTISERUM TO AVIAN LH (ANTI-CM2) ON PROGESTERONE SECRETION

BY CHICKEN OVARIAN GRANULOSA CELLS in Vitro

Treatment Progesterone released

(ng/mYIOs -r- (N) SEM)

Control (10 ~1 normal rabbit sera) 34.0 + (5) 1.9*** LH (50 ng) + (10 ~1 normal rabbit sera) 146.8 2 (5) 11.0 Anti-LH (10 ~1) 30.7 2 (5) 0.5 LH (50 ng) + Anti-LH (10 ~1) 30.7 k (5) 1.7***

*** P < 0.001 by analysis of variance compared to LH alone.

AVIAN OVARIAN STEROIDOGENESIS 5

centrations, from 2 to 1 mg/ml, was accom- panied by a lower production of proges- terone in the production of LH. Over the range of 0.2 to 1.0 mg glucose/ml, there were no significant effects of glucose con- centration on either basal or LH-stimulated progesterone production (Table 5). How- ever, reduction of the glucose level to 0.02 mg/ml (1% of that used throughout the study, and approximately 1% of the cir- culating glucose concentration in the fowl) was accompanied by a strong tendency of reduction in basal (significant by Student’s t test from 2, 1, 0.2 mg glucose/ml control, P < 0.05) and a significant decrease in LH- stimulated progesterone secretion. Indeed, there were no observable effects of LH on progesterone release (Table 5).

DISCUSSION The present study agrees with previous

studies in demonstrating that LH is a potent stimulator of progesterone production in domesticated birds (Shahabi et al., 1975; Camper and Burke, 1977). Furthermore, mammalian LH has recently been shown to stimulate the release of progesterone from chicken ovarian granulosa cells in vitro (Hammond et al., 1978; Huang and Nal- bandov, 1979; Huang et al., 1979). In the present study, preparations of LH from both mammalian and avian sources in-

creased progesterone secretion. Estimates of the relative ability of the avian gonado- tropin fractions to stimulate progesterone production, by granulosa cells, agreed well with their previously established LH potencies, in a radioreceptor system (Table 1). Indeed, there was a very close correla- tion between the estimated “potencies” of the avian gonadotropin fractions or the two systems [r(3) = 0.988, P < 0.011. In addi- tion, a tendency for a correlation between radioimmunoassay and progesterone- stimulating activities was observed [r(3) = 0.7981. A similar relationship between immunoreactive-LH potency for avian go- nadotropins and their activity in an in vitro avian bioassay has been observed [r( 11) = 0.811, P < 0.01; calculated from Jenkins et al., 19781.

Less attention has been placed on the effects of FSH on steroidogenesis in female birds. A high dose of mammalian FSH (10 &ml) has been found to stimulate proges- terone production by chicken follicular granulosa cells in vitro (Huang et al., 1979a,b). These authors did not, however, preclude the possibility that the effect ob- served with FSH fraction was due to the LH contamination. In the present study, mammalian FSH even at low doses pro- duced a small but significant increase in progesterone release. The stimulation of

TABLE 5 EFFECT OF GLUCOSE CONCENTRATION ON PROGESTERONE SECRETION BY

CHICKEN OVARY GRANULOSA CELLS in Vitro

Glucose level tmgiml) Treatment P, (ng/ml/105 cells)

2.0 Control 58.3 t (5) 3.7,,, Ovine LH (100 ng) 196.7 2 (5) 36.5

1.0 Control 72.5 k (5) 15.5*** Ovine LH (100 ng) 137.7 ? (5) 18.2”

0.2 Control 60.0 ? (5) 3.5*** Ovine LH (100 ng) 122.5 2 (5) 7.5”

0.02 Control 25.0 t (5) 2.6 N.S.

Ovine LH (100 ng) 26.7 k (5) 0.8*

U Differs from LH-2.0 mg glucose/ml (P < 0.05). * Differs from LH-2.0 mg glucose/ml: from LH-1.0 mgiml; and from LH-0.2 mgiml (P < 0.001). *P < 0.05, ** P < 0.01, *** P < 0.001, N.S., nonsignificant by analysis of variance.

6 SCANES AND FAGIOLI

progesterone production, by increasing doses of FSH, appeared to reach a maximal level which was much lower than that found with LH. It may be noted that the effective doses of FSH are within the physiological range. Circulating levels of FSH in the adult female chicken have been reported to be in the range 0.1 to 0.6 pg equivalents NIH- FSH-Sl/ml, by bioassay (Imai and Nalban- dov, 1971) or to be approximately 67 ng chicken FSH standard (DC3)/ml by radioimmunoassay. The latter has been es- tablished to be equivalent to approximately 0.45 pg equivalents NIH-FSH-Sl/ml (Scanes et al., 1977). Using a different radioimmunoassay system for FSH, Follett (1976) found a similar FSH concentration on a single sample of hen’s plasma (ap- proximately 50 ng FSH-RP- l/ml; equivalent to 25 ng chicken FSH-DC3/ml). The present studies suggest that although LH is proba- bly the major stimulator of progesterone secretion, the possibility that FSH has some physiological role in this control can- not be excluded.

It is interesting to note that, as in the present study, both LH and FSH have been reported to promote progesterone produc- tion on various lower vertebrate groups. There is strong evidence that in those am- phibians studied (bullfrogs and salaman- ders), LH is the predominant gonadotropin stimulating progesterone secretion (e.g., Licht and Crews, 1976; Licht et al., 1977). In reptiles there are reports that both LH and FSH stimulate progesterone produc- tion by preovulatory follicles. For example, in the turtle (Chrysemys picta), Callard and co-workers have found that only FSH stim- ulates progesterone release by dispersed ovarian follicular cells in vitro (Lance and Callard, 1978). However, both mammalian LH and FSH stimulated progesterone syn- thesis, from [3H]cholesterol by minced preovulatory follicular tissue (increase in progesterone with LH 291%; with FSH 110%) (Callard et al., 1976). In other reptil- ian species, Licht and Crews (1976) have

reported that LH and FSH of either mam- malian or reptilian origin stimulate proges- terone secretion.

ACKNOWLEDGMENTS We are grateful to Dr. A. Gilbert for advise con-

cerning the separation of the granulosa cells, to Dr. P. M. M. Godden for the preparation of some of the gonadotropin preparations, to Dr. R. Gibson and Pro- fessor B. K. Follett for the radioligand assays of the avian gonadotropins, and to the Bureau of Biological Research (Rutgers University) and the New Jersey State Experiment Station for the Financial Support.

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AVIAN OVARIAN STEROIDOGENESIS 7

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