BIOLOGY

OF

REPRODUCTION

28,

591-597

(1983)

Biphasic

Effect

of

Gonadotropin Releasing by Rat Granulosa

C. EKHOLM, H. BILLIG,

Hormone Cells
C. MAGNUSSON

on Progestin

Secretion

C. S. SHEELA

RANt,

and T. HILLENSJO2

Department University S -40033

of Physiology of GOteborg Goteborg, Sweden

ABSTRACT The effect of an agonistic gonadotropin Gly#{176}-NH3 -GnRH -ethylamide, GnRHa) on absence of follicle-stimulating hormone (FSH) releasing granulosa or luteinizing hormone (GnRH) cell steroidogenesis hormone (LH) -analog (D -Ala ,desin the presence or was studied. Granulosa

cells, isolated from preovulatory follicles of pregnant mares serum gonadotropin (PMSG) -treated immature rats or from the less mature follicles of untreated immature rats, were cultured for a period of 72 h with daily changes of medium, and progesterone and its metabolite, 20a -dihydro progesterone (20o-OHP), were assayed in the medium. In granulosa cells from preovulatory follicles, LH and FSH caused a much greater stimulation of steroidogenesis than did GnRHa. There appeared to be no interaction between GnRHa and FSH during the first 10 h, but at 24 h and later the presence of GnRHa clearly inhibited the steroidogenic response to LH and FSH. Steroidogenesis in granulosa cells from immature rats was considerably lower and the effects of GnRHa and FSH alone less pronounced. In these cells, FSH #{149}stimulated progesterone secretion was inhibited by GnRHa only at 72 h. In contrast, 20o-OHP secretion in the same cultures was potentiated by the combined presence of FSH and GnRHa. In conclusion, it seems as though the effects of GnRHa on granulosa cell steroidogenesis varies with exposure time, the initial response being stimulatory and the later inhibitory. Further more, the response is also to some extent determined by the maturational stage of the granulosa cells.

INTRODUCTION

In addition

to its

their potent

effects to

on synthetic

the

pituitary, analogs

GnRH usually

for a period of not less than 2 days, in the presence of FSH. On the other
from
-

GnRH
(GnRHa) on the 1981;

and are gonads

known (reviewed

have direct actions by Hsueh and Jones,

hand, stimulatory granulosa cells intact, for short not PMSG

effects were preovulatory rats (up to these response

observed when follicles of were incubated

treated

Sharpe, 1982). There have been reports of both stimulatory (Clark et al., 1980; Ekholm et al., 1981, 1982; Clark, 1982) and inhibitory

alone,
did a!.,

periods and under modify the

5 h)
conditions to FSH

with

GnRH
GnRH

or LH. of in-

(Hsueh

and

Erickson,

1979,

Hsueh

et

1980; Knecht on granulosa

different these were treated model studies. observed, rats

et al., 1981) effects cell steroidogenesis.

systems Thus, granulosa
had

of GnRH However,
used effects immature (DES) exposed to
-

It seemed granulosa cells hibition)

likely that to GnRH with time.

the response (stimulation or granulosa present

have cells

been from
and

in

used

and

varies with

type of In the

cell study,
granu-

when

inhibitory

carried out to test these possibilities, losa cells of different stages were for up to

hypophysectomized, been

diethylstilbestrol
used

3 days
and absence

with
was

GnRHa,
of FSH

cultured both in the

LH.

presence

or

The pro(20z
-

culture gesterone Accepted Received

medium and

20a

changed daily dihydroprogesterone

and

November 4, 1982. August 9, 1982. address: Dept. of of Bangalore Training

OHP)
Biochemistry,
560012, India. Grant from the

were

assayed

in the medium.

Indian
Sup
-

Institute ported

Science, by a Research Health Organization. 2Reprint requests:

World
Animals and

MATERIALS Culture

AND Procedure

METHODS

Dr. Physiology, University of S-400 33 Goteborg, Sweden.

T. Hillensj#{246}, Dept. of Goteborg, Box 33031,

Twenty-eight-day-old, rats (Anticimex Ltd.,

female Stockholm)

Sprague -Dawley were used either

591

592

SHEELA

RANI

ET AL.

20

GnRHo

llllhlkflt
,

10

Mi

#{149} GnRHa.Anf

NH3) GnRH-ethylamide, Sigma Chemical Co., St. Louis, MO) were diluted with culture medium to the desired concentration before use. GnRH antagonist ((D-pGLU, D-Phe2, D-Trp3J LRF, Boehringer-Mannheim Biochemicals, Indianapolis, IN) was dissolved in phosphate-buffered saline prior to use and diluted with culture medium. 3H-labeled progesterone and 20a-OHP used for radioimmunoassay (RIA) were from New England Nuclear, Boston, MA. Other chemicals were purchased from Sigma Chemical Co. Steroid Assay

20

15

Radioimmunoassay for progesterone and 20oOHP employed specific antisera (Lindner and Bauminger, 1974) kindly donated by Drs. Lindner and Kohen, The Weizmann Institute of Science, Rehovot, Israel. RIA was performed without prior extraction after dilution of the culture medium with et al., distilled 1981). water as described previously (Hillensj#{246}

II
1iI
FIG. 1. Effect of GnRHa (100 ng/mI) and GnRH antagonist (Ant, 10 Mg/mI) on progestin accumulation during a 24-h culture of granulosa cells obtained from PMSG - treated rats. Mean SEM of 5 observa tions are shown. xx=P<0.01 vs. control (C).
-

Statistical Analyses

The data presented are means SEM from at least three different experiments, the number of replicates in each experiment being 4-6. Analysis of variance was followed by Student-NewmanKeuls test and a P value less than 0.05 was considered significant.

RESULTS

Granulosa
without any pretreatment or after a s.c. injection of 10 IU PMSG 2 days earlier. The rats were killed by cervical dislocation, the ovaries removed, cleaned of adherent tissue and placed in sterile culture medium (see below). The follicles were incised with a microknife under a stereomicroscope and the granulosa cells expressed into the medium. Cells were collected and washed once with fresh medium after centrifugation for 5 mm at 50-100 X g. After determining the number of cells and checking their viability with the crypan blue dye exclusion test (50-60% viable cells in each experiment), the cell suspension was pipetted into multi -well culture plates (Falcon). Approximately 70-100 X io granulosa cells were cultured in 500 sl medium. Oocyte-cumulus complexes were collected separately and distributed 10 per well in Microtest plates (Falcon), 200 Ml final volume. The cultures were kept at 37#{176}Cin humidified air for 10 or 72 h with changes of media at 24 and 48 h.

Cells

from cells caused

PMSG cultured a

Treated for 24

Rats h, GnRHa increase in

In granulosa (100 ng/ml)

2.5 -fold

progesterone and a 4-fold increase in 20aOHP secretion compared to control cultures (Fig. 1). Other experiments showed that GnRHa concentrations between 1 and 100
ng/ml of produced similar while 0.1 levels ng/ml of had stimulation no signifianalog activity, progesterone,

cant effect of GnRH

(Table 1). The antagonistic (10 i.eg/ml) had no intrinsic

but
GnRHa

when

present
abolished

together
the

with

GnRHa

it
by

completely

stimulation

Culture

Medium
minimal essential medium mM Hepes, 10% fetal bovine (50 g/ml) was used. and Cbemicals (0.1 Mg/mi) and GnRHa of LH (NIH-S19), ((D-Ala,des-Gly10with
serum

Eagles salts, 10 gentamicin Hormones

Earles and

(Fig. 1). GnRHa stimulated progesterone secretion with a maximal response at 24 h, but a stimulation could already be seen at 10 h (Fig. 2). FSH and LH (100 ng/ml) both caused a more pronounced stimulation of progesterone than did GnRHa at all times of culture. The rate of progesterone secretion declined with time and the amount of progesterone secreted in response to FSH and LH was lower on Days 2 and 3 of culture than during the first 24-h period. Interactions between GnRHa and

Stock
FSH

solutions (NII-I-S13)

FSH

EFFECT

OF

GnRH

ON

GRANULOSA

CELL

PROGESTIN

SECRETION

593

50
DC

a a

#{149}
LH

lal

z
0

I-, c/)

o5

I:

GnRHO

-+

-+

-+

-+

-+

-+

-+

-+

-+

-+

-+

O-IOh

O-24h

24-48h

48-72h

FIG. 2. Progesterone accumulation during The cells were cultured in control medium (C), (+) GnRHa (100 ng/ml). The cultures designated were continued for 72 h with changes of media vs. corresponding control (without GnRHa and without GnRHa, and =P<0.01.

culture of granulosa cells obtained from PMSG - treated rats. with FSH (100 ng/ml) or LH (100 ng/ml), without (-) or with 0-10 b were terminated at 10 h, whereas the other cultures at 24 and 48 h. Mean SEM of 6-15 observations. xP<0.05 gonadotropin). xxP<0.01, *p<)()5 vs. corresponding group

LH were studied by combining GnRHa either gonadotropin from the start of culture. The progesterone accumulation during 10 h of culture with FSH was not different when GnRHa also was present. However, at 24 h GnRHa significantly reduced the gonadotropin - stimulated progesterone produc tion (Fig. 2). Maximal inhibition of FSHstimulated progesterone secretion (60%) had developed after 48 h and was sustained at 72 h. Significant inhibition of the FSH response at 24 h was obtained with GnRHa at 10 ng/ml while lower concentrations were ineffective (Table 1), but on Day 2 significant inhibition was obtained with 1 ng/ml (40% inhibition). The pattern of 20a-OHP secretion in response to hormone stimulation resembled that of progesterone, although the levels were severalfold higher (Fig. 3). GnRHa caused a maximal stimulation at 24 h, but after the stimulation declined and ceased at 72 h. FSH caused a moderate stimulation at 10 h, and at 24 h a pronounced stimulation was seen
and

or

with

both

gonadotropins,

sustained

at

later

times. GnRHa

No interaction was found and FSH during the first

between

48

of

TABLE 1. GnRHa on 24-h culture treated rats. GnRHa (ng/ml) 0 0.1 1.0 10.0 100.0 aValues servations.

Effect of progesterone of granulosa

different concentrations of accumulation during a cells obtained from PMSG -

FSH

FSH 49.0 42.1 39.6 28.8

(100

ng/mI)

5.6 6.4 11.8 11.5 14.2 shown

o.loa

3.33

0.38
121b 090b 1#{149}08b the mean

3.62 2.95 329b of 4-5 ob-

are

SEM

bp<ooi
0 ng/ml) formation. cp<OOS with

versus

appropriate of variance

control after

(GnRHa, log trans

-

analysis

594

SHEELA

RANI

ET AL.

DC

#{149}
LH
a a

a
a

-+

-+

-+

-+

-+

-+

-+

-+

-+

-+

-4.

0-0h
FIG. 3. 20a -OHP accumulation during conditions were the same as given in Fig. gonadotropin). =p<0.05 vs. corresponding

0-24h

24-48h
PMSG group

48-72h
-treated (without rats. Culture GnRHa and

culture of granulosa cells obtained from 2. xx=P<0.01 vs. corresponding control group without GnRHa, and =P<0.01.

culture

but

72
by more

h. The
GnRHa

a slight LH response
at 48 h

inhibition

occurred

at

was and

already

inhibited

this
3).

inhibition

was

pronounced Cells

at 72 h (Fig.

lated progesterone secretion at 48 h or later. 20a-OHP was stimulated by GnRHa for 48 h. However, the combination of GnRHa and FSH caused a synergistic stimulation after 24 h of culture (Fig. 4).

Cumulus

Cumulus cell mucification and progesterone secretion were initiated by FSH (10-100 ng/ml) as reported earlier (Hillensj#{246} et al., 1981). No effect of GnRHa (10-100 ng/ml) was found on any of these parameters under basal or FSH-stimulated conditions at culture times varying between 10 and 48 h (data not shown).
Granulosa Granulosa rats the of Cells from Immature Rats

DISCUSSION

In

the

present

study,

GnRHa

stimulated

had
effects

cells from untreated immature low rate of steroidogenesis and

the hormones were less procaused a transient stimulation secretion (Fig. secretion period, 4), while FSH during the this response

nounced.
stimulated entire

of GnRHa

progesterone

progesterone 3-day culture

both progesterone and 20a-OHP production by granulosa cells from both preovulatory and immature rat ovaries during the first 24-48 h of culture but not later. With increasing culture time, at 24 h and beyond, GnRHa partly inhibited the progesterone secretion stimulated by FSH or LH. Thus, GnRHa appears to have a biphasic or timedependent effect on granulosa cell progestin production. The finding that GnRHa by itself can cause stimulation of progesterone production in rat granulosa cells in vitro is in accordance with earlier reports on preovulatory rat granu
losa reports cells or included whole only follicles. short-term These (up earlier to 5 h)

being increased between 48 and 72 h. Presence of GnRI-la decreased the gonadotropin -stimu-

exposures

(Clark

et

al.,

1980;

Ekholm

et al.,

EFFECT

OF

GnRH

ON

GRANULOSA

CELL

PROGESTIN

SECRETION

595

GnRHa 2000-

Uffihl FSH
Lai

z
0

1500

GnRHaFSH

Co..,. 0 0

1000

500

r*I
0-24 h

r
24-48h

48-72h

U cF
1W

I-

cJ

O-24h

24-48h

48-72h

FIG. 4. Accumulation of progesterone (top) and 20o -OHP (bottom) during culture of granulosa cells obtained from untreated immature rats. The cells were cultured without hormone (C), or with GnRHa (100 ng/ml) and/or FSH (100 ng/ml). The media were changed after 24 and 48 h of culture. Mean SEM of 10-12 observa tions. x=P<0.05 vs. corresponding control group, and xx=P<.01, =P<0.01 vs. FSH alone.

1981; Clark, 1982; Hillensjo et al., 1982). Recently, an intrinsic stimulation of basal progestin secretion by GnRH was also reported for immature granulosa cells cultured for 2 days (Jones and Hsueh, 1982a,b). In one of these studies (Jones and Hsueh, 1982b), GnRH was found to have a stimulatory effect on basal pregnenolone synthesis while it inhibited the FSH stimulation on this parameter.

Furthermore, these authors suggested that the stimulatory effect required higher concentrations of GnRHa than did the inhibitory effect. We observed that during the first 24 h

of culture the concentration of GnRHa required to elicit its stimulatory effect on basal progesterone was lower than that required to elicit its inhibition of the FSH response. However, lower concentrations were inhibitory after prolonged culture. The inhibitory effects of GnRHa on granulosa cell steroidogenesis have previously been reported mainly for the FSH-responsive system, consisting of cells from immature, hypophysectomized, DES-treated rat ovaries cultured for 48 h or more (Hsueh and Erickson, 1979; Hsueh et al., 1980; Jones and Hsueh, 1981a; Knecht

596

SHEELA

RANI

ET AL.

et al., 1981, 1982). In the present study, it was shown that GnRHa causes an initial stimulation followed by an inhibition of the gonadotropin-induced progesterone synthesis. This was observed both for immature granulosa cells being responsive to FSH and for mature cells being responsive to both FSH and LH. Interestingly, it has been reported (Knecht and Catt, 1981; Knecht et al., 1981) that in a culture system similar to that employed by Hsueh and co workers (namely granulosa cells from immature, hypophysectomized, estrogen-treated rats), FSH causes a biphasic effect on cAMP production with an acute increase by 3 - 5 h and a second increase after 24 h. GnRHa, when present along with FSH, was found to have no effect on the early rise in cAMP, while it prevented the late rise. This late inhibition was prevented by the phosphodiesterase (PDE) inhibitor MIX (methyl isobutyl -xanthine), suggesting that the late inhibition of cAMP by GnRHa involves activation of PDE. That such an activation of PDE does occur in granulosa cells cultured with GnRHa and FSH has been demonstrated (Knecht and Catt, 1981). It is possible that this can explain also the late inhibition of steroidogenesis that we found. In all studies, including the present one, where GnRH stimulation of steroidogenesis has been observed, the magnitude of response has been much smaller than the comparable response to a gonadotropin, implying different mechanisms of action. In earlier studies, the progesterone production induced by GnRHa in short-term incubations of preovulatory follicles, or granulosa cells therefrom, was found to be associated with increased prostaglandin (PG) synthesis, while no changes were detected in cAMP levels under these conditions (Clark et al., 1980; Hillensj#{246}et al., 1982). Stimulation of PG production, however, was found not to be necessary for the steroido genic response to GnRH (Clark, 1982; Hillensj#{246} et al., 1982). The exact mechanism of this observed stimulation, although small, is not presently clear. One could consider the possible role of calcium in GnRH action. Since calcium has been suggested to be a mediator of GnRH action in pituitary gonadotrops (Conn et al., 1981) it is possible that a similar requirement for calcium may exist for GnRH action in the gonads. A role for the calcium-calmodulin system in the regulation of steroidogenesis has been reported
-

to reside at post- cAMP sites in adrenal and testicular cells (Hall et al., 1981a,b). Furthermore, we have recently observed that tnfluoroperazine, a neuroleptic drug which inhibits calmodulin, can abolish the granulosa cell response to GnRHa (Hillensj#{246} et al., unpublished observations). This finding supports the hypothesis that calcium -dependent steps are involved at least in the stimulatory actions of GnRH on steroidogenesis. However, more work is necessary to understand the mechanism of action of GnRH on granulosa cell steroidogenesis, both for its stimulatory and inhibitory effects. It is also interesting that the cumulus cells were not affected by GnRHa. Whether these cells possess receptors for GnRH is not clear at present. A difference that was observed between the responses of the two types of granulosa cells used was that GnRHa inhibited the gonadotropin -stimulated 20a-OHP secretion in cells from preovulatory follicles (between 48-72 h, Fig. 3), while it potentiated the FSH-stimulated 20a-OHP secretion in cells from immature rat ovaries (Fig. 4). A parallel activation of the progesterone metabolizing enzyme, 20a -hydroxysteroid dehydrogenase (20a-SDH), by GnRHa in cultured granulosa cells from immature, hypophysectomized, DES-treated rat ovaries, has been reported (Jones and Hsueh, 1981b). It was suggested that this increase in 20a-OHP accumulation could be one mechanism for the decrease in progesterone synthesis by granulosa cells cultured with FSH in the presence of GnRHa, but this cannot be the mechanism in the case of cells from preovulatory rats. Other mechanisms for a decline in progesterone synthesis by immature granulosa cells are direct inhibition of 3j3-hydroxysteroid dehydrogenase (Jones and Hsueh, 1982a) and possibly the cholesterol side -chain cleavage enzyme (Jones and Hseuh, 1982b). In conclusion, the present study clearly shows that, in granulosa cells from preovula. tory follicles, GnRH can cause both stimulatory and inhibitory effects which are timedependent. Similar time -dependent effects have been found on granulosa cell glycolysis (Billig et al., 1982). The presence of GnRHlike peptides (gonadocrinins) in ovarian tissue was reported by Ving et al. (1981), but later the same group could not reproduce this initial observation (Esch et al., 1982). A physiological interpretation of the direct

EFFECT

OF

GnRH

ON

GRANULOSA

CELL

PROGESTIN

SECRETION

597
cultured rat cumulus

stimulatory

and

inhibitory

must therefore await stration of endogenous in the ovary.

GnRH the conclusive GnRH -like

effects demon peptides

progesterone
-

synthesis

in

ACKNOWLEDGMENTS
This

Swedish
6350), Magnus Faculty
was in

study was supported Medical Research the Swedish Society

Bergvalls Foundation,

by
Council

of

grants from the (5650, 6154, Medical Sciences, and the Medical

University

of Goteborg.

C. S. Sheela

Rani

receipt of a Research Training Grant from the WHO. We thank Prof. K. Ahr#{233}n for support and interest and Mrs. Harriet Thelander for technical assistance. We thank Des. H. R. Lindner and F. Kohen for the antisera

and

the

National

Pituitary

Program

for

the hormones.

REFERENCES Billig, H., Magnusson, C., Ekholm, C. and Hillensj#{246}, T. (1982). Biphasic effect of a GnRH agonist on glycolysis in cultured rat granulosa cells. Biol. Reprod. Suppl. 1, 26:152A. M.

Clark,

K. (1982). Stimulation of progesterone prostaglandin E accumulation by luteinizing hormone-releasing hormone (LHRH) and LHRH analogs in rat granulosa cells. Endocrinology 110: 146-1 52. Clark, M. R., Thibier, C., Marsh, J. M. and LeMaire, W. J. (1980). Stimulation of prostaglandin accumulation by luteinizing hormone-releasing hormone (LHRH) and LHRH analogs in rat granulosa cells in vitro. Endocrinology 107:1723. Conn, P. M., Marian, J., McMiIlan, M., Stern, J., Rogers, D., Hamby, M., Penna, A. and Grant, E. (1981). Gonadotropin-releasing hormone action in the pituitary: A three step mechanism. Endocr. Rev. 2:174-185. Ekholm, C., Hillensj#{246}, T. and Isaksson, 0. (1981). Gonadotropin releasing hormone agonists stimu -

and

late oocyte meiosis and ovulation in hypophy sectomized rats. Endocrinology 108:2022-2024. Ekholm, C., Clark, M. R., Magnusson, C., Isaksson, 0. and LeMaire, W. J. (1982). Ovulation induced by a gonadotropin releasing hormone analog in hypophysectomized rats involves prostaglandins. Endocrinology 110:288-290. Esch, F., Ling, N., Ying, S-V., and Guillemin, K. (1982). Peptides of gonadal orgin involved in reproductive biology. In: Role of Peptides and Proteins in Control of Reproduction (S. M. McCann and D. S. Dhindsa, eds.). Proceedings of National Institute of Health Workshop. Bethesda, MD. Hall, P. F., Osawa, S. and Mrotek, 3. (1981a). The influence of calmodulin on steroid synthesis in Leydig cells from rat testis. Endocrinology 109:1677-1682. Hall, P. F., Osawa, S. and Thomasson, C. L. (1981b). A role for calmodulin in the regulation of steroidogenesis. 3. Cell Biol. 90:402-407. Hillensjo, T., Magnusson, C., Svensson, U. and Thelander, H. (1981). Effect of LH and FSH on

ceUs. Endocrinology 108:1920-1924. Hillensj#{246}, T. LeMaire, W. 3., Clark, M. R. and Ahren, K. (1982). Effect of gonadotrophin-releasing hormone (GnRH) and GnRH agonists upon accumulation of progesterone, cAMP and prostaglandin in isolated preovulatory rat follicles. Acta Endocrinol. 101:603-610. Hsueh, A.J.W. and Erickson, G. F. (1979). Extrapituitary action of gonadotropin-releasing hormone: direct inhibition of ovarian steroidogenesis. Science 204:854-85 5. Hsueh, A.J.W. and Jones, P.B.C. (1981). Extrapituitary actions of gonadotropin-releasing hormone. Endocr. Rev. 2:437-461. Hseuh, A.J.W., Wang, C. and Erickson, G. F. (1980). Direct inhibitory effect of gonadotropin-releasing hormone upon follicle-stimulating hormone induction of luteinizing hormone receptor and aromatase activity in rat granulosa cells. Endocrinology 106:1697-1705. Jones, P.B.C. and Hsueh, A.J.W. (1981a). Direct effects of gonadotropin releasing hormone and its antagonist upon ovarian functions stimu lated by FSH, prolactin, and LH. Biol. Reprod. 24:747-759. Jones, P.B.C. and Hsueh, A.J.W. (1981b). Direct stimulation of ovarian progesterone metaboliz ing enzyme by gonadotropin-releasing hormone in cultured granulosa cells. 3. Biol. Chem. 256: 1248-1254. Jones, P.B.C. and Hsueh, A.J.W. (1982a). Regulation of ovarian 33-hydroxysteroid dehydrogenase activity by gonadotropin -releasing hormone and follicle-stimulating hormone in cultured rat granulosa cells. Endocrinology 110:1663167 1. Jones, P.B.C. and Hsueh, A.J.W. (1982b). Pregnenolone biosynthesis by cultured rat granulosa cells: modulation by follicle -stimulating hormone and gonadotropin-releasing hormone. Endocrinology 111:713-72 1. Knecht, M. and Cars, K. 3. (1981). Gonidotropinreleasing hormone: Regulation of adenosine 3,5 -monophosphate in ovarian granulosa cells. Science 214:1346-1 348. Knecht, M., Katz, M. S. and Catt, K. J. (1981). Gonadotropin-releasing hormone inhibits cyclic nucleotide accumulation in cultured rat granulosa cells. J. Biol. Chem. 256: 34-36. Knecht, M., Amsterdam, A. and Cars, K. J. (1982). Inhibition of granulosa cell differentiation by gonadotropin-releasing hormone. Endocrinology 110:865-872. Lindner, H. R. and Bauminger, S. (1974). Production and characterization of antisera to steroid hormones. In: Recent Progress in Reproductive Endocrinology (P. G. Crosignani and V.H.T. James, eds.). Academic Press, New York, pp. 193-227. Sharpe, R. M. (1982). Cellular aspects of the inhibitory actions of LH - RH on the ovary and testis. 3. Reprod. Fertil. 64:517-527. S-V., Ling, N., Bohlen, P. and Guillemin, R. (1981). Gonadocrinins: Peptides in ovarian follicular fluid stimulating the secretion of pituitary gonadotropins. Endocrinology 108: 1206-1214.

Ying,