Develop. Growth and Differ.

, 29 (l), 1-12 (1987)

Review

: A Teleost
17a , 20~-Dihydroxy-4-pregnen-3-one
Maturation-Inducing Hormone
( 17a, 20~-dihydroxy-4-pregnen-3-one/maturation-inducing
hormone/oocyte maturation/
ovarian folliclelteleosts)

YOSHITAKA NAGAHAMA
Laboratory of Reproductive Biology, National Institute for
Basic Biology, Okazaki 444, Japan

1. Introduction
In most vertebrates, full-grown postvitellogenic oocytes in the ovary are still in prophase
I of meiosis and cannot be fertilized. For the oocytes to be fertilized they must undergo final
oocyte maturation. This process consists of the breakdown of the germinal vesicle (GVBD),
chromosome condensation, assembly of the first meiotic spindle, and extrusion of the first
polar body. Three major mediators of oocyte maturation have been described in teleosts
and amphibians: gonadotropin, maturation-inducing hormone, and maturation-promoting
factor (1-6). These mediators function sequentially at the levels of the follicle layer, the
oocyte surface and the oocyte cytoplasm (Fig. 1). Among these three mediators, gonadotro-
pins have been purified in many vertebrate species. In contrast, although maturation-
inducing hormone and maturation-promoting factor have been known to exist for more than
15 years, only a limited progress has been made for their purification and characterization.
Over the past several years, a series of studies in our laboratory using a salmonid fish, the

Gonadotropin

Fig. 1 . Hormonal control of oocyte maturation in

amphibians and teleosts. Three major mediators,
Maturation-inducing hormone gonadotropin, maturation-inducing hormone and
t maturation-promotingfactor are involved.
1 I Receptor I
I / .c
Maturationpromoting factor

, Oocyte maturation /
I
1
 2 Y.NAGAHAMA

amago salmon, Oncorhynchus rhodurus, as a main experimental organism has provided

important new information about the chemical nature of maturation-inducing hormone and
its synthetic control by gonadotropin. As a result of these studies, we have identified the
maturation-inducing hormone of amago salmon, for the first time in any vertebrate, as
17a, 2Op-dihydroxy-4-pregnen-3-one (17a ,20P-diOHprog) (7). It is the purpose of this
article to review some of our studies on the identification of maturation-inducing hormone
and the mechanism by which gonadotropin regulates ovarian synthesis of 17a, 20P-diOHprog.

2. Primary Hormone Triggering Oocyte Maturation

The primary hormone involved in triggering oocyte maturation in salmonids, as in
other vertebrates, is gonadotropin (8). The number of gonadotropins present in the teleost
pituitary gland has been debated and is presently unresolved. A glycoprotein-rich gonadot-
ropin with a molecular weight of 25,000-40,000 has been purified in several teleosts (9, 10).
This type of gonadotropin, termed “maturational”gonadotropin, has been reported to
stimulate almost all gonadal activities. We purified a gonadotropin from a glycoprotein
fraction of acetone dried pituitaries of the chum salmon, Oncorhynchus ketu,using affinity
chromatography on Con-A Sepharose, ion exchange column on DEAE-Sephacel, and gel
filtration in Sephacryl S-200 (11). The molecular weight of this gonadotropin was estimated
to be 38,000-40,000by gel filtration and that of the two subunits to be about 15,000 and
20,000. This gonadotropin induces oocyte maturation and stimulates ovarian and testicular
steroidogenesis in amago salmon. A second gonadotropin, carbohydrate-poor (10) or
carbohydrate-rich (12) gonadotropin, has also been purified from the pituitaries of several
teleosts including chum salmon. It is unknown, however, whether these second gonadotro-
pins possess a function different from the “maturational” gonadotropin. In this review, the
term gonadotropin refers to the glycoprotein-rich “maturational” gonadotropin.

3 . Identification of Maturation-Znducing Hormone

Although it was known for some time that both gonadotropin and steroid hormones can
induce oocyte maturation and ovulation in vertebrate oocytes in vivo and in vitro, it was not
until the late 1960’s that in anuran amphibians, gonadotropins were found to stimulate the
follicular tissues to secrete a hormone, maturation-inducing hormone, which in turn acts on
the oocytes to initiate maturation. These findings have facilitated investigations of the
hormonal regulation of final oocyte maturation in a variety of vertebrates (13-16). While the
specific steroid produced by amphibian ovarian follicle cells to induce oocyte maturation in
response to gonadotropin has not yet been identified, a variety of evidence strongly suggests
that the maturation-inducing hormone of amphibians is progesterone or a similar steroid.
This hypothesis has been reinforced by the recent results of measurements of blood or ovarian
concentrations of progesterone during oocyte maturation (17-19).
Follicle-enclosed full-grown postvitellogenic oocytes of some teleosts undergo GVBD in
vitro when they are incubated with gonadotropin. However, denuded oocytes are incapable
of responding to gonadotropin (20). Cyanoketone, a specific inhibitor of 3P-hydroxy-
n5-steroid dehydrogenase, completely abolished the maturational effects of partially purified
chinook salmon gonadotropin (SG-G1 00) and pregnenolone, but not of steroids such as
progesterone or its metabolites (21). Thus, the action of gonadotropin in inducing oocyte
 MATURATION-INDUCING HORMONE 3

maturation appears to be dependent on the synthesis of a second steroidal mediator of meiotic

maturation. In agreement with this, we have shown that defolliculated oocytes of amago

Fig. 2. High-performance liquid chromatogra-

phy and maturation-inducing (MI) activity of the
50% methanol phase. (see NAGAHAMA and
ADACHI [7]for details).

5 10 15 20
Fraction number (3mVfr)
Sample

229 269

I
Lt J. - Y II 11 .

50 100 150 200 250 300 ( m l e )

Fig. 3. Mass spectra of fraction 10 (Fig. 2) of the 50% methanol phase and 17a, 20g-dihydroxy-4-
pregnen-3-one standard.
 4 Y.NAGAHAMA

salmon undergo GVBD when incubated in media in which folliculated oocytes had been
previously incubated with partially purified chum salmon gonadotropin (SGA, Syndel Lab.)
indicating media of folliculated oocytes contain a factor(s) which induces meiotic maturation.
We then attempted to purify and chemically characterize the natural maturation-inducing
hormone of amago salmon from these incubation media (7). In this study, maturation-
inducing activity of residues at various steps of purification was assessed by a homologous in
v i m assay. Ether extracts of the media from the incubates showed high maturation-inducing
activity. Yolk and oil droplets were first removed from the ether extract by partition with
equal volumes of 50% methanol and n-hexane. Maturation-inducing activity was found only
in the 50% methanol phase. Reversed-phase high performance liquid chromatography
(HPLC) was then employed to fractionate the 50% methanol phase. Of the 20 fractions
prepared, only one fraction exhibited maturation-inducing activity (Fig. 2). This fraction
had a retention time that coincided exactly with authentic 17a, 2OP-diOHprog standard and
differing appreciably from the retention time of the other steroid standards including 17a, -
20a-dihydroxy-4-pregnen-3-one.The purity and final characterization of the residues of this
fraction were further confirmed by a comparison with authentic 17a, 20P-diOHprog using thin
layer chromatography and mass spectroscopy. The mass spectrum of the fraction was
identical to that of standard 17a, 20P-diOHprog, the molecular ion peak being at m/e 332 and
base peak at m/e 287 (Fig. 3).
17a, 20P-DiOHprog was first identified in the plasma of mature adult sockeye salmon
(22). In collaboration with Dr. A. Kambegawa (Teikyo University), a specific radioimmu-
noassy for 17a, 20P-diOHprog was developed in our laboratory and has been applied to
measure blood concentrations of this steroid during the sexual maturation of female amago
salmon (23). 17a, 20P-DiOHprog levels were less than 0.5 ng/ml in vitellogenic females
(June-September) and in those with full-grown immature oocytes collected in early October.
A dramatic increase (50-70 ng/ml) occurred in mature and ovulated females collected in mid
October. This increase in plasma 17a,20P-diOHprog levels correlated well with a dramatic
rise in plasma gonadotropin levels. The peak of plasma 17a, 20P-diOHprog levels was

Precursor
FH3

.- OH

Po
1 7 0 -hydroxyprogesterone

Fig. 4. Maturation-inducing hormone of amago sal-

mon. 17a, 20,9-Dihydroxy-4-pregnen-3-oneis synthe-
sized from its precursor, 17a-hydroxyprogesterone, by
the action of the enzyme 208-hydroxysteroid dehyd-
rogenase.

1 7a,20p-dihydroxy-4-pregnen-d-one
 MATURATION-INDUCING HORMONE 5

observed 2-4 days prior to ovulation, coincident with the occurrence of GVBD of oocytes.
The relative effectiveness of a range of pregnene derivatives in inducing GVBD was
investigated in vitro using amago salmon oocytes. Of the steroids tested, 17a,20P-
diOHprog was found to be the most effective inducer of GVBD (24). Taken together, these
results indicate that 17a, 20P-diOHprog is the major naturally occurring maturation-inducing
hormone in amago salmon (Fig. 4). Further investigations from our laboratory and others
suggest that 17a, 20P-diOHprog functions as the maturation-inducing hormone common to
several species of salmonids (25-31). It is also possible that 17a, 20P-diOHprog acts as an
important steroidal mediator of oocyte maturation in some nonsalmonid teleosts (20, 24,
32-39).

4. Mechanisms of Gonadotropin Regulation of Follicular 17a, 20P-DiOHprog Biosynthesis

A. Two-cell type model
The identification of the maturation-inducing hormone in amago salmon has facilitated
research on gonadotropin control of ovarian synthesis of this steroid. Significantly elevated
levels of 17a, 20P-diOHprog were found in media in which folliculated oocytes of amago
salmon were induced to mature in vitro with gonadotropin (23,40), suggesting that the major
source of ovarian 17a, 20P-diOHprog biosynthesis is follicular tissue.
Ovarian follicles of teleosts, like those of other vertebrates, are composed of two major
layers, an outer thecal layer and an inner granulosa layer, which are separated by a substantial
basal lamina (41). The thecal layer consists of fibroblasts, collagen fibers and capillaries, and
in some species, large glandular cells known as “special thecal cells”, whereas the granulosa
layer consists of a single layer of uniform cells. Special thecal cells possess the ultrastructural
characteristics of a steroid-producing cell, such as abundant smooth endoplasmic reticulum,
mitochondria with tubular cristae, and lipid droplets. A restricted occurrence of various
steroidogenic enzymes has been histochemically demonstrated in these cells. In contrast,
granulosa cells contain organelles suggestive of protein synthesis, i. e., rough endoplasmic
reticulum, Golgi apparatus, and mitochondria with lamellae cristae, although histochemical
Gonadotropin

I GTH-receptor I
Cholesterol
0
Pregnenolone

17a -Hydroxyprogesterone
Fig. 5. Two-cell type model for the
production of 17a, 20b-dihydroxy-4-
pregnen-3-one in the ovarian follicle of
salmonids. (see the text for details).
G
0 17a-Hydroxyprogesterone

3 POP-HSD h
 6 Y. NAGAHAMA

evidence suggests the presence of steroidogenic enzymes in these cells of several teleosts.
Development of a simple dissection technique to separate the ovarian follicles of
salmonids into two layers, the thecal and granulosa layers, has made it possible to elucidate
the relative contributions of each layer and gonadotropin in the overall process of 17a, 20p-
diOHprog production in two species of salmonids, amago salmon and rainbow trout, Salmo
gairdneri (8, 42, 43). Intact follicles and co-culture preparations produced large amounts of
17a, 20P-diOHprog in response to salmon gonadotropin. Neither isolated thecal nor
granulosa layers alone were capable of producing substantial amounts of 17a, 20P-diOHprog
in response to gonadotropin, although in some cases thecal layer preparations produced
significant amounts of 17a, 20P-diOHprog in response to gonadotropin (SGA or SG-G100).
These results indicated that both thecal and granulosa layers are involved in the gonadotro-
pin-induced follicular production of 17a, 20P-diOHprog. Measurement of 17a-
hydroxyprogesterone concentrations of media from the same experiment revealed that
isolated thecal layers produced large quantities of 17a-hydroxyprogesterone,but granulosa
layers did not respond to gonadotropin. In contrast, levels of 17a-hydroxyprogesterone in
media from intact follicles and co-culture incubations, peaked at 12 hr and rapidly decreased
concomitant with a rapid rise in 17a,2OP-diOHprog levels. The presence of 20p-
hydroxysteroid dehydrogenase (20P-HSD), the key enzyme involved in the conversion of
17a-hydroxyprogesterone to 17a, 20P-diOHprog, has been demonstrated in the granulosa
layers, since this layer produced 17a, 20P-diOHprog when incubated with exogenous
17a-hydroxyprogesterone. When all these in vifro data are combined, a two-cell type model
has been proposed, for the first time in any vertebrate, for the follicular production of
maturation-inducing hormone, which is summarized in Fig. 5 . In this model, the thecal layer
produces 17a-hydroxyprogesterone that traverses the basal lamina and is converted to
17a, -20P-diOHprog by the granulosa layer where gonadotropin acts to enhance the activity of
20p-HSD. However, the extent to which thecal layers contribute to the production of
17a, 20P-diOHprog is still not clear, since in some cases thecal layer preparations produced
this steroid in response to gonadotropin. Although our granulosa layer preparations are
completely free of attached thecal layers, it is extremely difficult to obtain pure thecal layer
preparations from postvitellogenic follicles.
In previous in vitro studies, we have presented data indicating a similar interaction of
thecal and granulosa layers for the production of estradiol-17P by ovarian follicles of
salmonids during vitellogenesis (44, 45). In this model, the thecal layer contributes to
estradiol-17P production by synthesizing two aromatizable androgens (androstenedione and
testosterone) which are aromatized in the granulosa layer to estradiol-17P. Thus, the
production of two biologically important steroids, estradiol-17P and 17a, 20/?-diOHprog, in
salmonid reproduction depends on the interaction of the thecal and granulosa cell compart-
ments. It is of particular interest that in both cases the thecal layer secretes precursor
steroids, whereas the granulosa layer is the principal site of the conversion of the precursors to
the physiologically active steroids. Similar two-cell type models for follicular estradiol-17P
biosynthesis have been demonstrated in several species of mammals (46).
Studies of in v i m estradiol-17P and 17a, 20p-diOHprog production by amago salmon
follicles at different stages of development showed that vitellogenic follicles predominantly
secrete estradiol-17p in response to gonadotropin stimulation (47), whereas the capability of
 MATURATION-INDUCING HORMONE 7

the intact follicles to respond to gonadotropin by synthesizing and secreting 17a, 20P-
diOHprog was acquired immediately prior to the natural maturation period of oocytes (23).
These in vitro data is consistent with the observed shift from estradiol-17P to 17a,20P-
diOHprog in plasma immediately prior to or during oocyte maturation. In the light of the
newly proposed two-cell type model, we have attempted to explain mechanisms involved in
this steroidogenic shift. Our previous in vitro studies clearly showed that a sharp decrease in
the activity of the aromatizing enzyme in granulosa layers occurred after the completion of
vitellogenesis (48). In view of the enhanced ability of the thecal layer to produce testoster-
one in postvitellogenic follicles (47), the decreased follicular secretion can be explained by the
decreased aromatase activity in the granulosa layer. This is in contrast to the situation
reported in mammalian ovaries where the decreased secretion of estradiol-17P immediately
after ovulation has been explained by a diminished supply of aromatizable androgens due to
decreased activity of ovarian 170-hydroxylase and C17-C20 lyase (49). In amago salmon,
gonadotropin caused a slight, but significant stimulation of 20P-HSD activity (enhanced
conversion of exogenous 170-hydroxyprogesterone to 17a, 20P-diOHprog) in granulosa cells
collected from mid-vitellogenic follicles. However, the thecal layer only begins secreting
17a-hydroxyprogesterone immediately prior to the maturation of oocytes in vivo (Kanamori,
unpublished). Results from these in vitro studies can be interpreted to mean that in addition
to 20P-HSD activation in the granulosa cells by gonadotropin, the availability of 17a-
hydroxyprogesterone may also play a major role in the enhancing action of gonadotropin on
follicular biosynthesis of 17a, 20P-diOHprog.
B. Gonadotropin receptors
In the two-cell type model for the production of 17a, 20P-diOHprog described above,
gonadotropin has at least two sites of action, the thecal and granulosa layers. This suggests
the existence of gonadotropin receptors in both cell layers. We have investigated gonadotro-
pin binding to both thecal and granulosa layers of amago salmon. Purified chum salmon
gonadotropin (11) was iodinated with the aid of Iodogen (1, 3, 4, 6-tetrachloro-3a,
6a-diphenyl glycoluril). Crude membrane preparations derived from 23,000 g pellet of
homogenates of thecal or granulosa layer preparations were obtained from postvitellogenic
amago salmon follicles. The binding studies with 12’1- chum salmon gonadotropin demons-
trated its specific binding to both thecal and granulosa layers. The inability of other peptide
hormones of chum salmon such as growth hormone, prolactin and a-MSH to compete
effectively with iodinated chum salmon gonadotropin for binding sites further demonstrates
the high specificity of this receptor. Scatchard analysis of this binding suggests that thecal
and granulosa layers possess a single class of binding sites with similar characteristics
(Kanamori, unpublished). The physiological significance of the existence of these binding
sites in thecal and granulosa layers is unknown and is the subject of further investigation.
C . Adenylate cyclasekyclic AMP system
The earliest effect of gonadotropin on amago salmon thecal layers is receptor-mediated
activation of adenylate cyclase and formation of cyclic AMP (Kanamori, unpublished).
Gonadotropin action to enhance 17a-hydroxyprogesterone production by the thecal layer was
mimicked by forskolin, an adenylate cyclase activator, cyclic AMP analogs and phosphodies-
terase inhibitors such as 3-isobutyl-1-methyl-xanthine (IBMX) and theophylline. Both
gonadotropin and forskolin caused a rapid accumulation of cyclic AMP in the thecal layer
 8 Y. NAGAHAMA

with maximal levels at 30-60 min (Kanamori, unpublished). These findings are consistent
with the view that cyclic AMP is the second messenger for gonadotropin action in the thecal
layer. Our preliminary studies further suggest the major site of action is at the steroidogenic
step between cholesterol and pregnenolone.
The granulosa cells of salmonids provide a unique system in which to study gonadotropin
regulation of a steroidogenic enzyme, since precursor steroids cannot be produced by these
cells. This system allows the indirect quantification of 20P-HSD activity by the measure-
ment of 17a, 20P-diOHprog when these cells are incubated with exogenous 17a-
hydroxyprogesterone. The functional role of the adenylate cyclasekyclic AMP system in the
gonadotropin-induced activity of 20P-HSD in the amago salmon granulosa cells has been
investigated (50). When granulosa cells were incubated with forskolin in the absence of
17a-hydroxyprogesterone, 17a, 20P-diOHprog levels were very low. In contrast, in the
presence of 17a-hydroxyprogesterone,the production of 17a, 20P-diOHprog was strikingly
stimulated by forskolin. Similarly, dibutyryl cyclic AMP and phosphodiesterase inhibitors
strikingly enhanced the production of 17a, 20P-diOHprog when 17a-hydroxyprogesterone
was added to the incubation media, whereas dibutyryl cyclic GMP had no such action.
Furthermore, both gonadotropin and forskolin caused a rapid accumulation of cyclic AMP in
the granulosa layer with maximal levels at 30-60 min (Kanamori, unpublished). Taken
together, these findings suggest that in the amago salmon granulosa cells, the adenylate
cyclase/cyclic AMP system acts as an intracellular mediator in the activation of 20P-HSD by
gonadotropin.
D. Protein and RNA synthesis
An extremely pertinent area concerns the genetic control of follicular biosynthesis of the
maturation-inducing hormone. At the present time there exist no relevant data on this topic.
We have examined the effects of inhibitors of protein synthesis (cycloheximide and puromy-
cin) and RNA synthesis (actinomycin D, cordycepin and a-amanitin) on the production of
17a, 20P-diOHprog and its precursor, 17a-hydroxyprogesterone by gonadotropin (SGA) in
the amago salmon follicles (51). Both RNA synthesis and protein synthesis inhibitors
blocked gonadotropin-induced 17a, 20P-diOHprog production by intact follicles. These
results correlate well with earlier findings in rainbow trout which showed that gonadotropin-
induced GVBD in vitro was inhibited by both transcriptional and translational inhibitors (37).
Thus, the observed inhibition of GVBD can apparently be accounted for by the suppression
of follicular 17a, 20P-diOHprog production by these inhibitors. In contrast, gonadotropin-
induced 17a-hydroxyprogesterone production by intact follicles was not abolished by acti-
nomycin D, but was abolished by cycloheximide. Nevertheless, in rainbow trout actinomy-
cin D was reported to be effective in blocking gonadotropin-induced GVBD (37). Thus, it is
likely that in salmonids the significance of 17a-hydroxyprogesterone is its position as the
immediate precursor of 17a, 20P-diOHprog biosynthesis rather than it serving as a matura-
tion-inducing hormone. Furthermore, these in vitro data suggest that postvitellogenic
amago salmon ovarian follicles already contain the RNAs necessary for the biosynthesis of
precursors such as 17a-hydroxyprogesterone.
Effects of protein and RNA synthesis inhibitors on gonadotropin-induced 20P-HSD
activation were also determined (51). In this experiment, isolated granulosa cells were
incubated with various concentrations of actinomycin D, cordycepin, a -amanitin, cyclohex-
 MATURATION-INDUCING HORMONE 9

imide or puromycin in the presence of both SGA and 17a-hydroxyprogesterone,and 17a,

20P-diOHprog levels in the medium were determined. Gonadotropin markedly enhanced
17a, 20P-diOHprog production by granulosa cells incubated with 17a-hydroxyprogesterone.
The enhancing effect of gonadotropin was completely blocked by all of the five inhibitors
used. Further time course studies revealed that RNA synthesis inhibitors added - 1 , O , 3, and
6 hr after the addition of 17a-hydroxyprogesterone and gonadotropin, totally abolished
gonadotropin-induced 20/3-HSD, but not 9 hr after hormone addition. With cycloheximide
total inhibition was observed when added in the period of 1 hr before to 9 hr after the start of
the incubation. These time course results suggest that de n o w synthesis of 20P-HSD in v i m
in response to gonadotropin occurs, and consists of gene transcriptional events within the first
6 hr of gonadotropin exposure and translational events 6-9 hr after gonadotropin exposure.
More recent studies from our laboratory have also shown that both RNA synthesis and
protein synthesis inhibitors totally prevented dibutyryl cyclic AMP-induced 20P-HSD activa-
tion in granulosa cells (Nagahama, unpublished). Thus, these results suggest that gonadot-
ropin causes the de n o w synthesis of 20P-HSD in the amago salmon granulosa layer through
a mechanism dependent on RNA synthesis. Figure 6 summarizes our working hypothesis for
20P-HSD activation by amago salmon granulosa cells. The first step of the stimulatory
Precursor
( 17a-Hydroxyprogesterone)
I I GTH
- ...
- I
1
I
Y
Receptor
c Cell membrane
Adenylate cyclase

CAMP
e
Protein k inase
c
IPhosphorylation
’hosphorylation
-..

’-. mRNA
m=i
~ RINA
N A
4-pregnen-3-one k-....
I
I Nucleus

Granulosa cell
Secretion
Fig. 6. Diagram of 20B-hydroxysteroid dehydrogenase activation of the amago salmon granulosa
cell via the receptor-adenylate cyclase-cyclic AMP mechanism. ER, endoplasmic reticulum; GTH,
gonadotropin.
 10 Y.NAGAHAMA

effects of gonadotropin on granulosa cells appears to be receptor mediated activation of

adenylate cyclase and formation of cyclic AMP. It is generally believed that the elevated
levels of cyclic AMP induced by gonadotropin activate specific protein kinases which add
phosphate groups to key regulatory proteins. Since RNA synthesis and protein synthesis
inhibitors completely block both the gonadotropin- and cyclic AMP-induced 20P-HSD
enhancement, we suggest that gonadotropin promotes de novo synthesis of 20P-HSD by a
mechanism involving RNA synthesis. Thus, the induction of 20P-HSD activity by gonadot-
ropin in amago salmon granulosa cells is a good example of differentiated functions expressed
by the target cells in response to peptide hormone stimulation.

5. Conclusion
Pituitary gonadotropin is of primary importance in regulating the steroidogenic functions
of the gonads. The gonadal steroids, in turn, exert significant influences, both directly and
indirectly, in controlling germ cell growth and maturation. 170, 20P-DiOHprog was iden-
tified as the ovarian mediator of gonadotropin-induced oocyte maturation in amago salmon.
The interaction of two endocrine follicular layers, the thecal and granulosa layers, is necessary
for 17a, 20P-diOHprog secretion. The major action of gonadotropin for the production of
this steroid is to enhance the activity of 20P-HSD in the granulosa cells. This action of
gonadotropin is mediated by the adenylate cyclasekyclic AMP system. However, the nature
and the mechanism of action of the intracellular messengers generated by cyclic AMP are
totally obscure. Evidence presented in this review suggests that gonadotropin promotes de
novo synthesis of 20P-HSD by a mechanism involving RNA synthesis. Up to this point, we
know nothing about the molecular aspects of the gene expression process, but it seems that
the amago salmon granulosa cell system will provide an excellent model for the studies on
molecular mechanisms underlying gonadotropin-induced changes in cell function and also the
genetic control of cell differentiation.
At present, nothing is known about the mode of action of 170, 20P-diOHprog to induce
oocyte maturation. 170, 20P-DiOHprog has been found to be ineffective in inducing oocyte
maturation when microinjected into full-grown immature oocytes of goldfish, but was
effective when applied externally (Kishimoto and Nagahama, unpublished). These data
suggest that the site of action of steroidal inducers is at the oocyte surface. Certainly, the
oocyte surface receptor of 170, 20P-diOHprog merits further study. While our model
described in this article relates specifically to salmonids, it should provide a basis for our
understanding at the molecular action of gonadotropin in other vertebrates.

I thank Dr. T. F. Hourigan for reading the manuscript. The studies from our laboratory described in this article
have been aided in part by Grant-in-Aid for special Project Research (Project No. 581 19008 and 61 134041) and
Scientific Research from the Ministry of Education, Science and Culture. Japan.

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(Received November 4, 1986)