Table Of ContentZOOLOGICAL SCIENCE 11: 77-82 (1994) 1994Zoological SocietyofJapan
Regional Differences in Granulosa Cells of Preovulatory Medaka Follicles
Takashi Iwamatsu1*, Seiko Nakashima1 Kazuo Onitake2
, ,
Arito Matsuhisa1 and Yoshitaka Nagahama3
^Department ofBiology, Aichi University ofEducation, Kariya 448, 2Department of
Biology, Faculty ofScience, Yamagata University, Yamagata 990, and Reproductive
Laboratory, National Institutefor Basic Biology, Okazaki 444, Japan
—
ABSTRACT Theregionaldifferencesinmorphologyandsteroidproductionofgranulosacellsoftheanimalandvegetal
hemispheresofthepreovulatorymedaka(Oryziaslatipes)follicleswereinvestigated. Granulosacellsintheanimalpole
regionweredistinguishablefromthoseinthevegetalpoleregionintheirshapeanddistribution. Thedistributionoftall
granulosa cells was more compact in the vegetal pole area than in the remaining areas. The gonadotropin-induced
production of 17a,20/?-dihydroxy-4-pregnen-3-one by granulosa cells alone was at a level sufficient to induce in vitro
maturationoftheoocyte. Productionwassignificantlygreaterinthevegetalhemispherethanintheanimalhemisphere.
These results indicate that medaka follicles have morphological and physiological differences in granulosa cells
corresponding to regional differences along the animal-vegetal axis ofthe oocyte.
INTRODUCTION tics ofgranulosa cells surrounding the oocyte before matura-
tion. Atpresentthereislittleinformationonthedifferences
Eggs ofoviparous fishes generally have distinct polarity. in the distribution between the animal and vegetal hemis-
The polarity reflects the functional organization ofthe cellu- pheres and the structure of granulosa cells surrounding
lar constituents necessary for development. It must be fully-grown oocytes.
determinedbytheinteraction oftheoocytewithfolliclecells, Weinvestigatedthelocaldifferencesin thestructure and
especially the granulosa cells that are in contact with the function of granulosa cells of the medaka follicle. The
oocyte during oogenesis. Accessory structures such as results indicated that granulosa cells had regional differences
attaching filaments, which are located at the animal or the in morphology and steroid production along the animal-
vegetal pole side of the egg membrane (chorion) have been vegetal axis of the fully-grown oocyte.
found in a great number of teleost fishes (cf. [14]). In
medaka oocytes, the difference in the distribution of acces- MATERIALS AND METHODS
sory filaments on the chorion is detectable in the early stages
of oogenesis [5]. The differences in their morphology and Folliclepreparation
distribution seem to be established by the interaction with Preovulatory medakafollicleswere isolatedfrom the ovariesof
granulosa cells. mature females which had spawned every day under the light- and
Insometeleosts, oneofthematuration-inducingsteroids temperature-conditions controlled to induce reproduction. The
(MIS: [11, 16]), 17a,20/?-dihydroxy-4-pregnen-3-one (17a, follicleswereremovedfromtheovariesbyusingfineforcepsundera
binoculardissectingmicroscope (x20). The follicleswere handled
20/?-diOHp: cf. [13]) is producedandsecretedbyfollicle cells
inthepresenceofgonadotropin. Thematurationofmedaka tinhescaallinecel[l3].layFeirneanwdatcthhemakbears'esmefnotrcempesmwberraenteheonffusfedrotmopfeoelllictlhees
oocytes in vitro is also induced by the same steroid, which is starting at the vegetal pole area (VPA) identified by the long
produced by granulosa cells [6, 8-10, 15]. It is generally attaching filaments [4]. These oocytes surrounded by a granulosa
believed that whole surface of the oocyte is simultaneously celllayeralonewerecutpreciselywithsmallscissorsintotwohalves,
evenly stimulated by MIS from the granulosacells. Howev- theanimalandthevegetalhemispheres. Theooplasmwasremoved
er, there is no evidence that this is true. from the hemispherical chorion within the granulosa cell layer by
In the medaka MIS is produced by granulosa cells with washingthespecimensinsalinewithasmallpipette. Thus,prepara-
probablylittle orno participationofthethecalcells. There- tions consisted ofgranulosa cell layers on the chorion hemispheres.
fore, the spatial and functional differences in granulosa cells
may be very important for the establishment of oocyte Follicle incubation
Ten ofthe animal or vegetal hemispheresprepared above were
wpoelaarrietyinatnedreosotceydtienmtahteurreagtiioonnalinditfhfiserfeinshc.es iFnosrttehriosidsoegaesonne,- placed separately in 1 ml of culture medium (Earle's Medium 199,
Dainippon-seiyaku, Osaka) in each well of a 24-well culture dish
sis, particularly MIS production, and cytological characteris- (Cell Wells: Corning, N.Y.) and incubated for24hr at 27°C.
Accepted December 14, 1993 Observations byfluorescenceandelectron microscopy
Received October20, 1993 Immediately after removal of the thecal cell layer and the
To whom reprint requests should be addressed. basement membrane, oocytes surrounded by a granulosa cell layer
78 T. Iwamatsu, S. Nakashima et al.
alone were pre-fixed in 4% glutaraldehyde, rinsed in 0.1M phos- were measured in the samples ofculture medium using radioimmu-
phatebuffer(pH7.4)andpost-fixedinosmiumtetroxideat4°Cfor2 noassay (RIA) as described in a previous report [9, 10]. The
M
hr. These samples were washed in 0.01 phosphate buffer (pH sensitivity ofeach assay was 30pg/ml.
7.4),dehydratedinagradedseriesofacetone,driedaccordingtothe All experimentswere performed in triplicate and the datawere
critical point method with C02 and coated with gold. They were analysed by statistical comparison (the Student's f-test) with control
observed with a JEOL scanning electron microscope (SEM). values.
Fortransmissionelectronmicroscopy (TEM), thesimilarlyfixed
samplesweredehydratedinethanolfollowedbypropyleneoxideand RESULTS
embedded in Epon 812. Ultrathin-sections were observed after
staining with lead citrate and uranyl acetate. 1 Morphology ofgranulosa cells
As shown in the TEM images (Figs. 1 and 2), granulosa
Measurementofsteroids cellson the chorion ofpreovulatory oocytesdiffered inshape
and number between the animal and vegetal hemispheres.
Animalandvegetalhemispherespreparedabovewereincubated
in the presence of 100IU pregnant mare serum gonadotropin Shortcylindricalgranulosacells (diameterca. 8//m, height 13
(PMSG)or100ng/mlprogesteronefor24hrat27°C. Onemilliliter fim) were aligned in a monolayer on the chorion (Fig. 1),
ofculture medium fromeachwell wasthen collected and assayedto except in the vegetal pole area(Fig. 2) (VPA, diameter ca.
compare theproductionof17a,20/?-diOHpandestradiol-17/?(E2)by 430/um, [5]). The nucleus was located centrally in these
granulosa cells of the two hemispheres. 17a,20/?-DiOHp and E2 short cylindrical cells. The cells contained well developed
Fig. 1. Granulosa cells in the animal hemisphere of the preovulatory medaka follicle. The granulosa cell contains many
mitochondria, rough endoplasmicreticula and Golgi apparatusin thecytoplasm. Note microvilli (arrows) without these
organelles at the apical surface ofthe cell. Asterisks, non-attaching filaments; CH. chorion; N, nucleus, (bar 1 //m)
Regional Differences of Granulosa Cells 79
Fig.2. Granulosa cells in the vegetal pole area of the preovulatory medaka follicle. Tall granulosa cells contain a
mitochondrialmass(m),dilatedendoplasmicreticula(vesicles),anddevelopedGolgilammellaeintheapicalregionofthe
cell. Note the breb-like apices ofthe granulosa cellswhich contain many cytoplasmic inclusions. Asterisks, attaching
filaments; CH, chorion; N, nucleus, (bar 10fxm)
rough endoplasmic reticula (ER), some of which were di- animal hemisphere. The nucleus was basally located near
lated, mitochondria with an electrondense matrix, Golgi the chorion, and the conspicuously crowded mitochondria
complexes with very small vesicles, and large lysosomal were in the apical region. Othercell organelles in these tall
vesicles. The granulosa cells in the VPA were slimmer and cells were similar to those contained in the short cylindrical
taller (diameterca. 5/um, heightca. 37fj.m) than thoseinthe cells.
80 T. Iwamatsu, S. Nakashima et al.
A. *<
'
Fig. 3. Scanningelectronmicrographsofthesurfaceofthegranulosacelllayerintheanimalandvegetalhemispheres. A. B:
Intheanimalpoleregion(asterisk)(A;bar50//m),granulosacellsinteractwitheachotherbythread-likemicrovilli(B;bar
10,«m). nf.Non-attachingfilaments; arrows, flatgranulosacells. C, D: Inthevegetalpoleregion(asterisk) (C;bar 100
//m), which is encircled by the long distal portions of attaching filaments (af), the breb-like tips of the granulosa cells
exhibit smooth surfaces (D; bar 10//m) except for a few special cells with long microvilli.
The animal pole ofthe oocyte could be easily recognized and3B). The apical surface ofthese irregular-shapedgranu-
by its position opposite the location ofthe attachingfilaments losa cells was flat and smooth, as previously reported for the
on the chorion and by the bending direction of the non- intact follicle [6, 7]. These flatgranulosacellswere adherent
attaching filaments (Fig. 3A). Granulosa cells at the animal to eachother. The surface ofthe granulosacells in the VPA
pole region had many microvilli on the cell surface (Figs. 1 is shown in Figs. 3C and D. In the VPA, tall granulosa cells
Regional Differences of Granulosa Cells 81
Fig.4. Distribution of the nuclei of granulosa cells in the preovulataory follicle. After fixation with glutaraldehyde, the
follicle was stained with lO/zg/ml Hoechst for 30min (bar 100/urn). A: The animal hemisphere showing the nuclei of
granulosacells. B:Thevegetalpoleregionshowingthenucleiofgranulosacellsandattachingfilaments(arrows). Bars,
100txm.
Table1. Difference in steroid production by the animal and the vegetal hemispheres of medaka
follicles
Estradiol-17/9 17a,20/?-Dihydroxyprogesterone
Group (pg/ml) (pg/ml)
AH VH AH VH
PMSG (lOOIU/ml) 666±57 745+70 405±54 615+29*
Progesterone (lOOng/ml) 723+52 868+112 667+32 720+110
Control (no hormone) 200+28 300+49 6+4 31+29
* Significantdifference(P<0.05)betweentheanimal(AH)andvegetal(VH)hemispheres. Ineachgroup,
values represent mean+S.E. of three incubations.
were compactly distributed, although it was difficult to pre- pg/ml; [9]) of 17a,20^-diOHp were sufficient to induce
cisely determine their size and shape by SEM. oocyte maturation, and significantly greater in the vegetal
The apical portions of the tall granulosa cells were hemisphere than in the animal hemisphere in the presence of
lobular in shape (Figs. 2 and 3D). Many proximal portions PMSG.
of attaching filaments were observed in the VPA. The
nuclei of the granulosa cells when observed by Hoechst
DISCUSSION
staining appeared to avoid the areas of non-attaching fila-
ments on the chorion (Fig. 4), because the nuclei existed out The granulosa cells are in contact with the oocyte via
of focus. Calculated by the number of nuclei, the mean cytoplasmicprocessesthatpassthroughtheporecanalsinthe
number of granulosa cells was about 1.5XlO4 in the animal chorion in the medaka [2] and the pipefish [1]. The present
hemisphere. This coincided with the value determined by observations revealedmorphological differences in granulosa
calculation from the cell size (assuming the diameter as ca. 8 cells localized at special regions (VPA) surrounding the
fim). The mean number of cells in the vegetal hemisphere oocyte. These differences seem to reflect the different in-
includingtheVPAwithitstallcells(meandiameterca. 5fim) teractions ofthe granulosa cells with the oocyte, the polarity
was 1.7X104 as determined by the calculation from the cell ofwhich is established during oogenesis. In the early stage
size. of oogenesis, the VPA is determined by the position of the
Balbiani bodyin theooplasm, andthegranulosacellsaligned
2 Production ofsteroids by granulosa cells compactly on the VPA (Iwamatsu, unpublished data). The
E2 and 17a,20/?-diOHp produced by cultured granulosa attaching filaments differentiate and elongate on a restricted
cellsweremeasuredandthevaluesfortheanimalandvegetal region of the chorion in the VPA, and then spirally wind
hemispheres were compared. The production of E2 was around the VPA probably due to rotation of the oocyte and
stimulated by the presence of 100IU/ml PMSG and 100 ng/ granulosa cells [6].
ml progesterone (Table 1). No significant difference (P> We recently found that growing oocytes with granulosa
0.05) inE2productionwas measuredbetween the animal and cells may rotate within the basement membrane [5]. The
thevegetalhemispheres. Theproduction of17a,20/?-diOHp granulosa cells in the animal pole region possess many
by granulosa cells was also stimulated by the presence of microvilli on their apical surfaces, but those in the remaining
PMSG orprogesterone. The concentrations (more than 0.4 area do not. The difference in the distribution of the
82 T. Iwamatsu, S. Nakashima et al.
granulosa cells with microvilli may be related to a difference proteins in pipefish oocytes originate within the follicle and are
in the interactions with the basement membrane, or move- associated with the Z3 layer. J Exp Zool 251: 56-73
ment of granulosa cells beneath the basement membrane. 2 Hirose K (1972) The ultrastructure of the ovarian follicle of
Whether the morphological differences in the granulosa cells medaka, Oryzias latipes. Z Zellforsch 123: 316-329
depend on regional differences in the chorion regions with 3 IwamatsuT(1975) Medakaasateachingmaterial. II. Matura-
tion and fertilization of oocytes. Bull. Aichi Univ. Educ, 24
which they are in contact is not clear from the present study, (Nat. Sci.): 113-144 (in Japanese)
but it was suggested that the patterns of spiral structures on 4 IwamatsuT(1980) Studiesonoocyte maturationofthe meda-
thechorion are due tothe movement offollicularcellsduring ka, Oryzias latipes. VIII. Role offollicle cells in gonadotropi-
oogenesis [6]. nand steroid-induced maturation of oocytes in vitro. J Exp
The steroidogenic response to exogenous hormones of Zool 206: 355-364
granulosa cells from the animal pole hemisphere differed 5 IwamatsuT(1992) Morphology offilamentson the chorionof
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fromthatofcellsfromthevegetalhemisphere. Theproduc- 6 IwamatsuT,NakashimaS, OnitakeK(1993) Spiralpatternsin
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observed in the steroid production by progesterone- and follicle cells around the time of ovulation in the medaka,
slyt.imuTlhateeddifoflfleircelensc,eamltahyougbheitduweastnootthseigndiiffifcearnetnscteatiisnticcaell-l 8 tIOhrweyazmmiaeatdssaulkatTai,peOOsr.hytziaAansT,nloaOttsiphZeiso.mola—JE,aSpStoaankgae5si4:oNf17(o1-o92c89y8t)e dOeovgeelonpemseinsti.n
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causeofthedifferencemayrelatetothehighabilityofthetall andinhibitoryactionsofalowmolecularweightserumfactoron
granulosa cells with crowded mitochondria to produce ster- in vitro maturation ofoocytes ofthe medaka. Biomed Res 8:
313-322
oidsinthevegetalpole area. Thecellsthatproducesteroids 10 IwamatsuT,TakahashiSY, SakaiN, Onitake K(1987) Induc-
are generally filled with mitochondria with well-developed tion and inhibition ofin vitrooocyte maturation andproduction
tubularcristae andtubularordilatedER(see [12]). Howev- ofsteroids in fish follicles by forskolin. J Exp Zool 241: 101—
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thetwohemispheres, inspiteofthedifferenceincellnumber. 11 Masui Y, Clarke HJ (1979) Oocyte maturation. Intern Rev
TcehlilsinmathyeivnedgiectaatlehtehmaitstphheerEe2isprnoodtudcitfifoenrebntyferaocmhtghraatniunlotshae 12 NICnyatg"oFalihs5ah7m:Pah1yY8s5i-(o21l98o82g3y)",vFouln.cItiXoAna(lWmSorHpohaorl,ogAyJoRfatnedlaelolstagnodnaEdsM.
animal hemisphere during the maturation period. There- Donaldson, eds), pp. 223-275, Academic Press, New York
fore, the granulosacellswith gonadotropin receptorsseem to 13 Nagahama Y, Adachi S (1985) Identification of maturation-
differ physiologically or distributively along the animal- inducing steroid in a teleost, the amago salmon (Oncorhynchus
vegetal axis of the oocyte. rhodurus). Develop Biol 109: 428-435
14 RiehlR,AppelbaumS(1991) Auniqueadhesionapparatuson
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ACKNOWLEDGMENTS Jap J Ichthyol 38: 191-197
15 Sakai N, Iwamatsu T, Yamauchi K, Suzuki N, Nagahama Y
The authors thank Dr. Cherrie A. Brown, California Regional (1988) Influence of follicular development on steroid produc-
PrimateResearchCenter, UniversityofCalifornia,Davis,forcritical tion ofthe medaka (Oryziaslatipes) ovarian follicle in response
reading ofthe manuscript. to exogenous substances. Gen Comp Endocrinol 71: 516-523
AW
16 Schuetz (1967) Action of hormoness on germinal vesicle
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