The Distribution of Contractile Cells in the Apex of the Preovulatory Hamster Follicle

BIOLOGY OF REPRODUCTION 20, 205-213 (1979)

205

The Distribution of Contractile Cells

in the Apex of the Preovulatory Hamster Follicle

PAULA B. PENDERGRASS’ and PRUDENCE TALBOT2

Department of Anatomy’,

Wright State University,

School of Medicine,

Dayton, Ohio 45435

and

Department of Biology2

University of California,

Riverside, California 92521

ABSTRACT

The apical regions of maturing hamster follicles were examined by electron microscopy at 14,

9, 5, and 3 h prior to expected ovulation. At all time periods, a discrete layer of smooth muscle

cells was found within the theca externa of the maturing follicle. In addition, bundles and clusters

of filaments were found in the surface epithelial cells of maturing hamster follicles throughout the

preovulatory period. A contractile role in ovulation is suggested for both the smooth muscle and

surface epithelial cells.

INTRODUCTION

Vertebrate ovulation is a complex phenome-

non involving rupture of the Graafian follicle

and emergence of the oocyte. This process

has been attributed to: 1) build up of intrafol-

licular pressure, an idea now largely discounted

(Blandau and Rumery, 1963; Espey and Lipner,

1963); 2) enzymatic digestion of the follicle

wall (for a review, see Espey, 1974); 3) inflam-

matory reactions in the follicle wall (Parr,

1974; Espey and Coons, 1976) and 4) contrac-

tion of ovarian smooth muscle (e.g., see Wright

et al., 1976). Although ovulation probably

involves more than one of these mechanisms,

this paper will be limited to a discussion of the

role of contractile cells in ovulation.

The idea that a contractile mechanism is a

general feature of vertebrate ovulation is

supported by studies involving both lower

vertebrates and mammals. In the fish, Oryzias,

intact follicles freed from ovarian musculature

ovulate by a contractile process which has

been attributed to microfilament-bearing cells

in the theca and which is reversibly inhibited by

cytochalasin B (Pendergrass and Schroeder,

1976; Schroeder and Pendergrass, 1976);

Accepted July 6, 1978.

Received November 21, 1977.

cytochalasin B has been shown to be an inhibi-

tor of microfilamentous systems (e.g., Wessels

et al., 1971; McGuire and Moellmann, 1972;!’.

Schroeder, 1970). A similar inhibition of

ovulation by cytochalasin B has been observed

in the frogs, Rana and Hyla, (Schroeder, 1973;

Larsen et al., 1977) and partial inhibition

occurs in the trout, Salino (Jalabert and Szol-

losi, 1975). Moreover, during frog ovulation,

microfilament-bearing cells of the theca shorten

to bring about oocyte extrusion (Larsen et al.,

1977) and in the trout, ovulation can be

induced by prostaglandin F2a, a smooth muscle

stimulant (Jalabert and Szollosi, 1975). These

observations are consistent with microfilament-

bearing cells and/or smooth muscle cells being

involved in ovulation.

Many reports of smooth muscle and smooth

muscle-like cells in the walls of ovarian follicles

(Fumagalli et al., 1971; Burden, 1973; Bjersing

and Cajander, 1974; O’Shea, 1971; Walles et al.,

1975; Osvaldo-Decima, 1970; McReynolds et

al., 1973) suggest that a contractile mechanism

is important in mammalian ovulation. This idea

is strengthened by the observation that rabbit

ovulation is inhibited after injection of �3

receptor blockers into either the ear vein or

ovarian artery (Foda et al., 1975; Virutamasen

et al., 1971) and by the recent demonstration

that ovarian contractility increases before

expected ovulation in the rabbit (Wright et al.,

206 PENDERGRASS AND TALBOT

1976; Virutamasen et al., 1976). However,

there is disagreement about the probable role of

this mechanism and direct evidence that smooth

muscle contractions result in mammalian

ovulation is needed.

With the above evidence in mind, we have

asked the questions: Do contractile cells

function in mammalian ovulation and if so,

how are they involved? We have initiated

studies to answer these questions using the

hamster as an experimental animal. The pur-

pose of this report is to describe the location

and development of contractile elements found

both in the theca externa and in the surface

epithclium of the apex of the maturing preovu-

latory hamster follicle. By establishing the

presence and nature of such contractile cells,

we hope to provide a framework for interpreta-

tion of ultrastructural and physiological studies

designed to demonstrate that contractile

elements in the mammalian follicle do play a

role in ovulation.

MATERIALS AND METHODS

Female golden hamsters were injected with 25 lUhuman chorionic gonadotropin (hCG) on Day 3 of a 4

day cycle to synchronize follicular development, with

the completion of ovulation expected 14 h post-hCG

injection. At 14, 9, 5 and 3 h prior to expected

ovulation, the left ovary and oviduct were removed

surgically and processed for transmission electron

microscopy. Hamsters were allowed to recover from

surgery. At 2 h after expected ovulation (16 h post-

hCG injection), the intact right tract was examined for

signs of ovulation (cumulus masses in the oviduct).

The only follicles studied were those from hamsters inwhich ovulation was verified in the right ovary.

Fixation was in 3.5% glutaraldehyde-paraformal-

dehyde buffered in 0.2 M cacodylate (Karnovsky,

1965). The largest 3 or 4 follicles in each ovary were

dissected out and placed into fresh fixative for 3 h.

After several buffer rinses, follicles were postfixed in

1% 0504 buffered with cacodylate, dehydrated in a

graded series of acetone, infiltrated in 11 acetone-lowviscosity plastic (Spurr, 1969) and embedded. Follicles

were oriented sideways in blocks so that radial sec-

tions could be taken from the apical region and thick

sections were used to confirm location in the follicles.

Thin sections were made on an MT-2 ultramicrotome,stained with uranium and lead salts and examined witha Siemans 101 electron microscope.

RESU LTS

The hamster follicle is comprised of an outer

layer or surface epithelium which is separated

from the underlying tunica albuginea by a

basement membrane; beneath the tunica

albuginea are the theca externa and theca

interna which are separated from the granulosa

cells by a basement membrane (Figs. 1, 2). For

orientation, the apical region studied in follicles

is shown in the inset in Fig. 1.

In 14, 9, 5 and 3 h preovulatory hamster

follicles, the theca externa is comprised of a

layer of 4-6 cells containing numerous fila-

mentous cells (shown in Fig. 1 and 2 for 14 and

3 h preovulatory). At higher magnification,

these cells have the characteristics of smooth

muscle cells. Filaments (60-80 A), deposits

resembling ii glycogen and caveolae are numer-

ous (Figs. 3-5); dense attachment plaques

within the filament masses are present, although

quite sparse (Fig. 6); the smooth muscle cells

are attached to each other laterally by special-

ized junctions (Fig. 7). The structure of any

given cell is quite varied along its length (Fig.

2); thus, a particular section may show features

of smooth muscle cells, microfilament-bearing

cells or cells which appear to be intermediate.

However, correlation of low and high magnifi-

cation micrographs verified that only one

filament-bearing cell type, the smooth muscle

cell, is present in the theca externa.

In the surface epithelium, groups of filaments

(80-100 A) are seen in follicles throughout the

preovulatory period. Occasionally the filaments

appear in bundles, but more commonly they

appear as loose clusters (Fig. 8). Other than the

presence of filaments, the surface epithelium

has none of the characteristics of smooth

muscle cells.

DISCUSSION

The distribution of cells which morphologi-

cally appear capable of contraction, i.e., fila-

ment-bearing cells, was examined during various

stages of preovulatory development in hamster

follicles. Our results indicate that in the apex of

the preovulatory hamster follicle: 1) the

theca externa contains a discrete layer of

smooth muscle cells and 2) that as the follicle

matures and prepares to undergo ovulation,

bundles and clusters of filaments appear in the

surface epithelium.

We have referred to cells in the theca externa

as smooth muscle cells because the appropriate

distinguishing features of smooth muscle

are present, i.e., filaments, caveolae, glycogen-

like particles and electron dense attachment

plaques (see Gabella, 1973, 1975; Gillespie and

Lullmann-Rauch, 1974). Depending on the

section, we have also found fibroblast-like and

intermediate cells such as have been reported in

CONTRACTILE CELLS OF THE HAMSTER FOLLICLE 207

FIG. 1. Hamster follicle 14 h before ovulation showing surface epithelium (SE), basal lamina (BL), tunica

albuginea (TA), theca externa (TE) and theca interna (TI). X 10,000.

cat (Fumagalli et al., 1971; Burden, 1973),

sheep (O’Shea, 1971), cow (Walles et al., 1975)

and hamster and gerbil (McReynolds et al.,

1973). However, careful correlation of high and

low magnification micrographs in the hamster

reveals considerable variation among processes

of a given cell such that while some portion(s)

always have smooth muscle characteristics,

other portions may have features suggestive of

fibroblasts or intermediate cell types. In con-

trast, the cells of the hamster surface epitheli-

urn, although filament bearing, do not have the

other distinguishing characteristics of smooth

muscle and accordingly we have not classified

them as such.

Our results are in close agreement with those

of other workers who have reported perifollicu-

lar contractile cells in mammals (Fumagalli et

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FIG. 2. Hamster follicle 3 h before ovulation showing surface epithelium (SE), basal lamina (BL), tunics

albuginea (TA) and theca externa (TE). X 10,000.


FIG. 3. Smooth muscle of hamster theca externa 14 h before ovulation showing filaments (arrows) and

glycogen (Gly). X 80,000.

210

I.

PENDERGRASS AND TALBOT

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FIG. 4. Smooth muscle of hamster theca externa 3 h before ovulation showing filaments (arrows) and cavelo-lae (Ca). X 80,000.

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FIG. 5. H.smster smooth muscle cell showing abundant caveolae. X 25,000.

FIG. 6. Hamster smooth muscle cell showing dense attachment plaques (arrows) in the filament

mass. X 25,000.FIG. 7. Specialized �unctiQn (arrow) connecting 2 smooth muscle cells laterally. X 25.000.

FIG. 8. Surface epitheliuin of hamster follicle showing filament clusters (a) and bundles (b). X 50,000.


al., 1971; Burden, 1973; Bjersing and Cajander,

1974; O’Shea, 1971; Walles et al., 1975; Osvaldo-

Decima, 1970; McReynolds et al., 1973). They

are in particular agreement with those of

McReynolds et al. (1973) who reported smooth

muscle-like cells, as well as fibroblast-like cells

and an intermediate cell type, in the theca

externa of mature hamster and gerbil follicles.

Our results add to theirs in that we have con-

sistently found a 5-6 cell layer of smooth

muscle cells connected by specialized junctions

in the apex of the hamster follicle, not only at

the approximate time of ovulation, but also as

early as 14 h before ovulation. Smooth muscle

in the apex of mammalian follicles has previous-

ly been reported only for rabbit (Bjersing and

Cajander, 1974) and rat (Burden, 1973). Thus,

it appears from combined studies that the theca

externa around the entire hamster follicle

probably contains a discrete 5-6 cell layer of

smooth muscle. This layer of cells is certainly

present in sufficient quantity and in the correct

location to influence ovulation.

That contractile cells play a role in hamster

ovulation is further strengthened by our report

of bundles and clusters of filaments appearing

in the surface epthelium during the preovula-

tory period. The presence of filaments in the

preovulatory surface epithelium is highly

suggestive and implies that these cells possess

contractile capabilities which could assist in the

ovulation process. It is likely that the smooth

muscle cells and surface epithelial cells are both

involved in ovulation, although perhaps different

aspects. For example, surface epithelial cells

may contract to assist in stigma formation and

rupture of the stigma subsequent to enzymatic

digestion or inflammation, while the smooth

muscle cells may form a ring of constriction

and cause extrusion of the oocyte.

Considering recently developed models in

both fish and frog where contractile cells in the

theca of ovarian follicles function in ovulation

(Pendergrass and Schroeder, 1976, Schroeder

and Pendergrass, 1976; Schroeder, 1973;

Larsen, Schroeder and Waldo, 1977) and

considering the disposition of contractile cells

in the theca of the apex of the hamster follicle,

it is quite likely that contractile mechanisms are

a general feature of vertebrate ovulation,

although additional data, in particular for

mammals, is required to establish this point and

to explain the precise role of contractile cells in

ovulation.

ACKNOWLEDGMENTS

This research was supported by an Institutional

Research Grant from Texas Woman’s University andby NIH grant HD-11008. The cooperation of Dr.Luther Franklin in making research facilities availableat the University of Houston, Houston, Texas is

gratefully acknowledged.

REFERENCES

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of tunics albuginea and theca externa of rabbitGraafian follicles prior to induced ovulation. Cell

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Blandau, R. and Rumery, R. (1963). Measurements of

intrafollicular pressure in ovulatory and preovula-

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341.

Burden, H. (1973). The distribution of smooth muscle

in the cat ovary with a note on its adrenergic

innervation. J. Morph. 140, 467-476.

Espey, L. (1974). Ovarian proteilytic enzymes and

ovulation. Biol. Reprod. 10, 216-23 5.

Espey, L. and Coons, P. (1976). Factors which influ-

ence ovulatory degradation of rabbit ovarian

follicles. Biol. Reprod. 14, 233-245.

Espey, L. and Lipner, H. (1963). Measurement ofintrafollicular pressure in the rabbit ovary. Amer.

J. Physiol. 205, 1067-1072.

Foda, M., Darwish, N. and Shafeek, A. (1975). Theeffect of some p-adrenergic blocking agents on

ovulation in rabbits. Contraception 21, 53-58.

Fumagalli, Z., Motta, P. and Calvieri, S. (1971). Thepresence of smooth muscular cells in the ovary of

several mammals as seen under the electron

microscope. Experientia 27, 682-683.

Gabella, G. (1973). Fine structure of smooth muscle.

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Gabella, G. (1975). Hypertrophy of intestinal smooth

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Gillespie, J. and Lullman-Rauch, R. (1974). On the

ultrastructure of the rat anococcygenus muscle.

Cell Tiss. Res. 149, 92-103.Jalabert, B. and Szollosi, D. (1975). In vitro ovulation

of trout oocytes: effect of prostaglandins on

smooth muscle-like cells of the theca. Prostaglan-

dins 9, 765-778.

Karnovsky, M. (1965). Aformaldehyde-glutaraldehyde

fixation on high osmolarity for use in electron

microscopy. J. Cell. Biol. 27, 137a.Larsen, J., Schroeder, P. and Waldo, A. (1977).

Structure and function of the amphibian follicu-lar epithelium during ovulation. Cell Tiss. Res.

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McGuire, J. and Moellmann, C. (1972). Cytochalasin

B’ Effects on microfilaments and movement of

melanin granules within melanocytes. Science

175, 642-644.

McReynolds, H., Siraki, C., Bramson, P. and Polloch,R. (1973). Smooth muscle-like cells in ovaries ofthe hamster and gerbil. Z. Zellforsch. Mikrosk.Anat. 140, 1-8.

O’Shea, J. (1971). Smooth muscle-like cells in thetheca externa of ovarian follicles in the sheep. J.Reprod. Fert. 24, 282-285.


Osvaldo-Decima, L. (1970). Smooth muscle in the

ovary of the rat and monkey. J. Ultrastruct. Res.29, 218-237.

Parr, E. (1974). Histological examination of the rat

ovarian follicle wall prior to ovulation. Biol.

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Schroeder, P. (1973). The inhibition of amphibianovulation in vitro by cytochalasin B. Experientia

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Schroeder, P. and Pendergrass, P. (1976). The inhibi-

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Schroeder, T. (1970). The contractile ring. I. fine

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Local ovarian effects of catecholamines onhuman chorionic gonadotropin-induced ovulation

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Virutamasen, P., Smitasira, P. and Fuchs, A. (1976).

Intraovarian pressure changes during ovulation in

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Walles, B., Edvinsson, L., Falck, B., Owman, C.,Sjoberg, N. and Sevensson, K. (1975). Evidence

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fluorescence and electron microscopy and effectsof thyramine on follicle strips. Biol. Reprod. 12,239-248.

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Wright, K., Wallach, E., Fromm, E. and Jeutter, D.

(1976). Studies of rabbit ovarian contractility

using chronically implanted transducers. Fertil.

Steril. 27, 310-318.

RECOMMENDED REVIEWS

Bjersing, L. and Cajander, S. (1974). Ovulation and

the mechanism of follicle rupture. V. Ultrastruc-

ture of tunics albuginea and theca externa of

rabbit Graafian follicles prior to induced ovula-

tion. Cell. Tiss. Res. 153, 15-30.

Larsen, J., Schroeder, P. and Waldo, A. (1977).

Structure and function of the amphibian follicular

epithelium during ovulation. Cell Tiss. Res. 181,

505-5 18.Wright, K., Wallach, M., Fromm, E. and Jeutter, D.

(1976). Studies of rabbit ovarian contractility

using chronically implanted transducers. Fertil.

Steril. 27, 310-318.

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The Distribution of Contractile Cells in the Apex of the Preovulatory Hamster Follicle