Molecular Human Reproduction vol.2 no.8 pp. 541-548, 1996
Human granulosa cells in culture exhibit functional cyclic AMPregulated gap junctions
C.Furger1-5, LCronier2, C.Poirot3 and M.Pouchelet4
^NSERM U361 and Clinique Universitaire Baudelocque, Paris, 2 Laboratoire de Physiologie Cellulaire, CNRS URA 1869,
Poitiers, 3 Laboratoire de Biologie de la Reproduction, Hopital Cochin, Paris and 4INSERM Service Cinematographie,
Le Vesinet, France
5
To whom correspondence should be addressed at: Garvan Institute of Medical Research, Cancer Biology Division, St
Vincent's Hospital, 384 Victoria Street, Darlinghurst, Sydney, New South Wales 2010, Australia
Numerous gap junctions exist between granulosa cells, between cumulus cells and between cumulus cells
and the oocyte. They may play a role in the regulation of both follicular development and oocyte status. We
used primary cultures of human granulosa cells to study the molecular nature and functionality of these gap
junctions. As shown by a cinemicrographic technique, during the first 3 days of culture, cells flattened and
extended in several directions by means of cytoplasmic extensions. An ultrastructural study showed the
presence of both intercellular and annular gap junctions after 48 h of culture. As revealed by immunodetection
analyses, connexin 43 was present. An analysis using a functional procedure, the gap fluorescence recovery
after photobleaching (FRAP) method, indicated that: (i) diffusional communication existed among granulosa
cells; (ii) the communication was delayed by treatment with 1-heptanol, a well-documented inhibitor of gap
junction permeability; and (iii) permeability was up-regulated by incubation with 8-Br-cAMP, an analogue of
cyclic AMP. The detection of connexin 43 and functional gap junctions in networks of cytoplasmic extensions
indicated junction formation among cells during culture. In conclusion, our results show that human granulosa
cells in culture exhibited functional gap junctions. Connexin 43 was present and the permeability of the gap
junctions was up-regulated by cyclic AMP, an important modulator of human granulosa cell function.
Key words: connexin 43/cyclic AMP/FRAP method/gap junction/human granulosa-lutein cell
Introduction
The follicle is the main functional unit of the mammalian
ovary and provides the support system for oocyte development.
Granulosa cells (GC) surrounding the oocyte contribute in
several ways towards accomplishing this function. They provide nutritive requirements for the growing oocyte, produce
steroid hormones and control both nuclear and cytoplasmic
maturation of the oocyte selected for ovulation. During follicular maturation, the function and differentiation of GC are
regulated by the coordinated actions of the pituitary gonadotrophins through their ability to stimulate the production of
second messengers such as cyclic AMP (cAMP; Marsh, 1975).
GC can communicate through the local production of intraovarian factors such as cytokines (Adashi, 1992) or growth
factors (Adashi et al., 1991) which act as paracrine and/or
autocrine modulators. Furthermore, an intimate anatomical
relationship links the GC. Numerous gap junctions have been
observed among GC (Albertini and Anderson, 1974) and
between the oocyte and the GC of the cumulus (Anderson and
Albertini, 1976). Gap junction channels connect the cytoplasms
of contacting cells, while the junctional aqueous pore (~14 A
in diameter) enables the transfer of small molecules (mol. wt
=£1.5 kDa) (for a review see Kumar and Gilula, 1996).
Functional involvement of gap junctional communication during follicular development has been reported (for a review see
Larsen et al., 1991). Gap junctions enable the passage of
© European Society for Human Reproduction and Embryology
nucleotides, amino acids and sugars from GC to the oocyte
for its growth and development (Eppig, 1979; Heller et al.,
1981; Brower and Schultz, 1982). Furthermore, an intracellular
factor, possibly cAMP, could in this manner control the status
of oocyte meiosis (Dekel and Beers, 1978; Dekel et al., 1981).
Therefore, the disappearance of gap junctions observed near
the time of pre-ovulatory cellular dissociation might be responsible for meiotic resumption (Larsen et al., 1981, 1987;
Racowsky et al., 1989).
In the human, mechanisms controlling gap junctional communication during follicular development are unknown. In the
rat, some data suggest that the synthesis and turnover of gap
junctions are promoted by gonadotrophin hormones (Burghardt
and Anderson, 1981; Burghardt and Matheson, 1982). Following the pre-ovulatory luteinizing hormone (LH) surge or human
chorionic gonadotrophin (HCG) hormone-induced ovulation,
a decrease in gap junction number is observed (Bjersing and
Cajander, 1974). Simultaneously, an increase in characteristic
cytoplasmic double-membrane organelles, termed annular gap
junctions, is reported (Bjorkman, 1962; Espey and Stutts,
1972). This observation supports the hypothesis that membrane
junction breakdown may occur after internalization, via an
endocytotic process of the entire junction into one of the two
cells (Larsen and Tung, 1978; Wert and Larsen, 1990).
Recently, connexin 43 (cx43), a component of the gap
junction protein family, was detected in rat (Beyer et al., 1989;
541
C.Furger et al.
Figure 1. Typical computer-generated fluorescence images of cultured human granulosa cells. Cells were loaded for 10 min with the
fluorescent dye CFDA, before laser scanning and the photobleaching procedure. Fluorescence images represent selected areas corresponding
to human granulosa cells: (a) before photobleaching; (b) just after photobleaching of cells 1 and 2; and (c) 12 min after photobleaching.
(d) Same field seen by phase-contrast microscopy.
Wiesen and Midgley, 1993) and cattle (Sutovsky et al., 1993)
ovaries. The expression of cx43 mRNA and protein seems to
depend on the stage of follicular maturation and on intrafollicular cell position (Wiesen and Midgley, 1993, 1994).
The emergence of in-vitro fertilization (IVF) programmes
has enabled the assessment of gap junctional communication
to be made in human GC. Our investigations were designed
to examine the presence of gap junctions and gap junctional
communication in human GC. The use of human GC in
primary culture requires a detailed knowledge of their in-vitro
development process. Therefore, an analysis of their evolution
in culture was performed by time-lapse cinematography. The
presence of gap junctions was investigated by ultrastructural
techniques and cx43 immunolocalization. The presence of
a functional gap junction communication, and its possible
modulation by the 8-Br-cAMP (a membrane-permeant analogue of cAMP), were investigated by fluorescence recovery
after photobleaching (gap FRAP).
Materials and methods
Collection and culture of human GC
GC were aspirated along with follicular fluid and oocytes from patients
participating in an IVF programme. Patients were stimulated by com542
bined therapy with [D-Trp6]-gonadotrophin-releasing hormone (Decapeptyl; Ipsen-Biotech, Paris, France) and human menopausal
gonadotrophin(HMG; Humegon; Organon, Serifontaine, France). Ovulation was triggered with HCG (5000 IU; Organon). The cumulusoocyte complex was used in the IVF programme. Follicular fluids from
several follicles of several patients were collected. Follicularfluidwas
removed after sedimentation of the cells by centrifugation (800 g, 10
min, 20°C) and replaced by phosphate-buffered saline (PBS; Sigma,
St-Quentin-Fallavier, France). Red blood cells were removed by centrifugation on a preparation of Ficoll and sodium diatrizoate (Histopaque1077; Sigma; 300 g, 35 min, 20°C). After mechanical dispersion, cells
were counted in a haemocytometer and cultured (50 000 cells/cm2) in
35 mm plastic culture dishes (Poly Labo, Strasbourg, France) or 4-well
glass slides (LAB-TEK; Poly Labo). Cell viability was assessed by
Trypan Blue exclusion in a protein-free medium and was ~80%. Cells
were then cultured in Menezo B 2 medium (BioMerieux, INRA, France)
supplemented with 10% fetal calf serum and maintained for 48 h at
37°C in a humidified environment with 5% CCh and 95% air. After 24
h and before replacing the medium, three washings with PBS at 37°C
enabled the removal of dead granulosa and red blood cells. The morphology of the cells was similar after 48 h of culture in plastic or glass
substrates. For comparable results, all the experiments were performed
with the same cell density (50 000 cells/cm2). The cell density varied
throughout the culture dish, but the same immunostaining and fluorescence recovery curve types were observed between cells grown from
clusters and between well-separated cells.
Functional gap junctions between human GC
Figure 2. Behaviour of human granulosa cells in culture. Micrographs represent the selected images of a 16 mm film after (a) 5, (b) 20,
(c) 40 and (d) 60 h of culture. Note the increase in the intercellular contact number with time, and the long cytoplasmic extensions emitted
by cells during their spreading. Bars = 50 |im.
Time-lapse cinemicrography
Cell culture was performed using a 'Rose chamber', an airtight culture
chamber comprising two circular glass coverslips separated by a silicon
joint, the latter being pierced by two needles that allow the medium to
be changed during culture (Rose, 1954). Cell behaviour was recorded
5 min after the introduction of collected cells in the Rose chamber. A
record of cell behaviour over 72 h was taken with a Zeiss inverted
microscope (ICM 405) equipped with a 16X Ph N.A. 0.40 Neofiuar
objective and a 16 mm Arriflex standard cinecamera driven by a handmade time-lapse unit. The microscope was placed in a box maintained
at 37°C with the use of an integrated temperature controller device
(Multitop, Chauvin Arnoux, France). Frames were taken every minute.
Between two successive frames, the light was turned off to avoid cellular
damage. Agfa Copex Rapid AHU film was used. Cell behaviour was
analysed by an analysis projector.
Transmission electron microscopy
After 2 days of culture, cells grown in 35 mm culture dishes were
fixed in the dishes by 3% glutaraldehyde in Sorensen buffer, pH 7.3,
for 1 h at room temperature. The cells were then scraped and
centrifuged. The pellet was post-fixed with 4% OSO4 in the same
buffer for 1 h at 4°C, dehydrated in a graded series of ethanol and
propylene oxide, and embedded in Epon. Thin sections were stained
with uranyl acetate and lead citrate and examined using a Zeiss
electron microscope EM 10C/CR.
Immunofluorescence
After 2 days of culture, cells grown in four-well glass slides were
washed three times with PBS and fixed for 10 min with absolute
methanol at -20°C. After rinsing with PBS, cells were incubated
overnight at 4°C in a light-tight humidified box with either cx43specific rabbit antibody or preimmune serum (El Aoumari el at.,
1990) [both diluted 1:20 in PBS-0.1 % bovine serum albumin (BSA)].
Cells were washed three times with PBS for 10 min each, followed
by incubation with a fluorescein isothiocyanate (FITC)-conjugated
goat anti-rabbit immunoglobulin G (Nordic; Tebu, Le Perret en
Yvelines, France) diluted 1:10 in PBS-0.1% BSA for 1 h at 20°C.
Slides were washed three times with PBS for 10 min each and
coverslips were mounted with a mixture of glycerol and mowiol.
Photographs were taken with a microscope (DMRB; Leica,
Rueil-Malmaison, France) equipped with an F1TC filter (1C; Leica)
or an interference contrast system.
Fluorescence recovery after photobleaching (gap FRAP)
method
The cell-cell diffusion of a fluorescent dye was measured by the gap
FRAP method, developed by Wade et al. (1986), using an interactive
laser cytometer (ACAS 570; Meridian Instruments, Okemos, MI,
USA), which allows for convenient digital video imaging and analysis.
After 2 days of culture in 35 mm culture dishes, the medium was
replaced by Tyrode solution containing (in mM) 144 NaCl, 5.4 KC1,
2.5 CaCl2, 1 MgCl2, 0.3 NaH2PO4, 5.5 glucose and 5 HEPES buffer
at pH 7.4. Cells were loaded for 10 min at room temperature with
6-carboxyfluorescein (CF) diacetate (7 (ig/ml in 0.25% dimethylsulphoxide), a membrane-permeant molecule which is hydrolysed by
cytoplasmic esterases to CF, a hydrophilic derivative that remains
trapped in the cells. The fluorescence of some selected cells adjacent
to other cells was photobleached by applying strong light pulses from
an argon laser set at 488 nm. The fluorescence intensity (excited by
weak laser pulses) was recorded before and after photobleaching
(Figure 1) and stored for analysis. If cells were interconnected by
permeable gap junctions, fluorescence recovery following a slow
exponential time course was observed in bleached cells because of
the diffusion of CF from adjacent unbleached cells. The procedure
for the local perifusion of 1-heptanol has been described previously
(Cronier el al., 1994). Briefly, a needle was moved close to the
selected cells under microscopic control and 1 -heptanol (3 mM) was
injected continuously over a 2 min period. At the end of the injection,
I-heptanol was not removed but its local concentration rapidly
decreased because of dilution in the medium. For the cAMP study,
cells showing a typical fluorescence recovery curve were chosen. GC
were cultured in the presence of 8-Br-cAMP (5X10"4 M) at 37°C in
543
C.Furger et al.
100
100
Figure 7. Corrected fluorescence recovery curves corresponding to
two photobleached cells, one isolated (O), and another in contact
with other cells ( • ) . No fluorescence recovery was observed in the
isolated cell. ( • ) corresponds to an isolated unbleached cell used as
control.
constant k was determined from recovery curves using the
following equation:
Figure 8. Corrected fluorescence recovery curves showing the
effect of a 2 min local perifusion of 1-heptanol (Hep), an inhibitor
of gap junctional permeability. ( • ) and (D) represent
photobleached and control cells respectively. The low rate of
fluorescence intensity in the bleached cell persists as long as
heptanol is present in the medium.
( f i - Ft)/(F{ - Fo) = e"*
Table I. Increase in the relative rate constant by treatment with 8-Br-cAMP
where Fh Fo and Ft are fluorescence intensities in tested cells
before photobleaching, just after photobleaching and at time t
respectively. The determination of k was performed during the
4 min following the photobleaching (Figure 6, inset). The
mean value of rate constant k of cells that recovered (n = 24)
was 11.80 ± 1.33X10"2 min"1. When isolated cells were
photobleached, no fluorescence recovery was observed (Figure
7,O).
Aliphatic alcohols with chain length C6-C9 rapidly and
reversibly interrupt the cell-cell communication through gap
junctions (Johnston et al., 1980; Deleze and Herve, 1983). To
ascertain junctional communication type, an experiment was
performed with 1-heptanol. A 2 min local perifusion of 1heptanol delayed the recovery of fluorescence for 2 min (Figure
8), confirming the gap junctional nature of the dye diffusion.
To test the short time effect of cAMP on gap junctional
communication between human GC, we measured the rate
constant k of four selected areas before and after incubation
with 8-Br-cAMP (5 X10^ M) for 1 h. For all tested areas,
8-Br-cAMP increased the rate constant k (Table I), suggesting
a modulating role for cAMP in gap junctional communication
between GC.
Discussion
Our results demonstrate for the first time that human GC in
culture exhibit contact-dependent communication by means of
gap junctions. Cinematographic studies revealed that cytoplasmic extensions appear a few hours after the beginning of
the culture, and that the cell-cell contact surface increases
significantly with time. Cx43 and gap junctional communication are observed between GC connected by only one thin
546
Experiment no.
1
2
3
4
Mean ± SD
(lO"2 min"1)
GO"2 min"1)
13.63
8.98
15.48
11.61
12.42 ± 2.78
16.29
15.92
18.82
18.22
17.31 ± 1.42
Percentage of
increase (%)
19.52
77.28
21.57
56.93
a = relative rate constant measured under control conditions; (3 = relative
rate constant measured on the same cells after 8-Br-cAMP incubation for 1
h.
cytoplasmic extension. This argues for gap junction formation
during cell spreading.
The ultrastructural analysis confirms the presence of the
typical gap junctions reported previously in the same model
(Amsterdam et al., 1989). Furthermore, it revealed that the
two GC maturation states observed in fresh undissociated
human GC, using cytoplasmic organelle development as a
criterion of cell differentiation (Rotmensch et al., 1986),
are also present in our in-vitro system. Therefore, we can
hypothesize that both peri-ovulatory and less mature GC coexisted in our experiments. These different maturation stages
are likely to reflect the diversity of the cell origin inherent in
the IVF procedure. GC originated from several follicles of
different sizes and probably different follicular compartments.
Our data demonstrate for the first time the expression
of cx43 in human GC. In rat ovarian follicles, cx43 was
immunolocalized within the GC layer (Beyer et al., 1989).
Recently, immunolocalization and in-situ hybridization were
used to specify cx43 distribution within the developing follicle
of the rat ovary (Wiesen and Midgley, 1993). The protein and
its mRNA were localized in the GC mural part of the mature
Functional gap junctions between human GC
follicle, but were lacking in the corpus luteum (Wiesen and
Midgley, 1993) and atretic follicles (Wiesen and Midgley,
1993, 1994). A dramatic loss of cx43 was also observed at
the time of ovulation (Wiesen and Midgley, 1993). In our
study, the use of a cx43-specific antibody revealed intercellular
and macular stainings. Such localization supports the model
of ultrastructural gap junction topology and is consistent with
our ultrastructural localization. Both procedures indicate that
gap junction components are present not only among cellular
bodies, but also within networks of cytoplasmic extensions
and between cytoplasmic extensions and cellular bodies. Moreover, it is likely that the intracellular macular fluorescent
staining observed within some cells reflects the presence of
the very common intracytoplasmic annular gap junctions
observed by electron microscopy. The same pattern of cx43
intracytoplasmic staining was reported recently in both baboon
and human luteal cells in culture using a different cx43
antibody (Khan-Dawood et al., 1996). However, one cannot
exclude the possibility that cytoplasmic staining is caused by
the presence of free hemi-connexons or free cx43 proteins
localized within specific cytoplasmic compartments like the
Golgi network.
The gap FRAP method, which was used successfully to
investigate intercellular communication in rat primary (Stein
et al., 1991) and spontaneous immortalized (Stein et al., 1993)
GC, in bovine cultured luteal cells (Redmer et al., 1991) and
in other endocrine cells (Cronier et al., 1994), has enabled us
to demonstrate the presence of a cell-cell communication
through gap junctions in human GC in culture. The rapid,
reversible uncoupling observed after heptanol addition in the
control medium confirmed that the initial dye diffusion, which
follows an exponential time course, occurs via gap junctions.
This finding was substantiated by the ultrastructural observation
of gap junctions between GC membranes and by immunolocalization of cx43. Although dye coupling between contacting
cells is not a direct measure of the flow of biologically
active molecules from one cell to another, it unequivocally
demonstrates the intercellular transfer of hydrophilic molecules
(Wade et al., 1986). It is very likely that biologically important
molecules of low molecular weight are also exchanged between
cells that exhibit dye coupling. Our results show that the
rate of transfer of molecules across human GC junctions is
significantly and rapidly increased by the presence of
8-Br-cAMP (5X10^* M). Previously this nucleotide has been
associated with the modulation of gap junction communication
in different cell types (Flagg-Newton et al., 1981; Saez
et al, 1986; Giaume et al., 1991). In swine cultured GC,
communication was reduced sharply after the intracellular
microinjection of protein kinase A inhibitor, but resumed with
the injection of protein kinase A catalytic subunit or exposure
to follicle stimulating hormone (FSH; Godwin et al., 1993).
These results and ours indicate that cAMP, which has been
implicated recently in the action of gonadotrophin hormones
in human GC (Furger et al., 1996), could regulate its own
diffusion rate through gap junctions within the GC layer.
Moreover, one can hypothesize that the cAMP-dependent
increase in junctional permeability is the consequence of direct
protein kinase A-dependent phosphorylation of the channel-
forming protein cx43. This protein has been found to be
present in different phosphorylation states (Kadle et ai, 1991;
Musil and Goodenough, 1991) regulated by the cAMP pathway
(Burt and Spray, 1988; Granot and Dekel, 1994). However,
gonadotrophin-releasing hormone and 12-D-tetradecanoyl
phorbol-13-acetate (TPA) have also been shown to regulate
the phosphorylation state of cx43, and protein kinase C is also
likely to be involved in that process (Granot and Dekel, 1994).
In summary, we show that human GC in culture exhibit
functional gap junctions, the permeability of which is
up-regulated by a cAMP analogue. It is likely that cAMP,
which mediates the intracellular effect of LH and FSH, allows
the gonadotrophins to control gap junctional status in human
GC. Furthermore, the resumption of meiosis appears to be
temporally correlated with the loss of gap junctions occurring
in granulosa but not cumulus cells during the peri-ovulatory
period (Moor et al., 1981; Eppig, 1982). Gap junctions between
GC at a distant position from the oocyte may occupy a critical
location in the follicle by providing a bridge between the
entire oocyte-cumulus complex and the remaining membrana
granulosa (Racowsky etai, 1989). Loss or closure of junctional
channels could isolate the oocyte and some companion somatic
cells from the regulatory influence of the underlying membrana
granulosa. Further studies of gating mechanisms and connexin
expression regulation in human GC in culture will contribute
to a better understanding of pre-ovulatory intra-ovarian
modifications.
Acknowledgements
The authors are very grateful to Dr Daniel Gros (CNRS UMR C9943,
Luminy, Faculte des Sciences, Marseille, France) for providing human
cx43-specific antibody, and to Dr Andre Malassine for constructive
discussions. We also thank Gerard Delrue and his group (INSERM SC
6) for iconographic assistance, Nelly Gouhier for the cinematographic
records and Mylene Navarro for providing the DMRB Leica fluorescence microscope. This work was supported by grants given by
INSERM.
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Received on March 22, 1996; accepted on June 6, 1996