Human Reproduction vol 11 no 10 pp 2211-2214, 1996
Flow cytometric analysis of granulosa cells from
follicular fluid after follicular stimulation
D.De Neubourg1*3, A.Robins2, S.Fishel1 and
L.Gibbon 2
'NURTURE (Nottingham University) Department of Obstetrics and
Gynaecology, 2Department of Immunology, University Hospital,
Queen's Medical Centre, Nottingham, NG7 2UH, UK
•'To whom correspondence should be addressed at: Division of
Reproductive Medicine, University Hospital of Antwerp,
Wilnjkstraat, 10, 2650 Edegem, Belgium
Granulosa cells are not easily accessible, unless they are
examined in follicular fluid after oocyte retrieval. These
samples are usually contaminated with blood. We have set
up a general technique for analysis of granulosa cells
without physically separating them from blood cells. The
sample is stained with CD45, which is a pan-leukocyte
marker, and granulosa cells are consecutively selected as
CD45 negative during flow cytometric analysis. Analysis
of forward scatter of the granulosa cells, which is correlated
to cell size, shows a wide size range throughout the
whole population rather than two distinct populations as
previously suggested.
Key words: flow cytometry/follicular fluid/granulosa cells
Introduction
In-vivo investigation of follicular dynamics could bring an
important contribution to our understanding of folliculogenesis
and steroidogenesis, but is usually not feasible because of the
inaccessibility of the follicle. Granulosa cells in follicular fluid
after oocyte retrieval provide a source of large amounts of
fresh material from dominant follicles. Flow cytometry is a
technique whereby cells from a single cell suspension are
analysed individually when intersecting an illuminating beam.
Scatter characteristics provide information on cell size and
granularity, whereas the use of fluorescent probes gives
information on cell surface, cytoplasmic or nuclear determinants.
The cellular content of follicular aspirates and flushes
consists of granulosa cells, single and in clumps, leukocytes
and large epithelial cells. Initially density gradients were used
to physically separate red and white blood cells from granulosa
cells (Whitman et al, 1991; Seifer et al., 1992). Both Ficoll
and Percoll gradients showed separation of red blood cells
and lymphocytes but the interphase fraction containing the
granulosa cells still had an important number of granulocytes.
For this reason and because of the possibility of losing
cell fractions in the gradient, this method was not adopted.
Granulosa cells from the human ovanan follicle have been
© European Society for Human Reproduction and Embryology
separated according to their isopycnic properties. It was
proposed that cumulus cells compose the least dense band
and that another band may be made up with antral cells
(Hartshome, 1990).
The use of a marker for granulosa cells was put forward
and cytoskeletal as well as endocrine markers were considered.
However, cytoskeletal markers were not exclusive (Czemobilsky et al., 1985) and required cell permeabilization (Larsen,
1994). The use of hormone receptors, whether cytoplasmic or
nuclear, was considered, but oestrogen receptors have only
recently been demonstrated on granulosa cells (Hurst et at.,
1995) By their very nature cyclic changes may be expected
and therefore hormone receptors were considered to be too
variable if the technique was to be used in other settings.
Finally, CD45 was used to mark leukocytes, and granulosa
cells were identified as CD45 negative cells during flow
cytometric analysis.
Materials and methods
Patients
Follicular fluid was collected during oocyte retrieval after informed
consent had been obtained from 13 patients attending the Nottingham
University Hospital (NURTURE), UK for male and female infertility
treatment Pituitary desensiuzation was obtained with gonadotrophinreleasing hormone analogues (GnRHa) (Suprefact: Hoechst,
Hounslow, Middlesex, UK) which were started during the midluteal
phase of the pretreatment cycle for a minimum of 2-3 weeks. Daily
l.m. injecuons of human menopausal gonadotrophins (HMG, Pergonal:
Serono SA, Aubonne, Switzerland; Humegon: Organon, Cambridge,
UK) were used as follicular sumulation. Ovulation was induced with
human chononic gonadotrophin (HCG) 10 000 IU l m (Profasi
Serono SA). Oocytes were retrieved 36 h later under transvaginal
ultrasound guidance. All the follicular fluid and flushes (with Hartman's solution containing 2 IU of hepann/ml) collected from one
patient were pooled.
Sample preparation
All the fluid was centnfuged at 300 g for 5 nun and cell pellets were
transferred into one tube and resuspended in RPMI 1640 (Sigma UK,
Poole, Dorset, UK) + 1% fetal calf serum (FCS) A sample analysis
and count was done with 0 04% of Gentian Violet m 3% acetic acid
This preparation showed granulosa cells, single and in clumps,
leukocytes and large epithelial cells Epithelial cells were very large
compared to the population of blood cells and granulosa cells. During
flow cytometnc analysis (see below), the gate for analysis was set
for the size of blood cells and granulosa cells, thereby excluding the
epithelial cells from analysis Fibroblasts and luteal cells were not
identified as a distinct population when a whole sample was stained
(see below) and they certainly were not present in large quantity The
clumps of granulosa cells were mechanically dissociated by gently
2211
D.De Neubourg et aL
pipetting the sample several times. Cell concentration varied between
2 5 and 25X10 6 cells/ml. This preparation was then adjusted to
1X10 6 cells/ml with RPMI1640 + 1% FCS Viability was tested using
Trypan Blue and revealed a minimum of 98% viable granulosa cells.
Staining procedure
CD45-fluorescence isothiocyanate (FITC) 5 Jil (Sigma) was pipetted
into Falcon tubes. Ahquots of 100 ul of the sample of 1X10 6 cells/
ml were added and gently mixed; the sample was incubated for
15 min in the dark at room temperature FACS lysing solution
1 ml [containing diethylene glycol and formaldehyde for use with
fluorescence-activated cell sorting (FACS); Becton Dickinson UK,
Cowley, Oxford, UK] for lysis of red blood cells was added and kept
for 10 min in the dark. It was then removed by centnfuging at 300
g for 5 min and discarding the supernatant. Cells were washed twice
by resuspension in 1 ml of RPMI 1640 + 1% FCS followed by
centrifugation at 300 g for 5 min. The sample was fixed by adding
300 jxl of 0.5% formaldehyde and stored in the dark until analysis.
Control samples included an autofluorescence control and an
isotype matched-FITC conjugated control antibody-treated sample
Flow cytometrie analysis
Flow cytometric analysis was performed on FACScan (Becton
Dickinson) using standard settings: fluorescence 1 (FL1), 4 decades
(logarithmic), detector 648 V, log amplifier, compensation 1.1%;
fluorescence 2 (FL2), 4 decades (logarithmic), detector 496 V, log
amplifier, compensation 22.8%. Data analysis was performed using
Lysis software (Becton Dickinson). For each sample, since analysis
was based on measurement of 10 000 cells, the influence of cells
such as fibroblasts and lutea] cells, which were not identified as
distinct populations in whole sample staining, was felt to be negligible.
Cell sorting
A sample stained with CD45-FTTC was submitted to cell sorting on
the basis of CD45 presence or absence using a FACS IV cell sorter
(Becton Dickinson). Aliquots from each sorted population were
cytospun into a monolayer using cytospin 3 (Shandon, Runcom,
Cheshire, UK) and stained with Wright staining
Results
Identification of granulosa cells
To assess whether CD45 was a suitable marker for identification
of granulosa cells in the sample, the latter being CD45 negative,
a sample of granulosa cells only was analysed. A clump of
granulosa cells was picked out of the whole sample of follicular
fluid under the dissecting microscope, mechanically dissociated
by pipetting and stained with CD45 as described above.
Flow cytometric analysis of the sample showed absence of
fluorescence for CD45 (Figure 1). This feature was checked
on the whole sample of follicular fluid with cell sorting on
the basis of CD45 presence or absence. After cytospinning the
CD45 negative and CD45 positive population and Wright
staining it was found that CD45 negative cells were indeed
granulosa cells and CD45 positive cells were predominantly
lymphocytes and neutrophils. This discriminatory parameter
was further used for flow cytometric analysis of granulosa cells.
Scatter characteristics of granulosa cells
Initially it was evaluated whether scatter characteristics of the
granulosa cells could be used as a discriminatory parameter.
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Figure 1. Analysis of a clear population of granulosa cells. Dot
plot of forward scatter (FSC) versus side scatter (SSC) for the
sample, region of interest is delineated as region Rl (A). Dot plot
offluorescence1 (FL1) (CD45) versusfluorescence2 (FL2)
showing absence of fluorescence 1 in Rl (B).
However, when scatter characteristics of the CD45 positive
and negative populations were compared an important overlap
for both forward scatter (cell size) and side scatter (granularity)
was encountered for these populations, showing that these
parameters could not be used reliably to discriminate between
leukocytes and granulosa cells.
On a dot plot of FL1 (CD45) versus FL2, the CD45 negative
population was gated (Region 1/R1) and a histogram for
forward scatter (FSC) in Rl was drawn and compared to a
histogram of FSC for the whole population. FSC of the CD45
negative cells in Rl showed cell size to be equally distributed
over a variety of sizes after an initial peak of debris. When
the whole sample was analysed, i.e. CD45 positives and
negatives, two populations could be distinguished apart from
the debris peak. The presence of an important leukocyte
population may have imposed its own scatter characteristics
on the granulosa cell population, causing a peak of smaller
lymphocytes and larger granulocytes. Analysis of side scatter
(SSC) in Rl and in the whole population showed equally
distributed granularity after an initial debris peak in both Rl
and the whole population of cells present in follicular fluid
(Figure 2).
Discussion
We have succeeded in setting up a technique for flow cytometric
analysis of granulosa cells from follicular fluid. Our aim was
to set up a general technique which can m turn be adjusted
for other types of investigations or different sampling methods.
Our major concern was dealing with white blood cell contain-
Flow cytometric analysis of granulosa cells
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Figure 2. Analysis of forward scatter (FSC) and side scatter (SSC)
characteristics in CD45 negative cells and in the whole cell
population Dot plot of fluorescence 1 (FL1) (CD45) versus
fluorescence 2 (FL2) where CD45 negative cells are delineated in
region 1 (Rl) (A). Histogram of FSC in Rl (B). Histogram of FSC in
whole population ( O . Histogram of SSC in Rl (D) Histogram of
SSC in whole population (E)
ination, since this was present in a variable but prominent
quantity (30-60% of the lysed population). Its main origin
was blood contamination during oocyte retrieval which is
inherent to the procedure. The large vaginal epithelial cells
could be distinguished during flow cytometric analysis by their
forward scatter in comparison with granulosa cells The whole
sample was analysed and a gate for a group of cells of a
particular size including granulosa cells and leukocytes was set
From this moment onwards these settings remained unchanged
There were two routes that could be applied to gate out the
granulosa cells. The first approach was to look for a membrane
marker for the granulosa cells in analogy to the presence of
membrane antigens on white blood cells. A constant expression
on all cells of the population was a prerequisite to be considered
as such. By their very own nature endocrine markers in or on
granulosa cells would be convenient to look for. However,
because of the endocrine dynamics in this period of the cycle
they are not constant and may differ among patients. The
measurement of nuclear oestrogen receptors was taken into
account; these receptors have been analysed with flow cytometry in breast cancer cells (Brotherick et al, 1995). Yet
controversy on the presence of oestrogen receptors in granulosa
cells has arisen, probably because of their very low numbers,
receptor heterogeneity, cyclic changes and absence in nonhuman primates. Recently polymerase chain reaction has
shown the presence of oestrogen receptor mRNA (Hurst et al,
1995) and these authors postulate that a low but biologically
significant amount of receptor may be expressed. Cytoskeletal
markers were taken into consideration. Vimentin has been
shown to be present throughout the whole lifespan of a follicle,
and therefore in the granulosa cell (Santini et al, 1993; Park
and Kim, 1994). However, this cytoskeletal marker is generally
present in meso-denved tissue and therefore also in white
blood cells (Alberts et al, 1994). Cytokeratins 8 and 18 have
been shown present until the time of ovulation by some authors
(Ben Ze'ev and Amsterdam, 1989), but disappearing before
that time according to others (Czemobilsky et al, 1985);
doubts about their presence at ovulation limit their likely
role as discriminatory markers. Apart from these theoretical
considerations, there was an important practical problem in
that cells need to be permeabilized in order to give access for
the antibodies to reach the cytoskeletal antigens, which may
possibly alter and/or damage membrane antigens.
Recently, Honda et al. (1995) reported a new munne
monoclonal antibody OG-1 raised against human granulosa
cells and showed that the OG-1 antigen is a cell surface
differentiation-related molecule expressed on granulosa cells
of follicles and large luteal cells. Partial amino-acid sequencing
of OG-1 revealed it to be identical to the heavy chain of
human integrin a6 This antibody was not available for
evaluation as a discriminatory marker.
We therefore decided to look at the population of granulosa
cells by staining the sample with CD45 which is a panleukocyte marker We then selected granulosa cells on being
CD45 negative We have shown that granulosa cells are CD45
negative when a pure population of clumps of granulosa cells
were stained with CD45. On the other hand we showed that
if a whole sample of follicular fluid was stained with CD45
2213
D.De Neubourg et al
and cell sorting was performed on this basis, CD45 negatives
were indeed granulosa cells. With this method we analysed
the scatter characteristics of the population of granulosa cells
as it has been suggested by Whitman et al. (1991) that two
populations of granulosa cells exist in follicular fluid from invitro fertilization (FVF) patients after removal of lymphocyte
contaminants. We analysed a sample which was stained with
CD45-FTTC and analysed forward scatter of the CD45 negative
population. We found comparable amounts of cells with a
whole variety of FSC signals but not two distinct populations
as mentioned by Whitman et al. (1991). On the other hand,
when the whole sample was analysed, i.e. CD45 positives and
negatives, two populations could be distinguished apart from
the debris peak. We suggest that the presence of an important
leukocyte population in this sample may have imposed its own
scatter charactenstics on the granulosa cell population causing
a peak of smaller lymphocytes and larger granulocytes. This
phenomenon could be confirmed in the 13 patients' samples
analysed. From our data at least, there were no arguments in
favour of two populations of lutein-granulosa cells, rather a
wide size range was encountered.
In conclusion, negative labelling of granulosa cells with
CD45-FTTC can be used to identify granulosa cells in follicular
fluid without physically separating them. With the addition of
other fluorochromes, charactenstics such as membrane antigens, cytoplasmic determinants or nucleic acid content can be
examined. The proposed technique can be applied to single
follicles in stimulated and unstimulated cycles.
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Received on March 21, 1996, accepted on July 30. 1996
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