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Endocrinology 143(8):2905–2912
Copyright © 2002 by The Endocrine Society
Water Permeability of an Ovarian Antral Follicle Is
Predominantly Transcellular and Mediated
by Aquaporins
NISHA A. MCCONNELL, RAHEELA S. YUNUS, STEPHEN A. GROSS, KENNETH L. BOST,
MARK G. CLEMENS, AND FRANCIS M. HUGHES, JR.
Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223
Ovarian folliculogenesis is characterized, in part, by the formation and expansion of the fluid-filled antrum. Development
of this cavity requires water influx, which may occur by transcellular or pericellular transport mechanisms. To assess the
contribution of these mechanisms to the water permeability
of an antral follicle, the rate of 3H2O and 14C-inulin (a complex
sugar restricted to the extracellular compartment) uptake
into isolated follicles was determined. The rate of H2O movement was 3.5-fold greater than that of inulin, suggesting that
water enters a follicle primarily by transcellular pathways.
Preincubation of the follicles with 50 ␮M HgCl2 [a nonspecific
aquaporin (Aqp) inhibitor] decreased H2O movement to levels
D
EVELOPMENT OF FEMALE germ cells in vivo is dependent on the concomitant development of the surrounding follicle in the mammalian ovary, which takes place
in two major steps (for reviews of folliculogenesis, see Refs.
1–5). The mature follicle begins as a primordial follicle consisting of the immature oocyte surrounded by a monolayer
of undifferentiated epithelial cells. The primordial follicle
develops into a preantral follicle characterized by a distinct
proliferation and differentiation of the granulosa cell layer.
The multilaminar granulosa cell layer is surrounded by a
basement membrane and further surrounded by a layer of
encapsulating theca cells (4). As a result of gonadotropin
stimuli, development then continues to the antral or secondary stage, in which granulosa cell proliferation lends an insignificant contribution to the increase in follicle size. This
phase of development is primarily distinguished by the formation of a single fluid-filled cavity, termed the antrum,
within the follicle. Increase in the diameter of the follicle
during the antral stage is characterized by the increase in the
size of the antral cavity (4).
Antrum formation, after gonadotropin stimulation, is a
very rapid process, with the rate of follicle expansion approximately 50-fold higher than during the preantral phase
of follicle growth (6). This dramatic increase in size necessitates a rapid and massive transport of water. Because granulosa cells have been previously characterized as a so-called
leaky epithelia, it is reasonable to predict that water moves
from the follicular blood supply to the antrum by simply
flowing around the granulosa cells, a process known as periAbbreviations: Aqp, Aquaporin; Irrel Ab, irrelevant antibody; MIP,
major intrinsic protein; No Ab, without primary antibody; PMSG, pregnant mare’s serum gonadotropin; TCA, trichloroacetic acid.
seen with inulin, indicating that transcellular water movement is mediated through Aqp. To demonstrate the functional
presence of Aqp in granulosa cells, we show that swelling in
response to a hypotonic insult is attenuated by preincubation
with 50 ␮M HgCl2. Flow cytometry demonstrated the presence
of Aqps-7, -8, and -9, thus identifying candidate Aqp potentially mediating water movement into antral follicles. These
results suggest that water permeability of antral follicles occurs primarily through transcellular mechanisms, which may
be mediated by Aqps -7, -8, and/or -9 in granulosa cells. (Endocrinology 143: 2905–2912, 2002)
cellular transport (6). However, large plasma proteins are not
found in follicular fluid (7), whereas large proteins produced
by granulosa cells, such as proteoglycans, are confined to the
antrum (8), suggesting granulosa cells do serve to provide a
blood-follicle barrier. Indeed, Powers et al. (9) and Hess et al.
(10) have found that follicles do maintain a selective barrier
between the antral space and the follicular blood supply,
which is exclusive even to ions. Furthermore, the large
amount of water uptake by a developing antral follicle seems
unlikely to occur by pericellular transport through the multilaminar granulosa cell layer, because this would likely be
slow and circuitous. Thus, a more direct and considerably
shorter pathway for water to pass into the antral cavity of the
follicle would be through the transcellular pathway directly
through the cytoplasm of the granulosa cells.
Transcellular water movement can occur by two mechanisms. The first is by simple diffusion through the hydrophobic interior of the membrane, which is a slow and unregulated process. The second mechanism is through
proteinaceous water channels, termed aquaporins (Aqps),
which are able to support a large volume of water flow (11).
Aqps are members of the major intrinsic protein (MIP) family
of integral, channel-forming proteins (12, 13). As of today, 11
Aqps have been identified in mammalian tissues (Aqps 0 –10)
and have been extensively studied in tissues in which water
movement is a predominant feature, such as the kidney and
lung (for reviews of Aqps, see Refs. 12, 14, 15). All mammalian Aqps contain six trans-membrane-spanning domains
and form a pore between two hemichannels that orient 180degrees relative to one another to form an hourglass structure (16). Each of the mammalian Aqps, excluding Aqps-4
and -7, contains extracellular mercurial-sensitive sites in
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which Hg2⫹ binds an extracellular cysteine residue and inhibits water movement through the pore (17).
In the present study, we have assessed the contribution of
pericellular and transcellular mechanisms to the permeability of rat antral follicles by measuring the ability of a follicle
to incorporate 3H2O and 14C-inulin in vitro from the surrounding environment. We have then assessed the contribution of Aqps to transcellular water transport using the
general Aqp inhibitor HgCl2. In addition, we have determined the functional presence of Aqps in isolated granulosa
cells by measuring changes in cell volume in the presence or
absence of HgCl2. Finally, we have identified the expression
of Aqps-7, -8, and -9 in granulosa cells by flow cytometry.
Materials and Methods
Experimental animals
The animals used in all studies were 21- to 24-d-old female Sprague
Dawley rats, injected sc with 10 IU pregnant mare’s serum gonadotropin
(PMSG; kindly provided by Dr. A. F. Parlow and NIDDK’s National
Hormone and Pituitary Program) and killed, 48 h later, by gassing in a
CO2 chamber. This time period was used to ensure a synchronous point
in follicular development in which the antrum is rapidly expanding (18)
and, therefore, water movement is likely to be near maximal. Ovaries
were collected in isolation/transfer medium consisting of serum-free
McCoy’s 5a medium, containing 50 U/ml penicillin and 50 ␮g/ml streptomycin sulfate, and were cleaned of surrounding tissues. Antral follicles, 600 – 800 ␮m in diameter, were dissected, using fine point forceps,
under a stereomicroscope. Granulosa cells were harvested from ovaries
by needle puncture, as previously described (19). All experimental protocols were performed in accordance with the guidelines set forth in the
NIH Guide for the Care and Use of Laboratory Animals, published by
the Public Health Service.
Follicle permeability assay
Three follicles were transferred into a solution containing 480 ␮l PBS,
10 ␮l 3H2O (5 ⫻ 10–5 mCi), and 10 ␮l 14C-inulin (5 ⫻ 10–5 mCi) and then
incubated for 5, 10, or 20 min. After incubation, the solution with the
follicles was layered on top of a step gradient consisting of 500 ␮l 3:7
bromodecane:1-bromododecane and 500 ␮l 100% trichloroacetic acid
(TCA). Follicles were then centrifuged in a swinging-bucket rotor microcentrifuge (Fisher Scientific, ⬃7000 ⫻ g, for 2 min) through the oil
layer (which serves to strip away extrafollicular water molecules) and
lysed into the TCA. The TCA layer was then recovered through a hole
punctured in the bottom of the microcentrifuge tube, and the radioactivity contained in 400 ␮l was counted in a dual-channel scintillation
counter (Beckman Coulter, Inc., Fullerton, CA). To examine the effects
of Hg2⫹, isolated follicles were preincubated in 500 ␮l PBS containing
50 ␮m ⌯gCl2, for 25 min, before being transferred and incubated in
solution containing PBS, 3H2O (5 ⫻ 10–5 mCi), and 14C-inulin (5 ⫻ 10–5
mCi), for 10 min, as described above. Follicles were then centrifuged
through the three-layer step gradient, and 400 ␮l of the TCA solution was
collected and measured for quantity of 3H and 14C emissions.
Calculations
Total moles of H2O and inulin in the incubation, and that which had
moved into the follicles, was calculated, based on the specific activity of
the indicators. To allow direct comparison between these two molecules,
indifferent to the fact that there are many more H2O molecules than
inulin molecules available for diffusion into the follicle, the influx of H2O
and inulin was estimated by calculating the molar fraction of each
molecule that moved into the follicle. This was accomplished by dividing the moles of each molecule that moved into the follicle by the total
moles in the incubation medium at the beginning of the experiment.
These results are expressed as the fractional influx of each molecule
(moles in the follicle/moles in the medium). Molecules that traverse the
same pathway in entering the follicle would be expected to have the
same fractional influx, whereas a molecule that is relatively restricted
McConnell et al. • Aquaporins in Folliculogenesis
would have a lower fractional influx. Thus, if H2O enters via pericellular
pathways only, it would be predicted to have the same fractional influx
as inulin. On the other hand, if H2O has access to an additional transcellular pathway, its fractional influx would be predicted to be greater
than that of inulin.
Swelling assay
Granulosa cells were resuspended (106 cells/ml) in serum-free McCoy’s 5a medium, containing 50 U/ml penicillin and 50 ␮g/ml streptomycin sulfate, and then infused into incubation chambers constructed
on poly-l-lysine-coated slides by attaching two pieces of double-stick
tape and suspending a cover slip between them (Cunningham chambers
(20). Cells were allowed 1 h to adhere (37 C, in 95% air-5% CO2), washed
with medium, and then incubated an additional 15 min in medium with
or without 50 ␮m HgCl2. Slides were then washed again (no HgCl2) and
placed on a microscope stage. An initial photograph was taken, hypotonic medium (70% H20/30% McCoy’s 5a) infused into the chamber, and
a second photograph taken 30 sec later. The diameters were measured
and volumes calculated based on the assumption that the granulosa cell
approximates a complete sphere. Data are presented as the percent of
initial volume.
Antibodies, sources and preparation
Antibodies to Aqps-1, -2, -3, -4, -5, -6, and -7 were purchased from
Alpha Diagnostics (San Antonio, TX). All antibodies were rabbit-antirat
antibodies except for anti-Aqp-6, which was a rabbit antihuman antibody. Antipeptide antibodies to Aqps -8 and -9 were raised in our
laboratory using standard immunization techniques. Peptides were synthesized (Research Genetics, Inc., Huntsville, AL) that corresponded to
an extracellular portion of Aqp-8 (loop C between the third and fourth
transmembrane segments; amino acids 136 –155; KRFWNASGAAFAIVQEQEQVA) or the intracellular C terminus of Aqp-9 (amino acids
275–296; DMKAEPSENNLEKHELSVIM). Peptide was conjugated to
keyhole limpet hemocyanin (21), and rabbits were immunized with the
conjugate (Covance Laboratories, Inc., Denver, PA).
Peptide-absorbed antibodies were prepared by combining 1 mg peptide with 1 ml of the appropriate immune serum and incubating for 1 h,
at room temperature, with constant rocking. Serum was then aliquoted
and stored at ⫺70 C until used. For flow studies, irrelevant peptideabsorbed antibodies (Irrel Ab) were prepared by combining the Aqp-8
peptide with anti-Aqp-9 serum and the Aqp-9 peptide with anti-Aqp-8
serum. In all flow studies, the control (Irrel Ab) antibody was a rabbit
antimouse interleukin-18 antibody purchased from R & D Systems Inc.
(Minneapolis, MN).
ELISA
To assess the specificity of the anti-Aqp antibodies, an antipeptide
ELISA was performed as previously described (21). Briefly, Maxisorp
Immunoplates microtiter plates were coated with 100 ␮g/well (in 50 ␮l
0.1 m NaHCO3) of the Aqp peptide used for immunization or one of four
separate Irrel Abs (peptide no. 1, ELCDDDPK; peptide no. 2, KGVHLRAPLLA; peptide no. 3, RVGHFVEAPALRHALLPARHL; or peptide
no. 4, KITSATCTIDPEVNGK) by incubating overnight at 4 C. The plates
were then blocked for 1 h at 4 C with PBS containing 1% BSA and 0.02%
Tween 20. Increasing dilutions of antiserum [or an Irrel Ab raised against
KLILLKKKDENGDKSVMFTLTNLHQS conjugated to keyhole limpet
hemocyanin (21)] were incubated in the wells, for 1 h at 4 C, before
washing three times with PBS containing 0.02% Tween 20. Secondary
antibody (HRP-conjugated donkey antirabbit IgG; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was then added to the wells
and incubated, for 1 h at 4 C, before substrate (tetramethylbenzidine;
Promega Corp., Madison, WI) was added. Color development was
stopped by addition of 0.5 m H2SO4, and absorbances at 450 nm were
measured.
Antibody labeling for flow
Cells were labeled with anti-Aqp antibodies using a Cytofix/Cytoperm kit purchased from PharMingen (Franklin Lakes, NJ). Briefly, cells
were harvested from d-2 PMSG-treated rats, pelleted (1 ⫻ 106 cells/
McConnell et al. • Aquaporins in Folliculogenesis
Endocrinology, August 2002, 143(8):2905–2912 2907
sample), resuspended in 150 ml Cytofix/Cytoperm solution, and incubated for 20 min on ice. Cells were then washed twice with 200 ␮l 1⫻
Perm/Wash solution (PharMingen). Cells were resuspended in 0.5 ml
Perm/Wash solution, and antiserum was added to a final dilution of
1:500. For control [No Ab (without primary antibody)] samples, antibody was not added. After a 1-h incubation on ice, cells were washed
twice, as described above, and resuspended in 1 ml Perm/Wash solution
containing a 1:1000 dilution of phycoerythrin conjugated goat antirabbit
IgG (Sigma, St. Louis, MO). No Ab samples were also incubated with this
secondary antibody. Samples were then incubated for 1 h, on ice, in the
dark. After this incubation, cells were washed twice more in Perm/Wash
solution before being resuspended in PBS. Fluorescence was analyzed
on an FACSCalibur flow cytometer (Becton Dickinson and Co.,
San Jose, CA).
Western blot
Membrane preparations for Western blot were prepared by lysing
granulosa cells in a hypotonic buffer [10 mm MgCl2, 20 mm Tris (pH 7.4)]
for 15 min, on ice, followed by centrifugation at 500 ⫻ g for 5 min at 4
C. Supernates were then centrifuged at 100,000 ⫻ g for 1 h at 4 C. Pellets
were resuspended in SDS-PAGE loading buffer (20 ␮l/106 cells) containing ␤-mercaptoethanol and boiled at 95 C, for 10 min, before being
subjected to standard SDS-PAGE and Western blotting procedures. Visualization was accomplished using the ECL enhanced chemiluminescence Western blotting system (Amersham Pharmacia Biotech, Piscataway, NJ).
Statistical analysis
All data are presented as the mean ⫹ sem for replicated experiments
performed on separate days. Data for Figs. 1B and 3B were compared
by Student’s t test. Data for Fig. 2 were analyzed by a two-way ANOVA
on ranks and Tukey’s test. All other statistical analyses were performed
by one-way ANOVA and Dunnett’s test. Differences were considered
significant at P ⬍ 0.05. All statistical analyses were calculated with
SigmaStat version 2.0.
Results
Water permeability of an antral follicle is
predominantly transcellular
To determine whether a follicle’s permeability to water is
primarily through transcellular or pericellular mechanisms,
antral follicles were incubated with 3H2O and 14C-inulin (a
complex sugar which moves through tissues exclusively via
a pericellular mechanism), and the fractional influx of H2O
and inulin that moved into the follicles was calculated as
described in Materials and Methods. The resulting measurements allowed accurate determination of the quantity and
rate of movement of water and inulin into the follicles over
time, indifferent of the quantity of each available. As shown
in Fig. 1A, after 5 min, a considerably larger fraction of water
had moved into the follicles, compared with inulin. The
amount of both molecules within antral follicles increased in
a time-dependent manner, although the increase in H2O was
much greater than that of inulin. The mean slope of the lines
from all replicates of Fig. 1A is equal to the rate of water and
inulin movement into the follicles, expressed as fractional
influx/minute. As shown in Fig. 1B, the rate of H2O movement into follicles was substantially greater than the rate of
inulin movement. Total movement of H2O into antral follicles occurred at a rate of approximately 0.14 ⫾ 0.04%/min,
whereas inulin moved at a rate near 0.04 ⫾ 0.01%/min. These
results indicate that total water permeability of an antral
follicle is 70% transcellular and only 30% pericellular.
FIG. 1. H2O movement into an antral follicle is predominantly transcellular. Antral follicles were isolated from d-2 post-PMSG-injected
rats and incubated in PBS containing 3H2O and 14C-inulin for varying
time intervals. Radioactivity entering the follicles was then assessed,
and the amounts of H2O and inulin (in terms of fractional influx) were
calculated as described in Materials and Methods. A, Representative
graphical sample of the fractional influx of H2O and inulin taken in
by the follicles over a period of 20 min. B, Graphical representation
of the rate of H2O and inulin movement into the follicles, expressed
as fractional influx/minute. Data were obtained by determining the
slope of the lines, as depicted in Fig. 1A, for six separate experiments
performed on separate days. *, P ⬍ 0.05, compared with the mean rate
of inulin movement.
Transcellular water permeability of antral follicles is
Aqp-mediated
Because the majority of a follicle’s permeability is mediated via transcellular mechanisms, we next assessed the role
Aqps may play in water movement into antral follicles. As
shown in Fig. 2, movement of H2O into follicles that were
preincubated with Hg2⫹ (a general Aqp inhibitor) was
greatly reduced, compared with follicles preincubated with
PBS alone (P ⬍ 0.05), and was approximately equivalent to
the movement of inulin. Movement of inulin was only
slightly, although significantly, attenuated by exposure to
Hg2⫹.
Aqps are functionally expressed in granulosa cells
To assess Aqp function, osmotic swelling of granulosa cells
was measured after a 15-min preincubation in the absence or
presence of 50 ␮m HgCl2. As shown in Fig. 3, control cells
(⫺Hg2⫹) demonstrate significant swelling in this assay,
reaching sizes that are 222 ⫾ 19.5% of normal. HgCl2 blunted
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Endocrinology, August 2002, 143(8):2905–2912
swelling to 123 ⫾ 6.0% of normal, indicating that functional
Aqps are expressed in granulosa cells.
Aqps-7, -8, and -9 are expressed in granulosa cells
Our results demonstrate the functional presence of Aqps
in granulosa cells and suggest an important role for these
proteins in mediating water movement during folliculogenesis. To assess the expression of individual Aqps, we have
labeled granulosa cells with Aqp-specific antibodies and analyzed staining by flow cytometry. Figure 4A presents the
labeling histograms obtained from the flow cytometer for
Aqps-1 through -7 and indicates the gate (M1) considered as
positive staining. Figure 4B is a graphical depiction of these
results. Labeling for Aqps-1, -2, -3, -4, -5, and -6 was similar
to labeling seen in No Ab cells, indicating the absence or low
level of expression of these homologs. However, approximately 31 ⫾ 6.6% of the population labeled positively for
Aqp-7, identifying this homolog as a potential candidate to
mediate water movement during antrum formation.
Specificity of anti-Aqps-8 and -9 antibodies for the peptides was then assessed by ELISA, as described in Materials
and Methods. As shown in Fig. 5A, both anti-Aqps-8 and -9
antibodies had titers of greater than 1:10,000 against the
McConnell et al. • Aquaporins in Folliculogenesis
immunizing peptide but did not react significantly against
several control peptides of similar length and composition.
Likewise, the immunizing peptide did not react significantly
with a control antibody, demonstrating the specificity of the
antibodies for this peptide. A Western blot was then performed to ensure that these antibodies were, in fact, reacting
with a protein of the appropriate molecular weight. As
shown in Fig. 5B, anti-Aqp-8 reacted with a protein with
molecular weight of approximately 28, the expected molecular weight of Aqp-8, based on sequence analysis (22),
whereas anti-Aqp-9 reacted with a protein with a molecular
weight of approximately 31.4 (23). The presence of higher
molecular weight bands on these blots may represent Aqp
aggregates (24, 25). Importantly, these antibodies did not
cross-react.
These antibodies were next used to label granulosa cells for
analysis by flow cytometry. As shown in Fig. 6, No Ab cells
or Irrel Ab cells displayed very little labeling (⬍5% of the
population). In populations of cells incubated with antiAqp-8 or -9 antibodies, approximately 45–50% of the population labeled positively, but this number was drastically
reduced when the antibody was preabsorbed with the immunizing peptide (Pep Abs). Preabsorption with an Irrel Ab
had little, if any, effect on cell labeling with these antibodies.
Discussion
FIG. 2. HgCl2 inhibits the transcellular pathway of H2O movement
into antral follicles. Antral follicles were incubated in the presence (⫹
Hg2⫹) or absence (⫺ Hg2⫹) of 50 ␮M HgCl2, for 25 min, before incubation with 3H2O and 14C-inulin as described in Materials and Methods. Data are expressed as the mean ⫹ SEM of five separate experiments performed on separate days. *, P ⬍ 0.05, compared with all
other samples.
FIG. 3. HgCl2 inhibits Aqps and prevents granulosa cell
swelling in hypotonic medium. Granulosa cells from d-2
PMSG-treated rats were immobilized on poly-L-lysinecoated slides and then incubated with or without 50 ␮M
HgCl2 for 15 min. Cells were washed and exposed to hypotonic medium (70% H2O/30% medium) for 30 sec. A, Photographs (⫻400) of typical cells before or 30 sec after exposure to hypotonic medium. B, Graph depicting the final
volume of the cells (as the percent of initial volume) after
30 sec in hypotonic medium. Measurements represent the
mean ⫾ SEM of at least 12 separate measurements from 4
separate experiments performed on different days. *, P ⬍
0.05, compared with cells cultured in the absence of HgCl2).
Antrum formation and expansion in the ovarian follicle is
a well-characterized process (26 –28); however, the exact
mechanism by which water moves into the antral follicle has
remained unclear. In this study, we have sought to define the
contribution of pericellular and transcellular mechanisms to
the permeability of antral follicles. The results indicate that
follicular water permeability is 70% transcellular in a large
antral rat follicle. The primary barrier to transcellular transport of hydrophilic molecules is the plasma membrane, and
water is known to cross this membrane by two mechanisms:
simple diffusion through the lipid bilayer, and through the
Aqps. As shown in Fig. 2, we have found that the water
permeability of antral follicles is significantly reduced by the
addition of the general Aqp inhibitor HgCl2. Movement of
inulin was slightly, yet significantly, reduced in the presence
of Hg2⫹. This phenomenon may be attributed to a reduction
of the osmolyte concentration gradient for follicles in a state of
impaired water permeability. Inhibition of the transcellular
pathway of water, approximately 70% of the total follicular
McConnell et al. • Aquaporins in Folliculogenesis
Endocrinology, August 2002, 143(8):2905–2912 2909
FIG. 4. Analysis of Aqps 1–7 expression in rat granulosa cells, by flow cytometry. Granulosa cells from d-2 PMSG-treated animals were labeled
with antibodies to Aqps 1–7, as described in Materials and Methods, and analyzed by flow cytometry. A, Histograms depicting the fluorescence
of the cell population labeled with each antibody. Any cells appearing under the M1 gate are considered positive. B, Quantitative analysis of
the percentage of the population expressing each Aqp homolog (mean ⫾ SEM of three separate experiments). *, P ⬍ 0.05, compared with No
Ab cells.
water permeability, by Hg2⫹ would result in an increase of
solute concentration within the follicle, compared with incubation in the absence of Hg2⫹. A reduction of the total solute
concentration gradient across the follicle wall would produce
an impaired driving force for solute flux into the follicle. Thus,
Hg2⫹ inhibition of water movement into an antral follicle
would, as expected, result in an attenuation of inulin flux across
the follicle wall. These results indicate that the transcellular
mechanism of water permeability is indeed mediated by Aqps.
Furthermore, the detection of Aqps-7, -8, and -9 in rat granulosa
cells suggests a role for these channels in mediating the transcellular pathway of water movement into antral follicles. The
multiple expression of more than one Aqp homolog in these
cells is suggestive of a redundant mechanism for water move-
ment and implies that water movement is greatly important for
this cell type. In this study, we have found that not only are
these specific Aqp types present in granulosa cells, but at least
one, or possibly more than one, homolog is also functionally
available for the transport of water across the cell membrane.
In the experiments shown in Fig. 3, we were able to significantly
suppress cell swelling in response to a hypotonic insult by a
brief preincubation with HgCl2, indicating the functional presence of Aqps in these cells. HgCl2 was unable to completely
block this response, suggesting, at first, that the residual swelling was attributable to simple diffusion of water through the
lipid bilayer. However, Fig. 4 revealed the presence of Aqp-7 in
these cells. Aqp-7 is one of only two HgCl2-insensitive Aqp
family members (Aqps-7 and -4), suggesting that the hypotonic
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Endocrinology, August 2002, 143(8):2905–2912
FIG. 5. Specificity of anti-Aqps-8 and -9 antibodies. A, Microtiter
plates were coated with the indicated peptide and incubated with
increasing dilutions of the indicated antibody, as indicated in Materials and Methods, to assess antibody specificity. Specificity of the
antiserum is indicated for the immunizing peptide as well as four
control peptides. In addition, the reactivity of the immunizing peptide
for an Irrel Ab is also indicated. -⽧-, Aqp peptide/anti-Aqp antibody;
-f-, Aqp peptide/control antibody; -Œ-, control peptide-1/anti-Aqp antibody; -⫻-, control peptide-2/anti-Aqp antibody; --, control peptide3/anti-Aqp antibody; -●-, control peptide-4/anti-Aqp antibody. B,
Western blot analysis of anti-Aqp-8 and anti-Aqp-9 reactivity performed as described in Materials and Methods. Anti-Aqps-8 and -9
react with proteins of expected molecular weight. The molecular
weights of rat Aqps-8 and -9 are 28 and 31.4, respectively.
McConnell et al. • Aquaporins in Folliculogenesis
swelling in the presence of HgCl2 may, in fact, result from water
movement through Aqp-7.
These studies provide the first evidence of Aqp expression
in ovarian tissues. A previous study by Ishibashi et al. (29)
found that Aqp-9 mRNA was not expressed in whole ovarian
extracts by a multiple-tissue Northern blot. However, in the
present study, Aqp-9 expression in granulosa cells was demonstrated using antipeptide antibodies, specific for this homolog. The apparent discrepancy in our data may be the
result of the presence of high background levels on Ishibashi’s blot, a low level of Aqp-9 mRNA expression in granulosa cells, or the use of whole ovarian extracts in his study
vs. isolated granulosa cells in the present study. We have
since confirmed the presence of Aqp-9 mRNA in d-2 PMSGtreated granulosa cells, using RT-PCR (data not shown).
It is important to note that the experiments detecting Aqp
expression were performed with isolated granulosa cells
only. However, our studies examining the water permeability of antral follicles were performed with whole isolated
antral follicles. Previous studies have indicated that these
isolated follicles contain an intact theca cell layer (30, 31),
which undoubtedly contributes to the regulation of water
permeability of an antral follicle. Therefore, further studies
to investigate the expression of Aqps in the theca cell layer,
by techniques such as immunohistochemistry, must be performed to fully understand the nature of antrum formation
in these follicles.
Previous studies have detected large plasma proteins, such
as albumin, in follicular fluid (32, 33); and several investigators have suggested that this fluid is a plasma filtrate
created as fluid moves across the basement membrane (6, 34).
However, other studies have found significant differences
between the composition of serum and that of follicular fluid
(7, 8), although most studies concur that some proteins derived from serum are present. Our studies have indicated the
presence of significant pericellular transport pathways into
follicles, providing a route for movement of such large molecules from the plasma. However, our data also suggest that
pericellular transport is only a minor pathway for water
movement and that the majority of water enters a follicle
through Aqp-mediated transcellular pathways.
Net transcellular movement of water through Aqps into a
follicle requires the presence of an osmotic gradient, because
these channels only mediate the passive flow of water across
a cell membrane. Previous studies have found that augmented levels of K⫹ and Na⫹ ions exist in the follicular fluids
of some species (33, 35–37), suggesting that these cations may
be actively transported into the follicular fluid by the follicular cells. A positive electrical potential within the follicle
was determined by McCaig (38), suggesting that, indeed,
these ions are actively transported into the antral space.
Furthermore, this electrical potential was found to increase
as folliculogenesis progressed (38). This suggests that the
cells of a follicle may create an osmotic gradient within the
antral space to drive the flow of water into the follicle for
antrum formation and expansion. Although ion movement
is most often associated with the formation of osmotic gradients, additional studies have suggested that other molecules may be involved. Two such studies, one by Zachariae
(39) and one by Zachariae and Jensen (40), have proposed
McConnell et al. • Aquaporins in Folliculogenesis
Endocrinology, August 2002, 143(8):2905–2912 2911
FIG. 6. Analysis of Aqps-8 and -9 expression in rat granulosa cells, by flow cytometry. Granulosa cells from d-2 PMSG-treated animals were
labeled with antibodies to Aqps-8 and -9, as described in Materials and Methods. Cells were either incubated without primary antibody (but
with secondary antibody) (No Ab), with an irrelevant antibody to interleukin-18 (Irrel Ab), or with the indicated anti-Aqp antibody. Two
important controls that were included are the anti-Aqp antibody preabsorbed with either the immunizing peptide (Aqp- Pep Abs) or an irrelevant
peptide (Aqp- Irrel Pep Abs). A, Histograms depicting the fluorescence of the cell population labeled with each antibody. Any cells appearing
under the M1 gate are considered positive. B, Quantitative analysis of the percentage of the population expressing each Aqp homolog (mean ⫾
SEM of five separate experiments). *, P ⬍ 0.05, compared with No Ab cells.
that the hydrolysis of polymeric glycosaminoglycans in the
antrum could increase the osmolality of follicular fluid and
could induce antral swelling. Despite the existence of several
studies revealing a significant increase in the osmolality of
follicular fluid relative to plasma (37, 41), other studies have
found the osmolalities of follicular fluid and plasma to be
near equilibrium (42). However, it is apparent that osmotic
gradients smaller than 1 mOsmol/kg can induce a substan-
2912
Endocrinology, August 2002, 143(8):2905–2912
tial amount of water movement (43). Furthermore, the evidence for the production of an osmotic gradient by follicular
cells can be deduced from studies where follicles are grown
in culture and induced to form and expand an antrum on
gonadotropin stimulation without the presence of a vascular
blood supply (27, 28, 44). This suggests that antrum formation may be an active process initiated by follicular cells, most
likely by the granulosa cells, as these cells line the antrum (3).
Because the activity of most Aqps can be regulated by a
variety of posttranslational alterations (45, 46), this data suggests, for the first time, that a significant proportion of water
movement into a follicle may be regulated independently
from solute movement.
Acknowledgments
Received January 28, 2002. Accepted April 18, 2002.
Address all correspondence and requests for reprints to: Francis M.
Hughes, Jr., Department of Biology, University of North Carolina at
Charlotte, 9201 University City Boulevard, Charlotte, North Carolina
28223. E-mail: [email protected].
This study was supported by the NIH (Grants HD-39683 and HD39234) and internal research funds from the University of North Carolina
at Charlotte.
McConnell et al. • Aquaporins in Folliculogenesis
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