Angiotensin II induces apoptosis in human mural granulosa-lutein cells, but not in cumulus cells.
ABSTRACT To test whether angiotensin II (AngII) could modulate apoptosis of human granulosa-lutein (GL) cells from gonadotropin-stimulated follicles.
In vitro assays on mural and cumulus granulosa cells.
University laboratory and private IVF practice.
One hundred six consecutive women undergoing 113 IVF cycles.
Purified human GL mural or cumulus cells were cultured in serum-free media in the presence or absence of AngII with or without the AngII receptor blockers saralasin and CGP42112A.
Detection of apoptosis using a fluorescent in situ marker for activated caspases.
Mural cells had approximately eightfold the amount of apoptosis compared with cumulus cells (average 0.23 vs. <0.03, respectively). With mural cells, AngII increased GL cell apoptosis versus untreated control samples (AngII 10(-)11 mol/L +6.5%; AngII 10(-9) mol/L +13.3%, and AngII 10(-7) mol/L +11.3%), an effect which was blocked by concurrent incubation with AngII receptor blockers. The AngII receptor blockers produced a significant decrease of apoptosis compared with control cultures (saralasin: 19.4%; CGP42112A: 28.9%). Neither AngII nor blockers had effect on cumulus cells.
Preovulatory concentrations of AngII, most likely via AT2 receptors, increase apoptosis of cultured mural GL cells but have no effect on cumulus cells. Granulosa cells appear to be differentially regulated by AngII.
Article: The incidence of apoptotic bodies in membrana granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs.[show abstract] [hide abstract]
ABSTRACT: To investigate the relationship between the incidence of apoptotic bodies in membrana granulosa and follicular steroid concentrations in human follicles. Case-controlled prospective study for 132 individual follicles. Procedures were performed in Yamagata University Hospital. Thirty-six normo-ovulatory women with tubal infertility underwent ovulation induction for IVF-ET with a conventional hyperstimulation method. Patients underwent follicle aspiration after the administration of hCG. The nuclei of recovered granulosa cells were examined by fluorescence microscopy, and the incidence of apoptotic bodies was tabulated. Intrafollicular steroids were evaluated mainly by RIA. These data were analyzed with respect to oocyte-retrieval, oocyte maturity, fertilization, and embryo quality. Membrana granulosa cells in the follicles from which oocytes were subsequently fertilized showed a significantly lower incidence of apoptotic bodies than those in follicles from which the oocytes did not fertilize. Membrana granulosa cells in the follicles from which oocytes were developed into good quality showed a significantly lower incidence of apoptotic bodies than those in the follicles from which oocytes developed into fair and poor quality. The incidence of apoptotic bodies was significantly higher in the mural granulosa cell region than in the cumulus cell region in most cases. Intrafollicular E2, P, and free T levels were not different between the oocyte groups. These results indicate that lower incidence of apoptotic bodies in individual follicles is associated with better outcomes for oocytes. Also, mural granulosa cells and cumulus cell in each follicle may show differentiation during follicular maturation.Fertility and Sterility 09/1997; 68(2):312-7. · 3.56 Impact Factor
Article: Incidence of apoptotic bodies in membrana granulosa of the patients participating in an in vitro fertilization program.[show abstract] [hide abstract]
ABSTRACT: To investigate the incidence of apoptotic bodies in mural granulosa cell masses and cumulus cell masses. Nonrandomized, prospective study. Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Yamagata, Japan. One hundred twenty-nine normally ovulating women underwent ovulation induction for IVF-ET with GnRH analogue (GnRH-a) and gonadotropins. Patients underwent follicle aspiration after the administration of hCG. The nuclei of recovered granulosa cells were examined by fluorescence microscopy and the incidence of apoptotic bodies was tabulated. The incidence of apoptotic bodies was significantly higher in mural granulosa cell masses than in cumulus cell masses in the entire group of 129 patients. Both incidence of apoptotic bodies of mural granulosa cell masses and cumulus cell masses were significantly higher in patients with less than six follicular oocytes compared with patients with six or more oocytes. Nonpregnant patients showed significantly higher incidence of apoptotic bodies in mural granulosa cell masses compared with pregnant patients. These results indicate that mural granulosa cell masses and cumulus cell masses may have different functions in follicular maturation. The incidence of apoptotic bodies in mural granulosa cell masses can be used as an indicator of success of IVF.Fertility and Sterility 03/1997; 67(2):302-8. · 3.56 Impact Factor
Article: Apoptotic cell death in human granulosa-lutein cells: a possible indicator of in vitro fertilization outcome.[show abstract] [hide abstract]
ABSTRACT: To determine whether women who do not conceive during an IVF treatment despite normal FSH levels have a higher rate of apoptosis in their granulosa-lutein cells than women who do conceive. Prospective. Large teaching hospital and fertility center. Patients with normal FSH levels undergoing an IVF treatment. None. Outcome of the IVF treatment and percentage of apoptotic granulosa-lutein cells. A significantly lower percentage of granulosa-lutein cells were apoptotic in patients who became pregnant compared with those who did not become pregnant. A higher basal FSH level was significantly correlated with the duration of the stimulation, and the number of follicles was significantly inversely correlated with the number of ampules of FSH used and the duration of the treatment. The number of embryos was significantly correlated with the number of oocytes, and significantly inversely correlated with the number of apoptotic granulosa-lutein cells. Fewer granulosa-lutein cells are apoptotic in women who have an ongoing pregnancy after IVF treatment than in women who do not conceive.Fertility and Sterility 11/1998; 70(4):747-9. · 3.56 Impact Factor
Angiotensin II induces apoptosis in human mural
granulosa-lutein cells, but not in cumulus cells
Elisa Acosta, M.Sc.,a? Oscar Pe~ na, M.Sc.,aFrederick Naftolin, M.D, D.Phil.,cJulio?Avila, Ph.D.,a
and Angela Palumbo, M.D., Ph.D.b
Reproduction, Tenerife, Spain; andcDepartment of Obstetrics and Gynecology, New York University, New York, New York
Objective: To test whether angiotensin II (AngII) could modulate apoptosis of human granulosa-lutein (GL) cells
from gonadotropin-stimulated follicles.
Design: In vitro assays on mural and cumulus granulosa cells.
Setting: University laboratory and private IVF practice.
Patient(s): One hundred six consecutive women undergoing 113 IVF cycles.
Intervention(s): Purified human GL mural or cumulus cells were cultured in serum-free media in the presence or
absence of AngII with or without the AngII receptor blockers saralasin and CGP42112A.
Main Outcome Measure(s): Detection of apoptosis using a fluorescent in situ marker for activated caspases.
(AngII 10?11 mol/L þ6.5%; AngII 10?9mol/L þ13.3%, and AngII 10?7mol/L þ11.3%), an effect which was
decrease of apoptosis compared with control cultures (saralasin: 19.4%; CGP42112A: 28.9%). Neither AngII nor
blockers had effect on cumulus cells.
Conclusion(s): Preovulatory concentrations of AngII, most likely via AT2 receptors, increase apoptosis of cul-
tured mural GL cells but have no effect on cumulus cells. Granulosa cells appear to be differentially regulated
by AngII. (Fertil Steril?2009;91:1984–9. ?2009 by American Society for Reproductive Medicine.)
Key Words: Granulosa-lutein cells, apoptosis, in vitro fertilization, angiotensin II, saralasin, AT2 receptors, poly-
cystic ovaries, oocyte
Exfoliated gonadotropin-stimulated mural granulosa-lutein
(GL) cells obtained with the follicular fluid (FF) of women
at the time of egg retrieval for IVF undergo programmed
cell death (apoptosis) (1–7). The rate of apoptosis is associ-
ated with several indicators of IVF outcome, such as the sub-
ject’s age and FSH and the embryo’s condition and fate (7).
The apoptosis rate in these cells is also modulated by intrao-
varian growth factors (7). We and others have previously
raised the possibility that terminal differentiation of develop-
ing granulosa cells is a product of FF androgens and proges-
tins and determines the fate of the follicle: atresia or growth
to ovulation (8). We proposed that follicular angiotensin
(AngII), which is induced by LH, is a cause of this terminal
differentiation of the granulosa cells (9).
the oogonia-oocyte. Their lineage from the yolk sac differs
from the rest of the ovary (10, 11). These cells secrete and
conduct the contents of the ovarian follicle. These include
a complete gonadotrophin-regulated plasminogen activator/
plasminogen activator inhibitor system (12), a complete
and functional gonadotrophin-sensitive ovarian renin-angio-
tensin system (OVRAS) (13–21), and both types of AngII re-
ceptors (AT1 and AT2) (21–24).
It is not known what role the OVRAS plays in the selection
and development of the follicular antrum and the develop-
ment of the specialized granulosa cells that contact the
oocyte, the cumulus oophorus. However, the activity of the
OVRAS in FF is increased when the plasminogen activator
inhibitors are suppressed by the rise of FSH at midcycle,
and this unleashes the AngII cascade that is part of the ovu-
lation mechanism (9, 14–17).
Angiotensin II has been shown to inhibit cell proliferation
and differentiation and promote apoptosis in several cell
types and tissues, including the nervous system (25–28),
thelial cells (33), cardiac cells (34–35), and the ovary (36–
37). This appears to be an AT2 receptor–mediated action
(23, 25–30, 33, 36–37).
Published evidence of a causative effect of AngII on apop-
tosis in the ovary is limited to the rat (36–37). Because high
levels of AngII are present in preovulatory FFof IVF patients
online August 5, 2008.
to disclose. J.A. has nothing to disclose. A.P. has nothing to disclose.
Supported by grant PI060772 from Fondo Investigaciones Sanitarias,
Spain; by an educational grant No 27303 from Fundaci? on Salud
2000, Serono, Spain; and by an educational grant from Caja Canarias,
Reprint requests: Angela Palumbo, M.D., Ph.D., Centro de Asistencia a la
Reproducci? on Humana de Canarias, C/ Doctor Zamenhof 14, E-38240,
La Laguna, Tenerife, Spain (FAX: 922 63 28 79; E-mail: apalumbo@ull.
Fertility and Sterility?Vol. 91, No. 5, Supplement, May 2009
Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
at the time of egg retrieval (13–15, 21), and because the gran-
ulosa-lutein cells purifiedfrom this FF undergoapoptosis, we
postulated that intrafollicular AngII might modulate apopto-
sis of GL cells. Therefore, we studied the in vitro effect of
AngII and its receptor blockers on apoptosis of cultured
human GL cells. We confirmed the effect of AngII on mural
cell apoptosis, but also found that the cumulus GL cells have
little apoptosis and are not affected by AngII.
MATERIALS AND METHODS
L-Glutamine, bovine serum albumin (BSA), amphotericin B,
AngII and its receptor blockers saralasin and CGP42112A,
sodium bicarbonate, streptomycin sulphate, penicillin G,
Percoll, modified McCoy’s 5A medium, and Medium 199
with Hanks’ salts were purchased from Sigma-Aldrich Co.
(Madrid, Spain). The fluorescent in situ marker caspACE
FITC-VAD-FMK was obtained from Promega Corporation
(Madison, WI). Dynabeads M-450 CD45 were from Dynal
ASA (Oslo, Norway), and four-well plastic culture plates
were from Nunc (Roskilde, Denmark).
Cells from 113 cycles in 106 consecutive women were ana-
lyzed under a protocol approved by the Ethics Committee
of the Universidad de La Laguna as part of the Institutional
Review Board approval of the study. The causes of infertility
were tubal factor in 14 patients (14 cycles), male factor in 41
patients (46 cycles), endometriosis in 6 patients (7 cycles),
polycystic ovary syndrome in 21 patients (21 cycles), un-
known in 8 patients (8 cycles), and high FSH in 3 patients
(3 cycles; a day 3 FSH of >10 IU/mL was considered to be
elevated, indicating diminished ovarian reserve). A further
14 specimens of GL cells were from 13 egg donors. The
mean age of this population was 32.73 ? 6.19 years (range
Induction of ovulation was carried out as usual, using
recombinant FSH (Gonal F; Serono, Madrid, Spain), com-
bined with recombinant LH (Luveris; Serono) or hMG
(hMG-Lepori; Farma-Lepori, Madrid, Spain; or Menopur;
Ferring, Madrid, Spain). Doses were adjusted to the individ-
ual patient’s response. Ultrasound-guided follicle retrieval
was performed 36 h after administration of 10,000 IU hCG
(hCG Lepori; Farma-Lepori). In all cases, the fertilization
method for the retrieved oocytes was intracytoplasmic sperm
injection. Embryo transfer was carried out with a Wallace
catheter, under ultrasound guidance. One to three embryos
were replaced in each cycle, depending on the patient’s age
and the embryo morphology.
Isolation of GL Cells
Exfoliated mural GL cells Cells were collected from FF
obtained during ultrasound-guided transvaginal oocyte
retrieval and purified as previously described (7). Briefly,
all FF from each patient was pooled and the GL cells lightly
centrifuged after removal of the oocyte. The cells were
washed in ‘‘isolation medium’’ (Medium 199, supplemented
with sodium bicarbonate [3.7 g/L], penicillin [59 mg/L],
streptomycin [100 mg/L], amphotericin B [25 mg/L], L-glu-
tamine [0.29 g/L], and BSA [0.1%]) and separated from red
blood cells using a 50% Percoll gradient. Leukocytes were
separated using antiCD45-coated magnetic beads (Dyna-
beads M-450 CD45). The proportion of GL cells to erythro-
resuspended in McCoy’s 5A medium.
Cumulus cells Four hours after oocyte retrieval, the individ-
ual oocyte-cumulus complexes were denuded with hyaluron-
idase (80 IU/mL.). Cumulus cells were collected, washed in
‘‘isolation medium’’ and maintained in culture in the same
conditions as the GL cells.
Viability Cellular viability for both types of cells was
Cell Cultures and Treatments
The GL cells were cultured for 48 h at 37?C under 5% CO2
and air in modified McCoy’s 5A medium supplemented
with L-glutamine (0.29 g/L), bovine serum albumin (0.1%),
penicillin (59 mg/L), streptomycin (100 mg/L), and ampho-
tericin B (25 mg/L). The following groups were analyzed:
1) untreated cells (control); 2) AngII (10?7, 10?9, and
10?11mol/L); 3) AngII blockers saralasin (AT1 and AT2
blocker) or CGP42112A (AT2 blocker) at a concentration
of 10?5mol/L; 4) a combination of AngII (10?7, 10?9, and
10?11mol/L) and either saralasin or CGP42112A 10?5
mol/L. A poly-L-lysine–coated cover slip was placed in
each well, and approximately 5 ? 104live cells were plated
in each well. Every treatment was performed in duplicate.
Detection of Apoptosis in GL Cells
After 48 h of culture, cells were washed with phosphate-
buffered saline (PBS) and stained with caspACE FITC-
VAD-FMK (10 mmol/L) for 20 min in the dark at 37?C under
5% CO2and air. All subsequent steps were carried out in the
dark at room temperature. Cells were rewashed with PBS and
fixed with ice-cold pure methanol, then washed three times
with PBS and incubated in PBS þ 0.1% Triton X-100 (2 ?
5 min). Nuclei were marked with propidium iodide (0.05
mg/mL; 15 minutes) and rewashed with PBS þ 0.1% Triton
X-100 (3 ? 10 min). The slides were observed at ?400 mag-
nification under a fluorescence microscope. One hundred
cells per slide were counted and classified as viable (red
nucleus and red cytoplasm) or apoptotic (red nucleus and
green cytoplasm). Each slide was scored by two blinded
observers. The coefficient of variation between observers
Results on each slide were calculated as the percentage
of apoptotic cells/total cells counted and expressed as
Fertility and Sterility?
mean ? standard error. Statistical analysis was performed
using the Student t, Mann-Whitney U, and Kruskal-Wallis
tests, as appropriate.
Untreated Cells (Control Cultures)
After incubation for 48 h without serum, apoptosis differed
between the cumulus cells and the mural GL cells. Cumulus
cells were included ineach experiment. Theformer had <3%
apoptosis, whereas the latter averaged 23.09% ? 9.53%
Mural cells from young versus old patients Young (<35
years old) otherwise normal women (tubal or male factor
and donors) had lower apoptosis rates compared with older
(R35 years old) patients with all causes of infertility in-
cluded (20.74 ? 0.88 [n ¼ 49] vs. 28.93 ? 1.75 [n ¼ 43],
respectively; p¼.01 [Mann-Whitney U]). As expected, the
pregnancy rates were 67.35% in the young group and
48.84% in the older group.
Cumulus cells Therewas no effect of AngII or blockers. The
average percentage apoptosis was always <3% (Fig. 1A).
Mural GL cells
All doses of AngII tested increased apoptosis compared with
untreated control cells. Whereas AngII 10?11mol/L did not
reach statistical significance on the t test, the effect of 10?9
mol/L and 10?7mol/L was statistically significant (untreated
21.93 ? 1.09 vs. AngII 10?11mol/L 23.36 ? 1.32 [n ¼ 49];
p¼.127; untreated 23.20 ? 1.25 vs. AngII 10?9mol/L 26.29
? 1.54 [n ¼ 47]; p<.05; and untreated 22.99 ? 1.36 vs.
AngII 10?7mol/L 25.59 ? 1.56 [n ¼ 48]; p<.05; Fig. 2).
A dose-dependent effect could not be demonstrated (apo-
ptosis in untreated cells 23.14 ? 0.74 [n ¼ 135]; AngII
10?11mol/L 24.6 ? 1.26 [n ¼ 45]; AngII 10?9mol/L 27.3
? 1.51 [n ¼ 44]; and AngII 10?7mol/L 26.4 ? 1.53 [n ¼
46]; Kruskal-Wallis: p¼.389).
AngII receptor blockers The concurrent incubation with the
AngII receptor blockers completely reversed the proapop-
blocker CGP42112A was more effective than the mixed
blocker saralasin (Fig 3A).
When cells were culturedinthe presence ofAngIIreceptor
blockers alone, a statistically significant decrease of apopto-
sis was observed in all cases, both with saralasin (untreated
25.62% ? 11.71% vs. saralasin 20.73% ? 10.86% [n ¼
33]; Student t: p<.05) and CGP42112A (untreated 21.79 ?
6.48 vs. CGP42112A 15.20 ? 5.74 [n ¼ 33]; p¼.003; Fig.
3B). The specific AT2 receptor blocker was significantly
more effective than saralasin in decreasing apoptosis
(28.9% vs. 19.4 %; Mann-Whitney U: p¼.035).
showing absence of apoptosis. (B) Mural granulosa-
lutein cells after 24-h incubation without treatment.
(C) Mural granulosa-lutein cells after 24-h incubation
with angiotensin II 10?9mol/L. Apoptotic cells are
identified by green cytoplasm. Note the increased
apoptosis caused by the addition of AngII.
Acosta. Angiotensin II–driven follicular cell apoptosis. Fertil Steril 2009.
Acosta et al.
Angiotensin II–driven follicular cell apoptosis
Vol. 91, No. 5, Supplement, May 2009
GL cells demonstrated that administration of AngII in doses
found in preovulatory hCG-treated follicles increases apopto-
sis in pooled mural human GL cells from individual IVF pa-
tients, but not in cumulus cells. The most effective AngII
trations normally found in preovulatory FF (14–15); the scat-
ter and irregular effect of the low-dose AngII seem to be the
reason for the lack of proving a dose-dependent effect. The
lowest concentration of AngII tested, 10?11mol/L, increased
apoptosis in most cases, but its effect was more variable than
that obtained with the higher doses. The action of AngII was
invariably reversed by the specific AT2 receptor blocker
CGP42112A and to a lesser degree by the nonspecific blocker
saralasin. Because AT2 receptors mediate apoptosis in other
tissues (23, 25–30, 33, 36–37), we postulate that the proapop-
totic effect of AngII in human GL cells is mediated by AT2
Comparing the outcomes in young uncomplicated patients
vs. older patients, the effectof AngIItreatments wereconcor-
dant with the pregnancy rates (38). However, it is too early to
accept that AngII-induced apoptosis could be an indicator of
successful cycles. This is especially true because the cells
from each cycle were pooled.
It is noteworthy that cumulus cells have a very low level of
apoptosis compared with mural cells (<3%) and that AngII
has no effect on cumulus cells apoptosis. The lack of apopto-
sis observed in cumulus cells compared with GL cells is in
agreement with similar findings reported in cows (39, 40),
where it has been demonstrated that paracrine factors
secreted by the oocyte, such as bone morphogenetic proteins
15 and 6, protect cumulus cells from apoptosis (41). At the
present time, we have no explanation for the lack of an effect
Increased apoptosis of mural granulosa-lutein cells
after 48-h culture in the presence of different doses
of angiotensin II (solid columns) compared with
untreated control cultures (clear columns).aMeans
are statistically different (Student t test).
Acosta. Angiotensin II–driven follicular cell apoptosis. Fertil Steril 2009.
(A) Blockade of the angiotensin II (AngII)–driven
incubation withAngII and saralasin (a blocker of both
AngII type 1 [AT1] and type 2 [AT2] receptors). (B)
Blockade of the AngII-driven increase of granulosa
cell apoptosis by simultaneous incubation with AngII
and the specific AT2 receptor blocker CGP42112A.
(C) Decrease of apoptosis by the AngII receptor
blockers alone: Note that the specific AT2 blocker is
more effective than saralasin.aMeans are
statistically different (Student t test).
Acosta. Angiotensin II–driven follicular cell apoptosis. Fertil Steril 2009.
Fertility and Sterility?
of AngII on cumulus cells, and further work is ongoing in our
laboratory to assess the expression of AngII receptors on the
different follicular cell types. However, because the periovu-
latory increase of AngII is linked to oocyte maturation and
ovulation (9, 42), it is not unreasonable to consider that these
cells would be protected against untoward AngII-driven
event.Certainly,the protectionofcumulus cellsfromapopto-
sis is a very interesting field for investigation of antrum
formation and cumulus-oocyte development, as well as
oocyte-protective roles that the cumulus cells may play
during the cycle.
The OVRAS has been shown to play key roles in ovarian
physiology (43). As mentioned above, the preovulatory in-
crease in intrafollicular AngII may facilitate oocyte matura-
tion (42) and ovulation (9, 44) or the production of
progesterone by human GL cells (45). Angiotensin II is in-
creased in both follicular atresia (36, 37) and corpus luteum
regression (46), events that are known to be linked to apopto-
sis (47–53). The latter action is very prominent in polycystic
ovarian syndrome (PCOS), in which high levels of AngII are
expressed in the cystic follicle’s mural granulosa cells (54,
55). Using a rat model of PCOS, we have shown that apopto-
work is needed to better elucidate the role of apoptosis in the
development of cystic follicles in human PCOS: indeed, pre-
liminary experiments conducted in our laboratory show that
the rate of apoptosis in PCOS tends to be higher than in
patients with other causes of infertility.
We have previously shown that younger patients have
lower apoptosis levels compared with older patients and
that patients who become pregnant have lower apoptosis
levels compared with those who do not get pregnant in the
IVF cycle studied (7).The results of the present study confirm
our previous findings, extending the observation to young
ported pregnancy rates, which are higher than the general
pregnancy rates observed in our program reflect, on one
hand, the inclusion of donors in the young group and, on
the other hand, the selection bias due to exclusion of all cases
with poor response in which we were unable to obtain suffi-
cient amounts of cells to carry on the experiments.
The OVRAS is a recently discovered intraovarian system
that has many effects on ovarian function and disease. It is
necessary to better understand the role of each compound
of the OVRAS, including angiotensin receptors and AngIII
(57) before determining the significance of the OVRAS in
the larger picture of human reproduction and attempting to
manipulate it clinically.
In conclusion: 1) AngII increases in vitro apoptosis of
cultured exfoliated mural human GL cells, but not cumulus
cells. 2) The changes in apoptosis occurred at concentra-
tions that are present in FF after ovarian stimulation for
IVF. 3) The effect of AngII was blocked by angiotensin
receptor antagonists in a manner consistent with an AT2
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Acosta et al.
Angiotensin II–driven follicular cell apoptosis
Vol. 91, No. 5, Supplement, May 2009