Effect of anabolics on bovine granulosa-luteal cell primary cultures.

Abstract

Granulosa cell tumours are observed with increased frequency among calves slaughtered in Northern Italy. The use of illegal anabolics in breeding was taken into account as a cause of this pathology. An in vitro approach was used to detect the possible alterations of cell proliferation induced by anabolics on primary cultures of bovine granulosa-luteal cells. Cultures were treated with different concentrations of substances illegally used in cattle (17beta-estradiol, clenbuterol and boldione). Cytotoxicity was determined by means of MTT test, to exclude toxic effects induced by anabolics and to determine the highest concentration to be tested. Morphological changes were evaluated by means of routine cytology, while PCNA expression was quantified in order to estimate cell proliferation. Cytotoxic effects were revealed at the highest concentrations. The only stimulating effect on cell proliferation was detected in boldione treated cultures: after 48 h treated cells, compared to controls, showed a doubled expression of PCNA. In clenbuterol and 17beta-estradiol treated cells PCNA expression was similar to controls or even decreased. As the data suggest an alteration in cell proliferation, boldione could have a role in the early stage of pathogenesis of granulosa cell tumour in cattle.

Full text

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Introduction
The European Community banned the use of anabolics
in Europe by means of laws 96/22/EC and 96/23/EC.
In other countries (e.g. USA, Canada, Australia and
New Zealand) natural hormones, such as testosterone,
17β-estradiol and progesterone, and synthetic hor-
mones, like trenbolone, zeranol and melengestrol
acetate, are legally used as growth promoters. Despite
the regulations, in Italy, exogenous sex hormones, cor-
tisone and β-agonists are widely and illegally used in
cattle for anabolic purposes during the last two months
of the fattening period. Researches carried out in our
Department on veal calves regularly slaughtered in our
region have revealed a high frequency of lesions of the
female genital tract, related to illegal use of sex hor-
mones, sometimes in association with corticosteroids,
or clenbuterol [1,2]. The reported prevalence of gran-
ulosa cell tumours in cattle revealed at the abattoir is
between 0 and 0.74% [3]. They are mainly described
in adult animals and, very rarely, in veal calves [4].
However, an earlier study in our Department looking
at veal calves regularly slaughtered in Piedmont (Italy)
revealed a dramatically higher prevalence of granulosa
cell tumours (2.54%) [5].
Several factors may have played a role in the devel-
opment of granulosa cell tumours in veal calves with
such a high prevalence. Among these, it is probable
that specific environmental factors may be a major fac-
tor in the development of granulosa cell tumours in
veal calves. In the literature, a strict correlation
between hormones and cancer has been revealed [6-8].
Sex hormones and gonadotropins are thought to be
important in the regulation of granulosa cell prolifera-
tion and maturation [9,10]. Hormones can stimulate
cell growth, even in mutated cells, so that they are con-
sidered co-carcinogens. Stimulating mitosis, hormones
increase the mutation risk, increasing the number of
cell divisions [6]. Usually, the greater part of the muta-
tions is corrected by DNA repair mechanisms and, as
these require prolonged times, it is assumed that the
increase of cell division speed can increase the risk of
mutations transfer to daughter cells. Consequently, the
hormones may behave not only as co-carcinogens, but
also as true carcinogens, able to provoke an increase of
the risk of mutation in their target cells and to stimu-
FOLIA HISTOCHEMICA
ET CYTOBIOLOGICA
Vol. 45, No. 3, 2007
pp. 265-271
Effect of anabolics on bovine granulosa-luteal cell
primary cultures
Paola Pregel, Enrico Bollo, Francesca Tiziana Cannizzo, Antonella Rampazzo,
Simonetta Appino and Bartolomeo Biolatti
University of Turin, Dipartimento di Patologia Animale, Grugliasco (TO), Italy
Abstract: Granulosa cell tumours are observed with increased frequency among calves slaughtered in Northern Italy. The
use of illegal anabolics in breeding was taken into account as a cause of this pathology. An in vitro approach was used to
detect the possible alterations of cell proliferation induced by anabolics on primary cultures of bovine granulosa-luteal cells.
Cultures were treated with different concentrations of substances illegally used in cattle (17β-estradiol, clenbuterol and
boldione). Cytotoxicity was determined by means of MTT test, to exclude toxic effects induced by anabolics and to deter-
mine the highest concentration to be tested. Morphological changes were evaluated by means of routine cytology, while
PCNA expression was quantified in order to estimate cell proliferation. Cytotoxic effects were revealed at the highest con-
centrations. The only stimulating effect on cell proliferation was detected in boldione treated cultures: after 48 h treated
cells, compared to controls, showed a doubled expression of PCNA. In clenbuterol and 17β-estradiol treated cells PCNA
expression was similar to controls or even decreased. As the data suggest an alteration in cell proliferation, boldione could
have a role in the early stage of pathogenesis of granulosa cell tumour in cattle.
Keywords: Cattle - Granulosa-luteal cells - Anabolics - PCNA
Correspondence: P. Pregel, Dipartimento di Patologia Animale,
Via L. Da Vinci 44, 10095 Grugliasco (TO), Italy; tel.: (+39011)
6709037, fax.: (+39011) 6709031; e-mail: paola.pregel@unito.it

Page 2

late, at the same time, the replication of the mutated
cells [8]. Sex steroid hormones may act as growth fac-
tors and activate signalling pathways that have been
shown to have a proliferative role in many cell lines
[11]. Furthermore, sex steroids seem to interfere with
apoptotic control mechanisms although contradictory
results have been reported. Some authors found that
β-estradiol inhibited apoptosis on granulosa cells
[12,13], while others showed a lack of influence of this
hormone [14]. As regards androgens, in some experi-
ments they have been shown to promote granulosa
cells apoptosis [15], while other authors have affirmed
that they preserved granulosa cells and follicles from
undergoing programmed cell death [14]. 17β-bolde-
none, esters of 17β-boldenone and boldione are openly
sold in Italy as anabolic preparations. 17β-aldosterone
improves the growth and feed conversion of cattle and
therefore might be abused to achieve more efficient
meat production. Boldione is also available on the Inter-
net for use by body builders as a product with an even
greater anabolic potency than 17β-boldenone itself.
Like the other androgenic steroids, 17β-boldenone is
classified by the International Agency for Research on
Cancer as a probable human carcinogen, with a carcino-
genicity index higher than that of other androgens [16].
In recent years, several models of primary granu-
losa cell cultures collected from different animal
species have been widely used to evaluate the effects
of many substances and hormones on cell proliferation
and steroidogenesis in vitro in cattle [17,18].
Following these considerations, it should be useful
to evaluate the possible involvement of anabolics in
the pathogenesis of granulosa cell tumours in veal
calves. Due to the difficulties of performing an in vivo
study, the aim of the present research was to perform a
preliminary assessment of the effects of anabolic sub-
stances on granulosa-luteal cell cultures, using an in
vitro approach. The main focus was to detect the pos-
sible alterations of cell proliferation induced by ana-
bolic substances, being this the first step reported in
carcinogenesis [8]. Morphological changes were eval-
uated by means of routine cytology, while PCNA
expression was quantified in order to estimate cell pro-
liferation. To exclude toxic effects induced by anabol-
ic substances and to determine the highest concentra-
tion to be tested, cytotoxicity was determined by
means of MTT test.
Materials and methods
Preparation of culture medium and reagents. All the reagents
were obtained from Sigma (St. Louis, MO, USA) and the dispos-
able plastics from Nunc (Naperville, IL, USA), unless otherwise
specified. The culture medium consisted of M199 medium, modi-
fied with Earl's salt, 25 mM Hepes and 1.2 g/L sodium bicarbon-
ate, supplemented with 1% (v/v) antibiotic-antimycotic solution,
2.5 mM L-glutamine, and 10% fetal calf serum.
Follicular cells harvesting. Ovaries were obtained from healthy
heifers and veal calves slaughtered at a local slaughterhouse
(Turin, Piedmont, Northern Italy). The collected ovaries were
transferred to the laboratory within 1 h in sterile phosphate
buffered saline (PBS) containing 1 mg/L gentamicin at 25-30°C.
Granulosa cells were aseptically collected using a syringe fitted
with a 18-gauge needle by aspiration of follicular fluid from small
(<5 mm) follicles.
Follicles with an opaque appearance, fragmented or viscous
membrana granulosa were classified as atretic, according to the cri-
teria of Kruip and Dieleman [19], and were discarded. The collect-
ed follicular fluid was then immediately placed in sterile tubes con-
taining culture medium. To avoid cell clumping heparin was added
(50 UI/mL). The cell suspension was decanted for a short time in a
water bath at 38.5°C, then aliquots of sediment were transferred in
a sterile 35 mm petri dish and diluted with fresh culture medium.
Oocytes were discarded using a stereomicroscope (Zeiss, Jena,
Germany, magnification ×120), under sterile laminar flow condi-
tions, in order to avoid any interference with the tests.
Aliquots of purified granulosa cells were then pooled and cen-
trifuged at 600 g for 5 min. The pellet was treated with 0.9% pre-
warmed sterile ammonium chloride at 38°C for 3 min to remove
red blood cells and centrifuged at 600 g for 5 min. The cells were
washed twice in fresh medium, counted using a haemocytometer
and viability was estimated by trypan blue (0.4%) exclusion
method.
Cell culture and addition of test substances. Cells were cultured
with a microdrop technique, seeded in 45 μL drops at a concentra-
tion of 4.5×106cells/mL, in 8-well chamber slides, to perform
morphological and immunocytochemical studies, or in 96-well tis-
sue culture plates, for MTT test.
The cell cultures were incubated in a water-saturated atmos-
phere of 5% CO2at 38.5°C for an initial 48 h period, to achieve
cell adhesion. Then, one-half of the exhausted medium was
replaced. The cultures were fed with fresh culture medium con-
taining appropriate treatments, "feeding" the culture to a final vol-
ume of 90 μL. Different concentrations (from 10-3M up to 10-9M)
of 17β-estradiol, clenbuterol or boldione were added. The plates
were returned to the incubator for further 24 or 48 h. Test sub-
stances were dissolved in ethanol, and the final concentration of
ethanol never exceeded 1%. Culture medium alone was added to
control wells, and culture medium containing ethanol was added to
relative control wells, to evaluate the influence of ethanol on cul-
tures. Tests were carried out in 3-5 replicate wells.
Estimation of cytotoxicity. Estimation of cytotoxicity of anabol-
ics on cell viability was determined by means of MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay,
according to the protocol of Mosmann [20], on cells grown on 96-
well plates, after 24 or 48 h of treatment. Absorbance at 595 nm
was read on a multiwell spectrophotometer (Bio-Rad, model 450,
Hercules, CA, USA), with a 655 nm reference filter.
In the MTT assay, each anabolic concentration was tested in
triplicate-quintuplicate measurements per experiment, and each
experiment was carried out at least three times. Percent survival
was calculated as the percentage absorbance of the treated wells
relative to the untreated wells ±SEM. (standard error of the
mean).
Cytology and immunocytochemistry.At the end of the treatment
period, medium was removed from the cultures carried out in
chamber slides and cells were formalin fixed. Slides were rinsed
twice in PBS and stained with haematoxylin and eosin or
immunocytochemistry for PCNA, to determine the proportion of
granulosa-luteal cells entering the cell cycle. Slides were incu-
bated with a solution of 3% H2O2in methanol to quench endoge-
nous peroxidase activity for 10 min. The slides were washed
266
P. Pregel et al.

Page 3

three times in PBS and submitted to immunocytochemistry for
PCNA, using the Vectastain ABC Kit (Vector Laboratories,
Burlingame, CA, USA) and a specific monoclonal antibody to
PCNA (Clone PC10, 1:50 in PBS, Dako, Glostrup, Denmark) for
3 h. Controls included the omission of the primary antibody and
the incubation with an irrelevant isotype matched antibody. After
rinsing, slides were treated with 0.5 mg/mL diaminobenzidine
tetrahydrochloride in PBS and 0.3% (w/v) H2O2. Slides were
counterstained with haematoxylin for 10 s, followed by washing
and blueing in running tap water for 10 min. Finally, the slides
were ethanol-dehydrated, cleared with xylene, and mounted. The
slides were examined with an Olympus BX41 (Tokio, Japan)
microscope and pictures were taken with an Olympus BP12 colour
digital camera.
The labelling index for PCNA was calculated by counting the
percentage of positive nuclei on the total number. Image analysis
was carried out by means of Image-Pro Plus software (Image-Pro
Plus, Media Cybernetics, Silver Spring, MD, USA). For each treat-
ment three random microscopic fields were counted at a magnifi-
cation of ×200. For each tested substance, cultures treated with
three dilutions of the anabolic were counted: the lowest (10-9M), a
middle one (10-6M), and the highest where a toxic effect of the
substance was not revealed by MTT assay (referring to the para-
graph Results). Results were expressed as a percentage of the con-
trol internal to the culture.
Statistical analysis. Statistical analysis was performed by means
of Statgraphics Plus software (Statistical Graphic Corp., Manugis-
tics, Inc., Rockville, MD, USA). One-way ANOVA was used to
evaluate differences between treated cultures and controls, and
Fisher's tests were subsequently used to make individual compar-
isons within a given treatment group providing the initial one-way
ANOVA indicated a significant effect of that treatment.
Unless stated otherwise, numerical values are presented as
means ±SEM. Data were normalized respect corresponding con-
trols, carried out in the same conditions of the tested cultures
(same inoculum, medium, time of culture) and expressed in per-
centage. Data were combined from three to six individual cul-
tures and p values below 0.05 were considered to be statistically
significant.
Results
MTTtest showed that boldione drastically reduced cell
viability, showing an extremely toxic effect at the
highest concentration tested (1 mM): a 25% reduction
of cell viability was shown after 24 h of culture in the
presence of the anabolic, whereas it reached 75% after
48 h. Concentrations lower than 1mM did not interfere
significantly with cell viability. In cultures incubated
with 1 mM clenbuterol, the MTT test showed a viabil-
ity reduction of 50% 24 h post treatment and 25%
48 h after, in comparison to the control examined at the
same time. As for boldione, lower concentrations did
not interfere significantly with cell viability. Finally, as
regards 17β-estradiol, MTT suggested that there was
no significant difference between treated cell viability
and controls.
Following the results obtained by cytotoxicity eval-
uation, the highest dilutions used to quantify PCNA
expression were 10-3M for 17β-estradiol, and 10-4 M
for boldione and clenbuterol, i.e. the highest non-toxic
concentrations for the cells.
The morphological appearance of treated cultures
differed substantially from control cultures, even if a
small level of apoptosis was detected even in control
cultures, especially after 48 h.
Cells treated with 17β-estradiol 10-3M appeared
foamy (Fig. 1d and 2d). The nucleus/cytoplasm ratio
was strongly reduced and, especially after 48 h, the
cells presented chromatin condensation, cytoplasm
enlargement, and, often, even cytolysis and karyor-
rhexis (Fig. 1d and 2d). At the lower concentrations,
the lesions progressively reduced, and in 10-9M treat-
ed cells the nucleus/cytoplasm ratio was almost nor-
mal. The foamy appearance persisted in 10-6M treated
cells, after 24 h, where a discrete number of pyknotic
nuclei was also present and even in 10-9M treated
cells, after 48 h cytoplasmic vacuolization could be
detected.
Cells treated with clenbuterol, in high concentra-
tions (Fig. 1c and 2c), or boldione (Fig. 1b and 2b), even
in lower concentrations but for a longer time (48 h), pre-
sented many pyknotic nuclei. In clenbuterol 10-4M
treated cells cytoplasmic vacuolization was conspicu-
ous after 24 h (Fig. 1c) and the maximum presence of
pyknosis was detected at the same concentration after
48 h (Fig. 2c). At 10-6M the number of pyknotic nuclei
was fairly higher after 48 h than after 24 h. With the
lowest concentration a level of chromatin condensa-
tion different from controls was revealed only after
48 h of treatment, while after 24 h the cells were com-
parable to control, a part form the fact that their nuclei
were elongated.
Chromatin aggregation, nuclear and cytoplasmic
condensation, cell shrinkage, partition of cytoplasm
and nucleus into membrane bound-vesicles and frag-
mentation of cells into smaller apoptotic bodies char-
acterized cells treated with boldione 10-4M, especial-
ly at 24 h (Fig. 1b and 2b). These features were also
present, even if to a lesser extent, in cultures treated
with any lower concentration of the anabolic. Cells
had a polygonal shape, rather different from the fibrob-
last appearance of controls, and cytoplasm was vac-
uolized.
Different degrees of staining were revealed by
immunocytochemistry (Fig. 3). In control cultures
(Fig. 3a), the percentage of PCNA positive nuclei was
25.32% ± 7.90 (range: 10.88 - 33.46) of the total num-
ber of nuclei. Fig. 4 shows the quantification for
PCNAstaining. The asterisks indicate statistical differ-
ence from controls. Boldione did not interfere with
PCNA expression 24 h post treatment, while it was
able to increase its expression in a dose-dependent
manner after 48 h.
PCNA expression in clenbuterol treated cultures
seemed almost constant in time, independently from
examined dose. The lowest tested dose, 24 h after the
treatment, provoked a reduction of about 35% in
267
Anabolics on bovine granulosa cell cultures

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268
P. Pregel et al.
Fig. 1. Morphological appearance of cultured granulosa-luteal cells, 24 h post treatment. Haematoxylin and Eosin staining (magnification
×400). a. control culture; b. cells treated with boldione 10-4M; c. cells treated with clenbuterol 10-4M; d. cells treated with 17β-oestra-
diol 10-3M. Arrow: cytoplasm enlargement, vacuolization, and karyorrhexis; arrowhead: pyknotic nuclei.
Fig. 2. Morphological appearance of cultured granulosa-luteal cells, 48 h post treatment. Haematoxylin and eosin staining (magnification
×400). a. control culture; b. cells treated with boldione 10-4M; c. cells treated with clenbuterol 10-4M; d. cells treated with 17β-estradiol
10-3M. Arrow: cytoplasm vacuolization, foamy appearance, and karyorrhexis; arrowhead: pyknotic nuclei.

Page 5

immunocytochemical staining. 17β-estradiol neither
showed enhancement effects on PCNA expression in
treated cultures; indeed, in some conditions (1 mM and
1 nM at 24 h, and 1 μM and 1 nM at 48 h) a reduction
of the immunocytochemical staining was evident,
reaching the 40%, compared to control.
Discussion
The aim of the present research was to evaluate the
possible influence of anabolics on granulosa cells pro-
liferation in veal calves, using an in vitro approach.
A complete culture medium model allowed to obtain
homogeneous cultures, with cells more viable even on
morphological examination compared to serum free
cultures. Serum deprived granulosa cells, in fact,
undergo spontaneous apoptosis [21] and after only 5 h
of serum deprivation, 20-30% of granulosa cells are
apoptotic. In the present investigation, the experiments
were conducted only on follicular fluid aspirated from
small (<5 mm) follicles, which are more responsive to
hormonal treatments [22], and oocytes were eliminat-
ed in order to avoid any interference with the tests. In
fact it has been reported that some factors released
form oocytes stimulate granulosa cell proliferation
[23], and interfere with their differentiation [24,25].
Culturing cells in drops, in addition to the high seeding
cell density and to cell hyperplasia phenomena,
allowed to obtain a sub-confluent status in 48 h,
despite the scarce granulosa cells proliferation ability
in vitro. Treated cultures appeared morphologically
different from control cultures. Chromatin aggrega-
tion, and nuclear and cytoplasmic condensation could
be detected in cultures treated with each one of the
anabolics, especially at higher concentrations, suggest-
ing a possible activation of apoptotic pathways. Cells
treated with high concentrations of 17β-estradiol and
clenbuterol were altered, and presented pyknotic
nuclei, as cultures treated with boldione, even in low
concentrations for 48 h. The foamy appearance and the
strong reduction of nucleus/cytoplasm ratio presented
by cells treated with high concentrations of 17β-estra-
diol could also be related to an increased production of
hormones, being the 17β-estradiol strongly involved in
granulosa-luteal cells metabolism in vivo. The propor-
tion of granulosa-luteal cells entering the cell cycle
was determined using the cell proliferation marker
PCNA, the cofactor of delta DNA polymerase, active
both in replicative DNAsynthesis, during S phase, and
in reparative DNA synthesis, associated to damaged
DNA repair mechanisms [26]. Increased PCNA
expression correlates with the earliest signs of granu-
losa cell growth and its immunoreactivity remains
prevalent in granulosa and theca cells of follicles in
269
Anabolics on bovine granulosa cell cultures
Fig. 3. Microphotographs of cultured granulosa-luteal cells. PCNA immunostaining (magnification x200). a. control culture, 48 h of cul-
ture; b. cells treated with boldione 10-4M, 48 h post treatment; c. cells treated with clenbuterol 10-9M, 24 h post treatment; d. cells treat-
ed with 17β-estradiol 10-3M, 24 h post treatment. Brown staining of PCNA positive cells.

Page 6

subsequent stages of follicle growth, with a progres-
sive decrease related to follicular atresia [27,28].
MTT test suggested a strong toxic effect for
boldione and clenbuterol in high concentrations, so we
excluded these cultures from the PCNA labelling
analysis. Boldione treated cells compared to controls
showed a higher degree for PCNA, after 48 h. Low
doses of clenbuterol reduced PCNA expression in
granulosa-luteal cells after 24 h, as 17β-estradiol, at
some dilutions. Clenbuterol (10-3M-10-8M) and
17β-estradiol (10-7M and 10-8M) effects have been
evaluated previously using a model represented by
human tumour mammary cells [29]. In that study, as in
the present one, the highest concentration of clenbuterol
(10-3M) caused a high cell mortality (84%), while at a
10-4M dose an antiproliferative effect was evident
(probably due to the toxicity of the anabolic). In addi-
tion, 10-7M and 10-8M concentrations significantly
increased cell proliferation in a dose-related way.
As our results never showed an enhancement of
PCNA expression for both clenbuterol and 17β-estra-
diol, but instead an antiproliferative effect, we would
exclude their role in the pathogenesis of granulosa cell
tumour in cattle. The dramatic enhancement of cell
proliferation due to the boldione treatment of cultures
for 48 h, as revealed by PCNA expression, would sug-
gest its role in the early stage of pathogenesis of gran-
ulosa cell tumour in cattle. The simultaneous presence
of apoptosis and proliferation could represent a mech-
anism of cell resistance to tumourigenic stimuli.
Afuture target could be the evaluation of other pro-
liferation markers involved in follicular development
in vivo, to confirm these preliminary results, and to
verify the effects provoked on primary granulosa-
luteal cell cultures by other illegally used anabolics
(and their cocktails). Encouraging preliminary results
suggest the value of continuing with the present
research in order to deepen our knowledge of anabolic
treatments, and better understand how to control the
illegal use of anabolics and to verify their potential risk
for the consumer health.
Acknowledgements: We would like to thank Dr. Simona Stac-
chezzini for help in culture methods, and Professor Mauro Dacas-
to for suggestions in choosing anabolic substances and dilutions.
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Submitted: 6 March, 2007
Accepted after revision: 1 April, 2007
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Anabolics on bovine granulosa cell cultures