Theriogenology 114 (2018) 14e24

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Theriogenology
journal homepage: www.theriojournal.com

Proliferative and apoptotic changes in the healthy canine

endometrium and in cystic endometrial hyperplasia
N. Reusche a, b, A. Beineke c, C. Urhausen a, b, M. Beyerbach d, M. Schmicke e, S. Kramer b,
A.R. Günzel-Apel a, b, *
a
Unit for Reproductive Medicine of Clinics, University of Veterinary Medicine Hannover, Germany
b
Small Animal Clinic, University of Veterinary Medicine Hannover, Germany
c
Department of Pathology, University of Veterinary Medicine Hannover, Germany
d
Department for Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Germany
e
Clinic for Cattle - University of Veterinary Medicine Hannover, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Proliferation and apoptosis play an important role in cyclic changes in the healthy canine endometrium.
Received 15 August 2017 Proteins of the Bcl-2-family are known to be involved in the regulation of apoptosis. However, only few
Received in revised form reports mention their expression patterns during cystic endometrial hyperplasia (CEH). In order to
2 March 2018
correlate proliferative and apoptotic processes, expression of the apoptosis-regulatory proteins Bcl-2
Accepted 13 March 2018
(anti-apoptotic) and Bax (pro-apoptotic) in healthy and cystic hyperplastic endometrial tissue as well
Available online 15 March 2018
as in pyometra was investigated. Uteri from 33 bitches were assigned to three groups: group 1 - healthy
endometrium (n ¼ 12), group 2 e CEH (n ¼ 17) and group 3 - pyometra (n ¼ 4). Markers for proliferation
Keywords:
Canine endometrium
(Ki-67) and apoptosis (activated caspase-3 and TUNEL-method) as well as expression of Bcl-2 and Bax
Proliferation were determined in all endometrial layers. For groups 1 and 2 this was done during endometrial gland
Apoptosis secretion in mid and late luteal phase (mLP, lLP), endometrial reparation in early anestrus (eAE) and in
Bcl-2 the regenerated endometrium in late anestrus (lAE). For group 3 only the late luteal phase was
Bax investigated.
Cystic endometrial hyperplasia In group 1, cyclic proliferation patterns were found predominantly in superficial glands (SG) and
stroma, whereas progesterone-mediated low expression levels coincided with high apoptosis rates in the
basal glands (BG). In eAE, higher apoptotic activity was detected compared to lLP and lAE. Bcl-2 and Bax
expression levels showed an inverse cyclic relationship in all tissue layers. In the stroma, in eAE, a rise in
proliferative activity and concomitant increase in anti-apoptotic Bcl-2 expression levels was found,
indicating that this layer serves as a source for endometrial regeneration.
In CEH, no or limited cyclic patterns of proliferation, apoptosis, Bcl-2 and Bax were found. Increased
proliferation rates coincided with deregulated apoptosis. Besides the glandular compartments, the
stroma played an important role in the pathogenesis of canine CEH. In case of pyometra, both prolif-
eration and apoptosis increased, indicating irreversible damage of the inflamed canine endometrium.
In conclusion Bcl-2 and Bax play a role both in the physiological regenerative processes of the cyclic
canine endometrium and in deregulating proliferation and apoptosis in CEH and pyometra.
© 2018 Elsevier Inc. All rights reserved.

1. Introduction [7,8]. Bcl-2 (B-cell lymphoma-2) and Bax (Bcl-2-associated X-pro-

Proliferation and apoptosis have been shown to be involved in
cyclic changes in the canine endometrium [1e6] and canine oviduct
* Corresponding author. Unit of Reproductive Medicine of Clinics, Bünteweg 15,
D-30559 Hannover, Germany.
E-mail address: anne-rose.guenzel-apel@tiho-hannover.de (A.R. Günzel-Apel).
tein) are members of the Bcl-2-family, consisting of a variety of
anti-apoptotic and pro-apoptotic proteins. The ratio of anti-
apoptotic to pro-apoptotic proteins is crucial for the survival or
death of cells [9e11]. Formation of a heterodimer complex of the
anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins leads to inhi-
bition of apoptosis [12,13], whereas the predominance of Bax/Bax
homodimers induces apoptosis via the mitochondrial pathway

https://doi.org/10.1016/j.theriogenology.2018.03.018
0093-691X/© 2018 Elsevier Inc. All rights reserved.
 N. Reusche et al. / Theriogenology 114 (2018) 14e24
[9e11]. In the feline endometrium, the regulation of cyclic changes
like growth and regression has been related to the combined effect
of Bcl-2 and Bax, as well as Survivin which is a member of the in-
hibitors of the apoptosis protein (IAP) gene family [14]. The crucial
role of members of the Bcl-2-protein-family in maintaining endo-
metrial homeostasis is described in women [15e18] and primates
[18,19].
Regeneration of the endometrium and preparation for the next
proliferation phase are mainly linked to apoptotic processes, where
imbalances in the expression of Bcl-2 and Bax may induce patho-
logical conditions like endometrial hyperplasia [16]. The develop-
ment of endometrial hyperplasia in women is supposed to be
caused by changes in proliferation [16,17,20], and intensified
through deregulation of apoptosis, mainly due to an overexpression
of Bcl-2 [15,17,21e24]. Therefore human endometrial hyperplasia is
considered to be a precancerous lesion with a potential for devel-
oping neoplastic processes [17,24,25]. Cystic endometrial hyper-
plasia (CEH) in dogs is not considered to be a precancerous lesion
because in this species the incidence of uterine tumors is low.
However, in almost all reported cases of uterine tumors and
endometrial polyps, CEH was also found [26e32]. Up to now, no
detailed study has been performed on the expression and role of
Bcl-2 and Bax in the healthy cyclic canine endometrium or their
role in pathological conditions of the canine endometrium with
respect to proliferation and apoptosis processes.
The primary aim of the present study was to investigate the
apoptosis-regulatory proteins Bcl-2 and Bax in proliferative and
apoptotic events during the periods of glandular secretion (mid and
late luteal phase), endometrial reparation in the initial phase of
ovarian rest (early anestrus), and in the repaired, intact endome-
trium in late anestrus. This was done with the purpose of gaining
insights into their physiological roles. Furthermore proliferative
and apoptotic processes and Bcl-2 and Bax expression were studied
in case of cystic endometrial hyperplasia and pyometra.
2. Material and methods
2.1. Animals
Uteri and ovaries of 33 bitches were used for this study, from
which 20 were either mongrels or different breeds. Animals were
presented to the Clinic of the University of Veterinary Medicine
Hannover, Germany as well as a private practice in Germany. In
addition, nine Beagle bitches from the University of Veterinary
Medicine Hannover were included in the study. Surgery was per-
formed as a preventative measure to stop reproduction or in case of
a uterine disease. The study was approved by the Lower Saxony
State Office for Consumer Protection and Food Safety, Germany
(Reference no. 33.9-42502-05-11A131).
Based on histo-pathological criteria, animals were divided into
the following groups: Group 1 - Healthy endometrium (n ¼ 12, 1e8
years) showed normal anatomical structures without inflammatory
and degenerative changes (Fig. 1A), group 2 - Cystic endometrial
hyperplasia/CEH (n ¼ 17, 2e14 years) was characterised by hyper-
trophic proliferating epithelial cells and cystic distension of uterine
glands (glandular cysts) (Fig. 1B and C), group 3 - Cystic endome-
trial hyperplasia with pyometra/PYO (n ¼ 4, 6e7 years) consisted of
hypertrophic and hyperplastic endometrial changes and glandular
cysts together with lymphoplasmacytic to neutrophilic infiltrations
in the endometrial stroma and accumulation of degenerated leu-
kocytes in the uterine lumen (Fig. 1D). In ten animals of group 1 and
all animals in groups 2 and 3 a complete ovariohysterectomy was
performed. In two bitches in group 1 the ovaries and the tips of
both uterine horns were removed at the request of the owner.
15
2.2. Assignment of bitches to estrous cycle stages
Four stages of the ovarian and endometrial cycle were investi-
gated, and samples were classified as follows: mid luteal phase
(mLP) and late luteal phase (lLP) as periods of progesterone-
mediated endometrial gland secretion, the initial phase of ovarian
rest (early anestrus, eAE) as a period of endometrial reparation and
a stage of ovarian rest (late anestrus, lAE) coinciding with a
repaired, intact endometrium [33,34]. The stage of the estrous cycle
was assigned by taking into account the time point of the last heat,
appearance of the vulva and vaginal mucosa as well as cytological
evaluation of a vaginal smear combined with the peripheral pro-
gesterone (P4) concentrations. Mid and late luteal phase were
classified by P4 concentrations >10 ng/ml and 1.0 to <10 ng/ml,
respectively (Tables 1 and 2). In group 1, three genital tracts per
estrous cycle stage were available. In group 2, the distribution was
as follows: mLP (n ¼ 2), lLP (n ¼ 10), eAE (n ¼ 2) and lAE (n ¼ 3). All
dogs in group 3 were found to be in the late luteal phase (Table 2).
2.3. Blood sampling and hormone analyses
For progesterone analysis, a single blood sample of 10 mL was
collected in a tube containing additive carrier and clot activator
(Sarstedt AG & Co. Nümbrecht, Germany). This was done immedi-
ately before surgery via an indwelling catheter which was intro-
duced intravenously for premedication of anaesthesia. Samples
were centrifuged (3030  g, 10 min), the blood serum was trans-
ferred to a clean tube and stored at 20  C until analysis.
Serum progesterone concentrations were determined by means
of a chemiluminescence-immunoassay (CIA) using the automated
Immulite®-system (Immulite® 1000, LKPG1, Siemens Healthcare
Diagnostics GmbH, Erlangen, Germany). The lowest progesterone
concentration that could be measured was 0.09 ng/mL. The intra-
and inter-assay variation coefficients ranged from 6.3 to 8.1% and
5.8e10.0%, respectively. CIA-values were confirmed using an
enzyme-linked immunosorbent assay (ELISA).
2.4. Tissue preparation, staining and evaluation
2.4.1. Histo-pathology
Immediately after excision, the uterus was fixed in phosphate-
buffered 4% formaldehyde. Longitudinal sections of both ovaries
and cross-sections of the cranial, mid, and caudal region of both
uterine horns and the mid uterine body were prepared and
embedded in paraffin. Sections of 2 mm were cut from each location
and mounted on slides (Starfrost®; Engelbrecht Medizin-und
Labortechnik GmbH, Edermünde, Germany), stained with
hematoxylin-eosin (HE) using an automated staining system (Leica
ST 4040; Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany),
mounted with Roti® Histokitt II (Carl Roth GmBH, Karlsruhe, Ger-
many) and covered with a cover slip.
Tissue sections prepared for the histo-pathological examination
were stained with hematoxylin-eosin (HE) using the automatic
staining system. These were then equipped with cover slips by
Promounter RCM 2000 (Medite GmbH, Burgdorf, Germany) using
Roti® Histokitt II (Carl Roth GmBH, Karlsruhe, Germany).
Classification of the 33 uteri into either healthy or showing signs
of CEH was performed by light microscopy. Out of the 17 uteri with
CEH, nine were classified as having a mild degree of CEH (one or
more dilated glands affecting <30% of the endometrium) (Fig. 1B),
five with a moderate degree (one or more dilated glands affecting
30% to <60% of the endometrium), and three with a severe degree
(one or more dilated glands affecting 60%e100% of the endome-
trium) (Fig. 1C). These were distributed according to the estrous
cycle stages as follows: mLP (moderate degree n ¼ 2), lLP (mild
16 N. Reusche et al. / Theriogenology 114 (2018) 14e24

Fig. 1. Hematoxylin-eosin stained sections of A - the healthy endometrium, B - mild cystic hyperplasia of superficial glands, C - severe cystic hyperplasia of superficial glands with
marked hypertrophy of epithelial cells, D - severe cystic hyperplasia of a basal gland with accumulation of degenerated leukocytes (pyometra). A, B: LE ¼ Lamina epithelialis. A, B, C:
SG ¼ superficial gland. A, D: BG ¼ basal gland. D: ( ) neutrophil granulocytes Myo ¼ myometrium. *cystic dilated gland.

degree n ¼ 8, moderate degree n ¼ 1, severe degree n ¼ 1), eAE
(moderate degree n ¼ 1, severe degree n ¼ 1), lAE (mild degree
n ¼ 1, moderate degree n ¼ 1, severe degree n ¼ 1). In all four cases
of pyometra, a severe degree of CEH was found (Fig. 1D).
2.4.2. Immunohistochemistry and TUNEL
Tissue samples from the left and right mid uterine horn (n ¼ 31)
or the cranial uterine horn (n ¼ 2) were used for immunolocalisa-
tion studies and the TUNEL assay after removal of paraffin during
30 min incubation at 70  C.
Immunohistochemical examination was performed for Ki-67 as
proliferation marker, cleaved caspase-3 (apoptosis) for characteri-
sation of apoptosis and the apoptosis regulatory proteins Bcl-2 and
Bax. This was done using a modified version of the avidin-biotin-
complex (ABC)-peroxidase-method (Vectastain® Elite ABC Kit,
Vector Laboratories Inc., Burlingame, CA, USA), where 3,30 -
diaminobenzidine-tetrahydrochloride (DAB) was used as chro-
mogen. Immunopositive cells were stained brown.
Non-specific binding sites were blocked with inactivated goat-
normal-serum (GNS) diluted 1:5 in phosphate-buffered saline
(PBS, pH 7.1). Serum primary antibodies were diluted in PBS sup-
plemented with 1% bovine serum albumin (BSA). Canine control
tissues were used to test for cross-reactions. Secondary antibodies,
which are listed in Table 3, were diluted 1:200 in PBS without BSA.
Negative controls (i.e., without incubation with primary antibody)
were performed on one additional section per uterine location,
whereas canine lymphatic tissue material was used as positive
control (Table 3).
The standard immunohistochemical protocol was modified for
each antibody (Table 3) and was performed as previously described
with minor modifications [35]. It included the following steps:

Table 2
Table 1 Progesterone concentrations in bitches with cystic endometrial hyperplasia (CEH)
Progesterone concentrations in bitches with healthy uterus (group 1) in different (group 2) and pyometra (PYO) (group 3) in different estrous cycle stages.
estrous cycle stages.
Estrous cycle stage Endometrial status n Progesterone (ng/mL)
Estrous cycle stage n Progesterone (ng/mL)
mean ± SD range
mean ± SD range
mLP CEH 2 24.9 8.1 19.2e30.6
mLP 3 28.9 8.7 19.7e37.1 lLP 10 3.9 2.5 1.7e9.2
lLP 3 1.1 0.2 1.0e1.4 PYO 4 5.6 3.9 2.0e9.8
eAE 3 0.5 0.3 0.2e0.8 eAE CEH 2 0.5 0.4 0.2e0.7
lAE 3 0.4 0.3 0.2e0.7 lAE 3 0.5 0.4 0.2e1.0

mLP (mid luteal phase), lLP (late luteal phase), eAE (early anestrus), lAE (late mLP (mid luteal phase), lLP (late luteal phase), eAE (early anestrus), lAE (late
anestrus). anestrus).
 N. Reusche et al. / Theriogenology 114 (2018) 14e24 17

Table 3
Antibody (AB)-specific modifications of standard immunohistochemical protocols.

Marker Primary AB Dilution Positive Negative controls Incubation Secondary

(Indication) controls (primary AB/ AB
negative controls) Dilution
1:200

Ki-67 Monoclonal mouse-anti-human Ki-67- 1:100 Lymph-nodes, 1:1000 Balb/c (Control RT 75 min GAM-b (Biotinylated Goat-
(Proliferation) antigena (Clone MIB-1) thymus Ascites Fluid-MA)e Anti-Mouse)-IgG,
(BA-9200)g
Cleaved Caspase-3 Polyclonal rabbit-anti-cleaved caspase-3 1:200 Spleen 1:3000 Rabbit serum RT 75 min GAR-b (Biotinylated Goat
(Apoptosis) (Asp175) Antibody (# 9661)b (Sigma R4505)f Anti Rabbit)-IgG,
(BA-1000)7
Bcl-2 Monoclonal mouse-anti-Bcl-2-oncoprotein 1:1000 Lymph-nodes 1:1000 Balb/c (Control RT 30 min, GAM-b (Biotinylated Goat-
(anti-apoptotic (Novocastra™ NCL-bcl-2-486, Clone 3.1)c Ascites Fluid-MA)e then 4  C overnight Anti-Mouse)-IgG,
protein) (BA-9200)g
Bax Polyclonal rabbit-anti-Bax- antibody 1:1000 Spleen 1:3000 Rabbit serum 4  C overnight GAR-b (Biotinylated Goat
(pro-apoptotic (sc-526)d (Sigma R4505)f Anti Rabbit)-IgG,
protein) (BA-1000)g
a
Dako, Jena, Germany.
b
Cell Signaling Technology®, Danvers, MA, USA.
c
Leica Microsystems Ltd., Knowlhill Milton Keynes, United Kingdom.
d
Santa Cruz Biotechnology, Inc., Heidelberg, Germany.
e
Cederlane®, Burlington, Canada.
f
Sigma-Aldrich Chemie GmbH, Steinheim, Germany.
g
Vector Laboratories Inc., Burlingame, CA, USA.

Removal of paraffin and rehydration, blocking of the endogenous
peroxidase via 30 min incubation with 0.5% H2O2 in 85% ethanol,
antigen retrieval by 20 min incubation in boiling citrate buffer (pH
6.0e6.5), incubation with blocking serum (120 mL per slide;
25 min at room temperature (RT)), incubation with primary anti-
body (120 mL per slide; 75 min at RT), incubation with secondary
antibody (120 mL per slide; 45 min at RT), incubation with ABC-
reagent (120 ml; 25 min at RT), incubation in DAB-solution supple-
mented with 0.03% H2O2 (5 min at RT, counterstain with hema-
toxylin for 25 s followed by dehydration and mounting with Roti®
Histokitt II (Carl Roth GmbH, Karlsruhe, Germany)).
Apoptosis was examined by the TUNEL-method by using the
ApopTag® Plus Peroxidase In Situ Apoptosis Detection Kit (Merck
Millipore, Billerica Millipore, MA, USA). The protocol was per-
formed in accordance with the instructions provided by the
manufacturer with minor modifications. This method detects DNA-
fragments which are formed during apoptosis [36].
Samples from healthy canine spleen tissue and rat mammary
gland were used for positive controls. Sections from the right
uterine horn served as negative control. Incubation with terminal
desoxyribonucleotidyltransferase (TdT) was omitted for the nega-
tive control. Incubations were done in a humidified chamber.
After removal of paraffin and rehydration, slides were treated
with 100 mL proteinase K-solution (20 mg/mL) for 10 min at RT. Then
endogenous peroxidase was blocked via incubation in 2% H2O2 for
5 min. The slides were incubated with equilibration buffer (75 mL)
for 10 min at RT, after which the equilibration buffer was replaced
by TdT-solution (54 mL), a cover slip was added and specimens were
incubated for 1 h at 37  C. After removal of the cover slips, slides
were transferred to a Coplin jar with stop/wash buffer and incu-
bated at 37  C for 30 min under continuous movement. After
washing, anti-digoxigenine-peroxidase (55 mL) was added, speci-
mens were covered and incubated for 30 min at RT. Reaction with
DAB, dehydration and mounting were done as described above.
Slides were evaluated by light microscopy using a 400
magnification. For each slide four randomly selected areas were
inspected. The lamina epithelialis (LE), superficial glands (SG), basal
glands (BG) and stroma were evaluated separately. In LE, SG and BG
a quantitative evaluation was performed.
A semi-quantitative score was used for the stroma. Frequency
(F-score) was classified as negative (0): no positive cells; scattered
(0.5): one to two positive cells; minor (1): three to five positive
cells; moderate (2): six to ten positive cells; high (3): >10 positive
cells in an area.
For Ki-67, cleaved Caspase-3 and TUNEL, the percentage of
positive cells was determined by counting. In the case of markers
for apoptosis (cleaved Caspase-3 and TUNEL), only cells were
counted which also exhibited morphological signs of apoptosis. For
Bcl-2 and Bax in LE, SG and BG, the quantitative histochemical
scoring method (H-score) was used as described in detail else-
where [37]. This score includes both the percentage of immune-
P
positive cells and staining intensity (H-score ¼ Pi  i, P ¼ per-
centage, i ¼ intensity of stained cells) [37]. Staining intensities were
classified as 0 ¼ negative, 1 ¼ weak, 2 ¼ moderate and 3 ¼ strong.
The H-score ranged from 0 (no positive cells) to 300 (100% strongly
stained cells).
In the stroma, an additional Intensity score (I-Score) of
1 ¼ weak, 2 ¼ moderate and 3 ¼ strong was assigned.
For all parameters, mean values were calculated from the left
and right horn of each uterus.
2.5. Statistical analysis
Statistical calculations were performed using the Statistical
Analysis System (Version 9.3, SAS, Cary/NC, USA). As the percent-
ages of Ki-67, cleaved Caspase-3 and TUNEL positive cells showed a
relatively low range (approximately 0) and a skew distribution,
they were logarithmised in order to obtain a normal distribution
and homogeneity of variance. The absolute value 0 was replaced by
0.01. The calculation of significances is related to logarithmised
values. Scores were calculated using the original values. For all
parameters, the occurrence of cyclic endometrial changes was
detected by one-factorial ANOVA. As Post hoc test for comparison of
the findings in the different estrous cycle stages the t-test (LSD,
least significant difference) was used. The latter was also performed
for comparing the results found in group 1 with the corresponding
findings in CEH (group 2) and pyometra (group 3). Differences
between the estrous cycle stages and between groups were
assessed as being significant if P < 0.05. The Pearson correlation
coefficient was determined for further characterisation of related
influences.
18 N. Reusche et al. / Theriogenology 114 (2018) 14e24
3. Results
3.1. Healthy endometrium (group 1)
3.1.1. Proliferation and apoptosis
In LE, the proliferation rates were low during mLP, lLP and eAE
and increased during lAE (lLP versus lAE P < 0.05) (Fig. 2a). In SG,
the highest proliferation rates were found for eAE and lAE, whereas
they were lowest in mLP (P < 0.05) (Fig. 2b). In BG, Ki-67 positive
cells were generally scarce, with no Ki-67 positive cells during mLP
and eAE, and only 0.08 ± 0.06% in lLP and 0.1 ± 0.08% in lAE (Fig. 2c).
In stroma, the pattern of Ki-67-positive cells was similar to the one
found in SG, with the highest frequency score in eAE and the lowest
Fig. 2. Proliferation (Ki-67) and apoptosis (TUNEL) in a) Lamina epithelialis, b) su-
perficial glands, c) basal glands and d) stroma during mid luteal phase (mLP), late
luteal phase (lLP), early anestrus (eAE) and late anestrus (lAE) in healthy endometrium.
Different letters between cycle stages per parameter indicate significant differences
(P < 0.05).
in mLP (P < 0.05) (Fig. 2d).
For SG, a negative correlation was found between the prolifer-
ation rate and P4-concentrations (r ¼ 0.75, P < 0.01).
The apoptosis markers cleaved Caspase-3 and TUNEL showed
similar patterns, and their expression levels exhibited positive
correlations in LE (r ¼ 0.65, P < 0.05) and BG (r ¼ 0.87, P < 0.001).
TUNEL appeared to be more sensitive. Apoptosis rates were low in
LE, SG and stroma in all stages of the estrous cycle. Significant
differences were present only in BG (Fig. 3c). The highest rates were
found in mLP and coincided with the presence of maximum P4-
concentrations. A lower apoptosis level was found both in lLP and
lAE (Caspase-3, TUNEL: P < 0.05). Despite a positive correlation
with P4 (TUNEL: r ¼ 0.62, P < 0.05), an increase in apoptotic cells
was observed under basal P4-concentrations in eAE (TUNEL:
P < 0.05) (Fig. 2c). In SG and stroma, the cyclic pattern was similar
but less pronounced (Fig. 3b,d).
3.1.2. Bcl-2 and Bax
For Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic), negative
correlations were found in LE and SG (both r ¼ 0.72, P < 0.01) and
BG (r ¼ 0.65, P < 0.05) (Fig. 3aec), indicating their opposite
functions in apoptotic processes. In mLP, Bcl-2 was not expressed in
LE and only detected in very few cells in SG and BG. Also, an
increased level was found in the later cycle stages (P < 0.05)
(Fig. 3aec). A similar pattern was seen for staining intensity, but not
frequency, in the stroma (Fig. 3d). Staining intensity of Bcl-2 posi-
tive cells also increased from SG to BG (Fig. 4A,B).
Bax was expressed in all endometrial layers and cycle stages
(Fig. 3aed) showing highest intensities in mLP. In LE and SG Bax
expression was significantly stronger (Fig. 3C) than in all other cycle
stages (Fig. 2aed; Fig. 3D). In BG Bax expression was significant
lower only in lAE. In stroma this was also the case in lLP (Fig. 3c and
d).
P4-secretion exhibited a negative correlation with Bcl-2 (LE:
r ¼ 0.91, SG: r ¼ 0.94, BG: r ¼ 0.96, each being P < 0.0001) and
a positive correlation with Bax (LE: r ¼ 0.79, P < 0.01; SG: r ¼ 0.86,
P < 0.001; BG: r ¼ 0.60, P < 0.05), indicating that expression levels
of Bcl-2 and Bax were regulated by cyclic changes in P4.
3.1.3. Relationships between proliferation, apoptosis, Bcl-2 and Bax
In SG, correlations were found between Ki-67 and Bcl-2 as well
as Bax (Bcl-2: r ¼ 0.75, Bax: r ¼ 0.72; each P < 0.01). Also, the
apoptosis rate (Caspase-3) correlated with Bax (r ¼ 0.62, P < 0.05).
The lowest Bcl-2/Bax-ratio was detected in BG during mLP (Fig. 3c),
which was correlated with the highest apoptosis rate determined
by the TUNEL method (Fig. 2c). Apoptosis rates in turn correlated
with Bcl-2 expression levels (r ¼ 0.68, P < 0.05).
3.2. Cystic endometrial hyperplasia (group 2) and pyometra (group
3)
3.2.1. Proliferation and apoptosis
Cystic endometrial hyperplasia was characterised by a reduction
in or complete loss of the cyclic patterns in the expression of
markers for proliferation and apoptosis. Compared with the healthy
endometrium, for CEH distinctive changes were observed in all
layers and various cycle stages in proliferation and apoptosis rates.
Ki-67 positive cells were completely missing in LE during eAE.
Furthermore, significantly lower numbers of positive cells were
found in lAE (Fig. 5a). In SG, no change in Ki-67 expression was seen
during mLP (healthy: 0.24 ± 0.11%, CEH: 0.31 ± 0.28%), whereas in
lLP mean Ki67 expression levels were higher (healthy: 0.69 ± 0.37%,
CEH: 3.24 ± 4.14%) (Fig. 5b). In BG, proliferation rates were higher
compared with healthy endometrium. This was observed in all
cycle stages. However, this effect was not significant, probably due
 N. Reusche et al. / Theriogenology 114 (2018) 14e24
Fig. 3. Expression of Bcl-2 and Bax in a) Lamina epithelialis, b) superficial glands, c)
basal glands and d) stroma during mid luteal phase (mLP), late luteal phase (lLP), early
anestrus (eAE) and late anestrus (lAE) in healthy endometrium. Different letters be-
tween cycle stages per apoptosis regulatory protein indicate significant differences
(P < 0.05).
to the high variation in expression levels in CEH (Fig. 5c). In the
stroma, where proliferation in the healthy endometrium was
relatively low, but elevated during eAE compared to mLP (P < 0.05)
(Fig. 2d), increased proliferative activity was also found during the
other three cycle stages (mLP: healthy 0.06 ± 0.06; CEH 0.30 ± 0.33;
lLP: healthy 0.19 ± 0.03, CEH 0.74 ± 0.50; lAE: healthy 0.13 ± 0.05,
CEH 0.21 ± 0.10; for lAE P < 0.05) (Fig. 5d).
As described above, apoptosis was detected more clearly (i.e.,
differences amongst locations and/or cycle stages) by the TUNEL
method. Similar differences between the healthy endometrium and
CEH were found by TUNEL and Caspase-3 stained tissue sections, as
indicated by their correlation (LE: r ¼ 0.49, P < 0.05; SG: r ¼ 0.66,
P < 0.001; BG: r ¼ 0.67, P < 0.001). Except for mLP, the percentage of
19
TUNEL-positive cells in LE was higher in CEH than in the healthy
endometrium. The difference was significant in the case of lLP,
where no apoptosis was detected under physiological conditions
(lLP: healthy 0.0, CEH 0.52 ± 0.58; P < 0.05) (Figs. 2a and 5a). In SG
the mean level of TUNEL-positive cells in CEH was lower in mLP
compared with the healthy endometrium (healthy 0.68 ± 0.03, CEH
0.38 ± 0.05; P < 0.05) (Figs. 2b and 5b). In eAE and lAE, no signifi-
cant changes were found. Apoptosis rates were constant during lLP
for both endometrial conditions. This was also the case for BG
during mLP, lLP and eAE. In lAE, however, a higher mean level was
detected in CEH (healthy 0.25 ± 0.09, CEH 1.36 ± 1.01; P < 0.05)
(Figs. 2c and 5c). In stroma, no differences in TUNEL positive cells
were found between healthy endometrium and CEH.
In pyometra, which was found in four bitches in lLP (group 3),
Ki-67 expression levels were higher in LE, SG and stroma than in
CEH (stroma P < 0.05) (Table 4). The apoptosis rate increased in
stroma compared to CEH (P < 0.05) (Table 4). This was also evident
in activated Caspase-3 (Frequency score: CEH 0.32 ± 0.3, PYO
0.79 ± 0.17; P < 0.05).
3.2.2. Bcl-2 and Bax
The expression of Bcl-2 in mLP was clearly stronger in CEH than
in the healthy endometrium in all endometrial layers. Differences
were only significant in SG and stroma (SG: healthy 4.2 ± 1.8, CEH
103.8 ± 2.1; stroma: healthy 0.9 ± 0.2, CEH 2.1 ± 0.2) (Fig. 3b,d,
Fig. 6b,d). In BG, Bcl-2 expression was lower in CEH during lLP, eAE
and lAE (eAE: healthy 206.2 ± 2.9, CEH 182 ± 6.2; P < 0.05) (Figs. 3c
and 6c). Bax expression in mLP was lower in LE and SG than in the
healthy endometrium (LE: healthy 241.4 ± 27.3, CEH 174.9 ± 55.8;
SG: healthy 252.0 ± 42.7, CEH 209.1 ± 39.0), whereas expression
levels were similar in the other cycle stages (Fig. 3a and b, Fig. 6a
and b). Higher Bax expression was detected in BG during mLP
(healthy 199.5 ± 6.2, CEH 242.2 ± 23.9; P ¼ 0.05) and eAE (healthy
178.6 ± 1.6, CEH 189.6 ± 5.7; P < 0.05) (Figs. 3c and 6c). In stroma,
no distinct changes in Bax expression were found (Figs. 3d and 6d).
In pyometra, Bcl-2 expression was significantly decreased in LE and
SG (P < 0.05) and a similar trend was seen in BG when compared
with CEH. In stroma, however, Bcl-2 expression exhibited similar
levels in CEH and pyometra (Table 4). Comparison of pyometra and
the healthy endometrium revealed a decreased expression level for
Bcl-2 in BG (healthy 202.7 ± 9.9, PYO 96.6 ± 22.8; P < 0.05) and an
increased expression level for Bax in stroma (healthy 1.0 ± 0.2, PYO
1.8 ± 0.5; P < 0.05).
In CEH, Bcl-2 expression and P4-secretion showed a correlation
in BG (r ¼ 0.42, P < 0.05). Correlations with Bax were reduced in
all layers (LE: r ¼ 0.39, SG: r ¼ 0.42, BG: r ¼ 0.41, each being
P < 0.05) compared to the conditions found in healthy
endometrium.
4. Discussion
The combined assessment of the time interval since the start of
the last heat, clinical signs of the external and internal genital tract
(appearance of vulva, vaginal mucosa, cells in vaginal smear) and
the peripheral P4-concentrations proved to be suitable for precisely
assigning the bitches to a particular estrous cycle stage. Mid luteal
phase is characterised by high P4-values, whereas late luteal phase
corresponding with luteal regression is indicated by decreasing P4-
concentrations [33,38,39]. Differenting the period of ovarian rest
between an early stage (eAE) coinciding with endometrial repara-
tion and a late stage (lAE) corresponding with an intact endome-
trium can be done by taking into account the time span from the
beginning of the last heat (for eAE four to five months, for lAE >5
months). Furthermore, here only basal cells are present in the
vaginal smear and a basal P4-concentration is detected.
20 N. Reusche et al. / Theriogenology 114 (2018) 14e24

Fig. 4. Bcl-2 expression in late luteal phase, A - in superficial glands (SG), B - in basal glands (BG); Bax expression in superficial glands (SG), C - in mid luteal phase, D e in late luteal
phase. A: thin black arrow ¼ weakly stained positive cells. B, C, D: thick black arrow ¼ positive cells stained in moderate intensity. C: thick white arrow ¼ positive cells stained in
high intensity. B: Myo ¼ myometrium. C: LE ¼ Lamina epithelialis. A, D: inserts are showing negative controls.

In the canine endometrium, maximum proliferation related to
follicular phase and high estrogen secretion has been described
before [3,38,40]. In the present study, immunodetection of Ki-67 as
a marker for proliferative processes, revealed clear cyclic changes in
different layers of the secreting (mLP, lLP), regenerating (eAE) and
completely repaired endometrium (lAE). High P4-levels have been
described as having a negative effect on proliferation in all endo-
metrial layers during mLP [3,6]. In addition, we found that the
subsequent decrease in P4-levels in lLP was accompanied by a
gradual increase in proliferation in SG and stroma. In eAE, prolif-
eration remained at this level in SG, which was maintained during
lAE. The increase in proliferation in LE during lAE which has been
described before [5] was in agreement with other findings. This was
probably due to oscillating basal E2-levels related to concomitant
changes in follicular activity [33,41e43] as well as expression of
estrogen receptors verified in the surface epithelium of the canine
endometrium [44]. High estrogen receptor expression was found
for the luminal epithelium and superficial gland epithelium in eAE
and lAE [45]. An increase in the expression of estrogen receptors in
stroma during lLP and anestrus has been reported [44e46] and may
contribute to the increasing proliferation during eAE.
In the human endometrium, which undergoes more pro-
nounced destruction during menstruation [47], proliferation in the
secretory endometrium is restricted to the stroma [48].
Tissue recombination studies have demonstrated the impor-
tance of the endometrial stroma with respect to regenerative pro-
cesses in the superficial layers. These studies indicated that the
proliferative effects of estrogens on endometrial epithelial cells are
mediated indirectly via interaction with the stromal cell estrogen
receptor a and downstream production of growth factors (such as
EGF, IGF-1) that mediate epithelial proliferation [49e51].
The rate of apoptotic cells as revealed by immunodetection of
activated Caspase-3 and by the TUNEL-method in our studies
showed similar cyclic patterns in LE and BG. Others found Caspase-
3 to be the more sensitive of the two parameters [2], while in the
present study TUNEL appeared to give better insights into apoptotic
processes. The increased appearance of apoptotic processes in BG
during mLP and the subsequent decrease in lLP are indicative of a
causal connection between apoptosis and P4-levels. This positive
correlation has been reported by other authors before [1,4,52,53].
The reincrease in apoptotic cells in BG which occurred irrespective
of elevation in P4-concentrations in eAE can be explained by higher
expression levels of the P4-receptor in eAE compared to lAE with
similar P4-concentrations [45]. Also, the decreased P4-receptor
expression in BG from mLP to lLP [54] may play a role in the abrupt
drop in apoptosis during luteal regression. The relatively constant
low level of apoptotic activity in stroma may be caused by the cyclic
expression of the P4-receptor which was found to be low during
high luteal activity and increased during luteal regression and eAE
[45]. The simultaneous increase in apoptotic cells in BG during eAE
and increase in proliferation in stroma coinciding with down-
regulated apoptotic activity indicate that the stroma serves as a cell
source for endometrial regeneration.
In the human endometrium, the dense stroma surrounding the
bases of endometrial glands has been shown to permanently
generate cells for forming a new functional layer every month
 N. Reusche et al. / Theriogenology 114 (2018) 14e24 21

Table 4
Ki-67- and TUNEL-positive cells and expression of Bcl-2 and Bax in cystic endo-
metrial hyperplasia (CEH) (n ¼ 10) and pyometra (PYO) (n ¼ 4) in lamina epithelialis
(LE), superficial glands (SG), basal glands (BG), and stroma during late luteal phase.

Parameter Ki-67 TUNEL Bcl-2 Bax

Endometrial layer mean ±SD mean ±SD mean ±SD mean ±SD

LE % H-score
CEH 2.67 4.55 0.52 0.58 58.9a 34.4 178.4 27.7
PYO 5.14 3.43 0.94 0.70 8.3b 14.9 146.0 18.4
SG % H-score
CEH 3.24 4.14 0.43 0.39 98.0a 40.4 199.7 19.4
PYO 9.00 9.35 0.39 0.09 18.2b 29.4 186.6 11.6
BG % H-score
CEH 5.95 11.35 0.77 0.95 142.0 77.4 192.4a 27.1
PYO 2.07 2.02 0.69 0.31 96.6 22.8 241.6b 53.0
Stroma Frequency score Intensity score
CEH 0.74a 0.50 0.29a 0.21 1.4 0.6 1.3 0.4
PYO 1.94b 0.67 0.65b 0.16 1.6 0.3 1.8 0.5

Different letters between CEH and PYO within parameters per layer indicate sig-
nificant differences P < 0.05.

period of ovarian rest coinciding with a completely repaired

Fig. 5. Proliferation (Ki-67) and apoptosis (TUNEL) cells in a) Lamina epithelialis, b)
superficial glands, c) basal glands and d) stroma during mid luteal (mLP, n ¼ 2), late
luteal phase (lLP, n ¼ 10), early anestrus (eAE, n ¼ 2) and late anestrus (lAE, n ¼ 3) in
cystic endometrial hyperplasia.
[47,55].
For dogs, endometrial desquamation following luteal regression
as part of endometrial regeneration has been described by various
authors [1,5,33,34,56]. However, this has been questioned by Van
Cruchten et al. [4] because of the low apoptosis rates in LE. With
regard to this it should be noted that the apoptotic phase is short
and that apoptotic bodies can be eliminated from the glandular and
mucosal epithelium via the uterine lumen [57e59].
When comparing endometrial regeneration in humans and dogs
it should be noted that the duration of the luteal phase lasts 12e14
days in women [60] and about two months in dogs [33,39].
Moreover, endometrial repair takes five to six days in women
[61,62] and two to 2.5 months in dogs [33,34]. Furthermore, the
endometrium has a duration of about one to two days in women
[60] and takes at least one month in dogs [39].
In our study we found a positive correlation of the anti-
apoptotic Bcl-2 with proliferation in SG and a negative correlation
with apoptosis in BG and with P4-concentrations in the upper three
layers of the endometrium. In contrast, expression of the pro-
apoptotic Bax was negatively correlated with Ki-67 in SG and
positively correlated with P4-levels in LE, SG and BG. The positive
correlation between Bax and apoptosis found in SG (Caspase-3)
also indicates the role of Bax in the regulation of endometrial
remodelling. The opposite effects and interplay of Bcl-2 and Bax
were also evident in mLP. In the presence of high P4-concentrations,
Bcl-2 expression was missing (LE) or weak (SG, BG), whereas Bax
expression was maximal. In BG, the lowest Bcl-2/Bax ratio and
highest apoptosis rate were foundas previously mentioned [52].
Similar cyclic patterns of Bcl-2 and Bax were documented for the
endometrium of cats [14], marmosets [18] and humans
[15e18,20,63e67].
In the human endometrium the decrease of Bcl-2 and Ki-67
levels in stroma coinciding with the increase and effect of P4 in
the secretory endometrial phase is described as functioning as a
trigger for disintegration and menstruation [63]. The persistent Bcl-
2 expression found in stromal cells of the human endometrium is
thought to account for the privilege of escaping from apoptosis-
inducing signals allowing reconstruction of the functional endo-
metrium after menstruation [63]. Our observations on increased
Bcl-2 expression during luteal regression and maintenance of
constant levels in the following estrous cycle stages may indicate
similar mechanisms. This is supported by the gradual shift from
pro-apoptotic to anti-apoptotic markers during lLP, eAE and lAE in
all layers of the canine endometrium, as well as their pronounced
presence from LE to stroma and the opposing patterns of Bcl-2 and
Bax.
With respect to the increased apoptotic rates in eAE coinciding
with high Bcl-2 expression it should be noted that irrespective of
the increase in P4-receptor expression [45] probably factors other
than P4 are involved. These include, for example, the second,
receptor-mediated pathway via the Fas-receptor and Fas-ligand
which has been described as playing a role in the canine endo-
metrium around implantation [68].
In CEH and pyometra, cyclic patterns as found in the healthy
endometrium were lost. Despite high standard deviations in CEH
(group 2) which may have been caused at least partly by the
22 N. Reusche et al. / Theriogenology 114 (2018) 14e24
Fig. 6. Expression of Bcl-2 and Bax in a) Lamina epithelialis, b) superficial glands, c)
basal glands and d) stroma during mid luteal (mLP, n ¼ 2), late luteal phase (lLP,
n ¼ 10), early anestrus (eAE, n ¼ 2) and late anestrus (lAE, n ¼ 3) in cystic endometrial
hyperplasia.
variation in CEH degree (mild, moderate, severe), the mean Ki-67
expression was higher in BG and stroma during the P4-dominated
phases, leading to non-physiological proliferation and apoptosis
patterns. The dysregulation of proliferation in CEH is also evident in
the missing proliferation in LE during eAE, higher proliferation
rates in BG during eAE and lAE and in stroma during lAE. The CEH-
related changes in endometrial proliferation are explained in part
by ovarian dysfunction, including prolonged follicular maturation
or delayed ovulation, resulting in an abnormal estrogen/proges-
terone ratio. Also, an abnormal expression of estrogen receptors
may be involved in this process [69,70]. Compared with the healthy
endometrium, estrogen receptor expression was found to be lower
in CEH in the luminal epithelium and higher in BG, reaching the
maximum in lLP [69]. Glands with cystic degeneration showed less
pronounced estrogen receptor expression. In the stroma, a higher
expression was found during the late secretory phase and anestrus,
whereas expression was lower during the early secretory phase
[69].
The permanent accumulation of cells in the deeper layers of the
endometrium may contribute to the formation of CEH, where the
endometrium is thickened and cystic hyperplasia of uterine glands
exists. In women, increased proliferation rates in relation to
endometrial hyperplasia have been previously described
[16,17,22,24,71].
In mLP in the presence of high P4 levels, apoptosis rates were
absent in LE and higher in lLP, eAE, and lAE as compared to the
healthy endometrium. Thus, in this case, proliferative and
apoptotic processes were concomitantly decreased in mLP and
increased in lLP, indicating abnormal conditions. Also, in SG a
lower apoptosis rate in mLP and higher rates in later stages were
found. The higher apoptotic activity in BG of CEH during lAE may
be a reaction to the permanently increased rate of proliferation. All
these differences in cyclic proliferation and apoptosis patterns
between the healthy endometrium and CEH indicate a loss in the
controlled endometrial regeneration in CEH. An abnormal
apoptosis/proliferation ratio is also reported for the hyperplastic
human endometrium [22,24].
The higher Bcl-2 expression during mLP in CEH illustrates its
role in the decreased apoptosis rates. Dysregulation of apoptosis in
CEH is also evident in the simultaneous decrease in Bcl-2 and in-
crease in Bax expression levels in BG during eAE compared with the
pattern observed in the healthy endometrium. In comparison to the
hyperplastic endometrium, pyometra (group 3) was characterised
by higher rates of both apoptosis and proliferation in stroma. The
increase in Bax expression in BG and the concomitant decrease in
the expression of the anti-apoptotic Bcl-2 in LE, SG and BG indicates
a severe dysregulation of proliferative and apoptotic processes in
pyometra. In stroma of pyometra almost no anti-apoptotic regula-
tion took place, whereas in the healthy endometrium increased Bax
expression can be identified as regulating induction of apoptosis.
Altogether, one reason responsible for the development of pyo-
metra is an uncontrolled increase in apoptosis in BG and stroma.
5. Conclusion
In the present study it was demonstrated that the apoptosis-
regulatory proteins Bcl-2 and Bax are involved in homeostasis of
proliferation and apoptosis in the healthy canine endometrium. In
early anestrus, the period following luteal regression, apoptosis is a
progesterone-independent mechanism leading to morphological
and functional changes in the canine endometrium. Furthermore,
as found for the human endometrium, the stroma seems to serve as
a source of cells needed for endometrial regeneration. As previously
commented on by other authors, these findings reiterate the ne-
cessity to clinically consider eAE as a separate stage of endometrial
tissue remodelling and to define it during the first two months after
complete luteal regression.
CEH is accompanied by severe changes in proliferation and
apoptosis, coinciding with a dysregulation in the expression of Bcl-
2 and Bax and loss of cyclic patterns. The alterations result in
excessive proliferation combined with deregulation of apoptosis,
which in turn results in insufficient endometrial regeneration as is
described for human endometrial hyperplasia. Besides the glan-
dular compartments, the endometrial stroma seems to be espe-
cially involved in the pathogenesis of canine CEH. The abnormally
increased proliferation and the concurrent intensification of
apoptotic activity in this layer observed in pyometra indicate severe
and irreversible damage of the inflamed canine endometrium.
 N. Reusche et al. / Theriogenology 114 (2018) 14e24
Conflicts of interest
None.
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