Domestic Animal Endocrinology 70 (2020) 106382

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Domestic Animal Endocrinology

journal homepage: www.journals.elsevier.com/
domestic-animal-endocrinology

Deficiency in proliferative, angiogenic, and LH receptors in

the follicle wall: implications of season toward the
anovulatory condition
G.M. Ishak a, b, G.A. Dutra a, G.D.A. Gastal a, M.E. Elcombe a, M.O. Gastal a,
S.B. Park c, J.M. Feugang c, E.L. Gastal a, *
a
Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, USA
b
Department of Surgery and Obstetrics, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq
c
Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS, USA

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

Article history: This study aimed to gain insight on the effect of different seasons of the year on the
Received 17 February 2019 expression pattern of growth factor and hormone receptors involved in follicle develop-
Received in revised form 12 June 2019 ment. A novel follicle wall biopsy technique was used to collect in vivo follicle wall layers
Accepted 24 July 2019
(ie, granulosa, theca interna, and theca externa) and follicular fluid samples from growing
dominant follicles, simultaneously and repeatedly, using the same mares during the spring
Keywords:
anovulatory (SAN), spring ovulatory (SOV), summer (SU), and fall ovulatory (FOV) seasons.
Follicle wall biopsy
The immunofluorescent expression patterns of epidermal growth factor receptor (EGFR),
Receptors
Season Ki-67, vascular endothelial growth factor receptor (VEGFR), and LH receptor (LHR) were
Ovary evaluated in each follicle wall layer, in addition to intrafollicular estradiol and nitric oxide
Horse (NO). Proliferative proteins (EGFR and Ki-67) were highly (P < 0.05–P < 0.001) expressed
during the SOV season compared with the SAN and FOV seasons. Lower (P < 0.05–P <
0.001) expression of both proteins was observed during SU compared with the SOV season.
The expression of VEGFR was greater (P < 0.05–P < 0.01) in the theca interna of dominant
follicles during the SOV season compared with the SAN and SU seasons. Similarly, in the
overall quantification, the VEGFR expression was greater (P < 0.001) during the SOV season
compared with the SU and FOV seasons. A higher (P < 0.05) LHR expression was detected
in the theca interna during the SOV season than the SAN season. Furthermore, a higher
(P < 0.05–P < 0.001) expression of LHR was observed in the granulosa, theca interna, and
in the overall quantification during the SOV season compared with the SU and FOV sea-
sons. Intrafollicular NO concentration did not differ (P > 0.05) among different seasons of
the year. The intrafollicular estradiol concentration was higher (P < 0.05) during the SU
compared with the SAN season and higher (P < 0.05) during the FOV season compared
with the SAN and SOV seasons. In conclusion, the synergistic effect of lower expression of
proliferative protein, angiogenic, and LH receptors in at least some of the layers of the
follicle wall seems to trigger dominant follicles toward the anovulation process during the
spring and fall transitional seasons.
Ó 2019 Elsevier Inc. All rights reserved.

1. Introduction

Unlike in other monovular species (eg, cows and

* Corresponding author. Tel.: þ1 618 453 1774; fax: þ1 618 453 5231. humans), the development and continuous growth of
E-mail address: egastal@siu.edu (E.L. Gastal). dominant follicles to preovulatory (POF) stage in mares are

0739-7240/$ – see front matter Ó 2019 Elsevier Inc. All rights reserved.
https://doi.org/10.1016/j.domaniend.2019.07.010
2 G.M. Ishak et al. / Domestic Animal Endocrinology 70 (2020) 106382
influenced by seasonal variations. Detailed studies have
been conducted in the past two decades in the mare to
understand mechanisms controlling reproductive season-
ality in relation to follicle dynamics and ovulatory compe-
tence [1–17]. However, in vivo studies using the same
individuals are needed to elucidate the expression pattern
of different hormone and growth factor receptors and
proteins in antral follicle wall layers (ie, granulosa, theca
interna, and theca externa), and their relationships with
intrafollicular fluid (FF) milieu during different seasons of
the year.
Regarding seasonal effects, antral follicular dynamics
are characterized by substantial variations not only be-
tween the ovulatory and anovulatory seasons but also
within different periods in the ovulatory season [5,7,14,16–
20]. In this regard, the use of color-Doppler ultrasonogra-
phy has shown that the first preovulatory period of the
reproductive season has less POF wall blood flow compared
with later ovulations during the summer (SU) [20]. Simi-
larly, it was reported in a recent study [17] that the POF has
less blood flow during the early ovulatory season (spring)
when compared with the late ovulatory period (fall
ovulatory [FOV] season). Furthermore, several reports have
shown that the POF diameter is regulated seasonally. In this
regard, POFs at the beginning of the ovulatory season are
larger in diameter compared with those of later periods
[16,17,19,20]. Moreover, the diameter of the POF preceding
the first ovulation of the season is greater than during the
second [13,20] and last [17] estrous cycles of the breeding
season.
More recently, with frequent use of color-Doppler ul-
trasonography, significant correlations have been found
between the vascularity and diameter of POFs. In this re-
gard, higher POF vascularization was positively correlated
with ovulation rate and subsequent corpus luteum (CL)
functionality in cows [21], with progesterone production
[22], and with pregnancy rates in mares [23] and heifers
[24]. Moreover, in mares, the capacity for ovulation during
the transition to the ovulatory season was closely related to
the vascularity of dominant follicles. For example, domi-
nant follicles during the spring ovulatory (SOV) season have
higher blood flow [9] and express greater amounts of
angiogenic factors [5] compared with dominant transi-
tional anovulatory follicles. Furthermore, several reports
indicated that a relationship exists between follicle diam-
eter and fertility in several species; a recent study indicated
that the size of POFs affects the diameter of subsequent CLs
in cows [21]. In addition, large follicles have produced
greater oocyte maturation rates compared with small fol-
licles [25]. Moreover, lower pregnancy rates and plasma
progesterone concentration were reported in sheep [26]
and cows [27,28] when small follicles were induced to
ovulate compared with larger follicles. Therefore, under-
standing the mechanisms controlling follicular vasculari-
zation and development in mares during different seasons
of the year has practical importance to the equine industry
to select optimum follicles for breeding. In addition,
studying the follicular environment during the final stages
of development provides more in-depth knowledge to
evaluate developmental competence of POFs in relation to
oocyte quality and the ovulation process.
The recent advent of the follicle wall biopsy (FWB)
technique in equine studies [29] provides a new approach
to study ovarian function at the cellular and molecular
levels within the same mares without jeopardizing ovarian
function. Therefore, the aims of this study were to use the
FWB technique to elucidate and compare expression pat-
terns of different receptors and proteins (epidermal
growth factor receptor [EGFR]), Ki-67, LH receptor [LHR],
vascular endothelial growth factor receptor [VEGFR], Bax,
and Bcl-2) and intrafollicular estradiol concentration in the
follicle wall of dominant follicles (30–34 mm) among
different seasons of the year (spring anovulatory [SAN],
SOV, SU, and FOV).
2. Materials and methods
2.1. Animals
Nine Quarter Horse mares (8–14 y old, weighing 400–
550 kg) were used in the present study to collect in vivo
FWB and FF samples during the SAN, SOV, SU, and FOV
seasons. The same mares were used in all seasons. How-
ever, 3 more mares were added to the FOV group to obtain
an additional number of samples in case some mares
entered the anestrous phase during or after sample
collection. Furthermore, all animals from the FOV group
were monitored by ultrasound after harvesting the FWB
and FF samples to confirm a subsequent ovulation, thus
ensuring that all samples were collected during the FOV
season. Some data evaluated in the present study were
collected as part of a previous study [29]. In the original
study, samples collected in vivo were not evaluated for any
seasonal influence. Herein, the effect of different seasons
(ie, SAN, SOV, SU, and FOV) on receptor and protein
expression was compared using growing dominant 30- to
34-mm follicles only. Therefore, from the original publica-
tion, only data (LHR and FF estradiol) of the largest follicle
category during the spring seasons were used. In addition,
in the present study, several unique potential markers
(EGFR, Ki-67, VEGFR, Bax, and Bcl-2) and FF concentration
of nitric oxide (NO) were evaluated. For the SU and FOV
seasons, data were obtained in a different study during the
same year and from the same mares in the respective
seasons of the year. The study was carried out in the
northern hemisphere (latitude, 37.7 N). The research pro-
tocol was approved by the Institutional Animal Care and
Use Committee of Southern Illinois University.
2.2. Ultrasonography, follicle tracking, and groups
Samples of FWB and FF were collected from dominant
growing follicles during March (SAN), May (SOV), July (SU),
and September (FOV). During the SAN season, transvaginal
ultrasound-guided ablations of all ovarian follicles 6 mm
were performed to induce a new follicular wave as
described previously [30]. However, during the SOV, SU,
and FOV seasons, follicle ablations were performed 10–11 d
after the ovulation of the previous cycle. Moreover, mares
were allowed to have at least one interovulatory interval
between seasons before being subjected to sample collec-
tion in the subsequent season.
 G.M. Ishak et al. / Domestic Animal Endocrinology 70 (2020) 106382 3

In all seasons, after induction of the new follicular wave,
a duplex color-Doppler ultrasound machine (Aloka SSD-
3500; Hitachi Aloka Medical America, Inc., Wallingford,
CT) equipped with a finger-mounted 3.5–10 MHz convex
array transducer (UST-995-7.5) was used for daily follicle
tracking until a growing follicle reached 30–34 mm in
diameter. The fate of the biopsied follicles (eg, regression,
luteinization, hemorrhagic anovulatory follicle [HAF], or
ovulation) was monitored by ultrasound after each biopsy
procedure for all seasons.
the best sections, with all follicle wall layers to be used for
the immunohistochemistry analyses (Figs. 1 and 2).
After harvesting the FWB samples, the tip of the 12 G
needle was kept inside the antrum of the follicle, and
leaking FF was collected directly into a Petri dish. Imme-
diately after collection, FF samples were centrifuged
(1,500 g for 10 min, at 4 C) and supernatants were stored at
80 C until analyses.
2.4. Immunohistochemistry

2.3. Follicle wall biopsy procedure and FF sampling
Antral follicle wall and FF samples were obtained in vivo
using an endoscopic biopsy forceps (5 FR gauge, 60 cm,
MPN# 27425Z, Karl Storz, Berlin, Germany) enclosed by a
12 G needle/cannula as described previously [29]. Briefly,
the biopsy forceps covered by a 12 G needle/cannula was
introduced into a needle guide mounted on a 5–10 MHz
transvaginal ultrasound-guided convex array transducer
(Aloka UST-987-7.5). As soon as the targeted follicle was
presented on the ultrasound screen, the 12 G needle was
introduced into the antrum of the follicle. Thereafter, the
biopsy forceps were propelled forward into the antrum of
the follicle by a second operator, and a FWB sample was
harvested from the follicle wall. Samples of the FWB were
fixed in 10% formalin solution for 4 h. Afterward, fixed
samples were preserved in 70% ethanol at 4 C for further
classical histological processing. Sections of the follicle
walls (5 mm) were analyzed using a light microscope (Nikon
E200, Tokyo, Japan) at 20 objective magnification to select
An immunofluorescence approach was used to evaluate
the expression levels of EGFR, Ki-67, LHR, VEGFR, Bax, and
Bcl-2 in each layer (ie, granulosa, theca interna, and theca
externa) of the follicle wall as previously described [29,31].
Briefly, slides were deparaffinized and subjected to antigen
retrieval for 30 min in a steamer using sodium citrate
buffer; sections were permeabilized using 1% Triton X-100
for 30 min, and nonspecific antibody binding sites were
blocked by 1% BSA solution for 60 min. Afterward, sections
of the follicle wall were incubated with respective primary
antibodies diluted (1:100) for 60 min (EGFR: sc-03-G, and
Bcl-2: sc-492, Santa Cruz Biotechnology, Dallas, TX; Ki-67:
PA5-19462, Thermo Fisher Scientific Inc, Waltham, MA;
LHR: LS-C312710, LifeSpan BioSciences, Seattle, WA;
VEGFR: ab15291, Abcam, Cambridge, MA; and Bax:
orb312174, Biorbyt, Berkeley, CA). Then, sections were
incubated in the dark with FITC-labeled secondary anti-
body (goat anti-rabbit conjugated; dilution, 1:200) for
60 min. Follicle wall sections were washed 3 times with PBS
between all steps, and all procedures were performed at

Fig. 1. Representative histological sections of follicle walls of growing dominant follicles during the (A) spring anovulatory season (SAN), (B) spring ovulatory
season (SOV), (C) summer (SU), and (D) fall ovulatory season (FOV). Granulosa, theca interna, and theca externa layers can be seen in the follicle wall samples.
Slides were stained with Periodic Acid-Schiff (PAS) and counterstained with hematoxylin. g, granulosa; ti, theca interna; and te, theca externa. Bar ¼ 100 mm.
4 G.M. Ishak et al. / Domestic Animal Endocrinology 70 (2020) 106382

Fig. 2. Representative micrographs of immunofluorescence protein detection in the follicle wall. (A) Negative control; (B) nuclei counterstained blue with DAPI;
(C) Epidermal growth factor receptor labeled green with FITC-conjugated secondary antibody; and (D) merged images. Granulosa, theca interna, and theca
externa layers can be seen in the follicle wall samples. g, granulosa; ti, theca interna; and te, theca externa. Bar ¼ 100 mm. (For interpretation of the references to
color in this figure legend, the reader is referred to the Web version of this article.)

room temperature. Sections were immediately counter-
stained with a DAPI-contained mounting medium, and
fluorescence evaluation was undertaken using a fluores-
cent microscope (EVOS FL Cell Imaging System, Thermo
Fisher Scientific Inc, Waltham, MA). Images were obtained
in 10 objective magnification and analyzed using ImageJ
software as described previously [29]. Negative control
sections were incubated with PBS without the primary
antibodies. All primary antibodies used in the present study
were successfully used and validated by the manufacturer
and previous studies with equine tissues [29,32–34].
2.5. Hormone assays
The estradiol concentrations in FF were determined
with a commercial ELISA kit (ERK E3009, Endocrine Tech-
nologies, Inc, Freemont, CA). The sensitivity of the assay
was 10 pg/mL, and the intra-assay CV was 9.0%. Samples
were diluted 1:3000 in the assay buffer.
Total NO concentrations were determined using a
colorimetric kit (EMSNOTOT; Thermo Fisher Scientific,
Vienna, Austria). The intra-assay CV was 4.1%, whereas the
sensitivity of the assay was 0.625 mM/mL. Samples were
diluted 1:2 in assay buffer.
2.6. Statistical analyses
All statistical analyses were performed using JMP soft-
ware version 13.0 (SAS Institute Inc., Cary, NC). A Shapiro-
Wilk test was used to verify the normal distribution of the
data set, and data for end points that were not normally
distributed were transformed to either rank or log. Outliers
were detected using Dixon's test and were excluded from
the data set before any statistical analysis. Data were
analyzed using one-way ANOVA. When a significant dif-
ference was identified, the difference was further analyzed
using Tukey's test. A student's t test was used when only 2
groups were compared. Data were expressed as mean
 SEM unless otherwise indicated. A probability of P < 0.05
indicated that a difference was significant, and P > 0.05
and 0.1 indicated that a difference approached
significance.
3. Results
3.1. Follicle wall biopsy and follicular fluid samples
A total of 39 FWB and FF samples were collected from
dominant growing follicles during the SAN (n ¼ 9), SOV (n ¼
9), SU (n ¼ 9), and FOV (n ¼ 12) seasons. Ultrasound
monitoring of the fate of biopsied follicles during the SAN
season showed that all follicles started to regress the day
after the FWB procedure. However, during the SOV and SU
seasons, all follicles were transformed to luteal tissue
within 3.7  0.7 and 3.6  0.6 d, respectively; in both sea-
sons, the luteal tissue regressed within 9.0  0.9 d after the
FWB procedure. During the FOV season, 53.8% of the bio-
psied follicles were transformed to luteal tissue within 3.5
 0.3 d, 38.5% were transformed to HAF within 3.5  0.3 d,
and 7.6% ovulated 3 d after the FWB procedure.
 G.M. Ishak et al. / Domestic Animal Endocrinology 70 (2020) 106382 5

3.2. Immunohistochemistry
3.2.1. Epidermal growth factor receptor
Expression of EGFR was greater (P < 0.05) in the gran-
ulosa layer of dominant follicles during the SOV season
compared with the FOV season (Fig. 3). Furthermore, the
EGFR expression during the SAN and SOV seasons tended (P
< 0.058–P < 0.067) to be higher compared with the FOV
and SU seasons, respectively. The theca interna layer in the
SOV season had a higher (P < 0.05) expression of EGFR than
in the other seasons. Moreover, in the theca externa layer,
although no difference (P > 0.05) was observed among
SAN, SOV, and SU seasons, SAN and SOV seasons had a
greater expression (P < 0.01) of EGFR than the FOV season.
In addition, when considering the overall quantification of
EGFR in all follicle wall layers, a higher (P < 0.05) expres-
sion was observed in follicles of the SOV season compared
with the other seasons. During the SAN season, follicles had
a higher (P < 0.05–P < 0.001) expression of EGFR than
follicles of the SU and FOV seasons.

4 Granulosa 8
ab bc
a
3 6
b
#
2 4
# #
ac
1 2
ab
b a
0 0

8 Theca Interna 8
b b

6 6

a # ab
4 4
a a
2 a a
EGFR relative expression

Ki-67 relative expression

0 0

3.5 Theca Externa 4

3.0 a
a ab 3
2.5 # #

2.0
2
1.5
1.0 1
0.5 b
0.0 0

18 Overall 10
b
16 b
14 8
12
a 6
10 a
8 a
4
6
4 c 2
c c
2
0 0
SAN SOV SU FOV SAN SOV SU FOV
Seasons Seasons

Fig. 3. Mean (SEM) immunofluorescence relative expression of EGFR and Ki-67 in the granulosa, theca interna, theca externa, and in all layers of the follicle wall
(overall evaluation) during the spring transitional anovulatory (SAN), spring ovulatory (SOV), summer (SU), and fall ovulatory (FOV) seasons. a,b,cBars with
different superscripts within an end point are different (P < 0.05). #Indicates a tendency for statistical difference between groups. EGFR, epidermal growth factor
receptor.
6 G.M. Ishak et al. / Domestic Animal Endocrinology 70 (2020) 106382

3.2.2. Ki-67
The granulosa layer of dominant follicles during the SOV
season had greater (P < 0.05–P < 0.01) fluorescence in-
tensity of Ki-67 compared with the SAN and SU seasons
(Fig. 3). In addition, during the FOV season, the expression
of Ki-67 was higher (P < 0.05) than in the SU season.
Similarly, greater (P < 0.05) Ki-67 expression was detected
in the theca interna layer during the SOV season compared
with the SAN and SU seasons. Furthermore, the theca
externa layer tended (P < 0.06) to express higher Ki-67
during the SOV and FOV seasons compared with the SU
season. The overall quantification of Ki-67 expression in all
follicle wall layers was greater (P < 0.05–P < 0.0001) in
dominant follicles of the SOV season than in those of all
other seasons. Moreover, dominant follicles of both the SAN
and FOV seasons had a higher (P < 0.05–P < 0.01)
expression of Ki-67 than those of the SU season.
3.2.3. Vascular endothelial growth factor receptor
The expression level of VEGFR in the granulosa and
theca externa layers was not different (P > 0.05) among
seasons (Fig. 4). However, the VEGFR expression was
greater (P < 0.05–P < 0.01) in the theca interna layer of
dominant follicles during the SOV season compared with

0.5 Granulosa 2.5

a
0.4 2.0
a
0.3 1.5

0.2 1.0

0.1 b 0.5
c
0.0 0.0

6 Theca Interna 10
b
b
5 8
4
6
3 c bc #
a 4
2
VEGFR relative expression

ac

LHR relative expression

1 c 2
a
0 0

1.4 Theca Externa 3.0

a
1.2 ab 2.5
1.0 2.0
0.8
1.5
0.6
bc 1.0
0.4
0.2 c 0.5
0.0 0.0

6 Overall 10
a
# a
5 a 8
4 ab
6
3
4
2 b b

1 2
b b
0 0
SAN SOV SU FOV SAN SOV SU FOV
Seasons Seasons

Fig. 4. Mean (SEM) immunofluorescence relative expression of VEGFR and LHR in the granulosa, theca interna, theca externa, and in all layers of the follicle wall
(overall evaluation) during the spring transitional anovulatory (SAN), spring ovulatory (SOV), summer (SU), and fall ovulatory (FOV) seasons. a,b,cBars with
different superscripts within an end point are different (P < 0.05). #Indicates a tendency for statistical difference between groups. VEGFR, vascular endothelial
growth factor receptor; LHR, luteinizing hormone receptor.
 G.M. Ishak et al. / Domestic Animal Endocrinology 70 (2020) 106382 7

the SAN and SU seasons. Furthermore, greater (P < 0.01) 25

expression of VEGFR was observed during the SU and FOV
seasons than the SAN season. The overall quantification of
a
VEGFR in all follicle wall layers showed higher (P < 0.001) 20

Bax/Bcl-2 relative expression

expression during the SOV season compared with the SU
and FOV seasons. In addition, the expression of VEGFR
tended (P < 0.06) to be greater during the SOV than the SAN 15
season.

3.2.4. Luteinizing hormone receptor 10

In the granulosa layer, the LHR expression was greater (P
< 0.05–P < 0.01) during the SAN and SOV seasons compared
with SU and FOV seasons (Fig. 4); no difference (P > 0.05) 5 b
was observed between the SAN and SOV seasons. Further-
more, dominant follicles of the SU season had greater (P <
0.05) LHR expression than those of the FOV season. In the 0
theca interna layer, dominant follicles had higher (P < 0.05– Anovulatory Ovulatory
Seasons
P < 0.01) LHR expression during the SOV season compared
with other seasons. In addition, the expression of LHR Fig. 5. Mean (SEM) immunofluorescence relative expression of the Bax/
during the SAN season was greater (P < 0.05) and tended (P Bcl-2 ratio in the granulosa layer during the anovulatory season (SAN) vs
< 0.08) to be greater than in the SU and FOV seasons, ovulatory combined (SOV þ SU þ FOV) seasons. a,bIndicates difference be-
respectively. Expression of LHR in the theca externa layer tween groups (P < 0.05).

was higher during the SAN season (P < 0.05) compared with
the SU and FOV seasons. In addition, follicles of the SOV 4. Discussion
season had a greater (P < 0.05) expression of LHR compared
with the FOV season. In the overall quantification, the LHR The present study compares the expression pattern of
expression in all follicle wall layers was greater (P < 0.05–P receptors and proteins related to cell proliferation (EGFR, Ki-
< 0.01) in both SAN and SOV seasons than in the SU and FOV 67), angiogenesis (VEGFR), endocrine function
seasons; no other differences were observed. (LHR), apoptosis (Bax/Bcl-2 ratio), and intrafollicular

3.2.5. Bax/Bcl-2 ratio

No differences were observed in the Bax/Bcl-2 ratio Estradiol
among different seasons in the granulosa and theca interna 3500 A c
B
layers, and the whole follicle wall. However, the Bax/Bcl-2
Concentration (ng/mL)

3000
ratio in theca externa layer was greater (P < 0.05) during bc
2500 b
the FOV season when compared with the SAN season (data #
not shown). Furthermore, when combined data of domi- 2000
nant follicles from ovulatory seasons (SOV, SU, FOV) were 1500
ab
compared with the anovulatory season (SAN) counterpart, 1000 a a
a higher (P < 0.05) Bax/Bcl-2 ratio was observed in the 500
granulosa layer of dominant anovulatory follicles (Fig. 5); 0
however, no other differences (P > 0.05) were observed in
the theca interna and the whole follicle wall. Nitric Oxide
30
C D
Concentration (μM/mL)

3.3. Intrafollicular hormone concentration 25

Intrafollicular estradiol concentration was greater (P < 20

0.05) during SU and FOV seasons than during the SAN 15
season (Fig. 6A). Similarly, estradiol concentration was
10
higher (P < 0.05) during the FOV season and tended (P ¼
0.08) to be greater during SU compared with the SOV 5
season. No differences were observed between the SAN and 0
SOV seasons or between the SU and FOV seasons. On the SAN SOV SU FOV Anovulatory Ovulatory
Seasons (SAN) (combined)
other hand, when data of ovulatory seasons (SOV, SU, and
Seasons
FOV) were regrouped and compared with the anovulatory
season (SAN), higher (P < 0.05) intrafollicular estradiol Fig. 6. Mean (SEM) intrafollicular concentration of estradiol and nitric
concentration was observed in the ovulatory group oxide during the spring transitional anovulatory (SAN), spring ovulatory
compared with the anovulatory group (Fig. 6B). Follicular (SOV), summer (SU), and fall ovulatory (FOV) seasons. Comparison among
individual seasons (A and C), and anovulatory season (SAN) vs ovulatory
fluid concentrations of NO did not differ (P > 0.05) among combined (SOV þ SU þ FOV) seasons (B and D) are presented. a,b,cBars with
seasons (Fig. 6C) nor for anovulatory vs ovulatory seasons different superscripts within an end point are different (P < 0.05). #Indicates
combined (Fig. 6D). a tendency for statistical difference between groups.
8 G.M. Ishak et al. / Domestic Animal Endocrinology 70 (2020) 106382
concentration of estradiol and NO among dominant
growing follicles collected during different seasons of the
same year (ie, SAN, SOV, SU, and FOV). Antral follicle wall and
FF samples used in the present study were obtained in vivo
from the same mares using the recently developed tech-
nique of antral FWB [29]. The results presented herein will
be vital in the understanding and designing of future studies
to investigate seasonal effect on key proteins that are
essential for the growth of dominant follicles, acquisition of
ovulatory capacity, and oocyte developmental competence.
The findings may also contribute to improving in vitro
oocyte maturation success rates and developing appropriate
protocols for superovulatory treatment in mares. Further-
more, the application of the FWB technique in the present
study allowed us to use samples collected from the same
mares in all seasons and therefore minimized potential
confounding effects of age or breed of the animals [35–37].
In the present study, proliferative proteins (EGFR and Ki-
67) were differentially expressed in follicle wall layers
among different seasons. In this regard, both EGFR and Ki-67
were highly expressed during the SOV season compared
with the SAN and FOV seasons, whereas a lower expression
of both proteins was observed during SU compared with the
SOV season. Furthermore, EGFR expression during the SU
season was lower than in the FOV season. Epidermal growth
factor (EGF), in association with FSH, has been reported to be
involved in continued follicle development in mares [33,34],
cows [38], goats [39], and sows [40–42]. Moreover, EGF has
been shown to play an important role in oocyte maturation
in mares, cows, and sows [43–48]. In addition, EGF reduces
apoptosis in granulosa cells [49] and modulates steroido-
genesis [50]. Conversely, it has been reported that Ki-67
expression is important for the continued growth of the
dominant follicle and acquisition of ovulatory capacity
during transition to the ovulatory season [5]. In the present
study, the expression patterns of EGFR and Ki-67 are
consistent with previous ultrasound reports that showed a
larger POF diameter at the beginning of the ovulatory season
(spring) compared with later periods (SU and fall)
[16,17,19,20,51]. Moreover, our results showed a higher
EGFR expression during the FOV season compared with the
SU season. Similarly, this finding is in agreement with the
results of ultrasound studies that showed a larger POF
diameter during the fall compared with the SU season
[51,52]. In addition, a greater ovarian follicular activity
during the spring season has been supported by a higher
incidence of minor and secondary waves of antral follicles
compared with the fall season [53]. Therefore, based on our
findings, it appears that the differential expression of EGFR
and of Ki-67 is associated with differences in follicular ac-
tivity and maximum POF diameter previously reported
among seasons. In addition, previous studies have shown
that differences in follicular activity among different sea-
sons of the year were associated with gonadotropin con-
centrations [2,11,53]. Therefore, further studies should be
undertaken to address the contribution of multiple factors
that might be involved in impacting activity within and
among seasons and their consequent relationships with
oocyte quality.
Results of our study demonstrated that VEGFR expres-
sion was greater in the theca interna of dominant follicles
during the SOV season compared with the SAN and SU
seasons. Moreover, in the overall quantification, the
expression level of VEGFR was greater during the SOV
season compared with the SU and FOV seasons. Our results
of lower expression of VEGFR during the SAN season are in
agreement with previous findings reported for pony mares
during the same season [5]. In this context, it has previously
been reported that VEGFR expression plays a key role in
developing new blood vessels and increasing vascularity of
the developing follicles [54], a process that is vital in
maintaining follicle health and development through the
delivery of nutrients, growth factors, oxygen, and gonado-
tropins to the follicle [54–57]. In addition, it has been re-
ported that dominant follicles are more vascularized, which
in turn results in preferential take-up of systemic gonado-
tropins compared with nondominant follicles [58]. More-
over, increased follicular vascularization results in a greater
release of intrafollicular hormones and steroids to the
general circulation [5], resulting in follicle survival and in-
hibition of atresia [59]. In contrast, reduced follicular
vascularity is one of the primary events that leads to
follicular atresia [60,61]. In the same context, our VEGFR
findings are in agreement with a color-Doppler ultrasono-
graphic study [9] that showed reduced blood flow in the
wall of dominant transitional anovulatory follicles
compared with dominant ovulatory follicles. Conversely, in
the present study, the lower VEGFR expression during the
SU and FOV seasons compared with the SOV season is
inconsistent with the color-Doppler reports that showed
greater follicle wall blood flow during the SU and FOV sea-
sons compared with the SOV season [17,20]. Therefore,
reduced VEGFR expression herein observed during the SU
and FOV seasons suggests that this could be one of the
earliest events leading to ovulatory incompetence (anov-
ulation) at the end of the breeding season.
This study showed differential LHR expression in every
follicle wall layer among different seasons of the year. In
this regard, a greater expression of LHR was observed in the
granulosa, theca interna, and theca externa layers, and in
the overall quantification during the SOV season compared
with the SU and FOV seasons. Results from previous studies
have confirmed the importance of LH for the establishment
of dominance after follicle deviation and further growth of
the largest follicle [62–64]. In addition, a higher systemic
LH concentration has been reported at the beginning of the
ovulatory season than during the SAN season [4,9,65]. In
this context, our present finding has also confirmed pre-
viously reported results that showed a higher mRNA
encoding LHR expression in the theca interna of dominant
follicles during the SOV season compared with the SAN
season [14]. Therefore, there is a general consensus that
dominant transitional follicles are ovulatory incompetent
and have a lower response to human chorionic gonado-
tropin treatment [18]. On the other hand, the differential
LHR expression, along with differential EGFR, and Ki-67
observed in the present study, may explain the difference
in follicle dynamics observed among the different periods
of the ovulatory season [16,17,20,51]. Moreover, it seems
that the lower LHR expression during the SU and FOV
seasons observed in the present study is one of the first
events in the cascade of the transition from the ovulatory to
 G.M. Ishak et al. / Domestic Animal Endocrinology 70 (2020) 106382
the anovulatory season. In this regard, a lower post-
ovulatory LH level was detected during the later compared
with the first part of the ovulatory season [66]. Further-
more, a lower LH concentration was observed after the last
ovulation of the reproductive season than after the pre-
ceding ovulation [67]. Moreover, during the last ovulatory
surge of the season, a 50% reduction in LH levels seems to
occur in most of the mares [3]. Therefore, more detailed
studies are required to elucidate the expression pattern of
LHR in the follicle wall, along with systemic and intra-
follicular concentrations of LH during the transitions into
and out of the ovulatory season.
The lower Bcl-2 expression observed in the present
study, demonstrated by a higher ratio of Bax/Bcl-2, revealed
early apoptosis signaling in the granulosa layer of dominant
anovulatory follicles compared with their ovulatory coun-
terparts. Surprisingly, the ratio of Bax/Bcl-2 in the theca
externa layer was inconsistent with that observed in the
granulosa layer. Results of previous studies demonstrated
the importance of Bcl-2 as a survival molecule through the
inhibition of apoptosis by interacting with Bax and there-
fore protecting the mitochondrial membrane [68–70].
Furthermore, a greater incidence of granulosa cell
apoptosis has been shown to be associated with lower
levels of Bcl-2 [71]. Similarly to our findings, higher
expression of Bcl-2 in growing follicles, and Bax in atretic
follicles, has been reported in quails [72].
In the present study, intrafollicular NO concentration
did not differ among different seasons of the year.
Furthermore, combined data did not reveal any difference
between the anovulatory and ovulatory seasons. In this
regard, a recent study [73] did not find any difference in
intrafollicular NO concentration between POFs and HAFs.
However, previous reports in human and small lab animals
had demonstrated the role of NO in follicle development,
steroidogenesis, angiogenesis, promoting the LH surge, and
ovulation (reviewed in the study by Dixit and Parvizi [74]).
Furthermore, mares with dominant follicles treated with
NO inhibitors showed delayed ovulation [75], whereas
dominant follicles from mares treated with an ovulatory
dose of human chorionic gonadotropin showed a higher
intrafollicular NO concentration compared with control
mares [76]. Therefore, more detailed studies are required to
examine the role of NO in follicle development, selection,
and the ovulatory process in mares.
The intrafollicular concentration of estradiol in the
present study was higher during the FOV season compared
with the SAN and SOV seasons. Similarly, higher estradiol
concentration was observed during the SU compared with
the SAN season and tended to be greater during the SU than
the SOV season, whereas no difference was observed be-
tween SAN and SOV seasons. Our finding for the SAN vs the
SOV season is in agreement with Bøgh et al [77] and
Donadeu and Schauer [12] and inconsistent with Watson
and Al-Zi'abi [5] and Acosta et al [9]. Intrafollicular fluid
samples of the SAN season were collected in the present
study near the end of the transitional anovulatory season
(around 30 d before the first ovulation of the ovulatory
season). In this regard, a progressive increase in estradiol
production has been observed as the mares advance to-
ward the ovulatory season [12,18]. Data from the regrouped
9
ovulatory seasons (SOV, SU, and FOV) demonstrated a
greater intrafollicular concentration of estradiol in com-
parison to the anovulatory season; the present finding
confirms previous reports that showed that transitional
anovulatory follicles are steroidogenically incompetent and
therefore produce less estradiol than ovulatory follicles
[6,78]. Our results showed that intrafollicular estradiol
concentration did not differ between SU and FOV seasons;
however, our result is inconsistent with previous results,
where a lack in follicular estrogen production and lower
aromatase activity was found during the transition to the
anovulatory season compared with the SU season [79].
Similarly, lower estradiol concentration relative to follicle
size was reported during the FOV season compared with
the SU season [52]. In the present study, FF samples of the
SU and FOV seasons were collected during July and
September, respectively. Therefore, with only a few inter-
ovulatory intervals separating the SU and FOV seasons, no
obvious difference between the 2 seasons was found. On
the other hand, the relationship between intrafollicular
estradiol concentration and the expression of estradiol re-
ceptors within the follicle wall still needs to be elucidated
during different seasons of the year.
In summary, the application of the FWB technique in the
present study allowed, for the first time, comparison of the
expression pattern of several receptors and proteins among
4 seasons using samples obtained in vivo from the same
individuals without jeopardizing their fertility and ovarian
function. Different receptor and protein expression pat-
terns among seasons were detected. The differences in
follicle dynamics reported throughout the year in previous
studies might be explained by the lower expression of the
EGFR, Ki-67, VEGFR, and LHR in the follicular wall during
the beginning and toward the end of the ovulatory season,
suggesting an important role of the studied proteins in
controlling ovarian activity in different seasons of the year.
The synergistic effect of lower expression of proliferative
proteins, lower expression of angiogenic receptors, and
inadequate expression of LHRs in at least some of the layers
of the follicle wall seems to support the concept of anov-
ulation of dominant follicles at the beginning of the spring
and end of fall transitional periods in the mare.
Acknowledgments
The authors are grateful to Hannah Moore and Kimberly
Perkins for their technical assistance and management of
the animals, and Christy Steadman for the technical sup-
port. The authors are also thankful to Maureen Doran from
Saffron Scientific Histology Services, Carbondale, IL for
helping with the histological processing.
This work was supported by Southern Illinois Univer-
sity, Carbondale, IL, USA; Ministry of Higher Education &
Scientific Research, Baghdad, Iraq; and USDA-ARS Bio-
photonics Initiative (8–6402-3-018). G.M.I. was the recip-
ient of a PhD scholarship from the Ministry of Higher
Education & Scientific Research, Baghdad, Iraq. G.A.D. and
G.D.A.G. were the recipients of a PhD sandwich and PhD
scholarships, respectively, from the Coordination for the
Improvement of Higher Education Personnel (CAPES; grant
#PPGMV-UFRRJ#88881.133485/2016-01) and The National
10 G.M. Ishak et al. / Domestic Animal Endocrinology 70 (2020) 106382
Council for Scientific and Technological Development
(CNPq; grant #246741/2012-0), Brazil.
Authors' contributions: G.M.I., J.M.F., and E.L.G.
contributed to conceptualization, methodology, and proj-
ect administration and were responsible for acquisition of
funding and resources. G.M.I., G.A.D., S.B.P., and E.L.G.
contributed to data curation. G.M.I. and E.L.G. contributed
to formal analysis and writing the original draft. G.M.I.,
G.A.D., G.D.A.G., M.E.E., M.O.G., S.B.P., J.M.F., and E.L.G.
contributed to investigation and reviewed and edited the
writing. J.M.F. and E.L.G. were responsible for supervision.
References
[1] King SS, Neumann KR, Nequin LG, Weedman BJ. Time of onset and
ovarian state prior to entry into winter anestrus. J Equine Vet Sci
1993;13:512–5.
[2] Irvine CH, Alexander SL, McKinnon AO. Reproductive hormone
profiles in mares during the autumn transition as determined by
collection of jugular blood at 6 h intervals throughout ovulatory and
anovulatory cycles. J Reprod Fertil 2000;118:101–9.
[3] Nequin LG, King SS, Roser JF, Soderstrom BL, Carnevale EM,
Neumann KR. Uncoupling of the equine reproductive axes during
transition into anoestrus. J Reprod Fertil Suppl 2000:153–61.
[4] Donadeu FX, Ginther OJ. Follicular waves and circulating concen-
trations of gonadotrophins, inhibin and oestradiol during the
anovulatory season in mares. Reproduction 2002;124:875–85.
[5] Watson ED, Al-Zi'abi MO. Characterization of morphology and
angiogenesis in follicles of mares during spring transition and the
breeding season. Reproduction 2002;124:227–34.
[6] Watson ED, Thomassen R, Steele M, Heald M, Leask R, Groome NP,
Riley SC. Concentrations of inhibin, progesterone and oestradiol in
fluid from dominant and subordinate follicles from mares during
spring transition and the breeding season. Anim Reprod Sci 2002;74:
55–67.
[7] Watson ED, Heald M, Tsigos A, Leask R, Steele M, Groome NP,
Riley SC. Plasma FSH, inhibin a and inhibin isoforms containing pro-
and-alphac during winter anoestrus, spring transition and the
breeding season in mares. Reproduction 2002;123:535–42.
[8] Nunes MM, Gastal EL, Gastal MO, Rocha Filho AN, Mellagi AP,
Ginther OJ. Follicle and gonadotropin relationships during the
beginning of the anovulatory season in mares. Anim Reprod 2005;2:
41–9.
[9] Acosta TJ, Beg MA, Ginther OJ. Aberrant blood flow area and plasma
gonadotropin concentrations during the development of dominant-
sized transitional anovulatory follicles in mares. Biol Reprod 2004;
71:637–42.
[10] Watson ED, Thomassen R, Nikolakopoulos E. Association of uterine
edema with follicle waves around the onset of the breeding season
in pony mares. Theriogenology 2003;59:1181–7.
[11] Ginther OJ, Woods BG, Meira C, Beg MA, Bergfelt DR. Hormonal
mechanism of follicle deviation as indicated by major versus minor
follicular waves during the transition into the anovulatory season in
mares. Reproduction 2003;126:653–60.
[12] Donadeu FX, Schauer SN. Differential miRNA expression between
equine ovulatory and anovulatory follicles. Domest Anim Endo-
crinol 2013;45:122–5.
[13] Ginther OJ. Folliculogenesis during the transitional period and early
ovulatory season in mares. J Reprod Fertil 1990;90:311–20.
[14] Watson ED, Bae SE, Steele M, Thomassen R, Pedersen HG, Bramley T,
Hogg CO, Armstrong DG. Expression of messenger ribonucleic acid
encoding for steroidogenic acute regulatory protein and enzymes,
and luteinizing hormone receptor during the spring transitional
season in equine follicles. Domest Anim Endocrinol 2004;26:215–30.
[15] Doyle LK, Hogg CO, Watson ED, Donadeu FX. Seasonal effects on the
response of ovarian follicles to IGF1 in mares. Reproduction 2008;
136:589–98.
[16] Pierson RA, Ginther OJ. Follicular population dynamics during the
estrous cycle of the mare. Anim Reprod Sci 1987;14:219–31.
[17] Ishak GM, Bashir ST, Gastal MO, Gastal EL. Pre-ovulatory follicle
affects corpus luteum diameter, blood flow, and progesterone pro-
duction in mares. Anim Reprod Sci 2017;187:1–12.
[18] Donadeu FX, Watson ED. Seasonal changes in ovarian activity: les-
sons learnt from the horse. Anim Reprod Sci 2007;100:225–42.
[19] Gastal EL, Gastal MO, Ginther OJ. The suitability of echotexture
characteristics of the follicular wall for identifying the optimal
breeding day in mares. Theriogenology 1998;50:1025–38.
[20] Gastal EL, Gastal MO, Donadeu FX, Acosta TJ, Beg MA, Ginther OJ.
Temporal relationships among LH, estradiol, and follicle vasculari-
zation preceding the first compared with later ovulations during the
year in mares. Anim Reprod Sci 2007;102:314–21.
[21] Pugliesi G, Santos FB, Lopes E, Nogueira É, Maio JR, Binelli M.
Improved fertility in suckled beef cows ovulating large follicles or
supplemented with long-acting progesterone after timed-AI. Ther-
iogenology 2016;85:1239–48.
[22] Tarso SGS, Gastal GDA, Bashir ST, Gastal MO, Apgar GA, Gastal EL.
Follicle vascularity coordinates corpus luteum blood flow and pro-
gesterone production. Reprod Fertil Dev 2017;29:448.
[23] Silva LA, Gastal EL, Gastal MO, Beg MA, Ginther OJ. Relationship
between vascularity of the preovulatory follicle and establishment
of pregnancy in mares. Anim Reprod 2006;3:339–46.
[24] Siddiqui MA, Almamun M, Ginther OJ. Blood flow in the wall of the
preovulatory follicle and its relationship to pregnancy establish-
ment in heifers. Anim Reprod Sci 2009;113:287–92.
[25] Brück I, Grøndahl C, Høst T, Greve T. In vitro maturation of equine
oocytes: effect of follicular size, cyclic stage and season. Ther-
iogenology 1996;46:75–84.
[26] Murdoch WJ, Van Kirk EA. Luteal dysfunction in ewes induced to
ovulate early in the follicular phase. Endocrinology 1998;139:
3480–4.
[27] Perry GA, Smith MF, Lucy MC, Green JA, Parks TE, MacNeil MD,
Roberts AJ, Geary TW. Relationship between follicle size at insem-
ination and pregnancy success. Proc Natl Acad Sci U S A 2005;102:
5268–73.
[28] Vasconcelos JM, Sartori R, Oliveira HN, Guenther JG, Wiltbank MC.
Reduction in size of the ovulatory follicle reduces subsequent luteal
size and pregnancy rate. Theriogenology 2001;56:307–14.
[29] Ishak GM, Bashir ST, Dutra GA, Gastal GA, Gastal MO, Cavinder CA,
Feugang JM, Gastal EL. In vivo antral follicle wall biopsy: a new
research technique to study ovarian function at the cellular and
molecular levels. Reprod Biol Endocrinol 2018;16:71.
[30] Gastal EL, Gastal MO, Bergfelt DR, Ginther OJ. Role of diameter dif-
ferences among follicles in selection of a future dominant follicle in
mares. Biol Reprod 1997;57:1320–7.
[31] Feugang JM, Greene JM, Sanchez-Rodríguez HL, Stokes JV,
Crenshaw MA, Willard ST, Ryan PL. Profiling of relaxin and its re-
ceptor proteins in boar reproductive tissues and spermatozoa.
Reprod Biol Endocrinol 2015;13:46.
[32] Herrera-Luna CV, Scarlet D, Walter I, Aurich C. Effect of stallion age
on the expression of LH and FSH receptors and aromatase P450 in
equine male reproductive tissues. Reprod Fertil Dev 2016;28:
2016–26.
[33] Aguiar FLN, Gastal GDA, Ishak GM, Gastal MO, Teixeira DIA,
Feugang JM, Figueiredo JR, Gastal EL. Effects of FSH addition to an
enriched medium containing insulin and EGF after long-term cul-
ture on functionality of equine ovarian biopsy tissue. Theriogenol-
ogy 2017;99:124–33.
[34] Gastal GDA, Aguiar FLN, Alves BG, Alves KA, de Tarso SGS, Ishak GM,
Cavinder CA, Feugang JM, Gastal EL. Equine ovarian tissue viability
after cryopreservation and in vitro culture. Theriogenology 2017;
97:139–47.
[35] Ginther OJ, Gastal MO, Gastal EL, Jacob JC, Siddiqui MAR, Beg MA.
Effects of age on follicle and hormone dynamics during the oestrous
cycle in mares. Reprod Fertil Dev 2008;20:955–63.
[36] Ginther OJ. Ultrasonic imaging and animal reproduction: book 2,
horses. Cross Plains, WI: Equiservices Publishing; 1995.
[37] Gastal EL. Recent advances and new concepts on follicle and
endocrine dynamics during the equine periovulatory period. Anim
Reprod 2009;6:144–58.
[38] Gutierrez CG, Ralph JH, Telfer EE, Wilmut I, Webb R. Growth and
antrum formation of bovine preantral follicles in long-term culture
in vitro. Biol Reprod 2000;62:1322–8.
[39] Celestino JH, Bruno JB, Saraiva MA, Rocha RP, Brito IR,
Duarte ABG, Araújo VR, Silva CM, Matos MT, Campello C. Steady-
state level of epidermal growth factor (EGF) mRNA and effect of
EGF on in vitro culture of caprine preantral follicles. Cell Tissue
Res 2011;344:539.
[40] Silva JR, van den Hurk R, de Matos MH, dos Santos RR, Pessoa C, de
Moraes MO, de Figueiredo JR. Influences of FSH and EGF on pri-
mordial follicles during in vitro culture of caprine ovarian cortical
tissue. Theriogenology 2004;61:1691–704.
[41] Zhou H, Zhang Y. Effect of growth factors on in vitro development of
caprine preantral follicle oocytes. Anim Reprod Sci 2005;90:265–72.
 G.M. Ishak et al. / Domestic Animal Endocrinology 70 (2020) 106382
[42] Wu J, Tian Q. Role of follicle stimulating hormone and epidermal
growth factor in the development of porcine preantral follicle
in vitro. Zygote 2007;15:233–40.
[43] Lorenzo PL, Liu IK, Carneiro GF, Conley AJ, Enders AC. Equine oocyte
maturation with epidermal growth factor. Equine Vet J 2002;34:
378–82.
[44] Goudet G, Belin F, Bezard J, Gerard N. Maturation-promoting factor
(MPF) and mitogen activated protein kinase (MAPK) expression in
relation to oocyte competence for in-vitro maturation in the mare.
Mol Hum Reprod 1998;4:563–70.
[45] Harper KM, Brackett BG. Bovine blastocyst development after
in vitro maturation in a defined medium with epidermal growth
factor and low concentrations of gonadotropins. Biol Reprod 1993;
48:409–16.
[46] Lorenzo PL, Illera MJ, Illera JC, Illera M. Enhancement of cumulus
expansion and nuclear maturation during bovine oocyte maturation
in vitro by the addition of epidermal growth factor and insulin-like
growth factor I. J Reprod Fertil 1994;101:697–701.
[47] Reed ML, Estrada JL, Illera MJ, Petters RM. Effects of epidermal
growth factor, insulin-like growth factor-I, and dialyzed porcine
follicular fluid on porcine oocyte maturation in vitro. J Exp Zool
1993;266:74–8.
[48] Ding J, Foxcroft GR. Epidermal growth factor enhances oocyte
maturation in pigs. Mol Reprod Dev 1994;39:30–40.
[49] Luciano AM, Pappalardo A, Ray C, Peluso JJ. Epidermal growth factor
inhibits large granulosa cell apoptosis by stimulating progesterone
synthesis and regulating the distribution of intracellular free cal-
cium. Biol Reprod 1994;51:646–54.
[50] Hsueh AJW, Welsh TH, Jones PBC. Inhibition of ovarian and testic-
ular steroiodogenesis by epidermal growth factor. Endocrinology
1981;108:2002–4.
[51] Ginther OJ, Pierson RA. Regular and irregular characteristics of
ovulation and the interovulatory interval in mares. J Equine Vet Sci
1989;9:4–12.
[52] Weedman BJ, King SS, Neumann KR, Nequin LG. Comparison of
circulating estradiol and folliculogenesis during the breeding sea-
son, autumn transition and anestrus in the mare. J Equine Vet Sci
1993;13:502–5.
[53] Ginther OJ. Major and minor follicular waves during the equine
estrous cycle. J Equine Vet Sci 1993;13:18–25.
[54] Reynolds LP, Redmer DA. Expression of the angiogenic factors, basic
fibroblast growth factor and vascular endothelial growth factor, in
the ovary. J Anim Sci 1998;76:1671–81.
[55] Tamanini C, De Ambrogi M. Angiogenesis in developing follicle and
corpus luteum. Reprod Domest Anim 2004;39:206–16.
[56] Ravindranath N, Little-Ihrig L, Phillips HS, Ferrara N, Zeleznik AJ.
Vascular endothelial growth factor messenger ribonucleic acid
expression in the primate ovary. Endocrinology 1992;131:254–60.
[57] Stouffer RL, Martınez-Chequer JC, Molskness TA, Xu F, Hazzard TM.
Regulation and action of angiogenic factors in the primate ovary.
Arch Med Res 2001;32:567–75.
[58] Zeleznik AJ, Schuler HM, Reichert Jr LE. Gonadotropin-binding sites
in the rhesus monkey ovary: role of the vasculature in the selective
distribution of human chorionic gonadotropin to the preovulatory
follicle. Endocrinology 1981;109:356–62.
[59] Shimizu T, Jiang J-Y, Sasada H, Sato E. Changes of messenger RNA
expression of angiogenic factors and related receptors during
follicular development in gilts. Biol Reprod 2002;67:1846–52.
[60] Greenwald GS. Temporal and topographic changes in DNA synthesis
after induced follicular atresia. Biol Reprod 1989;41:175–81.
11
[61] Jiang JY, Macchiarelli G, Tsang BK, Sato E. Capillary angiogenesis and
degeneration in bovine ovarian antral follicles. Reproduction 2003;
125:211–23.
[62] Gastal EL, Bergfelt DR, Nogueira GP, Gastal MO, Ginther OJ. Role of
luteinizing hormone in follicle deviation based on manipulating
progesterone concentrations in mares. Biol Reprod 1999;61:
1492–8.
[63] Ginther OJ. Selection of the dominant follicle in cattle and horses.
Anim Reprod Sci 2000;60:61–79.
[64] Gastal EL, Gastal MO, Nogueira GP, Bergfelt DR, Ginther OJ. Tem-
poral interrelationships among luteolysis, FSH and LH concentra-
tions and follicle deviation in mares. Theriogenology 2000;53:
925–40.
[65] Freedman LJ, Garcia MC, Ginther OJ. Influence of photoperiod and
ovaries on seasonal reproductive activity in mares. Biol Reprod
1979;20:567–74.
[66] Turner DD, Garcia MC, Ginther OJ. Follicular and gonadotropic
changes throughout the year in pony mares. Am J Vet Res 1979;40:
1694–700.
[67] Snyder DA, Turner DD, Miller KF, Garcia MC, Ginther OJ. Follicular
and gonadotrophic changes during transition from ovulatory to
anovulatory seasons. J Reprod Fertil Suppl 1979:95–101.
[68] Hussein MR, Bedaiwy MA, Falcone T. Analysis of apoptotic cell
death, Bcl-2, and p53 protein expression in freshly fixed and cry-
opreserved ovarian tissue after exposure to warm ischemia. Fertil
Steril 2006;85:1082–92.
[69] Namura S. Neuroprotectants: cell-death based. In: Primer on Cerebro-
vascular Diseases. 2nd ed. Academic Press, Elsevier; 2017. p. 189–92.
[70] Regan SL, Knight PG, Yovich JL, Leung Y, Arfuso F, Dharmarajan A.
Granulosa cell apoptosis in the ovarian follicleda changing view.
Front Endocrinol 2018;9:61.
[71] Choi D, Hwang S, Lee EY, Yoon S, Yoon B-K, Bae D. Expression of
mitochondria-dependent apoptosis genes (p53, Bax, and Bcl-2) in
rat granulosa cells during follicular development. J Soc Gynecol
Investig 2004;11:311–7.
[72] Van Nassauw L, Tao L, Harrisson F. Distribution of apoptosis-related
proteins in the quail ovary during folliculogenesis: BCL-2, BAX and
CPP32. Acta Histochem 1999;101:103–12.
[73] Bashir ST, Gastal MO, Tazawa SP, Tarso SG, Hales DB, Cuervo-
Arango J, Baerwald AR, Gastal EL. The mare as a model for luteinized
unruptured follicle syndrome: intrafollicular endocrine milieu.
Reproduction 2016;151:271–83.
[74] Dixit VD, Parvizi N. Nitric oxide and the control of reproduction.
Anim Reprod Sci 2001;65:1–16.
[75] Pinto CRF, Paccamonti DL, Eilts BE, Short CR, Godke RA. Effect of
nitric oxide synthase inhibitors on ovulation in hCG-stimulated
mares. Theriogenology 2002;58:1017–26.
[76] Pinto CRF, Paccamonti DL, Eilts BE, Venugopal CS, Short CR,
Gentry LR, Thompson DL, Godke RA. Concentrations of nitric oxide
in equine preovulatory follicles before and after administration of
human chorionic gonadotropin. Theriogenology 2003;60:819–27.
[77] Bøgh IB, Høier R, Synnestvedt B, Greve T. Steroid concentrations in
follicular fluid aspirated repeatedly from transitional and cyclic
mares. Theriogenology 2000;54:877–88.
[78] Davis SD, Sharp DC. Intra-follicular and peripheral steroid charac-
teristics during vernal transition in the pony mare. J Reprod Fertil
Suppl 1991;44:333–40.
[79] Seamans KW, Sharp DC. Changes in equine follicular aromatase
activity during transition from winter anoestrus. J Reprod Fertil
Suppl 1982;32:225–33.