Theriogenology 85 (2016) 1491–1498

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Theriogenology
journal homepage: www.theriojournal.com

Changes in intrafollicular concentrations of free IGF-1,

activin A, inhibin A, VEGF, estradiol, and prolactin before
ovulation in mares
S.T. Bashir a, G.M. Ishak a, M.O. Gastal a, J.F. Roser b, E.L. Gastal a, *
a
Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, Illinois, USA
b
Department of Animal Science, University of California, Davis, California, USA

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

Article history: Changes in intrafollicular growth factors and hormones were evaluated in vivo in post-
Received 13 July 2015 deviation and impending ovulation follicles. Mares (n ¼ 30) were randomly assigned to
Received in revised form 11 January 2016 five experimental groups based on target diameters of 25, 30, 35, 40 mm, and impending
Accepted 11 January 2016
signs of ovulation. Furthermore, data belonging to two or more proximal diameter groups
that were not different were combined and regrouped for each factor separately. Follicular
Keywords:
fluid-free insulin-like growth factor 1 was highest (P < 0.003) in 35-mm follicles, followed
Intrafollicular growth factor
by the 40-mm and impending ovulation follicle group, and the 25- to 30-mm follicle
Hormone
Follicle deviation group. However, concentrations of insulin-like growth factor binding protein 2 in follicular
Impending ovulation fluid did not differ (P > 0.05) among groups. Additionally, follicular fluid activin A tended
Mare (P < 0.06) to be higher in impending ovulation follicles when compared with the 25- to
40-mm follicle group. Concentrations of intrafollicular estradiol were higher (P < 0.0001)
in 40-mm and impending ovulation follicles than in the other follicle groups. Follicular
fluid concentrations of inhibin A and vascular endothelial growth factor were lower
(P < 0.05) in the 40-mm and the impending ovulation follicle group when compared with
the 25- to 35-mm follicle group. Systemic and intrafollicular prolactin levels were lower
(P < 0.05) in the impending ovulation group when compared with the 25- to 40-mm
follicle group. Prolactin concentrations were higher (P < 0.05) in the follicular fluid than
in the plasma. The novel findings of this study, a decrease in intrafollicular-free insulin-like
growth factor 1, inhibin A, vascular endothelial growth factor, and prolactin during the
final stages of follicular growth, document for the first time the occurrence of dynamic
changes among intrafollicular factors and hormones during the stages of follicle domi-
nance and as ovulation approaches.
Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction achieve higher success rates. Knowledge of basic physio-

In mares, although good progress has been made in the
use of assisted reproductive technologies like superovula-
tion, ovum pickup, embryo transfer, and intracytoplasmic
sperm injections [1], more information is required to
* Corresponding author. Tel.: þ1 618 453 1774; fax: þ1 618 453 5231.
E-mail address: egastal@siu.edu (E.L. Gastal).
logical mechanisms underlying ovulation, follicle matura-
tion, oocyte quality, and embryo production is vital for the
successful application (clinical and/or translational) of
advanced reproductive technologies in mares. Additionally,
the knowledge obtained from studying ovarian reproduc-
tive physiology in mares can be vital in understanding the
reproductive function and ovarian physiology in women.
Mares and women have significant similarities in ovarian
follicular dynamics [2]. Owing to the number of similarities,

0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2016.01.013
1492 S.T. Bashir et al. / Theriogenology 85 (2016) 1491–1498
several studies have used or advocated the use of the mare
as a model for study of ovarian function in women [3–9].
Therefore, studies of ovarian follicular dynamics in mares
provide a dual benefit in terms of advancement of knowl-
edge in ovarian function of the mare and women.
A follicular ovulatory wave in mares develops during the
latter half of an estrous cycle, during which a cohort of
growing follicles emerges, and usually one follicle is
selected to be dominant, which ovulates by the end of the
estrous cycle [10]. The process of selection of a single
dominant follicle is termed deviation [11]. During the
process of deviation, the dominant follicle continues to
grow, whereas the rest of the follicles from the cohort grow
at a reduced rate and then regress. The future dominant
follicle forms a characteristic anechoic layer under the
granulosa cell layer that can differentiate it from the second
largest follicle [12]. At deviation, the changes in follicle
growth rate and formation of the anechoic layer are
temporally associated with increase in intrafollicular
estradiol and free insulin-like growth factor 1 (IGF-1),
decrease in circulating FSH, and increase in LH [11–17]. In
mares, postdeviation growth of the dominant follicle is
attributed to LH [16]. The transition of the largest growing
follicle to dominant follicle and its subsequent growth is
associated with systemic FSH and LH and is also affected by
intrafollicular factors.
A dynamic interrelationship has been observed be-
tween follicle selection and intrafollicular factors, such as
free IGF-1, insulin-like growth factor binding proteins
(IGFBPs), IGFBP protease, activins, inhibins, vascular endo-
thelial growth factor (VEGF), steroids, GH, and others
[14,17–22]. Furthermore, intrafollicular factors, especially
free IGF-1 has been shown to be involved in continued
follicular development [17,20,23–25] and ovarian cyclicity
[22,26,27] in mares. Insulin-like growth factor 1 stimulates
the growth of ovarian follicles by granulosa cell prolifera-
tion and differentiation in later stages of follicular devel-
opment [24]. Moreover, IGFBPs play a vital role in
regulating the concentrations of free IGF-1. In mares,
IGFBP-2 and IGFBP-3 and their proteases are the most
important for controlling the levels of free IGF-1 [28,29]. In
addition, it has been observed that prolactin (PRL) might be
produced in the ovarian follicles [30], but its role in follicle
development and ovulation is not clear in mares.
Although intrafollicular factors have been extensively
studied during the process of deviation, knowledge is still
lacking concerning dominance and preovulatory (post-
deviation) phases of the estrous cycle. Most studies on
follicular fluid factors in mares have categorized the follicles
into ranges of small, medium, and large diameters
[11,14,17,18,20,21,23,25]. However, none of the studies have
compared changes in intrafollicular factors and hormones
during different stages (diameters) postdeviation and in
impending ovulatory follicles. Therefore, information about
intrafollicular growth factors and hormones during post-
deviation growth is critical for understanding the ovulatory
and postovulatory physiological mechanisms, such as oocyte
maturation, ovulation, and embryo development and sur-
vival that have important in vivo and in vitro applications.
It is evident from recent studies that intrafollicular
factors are important in the success of several assisted
reproductive technologies such as superovulation and
embryo production [1,7,31]. Additionally, recent studies
advocate the use of follicular fluid biomarkers for identi-
fying quality of oocytes [32] to produce superior results,
that is, production of offspring. A good intrafollicular
environment is important for proper development and
health of oocytes in several species, such as cattle [33],
horses [28,34,35], and humans [36]. Therefore, it is
important to properly understand the follicular microen-
vironment to comprehend the practical implications of
different factors present in follicular fluid. This knowledge
can be helpful in understanding follicular physiology and
can also be exploited to generate better outcomes in
assisted reproductive technologies.
This study aimed to identify the dynamics of some of the
important intrafollicular growth factors and hormones
involved in the growth of dominant and preovulatory fol-
licles from follicle deviation until ovulation. Identification
of these factors will help in designing more targeted ex-
periments in the future.
2. Materials and methods
2.1. Animals
Mares (n ¼ 30) were used during an ovulatory season
(April to October) in the northern hemisphere and handled
in accordance with the US Department of Agriculture Guide
for Care and Use of Agricultural Animals in Research. The
mares were large ponies, 2 to 20 years (9.8  0.9 years),
weighed 300 to 400 kg, and had docile temperament with
normal reproductive tract as determined by ultrasono-
graphic examination. Mares were kept under natural light
in pasture with free access to water and trace mineralized
salt.
2.2. Ultrasonographic examinations and groups
Transrectal ultrasonographic examinations were per-
formed after Day 12 of the estrous cycle (Day 0 ¼ ovulation)
until a growing dominant follicle reached 25 mm in
diameter. Subsequently, mares were randomly assigned
into groups based on target diameters of 25 (n ¼ 6), 30
(n ¼ 5), 35 (n ¼ 5), 40 mm (n ¼ 5), and follicles that showed
signs of impending ovulation (n ¼ 9) as described [37]. The
largest follicle was measured on each day of examination.
2.3. Collection of follicular fluid and blood samples
Follicular fluid was collected by ultrasound-guided
transvaginal follicle aspiration [14,38] when the dominant
follicle reached the target diameters. The aspirated follic-
ular fluid was processed immediately in a refrigerated
centrifuge (1500  g for 10 minutes), and the supernatant
was stored at 20  C until hormone assays were per-
formed. When the dominant follicle reached the target
diameter, a single blood sample was collected from the
jugular vein into heparinized tubes and immediately
placed in ice water bath and processed in a refrigerated
centrifuge (1500  g for 10 minutes), decanted, and stored
(20  C) until assay.
 S.T. Bashir et al. / Theriogenology 85 (2016) 1491–1498
2.4. Hormone assays
Concentrations of free IGF-1 were determined by a
sandwich-type ELISA (Diagnostic Systems Laboratories,
Webster, TX, USA); the interassay and intra-assay CVs were
4.4% and 6.1%, and the assay sensitivity was 0.05 ng/mL,
respectively. A double-antibody radioimmunoassay (RIA)
kit (Diagnostic Systems Laboratories) was used to quantify
the IGFBP-2 concentrations; the intra-assay CV was 1.4%,
and the assay sensitivity was 1.0 ng/mL. Follicular fluid
estradiol was assayed by means of a double-antibody RIA kit
(Diagnostic Products Corporation, Los Angeles, CA, USA;
[14]) without extraction, and the samples were diluted
1:6000 in assay buffer (PBS with 0.1% gelatin). Intra-assay
CV and assay sensitivity were 4.9% and 0.4 ng/mL, respec-
tively. Concentrations of inhibin A were measured by solid-
phase sandwich ELISAs (Oxford Bio-Innovation Ltd.,
Oxfordshire, UK); follicular fluid samples were diluted to
1:1000 in fetal calf serum. Intra-assay and interassay CVs
were 5.0% and 2.5%, and assay sensitivity was 1.0 ng/mL.
Activin A concentrations were measured by a solid-phase
sandwich ELISA (Oxford Bio-Innovation Ltd.); a sample
dilution of 1:250 in 5% BSA was used. Interassay and intra-
assay CVs were 14.9% and 4.1%, and the assay sensitivity
was 0.04 ng/mL. All the aforementioned hormone assays
have been used previously for mare follicular fluid samples
and had minimal cross reactivity with other hormones [18].
Concentrations of VEGF in the follicular fluid were
determined using a competitive ELISA kit (Neogen Corpo-
ration, Lexington, KY, USA) from which an assay has been
validated for use in mare follicular fluid [17]. The intra-
assay and interassay CVs for VEGF assay were 6.1% and
14.5%, and the sensitivity was 0.5 ng/mL.
Plasma and follicular PRL concentrations were deter-
mined using a modified homologous double-antibody RIA
as previously described [39]. The intra-assay and interassay
CVs were 6.0% and 10.0%, respectively, with a sensitivity of
0.25 ng/mL.
2.5. Statistical analyses
Data were tested for normal distribution using Shapiro–
Wilk test and were transformed to ranks if not normally
distributed. Outliers were identified using Dixon’s test and
excluded from any further statistical analyses. One-way
ANOVA was used to compare the data from multiple
groups, and t test was used in the case of two groups. To
identify significant changes in follicular fluid factors from
deviation to preovulatory stage that were not apparent
when the first overall analyses were done, the data were
reassigned into new groups separately for each factor. Two
or more adjacent groups not showing a difference were
combined and compared in overall and discrete analyses.
All the statistical analyses were performed using the SAS
statistical software (version 9.2; SAS Institute, Inc., Cary, NC,
USA). Systemic and follicular fluid PRL were compared
using paired t test, and correlation between them was
tested using Pearson’s correlation coefficient. A probability
of 0.05 indicated that a difference was significant, and
P > 0.05 or P < 0.1 indicated that the results approached a
significant difference.
1493
3. Results
3.1. Intrafollicular-free IGF-1 and IGFBP-2
Follicular fluid concentrations of free IGF-1 were
different (P < 0.02) among various follicle groups (Fig. 1A).
The 25-mm follicles had lower (P < 0.05) free IGF-1 when
compared with 35-mm and impending ovulation follicles.
Similarly, 30-mm follicles had lower (P < 0.05) concen-
trations of free IGF-1 compared with 35-mm follicles; no
other follicle groups showed any significant differences.
When the data were regrouped, free IGF-1 concentration
was lower (P < 0.05) in the 25- to 30-mm follicle group,
compared with the other groups. Furthermore, follicles
from the 40-mm þ impending ovulation group had lower
(P < 0.05) concentrations of free IGF-1 compared with 35-
mm follicles (Fig. 1B). On the other hand, concentrations of
IGFBP-2 did not differ among follicle groups (Fig. 1C, D).
3.2. Intrafollicular estradiol, inhibin A, activin A, and VEGF
Intrafollicular estradiol concentrations increased
(P < 0.0001) with an increase in follicle diameter (Fig. 2A, B).
Estradiol concentrations were lowest (P < 0.05) in 25-mm
follicles, followed by 30- and 35-mm follicles, and were
highest in 40-mm and impending ovulation follicles,
before or after combining similar groups. On the other
hand, concentrations of inhibin A did not show any dif-
ference among original follicle groups (Fig. 3A). However,
after regrouping analyses, concentrations of inhibin A
were higher (P < 0.02) in the 25- to 35-mm follicle group
when compared with the 40-mm þ impending ovulation
follicle group (Fig. 3B). Similarly, concentrations of activin
A did not differ among follicle groups (Fig. 4A); however,
after regrouping, the concentrations of activin A tended
(P < 0.06) to be higher in the impending ovulation group
than the 25- to 40-mm follicle group (Fig. 4B). Vascular
endothelial growth factor follicular fluid concentrations
were not different among the different follicular groups
(Fig. 3C). However, VEGF concentration in the 40-
mm þ impending ovulation follicle group was lower
(P < 0.002) than in the 25- to 35-mm follicle group
(Fig. 3D).
3.3. Systemic and intrafollicular PRL
Systemic concentration of PRL was higher (P < 0.05) in
35-mm follicles compared with 25-mm and impending
ovulation follicles (Fig. 4C). However, no difference
(P > 0.05) was detected in PRL concentrations in follicular
fluid of various follicle groups (Fig. 4C). Follicular fluid PRL
was higher (P < 0.05) in 40-mm and impending ovulation
follicles than concurrent systemic concentrations. Systemic
and follicular fluid concentrations of PRL were higher
(P < 0.05) in the 25- to 40-mm follicle group when
compared with impending ovulation follicles (Fig. 4D).
Also, intrafollicular concentrations of PRL were higher
(P < 0.05) in the 25- to 40-mm and impending ovulation
groups than the associated systemic concentrations.
Furthermore, no significant correlation between plasma
and follicular fluid PRL concentrations was observed.
1494 S.T. Bashir et al. / Theriogenology 85 (2016) 1491–1498

Intrafollicular IGF-1
A 50 B
G: P < 0.02 G: P < 0.003
a b

Concentration (ng/mL)
40
ab
abc c
30
bc
20 a
c

10

0
25 mm 30 mm 35 mm 40 mm Impending 25 - 30 mm 35 mm 40 mm -
ovulation Impending
ovulation
Follicle groups Follicle groups

Intrafollicular IGFBP-2
C 1200 D
G: NS G: NS
1000
Concentration (ng/mL)

800

600

400

200

0
25 mm 30 mm 35 mm 40 mm Impending 25 - 30 mm 35 mm 40 mm -
ovulation Impending
ovulation
Follicle groups Follicle groups

Fig. 1. Mean (SEM) intrafollicular concentrations of free IGF-1 and IGFBP-2 in (A, C) individual and (B, D) combined follicle groups in mares. Bars with different
superscripts within an end point are different (P < 0.05). The probabilities for an overall group effect are shown. IGF-1, insulin-like growth factor 1; IGFBP-2,
insulin-like growth factor binding protein 2.

Intrafollicular Estradiol

A 2500 B
G: P < 0.0001 G: P < 0.0001
Concentration (ng/mL)

2000 a a c

b
1500 b
b

1000 c a

500

0
25 mm 30 mm 35 mm 40 mm Impending 25 mm 30 - 35 mm 40 mm -
ovulation Impending
ovulation
Follicle groups Follicle groups

Fig. 2. Mean (SEM) intrafollicular concentrations of estradiol in (A) individual and (B) combined follicle groups in mares. Bars with different superscripts within
an end point are different (P < 0.05). The probabilities for an overall group effect are shown.
 S.T. Bashir et al. / Theriogenology 85 (2016) 1491–1498 1495

Intrafollicular Inhibin A
A B
600 G: NS G: P < 0.02
a
Concentration (ng/mL) 500

b
400

300

200

100

0
25 mm 30 mm 35 mm 40 mm Impending 25 - 35 mm 40 mm -
ovulation Impending
ovulation
Follicle groups Follicle groups

Intrafollicular VEGF
C 50
D
G: NS G: P < 0.002
Concentration (ng/mL)

40 a

b
30

20

10

0
25 mm 30 mm 35 mm 40 mm Impending 25 - 35 mm 40 mm -
ovulation Impending
ovulation
Follicle groups Follicle groups

Fig. 3. Mean (SEM) intrafollicular concentrations of inhibin A and VEGF in (A, C) individual and (B, D) combined follicle groups in mares. Bars with different
superscripts within an end point are different (P < 0.05). The probabilities for an overall group effect are shown. VEGF, vascular endothelial growth factor.

4. Discussion other hand, the concentrations of IGFBP-2 did not differ

Identification of novel and more contrasting dynamic
relationships of intrafollicular growth factors and hor-
mones than we had anticipated was discovered by the
combination of different proximal follicle groups. The
present findings will be essential in designing future
studies to investigate the role of the reported follicular fluid
factors and hormones in mechanisms of the establishment
of follicle dominance and growth of the dominant follicle
during the preovulatory and impending ovulation phases.
Additionally, a previous study [18] showed that intra-
follicular factors and hormones have more intricate differ-
ential dynamics rather than following similar or opposing
trends. Therefore, it is important to consider each individ-
ual follicular fluid factor or hormone based on its fluctua-
tions over time during follicular development.
Free IGF-1 continued to increase with increasing follicle
diameter from 25 to 35 mm. Afterward, a novel decrease
was observed in follicular fluid-free IGF-1 during the final
stages of follicle development and before ovulation. On the
between the postdeviation follicular development and the
time of impending ovulation. Previous studies have re-
ported the important role of free IGF-1 in follicle selection
in mares and heifers [17–20,40]. Furthermore, free IGF-1
has been shown to be important throughout continued
follicular development [23–25] and also in ovulatory folli-
cles [22,26,27] in mares. In spite of being one of the most
studied follicular fluid factors, the role of free IGF-1 in
postdeviation follicles and during impending ovulation is
not clear. A regression or cessation in growth of the pre-
ovulatory follicle has been observed 2 days before ovula-
tion in mares [37,41–44]. The current findings of a decrease
in concentrations of free IGF-1 in the 40-mm þ impending
ovulation groups could be a plausible reason a preovulatory
follicle ceases to grow or decreases in diameter before
ovulation. Our observation of no change in follicular fluid
IGFBP-2 is in agreement with a previous study [28].
In this study, intrafollicular inhibin A and VEGF con-
centrations were lower in 40-mm þ impending ovulation
follicles. Inhibin A has been shown to modulate the
1496 S.T. Bashir et al. / Theriogenology 85 (2016) 1491–1498

Intrafollicular Activin A

A B
350
G: NS G: P < 0.06
#
Concentration (ng/mL) 300

250

200

150

100

50

0
25 mm 30 mm 35 mm 40 mm Impending 25 - 40 mm Impending
ovulation ovulation
Follicle groups Follicle groups

Systemic and Intrafollicular PRL

C D Plasma
Follicular fluid
8
G: P < 0.1 (Plasma)
G: NS (Follicular fluid)
7 G: P < 0.05 (Plasma)
G: P < 0.05 (Follicular fluid)
6 # B
Concentration (ng/mL)

ab
Ba
5 B
a
Aa
4 Bb
Aab
b
3 Ab Ab

0
25 mm 30 mm 35 mm 40 mm Impending 25 - 40 mm Impending
ovulation ovulation
Follicle groups Follicle groups

Fig. 4. Mean (SEM) intrafollicular concentrations of activin A and systemic and intrafollicular concentrations of PRL in (A, C) individual and (B, D) combined
follicle groups in mares. Bars with different small superscripts (a, b) within an end point are different (P < 0.05). Differences between systemic and intrafollicular
PRL are indicated by different capital superscripts (A, B). The probabilities for an overall group effect are shown. #Indicates a tendency for statistical difference
between follicle groups or between systemic and intrafollicular PRL. PRL, prolactin.

responsiveness of granulosa cells to gonadotropins and activin A increased in the final stages of follicular devel-
steroidogenesis in vitro [45,46]. In addition, VEGF has been opment. Follicular fluid activin A has been involved in
responsible for increasing the vascularity of the follicles granulosa cell proliferation and in the upregulation of FSH
and the permeability of endothelial cells of the follicular receptors, estradiol synthesis, granulosa cell LH receptor
vasculature [47]. Furthermore, reports have shown that expression, and also in enhancing oocyte maturation
IGF-1 in vitro in cattle [48] and in vivo in mares [20] was (reviewed in Ref. [45]). Furthermore, IGF-1, when injected
able to increase concentrations of inhibin A and VEGF. into predeviation follicles in vivo, increased the concen-
Therefore, it is possible that the decrease in free IGF-1 trations of activin A in mares and heifers [20]; however, no
before ovulation might be responsible for the decrease in knowledge is available concerning how IGF-1 affects activin
inhibin A and VEGF, but this potential mechanism needs A in postdeviation follicles. Our novel findings indicated
further investigation. Hours before ovulation, an abrupt higher intrafollicular concentrations of activin A in
decrease in follicular wall blood flow is observed in the impending ovulation follicles although free IGF-1, VEGF,
ovulatory follicles in mares [8,9,49,50]. Therefore, it is and inhibin A concentrations were lower as ovulation
conceivable that both inhibin A and VEGF levels are approached. Furthermore, contrasting changes in systemic
decreased in ovulatory follicles before ovulation in mares. concentrations of inhibin B and activin A have been pre-
The concentration of activin A was higher in impending viously reported in cycling aging women [51]. Therefore,
ovulation follicles compared with the 25- to 40-mm follicle further investigation is required to elucidate the mecha-
group. In contrast to free IGF-1 and inhibin A and VEGF, nisms involved in contrasting intrafollicular dynamic
 S.T. Bashir et al. / Theriogenology 85 (2016) 1491–1498
changes of activin A compared with other follicular fluid
factors during the preovulatory period in mares.
Follicular fluid estradiol concentrations were higher in
40-mm and impending ovulation follicle groups followed
by the other follicle groups. Follicular fluid and systemic
estradiol increase with increasing follicle size has been
reported previously [14]. Moreover, IGF-1 and activin A can
increase in vitro production of estradiol by granulosa cells
(reviewed in Ref. [18]). Therefore, based on our findings,
activin A is a potential factor involved in continued pro-
duction of estradiol during the preovulatory period in
mares. A daily decrease in systemic estradiol concentration
has been reported preceding ovulation in mares
[41,42,52,53]. Our results of follicular fluid estradiol con-
centrations did not show any decrease as ovulation
approached. Therefore, potential mechanisms involved in
the decrease in systemic estradiol concentration, although
follicular fluid estradiol does not decrease before ovulation,
are: a decrease in vascular perfusion of the preovulatory
follicle wall, resulting in decreased permeability of intra-
follicular estradiol into the systemic circulation; a decrease
in follicle size, which reduces the total concentration of
intrafollicular estradiol; or both.
Plasma and follicular fluid PRL concentrations were
lower in impending ovulation follicles when compared
with all other follicle groups. Also, the mean follicular fluid
concentration of PRL was greater than the mean systemic
concentration when the groups were combined. No sig-
nificant detectable changes in systemic concentrations of
PRL during the estrous cycle have been reported in mares
[54]. However, several reports have related the beginning
of luteolysis with a rise in systemic concentrations of PRL in
the mare [55–57]. Periovulatory systemic PRL surge has
been reported in mares, but a significant variability among
animals and between seasons was also observed [58].
Furthermore, exogenous administration of estradiol
increased systemic concentrations of PRL in mares and
geldings [59–61]. Therefore, in the present study, lower
systemic PRL levels in the impending ovulation group
might have been associated with decreasing systemic
estradiol concentration as previously described in mares
[41,42,52,53]. A recent report [30] has suggested an ovarian
source for PRL in the mare, which might explain the higher
concentrations of PRL observed in follicular fluid when
compared with plasma. Prolactin plays an important role in
oocyte development and normal ovulation and enhances
in vitro oocyte maturation in rodents (reviewed in
Ref. [62]). Because the role of PRL in follicle development
and ovulation is not very well understood in mares, more
studies need to be undertaken to ascertain the source and
function of intrafollicular PRL.
In conclusion, after initial increasing levels during fol-
licle growth, lower concentrations of intrafollicular-free
IGF-1, inhibin A, VEGF, and PRL have been reported herein
for the first time during the final phase of follicular matu-
ration in mares. These findings could have been associated
with a physiological process of decrease or cessation in
follicular growth before ovulation. Also, the association
between increasing levels of intrafollicular activin A and
estradiol suggests that activin A might also be responsible
for continued production of estradiol in preovulatory
1497
follicles before ovulation. Furthermore, the higher con-
centrations of intrafollicular PRL compared with systemic
levels need to be further understood. Overall, the elucida-
tion of the role of follicular fluid factors and hormones and
their interactions needs to be studied further to allow a
better understanding of the ovulatory process in mares.
This knowledge will potentially help to obtain better out-
comes during the use of assisted reproductive technologies
and will also help to understand mechanisms of ovulatory
dysfunctions like the hemorrhagic anovulatory follicle/
luteinized unruptured follicle syndrome in the mare and
perhaps in other species.
Acknowledgments
The authors thank Dr M.A. Beg and Lil Sibley for technical
assistance with hormonal analyses and for reviewing the
manuscript, and Dr O.J. Ginther, University of Wisconsin,
Madison, USA, for allowing us to use some of the data
included in this publication.
References
[1] Roser JF. Superovulation in the mare: a work in progress. J Equine
Vet Sci 2012;32:376–86.
[2] Ginther OJ. The mare: a 1000-pound guinea pig for study of the
ovulatory follicular wave in women. Theriogenology 2012;77:818–
28.
[3] Ginther OJ, Gastal EL, Gastal MO, Bergfelt DR, Baerwald AR,
Pierson RA. Comparative study of the dynamics of follicular waves
in mares and women. Biol Reprod 2004;71:1195–201.
[4] Baerwald AR. Human antral folliculogenesis: what we have learned
from the bovine and equine models. Anim Reprod 2009;6:20–9.
[5] Adams GP, Singh J, Baerwald AR. Large animal models for the study
of ovarian follicular dynamics in women. Theriogenology 2012;78:
1733–48.
[6] Ginther OJ, Beg MA, Gastal EL, Gastal MO, Baerwald AR, Pierson RA.
Systemic concentrations of hormones during the development of
follicular waves in mares and women: a comparative study.
Reproduction 2005;130:379–88.
[7] Carnevale EM. The mare model for follicular maturation and
reproductive aging in the woman. Theriogenology 2008;69:23–30.
[8] Gastal EL. Ovulation: part 2. Ultrasonographic morphology of the
preovulatory follicle. In: McKinnon AO, Squires EL, Vaala WE,
Varner DD, editors. Equine reproduction. 2nd ed. Oxford, England:
Wiley-Blackwell; 2011. p. 2032–54.
[9] Gastal EL. Recent advances and new concepts on follicle and
endocrine dynamics during the equine periovulatory period. Anim
Reprod 2009;6:144–58.
[10] Ginther OJ. Selection of the dominant follicle in cattle and horses.
Anim Reprod Sci 2000;60-61:61–79.
[11] 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.
[12] Gastal EL, Donadeu FX, Gastal MO, Ginther OJ. Echotextural changes
in the follicular wall during follicle deviation in mares. Ther-
iogenology 1999;52:803–14.
[13] Gastal EL, Bergfelt DR, Nogueira GP, Gastal MO, Ginther OJ. Role
of luteinizing hormone in follicle deviation based on manipu-
lating progesterone concentrations in mares. Biol Reprod 1999;
61:1492–8.
[14] Gastal EL, Gastal MO, Wiltbank MC, Ginther OJ. Follicle deviation
and intrafollicular and systemic estradiol concentrations in mares.
Biol Reprod 1999;61:31–9.
[15] Gastal EL, Gastal MO, Ginther OJ. Experimental assumption of
dominance by a smaller follicle and associated hormonal changes in
mares. Biol Reprod 1999;61:724–30.
[16] Gastal EL, Gastal MO, Nogueira GP, Bergfelt DR, Ginther OJ.
Temporal interrelationships among luteolysis, FSH and LH con-
centrations and follicle deviation in mares. Theriogenology 2000;
53:925–40.
1498 S.T. Bashir et al. / Theriogenology 85 (2016) 1491–1498
[17] Ginther OJ, Gastal EL, Gastal MO, Checura CM, Beg MA. Dose-
response study of intrafollicular injection of insulin-like growth
factor-I on follicular fluid factors and follicle dominance in mares.
Biol Reprod 2004;70:1063–9.
[18] Donadeu FX, Ginther OJ. Changes in concentrations of follicular fluid
factors during follicle selection in mares. Biol Reprod 2002;66:
1111–8.
[19] Beg MA, Ginther OJ. Follicle selection in cattle and horses: role of
intrafollicular factors. Reproduction 2006;132:365–77.
[20] Ginther OJ, Bergfelt DR, Beg MA, Meira C, Kot K. In vivo effects of an
intrafollicular injection of insulin-like growth factor 1 on the
mechanism of follicle deviation in heifers and mares. Biol Reprod
2004;70:99–105.
[21] Ginther OJ, Gastal EL, Gastal MO, Beg MA. In vivo effects of
pregnancy-associated plasma protein-A, activin-A and vascular
endothelial growth factor on other follicular-fluid factors during
follicle deviation in mares. Reproduction 2005;129:489–96.
[22] Donadeu FX, Watson ED. Seasonal changes in ovarian activity: les-
sons learnt from the horse. Anim Reprod Sci 2007;100:225–42.
[23] Bridges TS, Davidson TR, Chamberlain CS, Geisert RD, Spicer LJ.
Changes in follicular fluid steroids, insulin-like growth factors (IGF)
and IGF-binding protein concentration, and proteolytic activity
during equine follicular development. J Anim Sci 2002;80:179–90.
[24] Silva JR, Figueiredo JR, van den Hurk R. Involvement of growth
hormone (GH) and insulin-like growth factor (IGF) system in
ovarian folliculogenesis. Theriogenology 2009;71:1193–208.
[25] Spicer LJ, Santiago CA, Davidson TR, Bridges TS, Chamberlain CS.
Follicular fluid concentrations of free insulin-like growth factor
(IGF)-I during follicular development in mares. Domest Anim
Endocrinol 2005;29:573–81.
[26] Donadeu FX, Schauer SN. Differential miRNA expression between
equine ovulatory and anovulatory follicles. Domest Anim Endo-
crinol 2013;45:122–5.
[27] Gentry LR, Thompson Jr DL, Gentry Jr GT, Davis KA, Godke RA,
Cartmill JA. The relationship between body condition, leptin, and
reproductive and hormonal characteristics of mares during the
seasonal anovulatory period. J Anim Sci 2002;80:2695–703.
[28] Gerard N, Duchamp G, Magistrini M. Relationships between follic-
ular fluid composition and follicular/oocyte quality in the mare.
Livest Prod Sci 1999;60:243–53.
[29] Ginther OJ, Gastal EL, Gastal MO, Beg MA. Critical role of insulin-like
growth factor system in follicle selection and dominance in mares.
Biol Reprod 2004;70:1374–9.
[30] King SS, Oberhaus EL, Welsh CM, Heath DL, Jones KL. Evidence for
local neuroendocrine signaling in ovarian prolactin regulation. J
Equine Vet Sci 2014;34:107–8 [abstract].
[31] Velazquez MA, Zaraza J, Oropeza A, Webb R, Niemann H. The role of
IGF1 in the in vivo production of bovine embryos from super-
ovulated donors. Reproduction 2009;137:161–80.
[32] Revelli A, Delle Piane L, Casano S, Molinari E, Massobrio M,
Rinaudo P. Follicular fluid content and oocyte quality: from single
biochemical markers to metabolomics. Reprod Biol Endocrinol
2009;7:40.
[33] Alves BG, Alves KA, Lucio AC, Martins MC, Silva TH, Alves BG, et al.
Ovarian activity and oocyte quality associated with the biochemical
profile of serum and follicular fluid from Girolando dairy cows
postpartum. Anim Reprod Sci 2014;146:117–25.
[34] Siddiqui MA, Gastal EL, Ju JC, Gastal MO, Beg MA, Ginther OJ.
Nuclear configuration, spindle morphology and cytoskeletal or-
ganization of in vivo maturing horse oocytes. Reprod Domest
Anim 2009;44:435–40.
[35] Siddiqui MA, Gastal EL, Gastal MO, Beg MA, Ginther OJ. Effect of hCG
in the presence of hCG antibodies on the follicle, hormone con-
centrations, and oocyte in mares. Reprod Domest Anim 2009;44:
474–9.
[36] Dumesic DA, Meldrum DR, Katz-Jaffe MG, Krisher RL,
Schoolcraft WB. Oocyte environment: follicular fluid and cumulus
cells are critical for oocyte health. Fert Steril 2015;103:303–16.
[37] Gastal EL, Gastal MO, Ginther OJ. Serrated granulosa and other
discrete ultrasound indicators of impending ovulation in mares. J
Equine Vet Sci 2006;26:67–73.
[38] Gastal EL, Kot K, Ginther OJ. Ultrasound-guided intrafollicular
treatment in mares. Theriogenology 1995;44:1027–37.
[39] Roser JF, Chang YS, Papkoff H, Li CH. Development and character-
ization of a homologous radioimmunoassay for equine prolactin.
Proc Soc Exp Biol Med 1984;175:510–7.
[40] Hunter MG, Robinson RS, Mann GE, Webb R. Endocrine and para-
crine control of follicular development and ovulation rate in farm
species. Anim Reprod Sci 2004;82-83:461–77.
[41] Ginther OJ, Gastal EL, Gastal MO, Beg MA. Dynamics of the equine
preovulatory follicle and periovulatory hormones: what’s new? J
Equine Vet Sci 2008;28:454–60.
[42] Gastal EL, Gastal MO, Ginther OJ. Relationships of changes in B-
mode echotexture and colour-Doppler signals in the wall of the
preovulatory follicle to changes in systemic oestradiol concentra-
tions and the effects of human chorionic gonadotrophin in mares.
Reproduction 2006;131:699–709.
[43] Koskinen E, Kuntsi H, Lindeberg H, Katila T. Predicting ovulation in
the mare on the basis of follicular growth and serum oestrone
sulphate and progesterone levels. J Vet Med A 1989;36:299–304.
[44] Palmer E, Driancourt MA. Use of ultrasonic echography in equine
gynecology. Theriogenology 1980;13:203–16.
[45] Knight PG, Glister C. Potential local regulatory functions of inhibins,
activins and follistatin in the ovary. Reproduction 2001;121:503–12.
[46] Armstrong DG, Webb R. Ovarian follicular dominance: the role of
intraovarian growth factors and novel proteins. Rev Reprod 1997;2:
139–46.
[47] 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.
[48] Glister C, Tannetta DS, Groome NP, Knight PG. Interactions between
follicle-stimulating hormone and growth factors in modulating
secretion of steroids and inhibin-related peptides by nonluteinized
bovine granulosa cells. Biol Reprod 2001;65:1020–8.
[49] Ginther OJ, Gastal EL, Gastal MO, Siddiqui MA, Beg MA. Relation-
ships of follicle versus oocyte maturity to ultrasound morphology,
blood flow, and hormone concentrations of the preovulatory follicle
in mares. Biol Reprod 2007;77:202–8.
[50] Gastal EL. Ovulation: part 1. Follicle development and endocri-
nology during the periovulatory period. In: McKinnon AO,
Squires EL, Vaala WE, Varner DD, editors. Equine reproduction. 2nd
ed. Oxford, England: Wiley-Blackwell; 2011. p. 2020–31.
[51] Reame NE, Wyman TL, Phillips DJ, de Kretser DM, Padmanabhan V.
Net increase in stimulatory input resulting from a decrease in
inhibin B and an increase in activin A may contribute in part to the
rise in follicular phase follicle-stimulating hormone of aging cycling
women. J Clin Endocrinol Metab 1998;83:3302–7.
[52] Ginther OJ, Jacob JC, Gastal MO, Gastal EL, Beg MA. Follicle and
systemic hormone interrelationships during spontaneous and
ablation-induced ovulatory waves in mares. Anim Reprod Sci 2008;
106:181–7.
[53] Gastal EL, Neves AP, Mattos RC, Petrucci BP, Gastal MO, Ginther OJ.
Miniature ponies: 1. Follicular, luteal and endometrial dynamics
during the oestrous cycle. Reprod Fertil Dev 2008;20:376–85.
[54] Becker SE, Johnson AL. Failure of metoclopramide-induced acute
hyperprolactinemia to affect time of ovulation or spontaneous
luteolysis in the cycling mare. J Equine Vet Sci 1990;10:275–9.
[55] 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.
[56] Ginther OJ, Pinaffi FL, Silva LA, Beg MA. Temporal relationships of a
pulse of prolactin (PRL) to a pulse of a metabolite of PGF2alpha in
mares. Theriogenology 2012;77:99–107.
[57] Ginther OJ, Beg MA. Concentrations of circulating hormones
normalized to pulses of a prostaglandin F2alpha metabolite during
spontaneous luteolysis in mares. Theriogenology 2009;72:1111–9.
[58] King SS, Roser JF, Jones KL. Follicular fluid prolactin and the peri-
ovulatory prolactin surge in the mare. J Equine Vet Sci 2008;28:
468–72.
[59] Thompson Jr DL, Garza Jr F, St George RL, Rabb MH, Barry BE,
French DD. Relationships among LH, FSH and prolactin secretion,
storage and response to secretagogue and hypothalamic GnRH
content in ovariectomized pony mares administered testosterone,
dihydrotestosterone, estradiol, progesterone, dexamethasone or
follicular fluid. Domest Anim Endocrinol 1991;8:189–99.
[60] Kelley KK, Thompson Jr DL, Storer WA, Mitcham PB, Gilley RM,
Burns PJ. Estradiol interactions with dopamine antagonists in
mares: prolactin secretion and reproductive traits. J Equine Vet Sci
2006;26:517–28.
[61] Thompson Jr DL, Mitcham PB, Runles ML, Burns PJ, Gilley RM.
Prolactin and gonadotropin responses in geldings to injections of
estradiol benzoate in oil, estradiol benzoate in biodegradable
microspheres, and estradiol cypionate. J Equine Vet Sci 2008;28:
232–7.
[62] Devi YS, Halperin J. Reproductive actions of prolactin mediated
through short and long receptor isoforms. Mol Cell Endocrinol
2014;382:400–10.