Supplementation of equine early spring transitional follicles with luteinizing hormone stimulates follicle growth but does not restore steroidogenic activity

ArticleinTheriogenology 75(6):1076-84 · February 2011with13 Reads
Impact Factor: 1.80 · DOI: 10.1016/j.theriogenology.2010.11.016 · Source: PubMed
Abstract

This study was conducted to test the hypothesis that supplementation of growing follicles with LH during the early spring transitional period would promote the development of steroidogenically active, dominant follicles with the ability to respond to an ovulatory dose of hCG. Mares during early transition were randomly assigned to receive a subovulatory dose of equine LH (in the form of a purified equine pituitary fraction) or saline (transitional control; n = 7 mares per group) following ablation of all follicles >15 mm. Treatments were administered intravenously every 12 h from the day the largest follicle of the post-ablation wave reached 20 mm until a follicle reached >32 mm, when an ovulatory dose of hCG (3000 IU) was given. Saline-treated mares during June and July were used as ovulatory controls. In a preliminary study, injection of this pituitary fraction (eLH) to anestrus mares was followed by an increase in circulating levels of LH (P < 0.01) but not FSH (P > 0.6). Administration of eLH during early transition stimulated the growth of the dominant follicle (Group x Day, P < 0.00001), which attained diameters similar to the dominant follicle in ovulatory controls (P > 0.1). In contrast, eLH had no effect on the diameter of the largest subordinate follicle or the number of follicles >10 mm during treatment (P > 0.3). The numbers of mares that ovulated in response to hCG in transitional control, transitional eLH and ovulatory control groups (2 of 2, 3 of 5 and 7 of 7, respectively) were not significantly different (P > 0.1). However, after hCG-induced ovulation, all transitional mares returned to an anovulatory state. Circulating estradiol levels increased during the experimental period in ovulatory controls but not in transitional eLH or transitional control groups (Group x Day, P = 0.013). In addition, although progesterone levels increased after ovulation in transitional control and transitional eLH groups, levels in these two groups were lower than in the ovulatory control group after ovulation (Group, P = 0.045). In conclusion, although LH supplementation of early transitional waves beginning after the largest follicle reached 20 mm promoted growth of ovulatory-size follicles, these follicles were developmentally deficient as indicated by their reduced steroidogenic activity.

Full-text

Available from: Stephanie Schauer, Aug 05, 2015
Supplementation of equine early spring transitional follicles with
luteinizing hormone stimulates follicle growth but does not
restore steroidogenic activity
S.N. Schauer
a
, C. Briant
b
, M. Ottogalli
b
, C. Decourt
b
, I.G. Handel
a
, F.X. Donadeu
a,
*
a
The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin BioCentre, Midlothian EH25 9PS, UK
b
INRA, UMR85, Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly/CNRS, UMR6175, F-37380 Nouzilly/Université de
Tours, F-37041 Tours, France
Received 23 August 2010; received in revised form 11 November 2010; accepted 11 November 2010
Abstract
This study was conducted to test the hypothesis that supplementation of growing follicles with LH during the early spring transitional
period would promote the development of steroidogenically active, dominant follicles with the ability to respond to an ovulatory dose
of hCG. Mares during early transition were randomly assigned to receive a subovulatory dose of equine LH (in the form of a purified
equine pituitary fraction) or saline (transitional control; n 7 mares per group) following ablation of all follicles 15 mm. Treatments
were administered intravenously every 12 h from the day the largest follicle of the post-ablation wave reached 20 mm until a follicle
reached 32 mm, when an ovulatory dose of hCG (3000 IU) was given. Saline-treated mares during June and July were used as
ovulatory controls. In a preliminary study, injection of this pituitary fraction (eLH) to anestrus mares was followed by an increase in
circulating levels of LH (P 0.01) but not FSH (P 0.6). Administration of eLH during early transition stimulated the growth of the
dominant follicle (Group x Day, P 0.00001), which attained diameters similar to the dominant follicle in ovulatory controls (P 0.1).
In contrast, eLH had no effect on the diameter of the largest subordinate follicle or the number of follicles 10 mm during treatment
(P 0.3). The numbers of mares that ovulated in response to hCG in transitional control, transitional eLH and ovulatory control groups
(2 of 2, 3 of 5 and 7 of 7, respectively) were not significantly different (P 0.1). However, after hCG-induced ovulation, all transitional
mares returned to an anovulatory state. Circulating estradiol levels increased during the experimental period in ovulatory controls but
not in transitional eLH or transitional control groups (Group x Day, P 0.013). In addition, although progesterone levels increased after
ovulation in transitional control and transitional eLH groups, levels in these two groups were lower than in the ovulatory control group
after ovulation (Group, P 0.045). In conclusion, although LH supplementation of early transitional waves beginning after the largest
follicle reached 20 mm promoted growth of ovulatory-size follicles, these follicles were developmentally deficient as indicated by their
reduced steroidogenic activity.
© 2011 Elsevier Inc. All rights reserved.
Keywords: Mares; Transitional period; LH; Follicles; Ovulation; Estradiol
1. Introduction
The seasonal pattern of reproductive activity in the
mare involves a reduction in ovarian activity during the
winter months followed by a period of transition during
spring when follicular growth re-initiates leading to the
successive development of up to 3 or more waves with
large dominant follicles, which do not ovulate [1]. This
leads to a remarkable waste of time and resources by
veterinarians and breeders, who unsuccessfully try to
get mares in foal early in the year. Although exposure
to artificial lights is a proven method to effectively
* Corresponding author: Tel.: (0)131 5274331; fax: (0)131 440
0434.
E-mail address: xavier.donadeu@roslin.ed.ac.uk (X. Donadeu).
Available online at www.sciencedirect.com
Theriogenology 75 (2011) 1076 –1084
www.theriojournal.com
0093-691X/$ see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2010.11.016
Page 1
hasten the onset of the ovulatory season in mares, there
has long been a need in the horse industry for reliable,
shorter-term interventions that reduce the length of the
spring transitional period resulting in early ovulations.
Various hormonal treatments have been used to this end
[reviewed in 2], including daily injection of low doses
of hCG [3], however, these treatments have often
proven to be efficient only during the latter stages of the
transitional period, when follicle activity is already
significant. Some success has been recently achieved
by using FSH in spring transitional mares. Specifically,
FSH injected twice daily early during the transitional
period promoted the development of ovulation-compe-
tent follicles in many treated mares [4 –5], however, not
surprisingly, many of these mares double-ovulated thus
increasing the risk of multiple pregnancy.
Based on earlier reports [6], a deficiency of LH,
rather than FSH, may physiologically underlie deficient
follicle development during the transitional period and
in that regard LH supplementation may provide a more
rational approach to induce early ovulations. Scientific
evidence clearly indicates that available circulating
FSH is not a limiting factor for follicle development
during the transitional period [7]. In contrast, the re-
newed growth of dominant follicles at the onset of the
spring transitional period is associated temporally with
an increase in LH pulsatility [7– 8]. In addition, during
both the fall and spring transitional periods, signifi-
cantly higher LH concentrations were associated with
follicular waves involving a dominant follicle than with
waves producing only smaller follicles [9 –10]. Another
report showed higher circulating LH concentrations
early during development of the first ovulatory follicle
of the season than during the development of large
anovulatory follicles during the spring transition [11].
Developmental deficiencies of transitional follicles in-
clude an underdeveloped and poorly vascularized theca
cell layer together with reduced follicular cell expres-
sion of steroidogenic enzymes and LH receptors as well
as low IGF-1 bioactivity. All of these are associated
with severely reduced steroid production by these fol-
licles and are attributable, at least partially, to low
circulating LH levels [6]. Taken together, these obser-
vations indicate that adequate LH levels are required
during the anovulatory season in mares, both for the
growth of dominant follicles and for the acquisition of
ovulatory competence by these follicles. This conclu-
sion is consistent with experimental evidence that LH is
required for the growth of dominant follicles during the
ovulatory season in mares [12–14] and in cycling cows
[15].
Based on the above evidence, the present study
wished to directly test the hypothesis that supplemen-
tation of growing follicles with LH during early tran-
sition would stimulate development of steroidogeni-
cally active dominant follicles with the ability to
respond to an ovulatory stimulus, and that this could
therefore potentially be used to efficiently hasten the
onset of the ovulatory season.
2. Materials and methods
2.1. Experimental animals and equine LH
preparation
Seasonally anovulatory mares of mixed breeding,
mainly Welsh pony cross, three to 14 years of age,
with weights of 250 –500 kg and a history of good
reproductive health were used in the Northern Hemi-
sphere (55° N; Edinburgh, UK). Mares were kept
under natural light during the study. All experimental
procedures were carried out under the UK Home
Office Animals (Scientific Procedures) Act 1986, af-
ter approval by the Ethical Review Committee, Uni-
versity of Edinburgh.
Ovarian ultrasonography was performed twice
weekly in all mares starting during mid-December us-
ing a 7.5Mhz rectal transducer on a DP-6600 Vet Dig-
ital Ultrasonic Diagnostic Imaging System (BCF Tech-
nology, Livingston, UK). This was done to ensure that
mares were in deep anoestrus before the start of the
experiment, i.e., that no follicles 25 mm or a CL had
been detected by ultrasound for at least the previous
month.
A purified pituitary fraction was prepared from
crude equine gonadotropin (CEG 1.98) as described
[16] and used as source of equine LH (eLH). This
fraction was previously shown to contain only residual
FSH and to effectively induce ovulation when injected
into estrus mares [16]. The preparation used in the
present study was diluted in physiological saline to
contain 160
g eLH /ml, and its eFSH content was
determined to be below assay sensitivity (see below),
i.e., 1.56
g/ml. Two different eLH doses were used
to test the effects of LH supplementation in early tran-
sitional mares, 0.4 and 0.1
g/kg BW; these doses were
5- and 20-fold lower, respectively, than a dose of the
same fraction reported to induce ovulation in cycling
mares [16]. The rationale for this was that the injection
of sub-ovulatory doses of eLH would effectively stim-
ulate follicle growth without inducing early ovulation
or luteinization.
1077S.N. Schauer et al. / Theriogenology 75 (2011) 1076 –1084
Page 2
2.2. Preliminary eLH dose-response analysis
Deep anestrus mares were fitted with a jugular cath-
eter during January and injected once with either 0.4 or
0.1
g eLH/kg BW (n 5 mares/group). Blood sam-
ples were collected through the same catheter into hep-
arinized tubes at -30, -15, 0, 5, 10, 15, 30, 45, 60, 90,
120, 150 and 180 min relative to injection of eLH,
centrifuged at 1500g for 10 min and stored at -20 °C
until hormone analyses.
2.3. Experimental treatments and data collection
Once the first follicle 25 mm in diameter was detected
in the experimental mares after deep anestrus (beginning
early February), all follicles 15 mm were ablated by
transvaginal ultrasound-guided follicle puncture to induce
a new wave of follicular growth, as described [17]. There-
after, ovarian ultrasonography was performed every day
and at each ultrasound session the diameter of follicles 10
to 15 mm was estimated by comparison with the gradu-
ation marks on the scanner screen and follicles 15 mm
were measured with the electronic calipers. Follicles were
measured in two planes and the average of length and
width from a frozen image was taken as the actual diam-
eter. Ablated follicles that refilled with fluid to 15 mm
were re-ablated. Once the largest follicle of the ablation-
induced wave reached 20 mm, corresponding to just be-
fore the emergence of the dominant follicle [18], mares
were randomly assigned to receive eLH or an equivalent
volume of physiological saline vehicle (n 7 mares per
group). In mares assigned to receive eLH, this was in-
jected at a concentration of either 0.4
g/kg BW (n 4
mares) or 0.1
g/kg BW (n 3 mares); because there was
no significant main effect of Dose or an interaction be-
tween Dose and Day for any considered follicular growth
or hormone level endpoints (P 0.4), data from the two
dose groups were combined for comparative analyses
with controls and are shown as such throughout the man-
uscript. Treatments were administered iv every 12 h con-
tinuing until a follicle reached 32 mm at which time
3000 IU of hCG (Chorulon®) were injected iv to induce
ovulation. If no follicle reached 32 mm, treatments were
stopped once the largest follicle of the wave had ceased
growing for at least three days. Twice daily eLH injections
were performed in an attempt to simulate the natural
pattern of LH secretion during late diestrus, when the
ovulatory wave typically begins to develop, and which
involves a mean of one pulse of circulating LH every 12 h
[8,19]. Daily ultrasound monitoring of animals continued
until the dominant follicle reached 32 mm followed by
twice daily monitoring between the time of hCG injection
and ovulation; this increased ultrasound frequency was
used to precisely determine the time of ovulation after
hCG and to distinguish true corpora lutea from potential
hemorrhagic anovulatory follicles [20 –21]. The day of
ovulation was assigned to the ultrasound scanning session
when a CL was first detected.
Some of the mares used during transition (n 7)
were also used during the ovulatory season (June and
July). In this ovulatory control group, all follicles 15
mm were ablated 10 days after ovulation and once a
follicle reached 20 mm, mares were given saline iv
every 12 hours until a follicle reached 32 mm, when
hCG was administered to induce ovulation. Ovaries
were monitored and plasma samples were taken as
described for experiments during early transition.
Throughout experimental periods during both early
transition and ovulatory season, blood samples were
collected daily into heparinized tubes starting before
the first eLH or saline injection and continuing up to 13
days after ovulation or, if the dominant follicle did not
ovulate, until the follicle stopped growing for at least 3
consecutive days. Immediately after collection, plasma
was harvested by centrifugation at 1500g for 10 min-
utes followed by storage at -20 °C.
2.4. Hormone analyses
Plasma concentrations of LH and FSH were mea-
sured by radioimmunoassay as described [13]. Intra-
assay CVs and sensitivities were 14.8% and 0.4 ng/ml
for LH and 14.0% and 12.5 ng/ml for FSH.
Plasma concentrations of estradiol and progesterone
were measured by commercial radioimmunoassay kits
(Siemens Healthcare Diagnostics, Surrey, UK) vali-
dated in our laboratory by showing parallelism between
serial dilutions of equine plasma and the provided assay
standard curves. Before assaying for estradiol, samples
were extracted with ether, as described [10]. The re-
covery rate after extraction, as determined from sam-
ples containing tritiated estradiol, was 78%. Intra-assay
and interassay CVs and sensitivity were 17.9%, 19%
and 0.1 pg/ml, respectively, for estradiol, and 9.5%,
13.7% and 0.015 ng/ml, respectively, for progesterone.
For all hormones, sensitivity was calculated by sub-
tracting two standard deviations from the mean cpm at
maximum percentage binding and averaging over all as-
says.
2.5. Statistical analyses
Before statistical analyses, follicular and hormone
data taken over time were normalized to the day the
future dominant follicle reached 20 mm (determined
retrospectively) or to the day of ovulation. All data
1078 S.N. Schauer et al. / Theriogenology 75 (2011) 1076 –1084
Page 3
were tested for normality using a Kolmogorov-Smir-
noff test (P 0.05) and log-transformed if necessary.
Data were then subjected to analysis of variance using
a GLS procedure (R version 1.10.1) which accounted
for the autocorrelation between samples taken over
time, and the effects of Group, Day and the interaction
were determined. Whenever there was a significant
main effect or interaction, individual means were com-
pared using Tukey’s test. Group means involving per-
centages were compared using Chi-square analyses.
Statistical significance was considered at P 0.05.
3. Results
3.1. Short-term circulating gonadotropin responses
Changes in circulating gonadotropin levels follow-
ing injection in anestrus mares of the two different
doses of eLH are shown in Figure 1. Circulating LH
levels increased after injection (Time, P 0.01) and
this increase was more pronounced in response to the
0.4
g/kg than the 0.1
g/kg dose (Group, P 0.03),
so that pre-treatment circulating LH levels were re-
stored (P 0.05) by 30 min after injection of 0.1
g
LH/kg but only by 150 min after injecting the larger
dose (Fig. 1a). In contrast, FSH levels did not change
significantly after injection of any of the two eLH doses
(Day, P 0.6; Fig. 1b).
3.2. Follicular growth and ovulation
Mean follicular responses to treatments are pre-
sented in Figure 2 (normalized to the day the future
dominant follicle reached 20 mm; Day 0) and Table 1.
Administration of eLH to early transitional mares stim-
ulated growth of the dominant follicle (Group x Day, P
0.0001; Fig. 2a). As a result, on Days 4 and 5 follicle
diameters in that group were larger (P 0.05) than in
Fig. 1. Mean ( SEM) values for plasma concentrations of a) LH and b) FSH obtained form frequent jugular sampling after injection of eLH to
deep anestrus mares at doses of 0.4
g/kg (solid line, closed square) or 0.1
g/kg (dashed line, open square; n 5 mares/group). Whereas there
were no main effects of Time and Dose or an interaction for FSH (P 0.4), there were significant effects of both Time (P 0.01) and Dose (P
0.03) for LH concentrations. For LH, Dose means were different within each time point between 10 and 90 min (P 0.05). In addition, within
the 0.1
g/kg dose, each of the means at 5, 10 and 30 min were different from the mean at 0 min (P 0.05) and within the 0.4
g/kg dose, each
mean from 5 to 120 min was different from the mean at 0 min (P 0.05).
1079S.N. Schauer et al. / Theriogenology 75 (2011) 1076 –1084
Page 4
transitional controls and similar (P 0.1) to ovulatory
controls. However, there were no differences in diam-
eter of the largest subordinate follicle (Fig. 2b) or
number of follicles 10 mm (Fig. 2c) during treatment,
as indicated by the absence of main effect of Group or
an interaction for each of the two endpoints (P 0.3).
As shown in Table 1, the stimulatory effect of LH on
follicle growth resulted in a higher proportion of tran-
sitional mares (P 0.05) developing a follicle 32
mm during eLH treatment (6 of 7) than during treat-
ment with saline (2 of 7). For reasons unrelated to the
experiment, injection of one of the eLH-treated mares
with hCG once the dominant follicle reached 32 mm
was not possible. Of the remaining 5 mares, 3 ovulated
in response to hCG, whereas the two transitional con-
trol mares that developed a 32 mm follicle also ovu-
lated after hCG injection (Table 1). For mares injected
with hCG, there were no differences among the 3 ex-
perimental groups in the diameter of the dominant fol-
licle at hCG injection (P 0.4) or the diameter at
hCG-induced ovulation (P 0.6), with overall means
of 33.9 0.3 mm and 37.7 1.9 mm, respectively.
However, as shown in Table 1, the interval from hCG-
induced ovulation to the next (spontaneous) ovulation
was significantly longer (P 0.02) in the two transi-
tional groups than in ovulatory controls. In addition, the
interval from beginning of treatment to onset of cyclic
ovulatory activity was not different between LH- and
saline-treated transitional mares (P 0.3).
3.3. Circulating steroid levels
There was a Group by Day interaction for plasma
estradiol levels (P 0.013; Figure 3). Estradiol levels
during transition did not increase in eLH- or saline-
treated mares but did increase in ovulatory controls
between Days 1 and 4 (P 0.05) so that levels were
higher in the ovulatory group than in each of the tran-
sitional groups between each of Days 3 to 5 (P 0.05).
Circulating levels of progesterone after ovulation
in transitional mares that ovulated in response to
hCG and in ovulatory controls are shown (Fig. 4).
Progesterone data from eLH- and saline-treated tran-
sitional groups (n 3 and 2 mares, respectively)
were combined for statistical analyses as their mean
levels were similar. Although overall progesterone
levels increased after ovulation (Day, P 0.00001),
there was a significant effect of Group (P 0.045)
due to higher levels in ovulatory controls than in
transitional mares.
4. Discussion
While it is known that follicular responsiveness to
gonadotropins naturally change throughout follicle de-
velopment and across seasons [17], previous attempts
to stimulate follicular activity and ovulation in season-
ally anovulatory mares have not considered follicular
Fig. 2. Mean ( SEM) values for a) diameter of the dominant follicle,
b) diameter of the largest subordinate follicle and c) total number of
follicles 10 mm, normalized to the day the dominant follicle of the
post-ablation wave reached 20 mm (determined retrospectively) in
transitional mares treated with saline (solid line, open circle) or eLH
(solid line, closed circle) and in saline-treated ovulatory control
mares (dashed line, closed circle; n 7 mares/group). There was an
interaction between Group and Day for diameter of the dominant
follicle (P 0.0001). An asterisk indicates a significant difference
between the transitional control group and each of transitional LH
and ovulatory control groups within a day (P 0.05).
1080 S.N. Schauer et al. / Theriogenology 75 (2011) 1076 –1084
Page 5
wave status at the beginning of treatments. In this
regard, this study used a novel approach by adminis-
tering follicle-stimulating treatments to specifically tar-
get the growing dominant follicle of early transitional
waves, an approach that led to 6 of 7 mares developing
an ovulatory-sized follicle at a mean of 5 0.5 days
after the start of eLH-treatments. This result demon-
strated that eLH supplementation can effectively stim-
Table 1
Follicular growth and ovulation endpoints in eLH and saline-treated early transitional mares and in saline-treated ovulatory control mares.
Groups
Endpoint Transitional saline Transitional LH Ovulatory control
Interval from:
ablation of all follicles to first 20 mm-follicle (d) 2.9 0.3 4.1 0.8 2.9 0.1
beginning of treatment to dominant follicle 20 mm (d)* 0.4 0.3 0.6 0.4 0.1 0.1
beginning of treatment to first 32 mm-follicle (d) 17.0 4.5
a
6.6 1.6
b
4.1 0.3
b
hCG administration to ovulation (h) 60.0 12.0 56.0 8.0 56.6 8.6
(n 2) (n 3) (n 7)
hCG-induced ovulation to next ovulation (d)** 63.0 19.0
a
52.7 22.7
a
22.5 1.2
b
(n 2) (n 3) (n 7)
start of treatment to onset of ovulatory season (d) 60.3 9.5 53.0 11.7 NA
Percentage of mares that:
developed a 32 mm-follicle during the experimental period 28.6%
a
86.7%
b
100.0%
b
(2/7) (6/7) (7/7)
ovulated during the experimental period***
of mares injected with hCG 100.0% 60.0% 100.0%
(2/2) (3/5) (7/7)
of total mares 28.6%
a
50.0%
a
100.0%
b
(2/7) (3/6) (7/7)
N 7 mares per group, otherwise, actual numbers of mares are indicated below corresponding mean value.
Group means or percentages with different superscript (a,b) within endpoints are significantly different (P 0.05).
* Determined retrospectively.
** Transitional groups combined versus ovulatory control group, P 0.02.
*** HCG injection of one of the mares that developed a 32 mm-follicle in response to eLH was not possible and that mare was therefore
removed from analyses.
Fig. 3. Mean ( SEM) values for plasma estradiol concentrations
normalized to the day the dominant follicle of the post-ablation wave
reached 20 mm in transitional mares treated with saline (solid line,
open circle) or eLH (solid line, closed circle) and in saline-treated
ovulatory control mares (dashed line, closed circle; n 7 mares/
group). There was an interaction between Group and Day (P
0.013). Within the ovulatory control group, there was a mean differ-
ence between Days 1 and 4 (P 0.05). An asterisk indicates a
significant difference between the ovulatory control group and each
of transitional LH and transitional control groups within a day (P
0.05).
Fig. 4. Mean ( SEM) values for plasma progesterone concentrations
normalized to the day of ovulation in transitional mares that re-
sponded to an ovulatory dose of hCG (solid line, closed circle; data
from eLH- and saline-treated mares combined; n 5 mares) and in
ovulatory controls (dashed line, closed circle; n 7 mares). There
were main effects of Group (P 0.045) and Day (P 0.00001).
1081S.N. Schauer et al. / Theriogenology 75 (2011) 1076 –1084
Page 6
ulate growth of the dominant follicle of early transi-
tional waves thus confirming previous evidence of an
essential role of LH in promoting growth of dominant
follicles during both the anovulatory season [3,9] and
the ovulatory season [12] in mares as well as in other
species [15].
The observed effects of treatment on follicle devel-
opment could be attributed primarily to LH rather than
to residual FSH levels in the pituitary fraction. This
conclusion is based on 1) injection of the pituitary
fraction to deep anoestrus mares, in which levels of
both LH and FSH are reported to be minimal [22–23],
resulted in a clear increase in LH but no detectable
changes in FSH for up to 180 min after injection and 2)
an increased incidence of co-dominant follicles and
double ovulation, which is a common observation dur-
ing administration of recombinant eFSH to stimulate
early ovulation in transitional mares [4,24], was not
observed in mares injected with eLH in this study;
specifically, a co-dominant follicle (28 mm) was de-
tected in the experimental wave in one single mare
from each of the transitional control and transitional
eLH groups and no mare double-ovulated in any group
during the study. In this regard, as physiological up-
regulation of LH receptors is presumed to occur exclu-
sively in the future dominant follicle (usually only one)
of a wave during selection in mares [18], it is expected
that supplementation with eLH, unlike treatment with
eFSH, will not stimulate growth of additional follicles.
Although eLH robustly stimulated follicle growth,
the experimental hypothesis that supplementation with
eLH would promote the development of steroidogeni-
cally active, hCG-responsive follicles was not sup-
ported. An effect (positive or negative) on ovulation
could not be demonstrated as, although only 3 of 5
mares in the LH group ovulated in response to hCG
(compared to 7 of 7 mares in the ovulatory control
group), the proportion of mares that ovulated in re-
sponse to hCG was not significantly different among
the 3 experimental groups. This result needs to be
confirmed in future studies involving a larger number
of mares. In a previous report [3], daily low-dose hCG
treatment of seasonally anovulatory mares successfully
resulted in ovulation of most mares when administered
in March (7 of 8) but not in February (1 of 7), although
responses to hCG in that study may have been com-
pounded by the formation of hCG antibodies. It should
be noted that in the present study the two transitional
control mares treated with hCG ovulated. Although
reports have shown that most late transitional mares
with an ovulatory-size follicle will ovulate in response
to hCG [3], no critical studies have been undertaken to
determine the response of early transitional follicles to
an ovulatory dose of hCG. In the present study, after
hCG-induced ovulation, transitional mares took 58
14 days to ovulate (averaged over eLH and saline
groups), compared with a mean interovulatory interval
of 22.5 days in ovulatory season controls. This indi-
cates that, consistent with previous attempts to hormon-
ally stimulate ovulatory activity during deep anestrus or
early transition [2], induced ovulations in early transi-
tional mares in the present study were followed by a
return to the anovulatory state. As a result, the mean
interval between beginning of treatment and onset of
the natural ovulatory season was similar in eLH- and
saline-treated transitional mares (53 11.7 and 60
9.5 days, respectively). These intervals were in turn
similar to the mean duration of the spring transitional
period (54 days) reported for pony mares [7], thus
confirming that mares in this study were indeed in early
transition at the time of eLH injections.
One of the most interesting observations in this
study was that although LH administration effectively
promoted the growth of dominant follicles during early
transition, it did not restore the steroidogenic capacity
of these follicles, as demonstrated by the absence of an
increase in circulating estradiol during eLH treatments.
Examination of estradiol profiles of individual mares
from the two transitional groups revealed no apparent
increase in estradiol during the treatment period regard-
less of diameter attained by the dominant follicle or
ovulation outcome. This was in contrast with a clear
increase in estradiol during growth of the dominant
follicle in ovulatory controls, which was expected [25].
In agreement with the present results, ovulatory folli-
cles induced by daily low-dose hCG injection in tran-
sitional mares produced lower levels of estradiol than
spontaneously growing ovulatoy follicles [3]. More-
over, in the present study, corpora lutea derived from
transitional follicles that ovulated tended to produce
lower progesterone than corpora lutea from ovulatory
season controls, as indicated by differences in circulat-
ing progesterone levels between the two groups of
mares. This latter result is consistent with a previous
study showing reduced circulating progesterone after
ovulation in mares stimulated with GnRH during deep
anestrus relative to seasonally ovulating mares [26].
Transitional dominant follicles are naturally ste-
roidogenically deficient due, to a large extent, to an
underdeveloped theca cell layer and low expression of
steroidogenic enzymes in theca and granulosa cells as
well as low expression of LH receptors [27,28]. Steroid
1082 S.N. Schauer et al. / Theriogenology 75 (2011) 1076 –1084
Page 7
production by such follicles increases during the late
spring transition and this increase is required for proper
follicle maturation and eventually leads to an LH surge
and the onset of ovulatory activity [29]. Indeed, the first
ovulatory follicle of the season has been shown to have
restored estrogen-producing capacity [25]. It seemed
therefore plausible in the present study that LH supple-
mentation of early transitional waves beginning just
before emergence of a dominant follicle (i.e., when the
largest follicle reached 20 mm) would efficiently pro-
mote follicle maturation including increased steroido-
genesis. In this regard, the failure of LH to stimulate
steroid synthesis despite its clear positive effects on
follicle growth may be linked to the observation that
restoration of full steroidogenic capacity during the
natural period of spring transition occurs progressively
over several follicular waves [30], presumably in re-
sponse to increasing circulating LH levels [8]. Conceiv-
ably, an important effect of the increasing LH during
this period is to stimulate development of follicular
theca [27] so that 1) androgen production can be re-
stored to provide adequate substrate for the synthesis of
estradiol by late transitional dominant follicles [31] and
2) theca-derived luteal cells can later on contribute to
adequate levels of progesterone secretion by the CL.
Based on this, it can be hypothesized that stimulation of
steroid synthesis by dominant follicles during early
transition may be more effectively achieved by LH
supplementation beginning at a developmental stage
earlier than 20 mm, for example once follicles reach 10
mm or earlier, as this would stimulate theca develop-
ment and steroidogenic activity early on so that ade-
quate levels of androgens could be made available later
to meet the high demands of the dominant follicle for
estrogen precursor, a hypothesis that should be tested in
future studies. Additionally, other factors, such as pro-
lactin, may be required in addition to LH for ovulation-
competent follicles to fully develop in transitional
mares. In that regard, prolactin secretion naturally in-
creases in the mare in temporal association with sea-
sonal reproductive recrudescence [32] and it has been
shown that experimentally increasing circulating pro-
lactin levels can hasten ovulatory activity during the
spring [33–35].
In summary, supplementation of a follicular wave
with exogenous eLH during the early spring transition
in mares stimulated the growth of ovulatory-size folli-
cles indicating that deficient LH levels are naturally
implicated in the reduced growth of follicles during the
equine anovulatory season. Supplementation with eLH,
however, did not hasten the onset of the ovulatory
season. In addition, LH failed to stimulate steroid pro-
duction by the early transitional follicles and this was
associated with reduced levels of progesterone pro-
duced by corpora lutea derived from hCG-induced ovu-
lation of these follicles. In conclusion, although LH
supplementation of early transitional waves beginning
after the largest follicle reached 20 mm promoted
growth of ovulatory-size follicles, earlier and/or addi-
tional supplementation with LH or exposure to other
trophic factors may be necessary to induce full matu-
ration of these follicles.
Acknowledgments
The authors are grateful to Graeme Milne and Paul
Wright for animal care, Catalina Diaz and Kate Walker
for assistance with animal work, D Guillaume (INRA)
for help with LH assay, Y Combarnous (INRA) for the
gift of anti-FSH antibody and A.F. Parlow (NHPP) for
provinding purified FSH and LH, and anti-LH anti-
body.
This study was funded by the Horse Betting Levy
Board (Vet/Prj/745) and the Biotechnology and Bio-
logical Sciences Research Council.
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    • "More recently, twice daily treatment of the same preparation at doses that were 4-to 16-fold lower than the ovulation-inducing dose used in the present report readily stimulated follicular growth in early transitional mares [12]. Those effects were attributable to the LH activity of the eLH preparation rather than to any residual FSH [12]. Consistent with that conclusion, in the present study no significant differences were found in FSH levels between eLH-and vehicle-treated mares, either in samples collected immediately after eLH administration or at daily intervals throughout the treatment period. "
    [Show abstract] [Hide abstract] ABSTRACT: There is evidence in several species that high circulating LH concentrations can interfere with normal follicle development and ovulation. In the mare, high LH levels after induction of luteolysis with PGF(2α) have been temporally associated with an increased incidence of anovulatory follicles. We hypothesized that a premature increase in LH levels during a follicular wave in mares would disrupt normal follicle maturation leading to ovulatory dysfunction. In experiment 1, all follicles >10 mm were ablated at midestrous cycle in pony mares followed by twice daily administration of equine LH (eLH; 1.6 μg/kg body weight) or saline (vehicle; N = 8 mares per group). When a dominant follicle reached >32 mm, an ovulatory dose of hCG was given. Treatment with eLH had no effects on ovulatory responses or progesterone levels during the posttreatment luteal phase. In experiment 2, after follicle ablation, mares were treated with eLH or vehicle (as above) or were given a single injection of PGF(2α) (N = 7 mares per group), followed by aspiration of a dominant follicle when it reached >32 mm. Administration of eLH induced an increase in circulating LH levels similar to that after PGF(2α) injection. Neither PGF(2α) nor eLH administration had significant effects on follicle growth or total number of follicles in the postablation wave. However, compared with mares treated with vehicle, the preovulatory follicle in the eLH and PGF(2α) groups had lower levels of androstenedione (P = 0.03) and higher levels of insulin-like growth factor I (P = 0.03). Further, levels of prostaglandin E2 in preovulatory follicles tended to be lower in the eLH and PGF(2α) groups (P = 0.06). In conclusion, exposure of developing follicles to high LH in mares did not have apparent effects on ovulation but it induced changes in follicular fluid factor levels which might reflect a disruption in follicle and/or oocyte maturation, indicating the need to further study the implications of using PGF(2α) for the control of fertility in farm animals.
    Full-text · Article · Nov 2012 · Theriogenology
    0Comments 5Citations
  • [Show abstract] [Hide abstract] ABSTRACT: Previous evidence from in vitro studies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132 and miR-212, in regulating ovarian follicle development. The objective of this study was to determine changes in the levels of these miRNAs in relation to follicle selection, maturation and ovulation in the monovular equine ovary. In Experiment 1, follicular fluid was aspirated during ovulatory cycles from the dominant (DO) and largest subordinate (S) follicles of an ovulatory wave, and the dominant (DA) follicle of a mid-cycle anovulatory wave (n=6 mares). Follicular fluid levels of progesterone and estradiol were lower (P<0.01) in S follicles than in DO follicles, whereas mean levels of IGF1 were lower (P<0.01) in S and DA follicles than in DO follicles. Relative to DO and DA follicles, S follicles had higher (P≤0.01) follicular fluid levels of miR-145 and miR-378. In Experiment 2, follicular fluid and granulosa cells were aspirated from dominant follicles before (DO) and 24h after (L) administration of an ovulatory dose of hCG (n=5 mares/group). Relative to DO follicles, L follicles had higher follicular fluid levels of progesterone (P=0.05) and lower granulosa cell levels of CYP19A1 and LHCGR (P<0.005). Levels of miR-21, miR-132, miR-212 and miR-224 were increased (P<0.05) in L follicles; this was associated with reduced expression of the putative miRNA targets, PTEN, RASA1 and SMAD4. These novel results may indicate a physiological involvement of miR-21, miR-145, miR-224, miR-378, miR-132 and miR-212 in the regulation of cell survival, steroidogenesis and differentiation during follicle selection and ovulation in the monovular ovary.
    No preview · Article · Jun 2013 · Reproduction
    0Comments 20Citations
  • [Show abstract] [Hide abstract] ABSTRACT: Relatively little is known about the physiological roles of microRNAs (miRNAs) during follicular development. Previous evidence from in vitro studies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to gain insight on the involvement of these miRNAs during follicle maturation. Follicular fluid was aspirated from dominant follicles (>32 mm) during the ovulatory season (July to October) and the anovulatory season (January to March) in each of 5 mares, and the levels of steroids, IGF1, and miRNAs were analyzed by immunoassays and quantitative PCR. Levels of progesterone, testosterone, and IGF1 were lower (P ≤ 0.05) in anovulatory than in ovulatory follicles. Relative to ovulatory follicles, anovulatory follicles had higher (P < 0.05) mean levels of miR-21, miR-23b, miR-378, and miR-202 and tended to have higher (P = 0.06) levels of miR-145. Levels of miR-224 and miR-383 could not be detected in follicular fluid. These novel results indicate a physiological association between increases in follicular miRNA levels and seasonal anovulation in mares; further studies should elucidate the precise involvement of miR-21, miR-23b, miR-145, miR-378, and miR-202 in follicle maturation in the mare.
    No preview · Article · Jul 2013 · Domestic animal endocrinology
    0Comments 11Citations
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