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Introduction
The low summer fertility of about 60% of the world dairy
cattle population is associated with high ambient tempera-
tures. However, during the autumn, when air temperatures
have decreased and cows are no longer exposed to thermal
stress, conception rates remain lower than in the winter
(Hansen, 1997). This observation may be explained by the
facts that ovarian follicles are susceptible to heat stress
(Badinga
et al
., 1993; Wolfenson
et al
., 1995) and that it
takes about 40–50 days for small antral follicles to develop
into large dominant follicles (Lussier
et al
., 1987). Thus,
exposure to summer heat stress during the early stages of
follicular development may impair later follicular function
and decrease fertility in the autumn. Clear delayed effects of
summer heat stress on follicular function have been
observed: (i) a markedly low quality of oocytes collected
after summer heat stress was associated with low develop-
mental capability of embryos
in vitro
during the autumn
(Roth
et al
., 1999); and (ii) alterations in the pattern of
growth and development of medium-sized follicles asso-
ciated with a marked increase in plasma FSH concentration
were found during the first follicular wave of the oestrous
cycle, subsequent to heat exposure (Roth
et al
., 2000a). A
seasonal study has shown that oestradiol concentration in
the follicular fluid and androstenedione production by
thecal cells were both lower in dominant follicles collected
in autumn than in those collected in winter (Wolfenson
et
al
., 1997). However, seasonal studies provided only limited
information regarding a delayed effect of heat stress on
follicular steroidogenesis owing to their multifactorial
nature and the fact that proper contemporary control
animals could not be used in such studies.
The aim of the present study was to examine a possible
delayed effect of acute heat stress on follicular characteristics.
Steroid production by granulosa and thecal cells was
Delayed effect of heat stress on steroid production in
medium-sized and preovulatory bovine follicles
Z. Roth
1
, R. Meidan
1
, A. Shaham-Albalancy
1
,
R. Braw-Tal
2
and D. Wolfenson
1
*
1
Department of Animal Science, Faculty of Agricultural, Food and Environmental
Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel; and
2
Institute of Animal Science, Agricultural Research Organization, The Volcani Center
Bet Dagan 50250, Israel
Reproduction
(2001) 121, 745–751
Research
During the autumn, the conception rate of dairy cattle in
warm countries is low although ambient temperatures
have decreased and cows are no longer exposed to
summer thermal stress, indicating that there may be a
delayed effect of heat stress on cattle fertility. Two
experiments were conducted to examine possible delayed
effects of heat stress on follicular characteristics and
steroid production at two distinct stages of follicular
growth: medium-sized and preovulatory follicles, 20 and
26 days after heat exposure, respectively. Lactating
cows were subjected to heat stress for 12 h a day in an
environmental chamber, during days 2–6 of a synchro-
nized oestrous cycle. In Expt 1, ovaries were collected on
day 3 of the subsequent cycle, before selection of the
dominant follicle, and medium-sized follicles were
classified as atretic or healthy. In Expt 2, on day 7 of the
subsequent cycle, PGF
2α
was administered and pre-
ovulatory follicles were collected 40 h later. In both
experiments, follicular fluid was aspirated, granulosa and
thecal cells were incubated, and steroid production was
determined. In healthy medium-sized follicles (Expt 1),
oestradiol production by granulosa cells and androstene-
dione production by thecal cells were lower (
P<
0.05) and
the concentration of progesterone in the follicular fluid
was higher in cows that had been previously heat-stressed
than in control cows (
P<
0.05). In preovulatory follicles
(Expt 2), the viability of granulosa cells was lower
(
P<
0.05) and the concentration of androstenedione in the
follicular fluid and its production by thecal cells were
lower (
P<
0.05) in cows that had been previously heat-
stressed than in control cows. In both experiments, the
oestradiol concentrations in the follicular fluids were not
altered by heat stress. These results demonstrate a delayed
effect of heat stress on steroid production and follicular
characteristics in both medium-sized and preovulatory
follicles; this effect could be related to the low fertility of
cattle in the autumn.
© 2001 Journals of Reproduction and Fertility
1470-1626/2001
*Correspondence
Email: wolf@agri.huji.ac.il
studied at two distinct stages of follicular growth: in
medium-sized follicles before selection of the dominant
follicle and in preovulatory follicles 3–4 weeks after heat
exposure ended.
Materials and Methods
Two experiments were conducted in the winter.
Experimental design, tissue collection and cell incubation
were identical in both experiments. Delayed effects of 5
days of heat exposure on steroid production were examined
in medium-sized (Expt 1) and preovulatory (Expt 2) follicles.
Animals
Mature, cyclic Holstein dairy cows were selected for the
study. All experimental cows were housed in identical
chambers in the same shelter under the same husbandry
conditions from 1 week before cows were assigned to the
experimental groups to eliminate a possible confinement
effect. The cows were in late lactation, yielding an average
of 25 kg milk per day. The cows were fed a complete mixed
ration containing 16.5% (w/v) crude protein and 1.65 Mcal
per kg dry matter. This study has been reviewed and
approved by the appropriate institutional animal care and
use committee.
Experimental protocol
For synchronization of oestrus, an intravaginal
progesterone insert (CIDR; Eazi Breed, Hamilton) was
inserted for 9 days and 500 µg cloprostenol, a PGF
2α
analogue (Estrumate; Coopers, Berkhamsted), was injected
i.m. 7 days after insertion of the intravaginal progesterone
implant. Cows were checked for signs of oestrus three times
a day for 30 min each time. Cows expressing oestrous
behaviour within 48 h after removal of the CIDR were
included in the experiment. On day 2 of the oestrous cycle,
after confirmation of ovulation by ultrasonography, cows
were assigned randomly to either control or heat-stressed
groups. All cows were kept in the same shelter and were
milked in their housing environment. Control cows were
housed in the chambers under normothermic conditions
and heat-stressed cows were kept in similar chambers in
which air temperature and relative humidity were increased
to 36⬚C and 60%, respectively. Heat-stressed cows were
exposed to thermal stress for 12 h a day, between 07:00 h
and 19:00 h, during days 2–6 of the cycle. This early stage
of the cycle was chosen because the first follicular wave is
considered to be highly predictable in terms of follicular
dynamics. Although a 5 day duration of thermal stress was
sufficient to induce a significant immediate effect on
follicular steroidogenesis, a possible delayed effect was not
determined (Wolfenson
et al
., 1997). Accordingly, it was
decided to examine the delayed responses to such a short
heat stress in the time frame of the subsequent cycle, within
4 weeks after the end of heat exposure. At the end of heat
exposure, on day 7 of the oestrous cycle, control and heat-
stressed cows were grouped together and housed under
normothermic conditions. In both experiments, on day 18
of the treated cycle, PGF
2α
analogue (500 µg was injected
and cows showed signs of oestrus, on average, on day 21 of
the cycle. This synchronization procedure enabled the
interval between the end of heat exposure and the days in
the subsequent cycle on which ovaries were collected to be
set at a similar duration for all cows. Blood samples were
collected once a day during days 2–19 in the treated cycle
in both experiments, and during days 1–3 and days 1–7 of
the subsequent cycle in Expt 1 and Expt 2, respectively.
Blood samples were centrifuged at 2000
g
for 20 min and
plasma was stored at –20⬚C for determination of oestradiol
and progesterone concentrations.
In Expt 1, ovaries from control (
n
= 6) and heat-stressed
(
n
= 5) cows were collected on day 3 of the subsequent
cycle, 20 days after heat exposure was ended. The day of
ovary collection was selected to obtain the maximal
number of medium-sized follicles of the first follicular wave
before the suppressive influence of the dominant follicle
(Ginther
et al
., 1989; Fortune
et al
., 1991). Follicular
development was examined at 2 day intervals, from day 15
of the treated cycle to day 3 of the subsequent cycle, using
an ultrasound instrument (model SSD-210DXII; Aloka,
Tokyo) equipped with a 7.5 MHz transducer. Positions and
sizes of follicles and corpora lutea in the ovaries were
traced at each scanning and the exact location of the
follicles was recorded. This procedure enabled individual
follicles to be identified accurately during dissection of the
ovaries and selection of medium-sized follicles that had
grown during the first follicular wave of the subsequent
cycle and not those from the previous (treated) cycle.
In Expt 2, heat stress was applied on days 2–6 of the
cycle, as described for Expt 1. On day 7 of the subsequent
cycle, PGF
2α
analogue (500 µg) was injected to induce
regression of the corpus luteum and development of the first
wave preovulatory follicle and, after a further 40 h, ovaries
were collected from both heat-stressed (
n
= 4) and control
(
n
= 5) cows. Comparison of the morphology of the ovaries
with the ultrasonography records obtained on days 2, 4, 6
and 8 of the subsequent cycle enabled easy identification of
the preovulatory follicles.
Tissue collection and cell incubation
Ovaries from experimental cows were collected at the
abattoir after the cows were killed. During dissection of the
ovaries, the follicular diameter was measured with callipers
and follicular fluid from each follicle was aspirated and
stored separately at –20⬚C for determination of steroid
concentrations. The granulosa and thecal cells were
isolated from medium-sized follicles (6–9 mm in diameter;
Expt 1) or preovulatory follicles (Expt 2), dispersed
enzymatically and cultured separately. The viability of
granulosa and thecal cells was determined with 0.1% (w/v)
trypan blue as described previously (Meidan
et al
., 1990;
Wolfenson
et al
., 1999, Shores
et al
., 2000). Long-term
746
Z. Roth
et al.
experience in our laboratory has shown that low viability of
granulosa cells, as assessed by trypan blue, is associated
closely with oestradiol:progesterone concentration ratios
< 1 in the follicular fluid. For example, Shaham-Albalancy
et al
. (2000) found that granulosa cell viability and
oestradiol and progesterone concentrations in the follicular
fluid of healthy and atretic follicles were 66%, 31 ng ml
–1
and 16 ng ml
–1
versus 35%, 1 ng ml
–1
and 77 ng ml
–1
,
respectively. Accordingly, in Expt 1, medium-sized follicles
that expressed low viability of granulosa cells were
classified as atretic follicles and were not subjected to
further examination; the cell viability of these follicles was
21.3 ⫾ 5.4 and 18.6 ⫾ 2.8% in control and heat-stressed
cows, respectively. Follicles that had > 50% viability of
granulosa cells were classified as healthy follicles and were
subjected to further examination. The status of the healthy
medium-sized follicles was verified later by the observation
of a > 1 ratio of oestradiol:progesterone concentrations in
the follicular fluid (Ireland and Roche, 1983). Cells (10
5
viable cells per well) from individual healthy follicles were
incubated in a final volume of 0.5 ml in a 24-well plate
(Nunc, Kampstrup) in Dulbecco’s minimum essential
medium with Ham’s F-12 1:1 (v/v) nutrient mixture (Gibco,
BRL Life Technologies, Gaithersburg, MD), containing 1%
fetal calf serum (Biological Industries, Beit Ha’emek). Cells
were incubated for 6 h to determine steroid production
before spontaneous luteinization; a previous study in our
laboratory had indicated that granulosa cells maintained
aromatase activity after 6 h of incubation (R. Meidan and D.
Wolfenson, unpublished). Cells were incubated at 38⬚C
under 5% CO
2
. Granulosa cells were incubated in medium
only or with the addition of testosterone (300 ng ml
–1
;
Sigma, St Louis, MO). Thecal cells that had been isolated
from preovulatory follicles were incubated in medium only
or with the addition of forskolin (10 µmol l
–1
; Sigma) or LH
(50 ng ml
–1
; USDA bLH-B-5, provided through the USDA
Animal Hormone Program, Beltsville, MD). Thecal cells
that had been isolated from medium-sized follicles were
incubated in medium only or with forskolin (but not with
LH, for technical reasons). Three replicate wells were used
for each treatment. At the end of incubation, media were
collected from all wells and stored separately at –20⬚C for
determination of steroid concentrations.
Hormone analyses
Plasma samples were extracted with diethyl ether as
described by Badinga
et al
. (1992). Concentrations of
oestradiol, androstenedione and progesterone in follicular
fluid and medium, as well as the concentrations of
progesterone in extracted plasma, were determined by
radioimmunoassays that had been validated previously in
our laboratory (Meidan
et al
., 1990; Wolfenson
et al
., 1997,
1999) with specific antibodies (Sigma). The sensitivities of
the assays were 1.92, 3.9 and 3.9 pg per tube for oestradiol,
androstenedione and progesterone, respectively. Intra- and
interassay coefficients of variation were 9.9, 13.3 and 4.6%,
and 13.2, 13.9 and 8.6% for oestradiol, androstenedione
and progesterone, respectively. Oestradiol concentrations
in extracted plasma samples were determined by means of a
radioimmunoassay described by Badinga
et al
. (1992) that
had been validated in our laboratory (Shaham-Albalancy
et
al.
, 1997; Roth
et al.
, 2000a). Assay sensitivity was 0.5 pg
ml
–1
, and the intra- and interassay coefficients of variation
were 3 and 5%, respectively.
Statistical analysis
The General Linear Models procedure of the Statistical
Analysis System was used (SAS, 1987). Data relating to
follicular characteristics and hormonal concentrations in
the follicular fluid and media were analysed by one-way
ANOVA, separately for each type of follicle and cell. The
data of steroid hormone concentrations in the media of the
three replicate wells for each treatment were averaged, and
were analysed separately for basal and stimulated oestradiol
and androstenedione production. Concentrations of steroid
hormones in the media are expressed as ng per 10
5
viable
cells. The statistical model for concentration of hormones in
plasma included effects of treatment (control versus heat
stress groups), cow (within treatment), day of oestrous cycle,
and treatment by day interaction. Data are presented as
mean ⫾
SE.
Results
Body temperatures of heat-stressed cows increased
gradually to a maximum of 40.7⬚C, which is a level that is
maintained by high milk-yielding cows under natural
conditions during hot summer months. Control cows
maintained normothermia (body temperature of 38.7⬚C).
Experiment 1: delayed effect of heat stress on medium-
sized follicles
On the day of examination (day 3 of the subsequent
cycle) in both control and heat-stressed cows, a mean of
one medium-sized follicle per cow was recognized as a
follicle from the preceding cycle. These follicles had not
been dissected. The number of medium-sized follicles that
had emerged during the first follicular wave of the
subsequent cycle was similar in control and heat-stressed
cows (Table 1). More healthy medium-sized follicles were
counted in control cows than in heat-stressed cows (56 and
38% of total follicles, respectively), but this difference was
not significant. Healthy medium-sized follicles in control
and heat-stressed cows did not differ in terms of follicular
diameter, follicular fluid volume or number and viability of
thecal and granulosa cells (Table 1). The concentration of
progesterone in the follicular fluid was higher in heat-
stressed cows than in control cows (
P<
0.05; Table 2).
Androstenedione and oestradiol concentrations in follicular
fluid did not differ between groups. As expected, the
oestradiol:progesterone concentration ratio and the
oestradiol:androstenedione concentration ratio in the
Delayed effect of heat stress on steroidogenesis
747
follicular fluid of healthy follicles were > 1 in both groups.
Forskolin-stimulated androstenedione production by thecal
cells was three times higher in control cows than in heat-
stressed cows (
P<
0.05; Fig.1a). Oestradiol production by
granulosa cells in the absence or presence of testosterone
was also two or three times higher, respectively, in control
cows compared with heat-stressed cows (
P<
0.05; Fig.1b).
The concentrations of progesterone and oestradiol in
plasma did not differ between control and heat-stressed
cows during heat exposure (days 2–6: 0.6 ⫾ 0. 2 versus 0.9
⫾ 0.3 ng progesterone ml
–1
and 1.3 ⫾ 0.5 versus 1.5 ⫾ 0.3
pg oestradiol ml
–1
in control and heat-stressed cows,
respectively), after heat exposure (days 7–19 of the treated
cycle: 1.7 ⫾ 0.4 versus 1.5 ⫾ 0.4 ng progesterone ml
–1
and
1.9 ⫾ 0.6 and 1.6 ⫾ 0.4 pg oestradiol ml
–1
in control and
heat-stressed cows, respectively) and during the subsequent
cycle (days 0–3: 0.06 ⫾ 0.03 versus 0.08 ⫾ 0.03 ng
progesterone ml
–1
and 2.8 ⫾ 0.9 versus 2.6 ⫾ 1.1 pg
oestradiol ml
–1
in control and heat-stressed cows,
respectively).
Experiment 2: delayed effect of heat stress on
preovulatory follicles
The diameter, volume of follicular fluid, total number of
granulosa cells and viability of thecal cells of the
preovulatory follicles, collected on day 9 of the subsequent
cycle, did not differ between control and previously heat-
stressed cows (Table 3). However, the viability of granulosa
cells was lower (
P
< 0.05) in heat-stressed cows than in
control cows. The similarly high concentrations of
oestradiol (> 550 ng ml
–1
), together with the oestradiol:
progesterone and oestradiol:androstenedione concentration
ratios > 1 in the follicular fluid (Table 4), indicate that the
follicles examined in both groups were oestrogenically
active preovulatory follicles. The concentration of
androstenedione in the follicular fluid was lower in heat-
stressed cows than in control cows (
P
< 0.05). LH-
stimulated androstenedione production was lower in thecal
cells obtained from previously heat-stressed cows than in
those from control cows (Fig. 2;
P
< 0.05). Forskolin-
stimulated androstenedione production, although decreased,
was not affected significantly by heat exposure. Oestradiol
production by granulosa cells did not differ between
previously heat-stressed and control cows in either the
absence or the presence of testosterone (Fig. 2). Basal
progesterone production by granulosa cells did not
differ between previously heat-stressed and control cows
(2.3 ⫾ 1.2 versus 4.4 ⫾ 1.1 ng per 10
5
cells, respectively).
Basal, forskolin- or LH-stimulated production of
progesterone by thecal cells (0.4 ⫾ 0.1, 2.7 ⫾ 0.5 and
3.3 ⫾ 0.9 versus 0.3 ⫾ 0.1, 2.5 ⫾ 0.5 and 3.7 ⫾ 0.8 ng per
10
5
cells, respectively) also did not differ between
previously heat-stressed and control cows. The concen-
trations of progesterone and oestradiol in plasma did not
differ between control and heat-stress groups during heat
exposure (days 2–6: 1.8 ⫾ 0.9 versus 0.7 ⫾ 0.9 ng proges-
terone ml
–1
and 1.4 ⫾ 0.2 versus 1.6 ⫾ 0.3 pg oestradiol
ml
–1
in control and heat-stressed cows, respectively), after
heat exposure (days 7–19 of the treated cycle: 2.4 ⫾ 0.7
versus 3.8 ⫾ 0.7 ng progesterone ml
–1
and 1.4 ⫾ 0.2 versus
1.4 ⫾ 0.2 pg oestradiol ml
–1
in control and heat-stressed
cows, respectively) and during the subsequent cycle (days
0–7: 0.4 ⫾ 0.2 versus 0.6 ⫾ 0.1 ng progesterone ml
–1
and
2.6 ⫾ 0.3 versus 2.7 ⫾ 0.4 pg oestradiol ml
–1
in control
and heat-stressed cows, respectively).
Discussion
The present study provides, for the first time, evidence for a
delayed effect of heat stress on follicular steroidogenesis.
The delayed effect was detected in both medium-sized and
preovulatory follicles, but was expressed differently in
granulosa and thecal cells within each class of follicles.
Two experiments were performed during the winter to
avoid any potential seasonal carry-over effects from the
748
Z. Roth
et al.
Table 1. Characteristics of healthy medium-sized follicles of
control and previously heat-stressed cows at day 20 after heat
exposure
Characteristic Control Heat-stressed
Number of cows 6 5
Total number of follicles 2.6 2.6
per cow
a
Number of healthy follicles 1.5 1.0
per cow
b
Diameter (mm) 6.7 ⫾ 0.4 7.7 ⫾ 0.7
Follicular fluid volume (ml) 0.3 ⫾ 0.1 0.4 ⫾ 0.2
Number of granulosa cells 1.5 ⫾ 0.5 2.0 ⫾ 1.0
⫻ 10
6
per follicle
Viability of granulosa cells (%) 54.2 ⫾ 4.8 60.6 ⫾ 5.8
Viability of thecal cells (%) > 90 > 90
Values are mean or mean ⫾ SE.
a
Follicles that were shown by ultrasonographic monitoring to have grown
during the previous (treated) cycle were not dissected; those grown during
the first follicular wave of the subsequent cycle were dissected and included
in the data set.
b
Viability of granulosa cells > 50% and ratio of oestradiol:progesterone > 1.
Table 2. Steroid concentrations in the follicular fluid of medium-
sized follicles of control and previously heat-stressed cows
Steroid Control Heat-stressed
Oestradiol (ng ml
–1
) 23.4 ⫾ 18.6 76.2 ⫾ 25.0
Progesterone (ng ml
–1
) 18.0 ⫾ 8.5 47.8 ⫾ 11.4*
Androstenedione (ng ml
–1
) 8.6 ⫾ 1.6 3.9 ⫾ 2.5
Oestradiol:progesterone ratio 1.3 1.6
Oestradiol:androstenedione ratio 2.7 19.5
Values are mean ⫾ SE (control:
n=
6; heat-stressed:
n=
5).
*Significantly different from control group (
P
< 0.05).
previous summer. In addition, the experimental design, in
which heat-stressed cows were compared with contem-
porary control cows, enabled the acute heat exposure to be
associated with alterations in follicular function in medium-
sized and preovulatory follicles, 20 and 26 days later,
respectively. It might be expected that during the processes
of development and selection, the healthiest follicle from
the cohort of impaired medium-sized follicles would be
selected to become the dominant follicle. However,
alterations in follicular function were also found in
preovulatory follicles from previously heat-stressed cows.
Calculations made according to Lussier
et al
. (1987)
showed that the medium-sized or preovulatory follicles
studied (7.7 mm or 15.2 mm in diameter, respectively) had
diameters of about 0.5–1.0 mm when the cows were heat-
stressed. Follicles in such an early stage of follicular growth
are characterized by a high mitotic index of granulosa cells
(Lussier
et al
., 1987) and might be particularly sensitive to
environmental changes. It remains to be determined
whether very small antral (< 0.5 mm in diameter) or pre-
antral follicles are also susceptible to heat stress.
In terms of steroid production, the thecal cells were
found to be more susceptible than granulosa cells to heat
stress, and expressed a delayed effect of heat stress in both
classes of follicle. The consistent decrease in andro-
stenedione production in both medium-sized and pre-
ovulatory follicles in previously heat-stressed cows correlated
with the decreased concentrations of androstenedione in
their follicular fluid. This delayed effect of heat stress on
Delayed effect of heat stress on steroidogenesis
749
1.2
0.9
0.6
0.3
Basal Forskolin
(a)
a
b
Androstenedione (ng per 10
5
cells)
1.2
0.9
0.6
0.3
Basal Testosterone
(b)
a
b
Oestradiol (ng per 10
5
cells)
a
b
Fig. 1. Delayed effect of heat stress on steroid production in
medium-sized follicles. (a) Basal or forskolin-stimulated (10 µmol l
–1
)
androstenedione production by thecal cells and (b) oestradiol
production by granulosa cells in the presence or absence of
testosterone (300 ng ml
–1
). Columns represent production of cells
obtained from follicles of control (䊐;
n=
6) or previously heat-
stressed (䊏;
n=
5) cows on day 3 of the subsequent cycle, 20 days
after heat exposure. Values are mean ⫾
SE.
ab
Different letters
indicate significant differences between groups (
P
< 0.05).
Table 3. Characteristics of preovulatory follicles of control and
previously heat-stressed cows at day 26 after heat exposure
Characteristic Control Heat-stressed
Number of cows (one follicle
per cow) 5 4
Diameter (mm) 18.2 ⫾ 1.4 15.2 ⫾ 1.5
Follicular fluid volume (ml) 2.6 ⫾ 0.4 2.4 ⫾ 0.5
Number of granulosa cells
⫻ 10
6
per follicles 12.3 ⫾ 2.3 14.6 ⫾ 1.6
Viability of granulosa cells (%) 42.0 ⫾ 4.9 25.0 ⫾ 5.4*
Viability of thecal cells (%) > 90 > 90
Values are mean ⫾ SE.
*Significantly different from control group (
P
< 0.05).
4
3
2
1
Basal Forskolin
(a)
a
b
Androstenedione (ng per 10
5
cells)
30
20
10
0
Basal Testosterone
(b)
b
Oestradiol (ng per 10
5
cells)
LH
0
Fig. 2. Delayed effect of heat stress on steroid production in
preovulatory follicles. (a) Basal, forskolin-stimulated (10 µmol l
–1
)
or LH-stimulated (50 ng ml
–1
) androstenedione production by
thecal cells, and (b) oestradiol production by granulosa cells in the
presence or absence of testosterone (300 ng ml
–1
). Production
of steroids by cells obtained from follicles of control (䊐;
n=
5)
or previously heat-stressed cows (䊏;
n=
4) on day 9 of the
subsequent cycle 40 h after PGF
2α
administration, 26 days after
heat exposure. Values are mean ⫾
SE.
ab
Different letters indicate
significant differences between groups (
P
< 0.05).
androstenedione production by thecal cells indicates
strongly that the marked decrease in androstenedione
production by dominant follicles observed in the autumn
(Wolfenson
et al
., 1997) was due to previous exposure of
cows to summer heat stress. It has been shown that early
atresia of bovine follicles is characterized by a decrease in
androgen production by thecal cells (McNatty
et al
., 1984).
Thus, the above findings may indicate that both classes of
follicle obtained from previously heat-stressed cows had
been in an early stage of atresia. Early atresia in medium-
sized follicles could also be associated with low oestradiol
production by granulosa cells and increased progesterone
concentrations in the follicular fluid of heat-stressed cows.
Unlike the effect on thecal cells, the decreased production
of oestradiol by granulosa cells that was noted in medium-
sized follicles after heat stress was not carried over to the
preovulatory stage. Nevertheless, the delayed effect of heat
stress on granulosa cells in preovulatory follicles was
expressed in terms of a significant decrease of cell viability.
The reason for the difference in the responses of the two cell
types to heat stress within each of the two follicular stages,
or between the two stages, is not clear. It could be related to
the fact that granulosa cells acquire the steroidogenic
capacity at a later stage during follicular growth than do
thecal cells (Bao and Garverick, 1998).
The mechanism by which heat stress induces a decrease
in androstenedione production in thecal cells is not clear.
Recent analyses of mRNA content for LH receptor in thecal
cells obtained from preovulatory follicles did not provide
any evidence for alterations of mRNA content related to
previous heat exposure (Roth
et al
., 2000b). However, the
significant decrease of LH-stimulated, but not of forskolin-
stimulated, androstenedione production by thecal cells
noted in the present study may indicate that heat exposure
induced impairment of LH receptor function. Nevertheless,
lack of steroid precursor or compromise of other cell
function should be considered. Although a possible delayed
effect of heat exposure on LH secretion was not examined
in the present study, its involvement in the attenuated
androgen production cannot be ruled out. Seasonal studies
that might have shed some light on this subject provided
conflicting results regarding plasma LH concentrations after
summer heat stress (Crister
et al
., 1983; Day
et al
., 1986;
Badinga
et al
., 1994). Moreover, the specificity of heat-
stress as the environmental factor responsible for the
seasonal variations in LH secretion has not been examined.
In the present study, the oestradiol content of the
follicular fluid of preovulatory follicles was not affected by a
previous exposure to acute heat stress. This finding is in
agreement with the findings of Ambrose
et al
. (1999).
Comparison of the present findings concerning steroid
concentrations in the follicular fluid with those of previous
seasonal studies (Badinga
et al
., 1993; Wolfenson
et al
.,
1997) indicates that a decrease in oestradiol concentration
may depend on the duration and severity of the thermal
stress to which cows are exposed. Decrease of oestradiol
concentration in the follicular fluid is more likely to occur
after exposure to long-term, chronic (summer) heat stress
than to acute heat stress as in the present study. This
response would be consistent with the finding that after
chronic summer heat stress an eight times decrease in
androgen production by thecal cells in the autumn was
accompanied by a significant decrease in oestradiol
concentration in the follicular fluid (Wolfenson
et al
.,
1997). However, a 5 day acute heat stress (present study)
induced a three times decrease in androgen production,
which was probably not enough to elicit a decrease in
oestradiol concentration in the follicular fluid. In addition,
the oestradiol content in the follicular fluid reflects the
balance between production of the hormone by the cells
and its clearance from the follicle to the circulation. Thus,
the discrepancy between steroid production and hormonal
follicular fluid content found in the present study could be
related to heat-stress-induced alteration in vascular
responses. Hyperthermia has been shown to decrease
ovarian blood flow (Lublin and Wolfenson, 1996) and to
inhibit angiogenesis (Fajardo
et al
., 1988). Blood flow and
vascular density determine the follicular perfusion rate,
which directly influences the rates of nutrient uptake and
hormonal release by the follicle. The relationships among
heat stress, vascularity and steroidogenic capacity require
further investigation.
In conclusion, exposure of cows to heat stress resulted in
impaired steroidogenesis 20 and 26 days later, in medium-
sized and preovulatory follicles, respectively. The delayed
effect was expressed in a different way in granulosa and
thecal cells within each class of follicles. Granulosa cells
expressed low oestradiol production in medium-sized
follicles and low viability in the preovulatory follicles. In
terms of steroid production, thecal cells appeared to be
consistently susceptible to heat stress and expressed a carry-
over effect on androgen production in both types of follicle.
Delayed effects of heat stress on follicular steroidogenic
capacity, together with its delayed effects on follicular
dynamics (Roth
et al
., 2000a), as well as on oocyte quality
and embryo development (Roth
et al
., 1999), could be
responsible for the low fertility of dairy cows during the
autumn.
750
Z. Roth
et al.
Table 4. Steroid concentrations in the follicular fluid of
preovulatory follicles of control and previously heat-stressed cows
Steroid Control Heat-stressed
Oestradiol (ng ml
–1
) 600 ⫾ 122 560 ⫾ 136
Progesterone (ng ml
–1
) 22.3 ⫾ 4.0 12.9 ⫾ 3.6
Androstenedione (ng ml
–1
) 10.2 ⫾ 1.6 4.8 ⫾ 1.4*
Oestradiol:progesterone ratio 27.0 43.0
Oestradiol:androstenedione ratio 58.8 116.7
Values are mean ±SE (control:
n=
5; heat-stressed:
n=
4).
*Significantly different from control group (
P
< 0.05).
The authors would like to thank Y. Graber for technical
assistance and the USDA for provision of bLH. This work was
supported by a grant from the US–Israel Binational Agricultural
Research and Development Fund (BARD).
References
Ambrose JD, Guzeloglu A, Thatcher MJ, Kassa T, Dias T and Thatcher WW
(1999) Long-term follicular dynamics and biochemical characteristics of
dominant follicles in dairy cows subjected to heat-stress
Journal of
Reproduction and Fertility Supplement
54 503–504
Badinga L, Driancourt MA, Savio JD, Wolfenson D, Drost M, De La Sota RL
and Thatcher WW (1992) Endocrine and ovarian response associated
with the first wave dominant follicle in cattle
Biology of Reproduction
47 871–883
Badinga L, Thatcher WW, Diaz T, Drost M and Wolfenson D (1993) Effect
of environmental heat stress on follicular development and steroido-
genesis in lactating Holstein cows
Theriogenology
39 797–810
Badinga L, Thatcher WW, Wilcox CJ, Morris G, Entwistle K and Wolfenson
D (1994) Effect of season on follicular dynamics and plasma concen-
trations of oestradiol-17β, progesterone and luteinizing hormone in
lactating Holstein cows
Theriogenology
42 1263–1274
Bao B and Garverick HA (1998) Expression of steroidogenic enzyme and
gonadotropin receptor genes in bovine follicles during ovarian follicular
wave: a review
Journal of Animal Science
76 1903–1921
Crister JK, Miller KF, Gunsett FC and Ginther OJ (1983) Seasonal LH profile
in ovariectomized cattle
Theriogenology
19 181–191
Day ML, Imakawa K, Pennel PL, Zalesky DD, Clutter AC, Kittok RJ and
Kinder JE (1986) Influence of season and estradiol on secretion of
luteinizing hormone in ovariectomized cows
Biology of Reproduction
35 549–553
Fajardo LF, Prionas SD, Kowalski J and Kwan HH (1988) Hyperthermia
inhibits angiogenesis
Radiation Research
114 297–306
Fortune JE, Sirois J, Turzillo AM and Lavoir M (1991) Follicle selection in
domestic ruminants
Journal of Reproduction and Fertility Supplement
43 187–198
Ginther OJ, Kastelic JP and Knopf L (1989) Intraovarian relationships
among dominant and subordinate follicles and the corpus luteum in
heifers
Theriogenology
32 787–795
Hansen PJ (1997) Effects of environment on bovine reproduction. In
Current Therapy in Large Animal Theriogenology
pp 403–415 Ed. RS
Youngquist. WB Saunders, Philadelphia
Ireland JJ and Roche JF (1983) Development of nonovulatory antral follicles
in heifers; changes in steroids in follicular fluid and receptors for
gonadotropins
Endocrinology
112 150–156
Lublin A and Wolfenson D (1996) Effect on blood flow to mammary and
reproductive systems in heat-stressed rabbits
Comparative Biochemistry
and Physiology
115A 277–285
Lussier JG, Matton P and Dufour JJ (1987) Growth rates of follicles in the
ovary of the cow
Journal of Reproduction and Fertility
81 301–307
McNatty KP, Heath DA, Henderson KM, Lun S, Hurst PR, Ellis LM,
Montgomery GW, Morrison L and Thurly DC (1984) Some aspects of
thecal and granulosa cell function during follicular development in the
bovine ovary
Journal of Reproduction and Fertility
72 39–53
Meidan R, Girsh E, Blum O and Aberdam E (1990)
In vitro
differentiation of
bovine theca and granulosa cells into small and large luteal-like cells:
morphological and functional characteristics
Biology of Reproduction
43 913–921
Roth Z, Arav A, Bor A, Zeron Y, Ocheretny A and Wolfenson D (1999)
Enhanced removal of impaired follicles improves the quality of oocytes
collected in the autumn from summer heat-stressed cows
Journal of
Reproduction and Fertility Abstract Series
23 78 (Abstract)
Roth Z, Meidan R, Braw-Tal R and Wolfenson D (2000a) Immediate and
delayed effects of heat stress on follicular development and its
association with plasma FSH and inhibin concentration in cows
Journal
of Reproduction and Fertility
120 83–90
Roth Z, Meidan R, Preger E, Levi N, Braw-Tal R, Wolfenson D (2000b)
Delayed effects of heat stress on steroidogenesis in bovine preovulatory
follicles
Proceeding of the 14
th
International Congress on Animal
Reproduction
, Stockholm 1:15 (Abstract)
SAS Inc (1987)
SAS User’s Guide 6
th
Edition.
SAS Institute Inc, Cary, NC
Shaham-Albalancy A, Nyska M, Kaim M, Rosenberg M, Fulman Y and
Wolfenson D (1997) Delayed effect of progesterone on endometrial
morphology in dairy cows
Animal Reproduction Science
48 159–174
Shaham-Albalancy A, Rosenberg M, Folman Y, Graber Y, Meidan R and
Wolfenson D (2000) Two methods of inducing low plasma progesterone
concentrations have different effects on dominant follicles in cows
Journal of Dairy Science
83 2771–2778
Shores EM, Picton HM and Hunter MG (2000) Differential regulation of pig
theca cell steroidogenesis by LH, insulin-like growth factor I and
granulosa cells in serum-free culture
Journal of Reproduction and
Fertility
118 211–219
Wolfenson D, Thatcher WW, Badinga L, Savio JD, Meidan R, Lew BJ,
Braw-Tal R and Berman A (1995) Effect of heat stress on follicular
development during the estrus cycle in lactating dairy cattle
Biology of
Reproduction
52 1106–1113
Wolfenson D, Lew BJ, Thatcher WW, Graber Y and Meidan R (1997)
Seasonal and acute heat stress effects on steroid production by dominant
follicles in cows
Animal Reproduction Science
47 9–17
Wolfenson D, Sonego H, Shaham-Albalancy A, Shpirer Y and Meidan R
(1999) Comparison of the steroidogenic capacity of bovine follicular
and luteal cells, and corpora lutea originating from dominant follicles of
the first or second follicular wave
Journal of Reproduction and Fertility
117 241–247
Received 27 October 2000.
First decision 21 December 2000.
Accepted 30 January 2000.
Delayed effect of heat stress on steroidogenesis
751
