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Phill Rasnick

TA: Nathan Garvin


Biology 473/Section 008
April 12, 2013

The Effect of Varying Reproductive Hormone Levels on the Brain-Ovarian Axis in Female Rats

Introduction
The regulation of reproduction and the hormones involved takes place through hormonal
feedback loops between the brain and gonads; in females, this relationship is known as the brain-ovarian
axis and is under control by the hormones gonadotropic releasing hormone (GnRH), follicle stimulating
hormone (FSH), luteinizing hormone (LH), estrogen, progesterone, and inhibin. The menstrual cycle is
initiated by an increase in gonadotropins from the anterior pituitary including FSH and LH. Under the
influence of FSH follicles within the ovary begin to grow and develop. FSH and LH also respectively
stimulate the granulosa cells and thecal cells of the follicles to secrete steroid hormones which are
converted to estrogen by the granulosa cells. Increasing estrogen levels has a negative feedback on the
pituitary FSH and LH secretion, preventing the development of additional follicles. Despite the
decreasing FSH and LH levels, the increasing estrogen levels stimulate a further increase of estrogen
through a positive feedback pathway with the granulosa cells. Uterine endometrium proliferates under the
influence of estrogen by increasing in cell number and enhancing blood supply to prepare for the
implantation of a fertilized egg. Once follicular development reaches a particular point, there a large surge
in estrogen levels with an accompanying secretion of inhibin and progesterone. During this time, estrogen
and progesterone enhance the pituitarys responsiveness to GnRH. While estrogen provides negative
feedback on gonadotropin release through inhibition of the hypothalamus and anterior pituitary,
persistently high estrogen levels provide positive feedback, leading to a surge in GnRH followed by
largely increasing section of LH and FSH. Inhibin secretion, however, inhibits FSH secretion resulting in
the FSH peak to be significantly reduced when compared to the LH peak.
The high levels of LH are responsible for ovulation to occur. This is marked by the egg being
released from the follicle and entering the fallopian tubes. Without the egg, the follicle develops into a
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structure known as the corpus luteum, responsible for producing increasing amounts of progesterone and
estrogen. These hormones exert negative feedback on the hypothalamus and pituitary suppressing
gonadotropin secretion in addition to the suppression by inhibin production. The corpus luteum has a
lifespan of only a few weeks and spontaneously undergoes apoptosis if pregnancy does not occur.
Estrogen and progesterone levels decline, stopping the negative feedback on the gonadotropins and causes
the levels of FSH and LH to rise again as the menstrual cycle restarts. Because the endometrium is
maintained by the presence of progesterone, decreasing levels of progesterone cause the endometrium to
break down resulting in menstrual discharge.
In this experiment, unilateral and bilateral ovariectomies are carried out in addition to sham
operations to observe the roles of these circulating reproductive hormones as well as the effect of varying
hormone levels on the female sexual structures in a rat. It is hypothesized that performing a unilateral
ovariectomy on the rat will result in an increased size of the second ovary when compared to the control
group. This is expected because with removal of an ovary, the other ovary may need to overcompensate
to ensure levels of estrogen are sustained. The single ovary in the unilateral rat will be exposed to a higher
concentration of gonadotropins because the other ovary is not there to make use of them. This should
result in the single ovary producing more estrogen than normal. Additionally, the decreased estrogen
levels should also result in less negative feedback on gonadotropin production, further increasing
circulating gonadotropin levels and also estrogen production in that single ovary. In terms of the uterine
horn, it is hypothesized that there should be no significant change in the size between sham operation and
unilateral ovariectomy groups because levels of estrogen in both groups are expected to be elevated
enough to sustain the endometrium. However, when comparing the bilateral ovariectomy results to the
control and unilateral data, it is expected that there should be a significant decrease in size of the uterine
horn of the bilateral experimental group due to estrogen production being diminished. When considering
the effect of varying reproductive hormone levels on the pituitary gland, it is expected that there will be
an indirect relationship between pituitary size and levels of circulating estrogen due to a decrease in
negative feedback on the pituitary resulting from diminished estrogen levels. As a result, it is expected
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that rats post sham operation will have a slightly smaller pituitary glands when compared to the unilateral
or bilateral ovariectomy groups. Also, because of the decreased negative feedback, it is expected that rats
from the unilateral test group will have pituitary glands larger than the control group but smaller than the
bilateral ovariectomy group. Thus, pituitary size in post bilateral ovariectomy rats is expected to be
greater in size than the pituitary gland for both the control and unilateral ovariectomy groups.
From a clinical standpoint, the data collected during this experiment can be useful in identifying
the effect of menopause on females. This is because conducting an ovariectomy is the surgical equivalent
to menopause, marked by the time when the ovaries can no longer be receptive of gonadotropin signals
thus producing no estrogen.
Methods
In this experiment ovariectomies were carried out to provide information on the effect of varying
levels of circulating reproductive hormones. Data was collected for both unilateral and bilateral
ovariectomies, with a sham operation providing results for the control. The term ovariectomy refers to the
surgical removal of an ovary. Unilateral ovariectomies revolved around the removal of one ovary, while
the bilateral procedure removed both ovaries from the rats. Experimental protocol was followed exactly
from the biology 473 Rodent Survival Surgery Protocol Handout. Sterility was maintained throughout the
entire procedure, and local anesthetics were used to ensure proper handling of the rats. Pre surgery,
Ketamine was first used to bring the animal to a dream-like state of mind followed by use of xylazine as
an analgesic and muscle relaxant. Bupivacaine was additionally added post-surgery to the sutured muscle
wall to provide further pain relief. To determine the effect of varying levels of reproductive hormones on
the brain-ovarian axis, the animals were euthanized 3 weeks post-surgical procedure, and gonad, uterine
horn, and pituitary weights were collected.
Two tailed t-test analysis was used to identify the significance of the results. To identify
significance, an experimental alpha value of 0.05 was used. Because 3 different tests are being carried
out, the Bonferroni method was used to correct the alpha comparison level to a value of 0.01.

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Results
Figure 1: The Effect of Ovariectomy on Ovary Size in Control and Post Unilateral Overiectomy Rats

Figure 1: Displays the mean ovarian weights for collected from sham operated control groups and
unilateral ovariectomy groups. The Ovariectomy appeared to have a positive effect on ovary size in the
post unilateral experimental group. Average ovary size in the control was found to be 0.109 grams while
the ovary in post ovariectomy rats averaged 0.148. Standard error was additionally calculated to provide
the approximate margin of error for each experimental group.

Based on the results displayed in figure 1, performing the ovariectomy resulted in an increase of
size for the remaining ovary of the unilateral ovariectomy group when compared to control data. T-test
calculations were used to indicate that the ovary size in the unilateral ovariectomy test group was
significantly greater than ovary size for the control treatment (t=3.065, df=9, 2=0.01).














0.000
0.020
0.040
0.060
0.080
0.100
0.120
0.140
0.160
0.180
Experimental Group
W
e
i
g
h
t

(
g
)
Effect of Ovariectomy on Ovary Size
Control
Unilateral
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Figure 2: The Effect of Ovariectomy on Uterine Horn Weight

Figure 2: Presents experimentally collected data for uterine horn weights post unilateral ovariectomy,
bilateral ovariectomy, and sham operated rats. No significance was found between weight differences
among control and unilateral experimental groups, however, the bilateral ovariectomy resulted in a
significant decrease in uterine horn weights when compared to either control or unilateral test groups.
Average weight of the uterine horn post sham operation, post unilateral ovariectomy, and post bilateral
ovariectomy were 0.602 grams, 0.569 grams, and 0.154 grams respectively. Standard error was
additionally calculated to determine the error interval for each experimental group.

Results from figure 2 indicate that unilateral ovariectomies had little to no effect on the size of the
uterine horn when compared to control groups. Statistical calculations show no significance difference in
uterine horn weights between control and unilateral operated groups (t=0.503, df=30, 2=0.01). Although
no difference was observed between the unilateral and control groups in terms of uterine horn size, t-test
analysis demonstrated that the uterine horn size had been significantly decreased in the bilateral group
when compared to the control (t=7.232, df=23, 2=0.01). Additionally, there was a significant decline in
uterine horn size in the bilateral experimental group in comparison to the unilateral test group (t=5.767,
df=23, 2=0.01)







0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
Experimental Group
W
e
i
g
h
t

(
g
)
Effect of Ovariectomy on Uterine Horn Size
Control
Unilateral
Bilateral
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Figure 3: The Effect of Ovariectomy on Pituitary Gland Weight

Figure 3: Presents the experimentally collected data for average pituitary size in the experimental rat
populations for the unilateral ovariectomy, bilateral ovariectomy, and sham operation test groups. For the
control, unilateral, and bilateral test groups, average pituitary weight collected was 0.016, 0.017, and
0.014 grams respectively. Standard error was calculated to approximate error bars and the interval for
error for each experimental group.

Based on the results in figure 3, no significant change was found in pituitary size between any test
groups. T-test identified no significant difference between pituitary size for control and unilateral test
groups (t=0.343, df=27, 2=0.01). Statistical analysis also identified no significant difference in pituitary
size between control and bilateral ovariectomy groups (t=0.646, df=20, 2=0.01). There was additionally
no significant difference determined for weight size between unilateral and bilateral test groups (t=0.962,
df=19, 2=0.01).
Discussion
In support of the hypothesis, the t-test showed that there was a significant difference in
ovarian size between the sham operation control group and the unilateral ovariectomy
experimental group. Figure 1 indicates that the unilateral ovariectomy had a significant effect on
ovary size, resulting in an increase in ovary size for the group in which unilateral ovariectomy
had been performed. This could be due to an increase in estrogen production for that single
ovary, resulting from excess stimulation from the gonadotropins. The cause of this could be a
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
Experimental Group
W
e
i
g
h
t

(
g
)
Effect of Ovariectomy on Pituitary Size
Control
Unilateral
Bilateral
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decrease of negative feedback on the pituitary resulting from lower levels of estrogen. These
results are in strong support of the hypothesis because of the indicated significance between the
two groups.
The t-test additionally showed strong support for the hypothesis involving the effect of
ovariectomies on the size of the uterine horn. A significant difference was observed between the
control and bilateral groups as well as between the unilateral and bilateral groups. According to
figure 2, the size of the uterine horn was significantly decreased in the bilateral experimental
group when compared to both control and unilateral groups. Because estrogen is responsible for
maintaining the endothelial lining of the uterus, it was expected that without estrogen, as occurs
in the bilaterally treated group, the endothelium will degenerate. No difference was observed
between unilateral and control groups in this test because the ovary in rats that had undergone the
unilateral procedure is still capable of producing enough estrogen to support the uterine
endothelium
Opposing the hypothesis, no difference was observed in pituitary weight between any of
the tested groups as shown in figure 3. Also known as the master gland, the pituitary gland is
responsible in controlling numerous processes in the body, secreting more hormones than just the
gonadotropins discussed. Some of these hormones include growth hormone for the regulation of
growth, thyroid stimulating hormone to regulate the thyroid and metabolism, as well as prolactin
which is involved in the production of breast milk. As a result, the size of the pituitary gland may
not be significantly affected by levels of estrogen due to the numerous other processes affected
by the gland. Due to the relatively small size of the pituitary gland, and the fact that the feedback
loop between the gonadotropins and sex hormones occur on the anterior pituitary, even if a
difference did occur it may not have been identifiable. Further tests using a bigger sample size
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and additional means of measuring size of the anterior pituitary could be better used to indicate
any differences in pituitary size due to changes in estrogen levels.
The main sources of error in this experiment are a limited sample size and issues
associated with collecting the organs post procedure for measurement. For the rats that had
undergone ovariectomies, it was identified that many of the uterine horns were inflamed and
swollen with excess scar tissue as a result of the procedure. Prior to collecting uterine horn
weights, much of the swollen tissue was removed. This could have resulted in a decrease in
uterine horn weight for the ovariectomy test groups, however, based on the results obtained it is
unlikely that this slight change accounted for the significant reduction in uterine horn size
identified in the bilateral experimental group. Additional error could have occurred with
collecting data on the pituitary gland. As indicated by data, several groups were incapable of
isolating the pituitary gland. Because of the small size of the pituitary gland, if the organ was not
collected intact, there would be a negative effect on the observed weight. As discussed, further
experiment should be used to help identify the effect of estrogen on pituitary size.
Overall, the results of this data can be used to identify issues that occur in post-
menopausal women because an ovariectomy is the surgical equivalent to menopause. After
menopause, ovaries lose the ability to be regulated by circulating gonadotropins resulting in an
absence of estrogen production. This is carried out in experiment through ovariectomy by
removing one or two ovaries causing a respective decrease or termination of estrogen production.
Due to the lack of estrogen, menopausal women are susceptible to varying symptoms including
hot flashes, genitalia and breast atrophy, and as well as osteoporosis. Because of the issues
associated with depletion of estrogen, menopausal women are often subject to hormone
replacement therapy which typically consists of both estrogen and progesterone. In more recent
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years, selective estrogen receptor modulators (SERMs) have been used to treat issues associated
with menopause by selectively binding to estrogen receptors at different affinities.

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References
Overview of Reproductive Hormones. PSU, Biol 473 Laboratory Sp 2013
Biol. 473 Rodent Survival Surgery Protocol Handout. PSU, Biol 473 Laboratory Sp 2013
Guidelines for Analyzing Your Reproductive Endocrinology Data. PSU, Biol 473 Laboratory Sp 2013
Silverthorn, Dee. Human Physiology an Integrated Approach. 6 ed. Glenview, IL: Pearson, 2013. Print.