Nick Wassam

BIO 473 Sec 004

Aaron Monanti
Reproductive Endocrinology
Introduction:
Reproductive endocrinology studies many of the effects that hormones have on the
reproductive system and how different organs in the system interact with the brain and rest of the
body. In mammals the hypothalamus, anterior pituitary and gonads are responsible for a majority
of the interactions that control spermatogenesis and ovulation. The main hormones involved are
gonadotropin releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing
hormone (LH), inhibin and estrogen (Clayton & Catt, 2013). All of these combine in a feedback
loop to regulate the reproductive system.
Gonadotropin releasing hormone is released by the hypothalamus in the brain. This
release stimulates the anterior pituitary gland to release both LH and FSH into the blood stream.
These two hormones then interact with the ovaries and stimulate ovulation (Krecek, 1978).
During ovulation, the follicle, which consists of the oocyte (egg) and surrounding cells, will
become the corpus luteum and maintain levels of estrogen and progesterone in the female. After
ovulation estrogen and inhibin levels, produced by the ovaries, will rise and interact with the
brain in a negative feedback loop (Clayton & Catt, 2013). This negative feedback inhibits the
release of more GnRH, LH and FSH from the hypothalamus and the anterior pituitary. Once
blood levels of estrogen and inhibin begin to fall, the brain will begin to secrete GnRH, LH and
FSH once more.
An ovarectomy is the surgical removal of one or both ovaries. The removal of one ovary
is a unilateral surgery and the removal of both is considered a bilateral surgery (Waters &
Tomicek, 2014). Since the ovary is a part of the ovulation cycle and the negative feedback loop
controlling the hormones, removal will have an effect on both systems. Studying these effects is
beneficial because if a female should need to have one, or both, ovaries removed, it is important
to know the effects on the body. Doctors will then know what treatments to administer, and if the
female will be able to continue menstruation and have children.
This experiment will look at the effects of ovarectomy in rats. There is three groups in the
experiment, the control, unilateral removal and bilateral removal. Three organs were studied, the
ovary, uterine horn, and pituitary gland. It is expected that is a difference in weight between the
control and unilateral treatments for each of the three organs. It is also hypothesized that the
control and bilateral groups will be different and to a larger extent than the control versus
unilateral group. It is expected that there is a difference between the unilateral and bilateral
groups but not to the extent seen with the differences compared to the control group.
Methods:
Ketamine and xylazine were administered to the rats via the abdominal cavity or an
intraperitoneal injection. Ketamine put the rat in a sedated state and xylazine is a muscle relaxant
and pain killer. Bupivacaine was also administered during surgery to provide additional pain
medication. These drugs were administered so the rat was comfortable and experienced minimal
pain throughout the procedure.
Sprague-Dawley rats were used to observe the effects of the control, unilateral and
bilateral treatment groups. An instruction demonstrated how to pick up and hold the rats and the
same procedure was followed by students when handling the animals. A sterile operating site
was used during the surgery and each surgical team had an anesthesiologist, non-sterile assistant,
sterile surgical assistant and sterile surgeon. The roles are further described in the laboratory
manual. Each surgical group was given only one rat to operate on. The group then performed a
control experiment or unilateral removal of an ovary. The bilateral surgeries were performed by
the course instructor. For a full protocol on the surgery procedures, please refer to the Physiology
Laboratory Manual (Waters and Tomicek, 2014.) There was then a two week recovery period
following the surgery before the rats were euthanized. An autopsy was then performed on the
rats. Organ weight will determine if there was a change in the organ function following the
surgeries. The ovaries, if any remaining, were removed and weighed. The uterine horn, and
pituitary glands were also removed and weighed. The data was pooled across sections and T-
tests were used to see if there are differences in the weights of the organs. Again for full
procedures and protocol refer to the Physiology Lab Manual (Waters and Tomicek, 2014.)
Results:
The raw data can be accessed through the course on Angel. The following is the tabulated
results and figures for the pooled data.
Control Ovary Uterine Horn Pituitary
Average 0.1201875 0.580125 0.017
Variance 0.001171996 0.054734897 0.000132
Standard
Deviation
0.034234426 0.233954903 0.011489125
Standard
Error
0.006988073 0.047755845 0.002345208
n = 24

Table 1. Shows the calculated values for the control experiment. It shows the mean (average) weight for each organ autopsied
and the variance, standard deviation, and standard error for each group. The number of rats is seen as n=24.


Unilateral Ovary Uterine Horn Pituitary
Average 0.147111111 0.568 0.023470588
Variance 0.001795399 0.015441059 0.000806015
Standard
Deviation
0.042372145 0.124262057 0.028390398
Standard Error 0.00998721 0.029288848 0.006691681
n = 18

Table 2. Shows the calculated results for the unilateral treatment group. It has the average weight, variance, standard deviation
and standard error for the ovaries, uterine horn, and pituitary glands. The number of rats in this group is seen as n=18.
Bilateral Ovary Uterine Horn Pituitary
Average N/A 0.184247991 0.014839671
Variance N/A 0.067790678 0.000173816
Standard
Deviation
N/A 0.26036643 0.013183932
Standard Error N/A 0.08678881 0.004394644


n=9
Table 3 shows the calculated results for the bilateral treatment group. It has the average weight, variance, standard deviation
and standard error for the ovaries, uterine horn, and pituitary glands. The number of rats in this group is seen as n=9.
Tables 1, 2 and 3 have the calculated data for the control, unilateral and bilateral treatment
groups, respectively. They show the values of the mean weight, variance, standard deviation and
standard error of the data groups.

0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
Control Unilateral
M
e
a
n

W
e
i
g
h
t

(
g
)

Treatment Group
Figure 1. Mean Ovary Weight in Control versus
Unilateral Treatments
Figure 1 is the mean weight of the ovaries in the control and unilateral treatments. There is no bilateral data
because there are no ovaries when both are removed.

Figure 2 is the mean weight of the Uterine Horn in the control, unilateral and bilateral treatments.


Figure 3 is the mean weight of the Pituitary in the control, unilateral and bilateral treatments.
Figures 1, 2 and 3 show a side by side comparison of the mean weights of the ovaries, uterine
horn and pituitary glands, respectively. As seen in the figures, there is very little difference
between the treatment groups. The only major difference seen is in the uterine horn weight,
where the bilateral group is much lower than the other two treatment groups.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Control Unilateral Bilateral
M
e
a
n

W
e
i
g
h
t

(
g
)

Treatment Groups
Figure 2. Mean Uterine Horn Weight of Control
versus Unilateral versus Bilateral Treatments
0
0.005
0.01
0.015
0.02
0.025
0.03
Control Unilateral Bilateral
M
e
a
n

W
e
i
g
h
t

(
g
)

Treatment Group
Figure 3. Mean Weight of Pituitary Gland in
Control versus Unilateral versus Bilateral
Treaments
observed t
Control vs uni Control vs bi uni vs bi
ovarian 2.27788032 N/A N/A
40 N/A N/A
Uterine 0.199391746 5.247858914 8.670639661
40 31 25
Pituitary 1.014462418 0.831824343 1.015981894
40 31 25
Table 4 is the calculated observed t-value of the comparison between the three treatment groups. The degrees of freedom are
found underneath the calculated values.
t-significance Control vs uni Control vs bi uni vs bi
ovarian no N/A N/A


Uterine no yes yes


Pituitary no no no
Table 5. This is the T-significance results of the data when compared to a tabulated t-value. No means there is no significant
difference between the groups, and yes means there is a significant difference between the groups.
Tables 4 and 5 show the calculated t-values and results to a t-significance test. The observed t
was calculated as instructed in the laboratory manual. As the procedure guidelines on page 99
state, the critical tabulated t-value was found by using alpha = 0.01 and a two-tailed T-test. If one
were to use alpha = 0.05, then only one value is changed to being significantly different, the
other values remain the same.
Figures 1, 2 and 3 along with Table 5 show that there is no significant difference between
the control and unilateral groups for any of the organs. There is also no difference between the
average pituitary weights between any of the treatment groups. The only difference seen is
between the control versus the bilateral, and the unilateral versus the bilateral for the uterine horn
weight.
Discussion:
The data from the experiment show that there was no difference between the control and
the unilateral treatment for any of the organs. This is not what was expected and rejects the
hypothesis that there would be a difference between the control and unilateral groups. The data
also show that there is only a difference between the uterine weight of the bilateral when
compared to the unilateral and control groups. This agrees with the hypothesis that there will be
a difference seen in the bilateral group when compared to the other groups. However, this data is
weak because there is no difference seen in any of the other comparisons.
The lack of significance could be due to the small sample size of each treatment group.
The control group had only 24 rats, and the experimental groups had even less with the unilateral
group having only 18 surviving rats and the bilateral only had 9 surviving rats. This makes the
data weaker than it could be and the comparisons are less representative of what a larger sample
size would produce. There is also one erroneous data point that if excluded may cause a larger
difference in the pituitary size and could possibly increase the difference between the groups.
The sample size could also be increased to help determine if the results are reliable. The two
week period between the surgery and the autopsy may also not have been long enough for the
body to adjust to different hormone levels. It could be that some of the rats already had high
levels of estrogen and inhibin circulating in their system so the removal of the ovary had little
effect on the hormone levels.
To further improve the results, the serum levels of hormones could be measured at
different time intervals leading up to the autopsy. This could help confirm if the hormone levels
are affected from the surgery. It could also show if the stress from the surgery had a noticeable
impact on the rats. A longer time from the surgery to autopsy may help see a bigger change in
organ function and weight. Performing this experiment earlier in the year would allow for more
time between the surgery and autopsy, and could improve the results.
Unfortunately the data from this experiment is not what was hypothesized. It is also not
consistent with literature on the topic (Channing, Hillensjo, & Schaerf, 1978). A change in organ
function was to be measured by a change in organ weight for this experiment. The results show
that there was no difference between the control and unilateral groups, indicating no change in
organ function. The results also only show a change in the control versus bilateral and unilateral
versus bilateral groups for the uterine horn weight. This is not strong evidence because a
resulting change in the pituitary should have been seen since the feedback loop, mentioned in the
introduction, can no longer function properly without the ovaries. In conclusion, the errors and
results of the experiment show there is little to no effect in organ weight after two weeks of
surgery.
References:
Channing, C., Hillensjo, T., & Schaerf, F. (1978). Hormonal control of oocyte meiosis, ovulation
and luteinization in mammals. Clinics in Endocrinology and Metabolism, 7(3), 601-624.
Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/215357
Clayton, R., & Catt, K. (2013). Regulation of pituitary gonadotropin-releasing hormone
receptors by gonadal hormones. Endocrinology, 108(3), retrieved from
http://press.endocrine.org/doi/abs/10.1210/endo-108-3-887
Krecek, J. (1978). Effect of ovarectomy of females and oestrogen administration to males during
the neonatal critical period on salt intake in adulthood in rats. Physiologia
Bohemoslovaca, 27(1), 1-5. Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/148053
Waters, J. and Tomicek, N. (2014). Physiology Laboratory Manual. Hayden-McNeil, LLC.
Plymouth, MI.