Rina Allawh

The effect of the type of reproductive treatment on levels of circulating reproductive hormones and on the
weight of the ovary, uterus and pituitary gland of a Rattus norvegicus.

Introduction:

The synthesis and release of reproductive hormones are vital in the maturation and growth of various

female reproductive structures, such as the uterus and the ovary.1 The production and secretion of female

reproductive hormones is intricately regulated by neural signals through a hypothalamus-pituitary-ovarian

axis.1 In fact, in the brain, the hypothalamus produces and releases a trophic hormone known as

Gonadotropin Releasing Hormone (GnRH), which triggers the release of the Follicle Stimulating Hormone

(FSH) and the Luteinizing Hormone (LH) from what is commonly referred to as the master gland, the

anterior pituitary gland.1

Once released from the anterior pituitary gland, FSH and LH travel through the bloodstream and act

synergistically on the ovary, the target organ.1 As peptide hormones, FSH and LH bind to the G-protein

coupled transmembrane receptors on the ovary and consequently, activating a cAMP signal transduction

system.1 More specifically, FSH in both males and females acts to initiate and maintain gametogenesis.1 LH

has differing roles in males and females, wherein in males, LH stimulates the secretion of testosterone from

Leydig cells.1 However, in females, LH stimulates the production of estrogen by follicular cells in the

ovary.1

Also, a surge of LH in females stimulates the creation of the corpus leuteum from the follicular

tissue following ovulation.1 If an egg is fertilized and implanted in the uterine wall, the embryo produces

Human Chorionic Gonadotropin (HcG), which prevents the disintegration of the corpus leuteum in order to

maintain progesterone and estrogen levels.1 In fact, progesterone and estrogen are two key female sex

hormones; progesterone is important for the development of the fetus and decreases the contractility of the

uterine smooth muscle, as well as, acting to inhibit lactation.2 Estrogen, which is secreted by the ovaries,

stimulates the growth of the uterus and vagina at puberty and is important for egg maturation.2 Interestingly,

estrogen is produced in males, yet its effect is overshadowed by androgens.2

 Additionally, estrogen helps to maintain reproductive hormone levels via a negative feedback

mechanism. Through negative feedback mechanisms, high levels of estrogen and inhibin, whereby inhibin is

either produced in the ovary in females or is secreted from sertoli cells in the seminiferous tubules in males,

collectively inhibit GnRH secretion from the hypothalamus and FSH and LH secretion from the anterior

pituitary gland.3 Thus, the release and production of reproductive hormones is tightly controlled as a result

of both negative feedback mechanisms and the relationship between the hypothalamus and the anterior

pituitary in the brain, as well as, the gonads.3

Moreover, in order to understand the effect of reproductive hormones on the maturation and the

development of the pituitary gland and reproductive structures, an experiment was conducted, whereby the

effect of different types of reproductive treatments were analyzed, which include the control treatment, a

unilateral ovariectomy and a bilateral ovariectomy.4 The purpose of the control treatment was primarily to

act as a comparison when analyzing the effect of the removal of one or both of the ovaries.4 In fact, when

comparing the control treatment and the unilateral ovariectomy, it was hypothesized that both the ovary and

the uterus would have a greater mean weight for the rats that had a unilateral ovariectomy because following

the removal of one ovary, the remaining ovary will attempt to compensate for this loss by increasing the

production of estrogen and as a result, the weight of the remaining ovary increases. 4

Since, there is an increase in estrogen production, the growth of the uterus is stimulated more in

comparison to the control rats and so, it was hypothesized that the mean uterine weight would be slightly

greater for the rats that had a unilateral treatment compared to the rats that had a control treatment. On the

other hand, since the pituitary gland is still regulated by the estrogen via a negative feedback mechanism, it

was predicted that the pituitary gland would remain the same weight between the control and the unilateral

treatment rats. To compare the control and bilateral treatments, it was hypothesized that the weight of the

pituitary gland would be the same in both treatments because the pituitary gland acts as a master gland and

regulates a number of other processes and thus, altering estrogen levels will fail to have a large effect on the
pituitary glands average weight. Also, it was hypothesized that the mean weight of the uterus would be less

for the rats that had a bilateral ovariectomy, as a result of the removal of the source of estrogen.

Additionally, to compare the unilateral and bilaterial ovariectomies, it was hypothesized that the

mean weight of the uterus will be less for that rats that had a bilateral ovariectomy in comparison to the rats

that had a unilateral ovariectomy. Similarly, it was predicted that the weight of the pituitary gland will be the

same for both treatments. In actuality, the experiment conducted is valuable in comprehending not only the

effect of the reproductive hormones on the development of reproductive structures, but also to understand the

endocrine pathway between the hypothalamus, the anterior pituitary and the gonads. More importantly, this

experiment provides insight to Biologists and physicians in regards to the interplay between the reproductive

hormones and the maturation of the reproductive system.

Methods:

The experiment conducted was characterized by two distinct phases, the surgery and the autopsy, in

which a laboratory rat (Rattus norvegicus) was used as a model organism.4 Each lab group performed either

a control experiment, in which only the fat pad was lightly touched or a unilateral ovariectomy, where one of

the ovaries was surgically removed.4 Despite the differences between the different types of treatments, each

treatment was considered to be a survival surgery because no euthanasia was administered to any of the

laboratory rats.4 In fact, in all of the surgeries performed, the same amount of anesthesia and analgesics were

administered either directly under the skin, into the muscle or into the abdominal cavity, depending on the

anesthetic drug injected.4

Additionally, the anesthetics utilized include Ketamine, Xylzazine and bupivicaine.4 Ketamine is a

psychoactive drug that commonly causes muscle rigidness, which is counteracted by Xylzazine, an anesthetic

and an analgesic that also acts as a muscle relaxant.4 Due to the fact that Ketamine is not an analgesic, it

cannot be utilized but itself, but must be supplemented with Xylzazine.4 Bupivacaine, an analgesic, was

administered after the suturing of the abdominal wall, whereas Xylzazine and Ketamine were given as a

single injection prior to surgery.4

 For each surgery, sterile techniques were emphasized in order to protect the laboratory rats from

post-surgery infections. The sterile techniques utilized include disinfecting the surgical area on the animal

by utilizing alcohol and iodine swabs in a specified order; alcohol-iodine-alcohol.4 Also, the surgeons wore

masks and sterile gloves throughout the duration of the surgery.4 All instruments utilized throughout surgery

were sterilized prior to surgery and only surgeons were able to handle these instruments.4 Also, the surgery

was performed on top of a sterilized blue drape.4 During the autopsy, the pituitary gland, the uterus and the

ovary were weighed, whereby for the control rats the average of the two ovary weights was calculated.4

Results:

In order to determine the effect of the type of surgical treatment and the relative levels of

reproductive hormones on the weight of the pituitary gland, ovary and the uterus, Figures 1, 2 and 3 were

constructed, which each illustrate the average weights of each reproductive structure analyzed for each type

of treatment conducted; a control treatment, unilateral ovariectomy or a bilateral ovariectomy. According to

Figure 1, the average weight of the ovary is significantly greater for the rats that had a unilateral ovariectomy

in comparison to the ovarian weights measured in the control rats (t-test value: 3.069; degrees of freedom:

35; 2=0.01; critical value: 2.724).

Figure 1: The Effect of the Type of Treament (Unilateral vs.

0.15
Control) on the mean ovarian weight
Mean Ovarian Weight (g)

0.1
standard
standard error:0.01
0.05 error: 0.008

0
Control Treatment 1Unilateral Treament
Type of Treatment
Figure 1 illustrates the data averaged from multiple groups in different sections. It is important to note that since both
ovaries were surgically removed in the bilateral ovariectomy, no data was obtained from that treatment in regards to the
mean ovarian weight. The error bars presented represent +/- the standard error, which was calculated from the standard
deviation (control: std. deviation=0.032; unilateral ovariectomy: std. deviation=0.044). The bar graph was constructed
using Excel, which was also utilized for the calculations performed.
 In order to assess the effect of the type of reproductive treatment on the weight of the uterus, Figure

2 was constructed. One observation based on Figure 2 is that the average uterine weight is slightly greater,

however, is not statistically significant for the rats that had the unilateral ovariectomy in comparison to the

control rats based on the t-test value calculated in comparison to the critical t-value (t-test: 1.36; degrees of

freedom: 37; 2: 0.01; critical t-value: 2.715).

To compare the control treatment and the bilateral ovariectomy, the mean uterine weight for the

laboratory rates in the bilateral treatments are significantly less than the mean uterine weight for the control

rats (t-test: 6.710; degrees of freedom: 26; 2:0.01; critical t-value: 2.779). Additionally, the average uterine

weight for the rats in the unilateral treatment was significantly greater than the average uterine weight for the

rats that had a bilateral ovariectomy (t-test: 5.770; degrees of freedom: 27; critical t-value: 2.771; 2:0.01).

Figure 2: The Effect of the Type of Treatment (Unilateral, Control, Bilateral) on the Mean Uterine Weight

0.800
0.700
Mean Uterine Weight (g)

Control Treatment
0.600
Standard
0.500 Error:
Standard 0.006
0.400 Error:
0.004
Unilateral Ovariectomy
0.300
0.200
0.100 Bilateral Ovariectomy
Standard Error:
0.000 0.0033
1
Type of Treatment
* The average uterine weights were derived from data compiled from multiple groups. The error bars represent +/- the
standard error, which was calculated utilizing standard deviation (control: std. deviation=0.180;unilateral ovariectomy:
std. deviation=0.267; bilateral ovariectomy: std. deviation= 0.1). The bar graph was constructed using Excel, which was
also utilized for the calculations performed.
Furthermore, Figure 3 compares the average weights of the pituitary gland between each of the three

treatments analyzed. According to Figure 3, there was no significant difference between the average weights

of the pituitary gland for the rats in the unilateral treatment in comparison to the control rats because of the

fact that the t-test value was below the critical t-value (t-test value: 0.0649; degrees of freedom: 32; critical t-

value: 2.738; 2: 0.01). Similarly, there was no significant difference between the mean weight of the

pituitary gland for the control rats and the rats in the bilateral treatment (t-test: 0.0282; degrees of freedom:
22; critical t-value: 2.819; 2: 0.01). Additionally, there was no statistically significant difference between

the mean weight of the pituitary gland for the rats in the unilateral treatment and the rats in the bilateral

treatment (t-test: 0.702; degrees of freedom: 22; critical t-value:2.819; 2:0.01).

Figure 3: The Effect of the Type of Treatment (Control, Unilateral, Bilateral) on the
Mean Weight of the Pituitary Gland
0.016
Mean Pituitary Gland Weight (g)

0.014
Control
0.012 Treatment
0.01
0.008 Unilateral
Ovariectomy
0.006
0.004
Bilateral
0.002 Ovariectomy
0
Type of Treatment
* The average weights of the pituitary gland were derived from data compiled from multiple groups. The error bars
represent +/- the standard error, which was calculated utilizing standard deviation (control: std.
deviation=0.004;unilateral ovariectomy: std. deviation=0.006; bilateral ovariectomy: std. deviation= 0.002). The bar
graph was constructed using Excel, which was also utilized for the calculations performed.

Discussion:
In this lab, the effect of the varying types of treatments performed on the relative levels of circulating

hormones and how the varying amounts of each reproductive hormone effects the weight of the pituitary

gland, the ovary and the uterus was analyzed.4 Based on the data presented in Figure 1, the mean ovarian

weight was greater for the rats that had a unilateral ovariectomy than the control rats because of the fact that

the remaining ovary produces an elevated amount of estrogen in order to compensate for the loss the other

ovary.5 In fact, the results depicted in Figure 1 strongly support the hypothesis initially presented.

According to Figure 2 and the statistical analyses performed, the average uterine weight for the rats

in the unilateral treatment was not greater than the average uterine weight of the control rats, whereby failing

to support one of the hypotheses presented. Indeed, there is an increase in the growth of one of the ovaries;

however, since the total amount of estrogen secreted by the one ovary in the rat that had a unilateral

ovariectomy is mostly likely comparable to the total amount of estrogen secreted by both ovaries in a control

rat, the uterus is not significantly affected by the removal of one of the ovaries.5
 However, it was observed that the weight of the uterus in the rats that had a bilateral ovariectomy

was significantly less compared to average uterine weight in rats that had a unilateral ovariectomy, as well

as, in comparison to the control rats, which both strongly support the hypothesis that the average uterine

weight is greater in control rats compared to rats in the bilateral treatment and the hypothesis that the average

uterine weight is greater in rats in the unilateral treatment compared to rats in the bilateral treatment.

Additionally, Figure 3 explores the effect of the differing types of treatments on the average weight

of the pituitary gland. First, comparing the results of the control rat and the rat that had a unilateral

ovariectomy, it was concluded that statistically there was no difference between the mean pituitary gland

weights for the rats in these two treatments, which strongly supports the hypothesis initially noted. Due to

the fact that the pituitary gland acts as a master gland and is regulated by a number of other hormones, the

weight of the pituitary gland would not be significantly altered when one ovary is no longer present.5 Also,

this reasoning can be applied when comparing a control rat with a rat that had a bilateral ovariectomy in that

there was statistically no difference between the mean weights of the pituitary gland for these two treatments,

which also strongly supports the hypothesis initially presented.

Statistically, it is concluded that there was no difference in the pituitary gland weights; however, the

mean pituitary gland weights for the control and unilateral treatment rats were not equivalent to each other

and this may have been due to errors in the data set, such as the fact that there were a few data points missing

and this may be attributed to the difficulty in locating and isolating the pituitary gland. Similarly, there was

no difference between the average weights of the pituitary gland for the rats that had a unilateral ovariectomy

and the rats that had a bilateral ovariectomy and thus, these results also strongly supports the hypothesis

previously noted.

To conclude, the results presented efficiently and successfully exemplified the effect of the type of

the reproductive treatment on the levels of circulating reproductive hormones and on the weight of the ovary,

uterus and pituitary gland . In fact, the results obtained strongly support the hypotheses initially presented.

In order to expand beyond what has been presented in this experiment, future experimentation can be
conducted. For example, an additional future experiment that would demonstrate the relationship between

the hypothalamus, the pituitary gland and the gonads, is to remove the pituitary gland, the source of FSH and

LH, and observe the effects on the weights of the ovary and the hypothalamus. By understanding the general

relationship between structures in the brain and in the reproductive system, Biologists and physicians will be

able to determine and treat several reproductive pathologies that are associated with varying hormone levels

and understand the consequences of the manipulation of reproductive hormones on the weight of the pituitary

gland, the ovary and the uterus.

References:

1. Silverthorne, Dee U. Human Physiology: An Integrated Approach. 5th ed. New York: Pearson, 2009.

377-81.

2. Pinon, Ramon. Biology of Human Reproduction. Las Angeles: University of Science Books, 2002.
32-33.
3. Knobil, Ernst, and Jimmy D. Neill. Knobil and Neill's Physiology of Reproduction. 3rd ed. Vol. 1.
Oxford: Gulf Professional Publishing, 2006. 12-15.
4. Waters, John R. Physiology Laboratory Manual. 2nd ed. New York: McGraw Hill, 2003. 91-102.

5. Waters, Sophie. The Female Reproductive System. Boston: The Rosen Publishing Group, 2007. 12-

20.