Sherry Zhao

BIOL 473

3/28/2012

Reproductive Endocrinology Lab

Introduction:
The hypothalamus, anterior pituitary gland, and gonads are often referred to as the braingonadal axis or hypothalamic-pituitary-gonadal axis because these regions of the brain and the gonads (which are endocrine glands) are regulated in a feed-back loop via several important hormones. This axis is a critical part of the development and regulation of many body systems, including reproductive and immune systems1. At the top of the axis, the hypothalamus receives input about the body and, depending on conditions throughout the body, can be inhibited or stimulated. When levels of the sex hormone estrogen are low, the hypothalamus releases its neurohormone Gonadotropin-releasing hormone (GnRH), which stimulates the anterior pituitary to make its important hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In females, FSH and LH travel to the ovary via the blood and stimulate maturation of the oocyte (egg) through formation of ovarian follicle; the ovarian follicle will eventually rupture and release the mature egg into the fallopian tubes for possible fertilization. The release of the egg is known as ovulation. FSH and LH induce the follicle to produce estrogen, and after ovulation a portion of the follicle, known as the corpus luteum, remains and continues producing estrogen and progesterone. High levels of estrogen, progesterone, and inhibin, another hormone produced by the ovary, negatively regulate the hypothalamus and anterior pituitary gland, thus inhibiting further release of GnRH, FSH, and LH. After menstruation, the levels of estrogen and progesterone decrease; the tonic secretion of GnRH and the corresponding secretion of FSH and LH resume. Estrogen also promotes thickening of the uterine lining in preparation of implantation of the fertilized egg and maintains female primary and secondary sexual characteristics3.

Sherry Zhao

BIOL 473

3/28/2012

Understanding this cycle is important for scientists and clinicians because ovarectomies, or oophorectomy, are common surgeries performed on humans and other mammals. In humans, unilateral and bilateral ovarectomies are done to remove cancerous ovaries, decrease estrogen in response to estrogen-sensitive cancers, and remove ovarian cysts, etc4. Because the ovaries are involved in the complex and interconnected brain-gonadal axis, removal of one or both organs may have a significant effect on the development of all of the organs or glands in the axis, and on the release of hormones to the rest of the body. Therefore, investigation of the repercussions of these procedures is of interest for patients, clinicians, and various researchers. In this experiment, ovarectomies were performed on laboratory rats where the rats were divided into groups of no ovarectomy (control surgery), unilateral ovarectomy, or bilateral ovarectomy. After the rats recovered from the surgeries, they were euthanized and dissected; each rats pituitary, ovary, and uterus were weighed. In comparing control and unilateral treatments, we expect that the pituitary of the unilateral treatments will have little to no change in weight with respect to the control, while the remaining ovary will have increased in weight (hypertrophy) in respect to the control. This prediction is based on the fact that after the removal of one ovary the other should develop more to compensate for the decrease in activity. Since hypertrophy of the remaining ovary should keep hormone levels constant, the pituitary should not be affected. In this case the size of the uterus is also expected to change little or not at all, as the remaining ovary will take over activity for the missing one. However, in the case of bilateral ovarectomies, we expect an increase in the weight of the pituitary gland due to dramatic decreases in levels of the negatively-regulating ovarian hormones estrogen, progesterone, and inhibin. The uterus should decrease in weight due to lack of those hormones, which positively regulate uterus proliferation. Pituitary weight from the unilateral ovarectomy should be less than

Sherry Zhao

BIOL 473

3/28/2012

the pituitary weight from the bilateral ovarectomy; uterus weight from the bilateral ovarectomy should be less than the uterus weight of the unilateral ovarectomy. The remaining ovary of the unilateral ovarectomy should be larger and heavier relative to ovaries from control surgeries.

Methods:
Sexually mature female rats were used in all three types of surgeries. Surgeries that were assigned as control had surgery teams cutting through skin and muscle wall to expose the fat pad and ovary, but no organ was removed. In unilateral surgeries, an ovarectomy was performed in which one ovary was surgically removed from the rat. Surgeries were done in accordance to procedures outlined in the Rodent Survival Surgery Protocol Handout, published by Pennsylvania State Universitys Department of Physiology5. Sterile field was maintained by having surgeons only operate with sterile gloves and instruments and by performing the surgeries on sterile drape. Ketamine was administered before surgery to give rats a disassociated state of mind, and Xylazine was administered as a whole-body analgesic and muscle relaxant. Bupivacaine, a local analgesic, was administered to the sutured muscle wall at the end of the surgery to supplement Xylazine. After the rats were anesthetized, the pituitary, one ovary or both (if unilateral or control), and uterus was excised and weighed.

Sherry Zhao

BIOL 473

3/28/2012

Results:
0.16 0.14 0.12 Ovary weight 0.1 0.08 0.06 0.04 0.02 0 type of surgery Control unilateral

Figure 1: Mean Ovarian Weights (g) of Control and Unilateral Surgeries Ovaries were weighed after autopsies were performed on euthanized animals. The mean ovary weight is greater for unilateral surgery than that of the control surgery by 0.02 grams.

0.7 0.6 Uterine Weight (g) 0.5 0.4 0.3 0.2 0.1 0 Type of surgery Control Unilateral Bilateral

Figure 2: Mean Uterine Weights of Control, Unilateral, and Bilateral Treatments

Sherry Zhao

BIOL 473

3/28/2012

The mean uterine weight of the unilateral surgery is the greatest by about 0.01 grams. The lowest mean uterine weight is from the bilateral surgery, which is significantly less than the weight of control and unilateral uteruses.

0.0180 0.0160 0.0140 Pituitary weight (g) 0.0120 0.0100 0.0080 0.0060 0.0040 0.0020 0.0000 Types of surgeries Control Unilateral Bilateral

Figure 3: Mean Pituitary Weights of Control, Unilateral, and Bilateral Treatments Similar to uterus weights, the greatest mean pituitary weight is that of the control surgeries; the least is that of the bilateral surgeries. The three means differ from each other by less than or equal to 0.002 grams; between all of the three cases there was no significant difference.

Table 1: Critical t-values for control vs. unilateral, unilateral vs. bilateral, and control vs. bilateral surgeries control versus unilateral control versus bilateral unilateral versus bilateral

mean critical t-value = ovarian 2.704 weights mean critical t-value = critical t-value critical t-value uterine 2.704 = 2.771 = 2.756 weights mean critical t-value = critical t-value critical t-value pituitary 2.704 = 2.787 =2.771 gland weights Critical values for all three comparisons were made by using =0.01 and degrees of freedom calculated by n1+n2-2.

Sherry Zhao

BIOL 473

3/28/2012

Table 2: Observed t-values and degrees of freedom for control vs. unilateral, unilateral vs. bilateral, and control vs. bilateral surgeries

mean ovarian weights mean uterine weights mean pituitary gland weights

control versus unilateral control versus bilateral obs t value = 2.283 df= n/a 40 obs t value= obs t value = 7.553 0.220 df=40 df=27 obs t value = 0.405 df= obs t value = 40 1.249 df=25

unilateral versus bilateral n/a obs t value = 6.543 df=29 obs t value = 0.984 df=27

Tests that yielded t-values greater than critical t-values are highlighted. Only control vs. bilateral and unilateral vs. bilateral tests for uterine weight had observed t-values greater than critical values; therefore, only control vs. bilateral and unilateral vs. bilateral tests for uterine weight had significant differences between groups.

Discussion:
This experiment supported our hypotheses, but not completely; only when comparing control versus bilateral and unilateral versus bilateral uterine weights was a significant relationship found. The mean of bilateral uterine weights was significantly less than the mean uterine weights of control and unilateral surgeries; this observation is consistent with the hypotheses that completely eliminating the ovaries and thus the supply of estrogen and progesterone would negatively regulate uterine size1. Mean pituitary weight from bilateral surgeries was less than the mean weights from control and unilateral surgeries, although not significantly (Figure 3). This observation is not consistent with the hypotheses that a decrease in ovarian hormones would decrease negative regulation on the pituitary, causing it to increase in size, but it does suggest a more complex relationship between gonads and pituitary that is not strictly based on negative feedback. As for unilateral relative to control; the means of uterine and ovary weight from unilateral surgeries was slightly (but not significantly) greater than the mean from control surgeries. This observation suggests that the remaining ovary did grow more to compensate for

Sherry Zhao

BIOL 473

3/28/2012

the loss of the other, and might have even generated estrogen/progesterone levels equal to or slightly exceeding levels in the control (Figure 2). The strongest evidence to support our hypothesis comes from the significant difference between bilateral uterine mean weight compared to control and unilateral uterine mean weight, as well as the fact that unilateral ovary and pituitary weights are equal to or slightly greater than the mean weights from control surgeries (Figures 1 and 2). These observations all indicate that uterine development is significantly affected by decreased ovarian hormone, and that the uterus is positively regulated by ovarian hormones. While unilateral versus control data is not significant, unilateral mean weights for ovary and uterus were comparable to those of the control, which already indicates that the remaining ovary can compensate for the others activity. Only the pituitary data completely contradicted the hypothesis, which could reflect the difficulty of completely excising the pituitary from the rat and the fact that measurements were likely altered by improper dissections. In future experiments, dissections of the pituitary should be done with extra care. Data on bilateral ovaries also contained less than half of the data points of the other groups, which may have affected pituitary and other groups statistical outcomes. Overall, this experiment illustrates how the remaining ovary can compensate for activity in the case of unilateral ovarectomies, and how bilateral ovarectomies affect uterine development. However, the data from this experiment is not complete, and further studies should aim to collect a larger data set with more reliably collected samples.

Sherry Zhao

BIOL 473

3/28/2012

References:
1.

Overview of reproductive hormones. Department of Physiology, Pennsylvania State University, University Park, Pennsylvania.

2. Waters, J. (2012). Reproductive Endocrinology: Experiment Design and Survival Surgery Training. Department of Physiology, Pennsylvania State University, University Park, Pennsylvania. 3. Silverthorn, D. U. (2012). Human physiology: An integrated approach. (5th ed., pp. 84749). San Francisco: Pearson Benjamin Cummings. 4. Ovarectomy definition. (2012). Retrieved from http://medicaldictionary.thefreedictionary.com/Ovarectomy 5. Waters, J. (2012). Rodent Survival Surgery Protocol Handout. Department of Physiology, Pennsylvania State University, University Park, Pennsylvania.