The Effects of Unilateral & Bilateral Ovariectomies on Murine Reproductive Endocrinology

Maxwell Kruse BIOL 473 TA: Nathan Garvan

Introduction The hypothalamic-pituitary-gonadal axis is essential to the function of both the male and female reproductive systems. This axis refers to the integrated function of the hypothalamus, pituitary, and gonads. The female menstrual cycle is initiated in the brain when the hypothalamus secretes gonadotropin releasing hormone (GnRH). GnRH then stimulates the anterior pituitary gland to begin secreting the gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH subsequently trigger the maturation of follicles within the ovaries, as well as the production of estrogen and progesterone by both the ovaries and the follicles themselves. This increase in circulating estrogen exerts negative feedback on the hypothalamus by inhibiting the secretion of additional GnRH, and thus prevents the release of gonadotropins from the anterior pituitary. Inhibition of FSH and LH secretion prevents the development of additional follicles during the same menstrual cycle. In addition, estrogen stimulates increased secretion of estrogen in a selfperpetuating positive feedback loop. As estrogen levels peak, it there is a shift in the negative feedback to the hypothalamus; the effect of estrogen on GnRH production becomes positive, and the secretion of FSH and LH is increased. Simultaneously, the follicle begins to secrete inhibin, a hormone that acts to suppress the production of FSH. The combined effects of estrogen and inhibin on the pituitary gland results in a LH surge that triggers ovulation; the oocyte is released from the follicle, and remnants of the follicle remain in the ovary to become the corpus luteum. The corpus luteum maintains and increases the production of estrogen and progesterone. The combined increase in estrogen, progesterone, and inhibin levels exert a negative feedback on the hypothalamus, and prevent further release of GnRH, LH, and FSH. In addition, rising levels of progesterone promote further thickening of the endometrium in preparation for implantation of a fertilized egg. The corpus luteum survives for about 12 days in the ovary; if fertilization does not occur, it will die and the
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subsequent reduction of progesterone and estrogen levels results in retraction of vasculature to the endometrium. Without a sufficient blood supply, cells on the surface of the endometrium die and are removed with menstrual discharge. At this point, negative feedback on the hypothalamic-pituitary axis has been removed due to diminished estrogen and progesterone levels, and the cycle can begin again with the continued production of FSH and LH. In this experiment the hypothalamic-pituitary-gonadal axis was manipulated in rats by conducting sham (control) ovariectomies, unilateral ovariectomies, and bilateral ovariectomies. In removing an ovary, the estrogen production by that ovary is also removed, and therefore cannot act to down regulate the production of GnRH, LH, and FSH by the hypothalamus and pituitary glands. The purpose of this experiment was to determine how interfering with the interaction between the ovaries and the hypothalamic-pituitary axis would affect the circulation of reproductive hormones as well as the size of the organs that produced and were affected by these hormones. Autopsies were carried out 28 days after the surgeries were conducted, and the pituitary gland, uterine horn, and remaining ovaries were then weighed. A bilateral ovariectomy should result in a drastic reduction of circulating estrogen in the rats. In the absence of estrogen there would be no inhibition of GnRH, LH, and FSH production, thus leading to elevated levels of these hormones. This increased gonadotropin production would likely increase stress on the pituitary gland, causing it to enlarge. A complete removal of estrogen would also prevent further thickening of the endothelium and may even cause it to atrophy. Thus, it was hypothesized that rats which received a bilateral ovariectomy would have pituitary glands that weighed more than those of the control rats, while the uterine horns of these rats would weigh less than those of the control rats. Unilateral ovariectomies do not result in a complete removal of estrogen. Initially after the removal of one ovary, estrogen levels will decrease, thus decreasing negative feedback and causing an
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increase in circulating FSH and LH levels. This subsequent increase in FSH and LH, however, would cause the remaining ovary to increase its estrogen production. The prolonged elevation of stimuli by gonadotropins on the ovary and elevated estrogen secretion would likely cause the ovary to undergo hypertrophy. It was hypothesized that rats treated with unilateral ovariectomy would exhibit an enlarged ovary when compared to control rats. It was also hypothesized that there would be no difference in weight of the uterine horns between unilateral and control rats, because estrogen was still present to maintain the endothelial lining. Likewise, it was expected that the uterine horns of the unilateral group would weigh more than those of the rats which received bilateral ovariectomies. Although estrogen continues to be produced after a unilateral ovariectomy, circulating levels may be depressed compared to control rats, and the down regulation of GnRH, LH, and FSH may be slightly weakened, resulting in a small increase in gonadotropin production. This change in gonadotropin production by the pituitary, however, was expected to be relatively small. It was therefore hypothesized that there would be no enlargement of the pituitary gland after unilateral ovariectomy, and there would be no difference in the pituitary weight between the unilateral ovariectomy groups and control groups. The rats which received the bilateral ovariectomy, however, were hypothesized to have enlarged pituitary glands when compared to the unilateral ovariectomized (OVX) rats. This experiment is clinically relevant because it can help explain the effects of menopause on the physiology of the hypothalamic-pituitary-gonadal axis. In menopause, the ovaries are no longer responsive to gonadotropins and therefore stop producing estrogen and progesterone. This not only results in a cessation of reproductive cycles, but it eliminates negative feedback between the gonads and the hypothalamic-pituitary axis, causing an increased gonadotropin secretion. An increased gonadotropin secretion and a lack of estrogen cause a number of adverse side effects in post-menopausal women. The results of this experiment pose to improve the understanding of the interactions between the brain and

ovaries, and thus could be applied in treating the symptoms associated with menopause with hormone replacement therapies. Methods Ovariectomy refers to the surgical removal of the ovaries. In this experiment female rats were ovariectomized (OVX), and then euthanized after 28 days to assess the effects of ovary removal on organ weights. Rats were subjected to one of three surgeries: a control (or sham) surgery, unilateral ovariectomy, or bilateral ovariectomy. The control groups underwent surgery, but the ovary was simply tapped with forceps, and nothing was removed from the rat. In the unilateral ovariectomies, a single ovary was removed from the rats, while both ovaries were removed in bilateral OVX rats. Surgical procedures were consistent across the three treatment groups, and were followed exactly as presented in the Biol. 473 Rodent Survival Surgery Protocol Handout. Rats were first sedated with ketamine, and then anesthetized with xylazine prior to surgery. After completion of the surgery, bupivacaine was applied directly to the sutured muscle wall as a local analgesic. Sterile field was maintained throughout all surgical procedures. After surgery, rats were monitored closely throughout their recovery. Rats were euthanized 28 days after undergoing one of the three surgical procedures, autopsies were carried out. The pituitary gland, uterine horns, and any remaining ovaries were removed and weighed. Two tailed t-tests were carried out to determine if the surgeries resulted in any significant changes in organ weight. An level of .05 was used in these t-tests; the Bonferroni Method was used to correct for compounded error resulting from multiple (3) comparisons, however, so observed t-values were compared to critical t-values using = .01. Standard error was also calculated for all measurements.

Results

Figure 1: The effect of unilateral ovariectomy on ovarian weight

The Effect of Ovariectomy on Ovary Weight

Ovary Weight (g)
0.200 0.150 0.100 0.050 0.000 Operation Type Unilateral Control

Figure 1 results are based on the mean ovarian weights of control and unilateral OVX rats 28 days after surgery. These results indicate that the ovarian weights of the rats that underwent sham operations were significantly less than the ovarian weights of unilateral OVX rats (t=3.07, df=29, =.01). Figure 1 displays the mean ovarian weights of the control and unilateral OVX groups 28 days after surgery, which were 0.109g and 0.148g respectively. T-tests indicate that the ovarian weight of the unilateral OVX rats was significantly greater than the ovarian weight of the control group (t=3.07, df=29, =.01). Figure 2: The effect of ovariectomy on the uterine horn weight

The Effect of Ovariectomy on Weight of the Uterine Horn

Uterine Horn Weight (g)
0.800 0.600 Control 0.400 0.200 0.000 Operation Type Unilateral Bilateral

Figure 2 results are based on the mean uterine horn weights of the control, unilateral OVX rats, and bilateral OVX rats 28 days after surgery. These results indicate that there was no significant difference in uterine horn weight between the control and unilateral OVX groups (t=.503, df=30, =.01). There uterine horn weight of the bilateral OVX rats was found to be significantly less than uterine horn weights of both the control group (t=7.23, df=23, =.01) and unilateral OVX rats (t=5.78, df=23, =.01). The average uterine horn weights of the three treatment groups 28 days after surgery are displayed in Figure 2. These data indicate that there was no significant difference in uterine horn weights between the control group (0.602 g) and the unilateral OVX group (0.569 g) (t=.503, df=30, =.01). The mean weight of

the bilateral OVX rat uterine horn (0.15 g), however, was found to be significantly less than the control groups mean uterine horn weight (t=7.23, df=23, =.01). The uterine horn of the bilateral OVX group was also found to weigh significantly less than the uterine horn of the unilateral OVX group (t=5.78, df=23, =.01). Figure 3: The effect ovariectomy on weight of the pituitary gland

Weight of Pituitary Gland (g)

The Effect of Ovariectomy on Weight of the Pituitary Gland 0.020

0.015 0.010 0.005 0.000 Operation Type Control Unilateral Bilateral

Figure 3 results based on the mean pituitary weights of the control, unilateral OVX rats, and bilateral OVX rats 28 days after surgery. These data show that that there was no significant difference in weight of the pituitary gland between any of the three surgical treatments. No significant difference was observed between the pituitary weights of the control and unilateral treatments (t=.343, df=27, =.01). Nor was there a significant difference in pituitary weights when comparing the control and bilateral treatments (t=.6426, df=27, =.01). The difference in pituitary weight between unilateral OVX and bilateral OVX rats proved to be insignificant as well (t=.962, df=19, =.01). The mean post-operative weights of pituitary glands in the control rats, unilateral OVX rats, and bilateral OVX rats are compared in Figure 3, and were found to be 0.016 g, 0.002 g, and 0.002 g respectively. None of these weights were significantly different from each other. The weight of the pituitary gland in control rats was not found to be significantly different from the weight of the gland in the unilateral OVX rats (t=.343, df=27, =.01), nor was it statistically different from the weight of the bilateral OVX rats (t=.6426, df=27, =.01). The difference between unilateral OVX rat pituitary gland weight and bilateral OVX rat weight was also found to be insignificant (t=.962, df=19, =.01). Discussion

In this experiment, sham operations, unilateral ovariectomies, and bilateral ovariectomies were performed on three different groups of female rats. The effects of these procedures on the weights of the pituitary gland, uterine horn, and remaining ovaries in these rats were then measured 28 days later. By removing one or both androgen-producing ovaries, and then measuring the subsequent effect this had on organ weights, the effect of these procedures on the relative amount of circulating reproductive hormones could be determined indirectly. As seen in Figure 1, the mean ovarian weights of the unilateral operation were found to be significantly greater than the ovarian weights of the control operation, thus confirming the initial hypothesis. This enlargement of the remaining ovary suggests that it was secreting a greater amount of estrogen and progesterone than ovaries in the control rats. It is likely that the unilateral ovariectomy caused an initial reduction of circulating estrogen levels, thus leading to a reduced inhibition of GnRH, LH, and FSH. A subsequent Increase in circulating gonadotropin levels then caused the remaining ovary to increase its hormone production. This increased activity likely caused the ovary to hypertrophy, which is reflected by the data obtained here. As hypothesized, there was no significant difference in uterine horn weight between the control and unilateral treatments, while the bilateral ovariectomy proved to result in a significant decrease in uterine horn weight when compared to the other two treatments; this is clearly depicted in Figure 2. This result is explained by the fact that estrogen is required for the maintenance and growth of the endometrial lining of the uterine horn. The bilateral ovariectomy resulted in a complete cessation of estrogen production and therefore caused the endometrium of the uterine horn to deteriorate. In contrast, estrogen production continued after both the control and sham operations, resulting in the maintenance of the endothelium lining the uterine horn. Although circulating estrogen levels may have been slightly lower after unilateral ovariectomies when compared to the control groups, the data suggests that the amount of estrogen produced in unilateral OVX rats appeared to be sufficient enough to maintain the endothelium of the uterine horn. Although there did appear to be a slight reduction in the weight of the uterine horn in
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unilateral treatments when compared to control groups (Fig. 2), this difference was found to be insignificant via t-tests. This indicates that although there may have been a slight reduction in estrogen and progesterone production in unilateral OVX rats, that this decrease androgen production was not significant and did not produce a significant effect on reproductive physiology of the unilateral OVX rats. No significant differences were seen when comparing the weights of the pituitary glands of the three treatment groups. Although there may have been a slight decrease in total estrogen production after unilateral ovariectomy, estrogen was still produced by the remaining ovary and the negative feedback to the pituitary glands production of gonadotropins was therefore maintained. Thus, as expected, there was no apparent increase in pituitary activity. This was reflected by the fact that the pituitary gland of the unilateral treatment group was not significantly greater than that of the control group. It was expected, however, that the pituitary gland of the bilateral OVX rats would exhibit a significant increase in the weight when compared to the pituitary weights of the unilateral and control treatments. This was expected because there was a complete removal of estrogen with the bilateral ovariectomy, and thus a complete lack of negative feedback on the hypothalamus and pituitary gland which would cause an increase in GnRH, LH, and FSH secretion. It was hypothesized that this increase in pituitary hormone secretion would cause an increase in the size of the gland. Although the significant reduction in weight of the uterine horn after bilateral ovariectomy suggests there was a significant reduction in estrogen production after bilateral ovariectomy, this decrease in estrogen did not produce a change in the weight of the pituitary gland as expected. The pituitary gland is essential in modulating many endocrine processes throughout the body in addition to the production of FSH and LH. Therefore even if an increase in gonadotropin secretion occurred with ovariectomy, it is likely that this effect would not be large enough in relation to the total activity of the gland to produce a significant increase in size. In addition, the pituitary gland is a relatively small secretory structure. It is likely that even if a small change in weight did occur that it would not be detected with these procedures due to variability in autopsy technique of the gland across different surgical teams. In
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addition, the pituitary gland was often damaged or only partially removed during autopsy. If autopsy of the pituitary glands was made more precise, perhaps significant differences between weights of the pituitary glands of each treatment group would be elucidated. Despite there being no increase in weight of the pituitary gland when comparing bilateral OVX rats with unilateral OVX rats and control rats, all other results of this experiment supported the initial hypotheses. There were no data that appeared to be obvious outliers, and our results therefore appeared to be strong. As mentioned previously, the data for pituitary gland weight after treatment could be improved by ensuring a more precise autopsy technique across surgical teams. Although the results obtained with this experiment appear to be strong, the experiment could be improved upon by ensuring that the autopsy techniques for the uterine horn and ovaries was more standardized across surgical teams as well. During autopsy, the decision of where to cut between the uterine horn and ovary, as well as where to cut on Y of the uterine horn for each surgeon was somewhat arbitrary. This may have decreased the accuracy of the results because some surgeons may have removed more or less tissue with the organs than others. In addition, the accuracy of these results could be improved by ensuring that all rats were operated on during the same stage of their menstrual cycle. Prior to operation, the relative stage of each rats menstrual cycle was unknown; therefore the rats may have had different starting levels of these circulating reproductive hormones, and may have responded differently to their respective surgical treatment. The ovariectomies performed in this experiment were successful in predictably manipulating the circulating levels of reproductive hormones, including LH, FSH, and estrogen. These OVX rats, especially the bilateral OVX rats, therefore could be useful in mimicking menopause, a condition in which the ovaries are no longer responsive to gonadotropins, and therefore no longer produce estrogen. This experiment could therefore be used as a basis for studying the effects of various hormone therapies in treating the symptoms of ovariectomized rats that exhibit similar physiological symptoms to women with menopause.

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References Biol. 473 Rodent Survival Surgery Protocol Handout, PSU, Bio 473 Laboratory Sp 2013. Biol. 473 Overview of Reproductive Hormones Handout, PSU, Bio 473 Laboratory Sp 2013. Biol. 473 Guidelines for Analyzing Your Reproductive Endrocrinology Data, PSU, Bio 473 Laboratory Sp 2013. Silverthorn , Dee Unglaub. "26 Reproduction and Development." Human Physiology An Integrated Approach - 6E. 6 ed. Glenview, IL: Pearson, 2013. 801-888. Print.

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