Mammalian reproductive cycle

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Different hormones are present in different quantities, and at different times during the mammalian reproductive cycle. These hormones all have a vital role to play in the structure and function of various reproductive organs. The hormones GnRH, oestradiol and follicle stimulating hormone (FSH) were investigated in this study to understand how these hormones affect uterine, ovarian and pituitary functions. Gonadotropin-releasing hormone (GnRH), is a tropic peptide hormone responsible for the release of FSH and LH from the anterior pituitary (Clayton, 1988). GnRH is quickly broken down and cleared from the blood, so pulsatile release from neurons within the hypothalamus cause brief pulses reaching the pituitary (Clayton, 1988). During reproduction, oestradiol is produced by the granulosa cells of the ovaries via the aromatization of androstenedione (produced in the theca cells) to oestrone, followed by the conversion of oestrone to oestradiol (Herath et al., 2007). However, the effects of each hormone were assessed indirectly. Deslorelin, oestradiol benzoate, and PMSG were used in treatment to understand the affects of GnRH, oestradiol, and FSH respectively. Deslorelin treatment in conjunction with suprelorin downregulates or desensitises the pituitary. Furthermore, oestradiol benzoate is hydrolysed to oestradiol, and pregnant mare Syrum Gonadotrophin (PMSG) made by the chorionic membranes of pregnant horses mimic FSH activity (Horsley, 2004).

The experimental protocol followed that in the notes, except the treatment group tamoxifen was removed. The female Swiss mice were housed together to promote synchronisation of oestrous cycles in order to decrease variability between results. Our group along with others assessed the effects of oestrogen and the deslorelin implant. Data from the remaining groups tested PMSG, and Deslorelin and PMSG. All results were collated for the final analysis. We have concluded that the hormones investigated in this study play different roles in terms of structure of reproductive organs. Both deslorelin and oestradiol decreased the length of the uterus; however deslorelin also reduced the width and weight of the uterus. Deslorelin laso greatly reduced the number of corpora lutea (CL) within the ovaries. These results indicate that FSH activity plays a major role in regards to structure, and therefore function.

The age at dissection (weeks) used after each treatment was noted and the body weight (g) of the mice for each treatment were recorded before organs were dissected. All the hormones used had no significant effect on bodyweight compared to the control (31.46 + 0.54, Table 1).

Our group used a microscope whilst dissecting the uterus for all treatments. The mice treated with deslorelin showed a significant (t=2.02, df=37, P<0.005) decrease in uterine length (20.38 + 0.90 mm) in contrast to the control (27.48 + 1.34 mm, Figure 1). The uterus from the deslorelin treated mice had constricted arterial supply, white and little vascularisation was obvious. The histological slides with a section of the deslorelin treated uterus had a small lumen, little endometrial tissue, and a greater amount of myometrium compared to stroma; in contrast to the control.Oestradiol also showed a significant (t=2.5, df=17, P<0.05) decrease in uterine length (22.2 + 1.64 mm) when compared with the control (Figure 1) .The oestradiol treated uterus had dilated arterial supply, and it was pink and well vascularised. The oestradiol histology sections had a thick epithelium compared to the control. This may be due to a gradual increase in the number of perpendicular oriented mitoses and in the proliferative activity in uterine epithelia of mice following single injection with estradiol (Voznesenskaya and Blashk, 2005). Oestradiol contracts vascular and myometrial smooth muscle in the uterus (Fried and Samuelson, 1991), causing it to contract lengthwise and several major angiogenic factors and their receptors are increased within hours after oestradiol treatment within the uterus (Johnson et al., 2006). This indicates that oestradiol plays a role in regulation of angiogenesis in the uterus. These findings also support several other studies which claim that LH acts on the theca cells to stimulate the synthesis of the precursor of oestradiol, therefore, with the lack of LH release due to deslorelin - may be the reason why very little vascularisation was present in the uterus of deslorelin treated mice. However, deslorelin may have caused the uterus to shrink due to a lack of nutrients which diffuse across the arteries from the blood into uterine tissue. On the other hand, treatment with PMSG showed no significant (t=-0.12, df=10, P=0.92) difference in uterine length compared with the control (27.82 + 2.79 mm, Figure 1). Furthermore, PMSG and deslorelin also gave no significant (t=0.31, df=7, P=0.76) difference in uterine length compared with the control (Figure 1). However the uterine tissue of the deslorelin and PMSG treated mice was very vascularised, which suggests that FSH also plays a role in angiogenesis. Sato et al. (2009) showed that angiogenic activity was increased in extracts of ovaries from mice treated with FSH.

Deslorelin also caused a significant (t=3.13, df=41, P=0.002) decrease in uterine width (1.6 + 0.17 mm) compared with the control (2.4 + 0.19 mm, Figure 2). This effect may be due to a lack of nutrients caused as a result of little vascularisation; which may also cause the uterus to contract in width. Oestradiol did not have a significant (t=-1.82, df=11, P=0.1) effect on uterine width (3.18 + 0.38 mm, Figure 2), however, it did cause the uterine width to increase by 33% compared to the control. This correlates with oestradiols ability to contract the uterine lengthwise subsequently causing the uterine width to thicken. An increase in estradiol causes the LH surge and ovulation to occur followed by a rise in progesterone (Gumen and Wiltbank, 2005). Progesterone and estrogen cause the lining of the uterus to thicken more, to prepare for possible fertilization (Gumen and Wiltbank, 2005). On the other hand PMSG had no significant effect on uterine width (2.6 + 0.33 mm, Figure 2), the uterus had very similar widths in mice with a deslorelin implant and those treated with deslorelin and PMSG (1.68 + 0.27, Figure 2). This suggests that FSH plays no role in contracting and dilating uterine tissue.

A microscope was used to dissect the ovary and to count the number of corpora lutea (CL). Mice with a deslorelin implant showed a significant decrease in uterus weight (t=4.02, df=42, P<0.005) and in the number of CL (t=2.66, df=33, P=0.011) compared with the control (Figure 3). The subsequent treatment groups displayed no significant difference of uterine weights and the number of CL, compared with the control, respectively (Figure 3).

Pituitary, decreases the amount of LH released from it . It is the mid-cycle surge of hormone LH that causes the dominant follicle to rupture and release the mature egg; subsequently forming the CL (Christenson and Stouffer, 1997). The number of ovulations is directly proportional to the amount of CL's formed (Christenson and Stouffer, 1997). So, little LH means a low ovulation rate and a small number of CL's formed. Structural and functional development of the corpus luteum (CL) involves tissue remodeling, active angiogenesis, and steroid production (Haimov-Kochman et al., 2005). Therefore, non-active angiogenesis and steroid production such as testosterone which thickens uterine tissue and increases mucus production for implantation of the blastocyst, may be the reason for the significant decrease in uterine weight following deslorelin treatment. Whilst observing the histology sections, the deslorelin treated uterine lumen was smaller compared to the control. Furthermore, the deslorelin treated vaginal smears indicated that the mice were in di-oestrus.

The right and left ovaries were removed whilst using the dissecting microscope. None of the treatments used in mice gave a significant change in ovary weight compared with the control (Table 2). The numbers of follicles were then counted in the ovary for both the control and treated mice (Table 2). No treatment reached significance. However, following oestradiol treatment resulted in a 65% decrease in follicles (9.6 + 3.3) within the ovaries, compared to the control (14.6 + 2.8). Previous studies in ewes by Barett et al. (2007) suggests supraphysiologic concentrations of estradiol suppress follicle wave development by truncating FSH peaks , but the effect may also be caused by oestradiol decreasing the expression of FSH receptors on granulosa cells. However, the extent to which estradiol directly regulates follicular development is not fully understood. Whilst looking at the oestradiol treated ovaries in the histological sections, the follicles looked larger, and there were more blood vessels compared to the control ovary sections. Furthermore, the oestradiol treated vaginal smears indicated that the mice were in pro-oestrus.

Subsequently, pituitary weights were examined at the end of the treatment period. No treatment had a significant effect on pituitary weight compared to the control; however, oestradiol did increase the weight of the pituitary gland slightly more than 2-fold (Figure 4). Ichihara (1998) suggested that this increase could be due to an increase in serum prolactin levels, but the effect may also be caused through levels of LH and FSH within the anterior pituitary and would therefore only increase the weight of the anterior side of the pituitary. Unfortunately, we did not separate the anterior and posterior sides of the pituitary so we could not test for this.

A failing of this study was the lack of an additional control such as mice with an implant but with no deslorelin applied to it, as the implant alone may have increased the variability between mice. Moreover, there was a large variability between the ages of the mice used for different treatments; this would increase the variability of hormones present between the mice, as the combination and amount of hormones coincide with age.

The exact targets of the hormones in Swiss mice investigated in this study can not be concluded from our results. The effects we viewed in reproductive organs may have been indirect. Further studies are needed to investigate where abouts the hormones investigated in this study act in vivo, and the significance of the different physical changes within and between each organ observed, in regards to function. This preliminary study has shown that various hormones work to change the structure of reproductive organs in different ways, and at different stages of the oestrous cycle.

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