The Effect of Tamoxifen Citrate on Granulosa Cells of Ovarian Follicles in Adult Female Rats: Light Microscopic Study.

The most common malignancy in women worldwide is breast cancer. Tamoxifen is a widely popular chemopreventive drug used to manage breast cancer.[ 1 ] Recent research found that tamoxifen can suppress brain metastasis arise from breast tumors.[ 2 ] Tamoxifen is one of the selective estrogen receptors modulator (SERM) family which inhibits the binding of estrogen to the receptor.[ 3 ] Tamoxifen is recognized to have different effects on organs ranging from estrogen antagonistic activity in breast tissue to beneficial estrogenic actions in bone and liver.[ 4 5 ] Tamoxifen is reported to be used in the stimulation of ovarian function in subfertile women.[ 6 7 ] Several researchers found that the treatment with tamoxifen leads to the growth of follicular cysts and an increased possibility of ovarian malignancy.[ 8 9 10 11 12 ] There are a rare case of hyperplastic lesions in the endometrium accompanied with endometriosis in a patient who received tamoxifen therapy.[ 13 ] Tamoxifen has deleterious effect on the endometrial mucosa, which may result in the development of endometrial cancer.[ 14 15 ] It had also harmful effects on the ovaries of developing embryo, so it should be excluded or used with great attention during pregnancy.[ 16 ] Some experimental research on rats found that tamoxifen had effect on fertility consisted of irregularity in the estrous cycle, decrease in the number of pregnancies, and decreased live embryo.[ 17 ] Tamoxifen had effects on the reproductive tract causing mucinous degeneration in vagina, shedding of endometrial cells, hyperplasia of granulosa cell (GC), and dysplastic lesions in the ovary and oviduct.[ 17 18 19 20 ] In line with that, the present study was undertaken to see the influence of tamoxifen Citrate on the ovary of the adult rats, particularly on the GCs of different developing follicles and the possible recovery after tamoxifen withdrawal.

Examination of sections of the ovary of both groups, either untreated or olive oil-treated group, showed normal ovarian histological structure. There were no apparent differences between the two groups. In these sections, the ovary showed follicles at various stages of development embedded in the compact cortical stroma [ Figure 1a ]. The secondary follicles (SF) had an oocyte enclosed by zona pellucida, antral cavities, and several layers of GCs [ Figure 1b ]. The mature Graafian follicles distinguished from other follicles by the presence of a single large crescent-shaped antrum bordered by thin layers of GCs [ Figure 1c ]. Hematoxylin and Eosin-stained ovarian sections from control group showing: (a) Cortex containing secondary follicles, mature Graafian follicle with compact stroma (S) between them (×50). (b) Secondary follicles with an irregular spaces filled with liquor folliculi surrounded by granulosa cells and theca folliculi cells (×200). (c) Graafian follicle containing an oocyte (O), zona pellucida, corona radiata (B), granulosa cells, antrum (A) (×400) In tamoxifen-treated rats, the ovaries revealed distinctly disorganized ovarian structure in the form of many degenerated atretic follicles [ Figure 2a ]. Some ovarian follicles had severely degenerated oocyte, disorganized GCs with vacuolated cytoplasm and pyknotic nuclei. The theca folliculi cells which surround the ovarian follicles appeared vacuolated. The cortical stromal cells (SC) between the follicles appeared vacuolated and arranged in groups [ Figure 2b ]. Hematoxylin and Eosin-stained ovarian sections from tamoxifen-treated group showing: (a) Many atretic follicles (→) with disordered granulosa cells, and congested blood vessels (×100). (b) Secondary follicle containing degenerated oocyte (O), a small cavity (C) Surrounded by disorganized granulosa cells, and peripheral vacuolated theca folliculi cells. Groups of vacuolated stromal cells can be seen (×400) Other ovarian sections revealed cystic ovarian follicles with dilated antral cavity containing acellular eosinophilic material surrounded by thin GC layers. Marked vascular congestion and hemorrhage also noticed in these sections [ Figure 3a and b ]. In addition, some ovaries showed multiple corpora lutea (CL) and reduction in ovarian follicles [ Figure 4a ]. Cysts of some corpus luteum were also noticed without ova inside the cystic cavity of the corpus luteum [ Figure 4b ]. Hematoxylin and Eosin-stained ovarian sections from tamoxifen-treated group showing: (a) Cystic ovarian follicles with dilated antral cavity containing eosinophilic material (A). The antrun surrounded by thin granulosa cell layers (→) (×50). (b) A higher magnification of Figure 3a showing part of cystic ovarian follicles lined by thin granulosa cell layers (→), marked vascular congestion and hemorrhage (stars) (×200) Hematoxylin- and Eosin-stained ovarian sections from the tamoxifen-treated group showing: (a) Multiple corpora lutea with few ovarian follicles (→) (×50). (b) Cysts of corpora lutea (C), There are multiple corpora lutea (stars), ovarian follicles (→) and dilated congested blood vessels (×100) The GCs of ovarian follicles of this group appeared variable in their size and shape. Their cytoplasm showed many changes in the form of cytoplasmic vacuolation, and some of them appeared with complete lysis of their cytoplasm. Some GCs were detached inside the antral cavity [ Figure 5a ]. The nuclei of GCs appeared irregular in shape with increased number of dark pyknotic nuclei [ Figure 5b ]. Some of the nuclei appeared fragmented into multiple heavily stained masses [ Figure 5c ]. The apoptotic bodies were observed among the highly vacuolated GCs and it appeared in the form of membranous structures containing many nuclear fragments [ Figure 5d ]. Hematoxylin and Eosin-stained ovarian sections from the tamoxifen-treated group showing: (a) Granulosa cells of ovarian follicles appeared variable in their size and shape. Their cytoplasm showed many vacuolation (stars). Detached granulosa cells in the antral cavity (→). Antrum (A), theca folliculi cells (×1000). (b) Granulosa cells of ovarian follicles with multiple dark pyknotic nuclei (→). Antrum (A) (×1000). (c) Vacuolated granulosa cells with fragmented nuclei (→). There are granulosa cells detached into the antral cavity (A) (×1000). (d) Apoptotic bodies among the highly vacuolated granulosa cells (→). Antrum (A), theca folliculi cells (×1000) The ovarian tissue showed slight improvement after withdrawal of the drug. The ovaries had normal primary follicles, SF, and many CL [ Figure 6a ]. Few atretic follicles with degenerated oocytes were observed. Hyperplasia of the SC inbetween ovarian follicles and dilated congested blood vessels (BV) were also noticed [ Figure 6b ]. Hematoxylin and Eosin-stained ovarian sections from recovery group showing: (a) Normal follicles like primary follicles and secondary follicles. Many corpora lutea (stars) and dilated congested blood vessels are also observed (×50). (b) A higher magnification of Figure 6a showing atretic follicles with degenerated oocyte (→), Hyperplasia of the stromal cells (S) and dilated congested blood vessels (×200)

The present study clearly exhibits that there are marked histopathological changes in the ovary of rats following exposure to tamoxifen. The benefits of tamoxifen in the management of breast cancer are well established, but it may induce severe ovarian side effects. Hence, it is recommended that when tamoxifen is used, frequent gynecological examination should be achieved to identify any complications. The gynecologist may then select the most appropriate dosage and period of treatment. Nil. There are no conflicts of interest.

Tamoxifen is one of a selective estrogen receptor modulators which has a role in the treatment of breast malignancy and it is also used to stimulate ovulation.[ 23 ] The present study revealed that intake of tamoxifen at dose of 5 mg/kg/d to adult albino rats for 4 weeks can lead to deleterious effects on the histological structure of the ovary. Looking at the obtained results from the present study, the ovaries of Group III showed an apparent increase in atretic ovarian follicles. This result is in agreement with previous work that noticed increases in the number of large atretic follicles.[ 17 ] Other investigators also found increased number of the atretic ovarian follicles in premenopausal women treated by tamoxifen as a breast cancer chemopreventive drug.[ 19 ] Dixon et al .,[ 24 ] revealed that atresia is a physiological process that lead to degeneration of many ovarian follicles and they also mentioned that many factors as Androgens, Interleukin-6, and tamoxifen can also induce atresia. Increased number of CL in the ovaries of Group III is consistent with earlier study that noticed the elevated number of corpus luteum and attributed that to the reduction of apoptosis by inhibition of prolactin secretion in rat.[ 17 ] De Quijada et al .[ 25 ] confirm this explanation as they found that tamoxifen suppresses normal prolactin synthesis in the rat. In contrast, some authors observed decrease in the total area of CL in the ovary of rats treated by tamoxifen or SERM.[ 26 27 ] The cytoplasm of GCs showed many changes in the form of cytoplasmic vacuolation and some of them appeared with complete lysis of their cytoplasm. These vacuoles may be signs of cellular necrosis[ 28 ] or due to lipid accumulation.[ 27 ] Many degenerated ovarian follicles were also observed in Group III and Some authors explained how SERM causes follicles degeneration by blocking of estrogen feedback to the hypothalamus, causing the persistent elevation in circulating luteinizing hormone levels which lead to sustained effect of this hormone in regulating ovarian development, resulting in degeneration of ovarian follicles.[ 24 27 29 30 ] In contrast with these results, Ting and Petroff[ 31 ] reported a beneficial effect of tamoxifen on the ovary, where they found that tamoxifen decreased the follicular loss of the rat ovary following exposure to chemotherapy drugs. In this study, the SCs of the ovarian cortex appeared with vacuolated cytoplasm and organized in groups. These vacuolated cells may be groups of theca cells which remain after the process of atresia.[ 27 ] Vacuolation of SCs may be due to lipid accumulation inside the cells due to inhibition of steroid synthesis.[ 24 ] Another critical observation is the appearance of apoptosis in the GCs of the follicles which appeared in the form of dark pyknotic nuclei, fragmented nuclei, and apoptotic bodies. These findings correspond with the previously recognized histological criteria of apoptotic cells and bodies.[ 28 32 33 34 ] Pyknosis is the process not exclusive to apoptosis, but it can be a part of morphological changes that occur with necrosis.[ 35 ] Zeiss[ 36 ] reported that it is hard to distinguish apoptosis from necrosis, and they may happen concurrently depending on causes like the extent of adenosine triphosphate reduction and the accessibility of caspases. Conversely, Piasecka-Srader et al .[ 37 ] revealed that follicular apoptosis is decreased by tamoxifen protecting the ovary from 4-hydroxycyclophosphamide-mediated toxicity. However, a previous study reported that the apoptosis of the postovulatory follicle is a physiological process, and it is essential to remodeling in the adult human body.[ 38 ] The apoptotic bodies were observed in this study as membranous structures holding several nuclear fragments is in consistent with a previous study by Yang and Rajamahendran[ 39 ] who observed the apoptotic bodies as detached structures containing multiple parts of condensed chromatin. Researchers explained the formation of apoptotic bodies by the stimulation of caspase 3-mediated cytoskeletal reconstruction.[ 28 40 ] The ovary of the tamoxifen-treated group in this study showed follicular and corpus luteum cysts. These follicular cysts were similar to those detected in the ovary of female rats treated by tamoxifen in the previous studies.[ 17 26 ] Furthermore, the growth of ovarian cysts is well documented in women treated with tamoxifen for breast cancer.[ 41 42 43 ] Some authors explained the appearance of follicular cysts during tamoxifen therapy by the hyperestrogenemia which may lead to a concurrent growth of several ovarian follicles and an increased risk of ovarian cyst formation.[ 44 45 ] Moreover, the corpus luteal cyst formation might be due to the accumulation of fluid or blood inside the follicle after ovulation.[ 24 ] After the withdrawal of drug in the present study, the ovarian tissue showed slight improvement with appearance of some atretic follicles with degenerated oocyte. The stroma showed marked cellularity and dilated congested BVs. These findings mean absence of complete recovery after the stoppage of tamoxifen. This could be due to the accumulative effect of tamoxifen because of its sustained serum half-life, which may persist in tissues for several months after stoppage of therapy.[ 46 ]

This experimental study was approved by the Institutional Review Board committee for animal research at Imam Abdurahman Bin Faisal University, Dammam, Saudi Arabia. Twenty-four adult female albino rats, weighing 250–300 g each, were obtained from the animal house of the Center of Research and Medical Consultation Studies. Rats were housed in well-ventilated stainless steel cages at ordinary room temperature and in 12-h light, 12-h dark cycle with strict care and hygienic measures. All rats were freely provided with water ad libitum and standard rat chow. Tamoxifen citrate (Nolvadex 10 mg) was obtained from AstraZeneca UK company, and it was available in tablets form. It was dissolved in olive oil and was given via oral gavage. Olive oil was purchased from the market (Bionaturae, Organic Extra Virgin Olive Oil, originally a product of Italy). The animals were randomly classified into four groups (six rats per group), as follows: Group I (control): Animals being kept without any treatment as control for all experimental groups Group II (placebo control): Animals received the same volume of olive oil which the experimental groups were received Group III (tamoxifen-treated): Animals received 5 mg/kg of tamoxifen dissolved in olive oil once daily for 4 weeks. The chosen dose was selected from the previous work done by Greaves et al .[ 21 ] Group IV (recovery group): Animals were sacrificed after 1 month of withdrawal of tamoxifen which was given daily for 4 weeks. Group I (control): Animals being kept without any treatment as control for all experimental groups Group II (placebo control): Animals received the same volume of olive oil which the experimental groups were received Group III (tamoxifen-treated): Animals received 5 mg/kg of tamoxifen dissolved in olive oil once daily for 4 weeks. The chosen dose was selected from the previous work done by Greaves et al .[ 21 ] Group IV (recovery group): Animals were sacrificed after 1 month of withdrawal of tamoxifen which was given daily for 4 weeks. At each time point, the rats of all groups were anaesthetized with intraperitoneal injection of xylazine (10 mg/kg) and ketamine (75 mg/kg). An incision was made and the wall of the abdomen was retracted, and the ovary was removed rapidly. Pieces of the ovary were removed and fixed immediately in a 10% formalin, dehydrated in rising grades of ethyl alcohol, cleared in xylene, impregnated and embedded in pure molten paraffin wax, sectioned on a rotary microtome at 5 μm thicknesses and mounted on a glass slide, then stained with Hematoxylin and Eosin (H and E) for studying the ovarian tissue structure.[ 22 ]