Effects of propolis on histopathological and immunohistochemical alterations in testes of thyroidectomized rat

The thyroid hormones, triiodothyronine (T3) and its prohormone, thyroxine, are secreted by the thyroid gland which are responsible for the regulation of metabolism in all body tissues. This work aimed to study the histopathological, immunohistochemical and biochemical changes in the adult rat testes after thyroidectomy, in addition to the role of propolis in reducing these changes. Twenty five male Wistar rats were assigned randomly into equal (5) groups; control, sham operated, shamed propolis, thyroidectomized, thyroidectomy treated with propolis. Levels in compare to control the thyroidectomized rats showed significantly decrease in serum T3 and increase in TSH levels. Degeneration and increase in the seminiferous tubules lumen also noticed. Immunohistochemical results showed a remarkable reduction of proliferating cell nuclear antigen (PCNA) and Ki-67 in spermatogonia of thyroidectomized rat testes as compared with control group. The results revealed that, propolis has an ameroliating role on thyroidectomized rats testes by decreasing damage and injury occurred as a result of thyroidectomy.


Introduction
Thyroid is a butterfly-shaped gland located at the base of the neck.It produces hormones that regulate every aspect of metabolism, from heart rate to calories burn.Removal of all or a part of thyroid gland is called thyroidectomy (Sakr et al., 2015).Thyroidectomy is used to treat thyroid disorders-such as cancer, goiter and overactive thyroid (hyperthyroidism).The growth, development, and metabolic functions of all organs and tissues are affected by thyroid hormones; which have been shown to play an important role in testicular physiology (Holsberger and Cooke, 2005).Gonadotrophin folliclestimulating hormone and luteinizing hormone mainly regulated testicular work (Tousson et al., 2011).Mendis-Handagama and Siril Ariyaratne )2005( postulated that thyroid hormones have been shown to play a vital role in the onset of Leydig cell differentiation and stimulation of steroidogenesis in postnatal rat testis.Many studies suggested that thyroid hormone required to maintain normal testicular morphology and spermatogenesis in the adulthood and also associated with some kind of sexual malfunction which accompanied by morphological testicular degeneration (Krassas and Pontikides, 2004;Faraone-Mennella et al., 2008).Triiodothyronine (T 3) regulates testicular function and development, Leydig cells, Sertoli cells, and the maturation of germ cells (Wagner et al., 2008).Disturbance in thyroid hormone level is leading to modulate functions of many tissues in mature animals by changing their antioxidant defenses (Das and Chainy, 2004;Chattopadhyay et al., 2007).Total thyroidectomy is a treatment for thyroid cancer, many studies postulated that total thyroidectomy reduces triiodothyronine, thyroxine, prolactin, insulin, and corticosterone levels, and also acid phosphatase activities, while increases cholesterol and glutamic oxaloacetic transaminase activities in rats (Panda andJindal, 1980, Gomez Dumm., et al 1985).
Bee glue or (Propolis) is a generic name for the resinous substance collected from different plant sources by honeybees (CHEMID, 1996).Monti et al., (1983) andCirasino et al., (1987) mentioned that propolis composed of 30% wax, 5% pollen, 50% resin and vegetable balsam, 10% essen-tial and aromatic oils, and 5% various other substances, including organic debris.Propolis have antiseptic, antimycotic, anti-inflammatory, bacteriostatic, anaesthetic and antioxidant properties (Marcucci, 1995 andDobrowolski et al., 1991) without side effects or toxicity (Jasprica et al., 2007 andBurdock ,1998).Propolis has an immunomodulatory effect restricted to macrophages, without any effects on the proliferation of lymphocyte (Dimov et al., 1991).The present work aimed to investigate the histopathological, immunohistochemical and biochemical changes effects of surgical thyroidectomy performed on the spermatogenesis and structure of the seminiferous tubules in adult rats testes, in addition to the possible ameliorating role of propolais .

i.Experimental animals & study design
Twenty five male Wistar rats weighing 130±5g and of 8-9 weeks age were used in this experiment, obtained from Zoology department farm-Tanta-Egypt.Rats were left for one week in the laboratory for accommodation and maintained on a standard rodent diet and water available ad libitum.The rats were divided randomly and equally into five groups as follows: Group (І) control group in which rats did not take any treatment.Group (II) Sham-operated group (control).Group (III) Sham-operated group treated with propolis (50mg/kg body weight) by a stomach tube for 4 weeks, propolis was dissolved in distilled water (Kinobe, et al., 2006).Group (IV) Rats surgically removed thyroid gland (Thyroidectomized) (Tenorio-Velasquez , et al., 2005).Group (V) Thyroidectomized rats treated with propilas (four weeks).

ii.Biochemical and histological study
After experimental period finished rats were euthanized with sodium pentobarbital, non heparinized blood samples were collected from the inferior vena cava of each rat and centrifugation was done for fifteen minutes at 3000 rpm.The concentration of triiodothyronine hormone (T 3 ) and thyroid stimulating hormone (TSH) were determined according to Chopra et al. (1971) and Engall et al., (1980) respectively.For histopathological examination, quickly after euthanization, animals were dissected, testes were removed from different groups, and routine method for histopathology was done according to Bancroft and Stevens (1990).

iii. PCNA, P53 and Ki-67 immunoreactivity:
Expression of PCNA, P53 and Ki-67 were examined in paraffin sections (5μm thick) of fixed testes of rats of different groups that mounted on gelatin chromalumcoated glass slides according to the methods described byb Tousson et al. (2011) and Hoeflich et al., (2009) .

iv.Statistical Analysis:
Data were expressed as mean values ± SE and statistical analysis were performed using SPSS statistical version 16 software package (SPSS ® Inc., USA).

3.1.biochemical results
Thyroidectomized rats showed a significant decreased value of (T3) compared with the control and sham groups (Table 1).On the other hand, (TSH) levels were significantly increased in thyroidectomized rats comparing with the control and sham groups.Thyroidectomized rats treated with propolis showed significantly decreased (T3) comparing with thyroidectomized rats.On the other hand TSH levels in thyroidectomized and propolis-treated animals were significantly increased compared with thyroidectomized or control groups (Table 1).

3.2.Histological observations:
The seminiferous tubules and interstitial tissues are the main structural components of the control testis.Figures (1A-1C) showed stages of spermatogenesis in testes sections of control, sham-operated and shamoperated treated with propolis groups.Two types of cells are identified in rat seminiferous tubules, the Sertoli cells and the spermatogenic cells (spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and sperms).The Sertoli cells, rest on the thin basal lamina (basement membrane) while the spermatogenic cells are arranged in many layers, namely the spermatogonia, primary and secondary spermatocytes; spermatoids and finally mature spermatozoa.However, many abnormalities were revealed in testis sections of the thyrodectomized rat as inhibition of spermatogensis, little numbers of Leydig cells, marked atrophy and degeneration with a significant decrease in the number of spermatogenic cells in the seminiferous tubules, degenerative Sertoli cell and an increase in the seminiferous tubules lumen with lack of sperms when compared with control group (Figs.1D&1E).Sections in testes of thyroidectomized rats that treated with propolis for 4 weeks revealed moderate improvement with regular distribution in spermatogenesis cycles, mild degeneration and atrophy with a significant increase in the number of spermatogenic cells in the seminiferous tubules, an increase in the sperm and Leydig cells numbers when compared with thyroidectomized rats (Fig. 1F).Tyroidectomized rats that treated with propolis with regular distribution in spermatogenesis cycles, mild degeneration and atrophy with a significant increase in the number of spermatogenic cells in the seminiferous tubules (Fig. 1F).

PCNA immuohistochemical alterations in testes:
The detection and distribution of PCNA immunoreactivity (PCNA-ir) in the testes sections in the different groups were revealed in figure 2. Testes sections in control, sham-operated and sham-operated treated with propolis groups showed strong positive reaction for PCNA in the spermatogonia (Figs.2A-2D).Testes sections in thyrodectomized rat revealed a marked decrease of PCNAir in many spermatogonia as compared to control group rats (Figs.2E).However, moderate numbers of spermatogonia that have positive reaction for PCNA-ir were detected in the testes of thyroidectomized rats that treated with propolis (Fig. 2F).

Ki-67 immunoreactivity:
Figure (3) showed the detection and distribution of Ki-67 immunoreactivity, testes sections in control (G1), sham-operated (G2) and sham-operated treated with propolis (G3) groups.A positive reaction for Ki-67was observed only in the spermatogonia (Figs.3A-3D).Marked decrease in Ki-67 immunoreactivity was detected in thyrodectomized rat testis sections compared to control group (Figs.3E).On the other hand the testes of thyroidectomized rats that treated with propolis showed moderate numbers of spermatogonia that have positive reaction for Ki-67 immunoreactivity (Fig. 3F).

P53 immunoreactivity:
Figure ( 4) showed the detection and distribution of P53 immunoreactivity, in rat testes of control rats(A), shamed(B) and shamed treated with propolis (C).Spermatogonia cells in the seminiferous tubules showing low reaction for the distribution of P53-ir, while in the thyroidectomized group a positive reaction was noticed (D,E), P53 showing low reaction in thyroidectomized group treated with propolis (F).

Fig (3):
showed the detection and distribution of Ki-67 immunoreactivity, testes sections in control (G1), shamoperated (G2) and sham-operated treated with propolis (G3) groups showed positive reaction for Ki-67 only in the spermatogonia (Figs.3A-3D).Marked decrease in Ki-67 immunoreactivity was detected in thyrodectomized rat testis sections compared to control group rats (Figs.3E).On the other hand the testes of thyroidectomized rats that treated with propolis showed moderate numbers of spermatogonia that have positive reaction for Ki-67 immunoreactivity (Fig. 3F).

Discussion
Hypothyroidism that produced by thyroidectomy or goitrogen treatment has many side effects on testicular function and reduction of gonadotrophins (Gomes, 1970 andBruni et al.,1975).Thyroid hormones regulate many body activities including growth, development, oxygen consumption, respiration, digestion strength, carbohydrate, lipid and protein metabolism, central nervous system function, and the actions of other endocrine glands (Toshihiro 2010, Massoud et al., 2012, Salama et al., 2013).
The present study investigated the protetive effect of propolis against testicular toxicity induced by thyroidectomy in male rats.Thyroidectomized rats showed depression of T 3 levels while TSH levels was increased in indicating that hypothyroidism have been done..This result is in agreement with Tousson et al., (2012Tousson et al., ( , 2014) ) and Ali et al., (2013) that used thyroidectomy to achieve hypothyroid state.Choksi et al., (2003) mentioned that the elevation of TSH level can be explained by the less production of T 3 from thyroid gland and that minimizes TSH feedback inhibition leading to an elevation of its value.Also it has been reported that serum FSH levels were reduced after chronic T3 administration (Aruldhas et al., 1982;Majdic et al., 1998).
Histopathological results showed that testes of thyroidectomy rats revealed a variety of histopathological lesions when compared with control or sham operating groups.Marked histological changes were detected in the testes of mature rats under prolonged deficiency of thyroid hormones (Wagner et al., 2009).Testes in thyroidectomized rats showed many of changes as incomplete of spermatogensis, little numbers of Leydig cells, marked degeneration and significant decrease in the number of spermatogenic cells and an increased in the seminiferous tubules lumen with lack of sperms when compared with control group.These results are in agreement with a number of studies which provided evidence that hypothyroidism causes an adverse effect on the reproduction (Tousson et al., 2011(Tousson et al., , 2012)).Prolonged hypothyroidism induced in rats by chemicals leads to arrest proliferation and differentiation of germ cells, leading to decreased germ cells number and the percentage of live sperm (Sahoo et al., 2007).
Previous reports indicated that both PCNA and Ki-67 used as a marker of cell proliferation in ameloblastomas (Meer et al., 2003).Immunohistochemical observations of the testes sections showed a marked decrease of the PCNA immunoreactivity after thyrodectomy.Strong reaction for PCNA was detected in the control and sham testes sections.Ki-76 immunoreactivity showed a little to moderate number of spermatogonia that have positive reaction in testes treated with propolis.These results are in agreement with Tousson et al. (2011) who reported that; proliferating cell nuclear antigen depress in spermatogonia of hypothyroid rats.The decrease in PCNA positive expression may be due to DNA damage in the abscence cell cycling (De olivera et al., 2008: Mighell 1995).Molina et al., (2013) reported that in his study higher percentage of PCNA compared to Ki-67 and these results are in agreement of this work as PCNA is more positive than Ki-67 in control, shamed and shamed with propolis.P53 was significantly increased in thyroidectomized rats, while decreased to be like normal in thyroidectomized rats treated with propolis.
p53-dependent apoptosis appears responsible for the initial phase of germ cell loss in experimental cryptorchidism based on a 3-day delay of apoptosis in p53-/-mice (Yin et al.2002).. Wang et al., (2005) reported that apoptosis is a protective mechanism to the body by destruction of genetically damaged cells.Many reports mentioned that thyroid hormones have antiapoptotic activity, Mukherjee et al., (2014) postulated that administration of T3 rescue hypothyroid liver cells from apoptosis done by oxidative stress.Thyroid hormone plays an important role in expression of MCLI, by which mitochondrial membrane destabilization was prevented ,leading to stop cytochrome c channels by which apoptotic induced Pietrzak and Puzianowska (2008).
The obtained results showed that treating thyroidectomized rats with propolis improved the testicular histopathological as well as immunohistochemical alterations.Moreover, T3 and TSH returned to its normal values.These results are in accordance with previous investigators who studied the ameliorative effects of propolis against testicular damage.El Mazoudy et al. (2011) postulated that administration of propolis increased seminal vesicles and prostate weights and reduced the damage effects of cyclosporine-A.Capucho et al. (2012) and Rizk et al. (2014) mentioned that propolis can increase the number of sperms in rats testis.Propolis protects the male reproductive system from testicular dysfunction, deterioration in semen quality and testosterone levels (Yousef and Salama, 2009;Ogretmen et al., 2014).Many investigators postulated that the amerolating effect of propolis against reproductive toxicity was attributed to its antioxidant activity Yousef and Salama, 2009.Therefore, the present study elucidated the therapeutic effects of propolis administered to minimize the effects of thyroidectomized on reproductive toxicity.

Figure 1 :
Figure 1: Photomicrographs of rat testes stained with hematoxylin and eosin (HE).Figs.(1A-1C) showed regular cycle of spermatogenesis in testes sections of control, sham-operated and sham-operated treated with propolis groups.The thyroidectomized rat, with a significant decrease in the number of spermatogenic cells in the seminiferous(black arrow) tubules, degenerative sertoli cell(white arrow) and an increase in the seminiferous tubules lumen with lack of sperms (Figs.1D&1E).Tyroidectomized rats that treated with propolis with regular distribution in spermatogenesis cycles, mild degeneration and atrophy with a significant increase in the number of spermatogenic cells in the seminiferous tubules (Fig.1F).

Figure 2 :
Figure 2: Photomicrographs of (PCNA-ir) in the testis sections of control rats(A,B), shamed(C) and shamed treated with propolis (D), spermatogonia cells in the seminiferous tubules (arrows) showing the distribution of PCNA-ir positive cells.(E), negative reaction of PCNA-ir in spermatogonia (arrows).(F), moderate reaction for PCNA-ir in spermatogonia (arrows).PCNA indicates proliferating cell nuclear antigen.

Fig( 4 )
Fig(4): showed the detection and distribution of P53 immunoreactivity, in rat testes of control rats(A), shamed(B) and shamed treated with propolis (C), spermatogonia cells in the seminiferous tubules showing low reaction for the distribution of P53-ir, while in the thyroidectomized group a positive reaction was noticed (D,E) apoptotic marker showing low reaction in thyroidectomized group treated with propolis (F).

Table (
a,b: means in the same column with different superscript are significantly different (P≤0.05).