Determine Cyp17a1 and Ki67 Expressions in Pcos Induced Rat Model Treated with Sepia pharaonis Ink Extract Proves Effective

Marine environment includes complex ecosystems and various organisms that are branded to possess bioactive substances as a general means of self-defence or for self-protection. Recently, many bioactive compounds have been taken out, categorized and purified from diverse marine animals such as cephalopods (Nair et al., 2011) Sepia pharaonis, is one among the most significant cephalopod fishery species in south-eastern Asia (Lee et al., 2016). Sepia pharaonis ink extract is known to have several beneficiary effects. It has been used for treatment purposes. It is to be noted that endocrine metabolic dysfunction affecting 5%-20% women of reproductive age is polycystic ovary syndrome (PCOS). According to the older data, identification of PCOS in women was ascertained with irregular periods, presence of polycystic ovaries and the hyperandrogenism, increased insulin level, hormonal dysfunction, stressand increased stimulation of adrenals are measured to be most important risk factors in the expansion of PCOS (Hamza et al., 2019).
       
Recent studies show that PCOS influences 10% of women of reproductive age and is most common in developed countries. The PCOS aetiology is not fully understood (Rodgers et al., 2019). PCOS is considered as the most common women endocrinopathy. Clomiphene citrate, which is widely used for treating PCOS condition (Sushma et al., 2016). The cuttlefish ink extracts have good antibacterial activities also. Using agar well diffusion technique, when Sepia pharaonis was tested against human pathogens, it showed an inhibitory effect against these pathogens (Senan et al., 2015).
       
Different species of squid and cuttlefish also contain nutraceutical properties together with the use as a food item. Astoundingly, the inks of these different species contain different nutraceutical properties. This ink is a great source for reducing various health problems (Hossain et al., 2018). Additionally, Quercetin, a flavonoid, had shown effects by diminishing body weight, diameter of ovary, cysts and repairing healthy follicle against the positive control. The effects of quercetin are fairly similar to metformin. Therefore, quercetin also has potentials to ease the metabolic and hormonal disturbances resulting in PCOS (Jahan et al., 2018).
       
Histopathological analysis of liver treated with Letrozole illustrated moderate degeneration of hepatocytes. Thus, the Sepia ink provides a healing effect and an antioxidant capacity on BDL rats that could restructure the complications of liver cholestasis (Saleh et al., 2015).
       
Even though, the ink extract possesses various pharmacological properties such as antioxidant, anticancer, antimicrobial and antiretroviral properties it could not be used as a pharmacological agent (Anisuzzaman et al., 2001). This present finding helps us to select this therapeutic study of SPIE on PCOS experimental model. Thus, the present study measured certain hormonal parameters to discover the action of Sepia pharaonis ink extract in female wistar albino rat model of polycystic ovary syndrome (PCOS). Moreover, it also discussed the histological and Immunohistochemistry changes in the ovaries in response to the respective treatment (Fig 1).
 

Test drug
 
Sepia pharaonis fishes were obtained from seashore, Tamil Nadu. It was informed by the Zoological Survey of India that these species belonged to the Sepia pharaonis (Ehrenberg, 1831). Fishes transferred to the laboratory and the ink was squeezed out from the ink sac by milking procedure and taken in a sterile glass ware. Then lyophilisation was done to make the ink into a black powder. The lyophilized black powder of SPIE was kept in sterile condition.
 
Experimental animals
 
Virgin female wistar albino rats aged three to four months were selected for the present study. The weights of the rats ranged from 200-240 gms and were allowed to get adapted to the condition for about a week. Under hygienic condition, the rats were kept in the cage and the temperature varied from (22 ± 3°C). They were kept in the artificial light and ensured 12 hours’ day and 12 hours’ night cycle. They had unrestricted access for food and water. The experiment was done in Biomedical Research Unit and Laboratory Animal Centre (BRULAC), Saveetha Dental College and Hospital, Velappanchavadi, Chennai-600077, Tamil Nadu, India in the year 2018 conferring to the strategies of Institutional Animal Ethical Committee (BRULAC/SDCH/SIMATS/IAEC/09-2018/012) of CPCSEA.
 
Experimental design
 
The female rats were categorized into five groups with 6 animals in each group. Only distilled water was given to the control group. For 21 days, letrozole 1mg/kg b.wt was administered to the subsequent groups to induce PCOS for other four groups. Subsequently, the PCOS model evaluation with an anovulatory estrous cycle was verified by analysis of vaginal smear, for which the first one-third of the vaginal wall was taken daily to evaluate the stage of the 4-day ovarian cycle during the examination of the study period. Group II is only PCOS induced. For 19 days, an allopathic drug, clomiphene citrate 1mg/kg was given to the standard treated group III. The different doses of SPIE (100 mg and 200 mg respectively) were administered to the next two groups along with water to test its beneficial action.
 
Collection of tissue and blood samples
 
All animals were weighed at the beginning and end of study. The rats were anesthetized with a single intraperitoneal injection of ketamine (80 mg/kg) and xylasine (12 mg/kg). The blood samples were obtained by retro-orbital puncture and transferred to EDTA-containing tubes. Plasma was separated by cold-centrifugation (4°C) at 3,000 rpm for 10 min. Ovarian tissues were excised for histological analysis and fixed in 10% formaldehyde (PBS 10 mM, pH 7.4). Plasma samples were stored in a freezer at -80°C for biochemical analysis.
 
Assay of biochemical parameters
 
Serum levels of estradiol, testosterone and progesterone were determined using a competitive enzyme-linked immunosorbent assay using commercially available ELISA kits (Elab Bioscience).
 
Histopathological study
 
Ovaries were fixed in 10% formalin at RT for a week. In a graded series of ethanol, the specimens were dehydrated, cleared in xylene and fixed in paraffin wax. Using a rotary microtome, tissue blocks were partitioned into 5 µm thickness. Sections were stained and viewed under the light microscope by a qualified pathologist.
 
Immunohistochemistry study
 
Immunohistochemically analysis was performed on cytochrome P450, 17alpha-hydroxylase protein staining in ovary tissues. The tissues of each group were immersed in the fixative solution for 4 h. The tissues were cryoprotected in 30% sucrose, embedded in tissue-freezing medium with liquid nitrogenand cut into frozen sections (3-5 µm) using a cryostat. Sections were stored under anti-freeze buffer. Parallel free-floating sections were subjected to endogenous peroxidase quenched with 1% H₂O₂ in PBS, followed by treatment with blocking buffer (5% normal chicken serum in PBS and 0.3% Triton X-100 for overnight at 4°C) and incubated with CYP17A1 primary antibody (dilution 1:500). After washing with PBS, tissues were incubated with a biotinylated goat anti-mouse secondary antibody (dilution 1:200). The tissues were subsequently exposed to an avidin-biotin peroxidase complex for 2 h. The peroxidase activity was visualized using a stable diaminobenzidine solution. All immunoreactions were observed using a compound light microscope.
       
The tissue sections were subjected to endogenous peroxidase quenched with 1% H₂O₂ in PBS, followed by treatment with blocking buffer (5% normal chicken serum in PBS and 0.3% Triton X-100 for overnight at 4°C) and incubated with ki67 primary antibody with the dilution 1:200. After washing with PBS, tissues were incubated with a biotinylated goat anti-mouse secondary antibody 1: 100 for 8hrs at 4°C. The tissues were subsequently exposed to an avidin-biotin peroxidase complex for 2 h. The peroxidase activity was visualized using a stable diaminobenzidine solution. All immunoreactions were observed using a compound light microscope.

Effect of SPIE on reproductive hormone levels
 
The data on the serum levels of progesterone, estradiol and testosterone in control and different treatment groups are presented in Fig 2. Data discovered a significant reduction in progesterone, estradiol accompanied by a rapid increase in the testosterone levels in rats with PCOS when compared to the healthy control group. Diseased rats treated with clomiphene citrate showed a remarkable decrease in the testosterone levels and significant increase in the estradiol and progesterone levels when compared to PCOS group. Consistently, a minor regression in the levels of testosterone accompanied by a rapid increase in estradiol and levels of progesterone were noticed in PCOS rats treated with SPIE 100 mg compared to PCOS rats. Similar findings were observed in diseased rats treated with SPIE 200 mg with a major regression in the levels of testosterone. Moreover, the reproductive hormone levels in SPIE treated PCOS rats were comparable to that of control, in which SPIE 200 mg treated PCOS rats exhibited good results when compared.
 

 
Effect of SPIE on ovary weight of rats in PCOS model
 
The ovary (Left and right) weight of test animals of both control and different treatment groups were depicted in Fig 3. PCOS group exhibited significant increase in ovary weight when compared with the control. Whereas clomiphene citrate treated PCOS rats showed a significant reduction in the ovary weight in contrast to PCOS group. SPIE treated PCOS rats were comparable to that of healthy rats. It has been shown from the results that SPIE with a dose of 200 mg considered to be safer than other treatment groups.
 

 
Effect of SPIE on ovary histology study
 
Fig 4A and 4C, healthy tertiary follicle in the normal control and standard drug treated rats. The theca layers and granulosa layers appear normal. Fig 4B follicle in the early process of atresia with apoptotic granulosa cells, most of which are in the inner parts of the granulosa layer in a polycystic ovary syndrome (PCOS) rat. Thin and elongated epithelioid cells form the inner surface of the wall. The cyst fluid contains macrophages. Fig 4 D and E from a SPIE (100 and 200 mg) treated PCOS rat with normal tertiary follicles. (H & E staining; index bars, 50 μm)
 

 
Effect of SPIE on CYP17A1 and Ki 67 protein expression in immunology-assay
 
In Fig 5 the level of CYP17A1 and Ki 67 expression in control group was low compared to PCOS group. PCOS+100 mg expressed moderately reduced level when compared to control and PCOS. PCOS+CC and PCOS+200 mg expressed good reduced levels of protein expression when compared to control and PCOS group.
 

       
Immunohistochemistry of CYP17A1 and Ki 67 pictures is depicted in Fig 6 and 7(A-E). 6 and 7A-Signifies control group showing decreased level of CYP17A1. 6 and 7B-Signify the PCOS group with increased levels of CYP17A1. 6 and 7C and 6 and 7E Signify the treatment groups PCOS+CC and PCOS+SPIE 200 mg highlights the reduced levels of CYP17A1. 6 and 7D-signify the PCOS+SPIE100 mg with slight reduction in CYP17A1 levels.
 

 

 
Effect of SPIE on body weight of rats in PCOS model
 
The body weight of PCOS was significantly rising compared to control group. PCOS+CC group also showed significant weight reduction compared to PCOS group. PCOS+SPIE 100 mg and PCOS+SPIE200 mg showed very progressive weight reduction compared to PCOS group as in Fig 8.
 

       
The most prevalent hormonal disorder found in reproductive aged women is PCOS. The weight of ovary gets decreased in PCOS rats (Song and Tan, 2016). On the contrary, a rapid increase in the ovary weight of PCOS group was observed in our study which was confirmed with the study of (De Leo et al., 2016). This was supported by the study of Louise et al., (2007) which showed an increase in ovary weight of Letrozole rats. Reduced aromatase effect might increase ovarian production of androgen and reduce oestrogen production, resulting to PCOS. Ovary weight found to be slightly higher in SPIE treatment rats when compared to the control in the present study. The present study also confirmed that SPIE treatment was mildly effective in normalizing the ovarian weight of rats when compared with PCOS group.
       
Many people with PCOS have higher insulin levels that can excite the ovaries to produce testosterone. Hyperan drogenism, particularly unbalanced testosterone levels are a key disturbance noticed in PCOS (Azziz et al., 2006). Testosterone has relatively strong bond with globulin (66-78%) and weak bond with albumin (20-32%). Only 1-2% of the total pool of testosterone is not protein-bound and thus biologically active (Mroczko and Medras, 2003). In PCOS women, free testosterone and free androgen index (FAI) are the markers of hyperandrogenism (Rotterdam, 2003; Conway et al., 2014). The levels of total testosterone in PCOS women might be normal or somewhat increased (maximal 5.2 nmol/L) (Sheehan, 2004). The present study confirmed higher testosterone levels in PCOS rats which was reduced by an allopathic drug and further normalized with SPIE treatment. Higher levels of total testosterone are typical for tumours that synthetize androgens (Conway et al., 2014). It was exposed that augmented levels of total testosterone in PCOS women, participates in the pathogenesis of atheroslerosis (Hak et al., 2002). Progression of several reproductive defects related with PCOS, with disruption of cyclicity, higher levels of testosterone levelsand expression of enzymes involved in androgen biosynthesis, as well as ovulatory dysfunction, were found in a rat model with targeted deletion of Pcos particularly in ovarian theca cells (Lan et al., 2017). NKB receptor antagonism in PCOS females were found to reduce LH pulse frequency and decrease serum LH as well as testosterone levels (George et al., 2016).
       
Progesterone is considered to be the primary GnRH pulsatility regulator. The present investigation confirmed a reduction in the levels of progesterone in PCOS rats further increased with standard drug and SPIE treatment. It is identified that PCOS patients exhibit a GnRH-generating pulse resistance to negative feedback by progesterone, which leads to higher LH pulses frequency and/or amplitude (Eagleson et al., 2000). Besides, LH excess is a variable PCOS feature, thus, it is hard to point the primary role to LH excess in the disease progression of all PCOS patients.
       
The effect of E₂ on granulosa cell GC function are not being clinically tested in PCOS women. Preclinical studies exhibited that higher estradiol concentration after ovarian stimulation, (Homer et al., 2014), negatively affects endometrial receptiveness. The current investigation showed reduced levels of estradiol in PCOS rats can be increased by different treatment. In contrast, studies have connected higher levels of estradiol with altered gene expression during implantation and asynchrony among endometrial gland as well as stroma development. Data from clinical observations recommend that higher concentration of estradiol has an adverse action on pregnancy outcomes, after fresh embryo transfer. Though, PCOS women usually have a greater ovarian response for the stimulation and usually have more oocytes, along with a higher estradiol level, than ovulatory women (Wei et al., 2018). The body weight of PCOS rats was significantly higher compared to control and treatment group (Karatekea et al., 2018). Similar results were found in our study.
       
The melanin in the sepia ink, a carrier of dopamine prevents excess dilution of ink after ejection in water. (Gabriella Fiore et al., 2004). Further studies implicit that Dopamine agonist effectively reduces the levels of Luteinizing hormone and testosterone levels (Dirami and Cook 1998). The SPIE also contains melanin and dopamine that must have acted on the PCOS ovaries to reduce the levels of androgenic hormone and induced ovulation.
       
Dopamine agonist cabergoline, a safe and well established medicine was useful in prevention of ovarian hyperstimulation syndrome in PCOS women undergoing assisted reproduction (Alvarezc et al., 2007). In the PCOS induced group the numbers of preantral follicles, antral follicles and corpus luteum decreased compared to the control groups. Treatment by Clomiphene citrate and SPIE increased the number of maturing ovarian follicles. Histological evaluation of ovaries revealed that thickness of granulosa and tunica albuginea increased, while thickness of theca layer deceased in the Pcos group compared to the control groups; however, consumption of SPIE and standard showed normal thickness of ovary layer. Immunoexpression of Ki-67 was higher in the theca interna cells and CYP17A1 in the interstitial and granulosa cells in the PCOS group (Leonardo Augusto Lombardi et al., 2014). Similarly, in our study PCOS group had more Ki-67 and CYP17A1 Immunoexpression compared to control and treatment group.
 

PCOS is a multifaceted disorder for which several treatment approaches are needed, depending on the reason for treatment. SPIE has exposed the best outcome study in treating infertility and also the data are limited concerning the treatment on PCOS. In the present treatment SPIE proved to be effective in controlling the hormones levels especially testosterone more effectively than standard drug treatment and oestrodiol and progesterone levels similar to treatment drug. Further confirmation by histopathological and Immunohistochemistry confirms that SPIE can be used as a drug of choice to effectively control PCOS in women of the present world and facilitate the healthy reproductive process. Advance studies are required to examine the mechanism of action and the effect of SPIE treatment.

I extend my hearty thanks to Saveetha Institute of Medical Sciences and Technology for providing a platform to conduct my research process in Biomedical Research Unit and Laboratory Animal Centre (BRULAC). I extent my sincere thanks to all technical staff in BRULAC for helping me in the research strategy.

All authors declare that they have no conflict of interest.