Action of Citrullus lanatus on the Cytoarchitecture of Testes in Wistar Rats Following Prolong Exposure to Methanolic Seeds Extract of Carica papaya

Aim: This study was carried out to examine the action of Citrullus lanatus on the cytoarchitecture and glycogen distribution in the testes of Wistar rats following prolong exposure to methanolic seeds extract of Carica papaya. Study Design: Eighteen adult male Wistar rats were randomly assigned into three groups of six (n=6): A, B, and C. Group A served as the control group while the other groups served as the treated groups. Place and Duration of Study: This study was carried out in the Animal Holdings of the Department of Anatomy, University of Ilorin, Ilorin, Nigeria between July, 2014 and October, 2014. Methodology: The Group A animals were given feed and water liberally throughout the study. Group B received 150 mgkg/bwt/d of Carica papaya methanolic seeds extract intraperitoneally for 8 weeks, Group C received 150 mgkg/bwt/d of Carica papaya methanolic seeds extract Original Research Article Amedu and Idoko; JALSI, 6(2): 1-7, 2016; Article no.JALSI.22956 2 intraperitoneally plus Citrullus lanatus orally for 8 weeks. The rats were sacrificed at the end of administration, histological and histochemical techniques were applied. Tissues were viewed under a light microscope. Results: Generally, the seminiferous tubules as well as interstitial spaces in Group A and C appeared normal but in Group B there was degeneration of interstitial spaces and congestion of seminiferous tubules. However, glycogen distribution was the same for all the groups. Conclusion: Citrullus lanatus has ameliorative effects on cytoarchitecture of the testes in wistar rats following prolong exposure to methanolic seeds extract of Carica papaya. It was also revealed that methanolic seeds extract of Carica papaya does not affect the glycogen distribution in testes especially at the dose used in this study.


INTRODUCTION
Carica papaya is a tasty fruit that is rapidly propagated in the tropics, it is believed to be a native of southern Mexico and neighbouring Central America [1]. It belong to the family of Caricacea [2]. It is commonly referred to as pawpaw (UK), papaya (France), papita (India), fruita bomba (Cuba) and mamão (Brazil) [3].
Distorted cytoarchitecture and reduction in interstitial cells has been linked to infertility following administration of unripe Carica papaya seeds [16]. Aqueous bark extract of Carica papaya has deleterious effects on both the seminiferous tubules and testicular interstitium [17]. Also, severe necrosis of the germinal epithelium of the testes has been reported following administration of root extracts of Carica papaya [12].
Citrullus lanatus is a tropical plant which belongs to the family of Cucurbitacea. It is grown all over South East Asia and most part of Africa [18]. It is commonly referred to as Egusi melon, egusi watermelon, West African watermelon, dessert watermelon, cooking melon [19,20]. Citrullus lanatus has fleshy parts that are red, orange, yellow or white in colour depending on the variety and green outer part [21,22]. The inner red part of Citrullus lanatus contains large quantity of beta carotene and is a significant source of lycopene [19,18]. Lycopene is rich in antioxidant and has potential health benefits. Citrullus lanatus plant has bioactive compounds such as cucurbitacin, triterpenes, sterols and alkaloids [23,24].
The extracts of Citrullus lanatus are used for various purposes such as; protection against prostate cancer [25,26] treatment of type 2 diabetes [27], improve blood vessel function [28] and reduction of blood pressure in hypertensive patients [29]. Others are: antimicrobial activities, antigiardial activity and anti-inflammatory activity [30,31]. Methanolic extracts of Citrullus lanatus seeds has proven to be a treatment option against lead-acetate induced oxidative stress and toxicity in testicular tissue [32].
As a result of earlier reported effects of Carica papaya on seminiferous tubules and testicular interstitium, this study was designed to examine the action of Citrullus lanatus on the cytoarchitecture and glycogen distribution in the testes of Wistar rats following prolong exposure to methanolic seeds extract of Carica papaya.

MATERIALS AND METHODS
Eighteen adult male Wistar rats (with an average weight of 240 g) were procured from department of anatomy, Ladoke Akintola University of Technology. The animals were acclimatized for two weeks at the animal holdings of Anatomy department, University of Ilorin, Ilorin before the commencement of the experiment. They were exposed to normal laboratory conditions of temperature, light and humidity. The rats were fed with pelletized feeds (produced by Vital feeds, Nigeria) and water ad libitum. The animals were given adequate care in accordance with the Principle of Laboratory and Animal Care prepared by the National Academy of Sciences and published by the National Institute of Health [33]. All the rats were carefully assessed and screened at the end of the acclimatization period. The investigation was conducted in accordance with the principles and guidelines for animal research.

Plant Materials and Extraction
Ripe Carica papaya was bought from Oja Oba market, Ilorin and it was authenticated at the Pharmacology department of university of Ilorin. The seeds were removed and air dried for 7 days. There were later pounded into powdery form using mortar and pestle. 80.65 g of the sample was immersed in 300 mL of methanol for 48 hours after which the solution was sieved and evaporated to dryness at 30°C. It was then weighed and refrigerated until use.
The Citrullus lanatus used was bought from Oja Oba market, Ilorin and it was authenticated at the Pharmacology department of university of Ilorin. The rind was separated from its inner part. The inner red part was chopped into small pieces then blended with an electronic blender. The juice was later concentrated using Rotary evaporator and then stored in a refrigerator at 4°C until use.

Experimental Design
Eighteen adult male rats were randomly divided into three (3) groups of six (6)

Termination of Treatment
At the end of 8 weeks, the rats were anaesthetized by cervical dislocation and then sacrificed. The testes were collected and immediately fixed in formal saline before embedding in paraffin wax. Sections were prepared using an electric rotatory microtome (YD-355AT) at 5 µm. The sections were stained with haematoxylin and eosin (H & E) stain, Periodic Acid-Schiff reaction (using the method of McManus, 1946) and feulgen DNA reaction (using the method of Feulgen and Rossenbeck, 1924) [34]. The stained sections were then viewed under the light microscope.

DISCUSSION
It has been reported that seed extract of Carica papaya causes infertility in male albino rats [14,35]. However, lycopene is believed to be a treatment for idiophatic infertility [36]. Citrullus lanatus is one of many plants that has lycopene content [18]. Lycopene is rich in antioxidant and has potential health benefits. Lycopene utilizes redox defence mechanism to fight against free radicals that could cause infertility [36]. Testes is believed to be an organ that store lycopene in human [36].
The testes is a part of male reproductive system, it has a dual function which is to produce spermatozoa and secrete testosterone [37]. Testosterone is a steroid hormone that belong to the androgen group. It is secreted primarily in the Leydig cells of the testes through stimulation of luteinizing hormone (LH) [38].
Among the structures that make up the testes are seminiferous tubules which produces the male reproductive cells and interstitial cells which occupy much of the space between the seminiferous tubules. The basal lamina of the testes is lined externally by myoid cells [37].
The present study (in Plate 1) shows that the interstitial space and seminiferous tubules with spermatocytes appeared normal in Group A and Group C. However Group B shows degenerated or disorganized interstitial space and congested seminiferous tubules. The degenerated interstitial space and congested seminiferous tubules seen in Group B was ameliorated in Group C possibly due to Citrullus lanatus administered which contains lycopene. Earlier findings supported this observation [32,17].

CONCLUSION
In conclusion, this present work has shown that Citrullus lanatus has ameliorative effects on cytoarchitecture of the testes in wistar rats following prolong exposure to methanolic seeds extract of Carica papaya. It has also revealed that Carica papaya methanolic seeds extract does not affect the glycogen distribution in the testes especially at the dose used in this study. Further studies is recommended to build on the findings of this work.

ETHICAL APPROVAL
All authors hereby declare that "Principles of laboratory animal care" (NIH publication No. 85-23, revised 1985) were followed, as well as specific national laws where applicable. All experiments have been examined and approved by the appropriate ethics committee. All authors hereby declare that all experiments have been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.