{"id":36958,"date":"2021-03-30T11:18:08","date_gmt":"2021-03-30T11:18:08","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=36958"},"modified":"2021-04-09T05:49:56","modified_gmt":"2021-04-09T05:49:56","slug":"ethanol-extract-of-young-papaya-seeds-carica-papaya-l-lower-the-level-of-testosterone-spermatozoa-count-and-expression-of-androgen-receptors-in-sertoli-cells-of-adult-mice-mus-musculus","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol14no1\/ethanol-extract-of-young-papaya-seeds-carica-papaya-l-lower-the-level-of-testosterone-spermatozoa-count-and-expression-of-androgen-receptors-in-sertoli-cells-of-adult-mice-mus-musculus\/","title":{"rendered":"Ethanol Extract of Young Papaya Seeds (Carica Papaya, L ) Lower the Level of testosterone, Spermatozoa Count and Expression of Androgen Receptors in Sertoli Cells of Adult Mice (Mus Musculus)"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

The Indonesian government deals with uncontrolled population growth via its Family Planning (KB) program.\u00a0The implementation of family planning aims to inhibit or postpone pregnancy for various reasons, including planning for pregnancy, limiting the number of children, and avoiding the medical risks of pregnancy1<\/sup>.<\/p>\n

Active participation of men in family planning in developing countries other than Indonesia varies, for example, Bangladesh 8%, Nepal 24%, and Malaysia 16.8% according to the 2007-2008 SDKI (Indonesian Demographic Health Survey)2<\/sup>.<\/p>\n

Family planning programs require the participation of various parties, both men and women.\u00a0However, the participation of men in family planning is lacking because in the family planning product and service domain, the contraceptives are aimed solely for women as the primary target compared to contraception for men. It means that, men have few options for contraception compared to women. Existing male contraceptive methods consist of interrupted intercourse (coitus interruptus),\u00a0<\/em>condoms, and vasectomy.\u00a0The interrupted intercourse (coitus interuptus)\u00a0<\/em>is the oldest natural contraceptive method performed by ejaculating sperm outside the vagina during a fertile period3<\/sup>.<\/p>\n

Research on plants that have the antifertility properties developed by gathering information from the knowledge of traditional medicine4<\/sup>. There are various types of plants that can be used as traditional medicine. The papaya plant (Carica papaya L.<\/em>) contained broad spectrum of phytochemicals, including polysaccharides, vitamins, minerals, enzymes, proteins, alkaloids, glycosides, fats and oils, lectins, saponins, flavonoids, steroids, etc5<\/sup>.<\/p>\n

Satriyasa&Pangkahila reported that methanol fraction and n-hexane fraction of young papaya seeds could inhibitspermatogoniadevelopment in male mice.\u00a0Based on this background,we prepared an experiment to investigate whether an ethanol-based extract of young papaya seeds (Carica papaya L.<\/em>) at a level of 20 mg\/20g of body weight lower testosterone levels, sperm count and the expression androgen receptors in sertoli cells in adult mice (Mus musculus<\/em>) compared to a control group6<\/sup>.<\/p>\n

Material and Method<\/strong><\/p>\n

This is an experimental study using a randomized post-test-only control group design,<\/em> conducted from July 2018 to February 2019. We use adult male mices (Mus musculus<\/em>) as experimental animals.<\/p>\n

The study has received ethical clearance\u00a0from the ethics committee of the faculty of\u00a0medicine, Udayana University\u00a0No: 1955\/UN 14.2.2.VII.14\/LP\/2018, datedon \u00a0September 3rd<\/sup>, 2018, and\u00a0has\u00a0received research implementation approval from the Integrated Biomedical Lab Unit, No: 403\/UN 14.2.2VII.6\/LT\/2018.<\/p>\n

Young papaya seeds\u00a0(Carica papaya L.) <\/em>were\u00a0obtained from farms in the Cau hamlet\u00a0area, Tua Village, Marga District, Tabanan Regency, Bali.\u00a0The extraction process was carried out by the maceration method with\u00a096% ethanol solvent.\u00a0Three thousand gram dried young papaya seeds\u00a0(Caricapapaya L.)\u00a0<\/em>produced from 1000 grams of dried organic matter. This organic matter was immersed in ethanol or five days and was stirred every 6 hours. Then, the solution was filtered, and the pulp was macerated again with a 25% mixture (2.5 liters) of solvent and macerated again with the same process.\u00a0Thin Layer Chromatography (TLC) tests were conducted at UGM Integrated Testing and Research Laboratory. The qualitative testof the ethanol extract of young papaya seed (Carica papaya L.<\/em>) showsa positive result for the presence of steroids.<\/p>\n

The ethanol\u00a0extraction\u00a0of\u00a0young\u00a0papaya\u00a0seeds\u00a0was\u00a0managed and performed at the Pharmacy Laboratory, Faculty of Medicine, Udayana University. The maintenance and treatment of the mices were carried out at the Laboratory Animal Unit, Pharmacology Division, Faculty of Medicine, Udayana University. Immuno histochemical\u00a0 (IHC)\u00a0 examination of androgen receptors on the sertoli cells was carried out\u00a0in the Integrated Biomedical Laboratory of Faculty of Medicine, Udayana University\u00a0and the Histology Division of the Faculty of Medicine, Udayana University. The testosterone and spermatozoa examination was carried out at the Reproductive or Anatomy Laboratory, Faculty of Veterinary Medicine, Udayana University.<\/p>\n

We study on adult male mices (Mus musculus<\/em>) aged 12-14 weeks,\u00a0Balb\/c strain, weighing 20-25 grams, from one offspring, healthy and not physically disabled. They were obtained and maintained at the Pharmacology Laboratory, Faculty of Medicine, Udayana University, following the principles of 3R, among others7<\/sup>. The total samples were 36 male mices,divided randomly and equally into the treatment and control group.\u00a0The animals were kept in a 40x30x15 cm cage with a 12\/12 hour light-dark cycle (dark from 8 pm to 8 am and light from 8 am to 8 pm).\u00a0The animals were given free access to food and sufficient water (ad libitum<\/em>).\u00a0The treatment group was givenethanol extract of\u00a0young\u00a0papaya\u00a0seeds\u00a0with\u00a0levels of steroids, flavonoids of\u00a020 mg\/20 g body weight, 0.5 ml, every day for 36 days, before treatment the\u00a0mice were\u00a0adapted for one week; on day 37, blood samples were taken from themices for hormone testing, sperm was collected for spermatozoa count, and testicular tissue was taken for examination of androgen receptor expression.<\/p>\n

Data normality was tested using the Shapiro-Wilk test because of the small sample size.\u00a0P-value>0.05 indicates the data is normally distributed; in contrast, p-value<0.05 indicates the data is abnormally distributed (skewed). Comparative tests were used to determine differences in\u00a0the levels of testosterone, spermatozoa count, and number of androgen receptors in sertoli cells in both groups. Itwas achieved using an unpaired t-test if the distribution of data was normal or a Mann-Whitney test if the data is abnormally distributed.<\/p>\n

Results<\/strong><\/p>\n

Variable levels testosterone,\u00a0<\/em>spermatozoa, and androgen receptor expression in sertoli cells\u00a0in the treatment and control groups were all plotted on a numeric scale so that data normality tests could be performed as a condition of bivariate analysis (Table 1).Data is normally distributed if the p-value>0.05.<\/p>\n

Table 1: Normality test of testosterone, spermatozoa and androgen receptor expression in sertoli cells in both groups<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\u00a0Variables<\/strong><\/td>\nGroup<\/strong><\/td>\nShapiro-Wilk<\/strong><\/td>\n<\/tr>\n
Statistics<\/strong><\/td>\ndf<\/strong><\/td>\nSig.<\/strong><\/td>\n<\/tr>\n
Testosterone<\/strong><\/td>\nTreatment<\/td>\n0.93<\/td>\n18<\/td>\n0.23*<\/td>\n<\/tr>\n
Control<\/td>\n1,00<\/td>\n18<\/td>\n1,00*<\/td>\n<\/tr>\n
Spermatozoa<\/strong><\/td>\nTreatment<\/td>\n0.96<\/td>\n18<\/td>\n0.61*<\/td>\n<\/tr>\n
Control<\/td>\n.97<\/td>\n18<\/td>\n0.87*<\/td>\n<\/tr>\n
Androgens Receptor Expressionin Sertoli cells<\/strong><\/td>\nTreatment<\/td>\n0.92<\/td>\n18<\/td>\n0.14*<\/td>\n<\/tr>\n
Control<\/td>\n0.94<\/td>\n18<\/td>\n0.23*<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*p-value<0.05= significant<\/p>\n

Table 2 shows the differences between testosterone levels in the treatment and control\u00a0groups. The mean levels of\u00a0testosterone in the treatment group were lower than the control group following the daily administration of the ethanol extract of young papaya seed\u00a0for 36 days.\u00a0The mean difference between the two groups was 17.02\u00a0%, with a p-value<0.001.<\/p>\n

Table 2: Differences\u00a0in\u00a0the testosterone\u00a0levels\u00a0between the two groups<\/strong><\/p>\n\n\n\n\n\n
Variable<\/strong><\/td>\nGroup<\/strong><\/td>\nAverage\u00a0\u00b1 SD<\/strong><\/td>\nMedian<\/strong><\/p>\n

Min-Max<\/strong><\/td>\n

Average difference<\/strong><\/td>\nCI 95%<\/strong><\/td>\np-value<\/strong><\/td>\n<\/tr>\n
Testosterone<\/td>\nControl<\/td>\n48.67\u00b11.81<\/td>\n48.66<\/p>\n

45.01\u00b152.35<\/td>\n

17.02<\/td>\n15.76-18.29<\/td>\n<0.001*<\/td>\n<\/tr>\n
Treatment<\/td>\n31.64\u00b11.91<\/td>\n31.36<\/p>\n

28.69\u00b135.28<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Table 3 and\u00a0figure 1 illustrate the differences between the number of spermatozoa in the\u00a0control\u00a0and\u00a0treatment\u00a0groups.\u00a0The number of spermatozoa in the treatment group is lower than that of the control group, following the daily administration of ethanol extract of young papaya seed\u00a0 for 36 days. The mean difference between the two groups was 33,17, with a p-value<0.001.<\/p>\n

Table 3: Differences\u00a0between spermatozoa\u00a0cell\u00a0counts in the two groups<\/strong><\/p>\n\n\n\n\n\n
Variable<\/strong><\/td>\nGroup<\/strong><\/td>\nAverage\u00a0\u00b1 SD<\/strong><\/td>\nMedian<\/strong><\/p>\n

Min-Max<\/strong><\/td>\n

Average difference<\/strong><\/td>\nCI 95%<\/strong><\/td>\np-value<\/strong><\/td>\n<\/tr>\n
Spermatozoa<\/td>\nControl<\/td>\n75.89\u00b14.71<\/td>\n76.67<\/p>\n

67,33\u00b184.00<\/td>\n

33.17<\/td>\n30.39-35.94<\/td>\n<0.001*<\/td>\n<\/tr>\n
Treatment<\/td>\n42.72\u00b13.33<\/td>\n42.50<\/p>\n

37.67\u00b149.67<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*p-value<0.05= significant<\/p>\n\n\n\n
\"Figure<\/a><\/td>\nFigure 1: The number of spermatozoa in the\u00a0treatment and control group.<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

The androgen receptor expression in\u00a0the\u00a0sertoli\u00a0<\/em>cells was\u00a0measured by IHC on testicular tissue using the\u00a0Rabbit Anti-phospho-Androgen Receptor Polyclonal Antibody (Ser578) kit.\u00a0<\/em>Alight microscope connected to\u00a0Optilab,\u00a0<\/em>with\u00a0a 400x\u00a0magnification\u00a0(40x objective and 10x ocular) was used.<\/p>\n

The results\u00a0of the IHC examination\u00a0of the androgen receptor expression in\u00a0the sertoli\u00a0cells of\u00a0mice in the control group and the treatment group over the 36-daytest period are presented\u00a0in\u00a0table 4 and figure 2.<\/p>\n

Table 4:\u00a0Differences in androgen receptor expression in sertolicells in both groups.<\/strong><\/p>\n\n\n\n\n\n
Variables<\/strong><\/td>\nGroup<\/strong><\/td>\nAverage\u00a0\u00b1 SD<\/strong><\/td>\nMedian<\/strong><\/p>\n

Min-Max<\/strong><\/td>\n

Average difference<\/strong><\/td>\nCI 95 %<\/strong><\/td>\np-value<\/strong><\/td>\n<\/tr>\n
Receptor Expression<\/td>\nControl<\/td>\n55.08\u00b12.49<\/td>\n55.60<\/p>\n

47.74\u00b159.22<\/td>\n

26.97<\/td>\n25.10-28.83<\/td>\n<0.001*<\/td>\n<\/tr>\n
Androgens in Sertoli cells<\/td>\nTreatment<\/td>\n28.11\u00b13.06<\/td>\n27.52<\/p>\n

23.81\u00b135.07<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Vol14No1_Eth_Dew_fig2\"<\/a><\/td>\nFigure 2: Amount of androgen receptor expression\u00a0 in\u00a0 sertoli\u00a0 <\/em>cells\u00a0in\u00a0both groups.Sertoli\u00a0<\/em>cell\u00a0whose cell nucleus has expressed\u00a0androgens (green arrows); sertoli cell whose cell nucleus has not expressed\u00a0androgens (red arrows).<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Figure\u00a02 shows that in the treatment group, the\u00a0appearance of\u00a0androgen\u00a0expression\u00a0(brown color cell nucleus) was less\u00a0than in the control group.\u00a0The green arrow indicates a\u00a0sertoli\u00a0<\/em>cell\u00a0whose cell nucleus has expressed\u00a0androgens, whereas\u00a0the red\u00a0arrow indicates a sertoli cell whose cell nucleus has not expressed\u00a0androgens.<\/p>\n

Discussion<\/strong><\/p>\n

Ethanol extract of young papaya seeds (Carica papaya L.<\/em>) lower\u00a0the testosterone levels\u00a0in adult mice (Mus musculus<\/em>)\u00a0<\/strong><\/p>\n

Testosterone is a primary steroid hormone in men derived from cholesterol precursor molecules, produced by the testes. Testosterone stimulates the development of primary sex organs of the embryo and encourages spermatogenesis. Besides, there are three main functions of testosterone, (1) maintaining primary and secondary genitalia, (2) maintaining the process of spermatogenesis to produce spermatozoa, and (3) ensuring the maturation of spermatozoa to be able to carry out fertilization.<\/p>\n

The results of this study are supported by previous studies, which show that natural ingredients have an antifertility effect by lowering testosterone levels. One study showed lower testosterone levels in the treatment group of papaya seed methanol extract and papaya seed methanol fraction at a dose of 100 mg\/kg body weight8<\/sup>.The administration of beluntas leaf active compound has the antifertility effect and decreasing testosterone levels9<\/sup>.<\/p>\n

The low level of testosterone in the treatment group is most likely due to low levels of FSH and LH. Ethanol extracts of young papaya seeds inhibit the secretion of FSH and LH simultaneously, which then inhibits the secretion of testosterone by leydig cells or often called interstitial cells.<\/p>\n

Apart from inhibiting LH secretion, the ethanol extract of young papaya seeds may have an effect of directly reducing testosterone in leydig cells involving alkaloids. Research has reported that alkaloids and steroids affect the permeability of leydig cell membranes, resulting in food transfer impairment as an energy source that failed the biosynthesis of testosterone9<\/sup><\/p>\n

Alkaloids papaya seed can also inhibit the function and development of leydig cells10<\/sup>.Also, alkaloids can inhibit the antioxidant system in leydig cells, thereby disrupting the intracellular redox system and causing disruption of leydig cell physiological functions11<\/sup>. When leydig cells are inhibited, the amount of testosterone produced decreases so that the ability of testosterone to stimulate the sertoli cells to carry out the process of spermatogenesis is also reduced.<\/p>\n

Ethanol extract of young papaya seeds (Carica papayaL.<\/em>) lower spermatozoa counts in adult mice (Mus musculus<\/em><\/strong><\/p>\n

The number of\u00a0spermatozoa\u00a0is widely used to test fertility, and also to test the antifertility effect of a particular compound.\u00a0Previously,\u00a0papaya\u00a0seed extract\u00a0has been extensively studied in terms of its effect as a natural ingredient with antifertility properties.<\/p>\n

The results of this study explain the antifertility mechanism of young papaya seed ethanol\u00a0extract\u00a0comprehensively, ranging from the low levels of FSH and LH, which then leads to\u00a0low levels of testosterone and low\u00a0spermatozoacell counts.\u00a0Testosterone is needed for the development of germ cells and the release of adult spermatids during stage VIII in mice12<\/sup>.\u00a0FSH, together with testosterone, supports the continuation of meiosis of cells and spermatids through intrinsic and extrinsic pathways13<\/sup>.<\/p>\n

In addition to the barrier effect of spermatogenesis through testosterone, LH, and FSH, the young papaya seed ethanol\u00a0also contains alkaloids that have a direct cytotoxic effect on spermatozoa.\u00a0Alkaloids in\u00a0papaya\u00a0seeds are\u00a0known to cause the degeneration of\u00a0spermatozoa\u00a0<\/em>cells\u00a0and reduce the number of\u00a0spermatozoa\u00a0<\/em>cells.\u00a0It indicates that during the process of spermatogenesis is disordered as a result of the presence of alkaloids in theethanol extract of young papaya seeds14<\/sup>.<\/p>\n

Ethanol extract of young papaya seeds (Carica papaya L.<\/em>) lower the number of androgen receptor expressions in sertoli cells in adult mice (Mus musculus<\/em>)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<\/strong><\/p>\n

The activation of androgen receptor (AR) by testosterone is essential for the maturation and function of leydig cells and the regulation of stereogenic enzymes15<\/sup>.\u00a0Androgen receptorautocrinesignaling in leydig cells protects the epithelium of the seminiferous tubules in mice and protects the leydig cell apoptosis in adult mice.\u00a0The number of receptors in a cell largely determines its reaction to hormones.\u00a0If enough hormones and receptors are in the cell, the ligand (testosterone) binds and activates the receptor16<\/sup>.<\/p>\n

This study is the first study to examine the effect of\u00a0young papaya seed ethanol\u00a0on androgen receptor expression.\u00a0The reduced expression of androgen receptor to cell types in the testes can be explained as follows: 1) loss of androgen receptor in sertoli cells mainly affects the function of sertoli cells to support germ cells.\u00a0As a result, spermatogenesis stops in the primary spermatocyte diplotene;\u00a02) due to the reduction ofandrogen receptor in leydig cells, there is steroidogenesis inhibition so that spermatogenesis stops at the spermatid stage;\u00a03) due to reduced androgen receptor in smooth muscle cells and peritubularmyoid cells there is a decrease in sperm;\u00a04) reduced androgen receptor gene in germ cells does not\u00a0affect spermatogenesis and fertility17<\/sup>.<\/p>\n

In this study, the expression of androgen receptorwas evaluated on sertoli cells; the results imply that young papaya seed ethanol\u00a0resulted in the cessation of spermatogenesis in diplotene primary spermatocytes17<\/sup>, resulting in a low number of\u00a0spermatozoa\u00a0as previously described.\u00a0It can becomprehensively asserted that young papaya seed ethanol\u00a0extract\u00a0causes infertility (and\/or lowers the number of sperm cells) in mice causedby FSH and LH secretion, which leads to lower testosterone and decreased expression of androgen receptor on sertoli cells.\u00a0Specifically, one mechanism that may be involved in the low expression of androgen receptors in mice given the young papaya seed ethanol\u00a0is the alkaloids content.\u00a0Previous studies showed that alkaloids in pepper (Piper longum<\/em>) also decrease the amount of androgen receptors in prostate cancer cell cultures.\u00a0The study showed that the alkaloids could decrease the expression of\u00a0full-length\u00a0<\/em>androgen receptors or by deletion in\u00a0the ligand-binding domain\u00a0<\/em>(LBD) indicates that domains other than the LBD in androgen receptors that acts as a mediator decreased expression of theseandrogen receptors.\u00a0The low number of androgen receptors expression due to alkaloids is probably due to the increased degradation of androgen receptors through the ubiquitin-proteasome pathway18<\/sup>.<\/p>\n

Research Novelty<\/strong><\/p>\n

This study is the first to examine\u00a0the antifertility activity effect of young papaya seed ethanol\u00a0extract\u00a0on the level of testosterone and the level of expression of androgen receptors in the sertoli cells of adult mice\u00a0(Mus musculus<\/em>).<\/p>\n

This study successfully\u00a0describes\u00a0the antifertility mechanism by which young papaya seed ethanol\u00a0extract\u00a0complexly mediated through decreased levels of testosterone, which then disrupts cell production\u00a0of spermatozoa.<\/p>\n

This study successfully demonstrates that\u00a0young papaya seed ethanol\u00a0also induces low levels of androgen receptors on sertoli cells, which also represents another antifertility mechanism of young papaya seed ethanol\u00a0extract.<\/p>\n

Conclusion<\/strong><\/p>\n

Young papaya seed ethanol\u00a0extract can be used as a natural contraceptive in adult mice by decreasing the level of testosterone, spermatozoa count, and the expression of androgen receptors on sertoli cells. It is hoped that human clinical trials will be carried out (in line with applicable rules) to assess if this extract can successfully lower human male spermatozoa counts, and ultimately benefit and support government family planning (KB) programs. Besides, young papaya seed ethanol\u00a0extract\u00a0is cost-effective and easy to produce and is likely to be widely accepted within the community.<\/p>\n

Acknowledgment<\/strong><\/p>\n

None<\/p>\n

Conflict of Interest<\/strong><\/p>\n

Authors declare there is no conflict of interest.<\/p>\n

References<\/strong><\/p>\n

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