Effect of Water Clover (Marsilea crenata) Ethanol Extracts on Follicle and Oocyte Diameter of Goat: In Vitro Study

Water clover (Marsilea crenata) is one of the herbal plants that has been using in alternative medicine. It possesses pharmacologically active compounds like flavonoid, which has cellular activities such as antioxidant and estrogenic activity. This study aimed to evaluate the impact of water clover ethanol extract (WCE) at different concentrations on the growth of follicles and oocytes based on follicles and oocytes diameter, respectively, after six days of culture. This experimental study used 24 isolated antral follicles (2.5-3.2 mm), which were randomly divided into four groups including control (without supplemented WCE) and experimental groups that supplemented with different concentrations of WCE (21.6, 43.2, and 86.4 μg ml-1) in culture medium for six days culture. The diameter of follicles was measured on days 0, 3, and 6. Additionally, oocytes diameter was also measured on day 6. The results indicate that the mean diameter of antral follicles and oocyte diameter of WCE 43.2 μg ml-1 was significantly increase compared to the other groups (P≤0.05). According to our results, WCE exerts its effect on the growth of the antral follicle and oocyte based on follicles and oocytes diameter respectively in a dose-dependent manner after six days of the antral follicle cultured.


INTRODUCTION 
Reproduction is a critical biological process and very important in all living systems to maintain species survival [1]. The ovarian follicle is the structural and functional unit of the female reproductive system, which plays a role in folliculogenesis [2]. Folliculogenesis is the physiological process that involves a complex interaction among endocrine, paracrine, and autocrine factors for activation, growth, and maturation of the ovarian follicles. So, it influences steroidogenesis, angiogenesis, oocyte maturation, as well as follicular atresia [3].
During folliculogenesis, reactive oxygen species (ROS) will be generated as a result of cellular metabolism. Cell living under aerobic need oxygen, so ROS such as superoxide anion radical (O2 -), hydroxyl radical (OH -), and hydrogen peroxide (H2O2) will be generated from oxygen [4]. Also, superoxide anion radical (O2 -) reacts with nitric oxide (NO-) to form peroxynitrite (ONOO -), so it will form reactive nitrogen species (RNS) [5]. The increase of ROS and RNS levels react with cellular lipids, protein, and nucleic acid, so that lead to significant damage of cell structure and thereby cause *Correspondence address: Sri Rahayu Email : yayuksrirahayu8@gmail.com Address : Dept. Biology, University of Brawijaya, Veteran Malang, Malang 65145 oxidative stress (OS). Several studies indicate that OS is involved in the initiation of apoptosis in antral follicles, so that affect of folliculogenesis and steroidogenesis [6]. However, the role of endogen antioxidants such as superoxide dismutase (SOD), glutathione peroxidase, and catalase reduced the increase of free radical and maintain the proper redox state of cells as the signalling molecules in normal physiological processes [5]. Folliculogenesis includes the growth and maturation of follicle which provides the microenvironment necessary for oocyte growth and development, so it will produce matured oocyte (metaphase II) [7]. The growth of follicles is characterized by increasing in follicular diameter due to an increase in oocytes diameter, the number of granulosa cells layer, and formation of the antrum (accumulation of follicular fluid) [ (Widhaningrum, et al.) 114 presence of flavonoids as an antioxidant in semen extender can improve the quality of semen from the effects of freezing [12]. Besides, isoflavone, the subclass of flavonoid also exerts estrogenic activity that plays an important role in folliculogenesis [13]. Therefore, the present study aimed to evaluate the impact of water clover ethanol extract (WCE) at the different concentrations on the growth of follicles and oocytes based on the follicles and oocytes diameter respectively, after the six days of the antral follicle cultured.

MATERIAL AND METHOD Extraction of Marcilea crenata
The leaves of WCE were collected in Surabaya, East Java, Indonesia. Leaves were washed thoroughly with water then dried in the greenhouse at room temperature (25°C) during 4-5 days. The dried leaves were ground into powder [14] then extracted with ethanol 70% through the maceration method with modification [15]. Ethanol extraction of phenolic compounds in WCE was carried out at the Biochemistry Laboratory, Faculty of Mathematics and Natural Sciences, Brawijaya University. The next step was freeze-dried for 24 hours to get the extracts in the form of pasta.

Isolated Antral Follicles and IVGC
This study used the antral follicle of Etawah Crossbred (PE) to analyze the effect of WCE on folliculogenesis. Goat ovaries were collected from slaughterhouse Malang City, East Java, Indonesia. The ovaries were washed with sterile 0.9% physiological saline and then were transported to the laboratory in a container kept at 37°C in sterile 0.9% physiological saline supplemented with antibiotics, which consist of 0.01 g streptomycin and 0.006 g penicillin (Meiji, Japan). After arrived at the laboratory, some fat tissues were removed from ovaries and washed again in sterile 0.9% physiological saline [16].
The antral follicles with diameter size of 2.5-3.2 mm (n=24) were isolated with the slicing technique and cultured for 6 days in 300 µL of culture medium in each well. The culture medium containing TCM-199 medium supplemented with 10% heat-activated Fetal Bovine Serum (FBS), 1% antibiotic Pen-Strep, 0.1 IU.mL -1 FSH, 1 IU.mL -1 HCG, 4% polyvinylpyrrolidone, then covered under sterilized paraffin oil and incubation at 37°C in 5% CO2 [17]. WCE were added in the treated groups at 21.6, 43.2, and 86.4 µg.mL -1 . And then, half of the culture medium (approximately 150 µL) was replaced every day, except the culture medium of the third day that was completely replaced.

Morphological Evaluation of Follicular Growth
Evaluation of the growth of antral follicles morphology was checked by stereo-microscope (SMZ 645 Nikon, Tokyo, Japan) that have been approved with a camera at x20 magnification and observed on days 0, 3, and 6. And then measured of follicular diameter were carried out by Image raster software [18].

Evaluation of Oocyte Quality
Evaluation of oocyte quality used oocyte diameter was carried out on the sixth day of culture. The oocytes in selected antral follicles were isolated with the slicing technique and then measured of oocyte diameter by an inverted microscope at x10 magnification [8].

Statistical Analysis
The data were presented in mean values and Standard Error (SE). The data were analyzed by one-way ANOVA using SPSS 16 for windows. If the result of ANOVA Test shows significantly different (P≤0.05) and followed by Posthoc Test using Duncan with a confident level of 95% (α=0.05).

RESULT AND DISCUSSION Effect of WCE on the diameter of cultured isolated antral follicles
One of the purposes of this study was to evaluate the impact of different concentrations of WCE on the growth of isolated cultured antral follicles for six days. The mean diameter in the sixth day cultured of the isolated cultured antral follicles, which were treated by WCE 43.2 µg.mL -1 was significantly increase compared to the other groups (Table 1, Fig. 1).
During folliculogenesis, the increasing of follicular diameter occurred due to the influence of gonadotropins, such as FSH promotes proliferation and differentiation of granulosa cell through cAMP-PKA and PI3K-AKT pathway [19]. Thus, the follicle able to form an antrum (follicular fluids were produced by the granulosa cell), and the follicular diameter will increase. In addition, 17β-estradiol through sex steroidogenesis also promotes proliferation and differentiation of granulosa cells in the presence of FSH stimulation [20]. The differentiation of granulosa cells can be divided into 2 groups, such as cumulus cells surrounding the oocyte to promote maturation oocyte and mural granulosa

ISSN. 2087-2852 E-ISSN. 2338-1655
Water Clover (Marsilea crenata) Ethanol Extracts on Follicle and Oocyte Diameter of Goat (Widhaningrum, et al.) 115 cell to produce sex steroid. So, the increase in the number of granulosa cells, oocyte diameter, and antrum formation cause the increase of follicular diameter [8].  µg.mL -1 (second, third, and fourth row respectively) on day 0, day 3, and day 6. Images were observed by the stereo microscope that has been approved with a camera at x20 magnification The increased follicular diameter after WCE treatment may occurred by antioxidant activity in WCE with binding to free radicals through the phenolic hydroxyl structure of flavonoid [21]. So, it can inhibit cell damage due to the increase of free radicals and can maintain the level of intracellular homeostasis between prooxidant and antioxidant [5]. In addition, flavonoids also regulate the activity of the antioxidant enzyme to inhibit cell damage and followed by apoptosis [22]. In addition, OS causes a decrease in follicular diameter, which can initiate apoptosis in antral follicles and affect follicular development and steroidogenesis [5]. Overproduction of ROS can impact the IVC of antral follicles because they act as the second messenger and induce the opening of a nonspecific pore in the inner mitochondria membrane. Thus, it releases cytochrome C and is followed by caspase activation to induce apoptosis and loss of follicular function [23].
The growth of the antral follicle also occurred due to the presence of phytoestrogens in WCE that produce estrogenic activity/agonist. So, there was a bind between phytoestrogen and estrogen receptor, such as ERα and ERβ in cytoplasm or nucleus through genomic and nongenomic action with lower binding compared to endogenous estrogen 17β-estradiol [24]. This complex yields estrogenic activity functioned as transcription factors that initiate the transcription activity of a gene [19][20][21]. This complex also yields a rapid cellular response, such as MAPK/ERK, PI3K/AKT, cAMP/PKA, PKC, tyrosine kinase pathway, which is also be used to increase transcription activity [25].
Whereas, the decreasing of follicular diameter occurred due to the phytoestrogens at certain dosage enable to yield antiestrogenic activity/antagonist, so they unable to promote proliferation and differentiation in follicle development of antral follicle. Based on the previous study, phytoestrogens at high doses inhibit the activity of cytochrome p450 (CYP11a1), which can convert cholesterol into pregnenolone, thus affect estrogen production [26]. Genistein, one of the phytoestrogens also alters the level of precursor hormones of estrogen, such as estrone, testoserone, DHEA, progresterone, and enables cause follicle atresia

Effect of WCE on the diameter of oocytes
The presence of WCE in the culture medium enables to increase oocyte maturation characterized by an increase of oocyte diameter at WCE 43.2 µg.mL -1 compared to the other groups ( Fig. 2 and 3). The increasing oocyte diameter may occur due to antioxidant activity in WCE. So, it can suppress free radical activity in oocytes. However, overproduction of ROS exceeds physiological limit, so it can induce destabilization of maturation promoting factor (MPF), decrease survival factor, and stimulate apoptosis in oocyte through mitochondria [28].  The decreasing of oocyte diameter occurred due to the phytoestrogens at high doses enable to inhibit the kinase activity of cyclin-dependent kinase 2 (cdk2)/cell division cycle 2 (cdc2) [29]. Thus, induce G2/M arrest in mammalian cell and maintain in meiotic arrested condition and followed by the inhibition of meiotic maturation [30].
The increasing of oocyte diameter related to the increasing of follicle diameter at WCE 43.2 µg.mL -1 (Fig. 1). It indicates the relationship between follicular diameter and oocyte maturation level characterized by the increasing of oocyte diameter [31]. It happened because there is an acquisition of competence development of oocyte that initiated by interaction between granulosa cells and oocyte through gap junction, su/ch as transfer of small molecules (cAMP, cGMP, RNA, and metabolite) so the oocyte able to regulate oocyte meiotic maturation and reach the final development competence stage [32].

CONCLUSION
According to our result, WCE exerts its effect on the growth of antral follicle and oocyte based on follicles and oocytes diameter, respectively in a dose-dependent manner after the sixth day in vitro culture of the antral follicle.