Primary culture of ovarian follicular cells of Sterlet, Acipenser ruthenus to develop an in vitro system

Primary culture of ovarian follicular cells of Sterlet, Acipenser ruthenus to develop an in vitro system

M. R. Nowruzfashkhami¹, ²*, M. Sudagar¹, M. Bahmani³, N. Salamat⁴, M. Mazandrani¹, M. A. Yazdani Sadati²

1- Breeding and Rearing Department, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan,  Iran

2- International Sturgeon Research Institute, Agricultural Research Education and Extension Organization (AEERO), Rasht, Iran

3- Agricultural Research Education and Extension Organization (AEERO), Tehran, Iran

4- Marine Biology Department, Marine Sciences Faculty, Khorramshahr University of Marine Sciences and Technology,

Khorramshahr, Iran

* Corresponding author’s E-mail: Nowruzfashkhami@yahoo.com

ABSTRACT

The aim of the present study was to develop an in vitro system for functional investigation of ovarian follicular cells in Sterlet, Acipenser ruthenus. Oocytes for the primary culture were obtained from the ovaries of a 6 years old Sterlet 729 g in weight and 47 cm in total length. The oocytes were in advanced vitellogenesis stage (PI >10). A part of the ovary (containing about 300 follicles) was  removed, ovarian follicles isolated by manually removing those from the interstitial tissue and washed with sterile phosphate buffered saline (PBS) containing antibiotics and Amphotericin B. Follicular cells were separated by treating oocytes with 0.25% trypsin-EDTA  in Ca²⁺ and Mg²⁺ free PBS and cultured in medium L-15 supplemented with 20% FBS, streptomycin sulphate (Gibco, 100 mg.ml^-1), penicillin G potassium (Gibco, 100 IU.ml^-1) and Amphotericin B (Gibco, 2.5 mg.ml^-1) at 22 °C. The concentrations of Testosterone (T), Estradiol-17β (E2), Progesterone (P4) and 17α-hydroxyprogestron (17αOHP) in the medium were measured at days 3, 5 & 7 by the Enzyme-Linked Immunosorbent Assay. According to the results, the ovarian follicular cells of Sterlet proliferated in L-15 medium were steroidogenically active as expressed by the secretion of T, E2, P4 & 17αOHP. Testosterone was the dominant hormone secreted by cultivated follicular cells, which was correlated closely with the end of vitellogenesis in the isolated oocytes. Decrease in production of these hormones was greater at days 3 & 4 in comparison with those at days 5 & 6. By successfully culturing ovarian follicular cells of Sterlet in L-15 culture medium, an in vitro system was developed which enables functional studies to be carried out similar to the in vivo situation in the ovarian follicles.

INTRODUCTION

Fish oocytes, like other vertebrates, are surrounded by follicular cells (Unal et al. 2005). These cells play a vital role in fish reproduction as well as in the synthesis of steroid hormones, growth and final maturation of oocytes (Nagahama 1994). Several studies have evaluated the synthesis and secretion of steroid hormones by follicular cells in fish (Nagahama et al. 1982; Lessman 1991; Venkatesh et al. 1992; Kime et al. 1992). It has also been documented that the fish follicular cells remained alive and continued to secrete hormones in culture medium (Stoklosowa & Epler 1985; Petrino et al. 1989 a, b; Salamat et al. 2010). The primary culture of ovarian follicular cells throughout the year can serve as a valuable tool to study the seasonal variations in hormones secreted by these cells (Galas et al. 1999). Reproduction in sturgeons is regulated by the hypothalamus-pituitary-gonadal (HPG) axis (Dettlaff et al. 1993; Doroshov et al. 1997). Gonadotropin releasing hormone (GnRH) is secreted from the hypothalamus under the influence of environmental factors (such as photoperiod and temperature). This hormone regulates the synthesis and release of gonadotropins (GTH I and GTH II) by the pituitary (Lescheid et al. 1995; Moberg et al. 1995; Querat et al. 2000). These hormones are transported to the follicular cells through bloodstream and induce them to secrete steroid hormones (Tyler et al. 1991). These hormones are involved in vitellogenesis, growth and final maturation of oocytes (Moberg et al. 1995).

With regard to the drastic decline in natural stocks of sturgeons during the recent years, the artificial breeding of these fish provides effective means for the restoration and sustainable use of sturgeon stocks. Artificial breeding of sturgeons first started with Sterlet, Acipenser ruthenus (Webb & Doroshov 2011). The Sterlet, belonging to the family Acipenseridae, is a native species in Volga River. It is also found in riverine regions of the Black, Azov and Balkan Seas. In their natural habitats, females attain sexual maturity between 4 and 7 years which can be reduced to 3 or 4 years under artificial rearing conditions. The eggs of Sterlet are 1.91 to 2 mm in diameter (Chebanov & Galich 2013). This species adapts easily to rearing conditions and does not require more special attention. It also feeds on a wide range of food items (Holcik 1989). Success in artificial breeding programs of sturgeon depends on the selection of female breeders that respond to hormone injection and produce the suitable quality post-vitellogenic oocytes, understanding of the factors that significantly affect the growth and maturation of oocytes, gamete quality, synthesis and secretion of the related hormones. Follicular cells are the key of the success in the sturgeon aquaculture. The present study was aimed at developing an in vitro system for functional studies of ovarian follicular cells in order to achieve suitable guidelines for the success in artificial breeding programs of sturgeons in the future. In this regards, the purpose of the present experiment was to culture the oocytes follicular cells in in vitro condition to study the viability and secretion of these cells. 

MATERIALS AND METHODS

 A sixty years old sterlet sturgeon (729 g in weight and 47.6 cm in length), was provided from International Sturgeon Research Institute, (Rasht,   north of Iran) in March 2015. Fish was anesthetized by clove powder (200 ppm), abdominal area was cleaned with 70% alcohol and dissected to remove a part of the ovary (containing about 300 follicles). The ovary was attained at advanced stage of vitellogenesis (PI>10) according to Chapman et al. (2007). The protocol developed by Stoklosowa and Epler (1985) was followed for isolating of ovarian follicular cells with some modifications. The follicles were washed in a petri dish containing 5 ml sterile phosphate-buffered saline (PBS), streptomycin sulphate (200 mg.ml^-1), penicillin G potassium (Gibco 200 IU.ml^-1) and Amphotericin B (5 mg.ml^-1). After a minute the follicles were transferred to another petri dish containing 5 ml L-15 Medium (Sigma), streptomycin sulphate (Gibco, 200 mg.ml^-1), penicillin G potassium (Gibco, 200 IU.ml^-1) and Amphotericin B (Gibco, 5 mg.ml^-1). After 10 minutes incubation at 22 °C, the culture medium was drained and 5 ml of fresh culture medium of the same composition was added to the petri dish.

The culture medium was drained after 10 minutes and 5 ml of fresh culture medium of the same composition was added to the petri dish. The interstitial connective tissue of the oocytes was removed manually under a stereomicroscope (Nikon). The culture medium was drained and the connective tissue free follicles were washed in PBS solution containing streptomycin sulphate (200 mg.ml^-1), penicillin G potassium (200 IU.ml^-1) and Amphotericin B (5 mg.ml^-1) for three times.  PBS was replaced by 5 ml 0.25 % Trypsin-EDTA (Gibco) in Ca²⁺ and Mg²⁺ free PBS (Gibco).  Trypsinization was carried out at the optimum temperature of 37 °C (Galas & Epler 2002).

The process of trypsinization and the state of follicular envelop were monitored by an inverted microscope (Nikon, Model Eclipse-Ti). Trypsinization was carried out twice for 25 minutes and once for 10 minutes (2 × 25' and 1 × 10'). After each incubation time, supernatant fractions (containing the follicular cells) were pooled and centrifuged, then the supernatant was discarded and 1 ml Fetal Bovine Serum (FBS) was added to the sediment to inhibit trypsin action. After the third trypsinization and centrifugation, supernatant was removed and the deposited follicular cells were re-suspended in the medium L-15 and then diluted to a concentration of 1.25 × 10⁶ cells.ml^-1 culture medium. After assessing viability of cells by trypan blue (0.4%) exclusion test (Doyle & Griffins  1998), the follicular cells were cultured (1.25 × 10⁶ cells.ml^-1) in medium L-15 supplemented with 20% FBS, streptomycin sulphate (Gibco, 100 mg.ml^-1), penicillin G potassium (Gibco, 100 IU.ml^-1) and Amphotericin B (Gibco, 2.5 mg.ml^-1) and then transferred into an incubator at 22 °C. Culture medium was collected on days 3, 5 and 7, centrifuged and supernatant was stored at -20 °C  (Epler et al. 1997) for further analysis of steroid hormones produced including testosterone (T),  progesterone (P4), 17α-hydroxyprogesterone (17αOHP) and estradiol-17β (E2). Steroids analyses were carried out by Enzyme-Linked Immune Sorbent Assay (ELISA).

After determining the concentrations of the steroids, the data were analyzed by One - Way analysis of variance (ANOVA) test (Holm 1979) and the significance of the differences between the means was determined using LSD test (Fisher 1935) performed by SPSS (Version 17) at 0.95% confidence level. Differences were considered significant at p<0.05 for all analyses. Graphs were plotted using Excel 2007 software package.  

RESULTS

By removing the interstitial connective tissue, separated oocytes surrounded only by the follicular layers were obtained (Fig. 1), and follicular cells were observed as a thin layer when oocytes were examined under an inverted microscope (Fig. 2). Oocytes and the follicular layers were expanded after first trypsinization (Fig. 3).

Fig. 1. Separated oocytes from interstitial tissue of the ovary in L-15 Medium.

Further trypsinization resulted in detaching of follicular layers from the rest of the oocytes and formation of the cell suspension containing theca and granulosa cells. The isolated follicular cells were observed under an inverted microscope as separated cells (Fig. 4A) and

Sometimes formed clumps (Fig. 4B). The number of obtained follicular cells was 1.25 × 10⁶ cells.ml^-1, and the viability of cells,   determined by the trypan blue exclusion test, was 92%. After 48 hours, follicular cells were attached to the bottom of the culture vessel and proliferated. The cells were mostly fibroblast- like in shape (Fig. 5).

Fig. 2. Sterlet oocyte (× 600).

Fig. 3. Trypsinized oocyte of Sterlet (× 600).

The propagation of cells increased in the following days (Fig. 6) and after one week the cultured cells had almost completely covered the bottom of the culture vessel (Fig. 7). Based on the results, the concentrations of T secreted by cultivated follicular cells after 2, 4 and 6 days were 15.93 ± 1.57, 10.30 ± 0.20 and 9.63±0.24 ng.ml^-1, respectively (Fig. 8). According to Fig. 8, T secretion in the culture medium decreased over time. T levels on days 5 and 7 were significantly lower than that on day 3. However T level on days 5 and 7 did not differ significantly. E2concentration in culture medium after 2, 4 and 6 days were recorded as 443.33 ± 12.01, 170.0 ± 11.54, and 130.0 ± 15.8 pg.ml^-1, respectively (Fig. 9). E2 levels gradually decreased in the culture medium and were significantly lower on days 5 and 7 than on day 3, although no significant differences were detected in E2 levels between days 5 and 7.

Levels of P4 in the culture medium increased gradually from 260.0 ± 17.32 pg.ml^-1 on day 3 to 303.33 ± 34.80 pg.ml^-1 on day 5 and further increased to 330.3 ± 36.05 pg.ml^-1 on day 7, although no significant differences were recorded in P4 levels between at the beginning and at the end of  the culture (Fig. 10).

Fig. 4. Follicular cells of Sterlet oocytes 1 h after culturing (A: × 120; B: × 1200).

Fig. 5. Fibroblast- like cells after two days of plating (× 300).

Similarly, 17αOHP levels in the culture medium also increased after days 2, 4 and 6. 17αOHP level was 106.67 ± 8.81 pg.ml^-1 on day 3 which significantly increased to 180.0 ± 5.78 and 193.33 ± 12.01 pg.ml^-1 on days 5 and 7, respectively (Fig. 11). No significant differences were detected between 17αOHP levels on days 5 and 7.  Based on the results obtained from this

study, cultivated follicular cells of Sterlet in
L-15 medium, containing 20% FBS, streptomycin sulphate (100 µg.ml^-1), penicillin G potassium (100 IU.ml^-1) and amphotericin B (2.5 µg.ml^-1) at 22 °C, responded and adapted to the culture, attached to the bottom of the culture vessel, proliferated and secreted steroids, T, E2, P4 and 17αOHP.

       Fig. 6. Fibroblast - like cells after four days (× 300).

Fig. 7. Fibroblast - like cells after a week (× 120).

                Fig. 8. Concentrations of T in the follicular cells culture medium (as mean ± SEM; P =0.700).

Fig. 9. Concentrations of E2 in the follicular cells culture medium (as mean ± SEM; P =0.001).

Fig. 10. Concentrations of P4 in the follicular cells culture medium (as mean ± SEM; P =0.010).

Fig. 11. Concentrations of 17α-OHP in the follicular cells culture medium (as mean ± SEM; P=0.287).