Primary culture and identification of rat ovarian granulosa cells
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Primary culture and identification of rat ovarian granulosa cells
Xu Chuan 1 / Shu Weiqun 1,  / Zhang Liang 1 / Cao Jia 2 / Zhou Xin 3
(1.Department of Environmental Hygiene, School of Military Preventive Medicine, Third Military Medical University, Chongqing 400038, China; 2. Department of Military Toxicology, School of Military Preventive Medicine, Third Military Medical University, Chongqing 400038, China; 3.Department of Burns, Southwest Hospital, Third Military Medical University, Chongqing 400038, China )
Culture and Identification of Granulosa Cells from Rat Ovary
XU Chuan1, SHU Wei-qun1, , ZHANG Liang1, CAO Jia2, ZHOU Xin3
(1. Department of Environmental Hygiene, School of Military Preventive Medicine, Third Military Medical University, Chongqing 400038; 2. Department of Toxicology, School of Military Preventive Medicine, Third Military Medical University, Chongqing 400038; 3.Institute of Burn Research, Southwestern Hospital,Third Military Medical University, Chongqing 400038, China)
Abstract BACKGROUND & OBJECTIVE: To investigate the method of culture and identification of rat ovarian high-purity granulosa cells, and to establish a simple and stable cell model. MATERIALS AND METHODS: SD female rats (21～25 d) were injected subcutaneously with pregnant horse serum gonadotropin (PMSG). After 48 hours, they were sacrificed by cervical dislocation. The bilateral ovaries were dissected and mechanical ovulation was used to release ovarian granules. The cells were lysed by 0.25% trypsin digestion in combination with low-speed centrifugation, and cultured in a DMEM/F12 medium containing 15% fetal bovine serum at 37 ° C in a 5% CO 2 incubator. The cultured ovarian granulosa cells were identified by HE staining and follicle stimulating hormone receptor (FSHR) immunohistochemical staining. The cell growth curve was drawn by MTT method, and the cell culture liquid estradiol (E2) and progesterone (P) were detected. The amount of secretion. RESULTS: The viability of isolated ovarian granulosa cells was >90%, and the cell purity was over 95%. The granulosa cells cultured in vitro were 48-96 h in logarithmic phase. The granulosa cells had normal estrogen secretion and cultured for 24 h. The secretion levels of E2 and P in the cell culture medium were (10.36±15.89) pg/ml and (77.91± 17.24) pg/ml, respectively. Conclusion: The ovarian granulosa cells isolated by mechanical separation combined with trypsin digestion and low-speed centrifugation have high purity and good activity. It is a simple and rapid method to identify granulosa cells by FSHR expression staining.
ã€Key words】 Rat; Ovary; Granulocyte; Culture; Identification
CLC number: R730.45 Document code: A Article ID: 1004-616X(2009)03-0234-04
[ABSTRACT] BACKGROUND AND AIM: To obtain and identify cultured granulosa cells from the ovary of rats so as to establish a convenient and stable experimental model. MATERIALS AND METHODS: Female SD rats were subcutaneously treated with pregnant mare serum gonadotropin (PMSG). -eight hours after dosing with PMSG, the animals were decapitated and the ovaries were aseptically removed. Granulosa cells were then released by mechanical method, trypsin digestion and low-speed centrifugation separation were used for granulosa cells isolation. Granulosa cells were diluted and incubated in Fresh DMEM-Ham's F-12 medium (1:1) containing 15% of fetal bovine serum at 37 °C in water-saturated environment of 5% CO2. Hematoxylin & Eosin (HE) and immunohistochemical staining of FSHR were used for ovarian granulosa cell Identification, Additionally, the growth curves of granulosa cells and hormone levels at different incubation times were evaluated as well. RESULTS: Over 95% of the cu Ltedd cells were ovarian granulosa cells. The exponential phase of growth was between 48 and 96 hours of incubation. CONCLUSION: More than 95% of highly purified granulosa cells could be obtained by mechanical method combined with trypsin digestion and low-speed centrifugation. Moreover, identifications of granulosa cells by HE and FSHR staining were quick and convenient approaches.
[KEY WORDS] rat; ovary; granulosa cell; culture; identification
Granulosa cells are the main functional cells of the ovary, and their proliferation and differentiation directly affect the ovarian function of follicle growth initiation, development, ovulation, corpus luteum formation and steroid secretion [1-2]. The reproductive toxicity and mechanism of toxic and harmful substances in humans and mammals in the environment are still being explored. The in vitro culture of granulosa cells is the basis for studying the reproductive toxicity of chemical poisons, and is an important means to explore the function and regulation mechanism of ovarian endocrine, and how to obtain High quality granulosa cells are the key to intensive toxicology research.
Although in vitro culture of mammalian ovarian granulosa cells has been reported in the literature [3-5], so far, there is no clear standard, efficient and stable culture method. This study aims to improve and improve granulosa cells. In vitro culture and identification methods lay the foundation for further research on in vitro reproductive toxicology of granulosa cells.
1 Materials and methods
1.1 Main reagents and instruments
DMEM/F12 medium was purchased from HyClone, USA, and no mycoplasma fetal bovine serum was purchased from Hangzhou Sijiqing Bioengineering Materials Co., Ltd., and pregnant horse serum gonadotropin (PMSG) was purchased from Hangzhou Animal Medicine Factory, tetramethylazozolium salt. (MTT), trypsin, dimethyl sulfoxide (DMSO) were purchased from Sigma, USA, DAB chromogenic kit, follicle stimulating hormone receptor (FSHR) rabbit polyclonal antibody was purchased from Wuhan Boster Company, Yihong, hematoxylin Purchased from Beijing Zhongshang Jinqiao Biological Engineering Co., Ltd. The estradiol (E2) and progesterone (P) radioimmunoassay kits were purchased from Beijing Atomic High-Tech Co., Ltd.
The main instruments include tissue culture microscope, optical microscope (Olympus), CO2 incubator (Thermo Forma), automatic microplate reader (Thermo), centrifuge (Hunan Xiangyi), GC-911 γ radioimmunoassay counter (Zhongjia photoelectric instrument) Branch) and so on.
1.2 Experimental animals
Ten 21- to 25-day-old (50-60 g) clean-grade healthy SD rats, female, were provided by the Experimental Animal Center of the Third Military Medical University.
1.3 Isolation and primary culture of ovarian granulosa cells
According to the method described by Lovekamp et al [3], female Sprague-Dawley rats were injected with 40 IU of PMSG subcutaneously, and were sacrificed by cervical dislocation after 48 h. Iodine and alcohol were disinfected. The ovaries were quickly dissected under sterile conditions and placed in pre-cooled sterile PBS to remove the surrounding tissues and surface envelope of the ovary. The cells were washed with PBS and placed in pre-cooled DMEM/F12 medium. The follicles were pierced with a 25-gauge needle under a dissecting microscope, and the granulosa cells were released into DMEM/F12 medium, and dispersed in a centrifuge tube to separate into individual suspension cells. Add 1 ml of 0.25% trypsin-0.02% ethylenediaminetetraacetic acid (EDTA), digest for 60 min at 37 °C in a 5% CO2 incubator, and take the suspension repeatedly from the incubator for 2 to 3 times with a pipette (to The tissue digestion is mucus-like, and the granule cell mass separation is taken as follows. The culture medium containing fetal bovine serum is added to terminate the digestion, and 200 mesh stainless steel cell sieve is used for filtration. Centrifuge at 800 r/min for 5 min, wash the filtrate, discard the supernatant and collect the cells. A single cell suspension was prepared by adding DMEM/F12 medium (containing 15% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin) to a loose cell mass deposited on the bottom of the centrifuge tube. Trypan blue staining, counting. The cells were diluted into a cell suspension at a concentration of 3.0×105/ml, inoculated into a culture flask or a culture plate, pre-incubated for 24 h at 37 ° C in a 5% CO 2 incubator, and then changed for one time to remove the unattached. Cells; then change the liquid once every other day.
1.4 Identification of granulosa cells
1.4.1 HE staining The cells of the 4th day of vigorous growth were digested with 0.25% trypsin-0.02% EDTA to prepare a cell suspension of 1×105/ml, which was planted in a small cover slip. The wells were placed in a CO2 incubator and cultured at 37 ° C, 5% CO 2 and fully saturated humidity. Slides were removed as the cells grew to 70% confluence and the coverslips were rinsed 3 times with PBS for 5 min each. 4% paraformaldehyde was fixed for 20 min. The cell slides were taken out, washed twice with PBS, stained with hematoxylin for 2 min, washed with water to remove the color, separated into hydrochloric acid alcohol for several seconds, rinsed with running water for 3 min, and the nuclei were clearly stained under the microscope, and the cytoplasm was not stained. 5% eosin staining for 2 to 3 minutes, the running water washed away the floating color, the gradient alcohol was dehydrated, the xylene was transparent, and the cell morphology was observed under the light microscope after sealing.
1.4.2 FSHR expression identification Granulocyte cell slides were made in the same way as 4% paraformaldehyde. Cell immunochemical staining was performed as follows: fixed cell slides were washed with PBS for 3 min×3 times; 3% H2O2 was incubated for 5 min at room temperature to eliminate endogenous peroxidase activity, and PBS was washed for 5 min×3 times; Add normal goat serum to block the unrelated antigen, incubate for 20 min at room temperature, and rinse without washing. A polyclonal antibody against FSHR rabbit (1:200) was added dropwise, overnight at 4 ° C, and washed in PBS for 5 min × 3 times. The secondary antibody IgG was added dropwise, incubated at 37 ° C for 60 min; PBS was washed for 5 min × 3 times. 50 μl of horseradish-labeled streptomycin working solution was added dropwise, incubated at 37 °C for 30 min; PBS was washed for 5 min×3 times. DAB color development (control color development time under microscope); tap water rinse, hematoxylin counterstaining for 10 min; tap water rinse 3 times; conventional gradient alcohol dehydration, xylene transparent, neutral resin sealing.
1.5 Observation of cell growth characteristics
The isolated cells were seeded at 1 x 104 per well in 96-well plates at 200 μl per well. The test group was tested once every 24 h during the culture, and 6 wells were tested each time until the 9th day after the inoculation, and the average value of each time point was taken. During the test, 180 μl of serum-free medium and 20 μl of MTT (5 mg/ml) were added to each well, and incubation was continued for 4 h at 37 °C. The wells were carefully aspirated, 150 μl of DMSO was added to each well, and shaken for 10 min. The absorbance value (A) at 490 nm was measured on an automatic enzyme-linked immunosorbent assay, and the cell growth curve was plotted with the absorbance value as the ordinate and the time as the abscissa.
1.6 Estradiol (E2) and progesterone (P) content
The vigorous growth cells were inoculated into 24-well culture plates, and the cells were cultured in DMEM/F12 medium containing 15% fetal bovine serum at a cell density of 1.0×105/ml, 1 ml per well, cultured at 37 ° C, 5% CO 2 . After 12 h and 24 h, the cell culture medium was collected (100 ng/ml FSH was added 3 h before the termination of culture), and the contents of estradiol (E2) and progesterone (P) were determined by radioimmunoassay to evaluate the cells. Functional status.
2 results
2.1 Morphological observation of ovarian granulosa cells
Trypan blue staining showed that the survival rate of the experimentally isolated culture cells was >90%. In the initial stage, the cells were round or elliptical in shape, and some of them were small and round (such as red blood cells). The ovarian granulosa cells grew in a single layer. After 24 hours of culture, the cells began to adhere and proliferate. After 48 hours of culture, the cells proliferated, and the adherence was good and the growth was strong (see Figures 1, 2). After HE staining, the adherent cells were intact under the microscope, the edges were clear, the size was uniform, and they were polygonal or fusiform. The dark blue staining was oval or irregular, and the cytoplasm was reddish (see Figure 3). . The morphological structure of ovarian granulosa cells is consistent with the literature.
2.2 FSHR expression identification of granulosa cells
FSHR immunocytochemical staining is a specific staining of granulosa cells in the ovary. The positive staining of FSHR is localized in the cytoplasm of the cells. It is stained with tan and the nucleus is dark blue. The positive rate of FSHR is >95% under the microscope (see Figure 4). Thus, the purity of the isolated granulosa cells was 95% or more.
2.3 cell growth curve
The cell growth curve is shown in Figure 5. It can be seen from the figure that the ovarian granulosa cell cultures proliferated with the prolongation of culture time, and the cells were in the adherent growth adaptation period for 24 h. The increase of A value was not significant. After 48 h of culture, the A value began to increase greatly, and the cells were judged to be rapid. In the logarithmic growth phase, the cell growth was extremely vigorous at 72 h, and the A value reached the maximum value; then it entered the plateau phase, gradually decreased on the 5th day, and began to decrease significantly on the 6th day until the end of the 9th day.
2.4 Secretion of estradiol (E2) and progesterone (P)
The secretion of E2 and P in ovarian granulosa cell culture medium at 12 h and 24 h is shown in Table 1. The secretion of E2 and P in culture medium increased with the prolongation of culture time. The average values ​​of E2 and P water after 24 h culture were respectively It reached 103.36 pg/ml and 77.91 pg/ml.
3 Discussion
With the deepening of reproductive toxicology research, various female reproductive damage caused by environmental chemical poisons have attracted people's attention. By effectively studying the effects of environmental toxic and harmful factors on the growth and development of ovarian granulosa cells and its regulation mechanism, Understand the mechanism of toxic effects of poisons on ovaries. At present, the application of ovarian granulosa cells in toxicology research is more and more extensive. How to separate, culture and culture ovarian granulosa cells simply, quickly and stably is the basis and necessary means to study the reproductive toxicity of chemical poisons.
In combination with the literature, this experiment used 21 ~ 25 d female SD rats to culture ovarian granulosa cells in vitro. Compared with adult rats, neonatal rat granulosa cells had not been exposed to endogenous gonadotropin before ex vivo. Stimulation, using PMSG to stimulate female SD rats to be in the early stage of estrus, obtaining a large number of granulosa cells at the same developmental stage. Furthermore, the mechanical separation method is used to release the granulosa cells, which not only reduces the damage to the cells, but also keeps the cell morphology intact and reduces the contaminating cells. Trypsin digestion combined with low-speed centrifugation to separate ovarian granulosa cells can remove other mixed cells (such as red blood cells) and tissue debris, and improve granulosa cell viability and purity. After 24 hours of in vitro culture, the granulosa cells began to grow adherently. The cells proliferated in 48-96 h, and the growth was vigorous, and the cell survival rate was high. The experimental results were consistent with the literature [3-4].
In view of the fact that granulosa cells are the only cells in the ovary that can express FSHR [6-7], the authors used the HE staining to observe the morphological characteristics of granulosa cells, especially the positive expression of FSHR cells as the standard for identifying granulosa cells. The results showed that in the cultured granulosa cells, the positive rate of FSHR can reach more than 95%, and the obtained high-purity granulosa cells can preserve the characteristics of the cells to the greatest extent, which will make the experimental data of serial reproductive toxicology more credible. Follicular granulosa cells play an important role in maintaining female reproductive function. The secretion of estrogen by granulosa cells provides an important microenvironment for the growth and development of egg cells. During the experiment, the secretion of E2 and P in the granulosa cell culture medium increased with time, indicating that the cultured cells possess and maintain normal physiological functions.
In summary, primary cultured granulosa cells with high purity and good activity can provide a good experimental basis for the evaluation of chemical toxicity. The cell culture system established in this experiment is feasible and ideal, and is suitable for related toxicological experiments.
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Received: 2008-10-27; Last revision date: 2008-12-24
Fund Project: National Natural Science Foundation of China (30630056); National Natural Science Foundation of China Youth Science Project (30800900); Chongqing Major Science and Technology Project (CSTC2006AA7003)
About the author: Xu Chuan (1980-), male, Jingmen, Hubei, attending physician, doctoral student, research direction: environmental toxicology. E-mail:
Correspondence to: SHU Wei-qun, E-mail: u.com.cn