Int J Clin Exp Pathol 2018;11(8):4236-4246 www.ijcep.com /ISSN:1936-2625/IJCEP0080020 Original Article Cytochrome P450 family proteins as potential biomarkers for ovarian granulosa cell damage in mice with premature ovarian failure Jiajia Lin1, Jiajia Zheng1, Hu Zhang1, Jiulin Chen1, Zhihua Yu1, Chuan Chen1, Ying Xiong3, Te Liu1,2 1Shanghai Geriatric Institute of Chinese Medicine, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China; 2Department of Pathology, Yale UniversitySchool of Medicine, New Haven, USA; 3Department of Gynaecology and Obestetrics, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China Received May 21, 2018; Accepted June 29, 2018; Epub August 1, 2018; Published August 15, 2018 Abstract: Premature ovarian failure (POF) is the pathological aging of ovarian tissue. We have previously established a cyclophosphamide-induced mouse POF model and found that cyclophosphamide caused significant damage and apoptosis of mouse ovarian granulosa cells (mOGCs). To systematically explore the molecular biologic evidence of cyclophosphamide-induced mOGC damage at the gene transcription level, RNA-Seqwas used to analyse the differ- ences in mOGC transcriptomes between POF and control (PBS) mice. The sequencing results showed that there were 18765 differential transcription genes between the two groups, of which 192 were significantly up-regulated (log2 [POF/PBS] > 2.0) and 116 were significantly down-regulated (log2 [POF/PBS] < -4.0). Kyoto Encyclopedia of Genes and Genomes analysis found that the neuroactive ligand-receptor interaction pathway was significantly up-regulated and metabolic pathways were significantly down-regulated in the POF group. Gene Ontology analy- sis showed that the expression of plasma membrane, regulation of transcription and ion binding functions were significantly up-regulated in the POF group, while the expression of cell and cell parts, catalytic activity and single- organism process functions were significantly down-regulated. Finally, protein interaction analysis reveals that in the ovarian steroidogenesis pathway, three Cytochrome P450 family proteins-Cyp1a1, Cyp11a1 and Cyp2u1-interact with Fdx1 to form an interactive network. These three proteins were down-regulated in POF cells, suggesting that they are likely direct regulatory targets of cyclophosphamide. RNA-Seq high-throughput screening analysis demon- strated that cyclophosphamide damage to mOGCs was achieved through its impacts on multiple pathways and on the transcription activities of multiple target genes. Among them, the protein network consisting of the cytochrome P450 family Fdx1, Cyp17a1, Cyp11a1 and Cyp2u1 is a potential new biomarker of mOGC damage in POF in mice. Keywords: Premature ovarian failure, ovarian granulosa cells, cyclophosphamide, RNA sequencing, transcriptomic differences Introduction POF [1-4, 6]. Our previous study found that the injection of cyclophosphamide could cause POF Premature ovarian failure (POF) is a common in female mice [4, 7, 8] and cyclophosphamide gynecological disease that causes female could significantly damage mouse ovarian gran- infertility [1-4]. The pathological features are ulosa cells (mOGCs) [4, 7, 8]. Although we also amenorrhea, anovulation, absence of mature revealed some mechanisms at the epigenetic follicles,significantly increased gonadotropin level, the regulatory mechanism at the level of levels,and significantly decreased estrogen lev- the entire genome is not yet clear. els in women before the age of 40 [1-4]. The mechanisms of POF are complex and diverse, RNA sequencing (RNA-Seq), also known as and genetic factors, endocrine factors, psycho- whole transcriptome shotgun sequencing (WT- logical factors, and autoimmune factors can all SS), uses next-generation sequencing to reveal lead to its occurrence [1-5]. In addition, there is changes in RNA transcription levels in biologic still no efficacious treatment or medicine for samples at a particular time point [9-13]. RNA- Biomarkers of mice POF determined by RNA-Seq Seq is often used to analyse changes in cellular exploit the related biomarkers, we used RNA- transcripts [9-13]. In particular, it focuses on- Seq technology to analyse the differences alternative splicing and transcription of genes, inmOGC transcriptomesbetween mice in the modification at the post-transcription level, POF group and control group (PBS). gene fusion, and transcription differences re- lated to single-nucleotide polymorphisms (SN- Material and methods Ps) and mutations [9-13]. Moreover, RNA-Seq can also be used to define the boundaries of Establishment of a mouse model of POF exons and introns of a gene as well as the bo- undaries of the previously annotated 5’ and 3’ Briefly [8], 10-week-old female C57BL/6 mice ends [9-13]. At present, RNA-Seq has been (n = 6) were purchased from the Experimental widely used in the field of genomic regulation in Animal Center of Shanghai University of Tra- embryonic development, disease mechanisms ditional Chinese Medicine, China. Mice were and screening of drug resistance genes [9-13]. randomized into two groups,with three mice in each group. POF mice were first injected intra- Ferredoxin 1 (Fdx1), an iron-sulfur protein, is a peritoneally with cyclophosphamide at 70 mg/ mono-oxygenase that promotes cytochrome kg (Sigma-Aldrich, St Louis, USA), followed by P450 enzymatic reactions. The gene encodes a intraperitoneal injection of cyclophosphamide protein that resides in the mitochondrial matrix, at 30 mg/kg once every 2 days for 3 consecu- and ferredoxin reductase transfers electrons tive weeks, to construct the POF mouse model. to mitochondrial cytochrome P450. There are In addition, the control group mice were inject- multiple Fdx1transcripts encoding different ed intraperitoneally with the same amount of subtypes due to alternative splicing. Fdx1 is PBS once every 2 days for 3 consecutive weeks. highly expressed in adult adrenal glands and The study was approved by the Ethics Co- ovaries [14-19]. Cyp2u1, which encodes poly- mmittee at the Shanghai Institute of Geriatrics peptide 1 of subfamily u in the cytochrome (SHAGESYDW2017008). All experiments are in P450 family 2, Cyp11a1, which encodes poly- line with China National Science and Technology peptide 1 of subfamily a in the cytochrome Commission animal laboratory regulations. P450 family 11, and Cyp17a1, which encodes polypeptide 1 of subfamily a in the cytochrome Isolation and culture of OGCs and establish- P450 family 17, all belong to the cytochrome ment of the in vitro injury model P450 family [20]. Tissue distribution of these three genes shows a significant preference, Briefly [8], 10-week-old female C57BL/6 mice mostly in the ovary, testis, and adrenal gland (n = 10) were purchased from the Experimen- [20]. Cytochrome P450 (Cyp) represents a large tal Animal Center of Shanghai University of family of self-oxidizing heme proteinsand is a Traditional Chinese Medicine. Mice were eutha- class of mono-oxygenases, named for its spe- nized by cervical dislocation, and ovarian tis- cific absorption at 450 nm [20, 21]. It partici- sues were isolated in sterile conditions and pates in the metabolism of endogenous sub- placed in PBS at 4°C. The ovarian tissues were stances and exogenous substances, including shredded and digested with 2.0 ml of hyaluroni- drugs and environmental compounds. Accord- dase (0.1%, Sigma-Aldrich, St Louis, MO, USA) ing to the degree of homology of the amino acid for 1 minute at 37°C. The tissue suspension sequence, its members are divided into the was gently pipetted,addedto 200 μlof fetal three levels of enzymes: family, subfamily and bovine serum (Gibco, Gaithersburg, MD, USA) individual [20, 21]. In cells, Cyp is mainly dis- to terminate the digestion, and then filtered tributed in the endoplasmic reticulum and the through a 200-mesh cell sieve. The filtrate was mitochondrial inner membrane, and it acts as added to 5.0 ml of PBS and mixed, then centri- a terminal oxygenase to participate in the syn- fuged at 1500 r/min for 5 min at 10°C. The thesis of steroid hormones in the body [20, 21]. supernatant was discarded, and the pellet was However, the relationship between the mem- resuspended in 5.0 ml of PBS and centrifuged bers of the cytochrome P450 enzyme family at 1500 r/min for 5 min at 10°C. The superna- and the development of POF is still unclear. tant was discarded, and the cell pellet was resuspended in DMEM:F12 (1:1) medium con- To systematically explore the molecular biology taining 15% fetal bovine serum, 10 ng/ml basic evidence of cyclophosphamide-inducedmOGC fibroblast growth factor (bFGF), 10 ng/ml epi- damage at the transcriptome level and to dermal growth factor (EGF), 2 mM L-glutamine, 4237 Int J Clin Exp Pathol 2018;11(8):4236-4246 Biomarkers of mice POF determined by RNA-Seq 10 ng/ml growth hormone (Gh) and 15 ng/ Flow cytometry (Quanta SC, Beckman Coulter mlestradiol (E2) (all reagents were purchased INC) was then used to analyse the cell cycle from Gibco, Gaithersburg, MD, USA). Cells were distribution of each group of cells (a total of seeded in 6-well-plates and incubated at 37°C 20,000), and data analysis was conducted with 5% CO2 until 80% confluent. MOGCs were using CellQuest software. divided into two groups, with 3 parallel controls in each group. The cells in each POF group were Co-IP treated with cyclophosphamide (IC concentra- 50 8 tion: 38.721 μM) for 24 hours. The PBS (con- Briefly [22], 1 × 10 /ml cells were lysed using trol) group cells were incubated with an equal western and IP cell lysate (Beyotime Bio- volume of PBS for 24 hours. technology). A total of 800 µl of total protein sample was taken, the protein concentration Hematoxylin-eosin staining was adjusted to 1 mg/ml, and 1 µg of IgG and 20 µl of fully resuspended protein a agarose Briefly [3], all fresh tissue was soaked in 4% (Beyotime Biotechnology, HangZhou, China) paraformaldehyde (Sigma-Aldrich, St. Louis, were added to the samplesand shaken slowly USA) for 30 minutes of fixation at room temper- at 4°C for 60 minutes, followed by centrifuga- ature, followed by ethanol gradient dehydra- tion at 2500 r/min for 5 minutes.