Int J Clin Exp Pathol 2019;12(5):1921-1931 www.ijcep.com /ISSN:1936-2625/IJCEP0091491 Original Article Chronic stress reduces spermatogenic cell proliferation in rat testis Pengxiang Tian1, Pin Lv1, Weibo Shi2, Minzhe Zhu1, Bin Cong2, Bo Wen3,4 1Institutes of Biomedical Sciences, MOE Key Laboratory of Metabolism and Molecular Medicine, School of Basic Medical Sciences, Fudan University Shanghai Medical College, Shanghai 200032, China; 2Department of Fo- rensic Medicine, Hebei Medical University, Hebei Key Laboratory of Forensic Medicine, Collaborative Innovation Center of Forensic Medical Molecular Identification, Shijiazhuang 050017, China; 3The Fifth People’s Hospital of Shanghai, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China; 4MOE Key Laboratory of Metabolism and Molecular Medicine and Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China Received January 18, 2019; Accepted February 20, 2019; Epub May 1, 2019; Published May 15, 2019 Abstract: Male reproductive dysfunction induced by mental stress and environmental factors has increased greatly in recent years. Previous studies of the male rat reproductive system under stress conditions evaluated changes in physiology and pathophysiology. However, no genome-wide study has been applied to such models. Here we studied the histopathologic changes in testes of rats under different durations of stress and used RNA sequencing (RNA- seq) to investigate the testicular transcriptome and detect differentially expressed genes. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and immunohistochemistry were used to verify these. Chronic stress resulted in significant histopathologic changes in seminiferous tubules and RNA-seq showed that growing numbers of genes were dysregulated with increasing stress exposure. Gene Ontology (GO) analysis showed that many biological processes of cell proliferation-associated terms were highly significantly enriched among down- regulated genes, from chronically stressed groups. Proliferating cell nuclear antigen (PCNA) was used as a key marker of cell proliferation. RT-qPCR and immunohistochemistry indicated that PCNA mRNA and protein expression levels were greatly decreased with prolonged stress, thereby contributing to the attenuation of spermatogenic cell proliferation in the rat testis. This could provide a new scientific basis for the study of male reproductive dysfunction caused by stress. Keywords: Stress, cell proliferation, testis, spermatogenic cell, RNA sequencing, PCNA Introduction the body reinforces its homeostatic ability, thus effectively reducing the impact of risk factors In modern societies, the rapid pace of life has on the body [2]. Moderate stress increases the greatly increased individual mental pressures. body’s ability to resist external risk factors, The mental disorders caused by such factors while excessive or long-term stress damages and their influence on reproductive function the body and can disrupt normal psychologic have attracted the attention of scholars. Not and physiologic functions [3]. Male fertility only female patients with reproductive dysfunc- depends on the production of large numbers of tion show significant increases in stress, but spermatozoa by spermatogenesis in the testic- also male patients with reproductive dysfunc- ular seminiferous epithelium, where germ cells tion face severe problems. are produced by sequential phases of mitosis and meiosis [4]. A previous study indicated that Stress is a comprehensive response of the enforced swimming (3 min for 15 days) in male body to cope with danger through the joint par- rats decreased the numbers of mature sperma- ticipation and regulation of the nervous, endo- tids, while fertility was not disrupted [5], proba- crine, and other systems [1]. Through proper bly because the duration of stress was shorter regulation of the metabolism of each system, than the spermatogenic cycle [6]. Other studies Chronic stress contributes to the attenuation of spermatogenic cell proliferation have indicated that stress induced changes in day. The process lasted for 3, 14, and 21 days the levels of corticosterone [7] and testoster- in the different RS+IS groups. The control rats one [8, 9], suggesting that germ cells might be were left in their cages for the same time with- damaged under stress by increased corticoste- out food or water. All rats were given food and rone and decreased testosterone levels. water ad libitum in the rest time. However, the detailed mechanism of action and the effects of stress on the proliferation of Tissue preparation spermatogenic cells are not clear, and no genome-wide study has been applied in rat tes- Sixty minutes after restraint stress and ice- tis under stress condition. Given that spermato- water swimming, the rats were anesthetized genesis is dependent on the proliferation and deeply. Tissue used for staining were harvested division of various spermatogenic cells, here and fixed immediately in 10% formalin. Sections we focused on identifying whether stress would were subsequently dehydrated in a graded eth- affect the proliferation of testicular germ cells. anol series and embedded in paraffin. They Using an RNA sequencing (RNA-seq) approach, were then prepared for hematoxylin and eosin quantitative reverse transcription polymerase (H&E) and immunohistochemical staining and chain reaction (RT-qPCR) and immunohisto- examined under a light microscope (Olympus chemistry, we investigated the changes in tes- IX73; Olympus, Tokyo, Japan). Tissues used for ticular spermatogenic cell proliferation and RNA-seq and RT-qPCR were removed rapidly, pathologic changes in rats with different dura- snap-frozen in liquid nitrogen then stored at tions of restraint and ice-water swimming, in -80°C. attempts to provide a theoretical basis for the RNA extraction and cDNA synthesis study of male reproductive dysfunction caused by stress. Total RNA was isolated from tissue samples Materials and methods using Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA) following the manufacturer’s protocol. Animals RNA concentrations and purity were deter- mined using a NANODROP 2000C spectropho- Male Sprague Dawley (SD) rats (Beijing Vital tometer (Thermo Fisher Scientific, Waltham, River Laboratory Animal Technology Co. Ltd.) MA, USA). For cDNA synthesis, 100-200 ng of initially weighing 200 ± 20 g were used in this total RNA was reverse transcribed using the study. The rats (four per cage) were housed at a Quantitect reverse transcription kit (Qiagen, constant temperature of 22°C and a relative Hilden, Germany). Samples were assayed three humidity of 50-60% (v/v) under a 12/12-h times, and mean values were recorded. light/dark cycle. The rats were given free access to food and water for at least 1 week RNA-seq library preparation prior to experimentation. All operations were approved by the Institutional Review Board for For each group, three testis samples were Animal Experiments at Fudan University. There taken, and 12 cDNA libraries were constructed. were four experimental groups: control, 3-days, Aliquots of 3 µg RNA per sample were used as 14-days and 21-days of restraint stress added initial material. Ribosomal RNA was removed to ice water swimming (RS+IS; n = 8 rats per using Epicentre Ribo-Zero™ Gold kits (Rat) group). (Epicentre, an Illumina company, Madison, WI, USA). The libraries were constructed according Models of restraint stress and ice-water swim- to the recommendations of NEBNext® Ultra™ ming Directional RNA Library Prep kits of Illumina. Briefly, RNA fragmentation and short RNA According to a previous study [10], models of strands were carried by using NEBNext First restraint stress and ice-water swimming were Strand Synthesis Reaction Buffer (5 ×). Using established. Briefly, the rats were placed in a random hexamer primers and M-MuLV reverse restrainer that allowed them to stretch their transcriptase, first-strand cDNA was synthe- legs but not move otherwise, with no access to sized. Second-strand cDNA was synthesized food and water for 8 h (from 09:00 to 17:00) subsequently with DNA polymerase I and each day. Then the restraint stressed rats were RNase H. Purified second-strand cDNA termi- placed in ice cold water to swim for 5 min each nals were repaired and then we added poly (A) 1922 Int J Clin Exp Pathol 2019;12(5):1921-1931 Chronic stress contributes to the attenuation of spermatogenic cell proliferation and an adapter. Fragments of approximately double distilled H2O, 1.5 mL of cDNA, and 10 300 base pairs (bp) were selected using UNG mL of 2 × Hi SYBR Green qPCR Mix (HaiGene). enzyme. Clustering was performed in a cBot Three samples were taken for each gene to per- cluster generation system by the TruSeq PE form the qPCRs, and the experiments were Cluster Kit v4-cBot-HS (Illumina). After cluster repeated three times for each. For quantifica- generation, the library was sequenced using tion of gene expression levels the 2-ΔΔCT meth- the Illumina HiSeq-4000 platform and 150 bp od was employed [16]. The internal control paired-end reads were generated. gene used for these analyses was that encod- ing β-actin. RNA-seq expression analysis Immunohistochemistry For data analysis, reads were quantified with aliment free software Kallisto, version 0.44.0 After deparaffinization and microwave antigen [11], and reference genome assembly Rnor_6.0 retrieval, sections were incubated for 30 min in downloaded from the