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Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx

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RNAi-mediated knockdown of inhibin a subunit increased apoptosis ingranulosa cells and decreased fertility in mice

Ishwari Kadariya a, Jiaxing Wang a, Zia ur Rehman a, Hamid Ali a, Hasan Riaz a,JiuYa He b, Dinesh Bhattarai a, Jia Jia Liu a, Shu Jun Zhang a,*aKey Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology,Huazhong Agricultural University, Wuhan 430070, People’s Republic of ChinabMRC-Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK

A R T I C L E I N F O

Article history:Received 4 March 2015Received in revised form 12 May 2015Accepted 14 May 2015Available online xxx

Keywords:RNA interferenceINHaGranulosa cellsApoptosisIn-vivo study

A B S T R A C T

Inhibin a (INHa), a member of TGFb superfamily, is an important modulator of reproductive functionthat plays a vital role in follicular changes, cell differentiation, oocyte development, and ultimately inmammalian reproduction. However, the role of inhibin a in female fertility and ovarian function remainslargely unknown. To define its role in reproduction, transgenic mice of RNAi-INHa that knock down theINHa expression by shRNAi were used. Inhibin a subunit gene was knocked down successfully at bothtranscriptional and translational levels by RNAi PiggyBac transposon (Pbi) mediated recombinantpshRNA vectors and purified DNA fragments were microinjected into mouse zygotes. Results showed thattransgenic female mice were sub-fertile and exhibited 35.28% reduction in litter size in F1 generationrelative to wild type. The decreased litter size associated with the reduction in the number of oocytesovulated after puberty. Serum INHa level was significantly decreased in both 3 and 6 weeks; whereas,FSH was significantly increased in 3 weeks but not in 6 weeks. Furthermore, suppression of INHaexpression significantly promoted apoptosis by up-regulating Caspase-3, bcl2, INHbB and GDF9 anddown regulated Kitl and TGFbRIII genes both at transcriptional and translational levels. Moreover, it alsodramatically reduced the progression of G1 phase of cell cycle and the number of cells in S phaseas determined by flow cytometer. These results indicate that suppression of INHa expression inRNAi-transgenic mice leads to disruption of normal ovarian regulatory mechanism and causesreproductive deficiencies by promoting cellular apoptosis, arresting cellular progression and alteringhormonal signaling.

ã 2015 Published by Elsevier Ltd.

Contents lists available at ScienceDirect

Journal of Steroid Biochemistry & Molecular Biology

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1. Introduction

Mammalian folliculogenesis is a complex process coordinatedby various intraovarian growth factors, pituitary gonadotropins,and steroidogenic hormones [1]. The fate of the follicle isdependent on a well-tuned balance of different factors that areresponsible for follicular growth and atresia. Preantral follicletransition stage is most susceptible to atresia, and is mainlyassociated with the formation and differentiation of granulosa cellsby locally produced growth factors independent of gonadotropins.After transition to the early antral stage, follicular survival relies onprecise gonadotropic and intra-ovarian signals. Continued growthbeyond these stages leads to follicular recruitment, selection, andovulation controlled by a variety of extrinsic and intrinsic factors

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38* Corresponding author. Tel.: +86 15071338061.E-mail address: sjxiaozhang@mail.hzau.edu.cn (S.J. Zhang).

http://dx.doi.org/10.1016/j.jsbmb.2015.05.0060960-0760/ã 2015 Published by Elsevier Ltd.

Please cite this article in press as: I. Kadariya, et al., RNAi-mediated knockdecreased fertility in mice, J. Steroid Biochem. Mol. Biol. (2015), http://

[2]. However, only few follicles undergo ovulation and most of thefollicles are lost before ovulation by granulosa cells apoptosis [3].Therefore, understanding these stage-specific molecular andcellular mechanisms of follicular growth and development arecrucial for the diagnosis of pathophysiological conditions and forthe development of new techniques to improve mammalianfertility.

INHa, a member of the TGFb super family, has a prominent rolein ovarian developmental dynamics and physiological functions.Inhibins are heterodimers composed of 18 kDa a-subunitdisulphide-linked to one of two 14 kDa b-subunits (bA and bB)[4–7]. INHa suppresses follicle-stimulating hormone (FSH)through endocrine feedback module [8]. Previous studies haveaccounted its involvement in folliculogenesis mostly in paracrinefashion and other reproductive functions [9–11]. Furthermore,inhibin mutation cause premature ovarian failure (POF) andother functional abnormalities [12,13]. Previous findings revealed that INHa knockout mice were infertile due to altered ovarian

down of inhibin a subunit increased apoptosis in granulosa cells anddx.doi.org/10.1016/j.jsbmb.2015.05.006

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enotype with granulosa cells tumor and defective folliculo-nesis [12,14,15]. Furthermore, deficiency of FSH and LH in INHaockout mice showed tumor formation that was gonadotropinpendent [16,17]. On the other hand, overexpressed INHaansgenic mice were sub fertile and exhibited 52% reduction inter size due to reduced numbers of ovulated oocytes [18].tracellular regulatory network of granulosa cells (GCs) are keyayers in ovarian function, which provide a key tool for thederstanding of molecular pathways [7]. Small hairpin RNAhRNA) has been routinely used to knockdown specific proteinsder both in vitro and in vivo conditions. RNAi in-vivo miceodel, to study the gene function has been well reviewed inevious study [19].To date, there are no reports on RNAi INHa subunit evaluatinge effect of GCs on mammalian reproduction through in-vivoouse model. However, previous works on RNAi INHa in in-vitro

mice [20], Sertoli cells [11,21], and pituitary cells [22],monstrated the significant role of inhibin on cell cycleogression, apoptosis, cell proliferation, hormonal regulation,d alteration in different genes. RNAi is used as powerful tool toaluate gene function due to its partial gene silencing efficiencyr specific phenotypes [23]. To understand the basis for howockdown of INHa using RNAi technique rescue the GCs lossrough the genetic makeup of TGFb superfamily member’s genes,e proposed to reveal the role of INHa subunit in cell cycle controld apoptosis of mice granulosa cells. Moreover, this study wouldovide new insights for better understanding of intraovaraingulatory mechanisms through RNAi-INHa to monitor the in vivole of the INHa in ovarian function, female reproductive system,d ultimately improving fertility in animals.

Materials and methods

. Construction of recombinant vectors and transgenic mice

The use of two plasmids PBi5-M containing INHa sequencelonging to mice (accession number: NM_010564.4), and Pbi5-Blonging to buffalo (EU884446.1) were derived from NCBI genenk database (Table 1). Four target sites were selected and forth groups (previously work done in our lab) according to siRNAogram [24,25] and only two sequence at positions 1016 and33 in the coding region and their specificity was furthernfirmed through the blast search engine. Briefly, to obtain shortirpin RNA, a typical oligonucleotide with 5 bases containing astriction site at its 50 end, 19–21 bases of sense strand, 7–9 bases

hairpin loop, 19–21 bases of antisense strand, 6 bases ofrminator, and 6 bases resulting in a total length of 65 bases. Theseere annealed and inserted into the BamHI and EcoRI sites of theAi-PiggyBac Transposon Vector (gift from Welcome Trust,nger Institute, UK). The recombinant plasmids were designated

RNAi-M and RNAi-B, respectively. Transgenic mice wereoduced through the injection of transgene DNA into pronucleus

mice zygotes. Microinjected eggs were transferred to theiducts of pseudo pregnant foster mothers. Genomic DNA waslated from tail biopsies of the founder mice born to confirmAi positive mice. RNAi-M and RNAi-B transgene mice wereentified and used to establish two-transgene line. However, due

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ble 1rget sequences of mouse INHa.

ame Target sequence (50 ! 30) cds position

NAi-M ACACCCTCCCAGTTTCATCTT 1016NAi-B AGTATGAGATGGTGCCCAA 1033

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to low genotypic and phenotypic efficiency of RNAi-B transgeneline, we used only RNAi-M line mutant for further experiment.

2.2. Experimental animal and ethics statement

RNAi-INHa Friend Virus B-Type N strain (FVB/N) micewere generated by CYAGEN Biosciences Inc. (ISO9001:2008),Guangzhou, Guangdong Province, China. The mice raised incontrolled room conditions of constant temperature (22–25 �C),humidity (60–75%) and 12-h light/dark cycles with food and waterad libitum. Animal studies were approved by the EthicalCommittee of the Hubei Research Center of Experimental Animals(Approval ID: SCXK (Hubei, 2008-0005)). The animal experimentwas performed according to the NIH guidelines.

2.3. Animal breeding and genotyping

FVB/N mice were used for breeding and sample preparation.Mating strategies were accomplished separately, where wild typemale � female and transgenic male � female kept in each boxduring entire breeding time. F1 offspring were used to measure thefertility performance and litter size of each female mouse wereaveraged. Genotyping of the mice performed by PCR using genomictail DNA with the primers as shown in Table 2. The PCR productswere separated and visualized on 1% agarose gel.

2.4. Isolation and culture of primary GCs

Preovulatoray GCs isolated by using the previously describedmethod [26]. Briefly, ovaries from 21-day-old female FVB micecollected and rinsed with cold PBS (Hyclone, UT, USA) and then GCsisolated mechanically by needle puncture method. After that, GCswere released by puncturing large antral follicles. To removeoocyte and cellular debris, cell suspension was filtered through a40-mm nylon mesh cell strainer. Separated cells were centrifugedat 15 �100g for five minutes three times, and then the supernatantwas aspirated and remaining pellet was used for culture. Trypanblue dye exclusion method used to check the viability of GCs. PCRdetection of CYP17A1 mRNA was performed for theca cells cross-contamination [27]. Finally, cells were seeded in 6-well cultureplates (Corning, Inc., USA) in Dulbecco’s modified eagle’s medium(DMEM/F12, Hyclone Laboratory, Inc., UT, USA), supplementedwith penicillin (50 IU/ml), streptomycin (50 mg/ml) and 10% fetalbovine serum (FBS; Hyclone Laboratory, Inc.). The cells wereincubated at density of 1 �106 cells/ml under serum freeconditions at 37 �C in 5% CO2 and 95% O2 for 48 h with mediumreplaced every 24 h.

2.5. RNA extraction and reverse transcription polymerase chainreaction (RT-PCR)

Total RNA extracted from GCs using the EZNA1 Total RNA Kit I.RNA preparation pure cell kits (OMEGA Biotech) according to themanufacturer’s instructions. Any residual genomic DNA contami-nation eliminated with DNase I. The concentration and purity ofRNA measured by spectrophotometric absorbance at opticaldensity of 260/280. One microgram of total RNA used to synthesizecomplementary DNA (cDNA) by using first strand cDNA synthesiskit (FSK-100; Toyabo Co., Osaka, Japan) according to themanufacturer’s recommended protocol.

2.6. Quantification of gene expression by quantitative polymerasechain reaction (qPCR)

RT-PCR was used to assess the transcriptional activity ofINHa and related genes as described previously [28]. Quantitative

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Table 2Sequence of gene primer pairs for genotyping and quantitative PCR.

Gene Primer sequence (50–30) Product length (bp) Annealing temperature (�C)

RNAi-B F: ACT TGT ACA GCT CGT CCA TG 284 58R: CTA CAA CAG CCA CAA CGT CT

RNAi-M F: AAGTTCATCTGCACCACCG 173 60R: TCCTTGAAGAAGATGGTGCG

GDF9 F: TCACCTCTACAATACCGTCCG 145 55.4R: AGTGGAGATGTTATGGCAGGC

CASP3 F: CTGACTGGAAAGCCGAAACT 103 53.8R: GGCAAGCCATCTCCTCATC

BCL2 F: GTCACAGAGGGGCTACGAGT 92 56.8R: CAGTGTCTCCCCGATGCTCA

Kitl F: TAGTGGATGACCTCGTGTTA 108 55.3R: AATCTTTCTCGGGACCTAAT

INHa F: GGGTGAGAAGGGTAGAAGAAG 164 54.9R: CCCACTCTTCCCATCTTCTTC

INHbB F: CTGAAACTGCTCCCCTATGTC 326 56.6R: GACTTTGACGAGGGGATACAG

TGFbRIII F: ATCTGAACCCCATTGCCTCC 144 55.1R: TAGACTTGGGGTAACGGAGG

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RT-PCR was carried out in 96-well plates (Light Cyber 480 multiwell Plate 96, Roche, IN, USA) using 20 ng of cDNA, specific primersets (Table 2), Syber green master mix (SYBR Green Real time PCRMaster Mix QPK-201; Toyobo Co.), using Bio-Rad iQ5 real time PCRsystem (Bio-Rad Laboratories, Inc.). Amplification was conductedto analyze mRNA relative expression levels. Moreover, meltingcurve analysis was performed after real-time PCR reactions tomonitor PCR product specificity. At least three biological replicateswere run for each sample, and fold change for relative quantifica-tion between amplified targets and control genes were calculatedby the formula: 2-DDCt method. Glyceraldehyde 3-phosphatedehydrogenase (GAPDH) gene served as control gene. Statisticaldifference was tested using one-way ANOVA followed by Turkey’sHSD test and significance was considered at P < 0.05 (SPSS version17, Chicago, IL).

2.7. Western blotting

For protein extraction, GCs were washed with ice cold PBSbuffer and lysed in RIPA buffer (Santa Cruze Bio Technology, Inc.,CA, USA) containing protease inhibitors cocktail (Santa Cruz)followed by incubation for 1 h at 40 �C and centrifugation at12,000 � g for 10 min to remove cellular debris, respectively. Totalprotein concentration was determined by BCA-assay (Pierce,Rockford, USA), and 40 mg of total protein was subjected to gelelectrophoresis. Total protein separated by 12% polyacrylamide gel(SDS-PAGE) before transferred to polyvinylidene difluride mem-brane (PVDF membrane) (Milli-Pore, Bedford, MA). After blockingin PBS supplemented with 5% nonfat milk (Sigma–Aldrich) and0.05% Tween 20 (Sigma–Aldrich), samples were immunoblottedovernight at 40 �C with polyclonal rabbit primary anti-antibodiesagainst anti INHa, (1:500, Santa Cruz, USA, sc-30146), anti Bcl2(1:500, Santa Cruz, USA, sc-492), anti caspase-3 (1:500, Santa Cruz,USA, sc-7148), anti inhbinbB (1:500, Santa Cruz, USA, sc-50287),anti GDF-9 (1:500, Santa Cruz, USA, sc-7407), anti Kitl2 (1:500,Santa Cruz, USA, sc-9132), anti TGFb RIII (1:500, Santa Cruz, USA,sc-28975) and anti GADPH (1:500, Santa Cruz, USA, sc-59540).Consequently, after incubation with the primary antibody over-night, membranes washed three times with PBS containing 0.1%

Please cite this article in press as: I. Kadariya, et al., RNAi-mediated knockdecreased fertility in mice, J. Steroid Biochem. Mol. Biol. (2015), http://

Tween-20, and further 1-h incubation was proceeded with horseradish peroxidase (HRP) labeled conjugated polyclonal goatanti-rabbit secondary antibody (Boster Co., China). Later on, itwas exposed to X-ray film to detect protein bands.

2.8. Detection of cell apoptosis

Fluorescence activated cell sorting analysis (FACS, Becton-Dickinson and Company, USA and Mod Fit LT for MacV3.0 software) was used to measure GCs apoptosis. GCs(1 �106 cells per 6 well plates) were cultured and collected after48 h, then washed with pre-warmed PBS, trypsinized withoutEDTA, incubated for 5 min at 370 �C and stained with Annex inV-FITC and propidium iodine (Apoptosis detection kit, Key GEN,Nanjing, China) in dark for 10 min. After completing all the stepsaccording to the manufacturers’ instructions, cells subjected toflow-cytometric analysis for apoptosis detection [29]. Viable cellswere negative for Annex in V-FITC and propidium iodide stain,early apoptotic cells showed positive with Annex in V-FITC stain,while, late apoptotic/necrotic cells double stained by Annex inV-FITC and propidium iodide. All experiments repeated fivetimes independently. For each determination, a minimum of10,000 cells analyzed.

2.9. Cell cycle analysis

GCs of respective experimental groups were harvested, washedwith PBS, fixed in ice-cold 70% ethanol overnight at 4 �C, washedagain in PBS and stained using propidium iodide/RNase A solutionat 37 �C in the dark chamber for 30 min. Flow cytometric analysiswere subjected a BD FACS Calibur (Becton, Dickinson andCompany, USA) and Mod Fit LT for Mac V3.0 software. Foreach determination, a minimum of 20,000 cells analyzed. Theexperiment repeated thrice independently.

2.10. Hormonal assays

Cycled female mice were used to collect serum for hormonalanalysis. Estrus cycle stage were determined by daily examination

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vaginal cytology and all samples were collected at same stage ofestrus. Mice anesthetized and blood was collected by cardiacncture into plastic centrifuge tubes containing EDTA (Sarstedt,mbrecht, Germany); after centrifugation, the plasma was stored

�20 �C until assays performed. Hormone assays for INHA andH performed on extracted serum samples. To examine potentialterations in hormone levels based on age, measurements wererformed on mice at 3 and 6 weeks of age. Serum hormoneeasurements were performed by ELISA kit (Wuhan Colorful Geneological Technology Co., Ltd., China). INHA and FSH results arepressed as nanograms per liter and international unit per liter/L). The lowest sensitivity of INHA and FSH was 3.01 �0.11 ng/Ld 0.37 � 0.01 IU/L. The inter assay coefficient of variation for allHA and FSH was 2.7% and 3.8%. All experiments were repeatedrice.

1. Fertility measurement

Mating strategies of mice were performed separately, whereild type male � female and mutant male � female housed in eachx until five times off springs produced from each pair. Later on,e litter size of each female mouse averaged. Age of mice variedm eight to nine weeks at the beginning of study and seven tone months at the end of the experiment. Initial age of male miceas 2 months and age at the end of breeding was 10 months. Totalice breeding time was 8 months. All male were at same age fromginning to end of experiment. Cages were monitored daily ande numbers of litters recorded.

2. Determination of onset of puberty

To determine the onset of puberty, the vaginal opening ofurteen mice in each group checked from PND 20 twice daily atAM and 8 PM, respectively. Through the regular observation ofgina, opening day was recorded and analyzed to see the effect ofAi INHa on wild type and mutant group of mice.

3. Superovulation and oocyte count procedure

Superovulation procedure employed as outlined by Luo et al.0] Three to nine weeks old, mice induced by intra-peritonealjection of 10 IU of pregnant mere serum gonadotropin (PMSG)igma Chemical Co.). After 46–50 h post PMSG injection, 10 IU ofG injected to each mouse (Sigma, CG-10, St. Louis, MO, USA).ter that, cumulus oocyte complexes (COCs) collected in cultureates by flushing (3 mg/ml BSA with PBS) the ampullae of oviducting micro dissecting forcep. COCs separated from oocyte usingaluronidase (0.3 mg/ml) by slight pipetting at room tempera-re. Finally, the number of oocytes was collected from right andft oviduct of each mouse and manually counted under a Nikonicroscope (Nikon, Natick, MA).

Results

. The RNAi-M induced silencing effect in GCs

To create the transgenic mice that can stably suppress thepression of INHa, two plasmids (PBi-5 M and PBi-5 B) carryinge hairpin DNA sequence corresponding to the RNAi-M andAi-B employed. Genomic DNA was isolated from the transgenicice off springs, and PCR performed to determine the RNAisitive mice (Fig. 1A). Furthermore, to check the suppressionficiency of INHa in RNAi-M and RNAi-B transgenic mice, GC cellsere isolated from ovary, then RNA and protein isolated, and downgulation efficiency was confirmed by measuring mRNA (by qPCR)d protein level (by Western blotting). The results showed that

Please cite this article in press as: I. Kadariya, et al., RNAi-mediated knocdecreased fertility in mice, J. Steroid Biochem. Mol. Biol. (2015), http:/

these vectors specifically reduced INHa and RNAi-M miceexhibited greatest down regulation effect (64%) as compared toRNAi-B (44%) (Fig. 1B and C). Greatest INHa knockdownrecombinant RNAi-M mice group and normal mice called controlgroup used for further progression of experiment.

3.2. Silencing of INHa altered the expression level of INHbB, bcl2,Caspase3, Kitl, GDF9 and TGFbIII in GCs

It has been reported that INHa is one of the representative genethat modulates ovarian function. To further confirm the regulatoryrole of INHa subunit in ovarian function, we quantified mRNA andprotein expressions of different apoptotic factors (Bcl2, Caspase3),intraovarain growth factors (Kitl, GDF9), one inhibin familymember (INHbB) and co-receptor of INHa (TGFbRIII). The resultsshowed that RNAi INHa subunit significantly down regulated theexpression of its proposed receptor, TGFBRII, indicating a receptormediated activity in GCs. In addition, suppression of INHa upregulated the mRNA and protein expressions of caspase3 and bcl-2(P < 0.05). These findings indicate that inhibin a might controlGCs apoptosis through mitochondrial dependent pathway.GDF9 transcript and protein expression significantly upregulated whereas; Kitl gene expression was significantly declined(Fig. 2A and B). These findings suggest that GDF9 and Kitl might beassociated with regulation of INHa in GCs. Interestingly,knockdown of INHa protein significantly up regulated (P < 0.05)the expressions of INHbB which might be due to homologousrelationship among inhibin family members.

3.3. Silencing of INHa induced mice GCs apoptosis

To assess the role of INHa subunit in regulation of apoptosis inmouse GCs, the exposure of phosphatidylserine on the cells’surface with Annex in V-APC/PI double staining between controland transgenic groups employed. Based on the flow cytometry, theresults showed a significant increase in the apoptotic cells inRNAi-M transgenic group as compared to control (Table 3). Theseresults indicate that INHa subunit is appeared to be an importantapoptotic regulatory factor of mouse GCs.

3.4. Knockdown of INHa altered the growth and proliferation of themouse GCs

To investigate the effects of INHa depletion on cell cycledistribution by flow cytometry, GCs were saturated withpropidium iodide (PI) that usually stains the nuclear contents ofa cell. Then, the cells subjected to fluorescent activated cell sorter(FACS). Findings of cell cycle distribution by flow cytometric areshown in Table 4. The results showed that in comparison with thecontrol group, INHa depleted GCs significantly (P < 0.05) increasedcell population at the G1 phase accompanied with the decreased Sphase and G2 phase cell populations, which indicated that the cellcycles were arrested at G1 phase induced by RNAi INHa. Theamount of cells in the G1 phase increased from 71.28% controlgroup GCs to 84.52%, in INHa knock down cells.

3.5. Knockdown of INHa altered the hormone secretions of INHaand FSH

To evaluate the effect of INHa knockdown on hormonalchanges, concentrations of INHa and FSH in blood of 3 and6 weeks of age were measured. Results showed that concentrationof INHa was significantly decreased (P < 0.05) in RNAi-M groups(Fig. 3A) for both 3 and 6 weeks. Whereas, release of FSH wassignificantly higher (P < 0.05) in RNAi-M group than control in3 weeks but it was non-significant in 6 weeks (Fig. 3B).

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Fig. 1. Detection and measure silencing efficiency of INHa in RNAi-M and RNAi-B in GCs of F1 generation mice. (A) RNAi positive mice determination through PCR analysisfrom genomic DNA. Lines 1, 2, 3 and 5, were RNAi-M INHa positive, line 4 was negative. (B) Expression of INHa subunit mRNA level in mice GCs detected by real timepolymerase chain reaction (RT-PCR) for RNAi-M, RNAi-B and control group. The statistical differences were tested using one-way ANOVA (P < 0.005). (C) For proteinestimation, Western blotting conducted and protein ratio normalized with relative protein level of b actin.

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3.6. Fertility studies

For fertility study, transgenic and control mice separately mated.Successive mating of transgenic mice revealed 35.28% decrease(P < 0.05) in litter size as compared to wild type for first five parity

Fig. 2. Effect of INHa knock down on the mRNA and protein expression of some genes

GDF9, Caspase3, bcl2, and Kitl and Betaglycan were quantified by real time PCR and wapoptosis related genes, Caspase3 and bcl2 were significantly up regulated (P < 0.05)GDF9 significantly up regulated, whereas mRNA level of beta-glycan and Kitl significantlybeta glycan quantified by Western blotting. The results showed that Caspase3 and bcl2,proteins of beta-glycan and Kitl down regulated by INHa silencing in RNAi-M with resgranulosa cells proliferation, apoptosis and cell cycle (P < 0.05).

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(Fig. 4A). RNAi-M transgenic females gave birth to an average littersize of 6.2 � 0.79 pups while control (non-transgenic) females hadan average litter size of 9.8 � 0.16 (P < 0.05). To assess whetherovulation defects were present in the RNAi INHa females, 3, 6 and9 weeks old females were super ovulated, and then the oocyte

related to apoptosis, folliculogenesis and oocyte development. The level of INHbB,estern blotting for RNAi-M and control groups respectively. (A) The mRNA level of

in RNAi-M as compared to control group. However, mRNA levels of INHbB and down regulated. (B) The protein level of INHbB, GDF9, Caspase3, bcl2, and Kitl and

INHbB and GDF9 proteins up regulated by INHa gene silencing. Nevertheless, thepect to control group. These results inferred that silencing of INHa affected mice

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Fig. 4. (A) Comparison of mean litter sizes between RNAi-M and the control group. Onlitters were born. The litter size of each mating pair averaged to yield the mean litter siwith variance indicated by SEM (P < 0.05). (B) Trend of litter size between RNAi-M andpairs averaged to yield the mean litter size per mating pair.

Table 3Measurement of apoptosis in mouse GCs by flowcyotmetric analysis of apoptoticmice GCs between RNAi-M and control group. There were significantly moreapoptotic cells and less vital cells in RNAi-M group as compared to those of controlgroup (mean � SEM, n = 5). “a” and “b” indicate level of significance in column(P < 0.05).

Type of mice Live cells (%) Apoptosis (%) Necrotic cells (%)

Control group 92.58 � 0.65b 3.4 � 0.42b 3.9 � 0.35b

RNAi-M 82.56 � 2.07a 9.7 � 0.69a 8.36 � 1.23a

Table 4Analysis of cell cycle by flow cytometry in mice GCs for RNAi-M and control group(mean � SEM, n = 5). All the results evaluated by one-way ANOVA. “a” and “b”indicate the level of significance in column (P < 0.05).

Type of mice G0/G1 (%) S (%) G2/M (%)

Control group 71.28 � 2.25b 19.80 � 0.94b 8.91 � 1.50b

RNAi-M 84.52 � 0.35a 12.70 � 1.4a 2.78 � 0.73a

Fig. 3. Concentration of INHa and FSH hormone measured in mice blood at 3 and 6 weand control group of mice. Knock down of INHa decreased the concentration of INHa

6 weeks of age. “a” and “b” represent level of significance (P < 0.05). (B) Concentration oINHa increased the concentration of FSH significantly in RNAi-M groups of mice as corepresent level of significance (P < 0.05).

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Please cite this article in press as: I. Kadariya, et al., RNAi-mediated knocdecreased fertility in mice, J. Steroid Biochem. Mol. Biol. (2015), http:/

release with induced gonadotropin in female mice analyzed. Theresults showed that at 3 weeks, transgenic mice produced theincreased number of oocyte as compared to control mice (Fig. 5). Incontrast, oocyte numbers were significantly reduced (P < 0.05) in6 and 9 weeks of age even after with the PMSG and hHCGadministration in transgenic mice. Litter size found on decline trendin RNAi-M mice as compared to control (Fig. 4B).

Decreased female fertility could be attributed to one or morereproductive dysfunctions, such as follicular defect, decreasedovulation, fertilization, or implantation and embryo survival. Theseresults indicate that INHa subunit may have an important role inthese reproductive events.

3.7. Delayed onset of puberty

The onset of puberty is an effective indicator of feedbackregulation and balanced steroid hormonal effects on reproductivesystem. Results showed that onset of puberty in transgenic micewere delayed (29.44 � 0.38 days) (P < 0.05) as compared to controlgroup (27.11 �0.49 days) (Fig. 6).

e male and one female defined as mating pair housed together until five parities ofze per mating pair. All the statistical analysis were performed using one-way ANOVA

control group of mice for five parities of F1 generation. The litter size of each mating

eks of age. (A) Concentration of INHa hormone in the blood serum of RNAi-M groupssignificant in RNAi-M group of mice as compared to the control group in both 3 andf FSH in the blood serum of RNAi-M group and control group of mice. Knock down ofmpared to control group in 3 weeks but in 6 weeks was non-significant. “a” and “b”

kdown of inhibin a subunit increased apoptosis in granulosa cells and/dx.doi.org/10.1016/j.jsbmb.2015.05.006

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Fig. 5. Gonadotropins induced superovulation between RNAi-M and control groupof female mice. All the statistical analysis were performed using one-way ANOVAwith variance indicated by SEM (P < 0.05).

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4. Discussion

To understand molecular and physiological function of INHa inmammalian reproduction, mechanism of GCs apoptosis, GCs cellcycle regulation, hormonal and fertility changes in RNAi-INHawere investigated in transgenic mice model. Using two differentkinds of INHa fragments as the shRNA DNA sequences, two mutantmice groups, PBi5-M (RNAi-M) and PBi5-B (RNAi-B) weregenerated and RNAi-M group was particularly selected for furtherstudies due to its significant silencing efficiency of INHa mRNA andprotein expression.

In this experiment, RNAi-INHa female mice appeared normal;however, the continuous mating study showed the subfertilitywith declining trends of litter size in different parities (Fig. 4A andB). These results are partially consistent with previous INHa

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Fig. 6. Determination of onset age of puberty in RNAi-M and control group of mice.Vaginal opening observed from birth to puberty. Findings revealed that onset ofpuberty was significantly higher (P < 0.05) in RNAi-M group as compare to controlgroup of mice.

Please cite this article in press as: I. Kadariya, et al., RNAi-mediated knockdecreased fertility in mice, J. Steroid Biochem. Mol. Biol. (2015), http://

knockout mice model; they demonstrated infertility and reducedfertility with abnormal ovarian function and follicle dynamics inboth mice and rat [15,17,18]. Results showed 35.28% decrease inlitter size and found significant oocyte number reduction at six andnine weeks after superovulation in RNAi-INHa female mice ascompared to wild type littermates. To understand the mechanismunderlying the reduced fertility in female RNAi-INHa mutantmice, phenotypic changes in mice were examined. RNAi-INHafemale mice showed significantly delayed onset of puberty (Fig. 6)and hormonal change which associate well with the reduced littersize, possibly due to the defects in hypothalamus–pituitary gonadaxis and other neuroendocrine pathways [14], thereby suggestingthat the INHa plays an important role in ovarian folliculogenesis.

These findings did validate that reduced ovulation after pubertyto the later age and altered folliculogenesis in RNAi-INHa femalemice, which could be the main cause of reduced fertility (Fig. 4A).Furthermore, the reduction of ovulation rate is probably attribut-able to the altered gonadotropin ratio (Fig. 3A and B), follicle, andoocyte related gene (Fig. 2A and B). The complication for thefertility effects of INHa deficient mice is due to greater bioactivityin TGFb pathway, which has been reported by previous findings[5]. For instance, INHa knock out affects ovary function byincreasing FSH production from pituitary, and INHbB and actbsubunit from the pool of homo and heterodimirization that alsohave the ability to stimulate efficient cell cycle progression insomatic cells [31]. Therefore, INHa knockout mice were not onlyinfertile, but also suffered with granulosa cell tumor and a cancercachexia-like wasting syndrome, with defects in hypothalamic–pituitary–gonadal axis regulation [14,32]. Experiment wasconducted to check the INHa knockdown could rescue GCs lossor not, whereas, INHa knockout mice model provide GCs loss andtumorigenic effect through the alteration of TGFb superfamilymember genes. To answer this question, the intraovarian growthfactors were analyzed, such as, GDF9, Kitl, and TGFb super familymember’s gene, INHbB and TGFBIII at both mRNA and proteinlevels in granulosa cells. Higher expression of GDF9 and INHbB andlower expression of Kitl and TGFbIII at both mRNA and proteinlevels suggest the control GCs proliferation and significant role ofINHa on regulatory mechanism in GCs led ovary function.

RNAi-INHa showed a significant increase in GDF9 and INHbBmRNA and protein levels compared with those from wild type(P < 0.05; Fig. 2A and B). In contrast, Kitl and TGFbIII mRNA andprotein expression were significantly lower (P < 0.05; Fig. 2A andB) as compared to wild type littermates. Consequently, there mightbe a coordinated mechanism to control GCs proliferation due to upregulation of GDF9, INHbB and down regulation of Kitl andTGFbRIII genes. TGFbRIII is commonly known as betaglycan, coreceptor for TGFbs and inhibins [33,34]. Which bind to the activinaugmenting the ability of inhibins to antagonize activin function[6]. Modification in inhibins and activins, that effect on adrenal cellfunction influencing adrenocortical steriodgenesis, adrenocorticaltumor formation and adrenomeduallry cell differentiation [35].The specific actions of RNAi-INHa in folliculogenesis andmammalian reproduction are still unclear, although knockout ofINHa resulted elevated activin, GDF9 and FSH suggesting theprominent role of INHa in gene regulation in local ovarian action[12]. It is noteworthy that, under the same conditions, there wereno significant differences in ovarian weight of 3 weeks old micebetween RNAi-INHa and control mice (data not shown). It isspeculated that lower level of Kitl could contribute to control theaberrant GCs proliferation and early tumerogenesis; yet, such micecan breed but the efficiency was poor. The decreased level of Kitlplays a role in initiation of follicle growth and prenatal folliculardevelopment [36]; therefore, it might be contributed torescue/control the GCs from tumerogenesis and maintain fertilityin mice to some extent. These findings is in agreement with

down of inhibin a subunit increased apoptosis in granulosa cells anddx.doi.org/10.1016/j.jsbmb.2015.05.006

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472 an473 tio474 la475 sim476 fa477 an478 Ea479 in480 ca481 ro482 th483 m484 re485 m486 of487 m488 [4489 sig490 th491 m492 pe493 re494 ca495 ca496 tio497 ca498 m499

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evious findings, Kitl is negatively regulated by GCs derived bothtivin [37,38] and oocyte-derived GDF9 [39].The expression of GDF9, Kitl, in pre-ovulatory stage and its role

early follicle growth has been well established [5,40]. Upgulation of INHbB and GDF9 in INHa knock down mice couldomote precocious follicle growth, so that oocyte releasenificantly increased in 3 weeks super ovulated mice [9]. One contrary, when superovulation employed after puberty, oocytembers significantly decreased in INHa mutant mice than controloup (Fig. 5). The loss of INHa subunit knockout mice results innificant increase in activin and inhibin b subunit [14]. Indition, overexpression of INHa subunit in MT a transgenicice had decreased inhibin b [18]. These findings demonstrate

existing auto-regulation mechanism of INHa-subunit genepression for folliculogenesis and oocyte maturation.It is well-established that the cross talk between cell death andrvival signals is very important for follicular developmenthether the follicle ultimately ovulates or undergoes atresia due toultiple ovarian factors, which regulate cell proliferation,fferentiation and apoptosis [41]. Interestingly, with regard toe RNAi-INHa on GCs apoptosis, our findings confirmed thatHa subunit knock down significantly promoted apoptosis inanulosa cells (Table 2); and therefore, INHa could play anificant role in the suppression of programmed cell death inese cells. Previous results have also supported our findings that,e apoptotic activity was higher in RNAi-INHa in atretic follicularanulosa cells [20]. Hence, we inferred that the INHa knockdownice would exhibit the modified gene expression pattern ofoptosis and cell cycle, perhaps explaining the differences inrum hormone levels and progression of GCs.Our results on mRNA and protein expression of Caspase3 andti-apoptotic factor bcl2 in GCs up regulated both at transcrip-nal and translational levels (Fig. 2A and B). Previous work in ourb on knockdown of INHa in anterior pituitary cells also showsilar results of up regulation of Caspase3, bcl2 and pro apoptotic

ctor bax [22].This indicate that INHa-subunit knockdown led to imbalance in the bcl-2/bax family and may promote apoptosis.rlier study reported caspase3 dependent repression of apoptosis

ovarian GCs when treated with inhibin analogue [42]. Thespases is a family of cysteine proteases that provide significantle in apoptotic pathways [43]. Caspase activation is regulatedrough, either extrinsic death receptor-mediated or intrinsicitochondrial signaling pathways [44]. However, Lindsay et al.ported that Bcl-2 family genes are expressed in theitochondrial membrane [45]. Evidence shows that the proteins

the Bcl-2 family play a pivotal role in regulation of mitochondrialembrane permeability and the intrinsic mitochondrial pathway6]. Along with the unbalance of the pro-apoptotic, anti-apoptoticnaling cascade, an attempt to allow cells to cope with stress. Inis scenario, both pro and anti-apoptotic signals converge atitochondrial membranes, then it becomes permeable. Thisrmeability of the mitochondrial outer membrane results thelease of intermediate apoptosis signaling molecule cytochrome cusing activation of caspase-9, which then cleaves and activatespase3, resulting in downstream apoptosis and DNA fragmenta-n [47]. Thus, increased protein levels of apoptosis markersspase3 in transgenic mice may be activated by mechanisms ofitochondrial mediated apoptosis pathway.INHa has been implicated as an autocrine/paracrine factor that

odifies follicular growth, atresia, gonadotropin responsivenessd steroidogenesis [48], Caspase3 dependent apoptosis activateshen follicle begins to leave in resting pool [49], while properuilibrium between bcl2/bax is important for GCs survival0,51]. Previously, experiment conducted related to apoptoticductor and neutralizers in GCs apoptosis in bovines [52,53]. Thelance between proliferation and apoptosis of granulosa cells is

Please cite this article in press as: I. Kadariya, et al., RNAi-mediated knocdecreased fertility in mice, J. Steroid Biochem. Mol. Biol. (2015), http:/

critical for the folliculogenesis both before birth and during thereproductive life [42]. However, knock down of INHa might not beable to further increase the apoptosis rate in RNAi-M group ascompared to control group in the normal culture of GCs. WhetherRNAi-INHa mechanism promotes cell survival or inhibitsapoptosis in mammalian GCs, an induction model of apoptosisis yet to be established.

As INHa has been involved in cell cycle regulation, therefore,we detected the cell cycle of GCs after INHa knock down. Findingsdisclosed that knock down of INHa-subunit led to a substantialarrest in G1 cell cycle phase, and controlled the cells in S/G2 phase(Table 4). Earlier report showed that RNAi-INHa could inhibit cellproliferation in pituitary cells of mouse [22]. Nevertheless, INHaknockout model could increase the proliferation of GCs and finallylead to the tumerogenesis after puberty [32]. However,results indicate that RNAi-INHa rescue the ovary from earlytumerogenesis through the control progression of GCs due toincreased level of apoptosis. Further, RNAi-INHa might be able tocheck uncontrolled cell proliferation to some extent, whichotherwise could lead to tumor development in advance agewhereas, our results showed the non-significant ovarian weightbefore puberty in RNAi-INHa mice group as compared to controlgroup (data not shown). Thus, the balance between apoptosis andproliferation must be strictly regulated for ovarian tissuedevelopment. By reason, uncontrolled proliferation can beassociated with a low level of apoptosis [54]. Taken together,these results indicate that INHa has a complex mechanism innormal cell cycle control, growth, proliferation and apoptosis inmice GCs. Further studies are required to explore the distinctpossible mechanisms of RNAi-INHa at cellular level.

Further, hormonal concentrations was also measured for FSHand INHa in RNAi-M and control group of mice. Loss of INHa favorsincreased FSH release due to loss of negative feedback by inhibin a.On the other hand, female mice overexpressing FSH demonstratehemorrhagic and cystic ovaries [16]. Although, the antral folliclepool expanded, superovulation of immature 3 weeks old femalemice might contribute to the increased ovulation rate in inhibin amutant mice (Fig. 5). With the well-documented function ofinhibins to suppress FSH, INHa knockout mice have increasedserum FSH levels compared to wild type mice [15]. Silencing ofINHa-subunit disrupted the inhibin-beta glycan signalingpathway, therefore increased the expression of GnRH receptors,and subsequently resulted in increased FSH hormone secretions inanterior pituitary cells [22]. Here, our results showed downregulation of beta glycan, which is consistent with the previousfinding. Activins and FSH are known regulators of granulosa cellproliferation [55]. Acceleration in granulosa cell proliferation inINHa deficient pre-pubertal mammals is not only a direct effect ofFSH, but more likely a magnitude of intraovarian growth factorssignaling [56].

5. Conclusions

Knock down of INHa in RNAi transgenic female mice, by 64%down-expression in vivo demonstrated sub fertility because ofreduced ovulations with cellular and molecular alterations. Thisstudy suggest that INHa subunit is an important regulator ofGCs apoptosis and cell cycle progression that confirmed bydetermining different apoptotic and growth factors. Moreover,results indicate that RNAi-INHa provide rescue operation tocontrol early tumerogenesis through the control progression ofGCs due to increased level of apoptosis. Findings further evidencethe role of INHa as key modulator of an ovarian function, playingan essential role in normal fertility and gametogenesis. Furtherstudies to evaluate the function of the somatic cells surroundingthe germ cells by RNAi transgenic mouse model would be of great

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interest to find out, how INHa plays the role in GCs proliferation,folicullogenesis and reproduction.

Author contributions

Conceived and design the experiments: SJZ and IK. Performedthe experiments: IK. Analyzed data: IK. Wrote the paper: IK andSJZ. SJZ was the major supervisor in this whole experiment.

Conflict of interest

The authors have declared no competing interests exist.

Acknowledgements

This study was supported grant from the Chinese Government(2014030709020305) and the Huanghe Yingcai Project, EUFP7 projects (PIIF-GA-2012-328205, PIIFR-GA-2012-912205, andFP7-KBBE-2013-7-613689). The funders had no role in studydesign, data collection and analysis, decision to publish, orpreparation of the manuscript.

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