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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 3  |  Issue : 3  |  Page : 133-140

Knockdown of the premature ovarian insufficiency candidate gene NUP107 in ovarian granulosa cells affects cell functions, including receptor expression and estrogen synthesis


1 Department of Obstetrics and Gynecology, Obstetrics and Gynecology Hospital of Fudan University; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Fudan University, Shanghai 200011, China
2 Department of Obstetrics and Gynecology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011; The Institute of Reproduction and Development, Obstetrics and Gynecology Hospital, Institute of Reproduction and Development, Fudan University, Shanghai 200438, China

Date of Submission09-May-2019
Date of Web Publication27-Sep-2019

Correspondence Address:
Bin Li
Department of Obstetrics and Gynecology, Obstetrics and Gynecology Hospital of Fudan University, No. 419 Fangxie Road, Shanghai 200011
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.268158

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  Abstract 


Objective: Mutations in NUP107 have been discovered in patients with premature ovarian insufficiency and may have tissue-specific effects in ovarian development. However, the role of NUP107 in human granulosa cell (GC) function and female fertility still remains unknown. In this study, we used RNA interference to investigate how NUP107 dysfunction influences GCs and ovarian development.
Methods: Immunohistochemical staining was used to detect the expression of NUP107 in ovaries. Cell counting kit-8 assay, real-time cell analysis, and flow cytometry were used to explore cell proliferation and apoptosis, and an enzyme-linked immunosorbent assay was used to assess the estrogen concentrations. Quantitative real-time reverse transcription-quantitative polymerase chain reaction and Western blot analyses were used to determine the expression of NUP107 and functional receptors.
Results: Knockdown of NUP107 expression had little effect on the growth and number of GCs. Further study confirmed that knockdown of NUP107 may interfere with estrogen synthesis in GCs and their sensitivity to the regulation of follicle-stimulating hormone (FSH) by decreasing the expression of estrogen synthesis-related genes AR, CYP17A1, CYP19A1, STAR, and NR5A1. Moreover, knockdown of NUP107 decreased the expression of AMHR2, FSHR, LHR, and ESR1 in GCs, but had no effect on the expression of ESR2.
Conclusions: These data revealed that NUP107 may impede follicle growth and maturation by regulating hormone synthesis, sensitivity to follicle-stimulating hormone, and expression of functional receptors in GCs, and may, therefore, interfere with female fertility.

Keywords: Estrogen; NUP107; Ovarian Granulosa Cells; Premature Ovarian Insufficiency


How to cite this article:
Liu XC, Han MX, Xu Y, Wang HY, Li B. Knockdown of the premature ovarian insufficiency candidate gene NUP107 in ovarian granulosa cells affects cell functions, including receptor expression and estrogen synthesis. Reprod Dev Med 2019;3:133-40

How to cite this URL:
Liu XC, Han MX, Xu Y, Wang HY, Li B. Knockdown of the premature ovarian insufficiency candidate gene NUP107 in ovarian granulosa cells affects cell functions, including receptor expression and estrogen synthesis. Reprod Dev Med [serial online] 2019 [cited 2019 Oct 17];3:133-40. Available from: http://www.repdevmed.org/text.asp?2019/3/3/133/268158




  Introduction Top


Premature ovarian insufficiency (POI) is characterized by premature amenorrhea (before the age of 40 years) that is accompanied by follicle-stimulating hormone (FSH) levels >25 IU/L (measured on two occasions more than 4 weeks apart).[1] Women with POI can present with hypoestrogenism, menopausal symptoms, infertility, and complications, such as high risks of cardiovascular and Alzheimer's diseases. As a heterogeneous disease, POI can be affected by many factors, including genetic mutations. More than seventy candidate genes related to oocyte meiosis,[2] oogenesis,[3] steroid hormone synthesis,[4] follicle recruitment and activation, and ovulation [5] have been reported to be associated with POI. Mouse and granulosa cell (GC) models have been used to demonstrate the functions of some candidate POI genes in ovarian development. However, most of the genes have not been extensively investigated in the POI population, and further study is necessary to conclusively determine as to how these mutations lead to POI.

Recent studies have highlighted the importance of nuclear pore complex (NPC) in the development of diseases. In addition to transporting mRNAs and proteins, the NPC may play critical roles in meiosis and transcriptional regulation.[6],[7],[8] NPC, a large and complicated protein complex, is made of more than thirty nucleoporins (NUPs). NUPs have been reported to have tissue-specific functions in developmental processes and various diseases, such as Huntington's disease,[9] nephrotic syndrome,[10] embryonic development,[11] and viral infection.[12] Recently, studies on several NUPs, such as Nup35,[13] Aladin,[14] and Nup154,[15] in mouse and Drosophila models, have revealed the relationship between NUPs and ovarian development. These studies have indicated that NUPs may have specific-tissue functions in female fertility.

NUP107 is a major component of the NUP107-160 complex, which regulates the recruitment of other NUPs and the formation of nuclear pores.[16] The NUP107 protein is highly conserved between humans and Drosophila at the amino acid level (35% identity and 54% similarity, with 5% gaps). Previous studies have reported several mutations of NUP107 in nephrotic syndrome and its reduced expression in senescent cells and mice.[17],[18] Functional studies have revealed colocalization of NUP107 and kinetochores as well as NUP107-associated dynamic changes in phosphorylation states during mitotic progression and regulation of nuclear translocation of the DNA damage response-associated factor Apaf-1, indicating a potential function of NUP107 in regulating cell cycle progression and DNA damage responses induced by genotoxic stress.[19],[20] NUP107 knock out (KO) Drosophila and mutant mouse models exhibit impairment of female fertility. There are striking differences in the female reproductive system among humans, Drosophila, and mice. Therefore, it is difficult to translate the information obtained from Drosophila and mice to humans without further validation in more relevant models.

The importance of ovarian GCs in follicle development and female fertility has been demonstrated in several studies. The failure of differentiation and proliferation of GCs may lead to oocyte death and depletion of follicles.[21],[22],[23] Moreover, factors secreted by GCs have been shown to regulate the activation and recruitment of follicles.[24] Mutations in the functional genes of GCs, such as FSH receptor (FSHR),[25] ESR1,[26] and GDF9,[27] have been found to be related to POI. In addition, specific KO of critical genes in GCs could lead to POI in mouse models.[28] Therefore, we wanted to determine whether NUP107 is critical for the survival and function of human GCs.


  Methods Top


Immunohistochemistry

We performed IHC to determine the cellular localization of NUP107 in the ovaries of a POI model. We used a cyclophosphamide (CTX)-induced POI mouse model established by Dr. X-Y Chen.[29] In brief, ovarian tissue was embedded in paraffin. Antigen retrieval was achieved by microwaving the slides in a 0.01-mol/L sodium citrate solution (pH 6.0) for 15 min and then cooling them to 25°C. The slides were blocked with 3% hydrogen peroxide in phosphate-buffered saline (PBS) for 10 min and 10% lamb serum in PBS for 1 h at 25°C. They were then incubated overnight at 4°C with a primary antibody against NUP107 (1:250, ab73290, Abcam, USA). Thereafter, on the next day, the slides were incubated for 1 h at room temperature with an HRP-conjugated goat anti-rabbit IgG secondary antibody (1:500, Guge, China). Immunoreactive signals were detected using a DAB Kit (Guge, China) at room temperature. The slides were counterstained with hematoxylin. The negative control was not incubated with the primary antibody. Immunostaining was observed, and images of the sections were captured using a Nikon Eclipse 50i microscope (Nikon Corp., Tokyo, Japan). The integrated optical density and area of staining were measured with Image-Pro Plus 6.0 (Media Cybernetics, Inc., MD, USA).

Cell culture

The human ovarian granulosa-like tumor cell line KGN contains functional FSHR and aromatizing enzyme activity, similar to normal human immature ovarian GCs. The cells were cultured in DMEM/F12 (with no phenolsulfonphthalein) and supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37°C in an atmosphere of 5% CO2. The cell line was bought from Cellcook. Ltd. (Guangzhou, China) and has been authenticated by short tandem repeat (STR) analysis in Biowing. Ltd. (Shanghai, China).

Plasmids and transfection

Cells were plated in 6- or 12-well plates, 12–24 h prior to transfection with 3 or 1.5 μg of pGPU6/Neo plasmid per well and Lipofectamine 3000 (Invitrogen, USA). Following transfection for 48 h, the cells were collected for various experiments. The RNA interference (RNAi) sequences were designed and verified by GenePharma. Ltd. (Shanghai, China). The sequences are listed in [Supplementary Table 1].



RNA isolation and quantitative real-time reverse transcription quantitative polymerase chain reaction analysis

Total RNA was extracted from the transfected cells using TRIzol (Invitrogen, USA). After assessment of the quantity and purity, the RNA was reverse transcribed using a PrimeScript RT Reagent Kit (Takara, Japan). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using a TB Green™ Premix Ex Taq™ II Kit (Takara, Japan). Specific pairs of primers were designed and verified by bioTNT. Ltd. (Shanghai, China) and used for gene amplification. For gene expression analysis, the data were analyzed using the 2−ΔΔCT method. The primers for the target genes are listed in [Supplementary Table 2]. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for quantification.



Western blot analysis

Proteins were extracted from placenta and trophoblast cells using radioimmunoprecipitation assay lysis buffer (Beyotime, China) supplemented with phenylmethanesulfonyl fluoride (PMSF, Beyotime, China) on ice. Equal amounts of protein extracts (30 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride membranes in transfer buffer (Beyotime, China) for 1–2 h according to the molecular weights of proteins. The membranes were blocked for 1 h in 5% skim milk in 1× TBST (1× TBS with 0.1% Tween-20) and then incubated overnight at 4°C with the following primary antibodies: GAPDH antibody (1:3,000, 60004-1-Ig, Proteintech, China), NUP107 antibody (1:3,000, 19217-1-AP, Proteintech, China), FSHR antibody (1:2,000, 22665-1-AP, Proteintech, China), anti-Mullerian hormone receptor 2 (AMHR2) antibody (1:1,000, ab197148, Abcam, USA), ERa antibody (1:1,000, SAB1402766, Sigma, Germany), and ERb antibody (1:1,000, ab3576, Abcam, USA). The membranes were then washed three times in 1× TBST for 10 min each. The blots were then incubated with the secondary antibody in 5% skim milk. The immunoreactivity was visualized with enhanced chemiluminescence reagents (Millipore, USA). GAPDH was used as the control.

Enzyme-linked immunosorbent assay

Cells were cultured and transfected with plasmids in 12-well plates. After 24 h, 10 ng/mL testosterone (T) was added alone or with FSH (100 IU/mL) into the medium, as a substrate and a regulator for estrogen synthesis. After 48 h of culture, the culture media were collected and stored at −80°C until the assay. The concentrations of estradiol (E2) were examined with an estradiol enzyme-linked immunosorbent assay (ELISA) kit (FR E-2000, LDN, Germany), with a sensitivity of 6.2 pg/mL and a range of 25–2,000 pg/mL.

Cell Counting kit-8 assays and real-time cell analysis

After transfection, cell proliferation was analyzed using cell counting kit-8 (CCK8) assays (Dojindo, China) and real-time cell analysis (RTCA) according to the manufacturers' instructions. Cells were seeded in 96-well plates at an initial density of 8 × 103 cells/well. At each time point, the cells were stained with 10% CCK8 for 1 h at 37°C. The absorbance was measured at 450 nm. The results were confirmed by manual cell counting. For RTCA, according to the manufacturer's instructions, cells were seeded in E-plates at an initial density of 3 × 103 cells/well in 100 μL of DMEM/F12 plus 10% FBS and incubated at 37°C in an atmosphere of 5% CO2. Continuous monitoring was done by the RTCA system (xCELLigence; ACEA Biosciences, USA), and data were recorded every 15 min for a total of 120 h.

Flow cytometry analysis of apoptosis and cell cycle distribution

After transfection for 48 h, the cells were harvested by trypsinization with 0.25% trypsin, washed with cold PBS, centrifuged at 800 g for 5 min, and resuspended in binding buffer. PE-Annexin V/7-amino-actinomycin (7-AAD) or PI was added, and the cells were incubated for 15 min or 30 min at room temperature or at 37°C in the dark (BD Pharmingen). Thereafter, 400 μL of binding buffer was added, and the samples were analyzed immediately on a CytoFLEX flow cytometer (Beckman Coulter, USA).

Statistics

The data were expressed as means ± standard deviation from at least three independent experiments. All experiments were performed in triplicate. Independent t-tests were used for statistical comparisons between the knockdown and control groups using SPSS 21.0 (SPSS Inc., Chicago, IL, USA), and the statistical significance levels were P < 0.05. All P values were two tailed.


  Results Top


NUP107 is downregulated in granulosa cells in a premature ovarian insufficiency mouse model

Although NUP107 has been identified as a POI-related gene, the cell type affected by the mutation remains unclear. Because of the difficulties in obtaining samples from POI patients, we investigated the expression levels of NUP107 by IHC in ovarian samples from CTX-induced POI model mice and their control littermates. To our surprise, significant differences in NUP107 levels between groups could be observed only in GCs, but not in non-GCs [Figure 1]. Therefore, we speculate that loss of NUP107 in GCs impairs their viability or function and leads to POI.
Figure 1: Decrease in the expression of NUP107 in GCs in a POI mouse model. (a-f) The expressions of NUP107 in GCs in negative control (a-c) and POI model (d-f) mice were examined by immunohistochemistry. (g) The expression of NUP107 in GCs was decreased in mice (n = 15) with POI induced by CTX compared to that in control mice; (h) no difference was observed in the expression of NUP107 in ovarian stromal cells between the groups. GCs: Granulosa cells; POI: Premature ovarian insufficiency; CTX: Cyclophosphamide; *: P < 0.05.

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Knockdown of NUP107 does not affect the proliferation of granulosa cells

To determine whether NUP107 impairment has any effect on the normal growth of GCs, we disrupted the expression of NUP107 by RNAi. For two of the three shRNAs used, a knockdown efficiency >50% was obtained [Figure 2]a, [Figure 2]b, [Figure 2]c. We used these two shRNAs for subsequent experiments. The cell viability and proliferation rate were measured by CCK8 assay and RTCA. The results of both these assays showed that the rates of proliferation were not affected by the knockdown of NUP107 [Figure 2]d and [Figure 2]e. To explore the function of NUP107 in GCs, cell cycle analysis was performed by flow cytometry (FCM) and RT-qPCR on cells, with or without NUP107 knockdown. No significant change in cell cycle distribution was identified upon NUP107 knockdown [Figure 2]f and [Figure 2]j.
Figure 2: Efficiency of NUP107 knockdown in KGN cells and cell proliferation after transfection. (a-c) Expression of NUP107 mRNA was detected by Western Blot after transfection for 48 h (a,b), and mRNA was detected by RT-qPCR after transfection for 12 h (c). (d) Cell viability was assessed by CCK8 in the NUP107 knockdown and control groups. (e) RTCA was used to monitor changes in cell amplification after transfection for 4 days. (f-i) Cell cycle distribution was analyzed by FCM after transfection for 48 h. (j) The RT-qPCR was used to detect CDK1, CDK2, CDK4 and CDK6 expression after transfection for 12 h. FCM: Flow cytometry; RTCA: Real-time cell analysis; RT-qPCR: Reverse transcription-quantitative polymerase chain reaction; *: P < 0.05.

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Knockdown of NUP107 does not affect the cell apoptosis or senescence of granulosa cells

FCM was used to detect the levels of the early apoptosis marker, PE-Annexin V, and the cell death marker, 7-AAD. The results suggested that the rates of apoptosis/death were not significantly different between control and NUP107 knockdown cells [Figure 3]a. These results indicated that NUP107 knockdown has no obvious effect on the replication and survival of GCs. In addition, we used a senescence-associated beta-galactosidase (SA-β-Gal) assay to measure the senescence rates of GCs. The basal senescence rate was very low in GCs, and NUP107 knockdown did not significantly changed the senescence rate [Figure 3]b. These data suggest that NUP107 knockdown does not change the cell apoptosis or senescence rates of GCs.
Figure 3: Knockdown of NUP107 has no effect on cell apoptosis or senescence state of GCs. (a) FCM was utilized to detect the apoptotic rate by PE-Annexin V/7-AAD staining after transfection for 48 h. Early apoptosis cells are stained positive for PE-Annexin V(+)/7-AAD(−), and late apoptosis cells are stained for PE-Annexin V(+)/7-AAD(+). (b) Representative micrographs (×100) of control and knockdown group cells subjected to SA-β-Gal activity assays after transfection for 48 h. Senescent cells were stained in blue. GCs: Granulosa cells; FCM: Flow cytometry; 7-AAD: 7-amino-actinomycin.

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Knockdown of NUP107 interferes with the expression of functional receptors in granulosa cells

After ruling out the effects of NUP107 on the viability and cell cycle progression of GCs, we investigated the effects of NUP107 on GC function. During the growth and development of follicles, GCs need to expand and differentiate into functional cells. These cells express receptors, including AMHR, FSHR, and luteinizing hormone receptor (LHR), which are required for hormonal regulation responses. To investigate whether NUP107 regulates the function of GCs, we measured the levels of a few receptors upon NUP107 knockdown. The results of RT-qPCR and Western blot analysis showed a marked decrease in the mRNA and protein levels of AMHR2, FSHR, and ESR1 after NUP107 knockdown [Figure 4]a and [Figure 4]b. These results indicate that loss of NUP107 may impair the expression of functional receptors in GCs.
Figure 4: Effects of NUP107 knockdown on receptors expression, estrogen production, and FSH sensitivity. (a) The mRNA expression of AMHR2, FSHR, LHR, ESR1 and ESR2 were detected by RT-qPCR after transfection for 12 h. (b) Expression of FSHR, AMHR2, ERa, and ERb at the protein level were tested by Western Blot. (c) Estradiol levels in cell culture media were detected by ELISA in the presence of 10 ng/mL testosterone (T) as a substrate after 48 h of transfection with and without 100 IU/mL rFSH. (d) The mRNA levels of AR, CYP17A1, CYP19A1, NR5A1, and STAR were detected by RT-qPCR. FSH: Follicle stimulating hormone; RT-qPCR: Reverse transcription-quantitative polymerase chain reaction; ELISA: Enzyme-linked immunosorbent assay.

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Knockdown of NUP107 decreases estrogen synthesis and attenuates sensitivity to follicle-stimulating hormone regulation in vitro

Low estrogen levels in POI patients can result in high risks of osteoporosis and dementia. GCs are one of the major cell types producing estrogen in female mammals. Under physiological conditions, GCs are stimulated by FSH and convert T into estrogen. Because we observed the downregulation of FSHR in GCs upon NUP107 knockdown, we further investigated whether NUP107 regulates estrogen synthesis.

Estrogen synthesis was measured under both basal and FSH-stimulated conditions. Under basal conditions, compared to control GCs, those with NUP107 knockdown exhibited reduced E2 levels. Importantly, FSH stimulation did not enhance estrogen synthesis in GCs with NUP107 knockdown [Figure 4]c. Consistent with the ELISA results, the mRNA levels of estrogen synthesis-related genes were downregulated by NUP107 knockdown [Figure 4]d. We previously found that NUP107 has no impact on the viability or the cell cycle of GCs. Therefore, the effects of NUP107 knockdown on estrogen synthesis might due to the changes in the function of cells.


  Discussion Top


In this study, we discovered the effect of NUP107 on the regulation of the growth and function of GCs. Our data revealed that reduced NUP107 expression did not affect the number of GCs but might have affected the expression of functional receptors and estrogen synthesis. These results indicate that NUP107 mutations may interfere with GC maturation and hormone synthesis.

NPCs were earlier considered as static structures that functioned only in regulating the nucleocytoplasmic transportation. In recent years, emerging evidence has indicated that NPCs are dynamic and that dysfunction of different NUPs may produce tissue-specific phenotypes. However, the influence of nucleocytoplasmic transport under the conditions of NUP107 knockdown remains controversial.[16],[30] Recent studies have revealed that different receptors may prioritize interactions with specific NUPs while passing through NPCs. When stimulated by hormonal signals, steroid receptors translocate into the nucleus and activate downstream pathways. As the sole regulator, the quantity of NPCs may affect the translocation of hormone receptors. However, whether interference with NUP107 expression impedes the translocation of hormone receptors remains to be explored. SMAD proteins are signal transducers and transcriptional modulators that act in multiple signaling pathways. Some Smad knockout mice show infertility or subfertility phenotypes. Currently, several NUPs are believed to selectively transport SMADs into the nucleus, which is a fundamental step in transforming growth factor (TGF) signal transduction.[31] Mutations in a number of TGF-β family genes, such as GDF9, BMP15, and AMH, have been identified in POI patients; these mutations may disturb GC proliferation, hormonal release, and follicle activation.[32],[33],[34] Similarly, some transcription factors associated with oocyte development, such as FOXO3a and NOBOX, can be blocked by the dysfunction of NPCs. The disconnection of signaling pathways may cause severe abnormalities in follicle development. As major nurse cells of oocytes, GCs support and communicate closely with oocytes. The influence of the crosstalk between GCs and oocytes still needs to be explored. NUP107 has also been discovered to be associated with cell senescence.[18] However, we did not find a difference in the percentage of senescent GCs between the knockdown and control groups, indicating that reduction in NUP107 could be a downstream event in the senescence of cells.

In recent decades, emerging evidence has indicated that NUPs play vital roles in regulating gene expression and epigenetics and are involved in chromatin organization.[30],[35] Our data suggest that the expression of functional receptor genes in GCs is regulated by NUPs, especially given the reduced expression of the AMHR2 and FSHR genes when NUP107 was knocked down. AMHR2 encodes the main functional receptor of AMH signaling, regulating the sensitivity of GCs to FSH and E2 levels and subsequently influencing primordial follicular growth and follicular selection.[36] Another essential membrane receptor of GCs is FSHR, which mediates FSH signaling and promotes the expression of LHR, stimulating folliculogenesis and beginning of ovulation at puberty. Mutations in FSHR have been discovered in women in whom follicular growth has been arrested.[25] Our results regarding reduced LHR expression and low sensitivity to FSHR in NUP107 knockdown GCs may partly have resulted from the decreased FSHR expression. Interestingly, our study demonstrates, for the first time, that NUP107 may participate in regulating estrogen synthesis by decreasing the expression of estrogen synthesis-related receptors and enzymes. However, the mechanism by which NUPs regulate gene expression remains unknown. Several studies have explored the mechanism and have revealed that NUPs may interfere with gene expression by regulating mRNA positions and interactions with other RNA regulation-related proteins or histone-modifying complexes.[37]

The Nup107-160 complex colocalizes with kinetochores at the initiation of mitosis and is one of the main regulators in chromosome segregation.[38] Glavy et al.[19] have demonstrated that NUP107 is phosphorylated on its N-terminal sites in vivo during mitosis and can be efficiently dephosphorylated by trimeric protein phosphatase 2A-B55 upon mitotic exit, suggesting that NUP107 is associated with the regulation of cell cycle progression.[39] However, in our study, we did not find any significant change in mitotic progress after knockdown of NUP107 in KGN cells. In females, all oocytes undergo a quiescent diplotene stage in prophase I during meiosis and can last for decades. Because chromosome segregation is one of the main events in meiosis, we suspect that dysfunction of NUP107 could obstruct oocyte meiosis and impede the maturation and fertilization of oocytes. In further studies, the impacts of NUP107 variants in oocytes will be explored.

In the present study, we have demonstrated that NUP107 may regulate the ability of estrogen synthesis and the expression of functional receptors in GCs and suggested that NUP107 might have tissue-specific function in follicular development.

Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.

Financial support and sponsorship

This study was funded by the National Natural Science Foundation of China (No. 81471423) to Bin Li. The funding bodies had no role in study design, collection, analysis, and interpretation of data or in manuscript preparation.

Conflicts of interest

There are no conflicts of interest.



 
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