|Year : 2019 | Volume
| 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
Xiao-Cheng Liu1, Meng-Xin Han1, Yan Xu1, Hong-Yan Wang2, Bin Li1
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 Submission||09-May-2019|
|Date of Web Publication||27-Sep-2019|
Department of Obstetrics and Gynecology, Obstetrics and Gynecology Hospital of Fudan University, No. 419 Fangxie Road, Shanghai 200011
Source of Support: None, Conflict of Interest: None
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 2020 Jan 25];3:133-40. Available from: http://www.repdevmed.org/text.asp?2019/3/3/133/268158
| Introduction|| |
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). 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, oogenesis, steroid hormone synthesis, follicle recruitment and activation, and ovulation  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.,, 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, nephrotic syndrome, embryonic development, and viral infection. Recently, studies on several NUPs, such as Nup35, Aladin, and Nup154, 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. 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., 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., 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.,, Moreover, factors secreted by GCs have been shown to regulate the activation and recruitment of follicles. Mutations in the functional genes of GCs, such as FSH receptor (FSHR), ESR1, and GDF9, have been found to be related to POI. In addition, specific KO of critical genes in GCs could lead to POI in mouse models. Therefore, we wanted to determine whether NUP107 is critical for the survival and function of human GCs.
| Methods|| |
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. 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).
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).
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|| |
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.|
Click here to view
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.|
Click here to view
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.|
Click here to view
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.|
Click here to view
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|| |
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., 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. 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.,, 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. 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., 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. 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. 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.
The Nup107-160 complex colocalizes with kinetochores at the initiation of mitosis and is one of the main regulators in chromosome segregation. Glavy et al. 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. 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.
| References|| |
European Society for Human Reproduction and Embryology (ESHRE) Guideline Group on POI, Webber L, Davies M, Anderson R, Bartlett J, Braat D, et al.
ESHRE guideline: Management of women with premature ovarian insufficiency. Hum Reprod 2016;31:926-37. doi: 10.1093/humrep/dew027.
Zhou Y, Qin Y, Qin Y, Xu B, Guo T, Ke H, et al.
Wdr62 is involved in female meiotic initiation via activating JNK signaling and associated with POI in humans. PLoS Genet 2018;14:e1007463. doi: 10.1371/journal.pgen.1007463.
França MM, Funari MF, Lerario AM, Nishi MY, Pita CC, Fontenele EG, et al.
A novel homozygous 1-bp deletion in the NOBOX gene in two Brazilian sisters with primary ovarian failure. Endocrine 2017;58:442-7. doi: 10.1007/s12020-017-1459-2.
Lourenço D, Brauner R, Lin L, De Perdigo A, Weryha G, Muresan M, et al.
Mutations in NR5A1 associated with ovarian insufficiency. N Engl J Med 2009;360:1200-10. doi: 10.1056/NEJMoa0806228.
Cordts EB, Santos MC, Santos AA, Mafra FA, Christofolini DM. FSHR polymorphisms are associated with premature ovarian insufficiency development. Fertil Steril 2013;100:S149. doi: 10.1016/j.fertnstert.2013.07.1541.
Tous C, Rondón AG, García-Rubio M, González-Aguilera C, Luna R, Aguilera A. A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors. EMBO J 2011;30:1953-64. doi: 10.1038/emboj.2011.109.
Oka M, Mura S, Yamada K, Sangel P, Hirata S, Maehara K, et al.
Chromatin-prebound Crm1 recruits Nup98-hoxA9 fusion to induce aberrant expression of Hox cluster genes. Elife 2016;5:e09540. doi: 10.7554/eLife.09540.
Chu DB, Gromova T, Newman TA, Burgess SM. The nucleoporin Nup2 contains a meiotic-autonomous region that promotes the dynamic chromosome events of meiosis. Genetics 2017;206:1319-37. doi: 10.1534/genetics.116.194555.
Grima JC, Daigle JG, Arbez N, Cunningham KC, Zhang K, Ochaba J, et al.
Mutant huntingtin disrupts the nuclear pore complex. Neuron 2017;94:93-107. e6. doi: 10.1016/j.neuron.2017.03.023.
Braun DA, Sadowski CE, Kohl S, Lovric S, Astrinidis SA, Pabst WL, et al.
Mutations in nuclear pore genes NUP93, NUP205 and XPO5 cause steroid-resistant nephrotic syndrome. Nat Genet 2016;48:457-65. doi: 10.1038/ng.3512.
Okita K, Kiyonari H, Nobuhisa I, Kimura N, Aizawa S, Taga T. Targeted disruption of the mouse ELYS gene results in embryonic death at peri-implantation development. Genes Cells 2004;9:1083-91. doi: 10.1111/j.1365-2443.2004.00791.x.
Kane M, Rebensburg SV, Takata MA, Zang TM, Yamashita M, Kvaratskhelia M, et al.
Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of M×2. Elife 2018;7. pii: e35738. doi: 10.7554/eLife.35738.
Chen F, Jiao XF, Zhang JY, Wu D, Ding ZM, Wang YS, et al.
Nucleoporin35 is a novel microtubule associated protein functioning in oocyte meiotic spindle architecture. Exp Cell Res 2018;371:435-43. doi: 10.1016/j.yexcr.2018.09.004.
Carvalhal S, Stevense M, Koehler K, Naumann R, Huebner A, Jessberger R, et al.
ALADIN is required for the production of fertile mouse oocytes. Mol Biol Cell 2017;28:2470-8. doi: 10.1091/mbc.e16-03-0158.
Gigliotti S, Callaini G, Andone S, Riparbelli MG, Pernas-Alonso R, Hoffmann G, et al.
Nup154, a new Drosophila
gene essential for male and female gametogenesis is related to the nup155 vertebrate nucleoporin gene. J Cell Biol 1998;142:1195-207. doi: 10.1083/jcb.142.5.1195.
Walther TC, Alves A, Pickersgill H, Loïodice I, Hetzer M, Galy V, et al.
The conserved Nup107-160 complex is critical for nuclear pore complex assembly. Cell 2003;113:195-206. doi: 10.1016/S0092-8674(03)00235-6.
Park E, Ahn YH, Kang HG, Miyake N, Tsukaguchi H, Cheong HI. NUP107 mutations in children with steroid-resistant nephrotic syndrome. Nephrol Dial Transplant 2017;32:1013-7. doi: 10.1093/ndt/gfw103.
Kim SY, Kang HT, Choi HR, Park SC. Reduction of Nup107 attenuates the growth factor signaling in the senescent cells. Biochem Biophys Res Commun 2010;401:131-6. doi: 10.1016/j.bbrc.2010.09.025.
Glavy JS, Krutchinsky AN, Cristea IM, Berke IC, Boehmer T, Blobel G, et al.
Cell-cycle-dependent phosphorylation of the nuclear pore Nup107-160 subcomplex. Proc Natl Acad Sci U S A 2007;104:3811-6. doi: 10.1073/pnas.0700058104.
Jagot-Lacoussiere L, Faye A, Bruzzoni-Giovanelli H, Villoutreix BO, Rain JC, Poyet JL. DNA damage-induced nuclear translocation of Apaf-1 is mediated by nucleoporin Nup107. Cell Cycle 2015;14:1242-51. doi: 10.1080/15384101.2015.1014148.
Gao F, Zhang J, Wang X, Yang J, Chen D, Huff V, et al.
Wt1 functions in ovarian follicle development by regulating granulosa cell differentiation. Hum Mol Genet 2014;23:333-41. doi: 10.1093/hmg/ddt423.
Ai A, Xiong Y, Wu B, Lin J, Huang Y, Cao Y, et al.
Induction of miR-15a expression by tripterygium glycosides caused premature ovarian failure by suppressing the Hippo-YAP/TAZ signaling effector Lats1. Gene 2018;678:155-63. doi: 10.1016/j.gene.2018.08.018.
Mereness AL, Murphy ZC, Forrestel AC, Butler S, Ko C, Richards JS, et al.
Conditional deletion of Bmal1 in ovarian theca cells disrupts ovulation in female mice. Endocrinology 2016;157:913-27. doi: 10.1210/en.2015-1645.
Li Q, McKenzie LJ, Matzuk MM. Revisiting oocyte-somatic cell interactions: In search of novel intrafollicular predictors and regulators of oocyte developmental competence. Mol Hum Reprod 2008;14:673-8. doi: 10.1093/molehr/gan064.
Bramble MS, Goldstein EH, Lipson A, Ngun T, Eskin A, Gosschalk JE, et al.
A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: A fertility application of whole exome sequencing. Hum Reprod 2016;31:905-14. doi: 10.1093/humrep/dew025.
Qin Y, Sun M, You L, Wei D, Sun J, Liang X, et al.
ESR1, HK3 and BRSK1 gene variants are associated with both age at natural menopause and premature ovarian failure. Orphanet J Rare Dis 2012;7:5. doi: 10.1186/1750-1172-7-5.
França MM, Funari MF, Nishi MY, Narcizo AM, Domenice S, Costa EM, et al.
Identification of the first homozygous 1-bp deletion in GDF9 gene leading to primary ovarian insufficiency by using targeted massively parallel sequencing. Clin Genet 2018;93:408-11. doi: 10.1111/cge.13156.
Kuo FT, Bentsi-Barnes IK, Barlow GM, Pisarska MD. Mutant Forkhead L2 (FOXL2) proteins associated with premature ovarian failure (POF) dimerize with wild-type FOXL2, leading to altered regulation of genes associated with granulosa cell differentiation. Endocrinology 2011;152:3917-29. doi: 10.1210/en.2010-0989.
Chen XY, Xia HX, Guan HY, Li B, Zhang W. Follicle loss and apoptosis in cyclophosphamide-treated mice: What's the matter? Int J Mol Sci 2016;17: pii: E836. doi: 10.3390/ijms17060836.
Sachani SS, Landschoot LS, Zhang L, White CR, MacDonald WA, Golding MC, et al.
Nucleoporin 107, 62 and 153 mediate Kcnq1ot1 imprinted domain regulation in extraembryonic endoderm stem cells. Nat Commun 2018;9:2795. doi: 10.1038/s41467-018-05208-2.
Schmierer B, Hill CS. TGFbeta-SMAD signal transduction: Molecular specificity and functional flexibility. Nat Rev Mol Cell Biol 2007;8:970-82. doi: 10.1038/nrm2297.
Hardy K, Mora JM, Dunlop C, Carzaniga R, Franks S, Fenwick MA. Nuclear exclusion of SMAD2/3 in granulosa cells is associated with primordial follicle activation in the mouse ovary. J Cell Sci 2018;131. pii: jcs218123. doi: 10.1242/jcs.218123.
Peng J, Li Q, Wigglesworth K, Rangarajan A, Kattamuri C, Peterson RT, et al.
Growth differentiation factor 9: Bone morphogenetic protein 15 heterodimers are potent regulators of ovarian functions. Proc Natl Acad Sci U S A 2013;110:E776-85. doi: 10.1073/pnas.1218020110.
Boyer A, Lapointe E, Zheng X, Cowan RG, Li H, Quirk SM, et al.
WNT4 is required for normal ovarian follicle development and female fertility. FASEB J 2010;24:3010-25. doi: 10.1096/fj.09-145789.
Raices M, D'Angelo MA. Nuclear pore complexes and regulation of gene expression. Curr Opin Cell Biol 2017;46:26-32. doi: 10.1016/j.ceb.2016.12.006.
Kevenaar ME, Themmen AP, Laven JS, Sonntag B, Fong SL, Uitterlinden AG, et al.
Anti-müllerian hormone and anti-müllerian hormone type II receptor polymorphisms are associated with follicular phase estradiol levels in normo-ovulatory women. Hum Reprod 2007;22:1547-54. doi: 10.1093/humrep/dem036.
Capitanio JS, Montpetit B, Wozniak RW. Nucleoplasmic Nup98 controls gene expression by regulating a DExH/D-box protein. Nucleus 2018;9:1-8. doi: 10.1080/19491034.2017.1364826.
Nakano H, Wang W, Hashizume C, Funasaka T, Sato H, Wong RW. Unexpected role of nucleoporins in coordination of cell cycle progression. Cell Cycle 2011;10:425-33. doi: 10.4161/cc.10.3.14721.
Mehsen H, Boudreau V, Garrido D, Bourouh M, Larouche M, Maddox PS, et al.
PP2A-B55 promotes nuclear envelope reformation after mitosis in Drosophila
. J Cell Biol 2018;217:4106-23. doi: 10.1083/jcb.201804018.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]