Dynein axonemal intermediate chain 2 plays a role in gametogenesis by activation of Stat3

Abstract We previously identified the mouse dynein axonemal intermediate chain 2 (Dnaic2) gene. This gene expresses a component of the axonemal dynein complex that functions in cilia or flagella. We found that overexpression of Dnaic2 results in female subfertility and male infertility. In this study, we generated Dnaic2 knockdown (KD) mice and identified the potential regulatory mechanisms involved in Dnaic2 function. For phenotype analysis, we found that body weight was lighter and size was smaller in Dnaic2 KD mice than in wild‐type mice. A total of 45% of these Dnaic2 KD mice were infertile due to sperm abnormalities in males, or had oocyte abnormalities and pathological changes in the tunica mucosa in the oviduct of females. Moreover, Dnaic2 overexpression enhanced the expression of proliferating cell nuclear antigen (PCNA) in the ovaries, which suggested that Dnaic2 stimulated proliferation of cells in the ovaries. However, PCNA expression in the testis of Dnaic2‐overexpressed mice was lower than that in controls. Additionally, the ratio of Bax/B‐cell lymphoma‐2(Bcl‐2) in the testis of Dnaic2‐overexpressed mice was higher than that in controls, which suggested that Dnaic2 inhibited cellular proliferation in the testis. To examine the molecular action of Dnaic2, immunoprecipitation analysis was used and showed that Dnaic2 protein interacted with signal transducer and activator of transcription 3 (Stat3). Molecular modelling analysis showed that Dnaic2 bound with the linker and SH2 domains of Stat3. Furthermore, overexpression of Dnaic2 promoted phosphorylation of Stat3. In conclusion, our study suggests that Dnaic2 plays a role in oogenesis and spermatogenesis by activation of Stat3.

spermatids through maturation of proliferation and differentiation.
Approximately 75% of these developed germ cells are generally lost by apoptosis or degeneration. 3 Moreover, regulation of proliferation and cell death of type A spermatogonia determines the number of differentiated spermatogonia that can enter spermatogenesis. 4 Therefore, cellular proliferation and apoptosis in gametogenesis are essential issues for mammalian reproductive development. Most signals or proteins that are involved in cellular proliferation and apoptosis, such as Bax, B-cell lymphoma-2 (Bcl-2), and stem cell factor (SCF)/c-kit, play important roles in gametogenesis. [5][6][7][8][9][10] Some defects in these proteins are serious enough to cause sterility.
Dyneins are one of three major cytoskeletal motors 11 and have a wide variety of cellular functions. 12 This minus end-directed motor is the only known candidate motor for moving cargoes anteriorly in the oocyte, and its function is essential for determination of oocytes. [13][14][15] Axonemal dyneins comprise the inner and outer arms of the eukaryotic axoneme, and are essential for cilia or flagella wave forms. 16,17 This function of dyneins contributes to oocyte transport of motile oviduct cilia and sperm movement, which are important for fertilisation. Defects in the function of inner and/or outer dynein arms may result in primary ciliary dyskinesia (PCD). 18 This disease phenotype results in lung damage, male infertility, and female subfertility. 19,20 Dynein axonemal intermediate chain 2 (Dnaic2) is a homologue of human DNAI2, which is associated with PCD. 21 Dnaic2 is highly expressed in oocytes and sperms. Dnaic2-overexpressed mice show lung damage, male sterility, and female subfertility or infertility. In the Dnaic2-overexpressed mouse, there are structural abnormalities of the outer and inner dynein arms. 22 Additionally, defects are found in oogenesis and spermatogenesis, indicating a role of Dnaic2 in gametogenesis. 22,23 However, no information is available regarding the potential regulation mechanism of Dnaic2.
This study aimed to determine the effect of Dnaic2 in mice with knockdown of Dnaic2 and to identify the potential regulatory mechanisms involved in Dnaic2 function. Our study shows the function of Dnaic2 in the lungs, testis, ovary, and oviduct. We also show an association between Dnaic2 and signal transducer and activator of transcription 3 (Stat3). These findings indicate that Dnaic2 plays a role in the lungs, testes, and ovaries by regulating Stat3.

| Mice
CD-1, C57BL/6, and C57BL/6 × CD-1 F1 hybrid mice were used in this study. The recipients were 6-week-old C57BL/6 × CD-1 F1 hybrid or CD-1 female mice that were sterilised by intraperitoneal injection of busulfan (30 mg/kg; resuspended in Dimethyl sulfoxide, DMSO) and cyclophosphamide (120 mg/kg). Controls were obtained by intraperitoneal injection of DMSO. All procedures were approved by the Institutional Animal Care and Use Committee of Shanghai, and were performed in accordance with the National Research Council Guide for Care and Use of Laboratory Animals.  Table S1. As shown in Fig-ure S1, amongst four shRNAs, shRNA2 and shRNA3 were more efficient than the others. ShRNA3 expression vector was selected for further study.

| Generation of Dnaic2-knockdown mice
Dnaic2 KD mice were generated as described by Zhang et al. 22 Briefly, we first isolated and then cultured female germline stem cells (FGSCs) from ovaries of CD-1 mice in the short-term. FGSCs were then cultured for 3-5 days and were transfected with the pRS-U6-shDnaic2-SV40-Puro vector using TurboFectin 8.0 transfection reagent according to the manufacturer's instructions (OriGene). After 48 h, the FGSCs were transplanted into ovaries of recipients sterilised by chemotherapy. The recipient mice were mated to wild-type mice. The genotypes were then identified by PCR and Southern blotting. PCR was performed using 1 μg genomic DNA from mouse tails with primer set (sense: 5′-TCGACCCTGTGGAATGTGT-3′; anti-sense: 5′-GGGCTTGTACTCGGTCAT-3′) specific for SV40 early promoter.
Southern blotting was used to confirm the results of Dnaic2 KD mice, and was performed according to the method mentioned above. 22 Finally, western blotting was carried out using rabbit anti-DNAI2 or anti-β-tubulin (see below).

| Histological analysis
Testes, ovaries, and lungs from Dnaic2 KD and wild-type mice were fixed with 4% paraformaldehyde, dehydrated, and embedded into paraffin-wax. The tissues were cut into 6 μm thick sections with a microtome and stained with haematoxylin and eosin for microscopic observation.

| Molecular modelling analysis
The crystal structure of Stat3 was reported in 2008 (pdb: 3CWG). 24 Dnaic2 was constructed using the SWISS-model with the default setting. [25][26][27] ZDOCK was used to dock Stat3 and Dnaic2. 28 2.10 | RT-PCR    Figure 1C). Finally, western blotting was used to show down-regulation of Dnaic2 protein in the testes, ovaries, and lungs of adult Dnaic2 KDs ( Figure 1D). Bodyweight was lighter and size was smaller in Dnaic2 KD mice compared to age-matched wildtype mice ( Figure 1E). We also found that 45% of Dnaic2 KD mice were infertile.

| Phenotype of Dnaic2-knockdown mice
On the basis of our previous finding that overexpression of Dnaic2 in mice led to defects of reproduction and lung disease, 22 we focused our analyses of 2 months old Dnaic2 KDs on reproductive capacity and histological characteristics of the lungs. Histological analysis showed that fewer elongated spermatids were found in some seminiferous tubules in testes from Dnaic2 KD mice compared to wild-type mice (Figure 2A). Additionally, 28.5% of sperm from the epididymis showed an abnormal morphology, including sperm without a head, sperm bent over in the middle part, formation of loops, and the head touching the middle ( Figure 2B). Irregular-shaped follicles were found in the ovaries of Dnaic2 KD mice compared to wild-type mice. Moreover, the number of follicles in the ovaries of Dnaic2 KD mice was less than that in the control groups and no mature follicles were found ( Figure 2C). In the fallopian tube lumen of Dnaic2 KD mice, the mucosal folds were disrupted, ciliated cells had become thinner, some cells had fallen off, and there was a lot of cell adhesions compared to wild-type mice ( Figure 2D). Additionally, histological analysis showed that the pulmonary alveolar structure of the lungs of Dnaic2 KD mice was damaged, and the alveoli had more serous exudate than in wildtype mice ( Figure 2E).

| Dnaic2 promotes cellular proliferation
To determine the effect of Dnaic2 on the growth of cells in vitro, the proliferation rate of NIH 3T3 cells was detected by BrdU.
Two days after transfection with pcDNA3.1-Dnaic2, the proliferation rate of NIH 3T3 cells with overexpression of Dnaic2 reached approximately 35%, whereas the corresponding control only reached approximately 26% ( Figure 3A). Additionally, the growth curve of NIH 3T3 cells with overexpression of Dnaic2 or control cells was analysed. NIH 3T3 cells that were transfected with pcDNA3.1-Dnaic2 grew faster than control cells after transfection ( Figure S2), which suggested that Dnaic2 promoted proliferation of NIH 3T3 cells.
To determine if Dnaic2 activates cellular proliferation in vivo, we analysed expression of PCNA, which is one of the potential proliferation markers in mouse testes, ovaries, and lungs. PCNA protein expression levels in the lungs and ovaries from Dnaic2-overexpressed mice were significantly higher than that in wild-type mice ( Figure 4A, B). This finding suggests that Dnaic2 actives cellular proliferation in mouse lungs and ovaries. Interestingly, a completely opposite result was found in the testes ( Figure 4A, B), and this was supported by Bax/Bcl-2 results (Figure 4C, D). These findings suggest that Dnaic2 inhibited cellular proliferation in mouse testis.

| Dnaic2 interacts with Stat3
To investigate proteins that are potentially associated with Dnaic2 function, immunoprecipitation analysis was performed. According to the sub-cellular location and function of Dnaic2, 10 proteins were selected to be tested, including the cell surface receptors TGFβ-RI, EGF-R, and c-kit. These receptors are important proteins in regulation of spermatogenesis and follicular development. We examined proteins in the JAK/Stat signaling pathway, which is responsible for signals from the cytoplasm to the nucleus, including JAK-2, Stat3, and SH 2 B. We also investigated members of the PI3K-Akt signalling pathway, which participates in transduction of multiple signals, including PI3K, Akt, and FAK. Amongst these proteins, Stat3 was pulled down together with Dnaic2 ( Figure 5A), which indicated that Stat3 was a binding partner of Dnaic2. Immunofluorescence double staining analysis showed a consistent expression pattern of Dnaic2 and Stat3 ( Figure 5B). Moreover, the docking complex between Stat3 and Dnaic2 is shown in Figure 5C. Dnaic2 was bound with the linker and SH2 domains of Stat3 ( Figure 5C).

| Dnaic2 activates Stat3
To determine the effect of Dnaic2 on Stat3, we first detected Stat3 expression in the testes, ovaries, lungs, and cells with overexpression of Dnaic2. In 293T cells, Stat3 expression did not change with a change in Dnaic2 expression ( Figure 6). However, phosphorylated

| DISCUSSION
In our previous study, we identified Dnaic2, and found that it was expressed in mouse ovaries and testes, and played a role in F I G U R E 7 Activation of Stat3 by Dnaic2 in vivo. A, Western blot analysis expression of Stat3 or phosphorylation of Stat3 in the testes and ovaries of adult wild-type or Dnaic2-overexpressed mice. Phosphorylation of Stat3 was exacerbated in Dnaic2-overexpressed mice. (B, C) Corresponding bar graphs of panel A. Values represent the relative levels of protein to β-tubulin (mean ± SEM, n = 3). *P < 0.05; **P < 0.01. C, wild-type mouse; OP, Dnaic2-overexpressed mouse oogenesis and spermatogenesis. 21,22 Dnaic2-overexpressed mice showed infertility in adults, partly due to some defects of gametogenesis and cilia or flagella in the oviduct or testis. However, not only defects of cilia or flagella occurred, but also disorders of gametogenesis and pathological changes in the oviduct were found in Dnaic2-overexpressed mice. 22 In the current study, we also observed similar symptoms in Dnaic2 KD mice, including defects in the testes, ovaries, and oviduct, as well as lung damage. These The potential regulatory mechanism of Dnaic2 in gametogenesis or cellular fate in the lungs is unknown. In this study, we detected proteins that are associated with the function of Dnaic2. First, we found that Dnaic2 promoted cellular proliferation in mouse lungs and ovaries, but had an opposite effect on cells in mouse testes, even if it increased cellular growth of NIH 3T3 cells in vitro. These results indicate that the regulatory mechanism of Dnaic2 in the mouse is complex. Further, our study showed that Dnaic2 was bound with Stat3. In particular, Dnaic2 could induce phosphorylation of Stat3.
This finding suggests that Dnaic2 plays a role in the testes, ovaries, and lungs by regulating Stat3.
Stat3 is a member of the Stat family proteins. Stat3 is activated in diverse signalling systems. In some cases, Stat3 can induce a set of important target genes, whereas in others it may function as a repressor or as a signalling adaptor, but without transcriptional function. [31][32][33][34] Therefore, the situation of Stat3 can promote proliferation, survival, or apoptosis in certain tissues. [35][36][37][38][39][40] Because of the multiple functions of Stat3, Dnaic2 had different effects on the lungs, testes, and ovary in our study. Many previous studies have reported that Stat3 plays essential and multiple roles in the lungs, including acute lung injury and various cancers. 36 Defects of Stat3 affect cytoprotection of the respiratory epithelium in the mouse during adenoviral infection. 41 Accumulating evidence has also demonstrated constitutive activation of Stat3 in multiple cancers, and downstream genes of Stat3 are biomarkers in human lung carcinomas. 36 In our previous study, cellular proliferation in the lungs from Dnaic2-overexpressed mice was increased. 22 In the mouse ovaries, Dnaic2 promoted cellular proliferation in our study, which is consistent with the finding that activity of Stat3 is required for mitosis of ovary cells. 42 Moreover, our previous study showed that the number of mature follicles was decreased in Dnaic2-overexpressed mice and Dnaic2-overexpressed mice showed female subfertility or even infertility. 22 A previous study reported that Stat3 participated in regulation of leptin in oocyte maturation in mammals. 43 Therefore, Dnaic2 might regulate maturation of oocytes through Stat3, which affects normal follicular development. Dnaic2 inhibited cellular proliferation in the mouse testes by regulating activity of Stat3. In the adult Dnaic2-overexpressed mice, overexpression of Dnaic2 inhibited cellular proliferation, affected spermatogenesis. 22 For further study, we will confirm that the opposing effects described between oocytes/lung and testis are STAT3 dependent or not by using condition knockout stat3 mice.
Taken together, our findings and previous studies suggest that Dnaic2 plays an essential role in the lungs, ovaries, and testes through affecting cellular proliferation by regulation of Stat3.