FOXP1 promotes the proliferation and self-renewal of spermatogonial stem cells via the nuclear factor kappa B pathway

Abstract

BACKGROUND

The proliferation and self-renewal of spermatogonial stem cells (SSCs) are essential for maintaining the sperm production process. The molecular mechanisms of self-renewal and differentiation of SSCs have not been fully clarified, although the functions of some regulatory factors in SSCs have been previously reported.

AIM

To investigate the role of forkhead box P1 (FOXP1) in regulating the proliferation and self-renewal of SSCs and its potential mechanism via the nuclear factor kappa B (NF-κB) pathway.

METHODS

Testicular tissue was collected from 31 patients, including 18 obstructive azoospermia (OA) and 13 nonobstructive azoospermia cases. Hematoxylin and eosin staining was performed to analyze the morphological difference in human testicular tissue between the OA and nonobstructive azoospermia groups. Immunohistochemistry staining was used to evaluate the expression of FOXP1 in human testicle tissues. FOXP1 knockdown lentivirus was injected into mice testes, and immunohistochemistry staining and immunofluorescence assays of ETS variant gene 5 and promyelocytic leukemia zinc finger were performed to investigate the effect of FOXP1 on spermatogonia in mice. A Cell Counting Kit-8 assay was used to assess the proliferation of SSCs. SC75741 was used to inhibit the NF-κB pathway, and proliferating cell nuclear antigen, ETS variant gene 5, promyelocytic leukemia zinc finger, and phosphorylated-NF-κB p65 were detected via western blot analysis.

RESULTS

FOXP1 was highly expressed in the testicular tissue of patients with OA. FOXP1 knockdown inhibited the proliferation and self-renewal of SSCs in mice and in vitro. Consistently, FOXP1 overexpression promoted the SSCs’ proliferation and self-renewal, which was regulated through the NF-κB pathway.

CONCLUSION

FOXP1 may promote the proliferation and self-renewal of SSCs via the NF-κB pathway. This research offers a theoretical foundation for male infertility treatment.

Key Words: Forkhead box P1; Spermatogonial stem cells; Proliferation; Self-renewal; Nuclear factor kappa B pathway; Male infertility; Azoospermia; ETS variant gene 5; Promyelocytic leukemia zinc finger

Core Tip: Spermatogonial stem cells (SSCs) are the basis of spermatogenesis in mammals and can self-renew or differentiate into the various cell types in the germ cell lineage. SSC homeostasis dysregulation is closely associated with male infertility. This study revealed the role of forkhead box P1 in regulating the function of SSCs via the nuclear factor kappa B pathway, thus offering a basis for the diagnosis and treatment of male infertility.

INTRODUCTION

Infertility has become a global problem that may seriously hinder human development. According to statistics, approximately 15%-20% of the world’s couples are infertile, of which male fertility dysfunction accounts for about half of all patients with infertility[1,2]. Nonobstructive azoospermia (NOA) is one of the most serious forms of male infertility and is caused by spermatogenesis failure[3,4]. Regrettably, due to the complexity of its pathogenesis, there remains a lack of effective treatment strategies[5]. In recent years, stem cell regenerative medicine has undergone rapid advancements, enabling the use of germinative stem cells to rehabilitate testicular dysfunction arising from impaired germ cells. Spermatogonial stem cells (SSCs) are the basis of spermatogenesis in mammals. SSCs have the ability to self-renew or differentiate into the various cell types in the germ cell lineage[6]. Their proper function is essential for maintaining male fertility and ensuring species continuity. SSC homeostasis dysregulation is closely associated with male infertility, particularly severe forms thereof, including NOA[7]. Therefore, understanding the regulatory mechanisms of human SSCs proliferation and self-renewal is crucial for attaining sustained in vitro cultivation and harnessing SSCs’ potential for the therapeutic management of male infertility.

The fate of SSCs is tightly regulated by transcription factors. These key regulators include promyelocytic leukemia zinc finger (PLZF), which is essential for SSC self-renewal[8,9]; glial cell-derived neurotrophic factor (GDNF) and its receptor GFRα1, which promote SSC maintenance[10]; and ETS variant gene 5 (ETV5), a downstream fibroblast growth factor signaling effector that supports SSC viability[11]. Forkhead box P1 (FOXP1), a member of the FOXP subfamily, plays various roles in cellular functions. FOXP1 is abnormally expressed in several cells, tissues, and organs. The FOXP1 protein contains DNA- and protein-binding domains and may function as a tumor promoter or inhibitor. FOXP1 was inhibited in breast[12] and gastrointestinal cancers[13] and promoted the progression of diffuse large B-cell lymphoma[14]. In addition, FOXP1 knockdown reduced the expression of stem cell-related genes and decreased the formation of spheroids and colonies, which indicates that FOXP1 plays a key role in regulating stem cells in chemotherapy-resistant tumors[15]. However, the effect of FOXP1 on the proliferation of SSCs has not been satisfactorily reported.

The nuclear factor kappa B (NF-κB) signaling pathway is known to participate in numerous cellular processes, including cell survival, proliferation, and inflammatory responses[16]. In the field of spermatogenesis, transcription factor Dmrt1 initiates the SPRY1-NF-κB pathway to maintain testicular immune homeostasis and male fertility[17]. Moreover, the Wuzi-Yanzong prescription alleviates heat stress-induced spermatogenesis disorders by acting on the protein kinase B/NF-κB signaling pathway[18]. In addition, the Bazibushen formulation can improve morphological changes and spermatogenic function in the testes of aged mice by regulating the Sirt6/NF-κB signaling pathway[19]. In germ cells, NF-κB activation has been linked to the GDNF-mediated migration of spermatogonial cells, thereby suggesting a potential role in SSC regulation[20]. However, it is currently unclear whether FOXP1 regulates the function of SSCs through the NF-κB pathway.

This study aimed to investigate the role of FOXP1 in regulating SSC proliferation and self-renewal, with a particular focus on its potential interaction with the NF-κB pathway. FOXP1 was upregulated in obstructive azoospermia (OA) testes samples with normal spermatogenesis compared with that of NOA samples. FOXP1 knockdown inhibited the proliferation and self-renewal of SSCs in mice. Furthermore, we found that FOXP1 promoted the proliferation and self-renewal of SSCs via the NF-κB pathway. This study revealed the role of FOXP1 in regulating the function of SSCs, thereby offering a basis for the diagnosis and treatment of male infertility.

MATERIALS AND METHODS

Human testicular tissue collection

Testicular samples were taken from biopsies of patients with NOA and OA. Testicular tissue from 31 patients was collected in this study, including 18 OA and 13 NOA, with weights ranging from 10 mg to 20 mg. The tissue samples were washed three times with phosphate-buffered saline, fixed with 4% paraformaldehyde, or frozen at -80 °C. All human tissue collection procedures were reviewed and approved by the Ethics Committee of Ganzhou People’s Hospital of Jiangxi Province (approval No. PJB2025-279-01). Written informed consent was obtained from all patients prior to sample collection.

Hematoxylin and eosin staining

Testicular tissue specimens from humans or mice were preserved using 4% paraformaldehyde for 2 hours, followed by embedding in Tissue-Tek OCT compound and subsequent cryosectioning. For paraffin-embedded sections, fixation was achieved with a 10% neutral buffered formalin solution. The tissue slices underwent hematoxylin and eosin staining. These prepared sections were then examined using an inverted microscope (DP70, Olympus, Tokyo, Japan).

Immunohistochemistry staining and immunofluorescence assays

For immunohistochemical experiments, the testicular sections of humans or mice were dewaxed with xylene, and ethanol gradients were used for fluid replenishment. Heat-induced antigen recovery was performed in a beaker with 0.01 M sodium citrate buffer at 98 °C for 18 minutes, and endogenous peroxidase activity was inhibited using 3% H₂O₂ (Zsbio, Beijing, China). The cell sections were then permeated with 0.25% Triton X-100 (Sigma, MO, United States) for 15 minutes and blocked with 5% bovine serum albumin at room temperature for 1 hour. The sections were incubated with the primary antibodies FOXP1 (1:800, #22051-1-AP, Proteintech, IL, United States), ETV5 (1:3000, #13011-1-AP, Proteintech, IL, United States), and PLZF (1:4000, #bs-5971R, Bioss Inc., MA, United States) overnight at 4 °C and then rinsed with phosphate-buffered saline. The sections were then incubated with the enzyme-labeled secondary antibody (#18B13B04, BOSTER Biotechnology, Wuhan, Hubei Province, China) at room temperature for 1 hour. The 3,3’-diaminobenzidine color development kit (#17J20B27, BOSTER Biotechnology, Wuhan, Hubei Province, China) was used for color development. Finally, hematoxylin staining was performed on the slices. For immunofluorescence, the testicular sections were combined with the secondary antibody and incubated with Alexa Fluor at 25 °C for 1 hour. The residual tissue portion served as a control to assess the primary and the secondary antibodies’ non-specific binding. The cell nucleus was stained with 4’6-diaminophenylindole and imaged with a microscope (Carl Zeiss, Oberkochen, Germany).

Cell transfection

The sh-FOXP1/negative control and FOXP1 overexpression (OE)/negative control plasmids were synthesized by Ribobio (Guangzhou, Guangdong Province, China). The SSCs were transfected with 100 nmol/L of the related plasmids using the Lipofectamine 2000 reagent (Invitrogen, CA, United States) according to the manufacturer’s instructions. The transfection efficiency was detected by quantitative polymerase chain reaction 48 hours after transfection.

Administration of lentivirus to animals

The sh-FOXP1 and control plasmids were cotransfected with the lentiviral vector into 293T cells, and the lentiviral particles were collected. The lentiviral particles were then microinjected into the lumen of the seminiferous tubules of 24-day-old C57BL/6 mice. After 48 hours, the expression level of FOXP1 protein in the testicular tissue of the mice was detected by immunofluorescence.

Mouse SSCs isolation and culture

The isolation and culture of mouse SSCs were performed as per a previously reported study[21]. Thy-1 served as a surface marker, as it is expressed on SSCs in the testes of mice at various life stages, including neonatal, pup, and adult phases[21]. The 8-day-old male C57BL/6 pups (Fujian Anburi Biological Company, Fuzhou, Fujian Province, China) were sacrificed, and the testicles were digested with trypsin-EDTA. The cells were magnetically sorted using Thy-1 microbeads (#130 049-101, Miltenyi Biotec, Germany) and inoculated into Sandos inbred mouse-derived 6-thioguanine resistance and wabane resistance incubators. Then, Thy-1+ germ cells were purified in the SSCs culture system. Germ cell cultures were maintained in a humidified environment with 5% CO₂ at 37 °C, using mouse serum-free medium containing 20 ng/mL of recombinant human GDNF, 150 ng/mL of recombinant rat GFRα1, and 1 ng/mL of recombinant human fibroblast growth factor 2.

Western blot analysis

A total of 20 μg of protein from the testicular tissue samples and mouse SSCs was subjected to sodium-dodecyl sulfate gel electrophoresis for protein separation. The separated proteins were then transferred onto polyvinylidene fluoride membranes and incubated overnight in Tris-buffered saline with Tween 20 (TBS-T) buffer and 5% non-fat dry milk at 4 °C. The membranes were subsequently incubated with the primary antibodies GAPDH (1:50000, #60004-1-lg, Proteintech, IL, United States), PCNA (1:18000, #ab29, Abcam, United Kingdom), PLZF (1:10000, #ab305064, Abcam, United Kingdom), ETV5 (1:10000, #66657-1-Ig, Proteintech, IL, United States), NF-κB p65 (1:15000, #10745-1-AP, Proteintech, IL, United States), and phosphorylated NF-κB p65 (p-NF-κB p65, 1:15000, #AF2006, Affinity, OH, United States) in TBS-T buffer with 5% nonfat dry milk for 3 hours at 25 °C. After washing with TBS-T buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, 1:2000, #SA00001-2, Proteintech, IL, United States) for 1 hour at 25 °C. The protein bands were visualized using an electrochemiluminescence detection system. All assays were repeated three times.

Cell Counting Kit-8 assay

The viability of SSCs was evaluated using the Cell Counting Kit-8 (CCK-8) assay kit (#A311-01/02, Vazyme, China), which was performed according to the guidelines provided by the manufacturer. Briefly, 10 μL of CCK-8 reagent was introduced into each well of a 96-well plate (3 × 10³ cells/well), and the plate was incubated for 1 hour at 37 °C. The absorbance was measured at 450 nm using a Multifunctional Microplate Reader (BioTek, VT, United States).

Statistical analysis

Data analysis was performed using GraphPad Prism v9.1 (GraphPad Software, La Jolla, CA, United States). For pairwise group comparisons, the Student’s t-test was employed, while comparisons involving three or more groups were analyzed using a one-way analysis of variance. Statistical significance was defined as P < 0.05.

RESULTS

FOXP1 was upregulated in human SSCs

The testicle tissues of patients with OA showed basically normal spermatogenic tubule structure and active spermatogenic process (with sperm in the lumen), while the testicles of patients with NOA revealed destruction of the spermatogenic tubule structure, absence of spermatogenic epithelium, and significant interstitial fibrosis (Figure 1A). In addition, immunohistochemical analysis showed that FOXP1 expression in the testicular tissues of patients with OA was higher than that in patients with NOA. The localization analysis of FOXP1 in the testicular interstitial tissue samples revealed that FOXP1 was predominantly found within the cell nuclei of the spermatogenic tubules, indicating its expression in spermatogonial cells (Figure 1B).

Figure 1 Forkhead box P1 was upregulated in obstructive azoospermia samples with normal spermatogenesis compared with non-obstructive azoospermia samples. A: Representative hematoxylin and eosin staining images of obstructive azoospermia and non-obstructive azoospermia testicular tissues; B: Immunohistochemical staining for the expression of forkhead box P1 in obstructive azoospermia and non-obstructive azoospermia testicular tissue samples. Scale bars = 100 μm, magnification 200 ×. OA: Obstructive azoospermia; NOA: Non-obstructive azoospermia.

FOXP1 knockdown inhibited the self-renewal of SSCs in mice

The sh-FOXP1 and scramble control vectors were microinjected into the lumen of the seminiferous tubules of mice. Hematoxylin and eosin staining indicated that spermatogenic cells at all developmental levels could be seen in the testicular seminiferous tubules of mice in the normal control group. There were a large number of mature sperm in the lumen, and the spermatogenic cells were neatly and regularly arranged. However, in the testicles of mice with FOXP1 knockdown, a large number of seminiferous tubules were atrophied and necrotized, the interstitial tissue was loose with a small amount of fibrous production, and some sperm cells had necrotized and undergone apoptosis. The epithelium of the seminiferous tubules became thinner, and the number of mature sperm in the lumen was significantly reduced (Figure 2A). Six weeks after infection with the sh-FOXP1 lentivirus, we found that the weight of both testicles in the knockdown group of mice was significantly lower than that in the normal control group (Figure 2B). SSCs, which were the most differentiated spermatogonia, exhibited stem cell markers such as PLZF, ETV5, and GDNF[22,23]. Immunohistochemistry staining and immunofluorescence assays indicated that the expression levels of PLZF and ETV5 in the sh-FOXP1 group were significantly lower than those in the normal control group (Figure 3). These results may indicate that FOXP1 knockdown inhibited the self-renewal of SSCs in mice.

Figure 2 Effect of forkhead box P1 inhibition on mice testicular morphology and weight. A: Hematoxylin and eosin staining of testicular tissues; B: Two testes were surgically excised and weighed 6 weeks after the mice were infected with sh-FOXP1 lentivirus. Scale bars = 100 μm, magnification 200 ×. n = 6, ^cP < 0.001. NC: Negative control; FOXP1: Forkhead box P1.

Figure 3 Forkhead box P1 knockdown inhibited the self-renewal of spermatogonial stem cells in mice. A and B: Immunohistochemical staining showing the protein level changes of ETS variant gene 5 and promyelocytic leukemia zinc finger in mice testes after transfection with sh-FOXP1 lentivirus; C and D: Immunofluorescence staining of ETS variant gene 5 and promyelocytic leukemia zinc finger in mice testes after transfection with sh-FOXP1 lentivirus. Scale bars = 100 μm. FOXP1: Forkhead box P1; NC: Negative control; ETV5: ETS variant gene 5; PLZF: Promyelocytic leukemia zinc finger; DAPI: 4’6-diaminophenylindole.

FOXP1 promoted the proliferation and self-renewal of SSCs via the NF-κB pathway

To investigate the function of FOXP1 in the regulation of SSCs, the SSCs were transfected with sh-FOXP1, scramble, and overexpression plasmids. The CCK-8 assay revealed that FOXP1 knockdown inhibited the proliferation of SSCs (Figure 4A), and FOXP1 overexpression promoted the proliferation of SSCs (Figure 4B). In addition, the proliferation and self-renewal proteins PCNA, PLZF, and ETV5 were downregulated in the sh-FOXP1 group and upregulated in the FOXP1 overexpression group compared with the control (Figure 4C and D). Furthermore, we found that FOXP1 inhibition reduced the expression of p-NF-κB p65, and FOXP1 overexpression increased the expression of p-NF-κB p65 (Figure 4E and F). Furthermore, we found that the p65 inhibitor SC75741 significantly reduced the increase in proliferation and self-renewal of SSCs caused by FOXP1 overexpression (Figure 4B and D). Thus, these results indicated that FOXP1 could promote the proliferation and self-renewal of SSCs via the NF-κB pathway.

Figure 4 Forkhead box P1 promoted the proliferation and self-renewal of spermatogonial stem cells via the nuclear factor kappa B pathway. A: Cell Counting Kit-8 assay showing the proliferation of spermatogonial stem cells (SSCs) in the control and sh-FOXP1 groups; B: Cell Counting Kit-8 assay showing the proliferation of SSCs in the control, nuclear factor kappa B (NF-κB) inhibitor (SC75741), FOXP1 overexpression (FOXP1-OE), and FOXP1-OE + NF-κB inhibitor groups; C: Western blot analysis showing the expression of proliferation and self-renewal proteins proliferating cell nuclear antigen, ETS variant gene 5, and promyelocytic leukemia zinc finger in the control and sh-FOXP1 groups; D: Western blot analysis showing the expression of proliferation and self-renewal proteins proliferating cell nuclear antigen, ETS variant gene 5, and promyelocytic leukemia zinc finger in the control, NF-κB inhibitor, FOXP1-OE, and FOXP1-OE + NF-κB inhibitor groups; E: Western blot analysis showing the expression of NF-κB p65 and phosphorylated-NF-κB p65 in the control and sh-FOXP1 groups; F: Western blot analysis showing the expression of NF-κB p65 and phosphorylated-NF-κB p65 in the control, NF-κB inhibitor, FOXP1-OE, and FOXP1-OE + NF-κB inhibitor groups. n ≥ 3, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. NS: Not significant; FOXP1: Forkhead box P1; NC: Negative control; ETV5: ETS variant gene 5; PLZF: Promyelocytic leukemia zinc finger; NF-κB: Nuclear factor kappa B; p-NF-κB p65: Phosphorylated nuclear factor kappa B p65; PCNA: Proliferating cell nuclear antigen.

DISCUSSION

SSCs are the source of sperm and continuously produce sperm throughout their life cycle. The abnormal self-renewal or differentiation of SSCs may lead to a decline in sperm quality or quantity[24]. Functional impairment of SSCs in mice can lead to abnormal sperm production and, in severe cases, the inability to produce germ cells[22]. For patients with NOA and testicular spermatogenic dysfunction, SSCs can promote spermatogenesis by repairing spermatogenic tubule damage and regulating the testicular microenvironment[25]. The prognosis of male infertility treatment is poor. However, SSCs are regarded as a promising alternative method for the regeneration of damaged or compromised sperm. SSC transplantation holds great promise as a therapeutic approach for the treatment of male infertility and restoration of spermatogenesis-related degenerative diseases[7]. However, the regulatory mechanism of SSCs remains to be studied.

Forkhead box family transcription factors exert a considerable influence on the developmental progression of spermatogonia and SSCs. For example, Foxa2 is expressed in rat spermatogonia and reported to be associated with genes enriched with spermatogonial stemness genes in the rat germline[26]. FOXO1 plays a crucial role in mouse SSCs and is indispensable for their preservation and triggering the onset of spermatogenesis[27]. Furthermore, FOXP4 was reported to promote the proliferation of human SSCs[28]. FOXP1 was also found to be enriched in rat undifferentiated spermatogonia[26]. In this study, we discovered a notable reduction in FOXP1 levels within the testicular tissues of patients with NOA compared with those of patients with OA. FOXP1 inhibition caused abnormal changes in the morphology and weight of the testes in mice. In addition, FOXP1 overexpression promoted the proliferation and self-renewal of mouse SSCs.

Furthermore, our study demonstrates that FOXP1 promotes SSC self-renewal via NF-κB activation, but the precise molecular mechanism warrants further investigation. FOXP1 is a transcription factor capable of protein-protein interactions and has been shown to cooperate with NF-κB in cancer models to enhance survival. FOXP1 cooperates with NF-κB to promote survival and proliferation in diffuse large B-cell lymphoma[29]. It is plausible that in SSCs, FOXP1 may serve as a transcriptional co-activator for NF-κB, thereby potentiating the expression of target genes, such as PCNA, ETV5, and PLZF. Conversely, it might repress the expression of negative regulators such as IκBα. Our finding that FOXP1 is enriched in undifferentiated spermatogonia[26] aligns with the possibility that it programs an NF-κB signaling permissive transcriptional landscape, which is known to be involved in germ cell migration and survival[20]. Future studies should explore whether FOXP1 physically interacts with p65 or modulates IκBα signaling in SSCs. In addition, NF-κB may itself regulate FOXP1 expression, thus forming a positive feedback loop that amplifies the self-renewal signal. Elucidating these interactions will enhance our understanding of SSC biology and may identify novel therapeutic targets for male infertility.

ETV5 and GDNF are both synthesized within Sertoli cells and are essential for sustaining the viability and promoting the self-renewal of SSCs in mice[30]. Endogenous factor PLZF plays key functions in the maintenance of SSC self-renewal[22]. Our immunohistochemistry results and immunofluorescence assays showed that the expression of PLZF and ETV5 was downregulated in the testicles of mice with FOXP1 knockdown. Moreover, their expression was upregulated in mice SSCs with FOXP1 overexpression. However, due to limitations in vitro expansion and culture of human SSCs, as well as the species differences between humans and mice, similar methods cannot be adopted to restore human spermatogenic function using SSCs. Therefore, there is an urgent need to clarify the mechanisms regulating the development of human SSCs, especially their self-renewal and proliferation mechanisms.

CONCLUSION

In conclusion, FOXP1 was upregulated in the testicles of patients with OA compared with those of patients with NOA. FOXP1 may promote the proliferation and self-renewal of SSCs via the NF-κB pathway. Our study offers a potential theoretical basis for the treatment of male infertility.