Role of miR-300-3p in Leydig cell function and differentiation: A therapeutic target for obesity-related testosterone deficiency

Jinlian Liang; Derong Chen; Ziyan Xiao; Siying Wei; Yuan Liu; Chengzhi Wang; Zhaoyang Wang; Yuqing Feng; Yaling Lei; Meirong Hu; Jingxian Deng; Yuxin Wang; Qihao Zhang; Yan Yang; Yadong Huang

doi:10.1016/j.omtn.2023.03.016

. 2023 Mar 23;32:879–895. doi: 10.1016/j.omtn.2023.03.016

Role of miR-300-3p in Leydig cell function and differentiation: A therapeutic target for obesity-related testosterone deficiency

Jinlian Liang ^1,^3,⁵, Derong Chen ^1,^3,⁵, Ziyan Xiao ^1,^3,⁵, Siying Wei ¹, Yuan Liu ¹, Chengzhi Wang ², Zhaoyang Wang ¹, Yuqing Feng ³, Yaling Lei ¹, Meirong Hu ¹, Jingxian Deng ³, Yuxin Wang ¹, Qihao Zhang ^1,³, Yan Yang ^1,^3,^∗, Yadong Huang ^1,^3,^4,^∗∗

PMCID: PMC10236194 PMID: 37273781

Abstract

MicroRNAs (miRNAs) regulate various cellular functions, but their specific roles in the regulation of Leydig cells (LCs) have yet to be fully understood. Here, we found that the expression of miR-300-3p varied significantly during the differentiation from progenitor LCs (PLCs) to adult LCs (ALCs). High expression of miR-300-3p in PLCs inhibited testosterone production and promoted PLC proliferation by targeting the steroidogenic factor-1 (Sf-1) and transcription factor forkhead box O1 (FoxO1) genes, respectively. As PLCs differentiated into ALCs, the miR-300-3p expression level significantly decreased, which promoted testosterone biosynthesis and suppressed proliferation of ALCs by upregulating SF-1 and FoxO1 expression. The LH/METTL3/SMURF2/SMAD2 cascade pathway controlled miR-300-3p expression, in which luteinizing hormone (LH) upregulated SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2) expression through methyltransferase like 3 (METTL3)-mediated Smurf2 N6-methyladenosine modification. The Smurf2 then suppressed miR-300 transcription by inhibiting SMAD family member 2 (SMAD2) binding to the promoter of miR-300. Notably, miR-300-3p was associated with an obesity-related testosterone deficiency in men and the inhibition of miR-300-3p effectively rescued testosterone deficiency in obese mice. These findings suggested that miR-300-3p plays a pivotal role in LC differentiation and function, and could be a promising diagnostic or therapeutic target for obesity-related testosterone deficiency.

Keywords: MT: Non-coding RNAs, Leydig cell, miR-300-3p, testosterone, METTL3, M6A methylation

Graphical abstract

Huang and colleagues reported that the expression of miR-300-3p is controlled by the LH/METTL3/SMURF2/SMAD2 cascade pathway. It plays a pivotal role in LC differentiation and function. Inhibition of miR-300-3p could effectively rescue low serum testosterone levels caused by obesity.

Introduction

Adult Leydig cells (ALCs) are located in the interstitium of the mammalian testis and are the main source of testosterone production. Testosterone plays a key role in male reproduction development,¹ spermatogenesis,² and maintaining secondary sex characteristics. During the postnatal development, ALCs are derived from stem Leydig cells (SLCs). There are two immediate stages between ALCs and SLCs, called progenitor Leydig cells (PLCs) at postnatal day 21 and immature Leydig cells (ILCs) between postnatal days 28 and 35.³ During puberty, proliferation of PLCs is required to increase the number of LCs. PLCs produce low amounts of testosterone, but testosterone production increases when PLCs differentiate into ALCs. LC dysfunction leads to male reproduction diseases, including testosterone deficiency, primary hypogonadism, and aspermatogenesis.⁴

Testosterone synthesis depends on the hypothalamic-pituitary-testicular axis. The hypothalamus impulsively secretes gonadotropin-releasing hormone (GnRH) to stimulate the secretion of gonadotropin-luteinizing hormone (LH) by the pituitary gland. LH binds to the LH receptor (LHCGR) on the surface of LCs to activate adenylyl cyclase (cAMP), leading to a cascade that stimulates testosterone production.⁵ LH is important for LC maturation,⁶ testosterone synthesis,² spermatogenesis,⁷ and sexual organ development in men.⁸ The disruption of the LH receptor gene can result in a drastic decrease in LC number and testosterone levels.⁹ In addition, the transcriptional regulation of steroidogenic proteins, including steroidogenic acute regulatory (StAR),¹⁰ cytochrome P450 family 11 subfamily A member 1 (CYP11A1),¹¹ 17α–hydroxylase (CYP17A1),¹² 3-beta-hydroxy-steroid dehydrogenase (HSD3β1),¹³ and 17beta-hydroxy-steroid dehydrogenase (HSD17β),¹¹^,¹³ also plays an important role in LC function.

MicroRNAs (miRNAs) interact with target genes by binding to the 3′ untranslated region (3′ UTR) or the coding sequence (CDS) of mRNAs and have been identified and characterized in the testis. Recent studies have demonstrated that miRNAs are important post-transcriptional regulators that play critical roles in the development and function of the testis.¹⁴^,¹⁵^,¹⁶ Multiple miRNAs are involved in steroid hormone synthesis that takes place in LCs.¹⁷ Indeed, miRNA expression profiling showed significant variations during PLC differentiation into ALCs, in which miR-199-5p, miR-29a-3p, miR-29b-3p, miR-29c-3p, and miR-7a-5p suppressed testosterone synthesis.¹⁴ For example, miR-1197-3p regulates testosterone production by targeting peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) in goat LCs,¹⁸ and the triclosan (TCS)-induced miR-142-5p inhibits CYP17A1 to downregulate testosterone levels.¹⁶ These studies demonstrate that miRNAs play an important role in androgen synthesis and development in LCs; however, the functions and mechanisms of miRNAs in LC regulation remain largely unknown.

In this study, we found that the expression level of miR-300-3p significantly varied at different developmental stages in LCs, with high expression in PLCs and low expression in ALCs. Previous studies showed that miR-300-3p plays a regulatory role in cell proliferation,¹⁹^,²⁰ differentiation,²¹ tumorigenesis, and metastasis.²²^,²³ However, the role of miR-300-3p in LC differentiation and testosterone synthesis has not been reported. Our results suggest that miR-300-3p overexpression inhibits testosterone synthesis and promotes PLC proliferation in vitro and in vivo. MEETL3-mediated m⁶A modification is critical for miR-300-3p expression, which is associated with obesity-related testosterone deficiency. Inhibition of miR-300-3p in testis of high-fat diet (HFD) mice rescued obesity-related testosterone deficiency, revealing that miR-300-3p is a potential therapeutic target for obesity-related testosterone deficiency.

Results

miR-300-3p suppresses testosterone synthesis in LCs by targeting Sf-1

We first assessed miR-300-3p expression in the testes using in situ hybridization. miR-300-3p expression was observed in both young (3-week-old) and adult (8-week-old) male mice, but was highly expressed in the testis interstitium of young mice (Figure 1A, Figure S1A). We isolated PLCs from young mouse testes and ALCs from adult mouse testes (Figure S1B) and used quantitative reverse transcription polymerase chain reaction (qRT-PCR) to detect the expression of miR-300-3p in these cell types. The level of miR-300-3p was significantly decreased in ALCs (Figure 1B). To determine whether the loss of miR-300-3p expression was accompanied by alterations in steroidogenic proteins, we examined the expression of steroidogenic factor-1 (SF-1), CYP11A1, and StAR in PLCs and ALCs. The results showed that SF-1, CYP11A1, and StAR levels were significantly higher in ALCs than in PLCs (Figures 1C and S1C). Moreover, the reduction in miR-300-3p was correlated with an increase in steroidogenic proteins and testosterone levels (Figure 1D), consistent with an inverse correlation between miR-300-3p expression and testosterone production in LCs. Therefore, we hypothesized that miR-300-3p plays a role in testosterone biosynthesis.

miR-300-3p directly targets *Sf-1* to suppress testosterone production

(A) *In situ* hybridization images illustrating that miR-300-3p is mainly localized in interstitial spaces in the testis (red arrow). Scale bar, 50 μm. (B) RT-qPCR results showing miR-300-3p expression in PLCs and ALCs. PLCs and ALCs were isolated from 3- and 8-week-old mouse testes, respectively. (C) Expression levels of steroidogenic proteins SF-1, CYP11A1, and StAR in PLCs and ALCs. (D) Radioimmunoassay assay (RIA) analysis of testosterone concentrations in PLC and ALC cell culture supernatants. (E and F) Testosterone concentrations in culture supernatant from LCs transfected with an miR-300-3p mimic or inhibitor. (G) and (H) Protein levels of SF-1, CYP11A1, and StAR in LCs transfected with an miR-300-3p mimic or inhibitor. (I) TargetScan predicted that miR-300-3p binds to the 3′-UTR of *SF-1*. (J) HEK293T cells were co-transfected with luciferase reporter vectors (psi-check 2 containing the wild-type or 3′-UTR *SF-1* mutants) and an miR-300-3p or NC mimic. The relative Renilla luciferase activity was measured and normalized to firefly luciferase activity. (K) Protein level of SF-1 in LCs co-transfected for 48 h with *Sf-1* overexpression vector and an miR-300-3p or NC mimic. (L) Protein levels of SF-1, CYP11A1, and StAR in LCs transfected with *Sf-1* siRNA. (M) Testosterone concentrations in culture supernatants from LCs transfected with *Sf-1* siRNA. All data are presented as the mean ± SD from at least three independent experiments. ∗p < 0.5, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns = not significant.

To investigate the function of miR-300-3p, ALCs were transfected with an miR-300-3p mimic to increase exogenous miR-300-3p, while PLCs were transfected with an miR-300-3p inhibitor to block miR-300-3p. The miR-300-3p mimic significantly suppressed testosterone production in ALCs (Figure 1E). In contrast, testosterone levels in PLCs increased in response to the miR-300-3p inhibitor (Figure 1F). Collectively, these results showed that miR-300-3p is involved in testosterone synthesis in LCs.

We also examined the expression of SF-1, CYP11A1, and StAR after LCs transfected with either the miR-300-3p mimic or inhibitor. The results showed that the miR-300-3p mimic downregulated the expression of SF-1, CYP11A1, and StAR (Figures 1G and S1B), while in contrast, the levels of SF-1, CYP11A1, and StAR were significantly upregulated in PLCs transfected with the miR-300-3p inhibitor (Figures 1H and S1C). To further reveal the molecular mechanism of miR-300-3p involvement in testosterone synthesis, TargetScan was used to predict the targets of miR-300-3p. Among the steroidogenic genes, Sf-1 was identified as a target of miR-300-3p (Figure 1I). Sf-1 is a key transcription factor of steroidogenic genes, and regulates steroidogenic gene expression and testosterone production in LCs.²⁴^,²⁵^,²⁶^,²⁷ To confirm if miR-300-3p can directly bind to the 3′-UTR of Sf-1, luciferase reporter vectors containing the wild-type (WT) or mutated 3′-UTR of Sf-1 were co-transfected with miR-300-3p mimic or negative control (NC) mimic into HEK293T cells. The miR-300-3p mimic significantly suppressed the luciferase activity of the WT reporter vector, whereas the miR-300-3p binding site mutants did not (Figure 1J). Exogenous overexpression of miR-300-3p specifically downregulated Sf-1 expression and this effect could be rescued by Sf-1 overexpression (Figure 1K). Similar to the effects of the miR-300-3p mimic, blocking the expression of SF-1 significantly downregulated the expression of steroidogenic proteins (CYP11A1 and StAR) (Figure 1L) and testosterone production (Figure 1M).

Western blotting also demonstrated that the miR-145-5p mimics downregulated CYP11A1 and StAR protein expression (Figures 2F–2H). To further support a role for miR-145-5p, we used miR-145-5p inhibitor to selectively block the activity of miR-145-5p in ILCs. Conversely, ILCs transfected with miR-143-3p inhibitor exhibited significant increases in levels of testosterone, compared with control cells. And the expression of steroidogenic genes and protein were significantly downregulated by miR-145-5p inhibitor.

miR-300-3p targets *FoxO1* to promote LC proliferation

(A–D) EdU assays show the effect of the miR-300-3p mimic and inhibitor on LC proliferation. NC-mimic = mimic negative control, NC-inhibitor = inhibitor negative control. Scale bar, 100 μm. (E) miR-300-3p was predicted to bind to the CDS region of *FoxO1* mRNA. (F) HEK293T cells co-transfected with luciferase reporter vectors (psi-check 2 containing the wild-type or mutated *FoxO1* CDS region) and miR-300-3p mimic or NC mimic. The relative Renilla luciferase activity was normalized to the firefly luciferase activity. (G and H) Protein levels of FoxO1, p-AKT, AKT, CCND1, and CCND2 in LCs transfected with miR-300-3p mimic, miR-300-3p inhibitor, or the respective NC. (I) Schematic diagram of the miR-300-3p activating AKT pathway. (J) Cell proliferation was assessed by EdU incorporation in PLCs and ALCs. Scale bar, 200 μm. Data are presented as the mean ± SD from at least three independent experiments. ∗p < 0.5, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns = not significant.

Results demonstrate that miR-145-5p regulates steroidogenic gene expression and testosterone biosynthesis. In addition, the level of free cholesterol that constitutes the pool used for steroidogenesis in LCs was analyzed, and Oil Red O staining showed that miR-145-5p overexpression resulted in lipid accumulation (Figure 2I). Moreover, transmission electron microscopy also revealed the formation of lipid droplet clusters in LCs overexpressing miR-145-5p (Figure 2J). In contrast, no obvious aggregation of lipid droplets was observed in cells treated with mimic-NC. These results suggest that miR-145-5p overexpression causes defects in cholesterol metabolism in Leydig cells, subsequently inhibiting testosterone production.

These results suggested that miR-300-3p expression decreased as LCs differentiated from PLCs into ALCs, and the reduction of miR-300-3p upregulated the expression of CYP11A1 and StAR by targeting the 3′-UTR of Sf-1, which subsequently promoted testosterone biosynthesis in ALCs.

miR-300-3p regulates LC proliferation by targeting FoxO1

To investigate whether miR-300-3p overexpression suppresses testosterone production by inhibiting PLC proliferation, an EdU incorporation assay was used to estimate LC proliferation after miR-300-3p mimic or inhibitor treatment. The miR-300-3p mimic increased the number of EdU-positive cells (Figures 2A and 2B) while the miR-300-3p inhibitor decreased the number of EdU-positive cells (Figures 2C and 2D). These results suggested that miR-300-3p overexpression had a suppressive effect on testosterone production that not due to cell proliferation inhibition. We further revealed the mechanism by which miR-300-3p promotes PLC proliferation and found that Forkhead box transcription factor O1 (FoxO1) is a putative target of miR-300-3p (Figure 2E). The miR-300-3p mimic significantly suppressed the luciferase activity of the WT reporter vector, whereas mutation of the potential miR-300-3p binding site did not (Figure 2F). FoxO1 is a growth-attenuating winged helix transcription factor protein that serves as a substrate for phosphoinositide protein kinase B (AKT) and mediates the feedback activation of AKT.²⁸ We assessed AKT and FoxO1 signaling by measuring the protein levels of AKT, phosphorylated AKT (p-AKT), FoxO1, cell cycle-associated proteins cyclin D1 (CCND1), and cyclin D2 (CCND2). We found that miR-300-3p overexpression inhibited FoxO1 expression and increased p-AKT, CCND1, and CCND2 expression (Figure 2G). The opposite effect was observed in cells transfected with miR-300-3p inhibitor (Figure 2H). These results suggested that miR-300-3p overexpression downregulated the expression of FoxO1, which increased p-AKT via a feedback loop that promotes the proliferation of LCs (Figure 2I). When we examined the proliferation of PLCs and ALCs, we observed more EdU-positive cells in PLCs than in ALCs (Figure 2J), which indicated that PLCs have a stronger proliferation ability than ALCs. The change in proliferation ability in PLCs and ALCs was consistent with the miR-300-3p regulation pattern. The loss of miR-300-3p induced the differentiation of PLCs into ALCs and promoted testosterone biosynthesis.

The functions of miR-300-3p in vivo

To further investigate whether miR-300-3p suppresses testosterone production in vivo, miR-300-3p agomir and antagomir were injected into the interstitium of adult mouse testes (Figure 3A), while control mice were injected with PBS and the mock control mice were injected with corresponding scrambled miRNAs. Two weeks after the treatment, hematoxylin-eosin (H&E) staining of teste tissue samples showed no obvious changes (Figures S2A and S2B); neither the miR-300-3p agomir nor antagomir resulted in a significant number of apoptotic cells (Figures S2C and S2D). These results suggested that the miR-300-3p agomir and antagomir did not exhibit cytotoxicity in the testes. We then confirmed the efficacy of miR-300-3p agomir and antagomir injections using in situ hybridization. The miR-300-3p agomir significantly elevated the expression of miR-300-3p in the interstitium of the testes, while the miR-300-3p antagomir successfully decreased the amount of miR-300-3p present in these tissues (Figures 3B, 3C, S2E, and S2F).

Serum was collected to determine testosterone concentrations 14 days after the miR-300-3p agomir or antagomir injection. The results showed that the miR-300-3p agomir significantly downregulated the level of serum testosterone, while the miR-300 antagomir dramatically increased serum testosterone production when compared with that of the controls (Figures 3D and 3E). Free cholesterol is a pool used for steroidogenesis in LCs and Oil Red O staining was used to analyze the level of free cholesterol. We found that a large number of lipid droplets accumulated in the interstitium of testes after miR-300-3p agomir treatment (Figure 3F) while miR-300 antagomir treatment decreased lipid droplet accumulation compared with normal control (Figure 3G). Immunofluorescence analysis revealed that the expression of CYP17A1 was upregulated by miR-300-3p agomir, which showed a consistent change in serum testosterone production (Figures 3H, 3I, and S2G). In addition, the miR-300-3p agomir significantly decreased the levels of SF-1, CYP11A1, StAR, and FoxO1; upregulated the expression of CCND1 and CCND2 in testis (Figures 3J and S2G). In contrast, the miR-300 antagomir significantly increased the protein levels of SF-1, CYP11A1, StAR, and FoxO1; and significantly decreased the protein levels of CCND1 and CCND2 (Figures 3K and S2H). Taken together, these results confirmed that miR-300-3p modulated testosterone synthesis and LC proliferation by targeting SF-1 and FoxO1, respectively. Overexpression of miR-300-3p in adult male testes may induce dysfunction of testosterone secretion in LCs.

miR-300-3p is a therapeutic target for obesity-related testosterone deficiency

We examined whether abnormally high miR-300-3p expression results in obesity-related testosterone deficiency. RIA was used to measure the serum testosterone levels of lean and obese men. The result showed that mean testosterone concentrations in lean men were 8.12 ng/mL, and obese men had subnormal levels of 6.00 ng/mL (Figure 4A). Circulating miR-300-3p in serum samples was detected by qRT-PCR, which showed that miR-300-3p was significantly increased in obese men compared with that in lean men (Figure 4B), suggesting that high miR-300-3p expression was associated with low testosterone levels (Figure 4C). These results demonstrated that abnormally high expression of miR-300-3p may be an important regulator of obesity-related testosterone deficiency.

miR-300-3p inhibitor rescue testosterone deficiency

(A) Serum testosterone concentrations in obese and lean adult men. (B) The expression level of serum miR-300-3p in obese and lean men was determined by qRT-PCR. (C) Analysis of the association between miR-300-3p expression and testosterone production in the serum. (D and E) Body weight change in chow-fed and HFD mice. (F) Serum testosterone levels in chow-fed and HFD mice. (G) Serum miR-300-3p expression in chow-fed and HFD mice. (H) Level of miR-300-3p expression in the testes of chow-fed and HFD mice. (I) Detection of miR-300-3p (red arrows) in testes of chow-fed and HFD mice by *in situ* hybridization. Scale bar, 50 μm. (J) miR-300-3p expression (red arrow) in testes after miR-300-3p inhibitor or NC inhibitor treatment. Scale bar, 50 μm. (K) Serum testosterone levels in HFD mice treated with miR-300-3p or NC inhibitors for 4 weeks. (L) The expression of SF-1 and FoxO1 in mouse testes after treatment with miR-300-3p or NC inhibitors. All data are shown as the mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and ns = not significant.

We further explored the role of miR-300-3p in obesity-related testosterone deficiency in male mice fed an HFD, 60% kcal fat) or normal chow (Figure 4D). After 12 weeks, the weight of HFD-fed mice (40.7 g) significantly increased by more than 40% compared with that of chow-fed mice (28.3 g) (Figure 4E). Serum testosterone concentrations in HFD mice was also dramatically reduced (Figure 4F) and miR-300-3p expression in serum was significantly elevated (Figure 4G) compared with that in chow-fed mice. In addition, qRT-PCR and in situ hybridization showed that the expression of miR-300-3p in the testes of HFD mice was significantly higher than that in the testes of chow-fed mice (Figures 4H and 4I). These data suggested that increased miR-300-3p expression in the testes of adult males may be involved in the modulation of obesity-related testosterone deficiency.

We further examined the effects of miR-300-3p on testosterone synthesis using an obese mouse model. HFD mice were treated with an miR-300-3p or NC inhibitors once a week for 4 weeks. Untreated mice fed normal chow were used as normal controls. The miR-300-3p inhibitor effectively downregulated the expression of miR-300-3p in HFD mice to levels that were comparable to that of the normal control (Figure 4J). Serum was collected for testosterone measurement after treatment with the miR-300-3p inhibitor, which showed that testosterone levels in HFD mice were similar to the normal levels (Figure 4K). The protein levels of SF-1 and FoxO1 in the testes were significantly downregulated in the HFD-fed mice; however, the miR-300-3p inhibitor rescued SF-1 and FoxO1 expression levels (Figure 4L). These results indicated that miR-300-3p silencing can rescue testosterone production in HFD mice and therefore, miR-300-3p is a potential therapeutic target in testosterone deficiency.

N6-adenosine methylation regulates the expression of miR-300-3p in LCs via the SMURF2/p-SMAD2 axis

The N6-adenosine methylation (m⁶A) modification is important for regulating the stability of m⁶A modified RNAs.²⁹^,³⁰ It has also been reported that m⁶A is involved in the regulation of hepatic lipid and glucose metabolism in HFD mice³¹; therefore, we examined whether m⁶A modification was involved in regulating miR-300-3p expression. Lower m⁶A levels were observed in HFD mouse testes than in chow-fed mice (Figure 5A). To further explore the enzymes that mediate m⁶A methylation in HFD, we examined the expression of methylation transferases (METTL3 and METTL14) and demethylase (ALKBH5) with western blotting. The level of METTL3 in the testes of HFD mice was significantly lower than that in the normal control mice, while the expression of METTL14 and ALKBH5 did not significantly change between HFD and chow-fed mice (Figures 5B and S3A). Thus, we hypothesized that miR-300-3p expression was modulated by METTL3. Mettl3 knockdown and overexpression in LCs were used to determine the role of METTL3 in the regulation of miR-300-3p expression. We found that Mettl3 overexpression significantly downregulated the expression of miR-300-3p (Figures 5C and 5D), while in contrast, Mettl3 siRNA increased miR-300-3p expression (Figures 5E and 5F). Moreover, we found that Mettl3 overexpression downregulated the expression of miR-300-3p, which was accompanied by decreased levels of phosphorylated SMAD2 (p-SMAD2) and increased levels of SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2) (Figures 5G and S3B). However, the knockdown of METTL3 led to the opposite results (Figures 5H and S3C). These data suggested that METTL3 controls the expression of miR-300-3p via SMURF2 and SMAD2.

m6A modification down-regulates the expression of miR-300-3p in LCs via the SMURF2/p-SMAD2 axis

(A) RNA m⁶A modification levels in testes of chow-fed and HFD mice were determined by colorimetry. (B) The protein levels of METTL3, METTL14, and ALKBH5 in testes of HFD mice and chow-fed mice. (C and D) The mRNA levels of *Mettl3* and miR-300-3p in LCs overexpressing *Mettl3* for 48 h. (E and F) The mRNA levels of *Mettl3* and miR-300-3p in LCs with si-*Mettl3* for 48 h. (G) The protein levels of METTL3, p-SMAD2, SMAD2, SMAD3, and SMURF2 in LCs transfected with the *Mettl3* overexpressed vector for 48 h. (H) The protein levels of METTL3, p-SMAD2, SMAD2, SMAD3, and SMURF2 in LCs with si-*Mettl3* for 48 h. (I) Schematic of the positions of the m⁶A motifs in *Smurf2* mRNA and (J) Schematic of mutated m⁶A motifs (GGAC to GGCC) in the 3′-UTR of *Smurf2* gene. (K) Luciferase reporter vectors and a *Mettl3* overexpression vector were co-transfected into HEK293T cell for 48 h before the relative Renilla luciferase activity was measured and normalized to firefly luciferase activity. (L and M) The mRNA levels of *Smurf2* in LCs transfected for 24 h with *Mettl3* overexpression vector or *Mettl3* siRNA. (N) The potential binding site of SMAD2 in the miR-300 gene. (O) The expression of miR-300-3p in LCs after *Smad2* overexpression. (P) Schematic of the mutated 5′ upstream region of miR-300 in the PGL-3basic vector used to investigate the promoter motif of miR-300. (Q) Luciferase reporter vectors, the *Smad2* overexpression vector, and the PRL-TK vector were co-transfected into HEK293T cells for 48 h before the relative firefly luciferase activity was measured and normalized to the Renilla luciferase activity. (R) METTL3 regulates the expression of miR-300-3p via the SMURF2/p-SMAD2 axis. All data are presented as the mean ± SD from three independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and ns = not significant.

To elucidate the regulatory relationship among METTL3, SMURF2, p-SMAD2, and miR-300-3p, we focused on the GGACU-binding motif of Mettl3, a subunit of m⁶A methyltransferase. The METTL3 binding motif “GGAC” was found in the 3′-UTR of Smurf2 (Figure 5I). Mettl3 overexpression significantly increased Smurf2 mRNA levels, while Mettl3 knockdown resulted in a reduction in Smurf2 expression. To confirm whether the Smurf2 transcript was regulated by Mettl3, we generated luciferase reporter vectors containing firefly and Renilla luciferases, followed by the 3′-UTR of Smurf2 WT or mutation sequences of each m⁶A site (i.e., “GGAC” to “GGGC”) (Figure 5J). The 3′-UTR luciferase reporter vectors of Smurf2 and pcDNA3.1-Mettl3 were co-transfected into HEK293T cells. Mettl3 overexpression significantly increased the luciferase activity of the WT vector (Figure 5K), while the mutation of sites 1 (MU1, NM_001362894.1:2853–2856) and 2 (MU2, NM_001362894.1:2929–2932) effectively attenuated this increase. Simultaneous mutations at all five sites (MU6) also downregulated the luciferase activity, which showed an overlay of multiple effects. These results suggested that Smurf2 mRNA is modified by m⁶A methylation.

Smurf2 is a negative regulator of the transforming growth factor (TGF)-β signaling pathway, which degrades phosphorylated SMAD2. Our results suggested Mettl3 overexpression increased the mRNA level of Smurf2, and inhibition of Mettl3 downregulated Smurf2 expression (Figures 5L and 5M). Moreover, the increased level of SMURF2 was associated with a decrease in p-SMAD2, while the expression of p-SMAD2 was increased by the inhibition of SMURF2. These results suggested that decreased METTL3 suppressed p-SMAD2 expression by upregulating the level of SMURF2. Moreover, we found Smad2 is a potential transcription factor that may bind to the promoter of the miR-300 gene (Figure 5N). The expression of miR-300-3p was significantly elevated by Smad2 overexpression (Figure 5O). To determine whether Smad2 can bind to the miR-300 promoter, we generated luciferase reporter constructs driven by the miR-300 promoter or the promoter with a mutated Smad2 binding site (Figure 5P). Luciferase activity was strongly increased by Smad2 (Figure 5Q); however, when the binding site was mutated, the fluorescence activity significantly decreased (Figure 5Q), confirming that Smad2 could bind to the promoter of miR-300 and subsequently initiate miR-300 transcription. These results confirmed that METTL3 regulates the expression of miR-300-3p via the SMURF2/SMAD2 axis (Figure 5R).

METTL3 is regulated by LH

LH is an important hormone that promotes the synthesis of LC and testosterone.⁹^,³² We examined the levels of serum LH and testosterone in male mice at different developmental stages. LH and testosterone levels progressively increased from 3 to 5 weeks after birth (Figures 6A and 6B). Moreover, RNA m⁶A levels and METTL3 expression also increased significantly during testis development between 3 and 5 weeks (Figures 6C, 6D, S4A, and S4B), while the expression of miR-300-3p decreased (Figure S4C). To investigate whether LH affects m⁶A modification levels in LCs, we measured the expression of METTL3 in LCs stimulated with LH at different concentrations (0, 5, 10, and 50 ng/mL) and found that the levels of METTL3 increased as LH concentrations increased. Upregulation of METTL3 expression also contributed to the increase in SMURF2, which then downregulated p-SMAD2 expression (Figures 6E and 6F) and eventually led to a reduction in miR-300-3p (Figure 6G). These data demonstrated that METTL3 was regulated by LH, which can drive changes in miR-300-3p expression in LCs.

LH regulates METTL3 expression in LCs

(A) Serum LH concentration in mice at 3, 4, and 5 weeks of age. (B) Serum testosterone of mice at 3, 4, and 5 weeks of age was measured by RIA. (C) A colorimetric method was used to detect the m⁶A modification level of LCs isolated respectively from 3-, 4-, and 5-week-old mice. (D) Western blotting showing the expression of METTL3, METTL14, and ALKBH5 in LCs isolated from 3-, 4-, and 5-week-old mice, respectively. (E) and (F) The protein levels of METTL3, SMURF2, p-SMAD2, and SAMD2 in LCs treated with different concentrations of LH for 24 h. (G) The expression of miR-300-3p in LCs treated with different LH concentrations for 24 h. All data are presented as the mean ± SD from three independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and ns = not significant.

In summary, the LH/METTL3/SMURF2/SMAD2/miR-300-3p pathway tightly controls LC proliferation, differentiation, and testosterone synthesis by targeting FoxO1 and SF-1. In ALCs, LH induced METTL3 and SMURF2 expression by promoting METTL3 mediated m⁶A modification of Smurf2, which suppressed miR-300-3p transcription by inhibiting p-SMAD2 levels. The reduction of miR-300-3p improved testosterone production and inhibited proliferation in ALCs by upregulating SF-1 and FoxO1. The abnormally high expression of miR-300-3p observed in ALCs was involved in the regulation of obesity-related testosterone deficiency (Figure 7).

Schematic of the LH/METTL3/SMURF2/SMAD2/miR-300-3p axis that mediates testosterone synthesis in LCs

LH level progressively increased from PLC to ALC. In ALCs, LH induced METTL3 and SMURF2 expression by promoting METTL3 mediated m⁶A modification of *Smurf2*, which suppressed miR-300-3p transcription by inhibiting p-SMAD2 levels. The reduction of miR-300-3p improved testosterone production and inhibited proliferation by upregulating SF-1 and FoxO1 expression in ALCs. The abnormally high expression of miR-300-3p observed in ALCs was involved in the regulation of obesity-related testosterone deficiency.

Discussion

LC differentiation and function depend on fine-tuning gene expression regulation.³³^,³⁴ LCs in mammals progressively improve their capacity to synthesize testosterone during development and their differentiation from PLCs to ALCs, peaking in ALCs.³⁵^,³⁶ Therefore, we hypothesized that some genes dynamically regulate LC function during LC differentiation. In this study, we observed that high miR-300-3p expression in PLCs progressively decreased with differentiation, suggesting that miR-300-3p is involved in regulating LC differentiation and function. However, the function of miR-300-3p in LCs has not yet been elucidated.

In this study, we found that overexpression of miR-300-3p in ALCs inhibited the expression of SF-1 and testosterone production by binding to the 3′-UTR of the Sf-1 gene, whereas inhibition of miR-300-3p upregulated SF-1 expression and promoted testosterone synthesis. SF-1 is an indispensable transcriptional regulator of LC development and survival that regulates the expression of steroidogenic genes²⁴^,²⁵^,²⁶^,³⁷ by binding to their promoters to enhance gene transcription. Ectopic overexpression of Sf-1 directly converted non-steroidal producing cells (e.g., mesenchymal cells, embryonic stem cells, and fibroblasts) into functional Leydig-like cells,³⁸^,³⁹^,⁴⁰^,⁴¹ illustrating the importance of SF-1 in testosterone synthesis and LC differentiation. In this study, we observed a gradual increase in SF-1 expression in LCs during their differentiation from PLCs to ALCs, suggesting that SF-1 is dynamically modulated by miR-300-3p during LC differentiation. We asked whether the abnormal increase in miR-300-3p expression in adulthood caused LC dysfunction and observed that low levels of serum testosterone were accompanied by a significant increase in miR-300-3p levels in both obese men and HFD mice. It has been reported that obese men have subnormal serum testosterone concentrations⁴²^,⁴³^,⁴⁴; however, the relationship between serum testosterone levels and obesity remains complex and the mechanism underlying obesity-induced testosterone deficiency has not been elucidated. We found that above-normal levels of miR-300-3p in obese men were correlated with testosterone deficiency, further confirming the role of miR-300-3p in obesity-related testosterone deficiency. The results from HFD mice treated with an miR-300-3p inhibitor suggested that miR-300-3p inhibition could effectively rescue low serum testosterone levels caused by obesity. These results highlight a vital role of miR-300-3p in regulating testosterone synthesis and could be a therapeutic target for testosterone deficiency.

In addition, miR-300-3p modulated LC proliferation by targeting the CDS region of the FoxO1 gene. In PLCs, high miR-300-3p expression downregulated FoxO1 expression by binding to its CDS regions, which facilitated PLCs proliferation. However, low miR-300-3p expression upregulated FoxO1 expression in ALCs and blocked their proliferation. Previous research suggested that miRNAs combine the targeting of CDS and 3′-UTR regions to flexibly tune the timescale and magnitude of their post-transcriptional regulatory effects.⁴⁵ These miRNAs inhibit translation by binding to sites located in the CDS of genes and trigger mRNA degradation by binding to the sites located in the 3′-UTR of genes.⁴⁶ While large numbers of miRNAs can regulate genes post-transcriptionally by binding to the target 3′-UTR regions, there are only a few reports of those that bind to CDS regions to modulate gene expression.⁴⁷ In this study, we found FoxO1 expression could be modulated through the binding of miR-300-3p to the CDS regions, which provides new evidence for this relatively uncommon mechanism. Moreover, the FoxO1 subfamily has emerged as a shared component among pathways that regulate diverse cellular functions, such as differentiation, metabolism, proliferation, and survival.⁴⁸ It has been reported that TGF-β1-modulated cardiac fibroblast differentiation is prevented by the downregulation of FoxO1 and enhanced by FoxO1 overexpression.⁴⁹ In addition, centrosomal protein 55 overexpression causes male-specific sterility in mice by suppressing FoxO1 nuclear retention through the sustained activation of PI3K/Akt signaling.⁵⁰ In our study, FoxO1-mediated feedback promoted AKT activation, to facilitate PLC proliferation. As a result of the differentiation of PLCs into ALCs, their proliferation was suppressed. Taken together, our study demonstrated that miR-300-3p directly targets and negatively regulates the expression of SF-1 and FoxO1 to modulate the differentiation and function of LCs.

We also investigated the molecular mechanisms underlying the expression of miR-300-3p. We found that METTL3 mediated m⁶A modification of Smurf2 mRNA as an important regulator of miR-300-3p expression. It has been shown that METTL3 is sufficient to promote miRNA maturation, and m⁶A modification is a key post-transcriptional modification that induces the initiation of miRNA biogenesis.⁵¹ Accumulating evidence has confirmed that reduced expression or loss-of-function mutations in METTL14 may result in a decrease in m⁶A levels. It has been reported that m⁶A is involved in regulating lipid metabolism in HFD mice.³¹^,⁵²^,⁵³ Moreover, m⁶A modification modulates testosterone synthesis in LCs.⁵⁴ In this study, a progressive increase in METTL3 expression was detected in LCs during their differentiation from PLCs to ALCs. The m⁶A mRNA modification level and METTL3 expression were significantly decreased in the testes of HFD mice compared with that in chow-fed mice. Overexpression of Mettl3 suppressed miR-300-3p expression in LCs, while its inhibition upregulated miR-300-3p expression, which demonstrated that METTL3 regulates the transcription of miR-300-3p. It was reported that m⁶A regulates gene transcription or protein expression by regulating the methylation levels at a specific motif, such as “RRAC.”⁵⁵ However, the "RRAC" motif was not found in the miR-300 mRNA, indicating that m⁶A may regulate miR-300-3p expression through other molecules. We identified the m⁶A methylation modification sites of Smurf2, a ubiquitin E3 ligase of SMADs that negatively regulates TGF-β signaling by promoting ubiquitin degradation of phosphorylated SMAD2.⁵⁶ SMAD2 binds to the promoter of miR-300, thereby increasing miR-300 expression, while METTL3 downregulated p-SMAD2 by increasing Smurf2 transcription, thus inhibiting the transcription of miR-300-3p. Finally, we demonstrated that METTL3 expression is regulated by LH. When LCs were stimulated with LH at different concentrations, the levels of METTL3 increased in response to increasing concentrations of LH and miR-300-3p decreased significantly. This trend is consistent with the expression pattern that increased MEETL3 is accompanied by a gradual increase in serum LH and testosterone levels during LC differentiation from PLCs to ALCs. However, the mechanism through which LH regulates METTL3 expression remains unclear. It would be interesting to explore this issue in future research.

Together, our results highlight the vital role of the LH/METTL3/SMURF2/SMAD2/miR-300-3p axis in regulating LC differentiation and testosterone synthesis through the targeting of FoxO1 and SF-1 expression. The aberrant expression of miR-300-3p induces LC dysfunction and, thus, may serve as a diagnostic marker or therapeutic target for obesity-related testosterone deficiency.

Materials and methods

Animals

Kunming (KM) mice and C57BL/6J mice were purchased from the Experimental Animal Center of Guangdong Province, China. KM mice were used to isolate primary Leydig cells for in vitro expriments and C57BL/6J mice were used for in vivo experiments. Animals were maintained under 12-h light/dart cycle at controlled temperature (25 ± 1°C) and relative humidity of 50%–60%. Corresponding feed and drinking water were freely accessible. All animal experiments were conducted according to the National Institutes of Health guidelines for the care and use of animals and approved by the Institutional Animal Care and Use Committee of Jinan University (Approval No. 20200327-68).

Adult male volunteers recruited

Thirty adult men (aged 20–40 years) were recruited and divided into lean (BMI ≤24 kg/m², n = 11) and obesity (BMI ≥28 kg/m², n = 11) groups according to their BMI. Five milliliters of venous blood was collected from each person for serum testosterone detection and circulating RNA isolation. All experiments were performed in accordance with the Medical Ethics Committee of Sun Yat-Sen Memorial Hospital (Approval No.2021-YW-036).

HFD induced obesity model

Forty 4-week-old C57BL/6 mice were randomly divided into two groups, chow group and HFD group. All mice were fed with standard diet (26% kcal from fat) for 2 weeks to acclimatize before the experiment started. Then, mice of the chow group continued to be fed with standard diet while the mice of the HFD group were switched to an HFD (60% kcal from fat, D12492). The experiment was performed for 12 weeks, until the HFD mice were 20% heavier than the chow mice.

LCs isolation and culture

In rodents, fibroblast-like PLCs appear in the testis by postnatal day 14–21, while ALCs in testis are formed on postnatal days 49–56. Therefore, PLCs were isolated from 3-week-old male KM mice and ALCs were isolated from 8-week-old male KM mice.⁵⁷ Briefly, decapsulated testes were digested with collagenase type IV (Biosharp, China) at 1 mg/mL for 7 min at 37°C shaking water bath. The isolated cells were cultured in DMEM (Gibco, USA) with 10% FBS (ExCell Bio, China) for 1 h, and then the complete medium was changed to remove blood cells, spermatocytes, and Sertoli cells that adhered slowly. Subsequently, the adherent cells were cultured in a cell incubator at 37°C in 5% CO₂. After 24 h, the cells were treated with hypotonic solution (20 mM Tris, pH 7.4) for 2 min to remove myoid cells. Cells were continually cultured for another 48 h to obtain LCs for subsequent experiments.

Western blot

The cells and testicular tissues were lysed in RIPA lysis buffer (Fude Bio, China) with 1 mM PMSF and protein phosphatase inhibitor (Roche, USA), and protein concentration was detected by BCA. Thirty micrograms of protein were electrophoresed on 12% SDS-PAGE and then transferred onto PVDF membranes. SF-1, CYP11A1, StAR, FoxO1, AKT, p-AKT, CCND1, CCND2, SMURF2, METTL3, METTL14, and ALKBH5 antibodies were added and incubated overnight at 4°C, and the blots were incubated with a secondary antibody labeled with horseradish peroxidase (HRP) for 1 h. Finally, the immunoreactions were detected by enhanced chemiluminescence (ECL) reagent. Antibody information is listed in Table S1.

Testosterone measurements

The concentration of testosterone in conditioned medium of LCs and serum testosterone concentration were measured with Iodine [¹²⁵I] Testosterone Radioimmunoassay Kit (Bejing North Institute of Biotechnology, China).

Serum miR-300-3p assay

RNA extraction

Total RNA was extracted from 300 μL serum samples using Trizol reagent (Life, USA). The collected RNA was dissolved in 20 μL RNase-free water, and used Nanodrop 2000 (Thermo Scientific, USA) to determine the quality. The purity range of the samples was 1.8–2.0 at wavelength 260/280, and the yield range was 400–1000 ng.

RT and quantitative real-time PCR

The RT and qRT-PCR were performed using 2 × All-in-One qPCR kit (Ribobio, China). DNA synthesized in 4 μL 5 × PAP/TR Buffer, 1 μL RTase Mix, 1 μL 2.5U/μL Ploy A Polymerase, and 400 ng RNA by performing incubation at 37°C for 60 min, and at 85°C for 5 min. Cel-miR-39-3p was used as external control to evaluate the relative expression of miR-300-3p. Two microliters of cDNA was used as template placed in 20 μL total reaction volume containing 10 μL 2 × All-in-One qPCR Mix, 2 μL adaptor primer, 2 μL miR-300-3p primer, and 4 μL nuclease-Free water. Then qRT-PCR was done using BioRad CFX manager system (BioRad, USA) at initial activation of 95°C for 10 min, followed by 40 cycles of 95°C for 10 s and 60°C for 20 s and 72°C for 10 s. The relative expression of miR-300-3p was calculated by the 2-^ΔΔCt method after normalization to cel-miR-39-3p.

Testicular injection of miR-300-3p agonist and antagonist

miR-300-3p agomir, antagomir, and their respective control (NC agomir and NC antagomir) were purchased from Ribobio (Guangzhou, China); 50 nmol of nucleic acid drugs were dissolved in 400 μL PBS. Each testis was injected with 15 μL of nucleic acid containing 1.875 nmol siRNA through the rete testes into the interstitial space using a microinjection pump (SPLab01, DK infusetek). A microinjector was inserted into one-third of the testis from outside the scrotum of mice, and the injection parameter was set to the flow rate of 15 μL/min. After injection, the needle was left for 30 s until the drug was fully absorbed. The sequence of miR-300-3p agomir and antagomir is listed in Table S2.

Histological analysis

The testis samples were fixed with 4% paraformaldehyde and embedded in paraffin wax following standard procedures. Sections of 5-μm thickness were prepared and stained with H&E for regular histological analysis. Briefly, dried sections were deparaffinized and stained with hematoxylin, then destained using 70% ethanol. Sections were then stained with eosin, destained, and dehydrated in ascending ethanol solutions. Sections were rinsed in xylene then cover-slipped with synthetic mounting media. Bright field images of the stains were obtained using using an ECLIPSE Ni-U upright microscope (Nikon).

For in situ hybridization (ISH) detection of miR-300-3p, the digoxigenin-labeled probe and in situ hybridization kit (MK1030) were purchased from Boster (Wuhan, China). In brief, the sections were dewaxed in xylene, rehydrated in gradient alcohol and distilled water, and endogenous enzymes were inactivated in 3% H₂O₂. mRNA in the tissue was exposed after pepsin digestion for 20 min. Prehybridization was conducted for 2–4 h at 42°C, followed by miR-300-3p probe hybridization overnight at 42°C. Then unhybridized probes were washed away with saline sodium citrate, and non-specific adhesion sites were blocked with 5% bovine serum albumin (BSA) for 30 min at 37°C. Tissues were then incubated with biotinylated anti-digoxin antibody for 60 min at 37°C, followed by incubation with streptavidin biotin-peroxidase complex (SABC) for 30 min at 37°C, and incubation with HRP for 20 min at 37°C. Finally, DAB was used for chromogenesis and hematoxylin for nuclear counterstaining. Images were captured using an ECLIPSE Ni-U upright microscope (Nikon) and analyzed by ImageJ software.

For immunofluorescence, frozen testis sections were fixed in acetone for 5 min and treated with 0.5% Triton X- for 10 min. Non-specific adhesion sites were blocked with 5% BSA at room temperature for 1 h. Primary and secondary antibodies were diluted in 5% BSA at 1:200. Tissues were incubated with primary antibodies overnight at 4°C, and then with secondary antibodies for 1 h at room temperature. Nuclei were stained with DAPI for 10 min. Fluorescence signals were captured and analyzed with an ECLIPSE Ni-U upright microscope (Nikon) and ImageJ software. Antibody details are listed in Table S1.

miR-300 promotor activity assay

MiR-300 promotor activity was measured by dual luciferase assay. Briefly, the potential transcription factors and promoter regions of miR-300 gene were predicted by JASPAR database. Then, the sequences containing miR-300 promoter sequences (−587/−4) were mutated, and the WT and mutation sequences were constructed into the PGL3-Basic vector respectively. HEK293T cells were transfected with WT or mutation PGL3-Basic-300 vector, together with pcDNA3.1-Smad2 and PRL-TK. Transcriptional activity was determined by a luminometer, using a dual luciferase assay kit. Renilla luciferase (R-luc) was used to normalize firefly luciferase (F-luc) activity to evaluate reporter translation efficiency.

m⁶A methylation modification detection by colorimetric method

Total RNA from LCs of mice at 3, 4, and 5 weeks of age were isolated by Trizol reagent. Then, m⁶A modification levels were detected by m⁶A RNA methylation quantification kit (Epigentek, USA) according to the manufacturer’s instructions.

Luciferase reporter assay

Luciferase reporter assay was performed using the Dual Glo Luciferase Assay System Kit (Promega, USA) according to the manufacturer’s instructions. Briefly, HEK-293T cells were co-transfected with luciferase reporter vectors (psi-check 2, psi-check 2-WT, psi-check 2-MU1, psi-check 2-MU2, psi-check 2-MU3. psi-check 2-MU4. psi-check 2-MU5, or psi-check 2-MU6) and pcDNA 3.1-Mettl3 in a 96-well plate. After transfection for 48 h, cells were analyzed with the Dual Glo Luciferase Assay System Kit. F-luc was used to normalize R-luc activity to evaluate reporter translation efficiency.

Statistical analyses

All experiments were repeated at least three times, and data were expressed as the mean values ±SD. More than six sections from each testis were analyzed for morphological evaluation. Statistical analyses were performed with unpaired Student’s t test or one-way ANOVA for more than two groups. GraphPad Prism 8 was used for data processing and plotting. P values less than 0.05 were considered significant and were marked as follows: p < 0.05 (∗), p < 0.01 (∗∗) and p < 0.001 (∗∗∗).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

This work was supported by National Natural Science Foundation of China (No. U22A20277, 32170865, and 82071634); Natural Science Foundation of Guangdong Province (No. 2022A1515012178, 2020A1515110963); Guangdong Clinical Research Center for Metabolic Diseases (2020B1111170009); The Science and Technology Plan Project of Guangzhou (No. 201508020001, 2016A020214013, 201803010044, 201704YG066, 2020A1515011203, 202102020261, 202103030003); Guangzhou Science and Technology Program Key Project (No. 201803010044).

Author contributions

J.L.L. and D.R.C. conceived this project; J.Y.X., S.Y.W., and Y.L. performed and analyzed the experiments; C.Z.W. provided clinical samples; Z.Y.W., Y.Q.F., Y.L.L., M.R.H., J.X.D., and Y.X.W. analyzed data and recorded the experiments; Q.H.Z. contributed analytic tools; Y.Y. and Y.D.H. designed the research and wrote the paper.

Declaration of interests

The authors declare no competing interests.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.omtn.2023.03.016.

Contributor Information

Yan Yang, Email: yangyan107@jnu.edu.cn.

Yadong Huang, Email: tydhuang@jnu.edu.cn.

Supplemental information

Document S1. Figures S1–S4 and Tables S1 and S2

mmc1.pdf^{(1.2MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(9MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S4 and Tables S1 and S2

mmc1.pdf^{(1.2MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(9MB, pdf)}

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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PERMALINK

Role of miR-300-3p in Leydig cell function and differentiation: A therapeutic target for obesity-related testosterone deficiency

Jinlian Liang

Derong Chen

Ziyan Xiao

Siying Wei

Yuan Liu

Chengzhi Wang

Zhaoyang Wang

Yuqing Feng

Yaling Lei

Meirong Hu

Jingxian Deng

Yuxin Wang

Qihao Zhang

Yan Yang

Yadong Huang

Abstract

Graphical abstract

Introduction

Results

miR-300-3p suppresses testosterone synthesis in LCs by targeting Sf-1

Figure 1.

Figure 2.

miR-300-3p regulates LC proliferation by targeting FoxO1

The functions of miR-300-3p in vivo

Figure 3.

miR-300-3p is a therapeutic target for obesity-related testosterone deficiency

Figure 4.

N6-adenosine methylation regulates the expression of miR-300-3p in LCs via the SMURF2/p-SMAD2 axis

Figure 5.

METTL3 is regulated by LH

Figure 6.

Figure 7.

Discussion

Materials and methods

Animals

Adult male volunteers recruited

HFD induced obesity model

LCs isolation and culture

Western blot

Testosterone measurements

Serum miR-300-3p assay

RNA extraction

RT and quantitative real-time PCR

Testicular injection of miR-300-3p agonist and antagonist

Histological analysis

miR-300 promotor activity assay

m6A methylation modification detection by colorimetric method

Luciferase reporter assay

Statistical analyses

Data availability

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Contributor Information

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

m⁶A methylation modification detection by colorimetric method