Fig 2: A model for spermatogenic arrest in xenografted testis. Sertoli cell secretory factors (GDNF, SCF, and INHBB), tight-junction transmembrane protein (CLDN11), and transcription regulators (WT1 and SOX9) expression create an intricate balance, which leads to proliferation and differentiation of spermatogonia (marked by UCHL1 expression). The gonadotropins FSH and LH play a critical role in the progression of spermatogenesis via their respective receptors (FSHR and LHR). Testosterone production in Leydig cells is regulated by LH, which controls steroidogenesis. In xenografts, elevation of SCF and GDNF expression leads to survival and proliferation of Sertoli cells but not spermatogonia, leading to loss of spermatogonia, as indicated by downregulation of UCHL1 expression. Downregulation of WT1 and SOX9 and elevation of CLDN11 indicate dysregulation of Sertoli cell-specific protein expression. An elevated serum FSH level of recipients and loss of FSHR from xenografts specifies disruption in the endocrine milieu. Dysregulation of INHBB expression may have caused an elevated FSH level. Although downregulation of LHR expression is apparent, LH and testosterone levels are restored in recipients. An elevated HSD3B level suggests hyperplasia of Leydig cell in xenografts responsible for the restoration of testosterone level in recipients.

Fig 3: Overexpression of Cldn11 enhances OB differentiation and its intracellular signaling.a–e Primary OB precursor cells were cultured in osteogenic medium containing AA (50 µg/mL) and ß-gly (10 mM). a Cells were cultured for the indicated times, and mRNA levels of Cldn11 were analyzed by quantitative real-time RT-PCR. b OBs transfected with pMX or pMX-Cldn11 were cultured for 7 days in osteoblastogenic medium. ALP-positive cells were monitored by ALP staining, and ALP activity was expressed a function of released p-nitrophenol. c Calcium deposits for matrix mineralization were measured by ARS staining after 21 days, and the intensity of staining was quantified with CPC. d Total cell lysates from OBs transfected with pMX or pMX-Cldn11 were analyzed by performing western blotting with the indicated antibodies. ß-actin was used as an internal control. e Cells were cultured for the indicated times, and the mRNA expression of the indicated genes was analyzed by performing quantitative real time RT-PCR. Data are presented as the mean ± SD of three independent experiments. **P < 0.01; ***P < 0.001 vs. pMX (control) of the indicated time point, respectively

Fig 4: The inhibitory effect of Cldn11 overexpression on OC differentiation and bone resorption was not associated with the expression of OPG and RANKL in OBs.a Primary OBs were transfected with pMX or pMX-Cldn11 and cultured in a medium containing IL-1 (10 ng/mL) or PGE2 (10−6 M) and VitD3 (10−8 M) for 1 day. Quantitative real time RT-PCR was performed to determine the mRNA expression of OPG and RANKL. b OBs and BMCs transfected with pMX or pMX-Cldn11 were co-cultured for 7 days in a medium containing IL-1 (10 ng/mL) or PGE2 (10−6 M) and VitD3 (10−8 M). After culturing, cells were fixed, and the number of TRAP-positive MNCs (nuclei >5) was counted. c The mRNA level of Cldn11 was assessed by quantitative real time RT-PCR during osteoclastogenesis in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL). d BMMs transfected with pMX or pMX-Cldn11 were cultured in the presence of the indicated concentrations of RANKL. After 4 days, GFP expression to confirm the efficiency of retrovirus was visualized under a fluorescence microscope, and cultured cells were stained for TRAP. e The number of TRAP-positive MNCs (nuclei >5) per well was counted. f Mature OCs transfected with pMX or pMX-Cldn11 from the co-culture system were seeded in a 48-well plate for 48 h, in a hydroxyapatite-coated plate for 24 h, or in dentin slices for 48 h. The cells attached to the 48-well plate were stained with TRAP solution (left), and those attached to the hydroxyapatite-coated plate (middle) and dentin slices (right) were removed. Then, the plates were photographed under a light microscope. g The number of TRAP-positive MNCs (nuclei >5) was counted (left), and relative resorption areas in the hydroxyapatite-coated plate and dentin slices were quantified using Image-Pro Plus (Ver 4.5) software (right). Data are presented as the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. (not significant) vs. the pMX (control)

Fig 5: The effect of Cldn11 in LPS-induced bone destruction and bone formation calvarial mouse models in vivo.a–d To evaluate the effect of Cldn11 on bone loss in vivo, LPS (500 μg/mouse) or an equal volume of PBS was injected subcutaneously into the calvaria of 5-week-old ICR mice. Recombinant protein of Cldn11 (0.6 mg/mouse) was injected subcutaneously daily for 9 days into the calvaria of LPS-treated mice. e, f Additionally, to investigate the effect of Cldn11 on bone formation in vivo, the same dosage of Cldn11 protein (0.6 mg/mouse) was injected subcutaneously daily for 21 days into the calvaria of normal ICR mice. a TRAP staining of the whole calvaria. b, e Measurements to determine the volume at the treated site and to assess bone formation were carefully performed using 3D image calculator software. The degree of bone density was arbitrarily determined in ROI as follows: low-density (threshold 1101–1600, blue), middle-density (threshold 1601–2100, orange), and high-density (threshold >2100, red). c Histological sections of calvarial bones were stained with hematoxylin and eosin (H&E) and TRAP. d The number of OCs per field was quantified using Image-Pro Plus (Ver 4.5) software. f Histological sections of calvarial bones were stained with H&E. The area of new bone formation was measured using histomorphometric analysis. Data are presented as the mean ± SD. n = 5–6 mice per group. *P < 0.05; **P < 0.01 vs. the control group, #P < 0.05; ###P < 0.001 vs. the LPS group