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. 2022 Oct 20;3(11):100792. doi: 10.1016/j.xcrm.2022.100792

Figure 1.

Figure 1

Testicular injection of AAV8-Lhcgr rescues Leydig cell function and recovers testosterone levels in pubertal Lhcgr−/− mice

(A) Schematic of the AAV vector used in the study.

(B) Experimental overview of the in vivo studies.

(C) Quantitative RT-PCR was used to quantify Lhcgr mRNA transcripts in testicular tissues from Lhcgr+/+ mice, Lhcgr+/− mice, and Lhcgr−/− mice injected with PBS or increasing doses of AAV8-Lhcgr (n = 4). β-actin was used for normalization.

(D) Representative images of LHCGR in the testes of Lhcgr+/+ mice, Lhcgr+/− mice, and Lhcgr−/− mice injected with PBS or AAV8-Lhcgr (n = 4). Nuclei were counterstained with DAPI. NC, negative control. Scale bar: 25 μm.

(E) The concentrations of serum testosterone were analyzed in Lhcgr+/+ mice (n = 5), Lhcgr+/− mice (n = 5), and Lhcgr−/− mice injected with PBS (n = 3) or increasing doses of AAV8-Lhcgr (8 × 109 gc/testis [n = 4], 4 × 1010 gc/testis [n = 5], 8 × 1010 gc/testis [n = 5], and 2 × 1011 gc/testis [n = 4]).

(F) The concentrations of intratesticular testosterone were detected in Lhcgr+/+ mice (n = 6), Lhcgr+/− mice (n = 6), and Lhcgr−/− mice injected with PBS (n = 5) or AAV8-Lhcgr (n = 5).

(G) The concentrations of serum dihydrotestosterone testosterone were analyzed in Lhcgr−/− mice injected with PBS or AAV8-Lhcgr (n = 5).

(H and I) The concentrations of serum LH (H) and FSH (I) were analyzed in Lhcgr+/+ mice, Lhcgr+/− mice, and Lhcgr−/− mice injected with PBS or AAV8-Lhcgr (n = 5).

(J) The concentrations of serum testosterone were analyzed before and 1 h after hCG injection in Lhcgr+/+ mice (n = 4), Lhcgr+/− mice (n = 4), and Lhcgr−/− mice injected with PBS (n = 5) or AAV8-Lhcgr (n = 5).

(K) CYP17A1 was evaluated by immunostaining of testes from Lhcgr+/+ mice, Lhcgr+/− mice, and Lhcgr−/− mice injected with PBS or AAV8-Lhcgr (n = 4). NC indicates negative control. Scale bar: 50 μm.

(L) CYP17A1+ cells were quantified in the different groups. Data are represented by boxplots, and whiskers show the minimum to maximum values. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant.