Figure 1.
Inducible Ablation of COUP-TFII at Pre-puberty Stage Leads to Infertility and Hypogonadism.
A) Scheme of inducible ablation of COUP-TFII at Pre-puberty stage. Tamoxifen or oil was intraperitonealy injected into P14 animals to induce the deletion of COUP-TFII gene, and mice were sacrificed at P90. B) Immunoblotting of COUP-TFII was performed to examine the deletion efficiency in mutant mice, and tissues were collected from the comparison littermates. F/F, Tam: COUP-TFflox/flox treated with tamoxifen: Cre/+ F/F, Oil: Cre-ERTM (+/−) COUP-TFIIflox/flox treated with oil and Cre/+ F/F, Tam: Cre-ERTM (+/−) COUP-TFIIflox/flox treated with tamoxifen. C) The photograph depicts the appearance of male reproduction organs from 3-month-old littermate. D) Relative weight of reproduction organs normalized with body weight. Results are expressed as the mean (±SD) of the ratios for each genotype. Statistical comparison was done with a student test. * P<0.05, ** P<0.01
Table 1.
Fertility Assessment and Epididymis Sperm Count.
Figure 2.
Arrest of Spermatogenesis at the Round Spermatid Stage in the Null Mice.
H&E staining of paraffin-embedded testes (A, A') and epididymis (B). A' is the large magnification of the box area in the A. (C) Quantitative realtime RT-PCR analysis of the germ cell differentiation markers. RNA was isolated from 3-months-old littermates. Expression levels of each gene were normalized to the levels of the 18sRNA (n = 6). Data in (C) indicate the mean±SD. * P<0.05; ** P<0.01
Figure 3.
COUP-TFII Null Mice Display Leydig Cell Hypoplasia.
(A) Immunohistological detection of COUP-TFII expression in the testes from adult wild type mice. Green, COUP-TFII; Blue: DAP1 (B) COUP-TFII deletion efficiency was examined by qRT-PCR. Testes were collected from the littermates. N = 6; ** P<0.01 (C) Serum testosterone, LH, and FSH levels in 3-month-old males: F/F; Tam, Cre/+ F/F; Oil and Cre/+ F/F; Tam. (n = 8, 10, and 7, respectively). (D-I) H&E staining of paraffin-embedded testes (D). Immunohistochemistry results of Leydig cell marker P450Scc (E), EST (F), 3β-HSD (G) and CYP19 (H) indicated that mutant mice display Leydig cell hypoplasia. However, Sertoli cells in the null mice are normal (I).
Figure 4.
Hypogonadism and Spermatogenesis Defects were Rescued by Testosterone Replacement.
Gross appearance of testes and accessory glands after testosterone treatment. The size of testes, epididymis, seminal vesicles (A) and prostate (B) grew markedly in Cre/+ F/F mice implanted with testosterone pellet compared with the null mice implanted with control pellet. H& E staining demonstrates the resumption of spermatogenesis in the null mine treated with testosterone. The elongated spermatid or spermatozoa could be observed in the mutant testes (C, D) and epididymis (E). In addition, loss of secretary protein in seminal vesicles could be observed in the rescued mice (F; arrow). A, anterior prostate; V ventral prostate; DL, dorsal-lateral prostate.
Figure 5.
Indispensable Roles of COUP-TFII in Progenitor Leydig Cell Differentiation.
A) Evaluation of Leydig cell differentiation in the mutant testes, indicated by Leydig cell marker 3β-HSD immunostaining. The samples were collected at P14, P21, P28, P60 and P90, which correspond to 0, 7, 14, 46 and 76 days after tamoxifen injection, respectively. B) Testosterone treatment could not rescue the hypoplasia of Leydig cells. There were no increases in the intensity of P450Scc (C) and EST (D) signals or localization changes of 3β-HSD (B) signals in the rescued animal. E) qRT-PCR analysis of the expression of Leydig cell marker. RNA was isolated from 3-months-old littermates implanted with control or testosterone pellet (n = 6). Expression levels of each gene were normalized to the levels of the 18sRNA. Data in (E) indicate mean±SD. * P<0.05; ** P<0.01
Figure 6.
COUP-TFII Plays Roles in Testis Organogenesis and Progenitor Leydig cell Formation.
A) Immunohistochemistry for COUP-TFII at embryonic 18.5 (E18.5) and P7. Tamoxifen was injected to pregnant mothers at E18.5. B) Immunohistochemistry for Leydig cell markers, 3β-HSD at E18.5, P14 and P21. Arrow indicated progenitor Leydig cells, and arrowhead was fetal Leydig cells. C) Quantitative results of progenitor Leydig cell number peri-seminiferous tubule. Data in (C) indicate mean±SD. * P<0.05; ** P<0.01. D) H& E staining of the testes and epididymis from P60 littermate of control and mutant mice. Spermatazoa was indicated by arrow. Immunohistochemistry result for Leydig cell markers, 3β-HSD and EST. E) The photograph depicts the appearance of testes from control and mutant mice at E18.5 and P14.
Figure 7.
COUP-TFII is not Essential for Maintenance of Leydig Cell Function.
A) Histological examination of testis and epididymis. Spermatazoa (arrow) were observed in testes and epididymis of 4-month-old control and Cre/+ F/F mice, which were treated with tamoxifen at the age of two months. B) Immunohistochemistry for Leydig cell markers, 3β-HSD, P450Scc and EST. C) Immunoblotting for COUP-TFII, 3β-HSD, P450Scc and EST. Tam OD indicates the day before tamoxifen injection when mice are two month old. Tam 60D means 60 days after tamoxifen injection.