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Essential roles of COUP-TFII in Leydig cell differentiation and male fertility.

Qin J, Tsai MJ, Tsai SY - PLoS ONE (2008)

Bottom Line: Notably, the rescued results also provide the evidence that the major function of adult Leydig cell is to synthesize testosterone.On the other hand, when COUP-TFII is deleted in the adult stage after Leydig cells are well differentiated, there are no obvious defects in reproduction and Leydig cell function.Taken together, these results indicate that COUP-TFII plays a major role in differentiation, but not the maintenance of Leydig cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.

ABSTRACT
Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII; also known as NR2F2), is an orphan nuclear receptor of the steroid/thyroid hormone receptor superfamily. COUP-TFII- mice die during the early embryonic development due to angiogenesis and cardiovascular defects. To circumvent the early embryonic lethality and investigate the physiological function of COUP-TFII, we knocked out COUP-TFII gene in a time-specific manner by using a tamoxifen inducible Cre recombinase. The ablation of COUP-TFII during pre-pubertal stages of male development results in infertility, hypogonadism and spermatogenetic arrest. Homozygous adult male mutants are defective in testosterone synthesis, and administration of testosterone could largely rescue the mutant defects. Notably, the rescued results also provide the evidence that the major function of adult Leydig cell is to synthesize testosterone. Further phenotypic analysis reveals that Leydig cell differentiation is arrested at the progenitor cell stage in the testes of mice. The failure of testosterone to resumption of Leydig cell maturation in the mice indicates that COUP-TFII itself is essential for this process. In addition, we identify that COUP-TFII plays roles in progenitor Leydig cell formation and early testis organogenesis, as demonstrated by the ablation of COUP-TFII at E18.5. On the other hand, when COUP-TFII is deleted in the adult stage after Leydig cells are well differentiated, there are no obvious defects in reproduction and Leydig cell function. Taken together, these results indicate that COUP-TFII plays a major role in differentiation, but not the maintenance of Leydig cells.

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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.
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pone-0003285-g006: 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.

Mentions: To examine whether COUP-TFII is important for early testis organogenesis and progenitor Leydig cell formation, the pregnant mothers (E18.5) received tamoxifen treatment to induce the deletion of COUP-TFII in pups. At P7, immunohistochemistry analysis was performed to examine the deletion efficiency of COUP-TFII. As shown in Fig. 6A, COUP-TFII was highly expressed in the mesenchymal cells of testes before tamoxifen injection (E18.5). There were comparable expression levels between the control and mutant mice. In contrast, COUP-TFII was efficiently deleted in the P7 mutant mice, whereas it was highly expressed in the control mice. In terms of progenitor Leydig cell formation, we used 3β-HSD as the Leydig cell maker combining with cell localization and spindle-like shapes to distinguish the progenitor Leydig cells (arrow) from fetal Leydig cells (arrowhead). It is known that fetal Leydig cells arises soon after testis differentiation at about E12.5 in the mouse, and are essential for masculinization of the fetus [11]. Concurrent with the postnatal appearance and differentiation of adult Leydig cells, the number of fetal Leydig cells diminishes after birth [14]. We examined the fetal Leydig cells population in E18.5 testis to ascertain the tamoxifen independent activation of the Cre recombinase, and there was no difference between the control and mutant mice. At P7 and P14, significantly less progenitor Leydig cells were observed in the mutant testis (Fig. 6B; arrow and data not shown). However, there was no clear difference at P21 (Fig. 6B; arrow). Further quantitative analysis demonstrated progenitor Leydig cell formation was delayed in the mutant testis (Fig. 6C). In addition, we also isolated the reproductive organs from P60 animals, in which COUP-TFII was knocked out at E18.5. As expected, the mutant male mice displayed spermatogenesis arrest, Leydig cell hypoplasia and hypogonadism defects. As shown in Fig. 6D, spermatozoa (arrow) was absent in the testes and epididymis of the mutant mice. Furthermore, the barely detectable signals of EST and spindle-shape positive cells of 3β-HSD indicated the failure maturation of Leydig cells in mutant testes (Fig. 6D). Interestingly, we found the size of testes harvested from the P14 mutant animal was much smaller in comparison with its littermate controls, indicating that COUP-TFII also played important roles in early testis organogenesis (Fig. 6E).


Essential roles of COUP-TFII in Leydig cell differentiation and male fertility.

Qin J, Tsai MJ, Tsai SY - PLoS ONE (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553269&req=5

pone-0003285-g006: 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.
Mentions: To examine whether COUP-TFII is important for early testis organogenesis and progenitor Leydig cell formation, the pregnant mothers (E18.5) received tamoxifen treatment to induce the deletion of COUP-TFII in pups. At P7, immunohistochemistry analysis was performed to examine the deletion efficiency of COUP-TFII. As shown in Fig. 6A, COUP-TFII was highly expressed in the mesenchymal cells of testes before tamoxifen injection (E18.5). There were comparable expression levels between the control and mutant mice. In contrast, COUP-TFII was efficiently deleted in the P7 mutant mice, whereas it was highly expressed in the control mice. In terms of progenitor Leydig cell formation, we used 3β-HSD as the Leydig cell maker combining with cell localization and spindle-like shapes to distinguish the progenitor Leydig cells (arrow) from fetal Leydig cells (arrowhead). It is known that fetal Leydig cells arises soon after testis differentiation at about E12.5 in the mouse, and are essential for masculinization of the fetus [11]. Concurrent with the postnatal appearance and differentiation of adult Leydig cells, the number of fetal Leydig cells diminishes after birth [14]. We examined the fetal Leydig cells population in E18.5 testis to ascertain the tamoxifen independent activation of the Cre recombinase, and there was no difference between the control and mutant mice. At P7 and P14, significantly less progenitor Leydig cells were observed in the mutant testis (Fig. 6B; arrow and data not shown). However, there was no clear difference at P21 (Fig. 6B; arrow). Further quantitative analysis demonstrated progenitor Leydig cell formation was delayed in the mutant testis (Fig. 6C). In addition, we also isolated the reproductive organs from P60 animals, in which COUP-TFII was knocked out at E18.5. As expected, the mutant male mice displayed spermatogenesis arrest, Leydig cell hypoplasia and hypogonadism defects. As shown in Fig. 6D, spermatozoa (arrow) was absent in the testes and epididymis of the mutant mice. Furthermore, the barely detectable signals of EST and spindle-shape positive cells of 3β-HSD indicated the failure maturation of Leydig cells in mutant testes (Fig. 6D). Interestingly, we found the size of testes harvested from the P14 mutant animal was much smaller in comparison with its littermate controls, indicating that COUP-TFII also played important roles in early testis organogenesis (Fig. 6E).

Bottom Line: Notably, the rescued results also provide the evidence that the major function of adult Leydig cell is to synthesize testosterone.On the other hand, when COUP-TFII is deleted in the adult stage after Leydig cells are well differentiated, there are no obvious defects in reproduction and Leydig cell function.Taken together, these results indicate that COUP-TFII plays a major role in differentiation, but not the maintenance of Leydig cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.

ABSTRACT
Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII; also known as NR2F2), is an orphan nuclear receptor of the steroid/thyroid hormone receptor superfamily. COUP-TFII- mice die during the early embryonic development due to angiogenesis and cardiovascular defects. To circumvent the early embryonic lethality and investigate the physiological function of COUP-TFII, we knocked out COUP-TFII gene in a time-specific manner by using a tamoxifen inducible Cre recombinase. The ablation of COUP-TFII during pre-pubertal stages of male development results in infertility, hypogonadism and spermatogenetic arrest. Homozygous adult male mutants are defective in testosterone synthesis, and administration of testosterone could largely rescue the mutant defects. Notably, the rescued results also provide the evidence that the major function of adult Leydig cell is to synthesize testosterone. Further phenotypic analysis reveals that Leydig cell differentiation is arrested at the progenitor cell stage in the testes of mice. The failure of testosterone to resumption of Leydig cell maturation in the mice indicates that COUP-TFII itself is essential for this process. In addition, we identify that COUP-TFII plays roles in progenitor Leydig cell formation and early testis organogenesis, as demonstrated by the ablation of COUP-TFII at E18.5. On the other hand, when COUP-TFII is deleted in the adult stage after Leydig cells are well differentiated, there are no obvious defects in reproduction and Leydig cell function. Taken together, these results indicate that COUP-TFII plays a major role in differentiation, but not the maintenance of Leydig cells.

Show MeSH
Related in: MedlinePlus