Use of Organ Culture to Study the Human Fetal Testis Development: Effect of Retinoic Acid

The bone morphogenetic protein (BMP) and growth and differentiation factor (GDF) signaling pathways have well-established and essential roles within the developing skeleton in coordinating the formation of cartilaginous anlagen. However, the identification of bona fide targets that underlie the action of these signaling molecules in chondrogenesis has remained elusive. We have identified the gene for the retinoic acid (RA) synthesis enzyme Aldh1a2 as a principal target of BMP signaling; prochondrogenic BMPs or GDFs lead to attenuation of Aldh1a2 expression and, consequently, to reduced activation of the retinoid signaling pathway. Consistent with this, antagonism of retinoid signaling phenocopies BMP4 action, whereas RA inhibits the chondrogenic stimulatory activity of BMP4. BMP4 also down-regulates Aldh1a2 expression in organ culture and, consistent with this, Aldh1a2 is actively excluded from the developing cartilage anlagens. Collectively, these findings provide novel insights into BMP action and demonstrate that BMP signaling governs the fate of prechondrogenic mesenchyme, at least in part, through regulation of retinoid signaling.

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168. TGFβ 2-BETAGLYCAN REGULATE FOETAL TESTIS DEVELOPMENT IN VITRO

Reproduction Fertility and Development ◽

10.1071/srb10abs168 ◽

2010 ◽

Vol 22 (9) ◽

pp. 86

Author(s):

M. A. Sarraj ◽

A. Umbers ◽

J. K. Findlay ◽

K. L. Stenvers

Keyword(s):

Target Cells ◽

Wildtype Mouse ◽

Culture Methods ◽

Agar Block ◽

Testis Development ◽

Fetal Testis ◽

Methods Development ◽

Development In Vitro ◽

Tgfβ Superfamily

Betaglycan is a co-receptor for the TGFβ superfamily, known to modulate TGFβ binding in target cells. We have previously found that betaglycan null murine testes at 12.5-13.5 dpc display poorly delineated seminiferous cords and disrupted Leydig cell development (1). Both TGFβs and inhibins are expressed by the fetal testis and it is currently unclear which regulate its development. We tested the hypothesis that loss of betaglycan compromises the functions of TGFβ2 in the differentiating fetal testis as TGFβ2 is known to bind poorly to its type II receptor in the absence of betaglycan. We tested the effect of TGFβ2 on betaglycan wildtype and null foetal gonad/mesonephros complexes using hanging drop or agar block culture methods. From each embryo, one gonad acted as a control; the other was treated. Gonads were cultured in the presence or absence of TGFβ2 (2.5-5 ng/mL) for 48 hours (n =3 pairs). In both culture methods, development in the absence of exogenous growth factor recapitulated normal cord development in wildtype testis and the disrupted cord phenotype in null testes. TGFβ2-treated cultures, 13.5 dpc wildtype mouse testes displayed a 14-35% reduction in total area compared to untreated cultures. Null testes exhibited significantly smaller reductions in gonadal area (2-13%; P < 0.01), indicating that betaglycan null testes exhibit reduced sensitivity to TGFβ2-mediated growth inhibition. However, preliminary observations suggest that TGFβ2 treatment partly rescued cord formation in two of three betaglycan knockout testes in vitro, with testis morphology confirmed by laminin and AMH immunostaining. These data support the notion that TGFβ2 acts via betaglycan to regulate cord development during foetal testis development. Supported by the New Investigator NHMRC (AUS) grant #550915 to MS, JKF Fellowship (#441101, #550915, #338516;#241000) and Victorian Government infrastructure funds. (1) Sarraj et al., 2010. Biol Reprod; 82(1): 153–62.

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