Differential expression of tissue inhibitor of metalloproteinases type 1 (TIMP‐1) during mouse gonad development

Abstract

In mammals, the gene Sry initiates signaling pathways triggering the differentiation of a testis from a sexually indifferent gonad. Assuming that these morphogenetic events may alter the proteolytic balance, the expression of matrix metalloproteinases (MMPs) and inhibitors (TIMPs) was investigated in gonads from 11.5 days postcoitum (dpc) onward, when testicular organogenesis occurs. Whereas selective MMPs and TIMPs (1-3) were detected in undifferentiated gonads (11.5 dpc) and in neonatal testes, a single TIMP (TIMP-1) was expressed in a sexually dimorphic manner from 12.5 dpc onward (i.e., after overt male gonad differentiation), demonstrated by using a semiquantitative reverse transcriptase-polymerase chain reaction and a Western blot analysis. To gain insight into the role of TIMP-1, the expression of gelatinases (mRNA levels and enzyme activity) was monitored. However, no sex differences could be evidenced, indicating that TIMP-1 was not inhibiting this class of MMPs during testis organogenesis. Apart from being an inhibitor of MMPs, TIMP-1 is known to display growth promoting activities. Of interest, testicular TIMP-1 (but not TIMP-2) levels were further enhanced up to 2 weeks of age, consistent with a role in the early postnatal testicular growth. We, therefore, established an organotypic culture system in which seminiferous cords may differentiate de novo and grow, depending on culture conditions. In that system and mimicking the in vivo situation, TIMP-1 immunolocalized strongly within the male gonadal territory and weakly in female gonads, in which no organization was evident. Experiments are now under way to determine to what extent timp-1 is a morphogenic gene involved in seminiferous cord formation and development. Developmental Dynamics 227:357-366, 2003.

Serine proteases and serine protease inhibitors in testicular physiology: the plasminogen activation system

“…Such a migration is accompanied by extensive restructuring. Accordingly, major sex-related differences in the distribution of ECM components and the expression of proteases and inhibitors have been reported, including the SERPINs E2 and A5 (Nef et al 2005, Wilhelm et al 2007, and TIMP-1 (Guyot et al 2003).…”

Fibroblast growth factor (FGF) 2 and FGF9 mediate mesenchymal–epithelial interactions of peritubular and Sertoli cells in the rat testis

“…The procedure for RNA extraction and RT-PCR has been described previously (Longin et al 2001, Guyot et al 2003. Total RNA was extracted from whole testes, liver and kidney recovered from 20-day-old Sprague-Dawley rats.…”

“…Proteins (10-20 µg/lane measured by BCA protein assay (Pierce-Interchim, Montluçon, France)) were electrophoresed at 4 C on 10% polyacrylamide gels containing 1 mg/ml gelatin (Sigma Chemical Co.) in the absence of any reducing agent (Longin et al 2001, Guyot et al 2003. Following electrophoresis, SDS was removed from the gel by exchange in Triton X-100 (two washes of 30 min at room temperature in 2·5% Triton X-100 followed by two washes of 20 min in distilled water).…”

“…SDS-PAGE and Western blotting were carried out as reported earlier (Longin et al 2001, Guyot et al 2003, using either (i) tenfold concentrated cell culture media for the analysis of TIMPs-1 and -2 and PAI-1 or (ii) cell lysates prepared using PBS containing 1% NP-40 and 5 mM EDTA for the analysis of TIMP-3. Briefly, proteins were separated by electrophoresis performed on 10% (15% for TIMP-2) polyacrylamide gels under reducing conditions.…”

A comprehensive survey of the laminins and collagens type IV expressed in mouse Leydig cells and their regulation by LH/hCG

“…Procedure for RNA extraction and RT-PCR has been described elsewhere (Longin et al 2001, Guyot et al 2003. Briefly, specific primers were designed using the Gene Jockey sequence processor (Biosoft, Cambridge, Cambs, UK) and primer3 software, and the optimal temperature of annealing was defined for each couple of primers (Table 1).…”