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MT1-MMP in rat testicular development and the control of Sertoli cell proMMP-2 activation

Juliette Longin, Patricia Guillaumot, Marie-Agnès Chauvin, Anne-Marie Morera and Brigitte Le Magueresse-Battistoni*

Inserm U329, Hopital Debrousse, 29 rue soeur Bouvier, 69322 Lyon Cedex 05, France



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  		Fig. 1. Immunohistochemical localization of MT1-MMP in 19-day-old (A,B), 33-day-old (C-F) and 60-day-old (G,H) rat testis. MT1-MMP was uniformly distributed in 19-day-old testis (A) but it concentrated in the apical portions of the seminiferous tubules in 33-day-old testis (C). Late pachytene spermatocytes (E) and early spermatids (F) are intensely stained in 33-day-old rats. In adult rats (G), immunoreactivity differed according to the stages indicated in roman numerals, with sperm cells in stage VIII being the most strongly labeled. Controls in B, D and H show no staining. Bar, 350 µm for A-D,G,H and 110 µm for E,F. Abbreviation: bv, blood vessel.

 


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  		Fig. 2. Immunohistochemical localization of MT1-MMP in adult rat testis at stages VIII (A,B), XIII (C), X (D), II-III (E), II (F), III-IV (G), IV-V (H,I). Controls are B, D, F and I. White asterisks indicate immunostained elongated spermatids; arrowheads point to Sertoli cells. Bar, 50 µm.

 


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  		Fig. 3. Western blot analysis of MT1-MMP in purified testicular cells. Cell lysates of 5-day-old cultured peritubular (C.Pc) and Sertoli (C.Sc) cells, and of freshly isolated Sertoli cells (FI.Sc), spermatocytes (FI.SPC), spermatids (FI.SPT), or mixed germ cells (FI.CGc) were prepared. 40 µg of proteins were loaded on to 7.5% SDS-PAGE gels. (+) and (-) indicate whether or not trypsin has been used in the course of the germ cell isolation.

 


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  		Fig. 4. Analysis of MT1-MMP gene expression during testicular development and in purified testicular cells by RT-PCR (A,B). Total RNA was prepared from (A) whole testis at various ages (17.5 dpc and 1, 10, 20, and 60 days old) and (B) from adult rat liver (positive control), Sertoli cells (Sc), peritubular cells (Pc), Leydig cells (Lc), spermatocytes (SPC), spermatids (SPT) and crude germ cells (CGc). The expected size of the PCR product is 329 bp. A DNA ladder has been included in the gels. (C) Northern blot analysis of MT1-MMP in 10-day-old (lane 1) and 15-day-old (lane 2) rat testis, in cultured Sertoli (lanes 3, 4) or peritubular cells (lane 5), and in crude germ cells (lane 6), using total RNA (10-20 µg) except in lane 3 (3 µg of polyA(+)). The positions of the 28S and 18S are indicated.

 


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  		Fig. 5. RT-PCR analysis of MMP-2 and TIMP-2 expression during testicular development (A) and in purified testicular cells (B). Total RNA was prepared from whole testes recovered from rats of different ages and from various purified testicular cells. Symbols are as used in Fig. 4. Rat uterus was used as a positive control. A DNA ladder has been included in the gels.

 


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  		Fig. 6. FSH and MMP-2 gene expression. Sertoli cells were treated or not (control (c)) with FSH (50 ng ml-1) or 8-bromo-AMP (10-3 M) for 48 hours, after which RNA was extracted and 3 µg of PolyA(+) were analyzed by northern blot (A). Membranes were successively probed with MMP-2 and GAPDH. A representative blot is shown in the inset (lane 1, control; lane 2, FSH; lane 3, 8-bromo-cAMP). Data yielded by scanning three independent blots were normalized to the GAPDH signal and expressed as mean±s.e.m. of three dishes. P values against the control: *, 0.05; **, 0.01. (B) MMP-2 antigen levels were analyzed by western blot in the 72-hour culture media of Sertoli cells treated with or without FSH.

 


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  		Fig. 7. FSH and MT1-MMP gene expression. Sertoli cells were treated or not (control (c)) with FSH (50 ng ml-1) or 8-bromo-AMP (10-3 M) for 48 hours, after which RNA was extracted and 3 µg of polyA(+) were analyzed by northern blot (A). Membranes were successively probed with MT1-MMP and GAPDH. A representative blot is shown. (B) MT1-MMP antigen levels were analyzed by western blot in the lysates of Sertoli cells treated with FSH (+) or not (-) for 24, 48 or 72 hours.

 


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  		Fig. 8. FSH and TIMP-2 gene expression. Sertoli cells were treated or not treated with increasing doses of FSH ranging from 0.1 ng ml-1 to 100 ng ml-1, after which total RNA was extracted and 20 µg were analyzed by northern blots (A). Blots were hybridized successively with the TIMP-2 and 18S probes, and quantified. A representative blot is shown in the inset (lanes 1-6 correspond to cultures exposed to 0-100 ng ml-1 FSH for 48 hours). Data are expressed as mean±s.e.m. of triplicate dishes; control is lane 1, with no FSH. P value against the control: *, 0.05. (B) TIMP-2 antigen levels were analyzed by western blot in the culture media of Sertoli cells treated with (+) or without (-) FSH for 24, 48 or 72 hours.

 


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  		Fig. 9. Gelatin zymography of Sertoli cell culture media (A) and the action of FSH (B). (A) Purified human MMP-2 (lane 1, 1 ng) and Mr standards (lane 3) were used to determine the size of the lytic bands present in a 3-day-old Sertoli cell culture media (lane 2). (B) Sertoli cells were treated with FSH (50 ng/ml) (+) or not treated (-) for 24, 48 and 72 hours, after which culture medium was collected and 40 µg of proteins were analyzed by gelatin zymography. The sizes of the lytic bands are indicated on the right.

 


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  		Fig. 10. MMP-2 activation in Sertoli cells co-cultured with germ cells. (A) Western blot analysis of TIMP-2 in Sertoli cells (Sc), peritubular cells (Pc) and crude germ cells (CGc). 40 µg of proteins were deposited per lane on 15% SDS-PAGE. (B) Western blot analysis of MMP-2 in Sertoli cells (Sc) and in Sertoli cells co-cultured with crude germ cells (CGc), spermatocytes (SPC) or spermatids (SPT). 40 µg of proteins were analyzed per lane on 7.5% SDS-PAGE. (C) Sertoli/crude germ cells co-culture media were analyzed by gelatin zymography. 80 µg of proteins were analyzed per lane in lanes 1 and 40 µg in lanes 2. The sizes of the lytic bands are indicated on the right.

 

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