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Spermatogenesis-preventing substance in Japanese eel

¹ Marine Bioresources Research Group, Field Science Center for Northern Biosphere, Hokkaido University, Hakodate 041-8611, Japan
² Division of Marine Biosciences, Graduate School of Fisheries Science, Hokkaido University, Hakodate 041-8611, Japan

View larger version (89K): [in a new window]   Fig. 1. Comparison of the deduced amino acid sequence of eSRS21 with the sequence of Müllerian-inhibiting substance (MIS) from chicken (Eusèbe et al., 1996), human (Cate et al., 1986) and mouse (Münsterberg et al., 1991). This alignment was performed using Clustal W software (Thompson et al., 1994). Numbering of the amino acid residues relates to eSRS21. Red characters indicate residues that are conserved throughout all sequences. Grey characters indicate residues that are the same for 3/4 sequences. Asterisks indicate seven conserved cysteine residues. Dash represents a gap in the sequence introduced to maximise alignment; arrowhead indicates the signal sequence cleavage site. Underlined residues denote a possible N-glycosylation site.

View larger version (47K): [in a new window]   Fig. 2. Characterisation of eSRS21 protein using an anti-eSRS21 antibody. (A) Western blot analysis of the immature cultivated eel testis under non-reducing (lane 1) and reducing (lane 2) conditions. (B) Testicular immunoprecipitation using anti-eSRS21 (lane 1). Lane 2 shows the negative control without testicular samples. H and L indicate heavy and light chains of IgG, respectively. (C) Western blot analysis of CHO cells transfected with pSD(X)/esrs21c19 (lane 1) and its conditioned medium (lane 2) under reducing conditions. Numbers on the left represent molecular size markers (kDa).

View larger version (59K): [in a new window]   Fig. 3. eSRS21 mRNA expression in developing testes. Northern blot analysis was performed using poly(A)+ RNA extracted from the testes 0, 1, 3, 6, 9, 12, 15 and 18 days after hCG treatment. Northern blot of elongation factor 1 (EF1), which serves as reference, is given underneath the figure.

View larger version (39K): [in a new window]   Fig. 4. Western blot analysis of eSRS21 protein expression during hCG-induced spermatogenesis using an anti-eSRS21 antibody. Samples were obtained from the testis at 0, 1, 3, 6, 9, 12, 15 and 18 days after hCG treatment. Numbers on the left represent molecular size markers (kDa).

View larger version (134K): [in a new window]   Fig. 5. Cellular localisation of eSRS21 mRNA in the immature cultivated eel testis by in situ hybridisation. (A) Section hybridised with antisense probes. (B) Section hybridised with sense probes as a negative control. Sections were stained nuclei by 0.2% Methyl Green, metachromatically. G and S indicate germ cell and Sertoli cell, respectively. Scale bar: 10 µm.

View larger version (55K): [in a new window]   Fig. 6. Northern blot analysis of mRNA from cultured testicular fragments for eSRS21. Testicular fragments were cultured without (lane 2) or with (lane 3) 10 ng/ml 11-ketotestosterone for 6 days. Lane 1 shows the initial control before culture. Northern blot of elongation factor 1 (EF1), which serves as reference, is given underneath the figure.

View larger version (79K): [in a new window]   Fig. 7. The effects of recombinant-eSRS21 (r-eSRS21) on 11-ketotestosterone-induced spermatogenesis in Japanese eel in vitro. (A-D) Microphotographs show the testicular sections from fragments cultured in basal medium alone (A), and with r-eSRS21 (B), 11-ketotestosterone (C) and r-eSRS21 and 11-ketotestosterone together (D). GA and GB indicate type A spermatogonia and late type B spermatogonia, respectively. Scale bar: 50 µm. (E) BrdU index. The number of positively immunoreacted germ cells is expressed as a percentage of the total number of germ cells. IC, initial controls; 11-KT, 11-ketotestosterone. Results are given as the mean±s.e.m. Values with the same lowercase letter(s) are not significantly different (P<0.05).

View larger version (87K): [in a new window]   Fig. 8. The effect of anti-eSRS21 antibody treatment on spermatogonial proliferation using a germ-somatic cell co-culture system. (A,B) Microphotographs show the pellets of germ cells/somatic cells cultured with anti-eSRS21 IgG and IgG from normal rabbit serum, respectively. GA and GB indicate type A spermatogonia and late type B spermatogonia, respectively. Scale bar: 50 µm. (C) The rate of appearance of proliferated germ cells (late type B spermatogonia). IC, initial controls; C, controls; 11KT, 11-ketotestosterone; anti-eSRS21, anti-eSRS21 IgG. Results are given as the mean±s.e.m. Values with the same lowercase letter(s) are not significantly different (P<0.05).

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