Figure 1.
HE staining of testicular tissues with different spermatogenic conditions.
(A–B) Only sertoli cells were observed in seminiferous tubules. Johnsen score was 2.(C–D) Sertoli cells and few spermatogonia were seen in seminiferous tubules. Johnsen score was 3.(E–F) Sertoli cells, spermatogonia and few spermatocytes but not spermatids or mature spermatozoa were found in seminiferous tubules. Johnsen score was 4.(G–H) Spermatogenic cells at different stages were observed in seminiferous tubules, but arranged irregularly. Spermatids and mature spermatozoa are not seen. Johnsen score was 5.(I–J) Spermatogenic cells were disorganized in seminiferous tubules, but the number decreased significantly; few early spermatids were seen in seminiferous tubules, without any spermatozoa. Johnsen score was 6. (K–L) Spermatogenic cells and many early spermatids were found, but with no late spermatids and spermatozoa. Johnsen score was 7.(M–N) Few of late spermatids and mature spermatozoa were found in seminiferous tubules. Johnsen score was 8.(O–P) Many late spermatids and mature spermatozoa can be seen. Johnsen score was 9. Figures of right side were amplified from the left ones. Sc, Sertoli cell. Spg, Spermatogonia. Spc, Spermatocyte. Spd, Spermatid. Sp, Sperm. Scale bar: 50 μm or 100 μm as indicated in the right-down corner.
Figure 2.
Differential expressions of HLA-G in testicular tissues with various spermatogenic conditions.
Expressions of HLA-G mRNA in testicular tissues with various Johnsen scores (A–B). HLA-G expressions were normalized with the reference gene GAPDH and with the sample which Johnsen score is 2. NS means no significant. *, ** and *** represent P<0.05, P<0.01 and P<0.001 respectively.
Figure 3.
HLA-G mRNA expression between sperm from normal semen and testicular tissues retrieved through testicular sperm extraction.
A total of 9 normal semen samples and 34 testicular tissues with Johnsen scores of 8–9 were enrolled. Expressions of HLA-G mRNA in normal sperm were normalized with GAPDH, and HLA-G expressions were standardized with one of the testicular tissue in which HLA-G expression was at the middle level. The small picture shown at top right corner demonstrated no significant difference of HLA-G mRNA expression between normal sperm and testicular tissues. NS represents no significant difference.
Figure 4.
Relationship between HLA-G expression in testicular tissues and oocyte fertilization, embryo cleavage or high-quality embryo rate after ICSI treatment.
Except for oocytes, the expression of HLA-G mRNA were detected in 3PN zygotes, embryos and blastocysts were detected (A). HLA-G mRNA expression in tissues from testicular extraction had no effect on fertilization rate (B), embryo cleavage rate (C), or high-quality embryo rate (D) after ICSI treatment. HLA-G mRNA expression was normalized with GAPDH, and all of the samples were standardized with the data used in Fig. 3. HLA-G mRNA expression was normalized with GAPDH, and HLA-G expression in embryos and blastocysts was standardized with that in zygotes. * means P<0.05.
Figure 5.
Gene expression in in vitro cultured and HLA-G silenced embryos.
(A) Expressions of HLA-G mRNA in one-cell zygotes and 2-to 4-cell embryos at 48 h post-fertilization. (B) HLA-G mRNA expression in embryos with different numbers of blastomeres at 72 h post-fertilization. HLA-G mRNA expression was normalized with GAPDH, and standardized with expression in one-cell zygotes 48 h post-fertilization (A) and 2–4 cell embryos 72 h post-fertilization (B). (C) The expressions of HLA-G, SLC2A1, PLAC8 and CCND2 were normalized with GAPDH, standardized with the expressions in si-CONTROL group. NS means no significant difference. * and ** represent P<0.05 and P<0.01 respectively.
Table 1.
Embryo cleavage after microinjection of HLA-G siRNA.