Histology and Hematoxylin and Eosin staining of teratoma tissue from ViO-ES9 cells showing differentiation into tissues indicative of the three germ layers (A) including secretory epithelium (i), articular cartilage (ii) and keratinized epithelium (iii).

<p>Immunoflorescent analysis of differentiation into all three germ layers (B): AFP (i); GATA-4 (ii); and NESTIN (iii). Secondary antibodies were labelled with Alexa Fluro® 488 (green) except for GATA-4 which was labelled with Alexa Fluro® 594 (red). Nuclei are stained with DAPI (blue). RT-PCR for Flk-1, VE-Cadherin, PECAM, Vimentin and Nestin (C).</p

Karyotype analysis of ES cells and live offspring derived from vitrified oocytes.

<p>A) Karyotype analysis performed on ViO-ES9 cells revealed a number of abnormalities. The male cell line has a chromosome count of 46. B) Karyotype analysis performed on the mice that were generated from vitrified oocytes revealed normal karyotypes, a representative karyotype for one of the male mice shows a normal 40 XY chromosome count with no abnormalities.</p

Survival, two-cell and blastocyst rates for vitrified mouse oocytes following IVF.

*<p>Fisher's test significantly lower than other treatments (P&lt;0.05);</p>**<p>Fisher's test significantly higher than other treatments within the same column (P&lt;0.001). IVF Data collected from 3 replicates.</p

Following in vitro fertilization and culture, both fresh (A1) and vitrified oocytes in 0.1 M trehalose (B1) were capable of developing to the two-cell (A2; B2), four-cell (A3; B3) and blastocyst (A4; B4) stages, respectively.

<p>Furthermore, blastocysts were able to hatch (A5; B5) without chemical or mechanical assistance and some were used to derive embryonic stem cells.</p

Survival, and live offspring rates for vitrified oocytes.

<p>Fisher's test indicated no significant differences between the treatments within same column (P&gt;0.05). Oocyte and live offspring data collected from 5 and 2 replicates respectively.</p

Survival, vitrified two-cell embryo and live offspring rates from either fresh or vitrified oocytes.

<p>Fisher's test indicated no significant differences between the treatments within same column (P&gt;0.05).</p

Analysis of the SSC population in Gilz KO testis.

<p>A: Immunohistochemistry for PLZF on testis sections from mice of the indicated postnatal ages (days). Higher magnification insets show the presence of PLZF⁺ cells in the periluminal region of day 6 tubules. B: Immunohistochemistry for SALL4 on testis sections from post-natal day 30 mice. Asterisks in A and B indicate day 14 and 30 tubules containing PLZF and SALL4-positive cells respectively. Scale bars represent 50 µM.</p

Effect of GILZ deficiency on FOXO1 activity during spermatogenesis.

<p>A: Day 6 old WT and Gilz KO mouse testes were used in immunofluorescence analysis to assess the localization of FOXO1 (green) and GILZ (red) using confocal microscopy (DAPI-stain nuclei shown in blue). High magnification inserts showing localization of FOXO1 and GILZ were also included. Scale bars represent 100 µM and insert scale bars represent 25 µM. B: Quantitative analysis of nuclear FOXO1 positive cells in day 6 old WT and Gilz KO testes. Data represents the mean ± SEM of 6 testes. **, p&lt;0.01. C: Quantitative PCR analysis of <i>Ret</i>, <i>Lhx</i>, <i>Egr4</i>, Sall4, Dppa4 and Bim mRNA expression in day 6, 10 and 14 old WT and Gilz KO testes. Results are expressed as the number of mRNA copies per 10⁶ 18 s rRNA copies. Data represents the mean ± SEM of 4–8 mice per group. **, p&lt;0.01 related to WT controls. D: Protein expression of BIM-EL (extra long) and GILZ was detected in day 20 old WT and Gilz KO testes using Western blotting. Representative images of two individual experiments.</p

Effects of GILZ deficiency on blood-testis barrier and testicular leukocyte.

<p>A: Immunostaining for the blood-testis barrier marker espin was performed on day 20 WT and Gilz KO testes using anti-espin antibody. Scale bars represent 100 µM. Single insert represents the no espin staining negative control. B: Blood-testis-barrier (indicated by arrows) in day 20 old WT and Gilz KO testes samples were examined using transmission electron microscopy. Scale bars represent 5 µM. C: The presence of leukocytes in adult (day 70) WT and Gilz KO testes was examined by immunohistochemistry using CD45 as a marker. Scale bars represent 100 µM.</p

Primer sequences for qPCR.

<p>Primer sequences for qPCR.</p