Incorporation of Phosphatase Inhibitor in Culture Prompts Growth Initiation of Isolated Non-Growing Oocytes

<div><p><i>In vitro</i> folliculogenesis of primordial and early preantral follicles is necessary for increment of reproductive efficiency in domestic animals, humans and endangered species. Recent study in phosphatase and tensin homolog (<i>Pten</i>) -knockout mice has revealed that this phosphatase acts as an inhibitory factor in follicle activation of primordial pool with the resultant inhibition of oocyte growth. To test <i>in vitro</i> effect of a phosphatase inhibitor on growth initiation of isolated non-growing oocytes in neonatal ovaries, we applied a specific inhibitor (bpV (HOpic)) for PTEN in culturing system. Non-growing oocytes isolated from the ovaries of newborn BDF1 (C57BL/6 × DBA/2) pups were divided to four culture groups. Five days after culture, the oocytes in 14 μmol/l bpV only, 14 μmol/l bpV plus 100 ng/ml Kit Ligand (KL), and 100 ng/ml KL groups showed significantly (<i>P</i><0.05) growth (19.3±0.55, 25.8±0.53 and 21.6±0.29 μm, respectively) compared with that of the control (no additive) (16.9±0.53 μm). In addition, western blotting in those groups showed enhanced expression of phosphorylated Akt. In conclusion, we clearly demonstrate that isolated non-growing oocytes develop in phosphatase inhibitor, especially to PTEN, incorporated culturing system, and show first as we know that oocytes with zona Pellucidae can be obtained <i>in vitro</i> from isolated non-growing oocytes.</p> </div

<i>In</i><i>vitro</i> growth initiation of non-growing oocytes in PhosSTOP incorporated culture.

<p>(A) <i>In </i><i>vitro</i> growth rates and (B) average diameters of non-growing oocytes 24 hours after culture both were significantly higher in PhosSTOP group compared to those in the control (*<i>P</i><0.05). (C) The non-growing oocytes in the control group showed survival up to day 4 of culture, while in the PhosSTOP group they did not. There was a significant difference between two groups (a^,b<i>P</i><0.05). (D) Microphotograph of non-growing oocytes in the PhosSTOP and control groups after 24 hours of culture. The oocytes in both groups were together transferred in single culture dish for comparing their figures in the photograph. The circles show the oocytes in the PhosSTOP group and the square brace shows ones in the control. Bar=10 µm. (E) Western blotting of phosphorylated Akt shows strongly detection in the specimen from the ovaries cultured with PhosSTOP.</p

<i>In</i><i>vitro</i> growth initiation of non-growing oocytes in PTEN specific inhibitor, bpV incorporated culture.

<p>(A) <i>In </i><i>vitro</i> growth rates trended increase dependent on concentration of bpV, and were significantly higher in 14 and 140 µmol/l bpV groups (a^,b<i>P</i><0.05) (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077533#pone.0077533.s001" target="_blank">Table S1</a> in detail). (B) The oocytes in 0, 0.14, 1.4 and 14 µmol/l bpV groups showed survival up to day 4 of culture, while in the 140 µmol/l bpV group most of them survived during only two days after culture. (C) Average diameters of the oocytes in 14 and 14 µmol/l bpV cultured groups increased significantly during culture. (D) Western blotting of phosphorylated Akt shows strongly detection in the specimens from the ovaries cultured with bpV for 24 hours dependent on bpV concentration.</p

<i>In</i><i>vitro</i> growth of non-growing oocytes in bpV and Kit Ligand incorporated culture.

<p>(A) <i>In </i><i>vitro</i> growth rates in 14 µmol/l bpV with 100 ng/ml KL culture group increased significantly compare to other groups (a^,b<i>P</i><0.05) (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077533#pone.0077533.s002" target="_blank">Table S2</a> in detail). (B) Among culture groups, survival rates in both 14 µmol/l bpV with 100 ng/ml KL and only 100 ng/ml KL groups showed significantly higher compared to others throughout first 3 days of culture, and afterward the latter group sustained higher survival rate. (C) Average diameters of the oocytes in 14 µmol/l bpV with 100 ng/ml KL group greatly increased compared to another groups 5 days after culture. (D) Microphotograph of typical morphology in growing oocytes that formed zona pellucidae 6 days after culture in 14 µmol/l bpV with 100 ng/ml KL group. Bar=10 μm. (E) Western blotting of phosphorylated Akt shows detection in the ovaries cultured in bpV and KL incorporated medium for 24 hours.</p

Abnormalities in placentae derived from cloned embryos.

<p>(A) Placental weights from ICSI-derived control embryos and CB- or LatA-treated cloned embryos. Error bars indicate SD. Asterisks indicate significant difference at p < 0.05. (B) Hematoxylin and eosin staining of placentae derived from ICSI-derived control and CB- or LatA-treated cloned embryos. Abnormal distortion of the boundary between the spongiotrophoblast and labyrinth layers was observed in placentas derived from both CB- and LatA-treated cloned embryos. Bar = 500 μm.</p

Histone modifications in 1-cell stage embryos.

<p>(A-D) acH3K9, acH3K14, H3K9me2 or H3K4me2 levels in ICSI-generated and CB- or LatA-treated cloned embryos. Bar = 30 µm. (E) The intensities of immunofluorescence for acH3K9, acH3K14, H3K9me2 and H3K4me2 relative to that of H2B. They are compared with the intensities in ICSI-generated control embryos. The acetylation or methylation levels of these regions were not different between LatA- and CB-treated cloned embryos.</p

Chromosome segregation in early embryonic development.

<p>(A) Chromosomes were misaligned at metaphase and lagging chromosomes were found at anaphase in embryos ACS was occured. (B-D) Merged and bright field images of embryos with normal chromosomal segregation (NCS) and abnormal chromosomal segregation (ACS). As an example, time-lapse images of chromosome segregation at the first, second and third mitosis are shown at the 2-cell, 4-cell and 8-cell stages, respectively. Arrows indicate chromosomal fragments appearing during the division. Green, EGFP-α-tubulin; red, mRFP–H2B. Bar = 30 µm. (E) The percentages of embryos with ACS at the 2-cell, 4-cell and 8-cell stages in ICSI-generated and cloned embryos. Values with different superscripts in the same category differ significantly between ICSI-generated, CB- and LatA-treated cloned embryos.</p

Aberrant Gene Expression and Sexually Incompatible Genomic Imprinting in Oocytes Derived from XY Mouse Embryonic Stem Cells <em>In Vitro</em>

<div><p>Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) <i>in vivo</i> and <i>in vitro</i>. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis <i>in vivo</i>; however, <i>in vitro</i> differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development. To address this issue, a mouse ESC line, capable of producing follicle-like structures (FLSs) efficiently, was established to investigate their properties using conventional molecular biological methods. The results revealed that the ESC-derived FLSs were morphologically similar to ovarian primary-to-secondary follicles but never formed an antrum; instead, the FLSs eventually underwent abnormal development or cell death in culture, or formed teratomas when transplanted under the kidney capsule in mice. Gene expression analyses demonstrated that the FLSs lacked transcripts for genes essential to late folliculogenesis, including gonadotropin receptors and steroidogenic enzymes, whereas some other genes were overexpressed in FLSs compared to the adult ovary. The E-Cadherin protein, which is involved in cell-to-cell interactions, was also expressed ectopically. Remarkably, it was seen that oocyte-like cells in the FLSs exhibited androgenetic genomic imprinting, which is ordinarily indicative of male GCs. Although the FLSs did not express male GC marker genes, the DNA methyltransferase, <i>Dnmt3L</i>, was expressed at an abnormally high level. Furthermore, the expression of sex determination factors was ambiguous in FLSs as both male and female determinants were expressed weakly. These data suggest that the developmental dysfunction of the ESC-derived FLSs may be attributable to aberrant gene expression and genomic imprinting, possibly associated with uncertain sex determination in culture.</p> </div

<i>In vitro</i> maturation culture of ESC-derived FLSs.

<p>(A) Size variation of FLSs emerged in 3-day differentiation cultures of clone-G ESCs. The FLSs were separated through membrane filters with three different pore sizes (40, 70, 100 μm). Scale bar, 100 μm. (B) Prolonged culture of FLSs. Following the 3-day differentiation culture, floating FLSs were collected and transferred to another dish for further cultivation. Scale bar, 100 μm. (C) Wide-field view of prolonged FLS culture on day 16. Scale bar, 100 μm. (D) FLSs in prolonged culture on day 6. Arrowhead indicates a collapsed FLS, which underwent abnormal development. Scale bar, 100 μm. (E) FLSs developing abnormally in collagen gels emerged after 10 days of culture. Scale bar, 100 μm. (F) Maturation culture of ovarian follicles using the ‘one-drop, one-follicle’ method. Secondary follicles isolated from mouse ovaries were cultivated for 14 days. Arrowhead indicates an antrum. Scale bar, 100 μm.</p

Expression of genes associated with sex determination in ESC-derived FLSs.

<p>(A) Semi-quantitative RT-PCR analysis was performed to examine the expression of male GC marker genes (<i>Nanos2</i>, <i>Miwi</i>, <i>Dnmt3L</i>) and sex determination factors of male (<i>Sry</i>, <i>Sox9</i>, <i>Dmrt1</i>) and female (<i>Foxl2</i>) gonads. Only representative images from three escalated cycles are presented. Total RNA was prepared from RW-4 ESCs, clone-G ESCs, clone-G ESCs-derived FLSs (day 3), adult mouse ovary and testis. <i>β-Actin</i> was analyzed as an internal control and water was used as a negative control. (B) Gene expression in clone-G ESC-derived FLSs (day 3) was compared to those in embryonic (E19.0) and neonatal (P5) ovaries and in neonatal testis by semi-quantitative RT-PCR analysis as shown in (A).</p