Loss- and gain-of-function analyses reveal the essential role of Cyp19a1 in ovarian determination of the red-eared slider turtle.

Estrogen signaling exerts a decisive role in female sex determination and differentiation in chicken and fish. Aromatase encoded by Cyp19a1 is the key enzyme that catalyzes the conversion of androgen to estrogen. Correlative analyses implicate the potential involvement of aromatase in reptilian sexual development, however, the direct genetic evidence is lacking. Herein, we found that Cyp19a1 exhibited temperature-dependent sexually dimorphic expression, and located in the medullary somatic cells in early female embryos of the red-eared slider turtle (Trachemys scripta elegans), before the gonad is distinct. To determine the functional role of Cyp19a1 in turtle ovarian determination, we established loss- and gain-of-function models through in ovo lentivirus-mediated genetic manipulation. At female-producing temperature, inhibition of aromatase or knockdown of Cyp19a1 in turtle embryos resulted in female-to-male sex reversal, with the formation of a testis-like structure and a male distribution pattern of germ cells, as well as ectopic expression of male-specific markers (SOX9 and AMH) and disappearance of ovarian regulator FOXL2. On the contrary, overexpression of Cyp19a1 at male-producing temperature led to male-to-female sex reversal. In conclusion, our results suggest that Cyp19a1 is both necessary and sufficient for ovarian determination in the red-eared slider turtle, establishing causality and a direct genetic link between aromatase and reptilian sex determination and differentiation.

ESR1 mediates estrogen-induced feminization of genetic male Chinese soft-shelled turtle†.

Exogenous estrogen have shown their feminization abilities during the specific sex differentiation period in several reptiles. However, the specific regulatory mechanism and downstream regulatory genes of estrogen remain elusive. In the present study, 17ß-estradiol (E2), as well as drugs of specific antagonists and/or agonists of estrogen receptors, were employed to figure out the molecular pathway involved in the E2-induced feminization in Chinese soft-shelled turtles, an important aquaculture species in China. E2 treatment led to typical female characteristics in the gonads of ZZ individuals, including thickened outer cortex containing a number of germ cells and degenerated medullary cords, as well as the disappearance of male marker SOX9, and the ectopic expression of ovarian regulator FOXL2 at the embryonic developmental stage 27 and 1 month after hatching. The specific ESR1 antagonist or a combination of three estrogen receptor antagonists could block the sex reversal of ZZ individuals induced by estrogen. In addition, specific activation of ESR1 by agonist also led to the feminization of ZZ gonads, which was similar to the effect of estrogen treatment. Furthermore, transcriptome data showed that the expression level of FOXL2 was significantly upregulated, whereas mRNA levels of DMRT1, SOX9, and AMH were downregulated after estrogen treatment. Taken together, our results indicated that E2 induced the feminization of ZZ Chinese soft-shelled turtles via ESR1, and decrease of male genes DMRT1, SOX9, and AMH and increase of ovarian development regulator FOXL2 might be responsible for the initiation of E2-induced feminization.

G protein-coupled receptor 30 mediates estrogen-induced proliferation of primordial germ cells via EGFR/Akt/ß-catenin signaling pathway.

In vertebrates, estrogens are required for the normal development and function of postnatal gonads. However, it remains unclear whether estrogens are able to modulate development of the fetal germ cells. Here, we show that, unexpectedly, chicken primordial germ cells (PGC) lacking estrogen receptor α/ß still proliferate in response to 17ß-estradiol (E(2)). This is due to the capacity of G protein-coupled receptor 30 (GPR30), existing on PGC, to directly bind E(2). Knockdown experiments suggest that GPR30 is required for E(2)-stimulated PGC proliferation. Furthermore, this estrogen-induced activation of GPR30 is revealed to occur through the Gßγ-subunit protein-dependent and through the matrix metalloproteinase-dependent transactivation of the epidermal growth factor receptor. Epidermal growth factor receptor activation results in a series of intracellular events, including activation of the phosphatidylinositol 3-kinase/serine-threonine kinase/ß-catenin pathway, which are followed by the induction of c-fos, c-myc, cyclin D1/E, and B-cell lymphoma 2 expression, and the inhibition of B-cell lymphoma 2-associated X protein expression and caspase3/9 activity. This eventually leads to decreased apoptosis and increased PGC proliferation. Collectively, these findings offer novel insights into the dynamic mechanism of estrogen action on PGC proliferation and suggest that E(2)/GPR30 signaling might play an important role in regulating fetal germ cell development, particularly at the stage before sexual differentiation.

Retinoic acid promotes proliferation of chicken primordial germ cells via activation of PI3K/Akt-mediated NF-κB signalling cascade.

As embryonic progenitors for the gametes, PGCs (primordial germ cells) proliferate and develop under strict regulation of numerous intrinsic and external factors. As the most active natural metabolite of vitamin A, all-trans RA (retinoic acid) plays pivotal roles in regulating development of various cells. The proliferating action of RA on PGCs was investigated along with the intracellular PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B; also known as Akt)-mediated NF-κB (nuclear factor κB) signalling cascade. The results show that RA significantly promoted PGC proliferation in a dose- and time-dependent manner, confirmed by BrdU (bromodeoxyuridine) incorporation and cell cycle analysis. However, this promoting effect was attenuated by sequential inhibitors of LY294002 for PI3K, KP372-1 for Akt and SN50 for NF-κB respectively. Western blot analysis showed increased Akt phosphorylation (Ser473) of PGCs after stimulation with RA, but this was abolished by LY294002 or KP372-1. Treatment with RA increased expression of NF-κB and decreased IκBα (inhibitory κBα) expression, which were inhibited by SN50. Blockade of PI3K or Akt activity inhibited NF-κB translocation from the cytoplasm to the nucleus. Finally, mRNA expression of cell cycle regulating genes [cyclin D1 and E, CDK6 (cyclin-dependent kinase 6) and CDK2] was up-regulated in the RA-treated cells. This stimulation was also markedly retarded by combined treatment with LY294002, KP372-1 and SN50. These results suggest that RA activates the PI3K/Akt and NF-κB signalling cascade to promote proliferation of the cultured chicken PGCs.

Induced multilineage differentiation of chicken embryonic germ cells via embryoid body formation.

Although the pluripotent and proliferative capacity of embryonic germ (EG) cells is thought to be equivalent to that of embryonic stem (ES) cells, there has been far less attention focused on the potential use of EG cells for applications in developing novel strategies of tissue transplantation in the treatment of degenerative diseases. In this study, EG cells were derived from primordial germ cells (PGCs) of genital ridges of 4-day-old chicken embryos. These cells satisfied the criteria previously used for defining chicken EG cells by using the expression of markers characteristic to ES cells. When injected subcutaneously, chicken EG cells could form teratomas that enable differentiation into a wide range of tissue types of all three primary cell lineages including neural cells, cartilage, forming bone, adipocytes, blood vessels, smooth muscle, and secretory epithelia in the recipients. Furthermore, cells in embryoid bodies (EBs) expressed lineage-specific markers of three germ layers and could be induced to differentiate into more advanced stages of various committed cell types, including dopamine and cholinergic neurons, astrocytes, oligodendrocytes, adipocytes, and hepatocytes, which were demonstrated by immunocytochemical staining or RT-PCR analysis. These findings support the multilineage differentiation capability of chicken pluripotent EG cells, thus confirming the presumption that chicken embryos may be used as a potential model for better understanding the mechanisms of tissue-specific differentiation and regeneration that will help to devise strategies based on the transplantation of stem cell-derived tissues for restoring function to damaged or diseased tissues.

Epidermal growth factor-induced proliferation of chicken primordial germ cells: involvement of calcium/protein kinase C and NFKB1.

Epidermal growth factor (EGF) has been shown to stimulate survival in diverse cells in vitro. In the present study, the effects of EGF and the EGF-related signaling pathway on proliferation of chicken primordial germ cells (PGCs) were investigated. Results showed that EGF (10-100 ng/ml) increased the number and area of PGC colonies in a time- and dose-dependent manner. EGF also activated PKC, a process that was inhibited by AG1478 (an EGFR tyrosine kinase inhibitor) and ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA; an intracellular Ca(2+) chelator). In addition, the degradation of NFKBIA and NFKB1 (p65) translocation was observed after EGF treatment, which was significantly blocked by pretreatment with AG1478, EGTA, H(7), or SN50 (NFKB1-specific inhibitor). Furthermore, we found that EGF-induced cell proliferation was significantly attenuated by AG1478, EGTA, H(7), and SN50, respectively. On the other hand, inhibition of EGFR, Ca(2+)/PKC, or NFKB1 abolished the EGF-stimulated increase in the expression of cyclins CCND1 and CCNE1, cyclin-dependent kinase 6 (CDK6), CDK2, and BCL2, and restored the EGF-induced inhibition of BAX expression and caspase 3/9 activity, indicating that EGFR, PKC, and NFKB1 signaling cascades were involved in EGF-stimulated DNA synthesis and antiapoptosis action. In conclusion, EGF stimulated proliferation of chicken PGCs via activation of Ca(2+)/PKC involving NFKB1 signaling pathway. These observations suggest that EGF signaling is important in regulating germ cell proliferation in the chicken embryonic gonad.

Ginsenosides promote proliferation of chicken primordial germ cells via PKC-involved activation of NF-kappaB.

The effect of ginsenosides on proliferation of chicken primordial germ cells (PGCs) was evaluated and involvement of nuclear factor (NF)-kappaB in the signaling pathway was investigated. PGCs were isolated from the genital ridge of 3.5-4 day embryos and cultured in Medium 199 supplemented with 5% FCS and 10 ng/ml LIF. PGCs subcultured on chicken embryonic fibroblast feeder were challenged with ginsenosides alone or in combination with PKC inhibitor H(7) or activator phorbol 12-myristate 13-acetate (PMA) for 24h. Moreover, the translocation of NF-kappaB and degradation level of IkappaBalpha were investigated by Western blot analysis. Results show that PGCs were identified by periodic acid-Schiff, alkaline phosphatase histochemistry as well as c-kit, SSEA-1 and Oct-4 immunocytochemistry. Treatment with ginsenosides at 1-100 microg/ml significantly increased the number and area of PGC colonies in a dose-dependent manner. However, this proliferating effect was obviously attenuated by combined treatment of H(7) (10(-7)-10(-5)M). Similarly, PKC staining of PGC colonies was more intensive after ginsenosides treatment compared with the control group. In addition, treatment with ginsenosides at 1-10 microg/ml stimulated the translocation of NF-kappaB (p65). However, the NF-kappaB translocation and the degradation of IkappaBalpha were significantly blocked by combined treatment with 10(-6)M H(7). These results indicated that ginsenosides promote proliferation of chicken PGCs through activation of PKC-involved NF-kappaB signaling pathway.

Pro-proliferating effect of homologous somatic cells on chicken primordial germ cells.

Many studies demonstrated that chicken primordial germ cells (PGCs) could maintain undifferentiated state on mouse embryonic fibroblast feeders supplemented with growth factors and cytokines. However, the xenosupport systems may run risk of cross-transfer of animal pathogens from the other animal feeder, matrix to the PGCs, then influencing later transgenic technology. In this study, chicken PGCs were identified by alkaline phosphatase, stage-specific embryonic antigen-1 and Oct-4 immunocytochemical stainings. Three different homologous somatic cell feeder layers (chicken embryonic fibroblast feeder layer, CEF; embryonic skeletal myoblast feeder layer; follicular granulosa cell feeder layer) were used to support growth and proliferation of PGCs to find a better supporting culture system. In addition, the effects of fetal calf serum (FCS), leukemia inhibitory factor (LIF) and the combination of insulin, transferring and selenite (ITS) on PGC proliferation were compared. Results showed that CEF was the best supporter for PGC growth and proliferation, which was verified by 5-bromo-2'-deoxyuridine incorporation stain. FCS alone or in combination with LIF could significantly promote PGC proliferation in the presence of CEF in ITS medium. This study will contribute to providing a safer supporting system for chicken PGC amplification in vitro, and may be applied in transgenic chicken production and transplantation therapy.