Prostaglandin-D synthetase induces transcription of the LH beta subunit in the primary culture of chicken anterior pituitary cells via the PPAR signaling pathway.

Downstream effects of prostaglandin-D synthetase (PGDS) in a primary culture of chicken (Gallus gallus) anterior pituitary cells were investigated to study how PGDS regulated laying in hens. Either PGDS downstream metabolite, PGD(2) or PGJ(2), elevated LHB mRNA and LHB protein levels in dose- and time-dependent manners, and treatment with arachidonic acid (1 microM) alone upregulated 15-deoxy-Delta(12,14)-PGJ(2) (15-d-PGJ(2); derived from PGJ(2))/PGJ(2), LHB mRNA, and LHB protein levels (P<0.05) in the primary culture of chicken pituitary anterior cells. Transfection of the plasmid Enhanced Green Fluorescent Protein-PGDS plasmid into these cells in medium containing 1 microM arachidonic acid additionally increased 15-d-PGJ(2)/PGJ(2), LHB mRNA, and LHB protein levels (P<0.05). In the hypothalamus/pituitary gland of laying hens, there was a high correlation (r=0.64; P<0.05) between PGDS and the peroxisome proliferator-activated receptor A (PPARA) mRNA level in a high egg production strain, the L2 Taiwan country chickens. In commercial Single-Comb White Leghorn layers, there were high correlation coefficients between PGDS and PPARA (r=0.65; P<0.05) and between PGDS and PPARG (r=0.67; P<0.01) mRNA levels. A broad-range PPARs agonist, GW9578 (5 to 500 nM), enhanced LHB mRNA and LHB protein levels (P<0.05). The PPARA-specific (GW6471) and PPARG-specific (T0070907) antagonists suppressed endogenous LHB mRNA and LHB protein levels (P<0.05); in addition, both antagonists attenuated arachidonic acid-induced LHB mRNA levels (P<0.05) and PGDS-induced (in the presence of 1 microM arachidonic acid) LHB mRNA and LHB protein (P<0.05) levels in the primary culture of chicken anterior pituitary cells. Higher LHB mRNA/LHB protein ratios in PGD(2)-, PGJ(2)-, arachidonic acid-, PGDS-, and GW9578-induced as well as GW6471- and T0070907-suppressed anterior pituitary cells suggested that LHB transcription occurred before translation. In conclusion, PGDS induced LHB transcription and subsequent translation via the PPAR signaling pathway.

Establishment of the long-term in vitro culture system for chicken primordial germ cells.

The objective of this study was to establish the long-term in vitro culture system for chicken gonadal primordial germ cells (gPGCs). Primitive gonads collected from 5.5-day-old chicken embryos were dissociated and explanted onto plates pre-coated with 0.1% gelatin. Each of the four different conditioned media from proliferating and mitotically inactivated chicken embryonic fibroblast (CEF) cells and murine embryonic fibroblasts (STO cells, CRL-1053, ATCC, USA), respectively, was supplemented with growth factors and used to support the growth of gPGCs. The result showed that all the conditioned media could promote the growth and colony formation of gPGCs in vitro, in particular the medium conditioned by inactivated CEF cells. The gPGC-derived colonies maintained in inactivated CEF cells-conditioned medium up to 281 days were positively stained by periodic acid Schiff reaction and antibodies specific to anti-SSEA-1, SSEA-3, SSEA-4, integrin alpha6 and integrin beta1. Their capacities of migration via vascular system and taking up residence in the primary gonadal ridge were further demonstrated by transferring to the dorsal aorta of stage 17 recipient embryos. These results suggested that our culture system is able to maintain chicken gPGCs for long-term in vitro culture without losing their capacity to express pluripotent markers and to integrate into the gonads.