Autophagy participates in germline cyst breakdown and follicular formation by modulating glycolysis switch via Akt signaling in newly-hatched chicken ovaries.

In females, the establishment of the primordial follicle pool is accompanied by a remarkable programmed oocyte loss for unclear reasons. In this study, the role of autophagy was investigated to serve as a protective mechanism for oocyte survival during chicken folliculogenesis. Inhibition of autophagy by 3-methyladenine (3-MA) led to a remarkable delay in germ cell cyst breakdown that resulted in fewer primordial follicles and retarded sequent follicular development either in vivo or in the ovarian organ culture. Furthermore, the glycolysis level was downregulated in ovaries treated with 3-MA, while Recilisib (a specific activator of Akt) reversed this inhibiting effect of 3-MA on primordial folliculogenesis. Collectively, these data indicate that autophagy functions to maintain germ cell cyst breakdown and primordial follicle assembly by regulating ovarian glycolysis involving Akt signaling in the ovaries of newly-hatched chickens.

Protective effect of rutin on ferroptosis-induced oxidative stress in aging laying hens through Nrf2/HO-1 signaling.

Oxidative stress is a major cause of ovarian aging and follicular atresia. There is growing evidence that showed potential roles of rutin in antidiabetic, anti-inflammatory, antitumor, antibacterial and antioxidant, although it is yet unclear what the underlying mechanism is. Here, we looked into the potential effects of rutin on oxidative stress in the prehierarchical small white follicles (SWFs) from 580-day-old (D580) laying chickens. According to the findings, aging D580 layer ferroptosis was much higher than it was for laying hens during the peak period (280-day-old, D280). In both naturally aged and d-gal-induced chicken SWFs, rutin treatment concurrently boosted cell proliferation and prevented apoptosis. In addition, rutin inhibited the increased ferroptosis in aging hens. Meanwhile, rutin markedly suppressed the elevated ferroptosis and descending antioxidant capacity of D280-culture-SWFs from chicken elicited by d-gal. Rutin's activation of the Nrf2/HO-1 pathway hastened the SWFs' verbal battle with oxidative damage and reduced ferroptosis. Furthermore, activation of the ferroptosis signal increased the oxidative damage in SWFs. In conclusion, rutin alleviated oxidative stress that was induced by ferroptosis in aging chicken SWFs through Nrf2/HO-1 pathway. These findings point to a novel mechanism by which rutin protects SWFs from oxidative stress by suppressing ferroptosis, which is presumably a fresh approach to slowing ovarian aging in laying hens.

TGF-ß1-induced collagen promotes chicken ovarian follicle development via an intercellular cooperative pattern.

Follicle development is a complex process under strict regulation of diverse hormones and cytokines including transforming growth factor ß (TGF-ß) superfamily members. TGF-ß is pivotal for the regulation of ovarian functions under physiological and pathological conditions. In this study, effect of TGF-ß1 on chicken follicle development was examined through investigating the accumulation and action of collagen, an indispensable member of the extracellular matrix (ECM) involved in this process. The granulosa cells (GCs) and theca cells (TCs) were separated from growing follicles of the laying chicken for treatment of TGF-ß1 and analysis of expression of ECM components and key proteins in intracellular signaling pathways. Results showed that collagen was mainly distributed in the follicular theca layer and was produced with the formation of the granulosa layer during ovarian development. Collagen accumulation increased with follicle growth and treatment of GCs with TGF-ß1 elicited an increased expression of collagen. After production from GCs, collagen was transferred to the neighboring TCs to promote cell proliferation and inhibit apoptosis. Treatment of collagen remarkably increased expression of p-ERK, mitogen-activated protein kinase (MAPK), and p-MAPK, but treatment with hydroxylase inhibitor (to break collagen structure) reversed these alterations. In conclusion, during follicle growth collagen was secreted by GCs under TGF-ß1 stimulation and was subsequently collaboratively transferred to neighboring TCs to increase cell proliferation and thus to promote follicle development via an intercellular cooperative pattern during development of chicken growing follicles.

Lactobacillus salivarius and Lactobacillus agilis feeding regulates intestinal stem cells activity by modulating crypt niche in hens.

Previously, we found that Lactobacillus salivarius, Lactobacillus agilis, and Lactobacillus aviarius were associated with excellent egg nutrition in native chicken. Next, the optimal Lactobacillus combination is worth studying. Here, a total of 120 HyLine hens (30 hens per group contained 3 replicate cells, 10 hens/cell) in the laying peak period were randomly divided into (1) control, (2) L. salivarius + L. agilis, (3) L. salivarius + L. aviarius, and (4) L. agilis + L. aviarius groups, fed with diet only or with corresponding Lactobacilli (108 colony-forming units/hen/day) for 30 days. As a result, L. salivarius + L. agilis feeding could (1) improve egg-laying rate, egg weight, and albumen's amino acid levels; (2) increase Lactobacillus abundance, decrease Escherichia coli abundance, upregulate the tryptophan metabolism pathway-related molecules, and downregulate the primary bile acid biosynthesis pathway-related molecules in intestinal contents; and (3) upregulate oxidative-phosphorylation pathway-related genes, reactive oxygen species levels, and mRNA abundance of Wnt3a, Dll1, Lgr5, CCDN1, and CDK2 in the crypt. Collectively, L. salivarius + L. agilis feeding in hens could improve intestinal microflora and metabolism profile, promote crypt's local energy metabolism and reactive oxygen species levels, and thus enhance Paneth cells and intestinal stem cells activity.Key points• Lactobacilli co-feeding could improve laying performance and egg nutrition.• Lactobacilli co-feeding could improve intestinal microflora and metabolism profile.• Lactobacilli co-feeding could enhance Paneth cells and intestinal stem cells activity.

TGF-ß1 sustains germ cell cyst reservoir via restraining follicle formation in the chicken.

The transforming growth factor ß (TGF-ß) superfamily members are important molecules that regulate many ovarian functions under normal physiological and pathological conditions. TGF-ß1 and its receptors are highly expressed in the ovarian cells of many species. However, the effect of TGF-ß1 on the capacity of the avian germ cell reservoir remains unknown. In this study, 5-day-old chicks were injected with TGF-ß1 (2.5, 12.5, and 62.5 µg/kg body weight) for 3 days to assess the effect of TGF-ß1 on early follicle development. Morphological analysis showed that treatment with TGF-ß1 (12.5 µg/kg) increased the number of germ cell cysts and reduced the number of primordial and growing follicles. The diameter and area of oocytes and follicles were decreased after TGF-ß1 treatment. Immunohistochemical staining of the proliferating cell nuclear antigen revealed that the ratios of the positive somatic and granulosa cells were decreased by 16.2% and 2.48%, respectively. Furthermore, more apoptotic cells were observed in the TGF-ß1 group than those of the control by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. In addition, we cultured the 5d chicken ovaries for 3 days in vitro and found that treatment with TGF-ß1 (10 ng/mL) manifested similar results as the in vivo experiment. However, the negative effect of TGF-ß1 on early ovary development was rescued by treatment with a TGF-ßR1 inhibitor SD208, resulting in increased expression of steroidogenic enzymes and cell cycle-regulating proteins. In conclusion, TGF-ß1 could maintain the germ cell reservoir by restraining follicle activation involving reduced cell proliferation and steroidogenic enzymes gene expression at the early stage of ovarian development.

Differentiation of adult human retinal pigment epithelial cells into dopaminergic-like cells in vitro and in the recipient monkey brain.

BACKGROUND: Cell therapy is proposed to be a potential treatment for Parkinson's disease (PD). Although fetal retinal pigment epithelial (RPE) cells have been tested in trials for treating PD patients, controversy has been raised over the issue of whether such cells can be reprogrammed into dopamine-producing cells for therapeutic efficacy. Here, we aim to investigate whether adult human RPE cells can be reprogrammed into dopamine-producing cells both in vitro and in the recipient monkey brain. METHODS: The RPE layer was isolated from frozen posterior eyeball tissue after penetrating keratoplasty surgery. The tumorigenicity of RPE cells was examined by G-banding and a tumor formation assay in nude mice. Immunogenicity was measured using a one-way mixed lymphocyte reaction (MLR) assay. Dopamine-production in chemically reprogrammed RPE cells was measured by HPLC. Finally, RPE cells were grafted into the brains of monkeys with MPTP-induced PD in order to investigate the potential of such cells treating PD patients in the future. RESULTS: RPE cell lines have been successively established from adult human eye tissues. Such cells can be chemically reprogrammed into dopamine-producing cells in vitro. Moreover, after being grafted into the brain caudate putamen of monkeys with MPTP-induced PD, RPE cells became tyrosine hydroxylase-positive cells, and recipient PD monkeys showed significant improvement of clinical conditions. CONCLUSIONS: This preclinical study using a primate model indicates that human adult RPE cells could be a potential cell source for the treatment of PD in the future.

Promotion of the prehierarchical follicle growth by postovulatory follicles involving PGE2 -EP2 signaling in chickens.

The postovulatory follicle (POF) in birds is an enigmatic structure, the function of which remains largely unknown. Previous studies on chickens have shown that removal of POFs leads to the postponement of oviposition and the disturbance of broody behavior. One suggestion is that POFs may secrete some crucial hormones or cytokines to act on reproductive organs. However, such secretions and their specific target organs remain to be identified. Here, we investigate the putative functions of POFs in promoting the development of prehierarchical follicles in chickens and explore the possible signaling mechanisms controlling these processes. Results show that POFs express steroidogenic acute regulatory protein (STAR), cholesterol side-chain cleavage enzyme (CYP11A1), cyclooxygenase 1 (COX1), and COX2 in granulosa cells (GCs), and, most notably, that POF1 produces more prostaglandin E2 (PGE2 ) or prostaglandin F2α than do the F1 follicle or the other POFs. Using coculture systems, we also found that POF1 or GCs from POF1 (POF1-GCs) significantly promote the proliferation of theca externa cells of small white follicles (SWFs, one phase of the prehierarchical follicle). Treatment with PGE2 significantly facilitates theca externa cell proliferation in SWFs. This POF-stimulating effect on SWF growth was prevented by treatment with indomethacin (COX inhibitor) or TG6-10-1 (PGE2 type 2 receptor [EP2] antagonist). Therefore, POF1 may secrete PGE2 to stimulate the progression of SWF by PGE2 -EP2 signaling. These results indicate that POF1 may serve as a transient supplementary endocrine gland in the chicken ovary that stimulates the development of the prehierarchical follicles through PGE2 -EP2 signaling.

Taurine regulates mucosal barrier function to alleviate lipopolysaccharide-induced duodenal inflammation in chicken.

The objective of the study was to investigate how taurine alleviates mucosal injury. Young chickens were fed with taurine for 1 week and then challenged with lipopolysaccharide. We found that, under lipopolysaccharide challenge, taurine could attenuate diarrhea and mucosal inflammation. Additionally, under LPS challenge, taurine could enhance epithelial proliferation and goblet cell function, could also decrease epithelial apoptosis by improving the mitochondrial membrane permeability. The high-performance liquid chromatography assay showed that taurine feeding could elevate taurine concentration in duodenum obviously. The antioxidant assay showed that taurine could reverse lipopolysaccharide-induced low GSH level, GSH/GSSG ratio, GSH-Px activity and SOD activity, high GSSG and MDA content. In summary, we suggested that taurine could enhance duodenal antioxidant status locally and further ameliorated lipopolysaccharide-induced chicken duodenal inflammation by improving mitochondrial membrane permeability and goblet cell function.

Effect of estrogen on chick primordial follicle development and activation.

Steroid hormones and their receptors play pivotal roles in avian reproduction and development. 17ß-estradiol (E2 ) is an essential mediator in ovarian development. In this study, Day 3 (D3) chicks were injected with E2 (0.025, 0.25 or 2.5 mg/kg body weight) for 4 days to assess the effects of estrogen on avian follicle development. Morphological analysis showed that treatment of E2 at 2.5 mg/kg increased the number of growing follicles by 28. 5% and activated follicles by 8.9%, compared with the group. Treatment of E2 also led to increased diameter and area of three kinds of follicles and oocytes, respectively. However, this stimulating effect of E2 was inhibited by combined treatment of the estrogen receptor antagonist tamoxifen, resulting in decreased number of the growing follicles by 14.3% and the activated follicles by 5.8%, respectively. Immunohistochemical staining of the proliferating cell nuclear antigen (PCNA) revealed that the ratios of the PCNA-positive granulosa and interstitial cells were increased by 14.0% and 21.5%, respectively. Western blot analysis of PCNA confirmed this stimulating effect of E2 . Expression of ERα mRNAs was elevated by administration of E2 in vivo and in vitro. Furthermore, E- and N-cadherin displayed increased expression after E2 treatment in vivo and in vitro. In conclusion, E2 can promote chicken primordial follicle development and activation involving its increased receptor and cadherin production at the early stage of ovarian development.

Involvement of Notch signaling in early chick ovarian follicle development.

The formation of primordial follicles is a crucial process in the establishment of follicle pools required for the female's reproductive life span. For laying hens, ample follicles are a prerequisite for high laying performance. Notch signaling plays critical roles in germ cell cysts breakdown and in the formation of primordial follicles. Here, we investigated the role of Notch signaling in the ovarian development of post-hatch chicks. Results showed that around post-hatch day 4 (H4), the germ cell cysts broke apart, oocytes became surrounded by squamous pregranulosa cells, and the primordial follicles were then formed. Subsequently, we detected the expression of Notch signaling-related genes including Notch receptors (Notch1, 2), ligands (Jag1, 2 and Dll1, 4), and target genes (Hes1, Hey1). These genes all showed expression at H4 and some of these genes were up-regulated during primordial follicle formation. To evaluate the Notch signaling requirement for early follicular development, we adopted an in vitro ovary culture system. Suppression of Notch signaling by γ-secretase inhibitor induced a decrease of primordial follicles and an increase of germ cells in cysts. Attenuating Notch signaling also inhibited the phosphatidylinositol 3-kinase/protein kinase B pathways and suppressed cadherin expression. These results suggest that Notch signaling is endowed with an indispensable role in primordial follicle formation in post-hatch chicks.

A biomimetic collagen/heparin multi-layered porous hydroxyapatite orbital implant for in vivo vascularization studies on the chicken chorioallantoic membrane.

BACKGROUND: The vascularization of an orbital implant is a key issue for reducing complications, such as exposure and infection. METHODS: Here, we developed a facile layer-by-layer assembly approach to modify porous hydroxyapatite (pHA) orbital implants with five collagen (COL)/heparin (HEP) multilayers. RESULTS: SEM characterization showed that the average pore size of the pHA/(COL/HEP)5 scaffold was 316.8 ± 77.1 µm. After being coated with five COL/HEP multilayers, the mechanical strength was improved compared with that of the pHA scaffolds. The in vitro assay displayed that the pHA scaffolds covered with COL/HEP multilayers resulted in a larger number of human umbilical vein endothelial cells after being cultured for 14 days. The macroscopic evaluation and semi-quantitative vascular density analysis of the chicken chorioallantoic membrane assay showed that the pHA/(COL/HEP)5 scaffolds resulted in more intense angiogenesis than the pHA scaffolds. CONCLUSIONS: These studies demonstrate that the biomembrane-mimicking coating of COL/HEP multilayers is a simple and effective strategy to endow combined biological performances of pHA orbital implants and to potentially reduce implant-related complications.

[Study on volatile components of Lonicerae Japonicae Flos in bud stage extended type].

Volatile components of Lonicerae Japonicae Flos in bud stage extended type Beihua 1 were determined by the headspace solid-phase micro-extraction, compared with traditional cultivar Damaohua. There are fifty-two volatile compounds were identified and the relative content of the volatiles was calculated by the area normalization method. Thirty-nine compounds were found in Beihua 1, whereas thirty-three components in Damaohua. Total twenty identical compounds existed in Beihua 1 and Damaohua. The contents of alcohols and hydrocarbons of Beihua 1 were higher significantly than that of Damaohua, while significantly lower than that of Damaohua in ketones content. Besides, twenty components were only detected in Beihua 1, such as methyl nicotinate, hexadecanoic acid, methyl ester,acetophenone, nonanoic acid.

Isolation and culture of chicken primordial follicles.

The establishment of a primordial follicle culture system is important for the study of follicular development. Hence, the objective of this study was to isolate chicken primordial follicles and establish culture methods. Ovaries from 2-wk-old chickens were treated with trypsin-EDTA, collagenase II, or collagenase type IA, along with a mechanical isolation technique. Isolated follicles were cultured under different conditions. Results showed a significant difference in the follicular recovery and survival rates among different enzymes and methods used. The maximal follicular yield was obtained by trypsin+EDTA and collagenase II digestion, followed by collagenase type IA digestion. However, the highest follicular viability rate was observed in groups of collagenase type IA digestion and the mechanical isolation method. Enzymatic treatment resulted in higher misshapen oocytes or follicles, though the diameters of the follicles were not significantly changed. In addition, our follicle culture results for different conditions showed maximal survival rates of primordial follicles in alginate hydrogel beads after 12 d of culture. Thus, we successfully established methods for isolating and culturing chicken primordial follicles. The present method will greatly facilitate investigation of the regulation of follicular development.

Bioactive Lignan Honokiol Alleviates Ovarian Oxidative Stress in Aging Laying Chickens by Regulating SIRT3/AMPK Pathway.

Aging is not only a key internal cause of age-related diseases in humans but also poses a threat to the productivity of farm animals with longer breeding cycles, such as laying chickens. Various measures were taken to prolong the laying period by reducing oxidative stress to improve poultry ovarian functions. Within the mitochondria, SIRT3, a member of the Sirtuin family, plays an important role in post-translational modifications and the regulation of protein activities involved in energy metabolism and oxidative response. This study aimed to investigate the alleviating effect of a bioactive lignan Honokiol (HKL) on oxidative stress in aging chicken ovaries in order to retard decline in egg production. The results showed that HKL treatment restored the abnormal balance between cell proliferation and apoptosis, and it enhanced the antioxidant capacity of the H2O2-induced small white follicles (SWFs) by activating the SIRT3/AMPK pathway. Moreover, HKL significantly increased total egg production, the number of yellow follicles, and the mRNA expression of yolk synthesis and deposition-related genes, serum estrogen, and antioxidant levels. These findings suggest that HKL holds promise in enhancing the egg productivity of aging laying chickens by promoting yolk deposition and reducing ovarian oxidative stress.

Nobiletin Ameliorates Aging of Chicken Ovarian Prehierarchical Follicles by Suppressing Oxidative Stress and Promoting Autophagy.

With the increase in the age of laying chickens, the aging of follicles is accelerated, and the reproductive ability is decreased. Increased oxidative stress and mitochondrial malfunction are indispensable causes of ovarian aging. In this study, the physiological condition of prehierarchical small white follicles (SWFs) was compared between D280 high-producing chickens and D580 aging chickens, and the effect of a plant-derived flavonoid nobiletin (Nob), a natural antioxidant, on senescence of SWFs granulosa cells (SWF-GCs) was investigated. The results showed that Nob treatment activated cell autophagy by activating the AMP-activated protein kinase (AMPK) and Sirtuin-1 (SIRT1) pathways in D-galactose (D-gal)-generated senescent SWF-GCs, restoring the expression of proliferation-related mRNAs and proteins. In addition, the expression of inflammation-related protein NF-κB was significantly enhanced in aging GCs that were induced by D-gal. Nob supplementation significantly increased the antioxidant capacity and decreased the expression of several genes associated with cell apoptosis. Furthermore, Nob promoted activation of PINK1 and Parkin pathways for mitophagy and alleviated mitochondrial edema. Either the AMPK inhibitor dorsomorphin (Compound C) or SIRT1 inhibitor selisistat (EX-527) attenuated the effect of Nob on mitophagy. The protective effect of Nob on natural aging, GC proliferation, and elimination of the beneficial impact on energy regulation of naturally aging ovaries was diminished by inhibition of Nob-mediated autophagy. These data suggest that Nob treatment increases the expression of mitophagy-related proteins (PINK1 and Parkin) via the AMPK/SIRT1 pathways to prevent ovarian aging in the laying chickens.

RALDH2, the enzyme for retinoic acid synthesis, mediates meiosis initiation in germ cells of the female embryonic chickens.

Meiosis is a process unique to the differentiation of germ cells and exhibits sex-specific in timing. Previous studies showed that retinoic acid (RA) as the vitamin A metabolite is crucial for controlling Stra8 (Stimulated by retinoic acid gene 8) expression in the gonad and to initiate meiosis; however, the mechanism by which retinoid-signaling acts has remained unclear. In the present study, we investigated the role of the enzyme retinaldehyde dehydrogenase 2 (RALDH2) which catalyzes RA synthesizes by initiating meiosis in chicken ovarian germ cells. Meiotic germ cells were first detected at day 15.5 in chicken embryo ovary when the expression of synaptonemal complex protein 3 (Scp3) and disrupted meiotic cDNA 1 homologue (Dmc1) became elevated, while Stra8 expression was specifically up-regulated at day 12.5 before meiosis onset. It was observed from the increase in Raldh2 mRNA expression levels and decreases in Cyp26b1 (the enzyme for RA catabolism) expression levels during meiosis that requirement for RA accumulation is essential to sustain meiosis. This was also revealed by RA stimulation of the cultured ovaries with the initiation of meiosis response, and the knocking down of the Raldh2 expression during meiosis, leading to abolishment of RA-dependent action. Altogether, these studies indicate that RA synthesis by the enzyme RALDH2 and signaling through its receptor is crucial for meiosis initiation in chicken embryonic ovary.

Prostaglandin E(2) and insulin-like growth factor I interact to enhance proliferation of theca externa cells from chicken prehierarchical follicles.

The interactive effect of insulin-like growth factor I (IGF-I) and prostaglandin E2 (PGE2) on the proliferation of theca externa cells (TECs) was investigated in the prehierarchical small yellow follicles of laying hens. IGF-I manifested a proliferating effect like PGE2 on TECs, but this stimulating effect was restrained by AG1024 (IGF-IR inhibitor), KP372-1 (PKB/AKT inhibitor) or NS398 (COX-2 inhibitor). AG1024, KP372-1 or NS398 abolished IGF-I-stimulated COX-2 expression and PGE2 production. Meanwhile, KP372-1, NS398 or AG1024 depressed the PGE2-stimulated expression of COX-2 and IGF-IR mRNA. Therefore, the IGF-I receptor pathway up-regulates COX-2 expression and PGE2 synthesis via PKB signaling cascade, and then PGE2 stimulates IGF-IR mRNA expression to promote TEC proliferation in an autocrine pattern. Overall, the reciprocal stimulation of intracellular PGE2 and IGF-I may enhance TEC proliferation and facilitate the development of chicken prehierarchical follicles.

Krüppel-like factor 5 activates chick intestinal stem cell and promotes mucosal repair after impairment.

The mucosal renewal, which depends on the intestinal stem cell (ISC) activity, is the foundation of mucosal repairment. Importantly, activation of reserve ISCs (rISCs) plays a vital role in initiating mucosal repair after injury. However, the underlying regulatory mechanism of rISCs activation in chickens remains unclear. In this study, immediately after lipopolysaccharide (LPS) challenge, mitochondrial morphological destruction and dysfunction appeared in the crypt, accompanied by decreased epithelial secretion (decreased Muc2 mRNA abundance and LYSOZYME protein level). However, immediately after mucosal injury, the mucosal renewal accelerated, as indicated by the increased BrdU positive rate, proliferating cell nuclear antigen (PCNA) protein level and mRNA abundance of cell cycle markers (Ccnd1, Cdk2). Concerning the ISCs activity, during the early period of injury, there appeared a reduction of active ISCs (aISCs) marker Lgr5 mRNA and protein, and an increasing of rISCs marker Hopx mRNA and protein. Strikingly, upon LPS challenge, increased mRNA transcriptional level of Krüppel-like factor 5 (Klf5) was detected in the crypt. Moreover, under LPS treatment in organoids, the KLF5 inhibitor (ML264) would decrease the mRNA and protein levels of Stat5a and Hopx, the STAT5A inhibitor (AC-4-130) would suppress the Lgr5 mRNA and protein levels. Furthermore, the Dual-Luciferase Reporter assay confirmed that, KLF5 would bind to Hopx promoter and activate the rISCs, STAT5A would trigger Lgr5 promoter and activate the aISCs. Collectively, KLF5 was upregulated during the early period of injury, further activate the rISCs directly and activate aISCs via STAT5A indirectly, thus initiate mucosal repair after injury.

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.

Basic fibroblast growth factor suppresses meiosis and promotes mitosis of ovarian germ cells in embryonic chickens.

Basic fibroblast growth factor (bFGF or FGF2) plays diverse roles in regulating cell proliferation, migration and differentiation during embryo development. In this study, the effect of bFGF on ovarian germ cell development was investigated in the embryonic chicken by in vitro and in vivo experiments. Results showed that a remarkable decrease in bFGF expression in the ovarian cortex was manifested during meiosis progression. With ovary organ culture, we revealed that meiosis was initiated after retinoic acid (RA) treatment alone but was decreased after combined bFGF treatment that was detected by real time RT-PCR, fluorescence immunohistochemistry and Giemsa staining. Further, no significant difference in mRNA expression of either RA metabolism-related enzymes (Raldh2 and Cyp26b1) or RA receptors was displayed after bFGF challenge. This result suggests that the suppression of bFGF on meiosis was unlikely through inhibition of RA signaling. In addition, as a mitogen, bFGF administration increased germ cell proliferation (via BrdU incorporation) in cultured organ or cells in vitro and also in developing embryos in vivo. In contrast, blockade of bFGF action by SU5402 (an FGFR1 antagonist) or inhibition of protein kinase C signaling showed inhibited effect of bFGF on mitosis. In conclusion, bFGF suppresses RA-induced entry of germ cells into meiosis to ensure embryonic ovarian germ cells to maintain at undifferentiated status and accelerate germ cell proliferation by binding with FGFR1 involving PKC activation in the chicken.