Autocrine/paracrine proliferative effect of ovarian GH and IGF-I in chicken granulosa cell cultures.

It is known that growth hormone (GH) and its receptor (GHR) are expressed in granulosa cells (GC) and thecal cells during the follicular development in the hen ovary, which suggests GH is involved in autocrine/paracrine actions in the female reproductive system. In this work, we show that the knockdown of local ovarian GH with a specific cGH siRNA in GC cultures significantly decreased both cGH mRNA expression and GH secretion to the media, and also reduced their proliferative rate. Thus, we analyzed the effect of ovarian GH and recombinant chicken GH (rcGH) on the proliferation of pre-hierarchical GCs in primary cultures. Incubation of GCs with either rcGH or conditioned media, containing predominantly a 15-kDa GH isoform, showed that both significantly increased proliferation as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, proliferating cell nuclear antigen (PCNA) quantification and ((3)H)-thymidine incorporation ((3)H-T) assays in a dose response fashion. Both, locally produced GH and rcGH also induced the phosphorylation of Erk1/2 in GC cultures. Furthermore, GH increased IGF-I synthesis and its release into the GC culture incubation media. These results suggest that GH may act through local IGF-I to induce GC proliferation, since IGF-I immunoneutralization completely abolished the GH-induced proliferative effect. These data suggest that GH and IGF-I may play a role as autocrine/paracrine regulators during the follicular development in the hen ovary at the pre-hierarchical stage.

Direct antiapoptotic effects of growth hormone are mediated by PI3K/Akt pathway in the chicken bursa of Fabricius.

Growth hormone (GH) is expressed in several extra-pituitary tissues, including the primary and secondary lymphoid organs of the immune system. In birds, GH mRNA and protein expression show a specific developmental distribution pattern in the bursa of Fabricius (BF), particularly in epithelial and B cells. Changes in the bursal concentration and distribution of locally produced GH during ontogeny suggest it is involved in B cell differentiation and maturation, as well as in a functional survival role in this organ, which may be mediated by paracrine/autocrine mechanisms. Here, we analyzed the anti-apoptotic effect of GH in BF and the intracellular signaling pathways involved in this activity. Also, we studied if this effect was exerted directly by GH or mediated indirectly by IGF-I. Bursal cell cultures showed an important loss of their viability after 4h of incubation and a significant increase in apoptosis. However, treatment with 10nM GH or 40 nM IGF-I significantly increased B cell viability (16.7 ± 0.67% and 13.4 ± 1.12%, respectively) when compared with the untreated controls. In addition, the presence of apoptotic bodies (TUNEL) dramatically decreased (5.5-fold) after GH and IGF-I treatments, whereas co-incubation with anti-GH or anti-IGF-I, respectively, blocked their anti-apoptotic effect. Likewise, both GH and IGF-I significantly inhibited caspase-3 activity (by 40 ± 2.0%) in these cultures. However, the use of anti-IGF-I could not reverse the GH anti-apoptotic effects, thus indicating that these were exerted directly. The addition of 100 nM wortmannin (a PI3K/Akt inhibitor) blocked the GH protective effects. Also, GH stimulated (3-fold) the phosphorylation of Akt in bursal cells, and adding wortmannin or an anti-GH antibody inhibited this effect. Furthermore, GH was capable to stimulate (7-fold) the expression of Bcl-2. Taken together, these results indicate that the direct anti-apoptotic activity of GH observed in the chicken bursal B cell cultures might be mediated through the PI3K/Akt pathway.

Expression and function of chicken bursal growth hormone (GH).

Growth hormone (GH) has several effects on the immune system. Our group has shown that GH is produced in the chicken bursa of Fabricius (BF) where it may act as an autocrine/paracrine modulator that participates in B-cell differentiation and maturation. The time course of GH mRNA and protein expression in the BF suggests that GH may be involved in development and involution of the BF, since GH is known to be present mainly in B lymphocytes and epithelial cells. In addition, as GH is anti-apoptotic in other tissues, we assessed the possibility that GH promotes cell survival in the BF. This work focused on determining the mechanism by which GH can inhibit apoptosis of B cells and if the PI3K/Akt pathway is activated. Bursal cell cultures were treated with a range of GH concentrations (0.1-100nM). The addition of 10nM GH significantly increased viability (16.7±0.6%) compared with the control and decreased caspase-3 activity to 40.6±6.5% of the control. Together, these data indicate that GH is produced locally in the BF and that the presence of exogenous GH in B cell cultures has antiapoptotic effects and increases B cell survival, probably through the PI3k/Akt pathway.

Neuro-protective effects of growth hormone (GH) after hypoxia-ischemia injury in embryonic chicken cerebellum.

Neuroprotection is a mechanism within the central nervous system (CNS) that protects neurons from damage as a result of a severe insult. It is known that growth hormone (GH) is involved in cell survival and may inhibit apoptosis in several cell types, including those of the CNS. Both GH and GH-receptor (GHR) genes are expressed in the cerebellum. Thus, we investigated the possible neuroprotective role of GH in this organ, which is very sensitive to hypoxic/ischemic conditions. Endogenous GH levels increased in the brain and cerebellum (30% and 74%, respectively) of 15-day-old chicken embryos exposed to hypoxia during 24h compared to normoxia. In primary embryonic cerebellar neuron cultures treated under hypoxia (0.5% O(2)) and low glucose (1g/L) conditions (HLG) for 1h, GH levels increased 1.16-fold compared to the control. The addition of 1nM recombinant chicken GH (rcGH) to cultures during HLG increased cell viability (1.7-fold) and the expression of Bcl-2 (1.67-fold); in contrast the caspase-3 activity and the proportion of apoptotic cells decreased (37% and 54.2%, respectively) compared to HLG. rcGH activated the PI3K/Akt pathway both under normoxic and HLG conditions, increasing the proportion of phosphorylated Akt (1.7- and 1.4-fold, respectively). These effects were abolished by wortmannin and by immunoneutralization, indicating that GH acts through this signaling pathway. Furthermore, the 15-kDa GH variant (10nM) significantly increased cell viability and decreased caspase-3 activity during HLG condition. Thus GH may act as a paracrine/autocrine neuroprotective factor that preserves cellular viability and inhibits apoptotic cell death.