Oxygen-sensitive regulation and neuroprotective effects of growth hormone-dependent growth factors during early postnatal development.

Perinatal hypoxia severely disrupts metabolic and somatotrophic development, as well as cerebral maturational programs. Hypoxia-inducible transcription factors (HIFs) represent the most important endogenous adaptive mechanisms to hypoxia, activating a broad spectrum of growth factors that contribute to cell survival and energy homeostasis. To analyze effects of systemic hypoxia and growth hormone (GH) therapy (rhGH) on HIF-dependent growth factors during early postnatal development, we compared protein (using ELISA) and mRNA (using quantitative RT PCR) levels of growth factors in plasma and brain between normoxic and hypoxic mice (8% O2, 6 h; postnatal day 7, P7) at P14. Exposure to hypoxia led to reduced body weight (P < 0.001) and length (P < 0.04) compared with controls and was associated with significantly reduced plasma levels of mouse GH (P < 0.01) and IGF-1 (P < 0.01). RhGH abrogated these hypoxia-induced changes of the GH/IGF-1 axis associated with normalization of weight and length gain until P14 compared with controls. In addition, rhGH treatment increased cerebral IGF-1, IGF-2, IGFBP-2, and erythropoietin mRNA levels, resulting in significantly reduced apoptotic cell death in the hypoxic, developing mouse brain. These data indicate that rhGH may functionally restore hypoxia-induced systemic dysregulation of the GH/IGF-1 axis and induce upregulation of neuroprotective, HIF-dependent growth factors in the hypoxic developing brain.

C1 Esterase Inhibitor Reduces BBB Leakage and Apoptosis in the Hypoxic Developing Mouse Brain.

Inflammatory pathways involved in blood-brain barrier (BBB) vulnerability and hypoxic brain oedema in models of perinatal brain injury seem to provide putative therapeutic targets. To investigate impacts of C1-esterase inhibitor (C1-INH; 7.5-30 IU/kg, i.p.) on functional BBB properties in the hypoxic developing mouse brain (P7; 8% O2 for 6 h), expression of pro-apoptotic genes (BNIP3, DUSP1), inflammatory markers (IL-1ß, TNF-alpha, IL-6, MMP), and tight junction proteins (ZO-1, occludin, claudin-1, -5), and S100b protein concentrations were analysed after a regeneration period of 24 h. Apoptotic cell death was quantified by CC3 immunohistochemistry and TUNEL staining. In addition to increased apoptosis in the parietal cortex, hippocampus, and subventricular zone, hypoxia significantly enhanced the brain-to-plasma albumin ratio, the cerebral S100b protein levels, BNIP3 and DUSP1 mRNA concentrations as well as mRNA expression of pro-inflammatory cytokines (IL-1ß, TNF-alpha). In response to C1-INH, albumin ratio and S100b concentrations were similar to those of controls. However, the mRNA expression of BNIP3 and DUSP1 and pro-inflammatory cytokines as well as the degree of apoptosis were significantly decreased compared to non-treated controls. In addition, occludin mRNA levels were elevated in response to C1-INH (p < 0.01). Here, we demonstrate for the first time that C1-INH significantly decreased hypoxia-induced BBB leakage and apoptosis in the developing mouse brain, indicating its significance as a promising target for neuroprotective therapy.

Differential regulation of angiogenesis in the developing mouse brain in response to exogenous activation of the hypoxia-inducible transcription factor system.

Angiogenesis due to hypoxic-ischemic (HI) injury represents a crucial compensatory mechanism of the developing brain that is mainly regulated by hypoxia-inducible transcription factors (HIF). Pharmacological stimulation of HIF is suggested as a neuroprotective option, however, studies of its effects on vascular development are limited. We analyzed the influence of the prolyl-4-hydroxylase inhibitor (PHI), FG-4497, and erythropoietin (rhEPO) on post-hypoxic angiogenesis (angiogenic growth factors, vessel structures) in the developing mouse brain (P7) assessed after a regeneration period of 72 h. Exposure to systemic hypoxia (8% O2, 6 h) was followed by treatment (i.p.) with rhEPO (2500/5000 IU/kg) at 0, 24 and 48 h or FG-4497 (60/100 mg/kg) compared to controls. In response to FG-4497 treatment cortical and hippocampal vessel area and branching were significantly increased compared to controls. This was associated with elevated ANGPT-2 as well as decreased ANGPT-1 and TIE-2 mRNA levels. In response to rhEPO, mildly increased angiogenesis was associated with elevated ANGPT-2 but also TIE-2 mRNA levels in comparison to controls. In conclusion, present data demonstrate a differential regulation of the angiopoietin/TIE-2 system in response to PHI and rhEPO in the post-hypoxic developing brain pointing to potential functional consequences for vascular regeneration and vessel development.

In vivo effect of growth hormone on the expression of connexin-43 in bovine ovarian follicles.

This study assessed the in vivo effects of recombinant growth hormone (rGH) administration on the expression of connexin-43 (Cx43) in bovine ovarian follicles. Two independent experiments were carried out using either estrous unsynchronized or synchronized multiparous Aberdeen Angus cows. rGH-treated animals were inoculated with a single dose of hormone (500 mg, intramuscular) while control animals were inoculated with hormone diluent. Five and 14 days after treatment (Experiments 1 and 2, respectively), ovarian Cx43 and apoptosis expression were assessed using immunohistochemistry. In both experiments primary, secondary, and tertiary follicles from rGH-treated and control groups distinctly expressed Cx43 protein. Primordial and atretic follicles were Cx43-negative. Interestingly, the number of Cx43 dots per granulosa cell did not show significant variation at different folliculogenesis stages neither in the rGH-treated nor in the control group. In unsynchronized animals, Cx43-positive follicles per total number of follicles ratio showed an interaction between stage of folliculogenesis and treatment due to significant differences between treatment groups in the early secondary follicle stage. In synchronized animals, there were significant differences between treatment groups and folliculogenesis stage. In both experiments, atretic follicles showed apoptosis-related DNA-fragmentation as determined by terminal uridin nick end labeling (TUNEL) assay. Tertiary follicles presented moderate TUNEL staining. Our results show significant increment in the number of ovarian follicles expressing the gap junction subunit Cx43 after in vivo rGH treatment. Therefore, we conclude that growth hormone can modulate in vivo gap junction assembly at early stages of folliculogenesis.

Growth hormone inhibits apoptosis in in vitro produced bovine embryos.

Growth hormone (GH) has recently been shown to exert distinct effects on the differentiation and metabolism of early embryos. Up to now, however, it is not clear whether GH is able to modulate apoptosis during early embryogenesis. Differential cell staining of 8-day-old bovine embryos cultured with 100 ng bovine recombinant GH (rbGH) per ml medium (synthetic oviduct fluid-polyvinylalcohol) demonstrated that GH significantly increased the number of inner cell mass (ICM) and trophectoderm cells in bovine expanded blastocysts. As shown by terminal deoxynucleotidyl transferase mediated dUTP labeling (TUNEL) supplementation of bGH decreased the percentage of 8-day-old embryos showing at least one apoptotic cell from 58 to 21%. The percentage of apoptotic cells in one blastocyst was significantly (P < 0.01) reduced from 4.6 to 1.1% by GH treatment. Incubation of the embryos with 150 mM vanillylnonanamide induced apoptosis in all embryos. Whereas in control embryos 14% of the embryonic cells were TUNEL-positive, the percentage of apoptotic cells declined to 2.7% in the GH treated embryos. Expression of immunoreactive bcl-2 in blastocysts was not affected by GH treatment. Synthesis of the bax protein which is known to promote apoptosis was reduced in embryos cultured with GH. Our results suggest that GH acts as survival factor during in vitro culture and reduces apoptosis by altering the bax to bcl-2 ratio during early embryogenesis.

Growth hormone-related effects on apoptosis, mitosis, and expression of connexin 43 in bovine in vitro maturation cumulus-oocyte complexes.

Pituitary LH and FSH are known to be the major regulators of ovarian function. In the last few years, however, there has been evidence that growth hormone (GH) is also involved in ovarian regulation. Therefore, the aim of our study was to elucidate the mechanisms of GH action during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). As shown by detection of the nuclear cell proliferation-associated antigen Ki-67, COCs matured in vitro in the presence of GH revealed a significantly (P < 0.05) higher proportion of proliferating cumulus cells (12.6%) compared with the COCs matured in the control medium TCM 199 (9.9%). In contrast, the percentage of proliferating cells was not increased by supplementation of the medium with a combination of GH and insulin-like-growth factor I (IGF-I). Apoptosis as determined by TUNEL (terminal doxynucleotidyl transferase mediated dUTP nick-end labeling) was significantly (P < 0.05) reduced in the cumulus cells by GH treatment. COCs matured with a combination of GH and IGF-I revealed the lowest percentage of apoptotic cells (11%). The localization and quantification of the gap junction protein connexin 43 (Cx 43) demonstrated that GH induced a significant decrease in the synthesis of the Cx 43 protein in the cumulus cells. Our results imply that GH increases cumulus expansion by promotion of cell proliferation and inhibition of apoptosis. Whereas the increase in cell proliferation is a direct effect of GH, the antiapoptotic effects of GH during in vitro maturation are modulated by IGF-I. Stimulatory effects of GH on oocyte maturation are correlated with changes in the synthesis of gap junction proteins.