The effect of placental growth hormone in mouse pregnancy

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Chapter 3: The effect of placental growth hormone in mice pregnancies

Preface

The following chapter contains data from a published article “The Placental Variant of Human Growth Hormone Reduces Maternal Insulin Sensitivity in a Dose-Dependent Manner in C57BL/6J Mice” (Appendix Ι). A version of this chapter was published in Endocrinology in 2016. Endocrinology.
In this chapter, a study was conducted to determine the dose-response relationship for human GH-V treatment in a mouse model of normal pregnancy.

Introduction

The GH and IGF-1 axis is a major regulator of mammalian growth. As discussed in Chapter 1, Two GH genes encode two 22 kDa GH proteins: pituitary GH (GH-N; GH1) and GH-V variant (GH-V; GH2). The protein sequences of GH-N and GH-V are highly conserved, differing by 13 out of 191 amino acids which are scattered throughout the protein (Alsat et al., 1998) but they have distinct expression profiles. GH-N is secreted in a pulsatile fashion from the pituitary, and is also expressed at extra-pituitary sites, while GH-V is secreted from the placenta in a nonpulsatile manner. The continuous secretion of GH-V into the maternal compartment is thought to contribute to maternal metabolic alterations during pregnancy (Eriksson et al., 1989). Both proteins bind the GHR with similar affinity and share similar physiological somatotrophic, lactogenic and lipolytic properties (Alsat et al., 1997; Verhaeghe, 2008). However, compared with GH-N, GH-V binds the prolactin (PRL) receptor poorly and its lactogenic affects are greatly reduced (Igout et al., 1995; MacLeod et al., 1991b).
During pregnancy, concentrations of GH-N in the maternal circulation decline, whilst GH-V expression increases from week five, gradually replacing GH-N completely at approximately 20 weeks (Eriksson et al., 1989). The increase in maternal circulating GH-V is positively associated with fetal growth and circulating IGF-1 concentrations during pregnancy (Chellakooty et al., 2004; Koutsaki et al., 2011; McIntyre et al., 2000; Mirlesse et al., 1993). A growth-promoting effect for GH-V has been demonstrated in vivo in non-pregnant hypophysectomized rats treated with GH-V and transgenic mice (Barbour et al., 2002; Caufriez et al., 1990a; MacLeod et al., 1991b; Selden et al., 1988a). Moreover, GH-V has pro-angiogenic properties (Struman et al., 1999) and stimulates trophoblast invasion in vitro and may therefore play a role in the process of placentation (Corbacho et al., 2002; Lacroix et al., 2005).
One of the characteristic features of the maternal adaptation to pregnancy is insulin resistance with resultant hyperinsulinemia (Catalano et al., 1991). This environment ensures adequate nutrient supply to the fetus. However, increased insulin resistance can lead to gestational diabetes. Placental hormones, and to a lesser extent increased fat deposition during pregnancy, may contribute to insulin resistance during pregnancy (Catalano, 2010; Ryan and Enns, 1988). Consistent with this, higher concentrations of circulating GH-V have been observed in pregnancies complicated by diabetes (Fuglsang et al., 2003; McIntyre et al., 2000). Furthermore, GH-V has been demonstrated to induce severe insulin resistance and alter body composition in non-pregnant transgenic mice that overexpress GH-V (Barbour et al., 2002).
Despite a proposed role for GH-V during pregnancy, the effects of GH-V administration on metabolic parameters and outcomes related to maternal and fetal growth are poorly understood. In the present study, the activity of GH-V in human and mouse cell lines was investigated, and the dose-response relationship for recombinant GH-V administration examined in a mouse model of normal pregnancy.

Hypothesis and aims

The hypothesis is that exogenous GH-V administration impacts fetal growth and maternal metabolic outcomes in mice.
The aims of this study are therefore:
a.To confirm activity of recombinant GH-V in cell lines.
b.To determine the effect of GH-V treatment on fetal growth;
c.To determine the effect of GH-V treatment on maternal metabolic outcomes, including maternal growth, body composition, and insulin sensitivity;
d.To elucidate the underlying mechanisms.

The effect of human GH-V on mice
Activation of the mouse GHR by GH-V

As mice don’t express placental growth hormone during pregnancy, experiments were conducted to confirm the activity of the human GH-V against the human and mouse GHR. The activation of STAT5 signal transduction was determined in human and mouse cell lines by Western blotting. Other studies have also shown that GH-V can activate the mouse GHR, but it was important to confirm this as the recombinant human GH-V used (from PLR Ltd) hadn’t been published before.
Both GH-N and GH-V stimulated STAT5 phosphorylation in the human prostate cancer cell line, LNCaP (Figure 3.1). GH can activate both the GH and PRL receptors. To determine whether GH-V activation of STAT5 occurred through binding to the GHR, PRL receptor expression was investigated. LNCaP cells have previously been demonstrated to only express very low levels of PRL receptor mRNA (22). I was unable to detect PRL receptor expression in LNCaP cells by semi-quantitative RT-PCR (Figure 3.2). Furthermore, induction of STAT5 phosphorylation by GH-N and GH-V was abrogated by the specific GHR antagonist, B2036, thus confirming that phosphorylation of STAT5 occurred through activation of the GHR (Figure 3.1). B2036 is the protein component of the pegylated peptide inhibitor, Pegvisomant (Pfizer). It is specific to the GHR and has no effect on the PRL receptor (van der Lely and Kopchick, 2006).
Both GH-N and GH-V stimulated STAT5 phosphorylation in the human prostate cancer cell line, LNCaP (Figure 3.1). GH can activate both the GH and PRL receptors. To determine whether GH-V activation of STAT5 occurred through binding to the GHR, PRL receptor expression was investigated. LNCaP cells have previously been demonstrated to only express very low levels of PRL receptor mRNA (22). I was unable to detect PRL receptor expression in LNCaP cells by semi-quantitative RT-PCR (Figure 3.2). Furthermore, induction of STAT5 phosphorylation by GH-N and GH-V was abrogated by the specific GHR antagonist, B2036, thus confirming that phosphorylation of STAT5 occurred through activation of the GHR (Figure 3.1). B2036 is the protein component of the pegylated peptide inhibitor, Pegvisomant (Pfizer). It is specific to the GHR and has no effect on the PRL receptor (van der Lely and Kopchick, 2006).

The effect of GH-V treatment on the tone of uterine arteries in mice

An animal study was conducted to determine whether there was a direct effect of GH-V on vessel tone using wire myography. Eight wild type female mice were mated and culled at gestational day 18.5. The uterine arteries were dissected and 4 segments were mounted on the myograph. Wire myography was performed as described in Chapter 2. Constriction was measured using phenylephrine (PHE; 10-10 to 10-5M). After performing the first dose-response curve to PHE, vessels were incubated with GH-V (500 ng/ml) for 30 min, and then the second dose-response curve to PHE was performed in order to test the effect of GH-V on vessel tone in response to vasoactive agents. Representative raw data are demonstrated in Figure 3.6. The effective concentration 80 (EC80) was calculated for individual vessel segments before and after GH-V treatment.

Chapter 1: Introduction
1.1. Structure of the placenta
1.2. Development of the placenta
1.3. The mouse as a model of human pregnancy
1.4. Pathological pregnancies
1.5. Growth hormone and pregnancy
1.6. Thesis objectives
Chapter 2: Methodology
2.1. Materials
2.2. Animals
2.3. Myography
2.4. Biochemical and molecular analysis
2.5. Cell culture
2.6. Statistical analysis
Chapter 3: The effect of placental growth hormone in mouse pregnancy
3.1. Preface
3.2. Introduction
3.3. Hypothesis and aims
3.4. The effect of human GH-V in mice
3.5. Discussion
Chapter 4: Comparison of pulsatile versus continuous administration of placental growth hormone in mice
4.1. Preface
4.2. Introduction
4.3. Hypothesis and aims
4.4. Recombinant GH-V protein stability
4.5. Effect of pulsatile versus continuous administration of GH-V treatment on mice
4.6. Discussion
Chapter 5: Optimisation of a GH-V ELISA and determination of the serum concentrations of GH-V and related proteins in pathological human pregnancies
5.1. Preface
5.2. The development of a GH-V ELISA
5.3. Maternal serum GH-V in inappropriate birth weight for gestational age pregnancies
5.4. Maternal serum GH-V in pregnancies complicated with gestational diabetes mellitus
5.5. Maternal serum GH-V in pregnancies complicated with pre-eclampsia
Chapter 6: Discussion
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The effect of placental growth hormone in pregnancy