The protein calmodulin binds Ca2+ in the cytosol and induces conformational
changes that enable the Ca2+-calmodulin complex to activate myosin light-chain ki-
nase enzymes. The myosin light-chain kinase enzymes phosphorylate myosin, thus activating myosin and enabling its interaction with actin, which results in SMC contractions (Vrachnis et al., 2011; Blackburn, 2014).
There are various pathways through which the entry of Ca2+ions into the cytoplasm
is mediated; one such pathway is through OT signalling (Michal & Schomburg, 2012, p. 292). The effects of OT are mediated by the OTR, a seven-transmembrane receptor. Like other seven-transmembrane receptors, OTR is located in the plasma membrane with both intra- and extracellular regions. The extracellular region of OTR contains the OT-binding site while its intracellular region is bound to a G- protein, which is composed of three subunits when inactive; Gαq/11, Gβ, and Gγ
(Gimpl & Fahrenholz, 2001).
When OT binds OTR, it induces conformational changes in the receptor that re- sult in the activation of the receptor. Once the receptor is activated, the G-protein separates into its constituent subunits. The Gαq/11 subunit binds to the enzyme phospholipase-C (PLC), activating the enzyme which goes on to hydrolyse a com- ponent of the cell membrane called phosphatidylinositol 4,5-bisphosphate (PIP2).
The hydrolysis of PIP2 results in the formation of inositol 1,4,5-trisphosphate (IP3)
and diacyl-glycerol (DAG), the latter of which aids in the activation of protein ki- nase C (PKC) and the mitogen-activated protein kinases (MAPK) cascade leading to the synthesis of prostaglandins. IP3, on the other hand, binds to the IP3 re-
ceptors located on the sarcoplasmic reticulum, opening Ca2+ channels thus releas-
ing Ca2+ ions from intracellular calcium stores. Intracellular Ca2+ binds calmod-
2.6. Parturition
ulin resulting in smooth muscle contractions as illustrated in Figure 2.15 (Vrachnis et al., 2011). Cytoplasm Plasma membrane Ca2+ Sarcoplasmic re5culum IP3 receptor Oxytocin Receptor β γ Gαq/11 Oxytocin PIP2
IP3 Ca2+ bound to calmodulin
Ac5vated kinase ATP ADP PLC DAG P P Phosphorylated myosin Thin filament Thick filament Voltage-‐gated channel Receptor-‐gated channel Ca2+
Figure 2.15: Activation of myosin by Calcium.
While OT induces the release of Ca2+ from intracellular stores, it also activates
plasma membrane voltage-gated calcium channels, through which most of the Ca2+
involved in smooth muscle contraction enters the cytoplasm. The extracellular Ca2+
binds to calmodulin, resulting in the activation of myosin and smooth muscle con- tractions (Garfield & Maner, 2007). Another mechanism by which Ca2+ can enter
the cytoplasm of SMCs is through channels that are sensitive to changes in the electrical properties of the SMCs. The depolarisation and repolarisation of the membrane through the movement of ions into and out of SMCs, generates bursts of action potentials that have been shown to influence the contractility of the uterus during labour (Laforet et al., 2011; Young, 2007; Lammers et al., 2008).
2.6. Parturition
The effects of P4, and P4 withdrawal, on the mRNA transcripts encoding the pro- teins involved in the activation and contraction of SMCs will be analysed in Chap- ter 8. The next chapter in this part of the thesis gives an overview of the rat model whose study is documented in this thesis.
Chapter 3
The Female Rat
The laboratory rat (Rattus norvegicus) has been used in experimental studies since the early 1900s (Sengupta, 2013). A lot of knowledge has since been gained through the use of rats as model animals (Hedrich, 2000). The work presented in thesis was conducted on tissues obtained from pregnant Spague-Dawley rats. As such, in this chapter, the anatomy and characteristics of the female rat reproductive system are described, as well as compared and contrasted with the human female reproductive system. In addition, the rat uterine and ovarian cycles are also compared with those of humans. The process of parturition in the rat is subsequently described, and the chapter concludes by summarising the aims of this thesis.
3.1
The Female Rat Reproductive System
The rat reproductive system contains all the constituent organs as those of the human reproductive system, i.e., the ovary, uterine tube, uterus, cervix, and vagina. The anatomy of the female reproductive system in the rat, however, differs from
3.1. The Female Rat Reproductive System
the human reproductive system in that the rat uterus is duplex, i.e., it is composed of two separate horns (Figure 3.1a). Each uterine horn is connected to an ovary, via a uterine tube, and has its own cervix. The uterine horns are joined at the vagina, forming a “Y-shape” (Kom´arek et al., 2000). Similar to human uterine walls, the walls of rat uterine horns consist of endometrial and serosal layers that sandwich the uterine muscle, but unlike human myometrium, the rat myometrial layer is composed of two distinct layers that are separated by a vascular layer as illustrated in Figure 3.1b. The inner layer of the myometrium, i.e., the one adjacent to the endometrium, is composed of SMC fibers that envelope the uterine horn in a circular fashion. The outer layer of the myometrium, which is sandwiched between the vascular layer and the serosa, contains SMC fibers that are arranged in “parallel” with the uterine horn (Jain et al., 2000). The inner layer is also called the circular myometrium, while the outer layer is called the longitudinal myometrium.
Unlike humans who menstruate, the uterine lining of the rat uterus is not shed and passed out of the body. Instead, the endometrial layer of the rat uterus is periodically transformed. The periodic changes in the endometrium, as well as changes that occur in the ovaries and vagina, are part of the rat estrus cycle (West- wood, 2008).
Uterine tube Ovaries Uterine horn Cervix Vagina Cervix Uterine horn Uterine tube (a) Endometrium Circular
myometrium myometrium Longitudinal
Vascular layer
Serosa
(b)
Figure 3.1: The female reproductive system in rats. (a) Illustration of the duplex uterus of the rat showing each horn with its respective ovary, uterine tube, and cervix, as well as the vagina. (b) Schematic of the walls of the uterine horns showing the distinct layers of the myometrium that are separated by a vascular layer, and sandwiched between the endometrium and serosa.