In the first trimester of human pregnancy specialised placental cells, termed cytotrophoblasts differentiate into extravillous trophoblasts (EVTs), which grow out from the placenta and invade into the maternal decidua, acting to physically attach the placenta to the decidua, and adapt the uterine spiral arteries to support pregnancy. A proportion of these EVTs also temporarily occlude the spiral arteries for approximately the first 10 weeks of gestation, preventing maternal blood flow to the placenta and creating a low oxygen environment in which placental and fetal development occur. The processes of trophoblast outgrowth and invasion, and the establishment of a low oxygen environment, are essential for the success of pregnancy, and inadequate trophoblast invasion into the uterus has been associated with recurrent miscarriage, pre-eclampsia and fetal growth restriction. However, the exact mechanisms that control the differentiation of cytotrophoblasts down the extravillous trophoblast lineage are poorly understood. Since the extent of this invasive process is unique to human implantation, animal models are of limited value in studying trophoblast invasion. Existing in vitro models have major limitations in that many are very difficult to quantify while others many not study the correct trophoblast population. The research in this thesis has focussed on the development of novel models by which to study cytotrophoblast differentiation, and the use of these models to further understand cytotrophoblast differentiation down the EVT lineage, and the regulation of EVT outgrowth by oxygen. Methods Outgrowth from first trimester villous explants was characterized using immunohistochemistry. Explant viability was investigated using dual staining with chloromethylfluorescin diacetate (CMFDA) and ethidium bromide, by examining DNA laddering, and by immunostaining sectioned explants over 96 hours of culture. The extended viability of cytotrophoblasts in multilayered cell islands in villous tips was exploited to isolate these cells using sequential trypsin digests of cultured villous explants. The trophoblast population obtained were characterized by immunohistochemistry. Finally, villous explants were cultured in either 1.5% or 8% oxygen and the frequency and area of outgrowths was quantified in order to determine the effect of gestation and oxygen on EVT outgrowth. Results Approximately 1/4 of explants cultured in 20% oxygen produced EVT outgrowth. Outgrowth formation and expansion resulted from proliferation of cells in the tips of anchoring villi, and EVTs within the outgrowth did not proliferate. The percentage of explants producing outgrowth declined as gestation increased from 8 to 12 weeks. Dual staining with CMFDA and ethidium bromide revealed degeneration of the syncytiotrophoblast by non-apoptotic mechanisms within 4 hours of culture, but this syncytiotrophoblast layer was able to be regenerated. The majority of cytotrophoblasts died within one week of culture, but despite this explants were able to produce EVT outgrowth for up to 3 weeks due to the extended survival of a specific set of cytotrophoblasts located in cell islands in the tips of anchoring villi. These surviving cells were able to differentiate into EVTs, but not regenerate the surrounding syncytiotrophoblast in the villus tip. Trypsinization of first trimester villi after extended explant culture resulted in the isolation of a viable population of ‘putative EVT progenitors’ that did not syncytialise in culture, but were able to proliferate. 20% of these cells differentiated down the EVT lineage within 96 hours of culture. The putative EVT progenitors expressed markers previously localised to cytotrophoblasts in cell islands of anchoring villi, including αvβ6 integrin and FGFR-2. The addition of exogenous FGF-4 did not affect the differentiation of these cells into EVTs, nor did FGF-4 alter the frequency of EVT outgrowth from explants. Culture in 1.5% oxygen significantly reduced the frequency and area of outgrowths in comparison to 8% oxygen. HLA-G and α1 integrin were both expressed throughout outgrowths with no difference in expression of these proteins between oxygen concentrations. Gestation influenced the response of explants to oxygen, with a significant differential response to oxygen concentration in placentae under 11 weeks of gestation but no differential response in placentae of 11 or 12 weeks. Conclusions In the first trimester, oxygen and gestational age regulate extravillous trophoblast outgrowth in both an independent and interdependent manner. The cytotrophoblast population in the first trimester does not consist of one homogenous bipotent population. Rather there are at least two separate populations: 1) EVT progenitors that exist in the tips of potential anchoring villi that are likely to be committed to EVT differentiation and 2) monolayer villous cytotrophoblasts which are likely to be committed to syncytiotrophoblast differentiation. The second population is easily isolated by traditional enzymatic digestion methods whereas the first much smaller population can be isolated by exploiting their prolonged survival in explant culture.

Testicular function is stimulated from the pituitary gland by the gonadotrophic hormones FSH and LH. The testis in turn regulates gonadotrophin secretion by negative feedback. The agents of feedback are steroids, and in addition, probably a protein hormone, which has been given the name inhibin. A method capable of detecting inhibin-like activity in bull seminal plasma (bSP) was developed. Administration of bSP at the time of castration to five-week old male rats inhibited the post-castration rise of serum concentrations of FSH and LH otherwise seen 24 h later. The degree of inhibition depended on the dose; 0.5 ml bSP or the equivalent amount of bSP-extract always suppressed FSH and LH to levels typical of intact rats. The rats' sensitivity to 0.2 ml bSP varied with the time of year, possibly reflecting seasonal changes in the onset of puberty. The time-course of action of bSP-extract was studied in intact rats. Serum FSH was suppressed 12, 24 and 48 h after a single injection, but not at 3 or 6 h. LH was suppressed at 6, 12, 24 and possibly 48 h.

A procedure was developed to purify ovine placental lactogen (oPL) from fetal cotyledonary tissue of late gestation. A prolactin radioreceptor assay using mammary tissue membranes from the late pregnant rabbit was established and used to monitor lactogenic activity throughout the purification.

This poststructuralist qualitative study explored the discourses constructing women’s choice for a caesarean section in the absence of clinical indications, in the talk and texts of women, midwives, an obstetrician, professional journals and the media publications. The study affirms inscriptions surrounding choice in childbirth are shaped discursively through a multiplicity of discourses underpinned by social and institutional practices. With advances in technology, childbearing women have a greater variety of options from which to choose. Controversial, is the option of a caesarean section, regardless of clinical need. The issue is depicted in both professional and popular discourse as contentious, complex and contradictory. Its momentum into the 21st century, as a new object of obstetric discourse, has been played out on a number of platforms. In this thesis I draw from the theoretical ideas of French philosopher Michel Foucault, to examine this complex debate. I argue there is a volatile moment in the history of childbirth in which an explosion of discourses have sculptured choice for a caesarean, in the absence of clinical indications, out of a repartee of autonomy, convenience, desire, fear and risk. In this precarious moment, new meanings joust with the old on a shifting terrain awash with rhetoric that co-opts, competes, and contradicts to bring about a caché of mutable ‘truths’. Whether caesarean, as an optional extra, can be explained in terms of a libertarian imperative, an embodiment of lifestyle, the satiation of desire, the attenuation of fear or the avoidance of risk, the democratisation of this choice has exposed a pathologising paradox, whereupon the normal emerges as the abnormal, and the abnormal emerges as the normal. The deconstruction of choice through a poststructuralist lens has enabled insight into how contradiction and contest befall the ‘order of things ’ and in so doing, provides new openings for contemplating the discursive positioning of women through the competing discourses of childbirth.

In the first trimester of human pregnancy specialised placental cells, termed cytotrophoblasts differentiate into extravillous trophoblasts (EVTs), which grow out from the placenta and invade into the maternal decidua, acting to physically attach the placenta to the decidua, and adapt the uterine spiral arteries to support pregnancy. A proportion of these EVTs also temporarily occlude the spiral arteries for approximately the first 10 weeks of gestation, preventing maternal blood flow to the placenta and creating a low oxygen environment in which placental and fetal development occur. The processes of trophoblast outgrowth and invasion, and the establishment of a low oxygen environment, are essential for the success of pregnancy, and inadequate trophoblast invasion into the uterus has been associated with recurrent miscarriage, pre-eclampsia and fetal growth restriction. However, the exact mechanisms that control the differentiation of cytotrophoblasts down the extravillous trophoblast lineage are poorly understood. Since the extent of this invasive process is unique to human implantation, animal models are of limited value in studying trophoblast invasion. Existing in vitro models have major limitations in that many are very difficult to quantify while others many not study the correct trophoblast population. The research in this thesis has focussed on the development of novel models by which to study cytotrophoblast differentiation, and the use of these models to further understand cytotrophoblast differentiation down the EVT lineage, and the regulation of EVT outgrowth by oxygen. Methods Outgrowth from first trimester villous explants was characterized using immunohistochemistry. Explant viability was investigated using dual staining with chloromethylfluorescin diacetate (CMFDA) and ethidium bromide, by examining DNA laddering, and by immunostaining sectioned explants over 96 hours of culture. The extended viability of cytotrophoblasts in multilayered cell islands in villous tips was exploited to isolate these cells using sequential trypsin digests of cultured villous explants. The trophoblast population obtained were characterized by immunohistochemistry. Finally, villous explants were cultured in either 1.5% or 8% oxygen and the frequency and area of outgrowths was quantified in order to determine the effect of gestation and oxygen on EVT outgrowth. Results Approximately 1/4 of explants cultured in 20% oxygen produced EVT outgrowth. Outgrowth formation and expansion resulted from proliferation of cells in the tips of anchoring villi, and EVTs within the outgrowth did not proliferate. The percentage of explants producing outgrowth declined as gestation increased from 8 to 12 weeks. Dual staining with CMFDA and ethidium bromide revealed degeneration of the syncytiotrophoblast by non-apoptotic mechanisms within 4 hours of culture, but this syncytiotrophoblast layer was able to be regenerated. The majority of cytotrophoblasts died within one week of culture, but despite this explants were able to produce EVT outgrowth for up to 3 weeks due to the extended survival of a specific set of cytotrophoblasts located in cell islands in the tips of anchoring villi. These surviving cells were able to differentiate into EVTs, but not regenerate the surrounding syncytiotrophoblast in the villus tip. Trypsinization of first trimester villi after extended explant culture resulted in the isolation of a viable population of ‘putative EVT progenitors’ that did not syncytialise in culture, but were able to proliferate. 20% of these cells differentiated down the EVT lineage within 96 hours of culture. The putative EVT progenitors expressed markers previously localised to cytotrophoblasts in cell islands of anchoring villi, including αvβ6 integrin and FGFR-2. The addition of exogenous FGF-4 did not affect the differentiation of these cells into EVTs, nor did FGF-4 alter the frequency of EVT outgrowth from explants. Culture in 1.5% oxygen significantly reduced the frequency and area of outgrowths in comparison to 8% oxygen. HLA-G and α1 integrin were both expressed throughout outgrowths with no difference in expression of these proteins between oxygen concentrations. Gestation influenced the response of explants to oxygen, with a significant differential response to oxygen concentration in placentae under 11 weeks of gestation but no differential response in placentae of 11 or 12 weeks. Conclusions In the first trimester, oxygen and gestational age regulate extravillous trophoblast outgrowth in both an independent and interdependent manner. The cytotrophoblast population in the first trimester does not consist of one homogenous bipotent population. Rather there are at least two separate populations: 1) EVT progenitors that exist in the tips of potential anchoring villi that are likely to be committed to EVT differentiation and 2) monolayer villous cytotrophoblasts which are likely to be committed to syncytiotrophoblast differentiation. The second population is easily isolated by traditional enzymatic digestion methods whereas the first much smaller population can be isolated by exploiting their prolonged survival in explant culture.

Testicular function is stimulated from the pituitary gland by the gonadotrophic hormones FSH and LH. The testis in turn regulates gonadotrophin secretion by negative feedback. The agents of feedback are steroids, and in addition, probably a protein hormone, which has been given the name inhibin. A method capable of detecting inhibin-like activity in bull seminal plasma (bSP) was developed. Administration of bSP at the time of castration to five-week old male rats inhibited the post-castration rise of serum concentrations of FSH and LH otherwise seen 24 h later. The degree of inhibition depended on the dose; 0.5 ml bSP or the equivalent amount of bSP-extract always suppressed FSH and LH to levels typical of intact rats. The rats' sensitivity to 0.2 ml bSP varied with the time of year, possibly reflecting seasonal changes in the onset of puberty. The time-course of action of bSP-extract was studied in intact rats. Serum FSH was suppressed 12, 24 and 48 h after a single injection, but not at 3 or 6 h. LH was suppressed at 6, 12, 24 and possibly 48 h.

A procedure was developed to purify ovine placental lactogen (oPL) from fetal cotyledonary tissue of late gestation. A prolactin radioreceptor assay using mammary tissue membranes from the late pregnant rabbit was established and used to monitor lactogenic activity throughout the purification.

In the first trimester of human pregnancy specialised placental cells, termed cytotrophoblasts differentiate into extravillous trophoblasts (EVTs), which grow out from the placenta and invade into the maternal decidua, acting to physically attach the placenta to the decidua, and adapt the uterine spiral arteries to support pregnancy. A proportion of these EVTs also temporarily occlude the spiral arteries for approximately the first 10 weeks of gestation, preventing maternal blood flow to the placenta and creating a low oxygen environment in which placental and fetal development occur. The processes of trophoblast outgrowth and invasion, and the establishment of a low oxygen environment, are essential for the success of pregnancy, and inadequate trophoblast invasion into the uterus has been associated with recurrent miscarriage, pre-eclampsia and fetal growth restriction. However, the exact mechanisms that control the differentiation of cytotrophoblasts down the extravillous trophoblast lineage are poorly understood. Since the extent of this invasive process is unique to human implantation, animal models are of limited value in studying trophoblast invasion. Existing in vitro models have major limitations in that many are very difficult to quantify while others many not study the correct trophoblast population. The research in this thesis has focussed on the development of novel models by which to study cytotrophoblast differentiation, and the use of these models to further understand cytotrophoblast differentiation down the EVT lineage, and the regulation of EVT outgrowth by oxygen. Methods Outgrowth from first trimester villous explants was characterized using immunohistochemistry. Explant viability was investigated using dual staining with chloromethylfluorescin diacetate (CMFDA) and ethidium bromide, by examining DNA laddering, and by immunostaining sectioned explants over 96 hours of culture. The extended viability of cytotrophoblasts in multilayered cell islands in villous tips was exploited to isolate these cells using sequential trypsin digests of cultured villous explants. The trophoblast population obtained were characterized by immunohistochemistry. Finally, villous explants were cultured in either 1.5% or 8% oxygen and the frequency and area of outgrowths was quantified in order to determine the effect of gestation and oxygen on EVT outgrowth. Results Approximately 1/4 of explants cultured in 20% oxygen produced EVT outgrowth. Outgrowth formation and expansion resulted from proliferation of cells in the tips of anchoring villi, and EVTs within the outgrowth did not proliferate. The percentage of explants producing outgrowth declined as gestation increased from 8 to 12 weeks. Dual staining with CMFDA and ethidium bromide revealed degeneration of the syncytiotrophoblast by non-apoptotic mechanisms within 4 hours of culture, but this syncytiotrophoblast layer was able to be regenerated. The majority of cytotrophoblasts died within one week of culture, but despite this explants were able to produce EVT outgrowth for up to 3 weeks due to the extended survival of a specific set of cytotrophoblasts located in cell islands in the tips of anchoring villi. These surviving cells were able to differentiate into EVTs, but not regenerate the surrounding syncytiotrophoblast in the villus tip. Trypsinization of first trimester villi after extended explant culture resulted in the isolation of a viable population of ‘putative EVT progenitors’ that did not syncytialise in culture, but were able to proliferate. 20% of these cells differentiated down the EVT lineage within 96 hours of culture. The putative EVT progenitors expressed markers previously localised to cytotrophoblasts in cell islands of anchoring villi, including αvβ6 integrin and FGFR-2. The addition of exogenous FGF-4 did not affect the differentiation of these cells into EVTs, nor did FGF-4 alter the frequency of EVT outgrowth from explants. Culture in 1.5% oxygen significantly reduced the frequency and area of outgrowths in comparison to 8% oxygen. HLA-G and α1 integrin were both expressed throughout outgrowths with no difference in expression of these proteins between oxygen concentrations. Gestation influenced the response of explants to oxygen, with a significant differential response to oxygen concentration in placentae under 11 weeks of gestation but no differential response in placentae of 11 or 12 weeks. Conclusions In the first trimester, oxygen and gestational age regulate extravillous trophoblast outgrowth in both an independent and interdependent manner. The cytotrophoblast population in the first trimester does not consist of one homogenous bipotent population. Rather there are at least two separate populations: 1) EVT progenitors that exist in the tips of potential anchoring villi that are likely to be committed to EVT differentiation and 2) monolayer villous cytotrophoblasts which are likely to be committed to syncytiotrophoblast differentiation. The second population is easily isolated by traditional enzymatic digestion methods whereas the first much smaller population can be isolated by exploiting their prolonged survival in explant culture.

Testicular function is stimulated from the pituitary gland by the gonadotrophic hormones FSH and LH. The testis in turn regulates gonadotrophin secretion by negative feedback. The agents of feedback are steroids, and in addition, probably a protein hormone, which has been given the name inhibin. A method capable of detecting inhibin-like activity in bull seminal plasma (bSP) was developed. Administration of bSP at the time of castration to five-week old male rats inhibited the post-castration rise of serum concentrations of FSH and LH otherwise seen 24 h later. The degree of inhibition depended on the dose; 0.5 ml bSP or the equivalent amount of bSP-extract always suppressed FSH and LH to levels typical of intact rats. The rats' sensitivity to 0.2 ml bSP varied with the time of year, possibly reflecting seasonal changes in the onset of puberty. The time-course of action of bSP-extract was studied in intact rats. Serum FSH was suppressed 12, 24 and 48 h after a single injection, but not at 3 or 6 h. LH was suppressed at 6, 12, 24 and possibly 48 h.

A procedure was developed to purify ovine placental lactogen (oPL) from fetal cotyledonary tissue of late gestation. A prolactin radioreceptor assay using mammary tissue membranes from the late pregnant rabbit was established and used to monitor lactogenic activity throughout the purification.