title: The Role of Amino Acids and the Threonine-SAM Pathway in the Development of Bovine Inner Cell Mass and Pluripotency

authors: Najafzadeh, Vahid

subjects: amino acid

abstract: Pluripotent stem cells (PSCs) are the foundation of all cell types in the body including functional gametes. They are derived either from the inner cell mass (ICM) of preimplantation mouse and rat embryos, known as embryonic pluripotent stem cells (ePSCs) or embryonic stem cells (ESCs) or via reprogramming of somatic cells to their pluripotent state, known as induced pluripotent stem cells (iPSCs). PSCs are excellent tools for gene editing and accelerated breeding in livestock animals. So far, germline-competent PSCs have only been isolated in mice and rats. Bovine ESCs are yet to be isolated due to the inadequate understanding of the mechanisms involved in bovine pluripotency. This study aimed to investigate the metabolic aspects of bovine ICM development and pluripotency. 
In this study, amino acids were screened via a targeted depletion approach to determine if any amino acid was critical for bovine ICM development and pluripotency. Experiments were conducted on in vitro fertilised day (D) 5 preimplantation bovine embryos, individually cultured in chemically-defined, protein- and glutamine-free synthetic oviduct fluid with variations of essential (E) and non-essential (NE) amino acids (AAs) until D8. Depleting (Δ) one (threonine, methionine), two (threonine_methionine, cysteine_methionine, cysteine_threonine, isoleucine_leucine, isoleucine_lysine, leucine_lysine), three (cysteine_methionine_threonine, isoleucine_leucine_lysine) or six (histidine_phenylalanine_arginine_valine_tryptophan_tyrosine) EAAs did not affect ICM formation, even when NEAAs were also removed from Δthreonine and Δmethionine groups. However, depleting another six (cysteine_isoleucine_lysine_leucine_methionine_threonine), nine (+cysteine_methionine_threonine, +isoleucine_leucine_lysine), eleven EAAs (+threonine, +methionine) or all twelve EAAs impaired blastocyst development. In vitro fertilised D1 preimplantation mouse embryos cultured from D1-4 were also not affected by a Δthreonine culture condition.
The role of the threonine dehydrogenase (TDH)-mediated threonine-s-adenosylmethionine (SAM) pathway in bovine ICM development and pluripotency in bovine embryos and cells was also investigated. This metabolic pathway has been shown to play a critical role in sustaining mouse pluripotency. TDH expression was probed at mRNA and protein levels, and its activity was knocked down using chemical inhibitors quinazolinecarboxamide (Qc1) and 3-hydroxynorvaline (3-HNV). The effect of TDH inhibition on embryo development, cell numbers, lineage-specific marker gene expression, histone trimethylation and autophagy was studied. 
TDH was present in trophectoderm (TE), ICM and 6 day old (D6) ICM outgrowths but absent in D6 TE primary cultures. In bovine embryos, Qc1 reduced blastocyst grade 1-3 (B¹⁻³) and 
B¹⁻² embryo development 1.5- and 2-fold, respectively, compared to the dimethyl sulfoxide (DMSO) controls. Qc1 also reduced ICM and TE cell numbers ~2-fold. This cell loss was due to increased autophagy. Qc1-treated ICM and TE nuclei were explicitly hypermethylated at histone (H)3 lysine (K)4 trimethylation (me3), while H3K9me3 and H3K27me3 were not affected. Hypoblast (PDGRFα), pluripotency-associated epiblast (NANOG, FGF4, and SOX2) and TE (CDX2) markers were not altered in the Qc1 group compared to DMSO controls. Qc1 also severely compromised mouse embryos cultured from day 1-4. In bovine cells, Qc1 compromised fresh ICM, D6 ICM outgrowths and fresh TE cells but failed to impair D6 TE primary cultures. 3-HNV severely compromised bovine embryo development, D1 ICM outgrowths, D6 TE primary cultures and bovine embryonic fibroblast (BEF) cells. However, it unexpectedly severely impaired TDH-free skin fibroblasts in a Δthreonine culture condition. 
The results indicate that the effect of amino acid starvation on embryos is different from that on individual cells. The findings also suggest that TDH is vital for bovine embryonic development via regulating H3K4me3 levels. Moreover, the results denote that TDH becomes restricted to cultured ICM cells and disappears from TE primary cultures. This may indicate that TDH is essential for maintaining bovine pluripotency. 3-HNV was claimed to be a specific TDH enzyme inhibitor; However, this study showed that it compromises TDH-free cells in a Δthreonine culture condition, suggesting that it does not specifically impair cells and embryos via the TDH-mediated threonine-SAM pathway.

text: The Role of Amino Acids and the Threonine-SAM Pathway in the Development of Bovine Inner Cell Mass and Pluripotency Pluripotent stem cells (PSCs) are the foundation of all cell types in the body including functional gametes. They are derived either from the inner cell mass (ICM) of preimplantation mouse and rat embryos, known as embryonic pluripotent stem cells (ePSCs) or embryonic stem cells (ESCs) or via reprogramming of somatic cells to their pluripotent state, known as induced pluripotent stem cells (iPSCs). PSCs are excellent tools for gene editing and accelerated breeding in livestock animals. So far, germline-competent PSCs have only been isolated in mice and rats. Bovine ESCs are yet to be isolated due to the inadequate understanding of the mechanisms involved in bovine pluripotency. This study aimed to investigate the metabolic aspects of bovine ICM development and pluripotency. 
In this study, amino acids were screened via a targeted depletion approach to determine if any amino acid was critical for bovine ICM development and pluripotency. Experiments were conducted on in vitro fertilised day (D) 5 preimplantation bovine embryos, individually cultured in chemically-defined, protein- and glutamine-free synthetic oviduct fluid with variations of essential (E) and non-essential (NE) amino acids (AAs) until D8. Depleting (Δ) one (threonine, methionine), two (threonine_methionine, cysteine_methionine, cysteine_threonine, isoleucine_leucine, isoleucine_lysine, leucine_lysine), three (cysteine_methionine_threonine, isoleucine_leucine_lysine) or six (histidine_phenylalanine_arginine_valine_tryptophan_tyrosine) EAAs did not affect ICM formation, even when NEAAs were also removed from Δthreonine and Δmethionine groups. However, depleting another six (cysteine_isoleucine_lysine_leucine_methionine_threonine), nine (+cysteine_methionine_threonine, +isoleucine_leucine_lysine), eleven EAAs (+threonine, +methionine) or all twelve EAAs impaired blastocyst development. In vitro fertilised D1 preimplantation mouse embryos cultured from D1-4 were also not affected by a Δthreonine culture condition.
The role of the threonine dehydrogenase (TDH)-mediated threonine-s-adenosylmethionine (SAM) pathway in bovine ICM development and pluripotency in bovine embryos and cells was also investigated. This metabolic pathway has been shown to play a critical role in sustaining mouse pluripotency. TDH expression was probed at mRNA and protein levels, and its activity was knocked down using chemical inhibitors quinazolinecarboxamide (Qc1) and 3-hydroxynorvaline (3-HNV). The effect of TDH inhibition on embryo development, cell numbers, lineage-specific marker gene expression, histone trimethylation and autophagy was studied. 
TDH was present in trophectoderm (TE), ICM and 6 day old (D6) ICM outgrowths but absent in D6 TE primary cultures. In bovine embryos, Qc1 reduced blastocyst grade 1-3 (B¹⁻³) and 
B¹⁻² embryo development 1.5- and 2-fold, respectively, compared to the dimethyl sulfoxide (DMSO) controls. Qc1 also reduced ICM and TE cell numbers ~2-fold. This cell loss was due to increased autophagy. Qc1-treated ICM and TE nuclei were explicitly hypermethylated at histone (H)3 lysine (K)4 trimethylation (me3), while H3K9me3 and H3K27me3 were not affected. Hypoblast (PDGRFα), pluripotency-associated epiblast (NANOG, FGF4, and SOX2) and TE (CDX2) markers were not altered in the Qc1 group compared to DMSO controls. Qc1 also severely compromised mouse embryos cultured from day 1-4. In bovine cells, Qc1 compromised fresh ICM, D6 ICM outgrowths and fresh TE cells but failed to impair D6 TE primary cultures. 3-HNV severely compromised bovine embryo development, D1 ICM outgrowths, D6 TE primary cultures and bovine embryonic fibroblast (BEF) cells. However, it unexpectedly severely impaired TDH-free skin fibroblasts in a Δthreonine culture condition. 
The results indicate that the effect of amino acid starvation on embryos is different from that on individual cells. The findings also suggest that TDH is vital for bovine embryonic development via regulating H3K4me3 levels. Moreover, the results denote that TDH becomes restricted to cultured ICM cells and disappears from TE primary cultures. This may indicate that TDH is essential for maintaining bovine pluripotency. 3-HNV was claimed to be a specific TDH enzyme inhibitor; However, this study showed that it compromises TDH-free cells in a Δthreonine culture condition, suggesting that it does not specifically impair cells and embryos via the TDH-mediated threonine-SAM pathway.

year: 2018

title: Accompanied by suspicion: An ethnographic account of negotiating gender tensions and positioning in counselling practice and researching child sexuality

authors: Flanagan, Paul G.

abstract: This chapter presents my experience of journeying into research on the contentious area of sexuality in childhood.  Reflecting on experiences from professional, personal and academic contexts, I expose occasions of discomfort, unease and public questioning of my integrity – because I am male, and because of the sensitive subject area of this research: children and sexuality. Revealing a number of discursive contexts (not only am I male, but my counselling practice has largely been with children and in the area of child sexual abuse and child sexual activity) the question is boldly presented – how do I balance gender tensions and positionings?  I recognise that gender tensions are an appropriate challenge within a research and public media gaze that calls for accountability for the extent and dominance of child and sexual abuse perpetrated by males. Responding to and within constraints of others’ perceptions and maligning remarks, I acknowledge that questions of gender and safety are appropriate and hold value for me, about how I step into researcher positioning taken up in this study. A sense of advocacy and social justice underpins my commitment to this research making suspicion an uncomfortable but valuable participant to negotiate in this process.

text: Accompanied by suspicion: An ethnographic account of negotiating gender tensions and positioning in counselling practice and researching child sexuality This chapter presents my experience of journeying into research on the contentious area of sexuality in childhood.  Reflecting on experiences from professional, personal and academic contexts, I expose occasions of discomfort, unease and public questioning of my integrity – because I am male, and because of the sensitive subject area of this research: children and sexuality. Revealing a number of discursive contexts (not only am I male, but my counselling practice has largely been with children and in the area of child sexual abuse and child sexual activity) the question is boldly presented – how do I balance gender tensions and positionings?  I recognise that gender tensions are an appropriate challenge within a research and public media gaze that calls for accountability for the extent and dominance of child and sexual abuse perpetrated by males. Responding to and within constraints of others’ perceptions and maligning remarks, I acknowledge that questions of gender and safety are appropriate and hold value for me, about how I step into researcher positioning taken up in this study. A sense of advocacy and social justice underpins my commitment to this research making suspicion an uncomfortable but valuable participant to negotiate in this process.

year: 2015

title: Pakeha students and a pro-Treaty analysis: Teaching issues in a diploma of clinical psychology programme

authors: Yensen, Helen

subjects: Maori

abstract: We ran Treaty/biculturalism workshops for the Diploma on an ad hoc basis in 1990, 1991, and 1992. This year we have contracted for a fuller package which involves work with first and second years and staff. Next year this will continue with third year students. We believe biculturalism training for non-Maori has two major aspects and, although they overlap in various ways we think that it is useful to separate them: the first one is awareness of Maori cultural practices, values, etc, which can perhaps be called ‘cultural sensitivity training’. This needs to be under the control of Maori and have major input from Maori. The second aspect is where our focus is, and that is awareness of the effect on the Maori world of the loss of sovereignty by Maori; of their marginalisation over the last one hundred and fifty years by Pakeha; of their oppressed status; loss of economic base, and the implications for change that flow from those events. We believe that, at least initially, Pakeha have the responsibility for educating themselves and other pakeha in this area.

text: Pakeha students and a pro-Treaty analysis: Teaching issues in a diploma of clinical psychology programme We ran Treaty/biculturalism workshops for the Diploma on an ad hoc basis in 1990, 1991, and 1992. This year we have contracted for a fuller package which involves work with first and second years and staff. Next year this will continue with third year students. We believe biculturalism training for non-Maori has two major aspects and, although they overlap in various ways we think that it is useful to separate them: the first one is awareness of Maori cultural practices, values, etc, which can perhaps be called ‘cultural sensitivity training’. This needs to be under the control of Maori and have major input from Maori. The second aspect is where our focus is, and that is awareness of the effect on the Maori world of the loss of sovereignty by Maori; of their marginalisation over the last one hundred and fifty years by Pakeha; of their oppressed status; loss of economic base, and the implications for change that flow from those events. We believe that, at least initially, Pakeha have the responsibility for educating themselves and other pakeha in this area.

year: 1993