Molecular Biology of an Autocrine Inhibitor of Milk Secretion

Bryson, Jane M (1997) Molecular Biology of an Autocrine Inhibitor of Milk Secretion. PhD thesis, University of Glasgow.

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Research over many years has shown that the rate of milk secretion is regulated by frequency and completeness of milk removal. The effect of milk removal occurs through local mechanisms within each mammary gland, and recent work indicates that local control is through feedback inhibition by a novel milk protein termed FIL, Feedback Inhibitor of Lactation. Evidence from studies in mammary cell culture suggests that FIL controls the rate of milk secretion, mediating the effect of frequency and completeness of milk removal, by inhibition of constitutive secretion, which involves reversible blockade of mammary membrane trafficking. Due to its effects on membrane trafficking, FIL may also regulate mammary differentiation. This may provide a mechanistic explanation for the developmental changes associated with sustained alterations in milking frequency or efficiency. For example, extended frequent milking elicits a significant increase in secretory cell differentiation as measured by mRNA abundance and activities of key enzymes involved in milk synthesis. However, neither the developmental changes at the level of gene expression or the mechanism underpinning these responses has been characterised in detail. The aim of this project was therefore to investigate whether frequency of milking does indeed control expression of key milk protein genes and to investigate the mechanisms underpinning the putative regulation of gene expression - specifically, to determine if FIL is competent to influence mammary gene expression. In the first phase of the project, manipulation of milking frequency and concomitant changes in the rate of milk secretion were found to be accompanied in the long term, but not in the short term, by changes in milk protein mRNA abundance. Treatments which did not change milk yield did not affect milk protein gene expression, indicating that changes in milk protein gene expression, like changes in milking frequency are dependent on effective manipulation of milk removal. To investigate the molecular mechanisms underpinning the increase in milk protein mRNA abundance, demonstrated in vivo, goat mammary cells m primary culture were treated with milk fractions and FIL to determine if this protein was indeed competent to modulate milk protein gene expression. These studies demonstrated that long term exposure to FIL decreases milk protein mRNA abundance in vitro, lending further credence to the theory that FIL is a regulator of mammary differentiation. Changes in gene expression in response to FIL, demonstrated in vitro, imply that FEL is involved in the developmental response of the gland to frequency of milk removal. Since FIL is itself a mammary gene product, it is also possible that FIL is an autocrine regulator of its own expression. Therefore, the next phase of this project was to clone the gene for FIL, and, if successful investigate the regulation of its gene expression. Several strategies were implemented to clone FII. including screening of goat mammary cDNA libraries with anti-FIL antibody and with synthetic oligonucleotides constructed on the basis of known FIL protein sequence. These strategies were not successful. Whether this was due to library composition, antibody specificity or excessive redundancy in the predicted nucleotide sequence of caprine FIL remains to be determined. In conclusion, the project has shown that the developmental responses to frequency and completeness of milk removal are associated with changes in expression of key milk protein genes, and experiments in cell culture suggest these changes may be elicited by FIL, as a long term consequence of its effects on mammary membrane trafficking.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Colin Wilde
Keywords: Molecular biology, Endocrinology
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-74768
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 13 Nov 2019 15:58
Last Modified: 13 Nov 2019 15:58

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