published_or_final_version / Zoology / Master / Master of Philosophy

These studies were conducted to determine the mechanisms by which prolactin (PRL) exerts its luteotropic effects on the mink corpus luteum (CL). Three experimental models were developed and utilized in these studies. In the first model, the ovaries from pregnant mink were collected at regular intervals throughout gestation, half the animals were treated with the dopamine agonist 2-bromo-$ alpha$-ergocryptine (bromocryptine), to suppress their endogenous PRL levels, and half were exposed to their endogenous PRL levels. The second model consisted of treating anestrous animals with exogenous gonadotropins to induce follicular development and ovulation, half the animals were then treated with PRL while the other half were left as untreated controls. In the third model, CL were collected from mink at several stages of mink gestation. The cells were enzymatically dispersed, placed in culture and incubated with different concentrations of PRL, luteinizing hormone (LH), follicle stimulating hormone (FSH) and (Bu)$ sb2$cAMP. Using these 3 models, the effects of PRL on P450 side chain cleavage (P450scc), 3$ beta$-hydroxysteroid dehydrogenase (3$ beta$-HSD), steroidogenic acute regulatory protein (StAR), luteinizing hormone receptor (LHr) and prolactin receptor (PRLr) mRNA were determined. Messenger RNA levels for P450scc did not vary significantly over the course of mink gestation and treatment of animals with bromocryptine did not alter the abundance. In the anestrous model, treatment of mink with PRL reduced P450scc mRNA levels below that of the untreated control, while treatment of cultured mink luteal cells with increasing concentrations of PRL had no effect on the abundance of P450scc mRNA. The abundance of 3$ beta$-HSD mRNA varied over the course of mink gestation. Levels were low during embryonic diapause, increased during CL reactivation and peaked during post-implantation gestation. Treatment of mink with bromocryptine prevented the pre-implantation rise in 3$ bet

We investigated possible sex differences in PRL secretory patterns and regulation by E$ sb2$ in gonadectomized Dorset x Leicester x Suffolk sheep kept under natural photoperiod (latitude 45$ sp circ 31 sp prime$). Patterns were assessed over 14 months in groups of 10 rams and ewes, half of which received E$ sb2$ replacement (Silastic implants) at the time of gonadectomy to maintain blood E$ sb2$ at ram breeding-season values. Mean monthly PRL level (based on two 3-h periods of blood sampling) was consistently elevated by E$ sb2$ in the ram (mean 19%), and during all but a few months in the spring and summer in the ewes (mean 90%). Sex differences in the mean PRL were most apparent for the E$ sb2$-treated sheep in August (rams 280 $ pm$ 54 ng/ml vs ewes 128 $ pm$ 18 ng/ml) and for the control sheep in November (rams 19 $ pm$ 5 ng/ml vs ewes 10 $ pm$ 2 ng/ml). Episodic PRL secretion (with 5-min sampling for 4 h) was assessed in every season. In all four groups, pulse amplitude and frequency and basal level were higher in summer than winter. E$ sb2$ treatment was associated with larger PRL pulses in both sexes in every season but summer, and with decreased (from 8.2 to 4.6 per 4 h) and increased (from 2.2 to 6.6 per 4 h) frequencies of pulses in rams in the spring and summer, respectively. The PRL response to TRH (two iv injections 50 ng/kg BW given 80 min apart) was also assessed in every season. Mean 40-min increases after injection were highest in spring and summer. E$ sb2$ treatment produced in both sexes a 2-3 fold larger increment 1 in every season, and increment 2 in specific seasons. Preinjection and increment values were positively correlated within animals of each group across seasons (r = 0.89 and 0.65). The Incr 2/lncr 1 ratio (mean 0.76 $ pm$ 0.10) was not affected by seasons or E$ sb2$, and did not denote a self priming effect of TRH. Diurnal patterns indicated that PRL mean levels during light and dark phases were not different from each other within

The Mac-T cell system was used to investigate the role of the prolactin (PRL) receptor in cell proliferation and the regulation of milk protein synthesis. This study was designed to investigate whether overexpressing the PRLR in the Mac-T cell line resulted in a change in its growth rate and an enhancement of its ability to produce milk proteins. To accomplish these goals, Mac-T cells were stably transfected with the rabbit prolactin receptor gene. Fifteen clones and a pool of transfectants were obtained. Of these, one clone and the pool were positive for the PRL receptor expression. The clone (S15) and pool (SP) cells were sorted into high (H), medium (M), and low (L) expressors, of the PRLR. The high expressors were used for all subsequent experiments. The presence of high levels of the PRLR on the surface of S15 and SP cells was further confirmed by receptor binding assay and Western Blot. Following the establishment of these cell lines, the cells were used to investigate the effect of increased levels of PRLR on cell proliferation and milk protein synthesis. / It was found that the growth rate of parental cells was depressed in the presence of 5 mug/ml of PRL. In contrast, the growth rate of the transfectants was enhanced by the addition of 5 mug/ml PRL to the culture medium. In addition, both "SP" and "S15" cells produced higher levels of STAT5 upon long-term (48 h) PRL stimulation. No effect on the synthesis of alpha S1- and beta-caseins was noted. It is likely that no differences in protein synthesis were observed because the cells have lost the ability to differentiate, even when cultured on collagen gels in the presence of lactogenic hormones.

A turkey prolactin (PRL) cDNA, encoding a 199 amino acid turkey PRL (tPRL), was cloned from a pituitary library. The mature PRL shared about 70% homology with mammalian PRLs and about 30% with fish PRLs. Areas of highest homology to other PRLs were located in the carboxyl terminus of the tPRL. Prolactin mRNA analyses, during the reproductive life of the turkey hen, confirmed that the high pituitary and plasma levels of PRL measured during the incubation phase are due to enhanced transcription of the PRL gene. Furthermore, tPRL mRNA levels were highly correlated with pituitary levels of tPRL. Recombinant tPRL (rctPRL), biologically and immunologically similar to pituitary tPRL, was purified from Escherichia coli cultures hosting an expression vector carrying the tPRL cDNA. Polyclonal antibodies raised against purified rctPRL behaved similar as antibodies raised against pituitary derived tPRL, in immunoblotting and immunocytochemistry experiments. Three tPRL isoforms (with estimated molecular weights of 27 kDa, 25 kDa and 24 kDa) were identified in turkey pituitary extracts. The relative proportion of the 27 kDa isoform increased while that of the 25 kDa decreased with increasing levels of total pituitary tPRL, during the reproductive life of the turkey hen. The partition of the immunoreactivity of tPRL into the three isoforms perhaps provides an additional control of the multitude functions of PRL.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 201-220). / Microfiche. / xiii, 220 leaves, bound 29 cm

Mammary gland development is controlled by hormones such as progesterone and prolactin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. This thesis investigates two molecules in detail, Elf5 and KIBRA, which were identified as potential prolactin targets in a transcript profiling screen for key members in this genetic program of mammary morphogenesis. We examined the effect of expression of Elf5, a transcription factor critical in alveolar differentiation, in a 3D culture model of non-transformed mammary epithelial MCF-10A cells. We discovered that Elf5 expression was selectively repressed over time in these cells when cultured on a basement membrane, and that Elf5 overexpression disrupted the architecture of acini resulting in luminal filling. This occurred due to an increase in the expression of epidermal growth factor receptor (EGFR) with repressed the induction of the pro-apoptotic molecule, Bim. We also observed that Elf5 is up-regulated with progesterone treatment, and that suppression of Elf5 expression in T47D breast cancer cells inhibits proliferation. Data obtained from the suppression of Elf5 expression in the presence of progesterone suggested that the role played by Elf5 in the Pg signalling pathway in T47D cells is relatively minor, and that rather than being a major downstream factor, the induction of Elf5 expression is utilised more to influence and potentiate other signalling pathways, such as the Prl pathway. We characterised expression of KIBRA in the mammary gland and breast cancer cell lines, and observed that KIBRA was also up-regulated with progesterone treatment. Using a bioinformatic approach, we identified the tyrosine kinase receptor DDR1 as a binding partner of KIBRA. We have demonstrated that the WW domains of KIBRA bind to a PPxY motif in DDR1, and that these molecules dissociate upon treatment with the DDR1 ligand, collagen. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodelling events of mammary gland development. Overall, the data presented in this thesis contributes to our growing knowledge of the genetic program responsible for mammary development and carcinogenesis.

Thesis (Ph. D.)--Florida State University, 2005. / Advisor: Dr. Marc E. Freeman, Florida State University, Program in Neuroscience. Title and description from dissertation home page (viewed June 8, 2005). Document formatted into pages; contains xi, 172 pages. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Lane J. Brunner. Vita. Includes bibliographical references. Also available from UMI.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 110-118).