Interactions of progesterone with zwitterionic dipalmitoyl phosphotidylcholine (DPPC) multilamellar liposomes (MLVs) were investigated as a function of temperature and progesterone concentration by using three non-invasive techniques of Fourier transform infrared (FTIR) spectroscopy, turbidity at 440 nm and differential scanning calorimetry (DSC). The results show that 1mol% of progesterone does not induce a significant change in the shape of thermotropic profile of DPPC. However as progesterone concentration increases, the main transition temperature decreases and phase transition curve broadens. Higher concentrations (12, 18 and 24mol%) also decreased the transition temperature but not as significantly as lower concentrations. The characteristic pretransition of DPPC was completely disappeared upon the addition of progesterone. Progesterone disorders the phospholipid membranes in a concentration dependent manner. Furthermore, low concentrations of progesterone (3, 6 and 9mol%) increase the fluidity of the system but high concentrations (12, 18 and 24mol%) stabilize the membranes by decreasing the mobility of the acyl chains. The opposite effect of progesterone on membrane dynamics of low and high concentrations was also supported by turbidity studies at 440 nm. DSC peaks broaden and shift to lower temperature degrees with increasing concentrations up to 9mol% of progesterone. At 6 and 12mol% of progesterone, the curve contains more than one peak. This indicates the existence of phase separation. The pretransition of liposomes was eliminated for all samples containing progesterone. Analysis of C=O stretching bond in FTIR spectroscopy showed that progesterone does not make any hydrogen bonds with the interfacial region of DPPC liposomes, instead it induces free carbonyl groups in the system. Ester groups were found to be disordered by the addition of progesterone and the effect is profound with 6 and 9mol% concentrations. The head group of liposomes were found to make hydrogen bonding in the vicinity of 3mol% of progesterone in both phases and of 6mol% of progesterone in liquid crystalline phase by infrared spectroscopy of PO- 2 stretching mode. This hydrogen bonding is made either with the hydroxyl group of progesterone or with the water molecules around the head group. With other concentrations of progesterone, there is no evidence of hydrogen bond formation.

Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains vii, 64 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 48-63).

Thesis--Ph. D., University of Hong Kong. / Mimeographed.

The goal of this research was to examine the role of the novel membrane progestin receptor alpha (mPR[alpha]) in the stimulation of sperm hypermotility by the progestin 17,20[beta],21-trihydroxy-4-pregnen-3-one (20[beta]-S) in two teleosts; the Atlantic croaker (Micropogonias undulatus) and the southern flounder (Platylicthys lethigstomata). In croaker, the expression, localization and hormonal regulation of mPR[alpha] in testis and sperm were investigated, as were the intracellular signaling pathways activated by 20[beta]-S and mPR[alpha] to induce croaker sperm hypermotility. In flounder, stimulation of sperm hypermotility by 20[beta]-S and binding of this steroid to flounder sperm membranes were examined. Finally, expression of mPR[alpha] was investigated in flounder testes and the expression and localization of this receptor in flounder testis and sperm was examined. In croaker sperm, mPR[alpha] was expressed on the plasma membrane and localized to the midpiece. Expression of mPR[alpha] was also shown to be associated with high sperm motility and regulated by gonadotropin. The signaling pathways activated by 20[beta]-S in croaker sperm were shown to involve activation of olfactory G-proteins (Golf). Subsequent activation of membrane adenylyl cyclases was also demonstrated and shown to be necessary for 20[beta]-S-stimulated cAMP production and 20[beta]-S-induction of sperm hypermotility. Furthermore, co-immunoprecipitation studies show mPR[alpha] and Golf physically associate with one another, establishing mPR[alpha] as the mediator of 20[beta]-S actions in croaker sperm. Finally, evidence was obtained for progestin-stimulation of sperm hypermotility and the presence of mPR[alpha] on sperm membranes in another marine teleost species belonging to a different family, the southern flounder. In addition, mPR[alpha] was shown to be expressed on flounder sperm membranes and also localized to the sperm midpiece. Results from the following studies support the hypothesis that mPR[alpha] is the mediator of 20[beta]S-stimulated sperm hypermotility in croaker and is a likely intermediary in southern flounder. Furthermore, these data provide a plausible mechanism by which 20[beta]-S and mPR[alpha] stimulate croaker sperm hypermotility. In addition, these results provide the first evidence of hormonal activation of Golf proteins for any species. Finally, mPR[alpha]-mediated mechanisms to increase sperm motility are suggested to be evolutionarily conserved in teleosts since they also likely exist in a non-sciaenid species, the southern flounder.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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No description available.

The anthracycline quinone, doxorubicin (Adriamycin) is an antineoplastic agent that has substantial therapeutic activity against a broad variety of human cancers. Unfortunately, the use of this agent is limited by its cardiac toxicity, which is associated with free radical formation leading to apoptotic cell death. The goal of this work is to improve our understanding about doxorubicin induced cardiomyopathy and to identify compounds to limit doxorubicin induced cardiomyopathy. The knowledge gained here will have a generalized impact on all cardiac diseases involving oxidative stress and apoptosis. We show that doxorubicin induced apoptosis in primary neonatal rat cardiomyocytes can be attenuated by progesterone (PG). The anti-apoptotic action of PG was blocked by a progesterone receptor antagonist, Mifepristone (MF), indicating a progesterone receptor dependent pathyway. Affymetrix gene analyses found that PG treated cardiomyocytes increased the expression of 180 genes. Among the genes upregulated is NAD(P)H: Quinone Oxidoreductase-1 (NQO1) gene. NQO1 is a flavo-enzyme that can catalyze a two-electron reduction of Dox to a more stable hydroquinone, thereby acting as a defense mechanism against oxidative stress. The induction of NQO1 mRNA and NQO1 activity in cardiomyocytes was observed in a dose and time-dependent manner with PG treatment and was blocked by MF. Induction of NQO1 by b-naphoflavone, an inducer of NQO1, resulted in a decrease in caspase-3 activity. However, inhibition of NQO1 by dicoumarol did not attenuate the cytoprotective effect of PG. This data indicates that although induction of NQO1 can decrease Dox induced apoptosis, this is not the primary mechanism of cytoprotection induced by PG. Microarray analyses revealed that PG induced an increase of Bcl-XL mRNA. Inhibiting the expression of Bcl-XL using siRNA reduced the anti-apoptotic effect of PG, suggesting that Bcl-XL is a key player in PG induced cytoprotection. Western blot analyses indicated that PG induced the expression of Bcl-XL in a dose and time dependent manner consistent with the protective effect of PG. Induction of Bcl-XL by PG was blocked by cyclohexamide, but was not blocked by Actinomycin D indicating that a transcriptionally independent mechanism is responsible for the induction of Bcl-XL by PG. The activity of a bcl-x 3'UTR reporter was induced by PG and blocked by MF. These data suggest that PG may induce stabilization of the Bcl-X mRNA. We further explored the mechanism of PG induced Bcl-XL gene expression by comparing the effect of PG to two other steroids: corticosterone (CT) and retinoic acid (RA). Both CT and RA attenuate Dox induced apoptosis in cardiomyocytes. CT, but not RA or PG induced the activity of a GRE reporter plasmid. Analysis of the 5' region of the Bcl-XL promoter indicated that RA and CT, but not PG induced the activity of the 0.9kb region of the Bcl-XL promoter. The induction of the 0.9kb reporter plasmid by CT was glucocorticoid receptor dependent, since it was inhibited by MF. The Bcl-XL promoter does not contain any glucocorticoid or retinoid response elements, but does have AP-1 and NFkB response elements. CT, but not RA or PG induced the activity of an AP-1 reporter plasmid. RA, but not CT or PG induced the activity of an NFkB reporter plasmid. The induction of the 0.9kb Bcl-XL reporter plasmid by CT was blocked by expression of a dominant negative c-jun, TAM67 as well SB202190 indicating a nongenomic effect of CT in activating the Bcl-XL promoter through a p38 MAPK mediated AP-1 mechanism. Therefore although all three types of nuclear receptor ligands induce bcl-xL expression, the effect of CT is mediated by transcriptional activation by AP-1 signaling while NF-kB transcription factor appears to be involved in RA indced bcl-xL transcription.

Menopausal symptoms, including sleep problems, occur as a result of reduced production of ovarian hormones in middle-aged women, and are often treated with replacement of these hormones. However, the efficacy of hormone replacement for improving sleep is controversial. We assessed sleep/wake patterns during baseline and recovery following 6 h of sleep deprivation in ovariectomized middle-aged rats treated with oil, estradiol, or estradiol and progesterone. We found that, at baseline, hormone administration reduced rapid eye movement (REM) sleep initiation and non-REM sleep amount, promoting wakefulness, particularly during the dark (active) phase, but that, during recovery following sleep deprivation, hormonal treatment reduced sleep intensity initially and lengthened REM sleep recovery. These results indicate that in middle-aged female rats ovarian hormones modulate baseline and recovery sleep differently, possibly by modulating circadian and homeostatic regulation of sleep in an age-dependent manner.

No description available.