The goal of the current experiments was to assess the role of estrogen receptor alpha (ERë±) in the ability of a prior, short-term exposure to estradiol in mid-life to enhance cognition and affect the hippocampus in aging, ovariectomized rats, as well as to investigate a possible mechanism involving the insulin-like growth factor-1 system by which ERë± may exert these effects. In Experiment 1, rats were trained on the radial maze, ovariectomized, and implanted with estradiol or cholesterol vehicle capsules for 40 days. Rats then continuously received JB1 or aCSF vehicle i.c.v. and were tested on delay trials on the radial maze. Using Western blotting, I determined effects of treatment on protein levels of ERë±, ChAT, IGF-1R, IGF-1BP2, and phosphorylated and total p42/p44 MAPK and Akt in the hippocampus. Antagonism of IGF-1 receptors blocked the ability of prior estradiol exposure to enhance cognition and increase ERë±, phosphorylated ERK/MAPK, and ChAT in the hippocampus. The second experiment was divided into two parts. In Experiment 2A, rats were trained on the radial maze and ovariectomized. After 40 days, rats underwent stereotaxic surgery to receive lentiviral delivery of the gene encoding for ERë± or control virus to the hippocampus, and were then tested on delay trials. Using Western blotting, I determined if proteins of interest previously found to be affected by a prior exposure to estradiol in Experiment 1 would be affected by treatments for all subsequent experiments. Lentiviral delivery of ERë± to the hippocampus was sufficient to enhance cognition and increase phosphorylation of ERK/MAPK in the hippocampus. In Experiment 2B, rats were trained on the radial maze, ovariectomized, and implanted with estradiol or cholesterol vehicle capsules for 40 days. Rats then continuously received an ER antagonist or aCSF vehicle i.c.v. and were tested on delay trials. Antagonism of ERë± blocked the ability of prior estradiol exposure to enhance cognition. In the third experiment, rats were trained on the radial maze, ovariectomized, and implanted with estradiol or cholesterol vehicle capsules for 40 days. Rats then underwent stereotaxic surgery to receive i.c.v. cannula. Infusion of an ERK/MAPK inhibitor or aCSF vehicle was administered every 12 hours during delay trials. Inhibition of ERK/MAPK did not block the ability of prior estradiol exposure to enhance cognition, although it did block an increase in levels of ERë± in the hippocampus. Altogether, these results indicate that ERë± plays an important role in the ability of prior estradiol exposure to enhance cognition and affect the hippocampus in aging, ovariectomized rats, and likely interacts with the IGF-1 system and its associated ERK/MAPK signaling pathway. / acase@tulane.edu

The only practical method for artificially breeding ewes with frozen semen is laparoscopic insemination into the lumen of the uterine horn. Like all surgical procedures, however, laparoscopic artificial inseminatiom has limitations. The procedure requires surgical skill and costly equipment. Repeated passage of the laparoscope through the abdominal wall causes adhesions. Depositing frozen/thawed semen at the os cervix results in low conception rates. The inability to pass an insemination pipette through the cervix has prevented artificial insemination in sheep from becoming a standard breeding method as in the cattle industry. This study compared laparoscopic and transcervical methods of insemination in nulliparous ewes and transcervical insemination in multiparous ewes after estradiol cypionate treatment. Forty nulliparous commercial-cross Rambouillet ewes were treated with pessaries containing fluorogesterone acetate to synchronize estrus. After 14 days, pessaries were removed and ewes were injected IM with 400 IU of pregnant mare serum gonadotropin (PMSG). Ewes were randomly divided into laparoscopic and transcervical treatment groups, 14 and 26 respectively. Ewes were inseminated with thawed semen (75x106 motile spermatozoa). Pregnancy rate with laparoscopic insemination was 85% (confirmed at 55 days with real-time ultrasound) .. With the transcervical method, the speculum could only be inserted into the vagina of five ewes and the insemination pipette could only be passed through the cervix in two ewes. The combined pregnancy rate for deep cervical and transcervical insemination was 40% at 55 days. Forty multiparous commercial-cross Rambouillet ewes were synchronized as previously described. Ewes were randomly separated into a control and estradiol cypionate treatment group of 20 animals each. Within each group 10 ewes were inseminated with frozen/thawed Suffolk semen and 1 Owith Rambouillet semen. Ten ewes within each sire genotype were treated with 1 mg IM of estradiol cypionate 16 hours prior to insemination. Treated ewes were inseminated into the uterine body 90% of the time and non-treated ewes 95%. There was no significant difference in cervical passage between these groups. Pregnancy rates for transcervical artificial insemination in the Suffolk genotype were 0.05% and 40% in the Rambouillet (pregnancy confirmed at 55 days by real-time ultrasound). Lambing rates for the Suffolk and Rambouillet groups were zero and 0.05%, respectively.

Im Rahmen der Arbeit wurde eine Methode für die Quantifizierung von freiem 17β-Estradiol, Estron sowie der hydroxylierten und methylierten Metabolite im Brustgewebe entwickelt. Aufgrund der geringen Probengehalte erforderte dies eine gezielte Isolierung der Analyte aus der Probenmatrix sowie eine effektive Aufreinigung und Aufkonzentrierung, so dass eine Extraktion mit anschließender Festphasenextraktion durchgeführt wurde. Zudem wurde eine empfindliche Mess-Methode etabliert, welche auf Grundlage einer multi-reaction-monitoring-Methode, mittels Gaschromatographie und gekoppelten Triple-Quadrupol-Massenspektrometer, entwickelt wurde. Die Anwendbarkeit der Aufarbeitungs- und Mess-Methode wurde überprüft, indem diese auf 30 Realproben übertragen wurde. Dabei sind die ermittelten Gehalte mit den publizierten Daten der Gewebekonzentrationen von 17β-Estradiol, Estron und deren Metaboliten verglichen und Korrelationen mit ausgewählten Brustkrebs-begünstigenden Risikofaktoren betrachtet worden. Um ein quantitatives Metabolitenprofil von 17β-Estradiol, Estron und deren Metaboliten im Gewebe zu erstellen, wurden mit Hilfe einer multi-reaction-monitoring-Methode für alle Metabolite ein spezifischer Quanti- und Qualifier-Übergang etabliert. Durch die Optimierung der Ionisierungs- und Kollisionsenergien sowie der Initial-, Transferline- und Ionenquell-Temperatur beziehungsweise der dwell-time wurden Methoden- und Geräte-bedingte Empfindlichkeitsverluste so weit wie möglich reduziert, so dass maximale Signalintensitäten aller Quantifier-Übergänge gewährleistet waren. Zur gezielten Isolation sowie Aufreinigung und Anreicherung der Analyten,... ...so dass trotz der geringen Anzahl analysierter Gewebe-spenden der Einfluss des Body-Mass-Index und die Einnahme oraler Kontrazeptiva auf die Gehalte von 17β-Estradiol in der prämenopausalen Frau deutlich wurden. Die entwickelte Mess-Methode ermöglicht den routinemäßigen Einsatz für die Quantifizierung von freiem 17β-Estradiol, Estron und deren Methyl-Catecholen in humanem Brustgewebe. Beim Vergleich der berechneten Nachweisgrenzen von Catechol-Estrogenen mit Literaturangaben wurde herausgestellt, dass empfindlichere flüssigchromatographische Methoden als Methode der Wahl bei deren Analytik heranzuziehen sind. Die Übertragung der in Standardlösungen durchgeführten Versuche zur enzymatischen Hydrolyse von Glucuronid-und Sulfat-Konjugaten auf Gewebematrix stellt für weiterführende Arbeiten den entscheidenden Ansatzpunkt dar, um ein quantitatives Metabolitenprofil von freiem und gebundenem 17β-Estradiol, Estron und den Metaboliten in Brustgewebe erstellen zu können. / The aim of the present work was to develop a method for the quantification of free estradiol, estrone, catechol estrogens and methylation products in breast tissue. Due to trace amounts in the samples it was necessary to isolate the analytes from the sample matrix, purify and concentrate them prior to tissue extraction and solid phase extraction. Additionally, a sensitive method of measurement based on multi reaction monitoring using gas chromatography and triple quadrupole mass spectrometry was developed. The applicability of the sample preparation and measurement method was tested in 30 tissue samples by comparing the measured levels with published data concerning concentrations of estradiol, estrone and their metabolites in breast tissue. Also it was necessary to find possible correlations between the analytes concentrations and selected risk factors for breast cancer. In order to develop a quantitative profile of estradiol, estrone and metabolites in tissue, qualifier and quantifier transitions were established using the multi reaction monitoring mode. By optimising the ionisation and collision energy, the initial, transferline and ion source temperatures and the dwell-time, it was possible to reduce the loss of sensitivity caused by both method and equipment and maximize the signal intensity of all quantifier transitions. For the isolation, purification and concentration of the analytes... ...have some influence on the levels of estradiol in premenopausal women. The developed measurement method enabled a routine usage for the quantification of free estradiol, estrone and methylcatechols in human breast tissue. The comparison between the calculated detection levels of catechol estrogens and the published data led to the conclusion that liquid chromatographic methods are more sensitive. The transference of these methods and experiments conducted, such as for the enzymatic hydrolysis of glucuronide- and sulfate-conjugates on tissue matrix in standard solution, seem to offer a starting point for future research projects. Especially, for the purpose of establishing a quantitative metabolite profile of free and conjugated estradiol, estrone and their metabolites.

Peripheral estradiol treatment enhances hippocampus-dependent memory and morphology in ovariectomized rats. Although these enhancements are traditionally thought to be due to circulating estradiol, recent data suggest these changes are brought on by hippocampus-derived estradiol, the synthesis of which depends on GnRH activity. The goal of the current work is to test the hypothesis that peripheral estradiol affects cognition and the hippocampus through brain-derived estradiol via hippocampal GnRH receptor activity. In Experiment 1, we investigated if peripheral estradiol exerts its effects through regulation of brain-derived estradiol. Intracerebroventricular infusion of letrozole, which prevents the synthesis of estradiol, blocked the ability of peripheral estradiol administration in ovariectomized rats to enhance hippocampus-dependent memory in a radial-maze task and blocked or attenuated estradiol-induced increases in hippocampal synaptic proteins. In Experiment 2, we investigated if peripheral estradiol affects cognition and the hippocampus through hippocampal GnRH receptors. Hippocampal infusion of antide, a long-lasting GnRH receptor antagonist, blocked the ability of peripheral estradiol administration in ovariectomized rats to enhance hippocampus-dependent memory in a radial-maze task and blocked estradiol-induced increases in hippocampal synaptic proteins and aromatase, the final enzyme involved in estradiol synthesis. In Experiment 3A, we investigated whether hippocampal GnRH infusion was sufficient to enhance hippocampus-dependent memory and affect the hippocampus in ovariectomized rats and whether this was dependent on neuroestradiol synthesis. Hippocampal infusion of GnRH enhanced hippocampus-dependent memory in a radial maze task, the effects of which were blocked by letrozole infusion. Unexpectedly, GnRH treatment decreased levels or had no effect on a hippocampal postsynaptic proteins or aromatase. Due to the unexpected molecular results of Experiment 3A, Experiment 3B investigated if shorter treatment of GnRH affects hippocampal proteins differently. As opposed to the 10-12 days of GnRH treatment in Experiment 3A, five days of GnRH treatment increased levels of synaptic proteins via neuroestradiol synthesis as coadministration of letrozole blocked these effects. Results indicate that peripheral estradiol-induced enhancement of cognition and hippocampal morphology is mediated by brain-derived estradiol via hippocampal GnRH receptor activity. / acase@tulane.edu

Graduation date: 1998

Thesis (M.S.)--Baylor University, 2005. / Includes bibliographical references (p. 61-68).

Objectives were to determine: 1) whether estradiol benzoate (EB) provides a superior alternative to GnRH for synchronizing emergence, growth and maturation of a new follicular wave for fixed timed AI (TAI) in Bos indicus-influenced cattle using CIDR-based protocols, 2) the effect of 48 h calf removal at CIDR removal on the rate of maturational synchrony of the dominant follicle and 3) the effect of varying the magnitude of peak plasma progesterone (P4) concentrations following CIDR insertion on the suppression of FSH and LH secretion in a CIDR-based protocol using EB. In experiment 1, sixty-four Braford (F-1) females were stratified by BCS, parity and days postpartum and assigned randomly to one of four groups in a 2 x 2 factorial arrangement of treatments: 1) Select-Synch + CIDR, 2) Select-Synch + CIDR with 48 h calf removal, 3) E-Synch + CIDR or 4) E-Synch + CIDR with 48 h calf removal. A greater number of cattle in the EB treated group exhibited NFWE compared to the GnRH group, 29 vs 17 cows for EB and GnRH respectively, (P<0.0006). Intervals to NFWE were also greater in EB treated cattle than in GnRH treated cattle, 4.2 vs 2.7 d for EB and GnRH treated cattle respectively, (P<0.0001). Proportions of GnRH- and EB-treated cows ovulating after CIDR removal did not differ. Post-CIDR suckling status did not affect ovulation frequency or interval to ovulation. In experiment 2, eight pubertal (F-1) heifers were used in a Latin Square design with four treatment levels of P4: 1) EB only, 2) EB and new CIDR, 3) EB and new autoclaved CIDR, 4) EB, new autoclaved CIDR and P4 injection at CIDR insertion. Treatments 2 through 4 increased (P < 0.01) mean plasma P4 concentrations compared to treatment 1, with treatment 4 creating the greatest increase in P4 with the longest duration. Suppression of plasma FSH was greatest in group 4 (P<0.08), with mean 60 h concentrations less than in all other groups. Mean concentrations of LH were lesser in group 4 than groups 1 and 2. Frequencies of occurrence of NFWE and ovulation and intervals to NFWE did not differ among treatments. Results indicate that the use of EB and CIDR to synchronize Brahman x Hereford females may provide better synchronization for TAI compared to GnRH and CIDR based protocols.

Objectives were to determine: 1) whether estradiol benzoate (EB) provides a superior alternative to GnRH for synchronizing emergence, growth and maturation of a new follicular wave for fixed timed AI (TAI) in Bos indicus-influenced cattle using CIDR-based protocols, 2) the effect of 48 h calf removal at CIDR removal on the rate of maturational synchrony of the dominant follicle and 3) the effect of varying the magnitude of peak plasma progesterone (P4) concentrations following CIDR insertion on the suppression of FSH and LH secretion in a CIDR-based protocol using EB. In experiment 1, sixty-four Braford (F-1) females were stratified by BCS, parity and days postpartum and assigned randomly to one of four groups in a 2 x 2 factorial arrangement of treatments: 1) Select-Synch + CIDR, 2) Select-Synch + CIDR with 48 h calf removal, 3) E-Synch + CIDR or 4) E-Synch + CIDR with 48 h calf removal. A greater number of cattle in the EB treated group exhibited NFWE compared to the GnRH group, 29 vs 17 cows for EB and GnRH respectively, (P<0.0006). Intervals to NFWE were also greater in EB treated cattle than in GnRH treated cattle, 4.2 vs 2.7 d for EB and GnRH treated cattle respectively, (P<0.0001). Proportions of GnRH- and EB-treated cows ovulating after CIDR removal did not differ. Post-CIDR suckling status did not affect ovulation frequency or interval to ovulation. In experiment 2, eight pubertal (F-1) heifers were used in a Latin Square design with four treatment levels of P4: 1) EB only, 2) EB and new CIDR, 3) EB and new autoclaved CIDR, 4) EB, new autoclaved CIDR and P4 injection at CIDR insertion. Treatments 2 through 4 increased (P < 0.01) mean plasma P4 concentrations compared to treatment 1, with treatment 4 creating the greatest increase in P4 with the longest duration. Suppression of plasma FSH was greatest in group 4 (P<0.08), with mean 60 h concentrations less than in all other groups. Mean concentrations of LH were lesser in group 4 than groups 1 and 2. Frequencies of occurrence of NFWE and ovulation and intervals to NFWE did not differ among treatments. Results indicate that the use of EB and CIDR to synchronize Brahman x Hereford females may provide better synchronization for TAI compared to GnRH and CIDR based protocols.

Endometrial glands are required for adult uterine function and develop post-natally in mammalian species. Therefore, studies were conducted using neonatal ewes as a model to determine: 1) the roles of estradiol-17-alpha and estrogen receptor-alpha (ER-beta) in endometrial gland development; 2) the role of ovaries in endometrial gland development; 3) the role of prolactin in endometrial gland development; and 4) factors regulating prolactin receptor expression in endometrial glands. Study one determined the effects of neonatal exposure of ewes to estradiol-17-alpha valerate (EV); EM-800, an ER-beta antagonist; or CGS-20267, an aromatase inhibitor on endometrial gland development. Results indicate E2-17-alpha does not regulate endometrial gland differentiation or development. Additionally, ER-beta does not regulate primary differentiation of glandular epithelium, but does influence coiling and branching morphogenesis of endometrial glands. Study two determined the effects of ovariectomy on endometrial gland morphogenesis. Results suggest that the ovary and, thus, an ovarian-derived factor(s) regulate, in part, the coiling and branching of endometrial glands. Expression of subunits of activin, follistatin, and inhibin in the neonatal ovine ovary in addition to modulation of the components of the activin/follistatin system in the uterus of ovariectomized ewes supports the hypothesis that the ovarian factors that influence endometrial adenogenesis in the neonatal ewe may be activin, follistatin, and/or inhibin. Studies three and four determined the role of prolactin in endometrial adenogenesis in the neonatal ewe. Studies in which either hypoprolactinemia or hyperprolactinemia were induced indicate that prolactin regulates ovine endometrial adenogenesis in the neonatal ewe. The aim of study five was to determine transcription factors that regulate the glandular epithelium specific expression of prolactin receptor. Prolactin receptor exon 2 was cloned and sequenced, but no identifiable exon 1 or promoter was found. Additionally, many bovine contigs containing portions of the prolactin receptor gene were identified suggesting the bovine genome will be a useful tool as it becomes more complete. These results indicate ER-beta, prolactin and prolactin receptor, along with an unidentified ovarian factor(s), influence endometrial gland development in the neonatal ewe; however, exposure of the neonatal ewe to exogenous estradiol-17-alpha prevents differentiation and development of endometrial glands.

No description available.