Having evolved longer than any other group of organisms, bacteria have been perfecting mechanisms of sensing their environments for billions of years. Recent advances in the field of biosensing have enabled miniaturization of existing biosensors, but the number of characterized biosensor elements remains limited. Sensing parts derived from bacteria make promising targets for integration into biosensor devices and could expand the repertoire of easily detectable compounds. Here, RNA sequencing screening was used to identify a novel TetR family 3-ketosteroid inducible transcription factor called SRTF1 (Steroid Responsive Transcription Factor 1) from the Gram-positive soil bacterium Pimelobacter simplex. This is the first transcription factor confirmed to be inducible by these steroids in-vitro. A potential regulon was identified using in-vitro chromatin immunoprecipitation sequencing, revealing a conserved 20 base pair long palindrome within several promoters in a region of the P. simplex genome highly differentially expressed on exposure to steroids. Biolayer interferometry and intrinsic tryptophan fluorescence were used to quantitatively characterize the transcription factor’s DNA binding strength and hormone induction specificity. Circular dichroism study of SRTF1 revealed it is primarily alpha-helical like almost all other TetR family transcription factors. Bases in a core GCCG repeat within the palindrome were identified as important for SRTF1 binding and a disrupted palindrome was generated that greatly increased a quantum-dot based SRTF1 biosensor’s sensitivity for progesterone. As the transcription factor displays cross reactivity to cortisol and aldosterone that is undesirable in a diagnostic device, a fluorescent reporter assay based on the transcription factor was constructed. This reporter assay showed a similar steroid induction profiles as purified SRTF1, and was used to select for mutant SRTF1 variants generated using error-prone polymerase chain reaction with reduced inducibility by these 11-hydroxy steroids. This pipeline for identifying novel transcription factors, characterizing their DNA and ligand binding profiles, and altering them through mutation of DNA and protein sequences could allow for an expanded number of biosensor parts.

Inadequate concentrations of progesterone during gestation can result in impaired embryonic growth and losses. These losses may be attributed to an overactive mechanism of progesterone catabolism or improper luteal function, which results in low concentration of progesterone. Progesterone catabolism occurs to the greatest extent by the liver, which holds a vast supply of cytochrome P450 enzymes and aldo-keto reductases that are involved in steroid inactivation. Insulin is a hormone produced by the pancreas that is involved in glucose uptake and metabolism. Progesterone catabolism is decreased in the presence of elevated insulin levels. Lipoic acid is a naturally occurring antioxidant and multienzyme cofactor which has been shown to increase insulin sensitivity and enhance glucose uptake in a number of species. The objectives of the current experiments were to 1) determine if administering a racemic mixture of lipoic acid by gavage at a dose of 32 mg/kg BW would increase peripheral progesterone concentrations, decrease progesterone clearance rates, or modulate cytochrome P450 2C (CYP2C), cytochrome P450 3A (CYP3A), or aldo-keto reductase 1 C (AKRIC) hepatic enzyme activity, and 2) determine if dosing lipoic acid directly into the rumen at 32 mg/kg BW or 64 mg/kg BW would increase progesterone in the blood, decrease progesterone clearance rates, or modulate insulin. In the first trial, Katahdin cross ovariectomized ewes were randomly assigned to a control or a lipoic acid treatment group. In this experiment, a controlled internal drug release (CIDR) device was inserted in all ewes and serum samples were collected daily for five days to determine progesterone. Liver biopsies were performed on day 10 to measure CYP2C, CYP3A, and AKRI C activity. Following liver biopsies, CIDRs were removed and an intensive blood sampling was performed to measure progesterone decay from peripheral circulation. We found that while lipoic acid does not have an effect on peripheral progesterone concentrations or hepatic enzyme activity, lipoic acid supplemented ewes have decreased progesterone clearance rates compared to control ewes. In the second trial, ovariectomized Katahdin cross ewes were randomly assigned to a control, low lipoic acid (32 mg/kg BW), or a high lipoic acid (64 mg/kg BW) treatment group. A CIDR was inserted in all ewes and blood samples were taken daily for 4 days. Following CIDR removal on day 11, an intensive blood sampling was performed to measure progesterone decay from peripheral circulation. One week following CIDR removal, ewes underwent an intravenous glucose tolerance test. It was found that lipoic acid supplementation did not affect progesterone concentrations, progesterone clearance, or insulin area under the curve. There was a treatment effect such that high lipoic acid dosed ewes had higher area under the curve for glucose when compared to control and low lipoic acid dosed ewes. Although no differences in progesterone concentrations were seen in the second trial, we speculate that the administration method rather than the efficacy of lipoic acid may account for the lack of differences observed. This theory is based on evidence from our first trial that oral lipoic acid supplementation did in fact reduce progesterone catabolism, as well as published data demonstrating that ruminally dosed lipoic acid is less effective than the equivalent oral dose.

No description available.

The objective of this study was to examine hepatic steroid inactivation and luteal function throughout bovine gestation. In pregnant beef cows, cytochrome P450 3A activity decreased from mid- to late-gestation, while progesterone concentrations tended to increase from mid- to late-gestation. Uridine diphosphate-glucuronosyltransferase activity per kg of body weight was increased in pregnant vs non-pregnant dairy cows. Total corpus luteum (CL) blood perfusion tended to be increased in pregnant vs non-pregnant dairy cows. Hepatic portal blood flow per kg of body weight was increased in pregnant vs non-pregnant dairy cows. Hepatic steroid inactivating enzyme activity, CL blood perfusion, and portal blood flow did not differ between pregnant and non-pregnant beef cows. There was no difference in progesterone concentrations in pregnant vs non-pregnant dairy or beef cows. The current study highlights the relevance of further investigation into steroid secretion and inactivation and their impact on the maintenance of pregnancy in cattle.

Establishment and maintenance of pregnancy is a central concern to the cattle industry, as it strongly impacts efficiency and profitability of beef cow-calf operations. The objective of this study was to determine if nutrient restriction impacts ovulatory follicle size and corpus luteum (CL) development and function of Bos taurus cows enrolled in estrous synchronization. A total of 26 Angus cows were housed in a facility equipped with a Calan gate system for individual animal intake. Cows were stratified by body weight (BW), and randomly assigned one of two nutritional treatments: 1) 100% of nutrient requirements (MTN; n=13) or 2) 70% of nutrient requirements (REST; n=13). Individual daily intakes were measured and adjusted weekly based on BW. Cattle underwent an acclimation period of 14 days and were exposed to nutritional treatments for 30 days prior to estrous synchronization. Body weight was measured daily using an automated scale and a conventional livestock scale at the beginning and end of the experiment. Cows were synchronized using a 7-day CO-synch + CIDR protocol beginning on day -10. Ultrasonography of the ovaries was performed at each event of the estrous synchronization protocol on days -10, -3, 0, 5, and 7. Blood samples were taken on days -10, -3, and daily from day 0 through 7 to observe changes in progesterone (P4). Data were analyzed as repeated measures using the MIXED procedure of SAS. Initial BW tended to differ between treatments (P = 0.07; MTN 597 ± 32 kg, REST 604 ± 32 kg), but MTN had greater final BW (P < 0.001; 687 ± 24 and 556 ± 27 kg, respectively) and greater average daily gain (1.35 ± 0.18 and -0.72 ± 0.21 kg/d, respectively, P < 0.001) than REST. Diameter of the largest follicle was similar (P = 0.851) between treatments at CIDR insertion (12.6 ± 0.6 mm) and CIDR removal (12.9 ± 0.4 mm) but was greater (P < 0.05) for MTN than REST cows at 60 hrs after CIDR removal (14.01 ± 0.6 and 12.37 ± 0.5 mm, respectively). Volume of CL was similar (P > 0.1) at 5 (3211 ± 113 mm3) and 7 (5280.3 ± 212 mm3) days after ovulation. Concentration of P4 did not differ on days -10, -3, or 0-5. However, on days 6 and 7, P4 was greater (P < 0.05) for MTN than REST (2.07 ± 0.15 and 1.65 ± 0.15, and 2.27 ± 0.15 and 1.83 ± 0.15 ng/mL, respectively). In conclusion, nutrient restriction to 70% of maintenance during estrous synchronization negatively affects diameter of the ovulatory follicle and circulating P4, but it did not affect CL volume in multiparous Bos taurus beef cows. / Master of Science / Ensuring successful pregnancy in beef cow-calf operations is crucial for the efficiency and profitability of the cattle industry. This study investigates the effects of nutrient restriction on ovulatory follicle size and corpus luteum (CL) volume in Angus cows undergoing estrous synchronization. A total of 26 cows were subjected to either a maintenance diet meeting 100% of nutrient requirements (MTN) or a diet providing 70% of nutrient requirements (REST). Intakes were updated weekly using computer software. The cows underwent a 30-day nutritional treatment before synchronization of ovulation. Results revealed that cows on the maintenance diet exhibited greater final body weight and average daily gain compared to those on the restricted diet. While estrus expression showed a numerical increase in MTN cows, the impact was not statistically significant. Analysis of ovulatory follicle size demonstrated that MTN cows had larger follicles 60 hours after synchronization compared to REST cows. Surprisingly, corpus luteum volume did not differ between the two groups at 5 and 7 days after ovulation. Additionally, circulating progesterone (P4) levels were affected by nutrient restriction, with notable differences observed on days 6 and 7. In summary, nutrient restriction during ovulation synchronization negatively influenced ovulatory follicle size and P4 levels, but did not affect corpus luteum volume in mature Angus cows. These findings contribute valuable insights for the cattle industry, emphasizing the importance of proper nutrition for optimal reproductive health in beef cows.

No description available.

No description available.

Maintenance of pregnancy in the ewe depends on progesterone synthesized by the ovaries during the first third of gestation and by the placenta during the last two-thirds of gestation. There have been few investigations of in vitro synthesis of progesterone by the ovine placenta or of mechanisms controlling placental steroidogenesis. Weekly blood samples were collected from ten gestating ewes that were sacrificed for collection of placental tissue on either day 80 (n=5) or day 115 (n=5) of gestation. All blood samples were analyzed for progesterone by radioimmunoassay. Immediately after each ewe was sacrificed, cotyledons were collected from different areas of each placenta, minced, and incubated in 2 ml of culture medium for either .5, 1, or 2 h. Minced tissue (100 mg) was incubated either in the presence or absence of pregnenolone and the tissue was subject to addition of either human Chorionic Gonadotropin (hCG), 3’, 5', cyclic adenosine monophosphate (cAMP), luteinizing hormone-releasing hormone (LHRH) or no additional treatment. Progesterone concentration in the medium after incubation were determined by radioimmunoassay. The temporal pattern of in vivo progesterone synthesized during gestation exhibited a biphasic increase with elevations occurring between days 70 and 80, and 95 and 115 of gestation. The difference in progesterone concentration in incubation medium after incubation of placental tissue collected on day 80 or day 115 of gestation was similar in magnitude to the difference in plasma progesterone concentrations of samples collected on those days. At day 80 and 115 of gestation, in vitro progesterone concentrations were 8.4 and 15.3 ng/ml, respectively, while in vivo progesterone concentrations at day 80 and 115 were 7.2 and 12.5 ng/ml, respectively. Progesterone accumulation in the medium of placental tissue collected at 115 days of gestation was greater than concentrations of progesterone in tissue collected at 80 days of gestation (P<.01). HCG had no effect on progesterone accumulation in the incubation medium; however, cAMP and LHRH significantly affected progesterone concentrations when pregnenolone was added to the medium of tissue collected on day 115. Cyclic AMP significantly increased progesterone concentrations in the medium after 2 h of incubation over that of tissue incubated alone (354.1 vs 402.6 ng/g). Mean progesterone concentrations in the medium of tissue incubated with LHRH were lower than control incubations after 1 h (288.8 vs 355.6 ng/g) and 2 h (306.1 vs 354.1 ng/g) of incubation. / M.S.

by Wang Siu Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 78-101). / Acknowledgments --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Table of Content --- p.vi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.4 / Chapter Chapter 3 --- Materials and Methods --- p.38 / Chapter Chapter 4 --- Results --- p.52 / Chapter Chapter 5 --- Discussion --- p.61 / Chapter Chapter 6 --- Conclusion and Further Studies --- p.75 / References --- p.78 / Appendix --- p.102 / Micrographs and Tables

RÉSUMÉ Les protéines associées à la gestation (PAGs) constituent une grande famille de protéinases aspartiques exprimées dans les cellules épithéliales de la couche superficielle du placenta des artiodactyles et plus précisément dans les granules des cellules binucléées. Des investigations par biologie moléculaire (clonage et séquençage) ont permis de montrer que les protéines de la gestation font partie de la grande famille des protéases aspartiques tout comme le pepsinogène, la pepsine, la chymosine, les cathepsines D et E, et la rénine. La détermination des concentrations en PAG par radioimmunoassay dans le sérum ou dans le plasma est actuellement employée comme méthode sérologique spécifique pour le diagnostic de gestation chez le bovin dès le 28e jour après la conception. Au-delà de ce délai, les dosages des PAGs peuvent également être utilisés pour assurer le suivi de la gestation notamment dans le cadre de létude de la mortalité embryonnaire précoce ou tardive et de la mortalité ftale. Au moment dentreprendre nos investigations expérimentales, une attention particulière a été portée à notre maîtrise des systèmes de dosages radioimmunologiques notamment dans les zones des faibles concentrations de PAG caractérisant le premier trimestre de la gestation chez les bovins. Cest ainsi que les deux premières études sur lesquelles repose ce travail de thèse ont visé à tester de nouveaux antisérums dirigés contre la PAG et à comparer leurs performances en milieu sérique ou plasmatique. Ensuite, cinq différents systèmes de dosages (utilisant différents antisérums) ont été comparés quant à leur sensibilité, spécificité, exactitude des diagnostics positifs et exactitude des diagnostics négatifs, sur des séries de plasmas provenant de femelles non gestantes et de femelles gestantes durant le premier trimestre de la gestation. Dans une troisième étude, les concentrations de PAG, LH et prolactine ont été interprétées en fonction dune classification basée sur le niveau de progestérone mesuré chez les femelles à jour 21. Dans la première étude, lobjectif a été de déterminer les paramètres de limite minimale de détection, reproductibilité, exactitude, spécificité et parallélisme des dilutions caractérisant les cinq systèmes radioimmunologiques différents : RIA-497, RIA-706, RIA-780, RIA-809 et RIA-Pool. Ensuite, leur capacité à distinguer les femelles non gestantes et gestantes prélevées au 30e jour suivant linsémination artificielle a été analysée dans le détail. Létude a été menée sur un effectif de 40 femelles de race Holstein-Frisonne dâge et de parité mélangés dont la gestation a été confirmée par exploration rectale réalisée entre le 2e et le 3e mois après linsémination. Un groupe contrôle composé de dix vaches maintenues en stabulation entravée confirmées non gestantes par examen échographique et exploration rectale a été utilisé simultanément. Les caractéristiques générales des différents systèmes étaient satisfaisantes. Leur spécificité était excellente à lexception du système RIA-809 ayant montré une très légère interférence de lacide syalique et de lhCG. Les concentrations mesurées dans le plasma des femelles non gestantes sont restées inférieures à 0,5 ng/mL excepté celles qui étaient mesurées dans les systèmes RIA-780 et RIA-809. Seuls les systèmes RIA-497, RIA-706 et RIA-Pool ont fourni des valeurs de sensibilité, spécificité, exactitude des diagnostics positifs, exactitude des diagnostics négatifs égales à 100%. Dans la deuxième étude, les cinq systèmes de dosage ont été utilisés pour déterminer les concentrations de PAG dans le plasma aux jours 30, 45, 60 et 80. Les corrélations ont été calculées entre les systèmes ainsi que les rapports de concentrations mesurées par chacun deux : les systèmes RIA-706, RIA-780, RIA-809 et RIA-Pool ont révélé des valeurs plus élevées que le RIA-497 à tous les stades étudiés. La troisième étude a visé à vérifier une hypothèse formulée à plusieurs reprises par différents auteurs : le niveau de concentration en progestérone atteint en début de gestation est-il susceptible dinteragir avec les concentrations de protéines trophoblastiques, de LH et de prolactine mesurées plus tard durant le premier trimestre de la gestation ? Dans cette étude, nous avons déterminé les concentrations de lensemble des paramètres sur les prélèvements correspondant aux jours 0, 21, 30, 45, 60 et 80 de 37 femelles gestantes. Les femelles ont été réparties en deux sous groupes : celles dont la progestérone au jour 21 était inférieure à la moyenne (Low-P4) et celles dont la même progestérone était supérieure à la moyenne au jour 21 (High-P4). Lorsque les deux sous groupes ont été suivis aux périodes ultérieures (jours 30, 45, 60 et 80), il est apparu que les concentrations de PAG ont eu tendance à être plus élevées dans tous les systèmes chez les vaches du groupe High-P4. Une différence significative est atteinte en utilisant le RIA-497 au jour 80. Les concentrations en LH et prolactine ont eu tendance à être plus faibles à toutes les périodes chez les vaches faisant partie du groupe High-P4. La différence a été significative pour la LH à jour 60 et pour la prolactine à jour 21. Dans leur ensemble, les résultats de ce travail montrent que le diagnostic de gestation peut être amélioré (sensibilité, spécificité, exactitude des diagnostics positifs, exactitude des diagnostics négatifs) en sélectionnant soigneusement les antisérums et en les mélangeant de façon appropriée. Ces systèmes sont utilisables pour suivre les concentrations de PAG durant le premier trimestre de la gestation, les résultats étant fortement corrélés tout en montrant des profils différents. Associées au dosage de la progestérone, de la LH et de la prolactine, les investigations sur les PAGs permettent de progresser dans létude des régulations qui relient lhypophyse, les gonades et le trophoblaste durant le premier trimestre de la gestation. SUMMARY Pregnancy Associated Glycoproteins (PAGs) constitute a large family of aspartic proteinases expressed in the outer epithelial cells layer of the placenta of eutherian species and more precisely in the granules of the binucleate cells. Investigations by molecular biology (cloning and sequencing) showed that the PAGs are part of the great family of the aspartic proteases like pepsinogen, pepsin, chymosin, cathepsins D and E, and renin. The determination of the concentrations in PAG by radioimmunoassay in serum or plasma is currently used as specific serological method for the diagnosis of gestation at day 28 after conception. Beyond this time, PAGs assays can also be used to ensure the follow-up of gestation in particular during the study of early or late embryonic mortality and early foetal mortality. When we started our experimental investigations, a special attention was paid to our control of radioimmunoassay systems especially in the zones with low concentrations of PAG which characterize the first trimester of bovine gestation. Thus the first two studies on which this work was based, aimed testing new antisera raised against PAG, and comparing their performances in seric or plasmatic medium. Then, five various assays (using different antisera) were compared as for their sensitivity, specificity, exactitude of the positive diagnoses and exactitude of the negative diagnoses, on series of plasmas from nonpregnant and pregnant females during the first trimester of gestation. In a third study, the concentrations of PAG, LH and prolactin were interpreted according to a classification based on the progesterone level measured in females at day 21. In the first study, the objective was to determine the parameters of minimal limit of detection, reproducibility, exactitude, specificity and parallelism of dilutions characterizing the five different radioimmunoassay systems: RIA-497, RIA-706, RIA-780, RIA-809 and RIA-Pool. Then, their capacity to discriminate non-pregnant and pregnant females investigated at day 30 after artificial insemination was also analyzed in detail. The study was undertaken on 40 Holstein-Friesian females of different age and parity, and whose gestation was confirmed by rectal exploration carried out between the second and the third month after insemination. A control group of ten cows maintained in stabling, confirmed non-pregnant by ultrasonography and rectal exploration, was used simultaneously. The general characteristics of the various systems were satisfactory. Their specificity was excellent except for system RIA-809 which shown a small interference of syalic acid and hCG. The concentrations measured in the plasma of non-pregnant females remained lower than 0.5 ng/mL except those which were measured in systems RIA-780 and RIA-809. Only the systems RIA-497, RIA-706 and RIA-Pool provided values of sensitivity, specificity, exactitude of the positive diagnoses, exactitude of the negative diagnoses equal to 100%. In the second study, all five systems were used in order to determine the concentrations of PAG in plasma at days 30, 45, 60 and 80. The correlations were calculated between the systems as well as the ratios of concentrations measured by each one of them: systems RIA-706, RIA-780, RIA-809 and RIA-Pool revealed higher values than the RIA-497 at all the studied stages. The third study aimed to check a hypothesis formulated by various authors: is the concentration level of progesterone reached at beginning of gestation likely to interact with the concentrations of trophoblastic proteins, LH and prolactin measured later during the first trimester of gestation ? In this study, we determined concentrations of the whole parameters on samples corresponding to days 0, 21, 30, 45, 60 and 80 of 37 pregnant females. The females were divided into two groups: those whose progesterone at day 21 was lower than the average (Low-P4) and those whose progesterone was higher than the average at day 21 (High-P4). When both groups were investigated at later periods (days 30 and 80), it appeared that the concentrations of PAG tended to be higher in all systems in the cows of the High-P4 group. A significant difference was raised by using RIA-497 at day 80. The concentrations in LH and prolactin lead to be weaker during all the periods in the cows of the High-P4 group. The difference was significant for LH at day 60 and for prolactin at day 21. In conclusion, the results of this work show that the diagnosis of gestation can be improved (sensitivity, specificity, exactitude of the positive diagnoses, exactitude of the negative diagnoses) by selecting the antisera carefully and by mixing them in a suitable way. These systems are usable to follow the concentrations of PAG during the first trimester of gestation, the results are strongly correlated while showing different profiles. Combined to assays of progesterone, LH and prolactin, the investigations on PAGs make progress in the study of the regulations between pituitary gland, gonads and the trophoblast during the first trimester of gestation.