Development and application of a bioassay for follicle-stimulating hormone : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Physiology at Massey University

Follicle-stimulating hormone (FSH) is involved in the regulation and maintenance of vital reproductive processes, such as gametogenesis, follicular development and ovulation. Produced in the anterior pituitary, FSH is a glycoprotein hormone that exists as a family of isohormones. Follicle-stimulating hormone concentrations have traditionally been measured by radioimmunoassay (RIA). However, results generated using RIA are a determination of the immunological activity of FSH. The potential of FSH to generate a biological response cannot be measured by RIA. Therefore, the identification of physiologically significant differences in the activity of these isoforms requires the use of assay systems that can differentiate between the biological activity of the FSH isoforms. Commonly used assays for measuring the biological activity of FSH are based on the measurement of aromatase activity in cultured rat Sertoli cells following stimulation with FSH. However, these assays have an inherently high ethical cost involved due to the use of primary tissue culture. In addition, the variation in these assays associated with differences between animals is difficult to eliminate. Recently a bioassay for human FSH has been described based on FSH stimulation of cyclic AMP production by a Chinese hamster ovary (CHO) cell line stably expressing the human FSH receptor (FSH-R). The purpose of this study was to evaluate the potential usefulness of this CHO FSH-R cell line expressing the human receptor for FSH to be used as a bioassay to measure the biological activity of ovine FSH. The receptor cell line bioassay described in this study is based on the ability of FSH to stimulate cAMP production by cultured CHO FSH-R cells. Optimisation of the culture system to enable the bioactivity of ovine FSH to be measured by bioassay was undertaken. This involved optimising the density of cultured cells, the time in culture and time exposed to FSH and the most suitable dose range for FSH. The influence of matrix effects, such as those exerted by serum was also investigated. The specificity of the assay towards FSH was also determined as was the sensitivity, accuracy and precision of the assay. No stimulation of cAMP production was seen in CHO FSH-R cells following treatment with α-FSH, β-FSH, LH, TSH, GH, prolactin or vasopressin at concentrations up to 10 μg/ml. Although the methodology used differed slightly depending on the presence or absence of serum, all assayed were performed using the following methods and materials. Freshly thawed FSH-R cells were bulked up in culture, and aliquots of 1 x 105 to 5 x 105 cells/well dispensed into 48 well culture dishes and incubated overnight at 37°C. The assay culture media was then replaced with 0.25 ml fresh media (α-MEM + 0.1% BSA + 0.25 mM 3-isobutyl-1-methyl-xanthine) containing varying doses of NIH-FSH-RP2 (RP2) FSH preparations or FSH containing samples, and the cells incubated for 4 hours at 37°C. The assay culture media was then removed and stored frozen at -20°C until assayed for cyclic adenosine monophosphate (cAMP) by RIA. Once optimal assay conditions were determined, the CHO FSH-R cell bioassay was used to measure FSH concentrations in ovine serum, pituitary extracts and medium from cultures of ovine pituitary cells. It was found that the concentrations of FSH in serum from intact sheep was close to the detection limit of the assay. Thus, while FSH concentrations could be measured in serum from some sheep, other animals had concentrations that were too low to be accurately measured by the bioassay in its present form. The assay was, however, well suited to measuring FSH concentrations in serum from sheep that had elevated concentrations of FSH. In one study, FSH concentrations measured by the bioassay were compared to those measured by RIA in sheep that had been ovariectomised and then hypophysectomised. It was found that the profile of FSH concentrations following hypophysectomy was similar whether measured by RIA or by bioassay (R2=0.7513), though absolute concentrations sometimes differed. This suggested that the immunoassay and bioassay were not always measuring the same characteristics of FSH. The assay was also used to measure FSH concentrations in samples of ovine hypophyseal venous blood. However, the results obtained for these samples indicated a poor correlation between FSH concentrations obtained by bioassay and RIA. Levels of bioactive FSH in hypophyseal venous blood fluctuated markedly and were up to 10-fold higher than the associated RIA concentrations. The CHO-cell bioassay was also found to be very suitable for measuring pituitary concentrations of FSH. In one study, pituitary extracts underwent chromatography and the separated isoforms of FSH were analysed by bioassay and RIA. Again, there was excellent correlation (R2=0.9328) between the concentrations of FSH measured both assay types. However, some differences were apparent suggesting a discrepancy in the biological and immunological characteristics of different FSH isoforms. The bioassay was also used to measure FSH concentrations in media from pituitary cells in tissue culture where serially diluted samples displayed good parallelism with the RP2 FSH standard curve. Results of this study demonstrate that the CHO FSH-R cell bioassay is suitable for measuring the biological activity of ovine FSH in a variety of biological fluids. The use of a permanent cell line eliminates the high ethical cost associated with primary tissue culture that other bioassay systems have. The inherent variation associated with culture systems utilising tissue from different sources is also avoided. The sensitivity of the bioassay is suitable for measuring FSH in surgically altered sheep or hypophyseal blood concentrations where FSH levels are generally higher than those in the peripheral circulation. In addition to blood samples, the bioassay is also excellent for monitoring FSH activity in pituitary extracts and in media from tissue culture. However, the sensitivity of the bioassay currently does not always allow measurement of bioactive FSH concentrations in serum samples with low FSH levels. In summary, the CHO FSH-R cell bioassay described in this study offers a useful alternative to RIA and other bioassays for monitoring the biological activity of ovine FSH and its isoforms in various biological fluids. It is concluded that this convenient and robust bioassay may have considerable application in future investigations of ovine FSH bioactivity

An analysis of LDEF-exposed silvered FEP teflon thermal blanket material

The characterization of selected silvered fluorinated ethylene propylene (FEP) teflon thermal blanket material which received 5 years and 9 months of exposure to the LEO environment on the Long Duration Exposure Facility is reported. X-ray photoelectron spectroscopy, infrared, and thermal analyses did not detect a significant change at the molecular level as the result of this exposure. However, various microscopic analyses revealed a roughening of the coating surface due to atomic oxygen erosion which resulted in some materials changing from specular reflectors of visible radiation to diffuse reflectors. The potential effect of silicon-containing molecular contamination on these materials is addressed

LDEF thermal control coatings post-flight analysis

The NASA Long Duration Exposure Facility (LDEF) provided a unique flight opportunity for conducting experiments in space and return of these experiments to Earth for laboratory evaluation. The results of one of these experiments, S0010, Exposure of Spacecraft Coatings, in which selected spacecraft thermal control coatings were exposed to the low-Earth orbital (LEO) environment on LDEF are reported. The objective of the experiment is to evaluate the response of thermal control coatings to LEO exposure, which includes atomic oxygen, ultraviolet and particulate radiation, meteoroid and debris, vacuum, and temperature cycling

Literacy Access through Storytime: An Ethnographic Study of Public Library Storytellers in a Low-Income Neighborhood

While early literacy achievement continues to be stratified by social class in the United States, public libraries often offer programs such as “storytime” in order to bolster the literacy development of youth in their communities. The purpose of the present ethnographic study was to explore how storytellers recruited and maintained participation in this free literacy program in a lower-income neighborhood. Via participant observations, semi-structured interviews, and artifact collection, storytellers recruited new patrons to storytime by (1) appealing to community members to enter the physical space of the library and (2) appealing to library patrons to attend storytime. Once patrons attended storytime, storytellers acted in order to maintain storytime attendance by (1) facilitating meaningful learning experiences, (2) fostering enjoyment through participation, (3) developing nurturing relationships, and (4) offering flexibility in storytime expectations. By exploring a contextualized account of the work of storytellers, the findings suggest important avenues through which public programs may contribute to more equitable access to literacy learning

Report of Test Excavations Along S.H. 16 in Bexar County 41BX502

Archaeological site 41BX502 is located in the center of San Geronimo, a hamlet in western Bexar County. The site is situated within the Texas Hill Country at an elevation approximately 1250 feet above mean sea level (384 meters). This site extends along a sloping hillside and terrace system immediately north of the juncture of Habey and San Geronimo Creeks and is cut on the west by the present route of State Highway 16. The site extends approximately 300 meters north and south along the present course of the highway and at least 300 meters east and west along Habey Creek (Figure 1). Only the western edge of the site is in the expanded right-of-way of State Highway 16. 41BX502 was not discovered during the cultural resource survey of the proposed route, presumably due to heavy vegetation cover. The survey personnel were not informed of the site, although it had long been known to local arrowhead collectors and pothunters. The site was brought to the attention of the archaeological section of the Texas State Department of Highways and Public Transportation by Mr. Don Fry, project engineer for the Department, after construction was halted but not before the site had been heavily damaged by construction activities and large numbers of people began inquiring about the future of the sit

Archaeological Testing of Site 41WM443 Williamson County, Texas

Testing of Site 41WM433 along FM 1431 in Williamson County, Texas, to determine eligibility for inclusion within the National Register of Historic Places and to determine site depth, cultural context, and archaeological significance was undertaken in April 1985. The site is located along a ridge line containing an outcrop of good quality Georgetown flint. Almost all the area within the right-of-way consisted of exposed bedrock ledges. One small incipient burned rock midden was present within the right-of-way. The midden measured 5meters in diameter, but about half of it had been destroyed by relic hunters. Results of the survey and testing indicate that portions of 41WM443 within the right-of-way contain a prehistoric component of unknown age and that the major activities were concerned with the procurement of flint and lithic reduction. No features, diagnostic artifacts, or organic remains were recovered although the testing procedures removed most of the burned rock midden. Evidence recovered does not support a determination of eligibility for inclusion within the National Register of Historic Places for the portions of the site within the right-of-way

Archaeological Testing of Site 41BX679 Bexar County, Texas

Testing of Site 41BX679 along Farm to Market Highway 2696 in Bexar County, Texas, to determine eligibility for inclusion within the National Register of Historic Places, cultural context, and archaeological significance was undertaken in April 1985. The site is located along a slight rise between Cibolo Creek and Muesebach Creek in northern Bexar County. It has been disturbed by previous construction or cutting of fire lines along Camp Bullis military reservation. Results of testing indicate that 41BX679 contained a minimal prehistoric component of unknown age. An intact hearth was uncovered but associated lithic debitage was almost totally absent. Evidence recovered does not support a determination of eligibility for inclusion within the National Register of Historic Places

Archaeological Testing of Site 41CI30 Childress County, Texas

The State Department of Highways and Public Transportation (SDHPT) conducted archaeological tests on Site 41CI30 in Childress County in May 1988. The site is located about 1.5 miles south of Childress at the intersection of Scatterbranch Creek and a county road and covers about one acre in the northeast quadrant of the intersection. The majority of it is located on private property outside the jurisdiction of the SDHPT. Testing indicated that the site is a low density prehistoric campsite. Lithic debris was largely limited to the upper 20-30 cm of orange, sandy soils. A total of four 1 meter squares were excavated within the right-of-way to a depth of 50 cm. These units exposed part of a hearth visible in the roadcut. A total of 47 flakes were recovered from 20 levels. No tools were found in the excavations and the only biological material recovered was an unburned hackberry seed from the hearth. Further research is not proposed because of the low artifact recovery rates and the eroded nature of much of the site. The portion of 41CI30 within the project right-of-way does not appear worthy of nomination as a State Archaeological Landmark. The area outside the project limits is less disturbed and located on more desirable landforms and may merit inclusion as a State Archaeological Landmark

Archaeological Testing of Site 41BU24 Burleson County, Texas

Testing of Site 41BU24 on State Highway 36 in Burleson County, Texas, to determine eligibility for inclusion within the National Register of Historic Places and to determine site depth, cultural context, and archaeological signbficance, was undertaken in January 1985. The site is located on a ridge overlooking Yegua Creek and extends into the creek floodplain. It has been heavily impacted by previous construction activities and the exact site limit is difficult to determine. Results of testing indicate that Site 41BU24 contains a minimal prehistoric component of unknown age which is thoroughly mixed with modern debris. Evidence recovered does not support a determination of eligibility for inclusion within the National Register of Historic Places