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Optimisation and validation of an in vitro bioassay as a tool for measuring luteinising hormone in several species of mammals

Nice, Penelope Angela (2013) Optimisation and validation of an in vitro bioassay as a tool for measuring luteinising hormone in several species of mammals. PhD thesis, Murdoch University.

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Understanding the underlying reproductive physiology of threatened and native species is crucial when developing conservation management strategies, especially in situations where ex-situ captive breeding programs are crucial to a species short-term survival. However, research into the reproductive biology of threatened species is hampered by the fact that it is difficult to measure the gonadotrophins, luteinising hormone (LH); and follicle-stimulating hormone (FSH), the chief controllers of reproduction in vertebrates. This project, therefore, aims to develop and validate a simple, rapid LH bioassay which is capable of measuring LH concentrations from a diverse range of mammalian species. The bioassay uses a clonal murine Leydig tumour cell line as an indirect method for measuring LH in mammals. The cell line responds to LH stimulation in vitro by producing the steroid progesterone, which is much easier to measure across species. It is envisioned that this assay would be a useful tool in situations where a high volume of samples from various species require analysis, such as in a zoo or wildlife breeding facility.

Firstly an optimisation of the conditions of the assay was conducted; specifically cell density/well and incubation time required for cells to produce progesterone was optimised to improve the sensitivity of the assay. An optimal cell density of 9,375 cells/well was discovered to have greatest sensitivity to physiological levels of LH after an optimal incubation period of 120 minutes. Using these optimised conditions, pituitary LH preparations from eight species of mammals (human, porcine, monkey, rabbit, bovine, canine, equine and, rat) produced good dose-response curves demonstrating the practicality of this assay to measure LH from diverse species. The sensitivity of the bioassay differed for each species, although there was no obvious phylogenetic relationship between species and sensitivity. The sensitivity ranged from 0.0625 – 1 ng/100 μl (NB: 100 μl of sample in each well) for most of the species tested, well within the normal physiological range, with only the bovine being slightly higher at 4 ng/100 μl, but still within the physiological range. There was no cross-reactivity with FSH in the bioassay. Therefore, it was concluded that the assay might be suitable for the analysis of samples from diverse mammalian species.

As standard available immunoassays are also often unsuitable for measurement of LH in marsupial species, this precludes assessment of the stage of their oestrous cycle. The application of the LH bioassay was investigated for its capability to act as an indirect method for measuring LH in marsupials, which has not been conducted previously as marsupial LH has often been described to be structurally and antigenically different from LH produced by mammals. The bioassay detected an increased circulating LH pre- and 25 minutes post- exogenous GnRH challenge in western grey kangaroos (P<0.05) and black-flanked rock wallabies (P<0.05), and also detected decreased LH levels in black-flanked rock wallabies treated with a contraceptive prior to GnRH challenge (P<0.01). Results obtained by this bioassay method were validated by comparison to those derived from an adapted enzyme immunoassay (EIA). There was no significant difference in change in LH concentrations as detected by either method in the western grey kangaroo or the control group of black-flanked rock wallabies, however, there was a significant difference in change in LH concentration between the two methods when assessing black-flanked rock wallabies on a contraceptive (P<0.05). It was concluded that the bioassay could be used to successfully measure changes in circulating levels of LH in two species of Macropodid marsupials, though it appears to be most effective at measuring LH concentrations at the mid to high end of the physiological range, and less reliable for ascertaining low physiological LH levels.

The aim of the next part of the study was to extend the species range of the bioassay to include elephants, which are currently the focus of a worldwide captive breeding program. The unique physiology and the presence of different hormone metabolites in the Asian elephant results in many commercially available hormone detection kits lacking sensitivity and/or suitability for application in elephants. Application of the bioassay to measure LH concentrations during the anovulatory (anLH) and ovulatory (ovLH) surges of the Asian elephant oestrous cycle was investigated with particular interest in its ability to distinguish between basal and surge LH concentrations. These results were validated by comparing them to those obtained by an established EIA. The bioassay successfully detected both anovulatory and ovulatory surges of LH during three consecutive cycles, with surge concentrations determined as those values which exceed the previous nadir by a minimum of three standard deviations (SD). In addition to this, the present study calculated the time taken from sample acquisition to progesterone determination as four hours suggesting the practicality of this method for obtaining rapid results making it more applicable to ‘real world’ situations where timing of the ovulatory surge is critical for artificial insemination to be successful.

The final part of the study used a unique animal model, the horse, to verify the LH bioassay against a definite LH surge-induced ovulation event. This is possible because the ovaries of the horse can be routinely assessed for signs of ovulation (presence of a corpus luteum) using rectal ultrasound. This is routinely done in large equine breeding facilities to assess the start of the breeding season. Horses are long day breeders, and the decreased daylight hours in winter are associated with decreased gonadotrophin secretion and hence decreased ovarian activity. The control of seasonal reproduction in the mare is primarily driven by photoperiod, yet other factors such as nutrition and body condition, environmental temperature, age and reproductive state can also have an effect. The aim of this part of the study was to investigate whether the bioassay could be used, firstly, to detect endogenous LH surges in the mare, and hence determine ovulation and the resumption of oestrous cyclicity; and secondly, to use the bioassay results to investigate whether other factors apart from photoperiod influence the resumption of oestrous cyclicity. The bioassay accurately detected a recent or imminent ovulation in 85% of cases suggesting that it is a highly reliable method, and could be adopted by equine reproductive specialists. It was also found that while photoperiod was the strongest influence on resumption of oestrous cyclicity, seasonal changes in pelage (hair loss and coat condition) also had a strong influence, as did a higher body condition or an increase in body condition.

Evidence presented in this thesis demonstrated that a clonal mouse Leydig tumour cell line could be developed and used as an in vitro LH bioassay, to accurately detect LH concentrations from blood plasma samples from diverse mammalian species. Hence, this bioassay method could be adopted by researchers working in mammalian reproductive endocrinology, especially those working on wildlife species for which commercial gonadotrophin immunoassays are not readily available.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Life Sciences
Supervisor(s): Miller, David, Matson, Philip and Rae, M.
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