Project topics and materials
Get Complete Project Material File(s) Now! »
Chapter 3 Radioimmunoassays
Testosterone determination
Plasma testosterone concentrations were determined using a Coat-A-Count total testosterone kit (Diagnostic Products Corporation, Los Angeles, California, USA). Extraction or chromatography was not required for this procedure. A 50µl sample in duplicate was used for the assay. The procedure entails solid-phase radioimmunoassay based on hormone specific antibody immobilised to the wall of a polypropylene tube. 125I-labeled testosterone competes for a fixed time with the specific hormone in the given sample for antibody sites. The tube is then decanted to separate bound from free and is then counted in a Cobra gamma counter.
The antiserum is highly specific for testosterone and has a low cross reactivity with other naturally occurring steroids except dihydrotestosterone, which is less than 5%.
The assay was validated by testing for parallelism using serial doubling dilutions of un-extracted plasma over the dilution range (1:1 to 1:64). The slope of the lines were compared and found not to differ significantly (ANCOVA F(1,6)=4.3 P>0.05) following a log-logit data transformation (Chard 1987). The sensitivity of the assay (90% binding) was 2.2 nmols/l. The intra-assay coefficient of variation was 2.5% (n=6).
Oestrogen determination
Oestradiol-17β was determined in mole-rat urine using a Coat-A-Count Oestradiol-17β kit (Diagnostic Products Corporation, Los Angeles, California, USA). A 100µl sample in duplicate was used for this assay. The method is a solid-phase radioimmunoassay that does not require purification of steroids or separation by chromatography. The antiserum is highly specific for oestradiol-17β, with a low cross reactivity with any other steroids present in the urine. The assay was validated by testing for parallelism using serial doubling dilutions of un-extracted urine over the dilution range (1:1 to 1:64) following log-logit transformation of the data (Chard 1987). The slope of the lines were compared and found not to differ significantly (ANCOVA F(1,6)=0.09, P>0.05). The sensitivity of the assay was 2 pmols/l. The intra-assay coefficient of variation was 9% (n=8).
Progesterone determination
Urinary progesterone concentrations were determined using a Coat-A-Count progesterone radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, California, USA), as described by Bennett et al. (1996). A volume of 100:1 of urine in duplicate was assayed without extraction.
he antiserum is highly specific for progesterone with a low cross reactivity to all other naturally occurring steroids except 20-α- dihydroprogesterone and 11-deoxycortisol with a cross reactivity of 2% and 2.4% respectively. A pooled urine sample (one with expected high concentrations from a pregnant queen) was double diluted from 1:1 to 1:64 and assayed.
In the assay were also included 6 samples at a dilution of 1:64 from a pool of low concentration progesterone. To these samples 100µl of progesterone in increasing concentrations (0.3, 1.6, 6.4, 31.8, 63.6 and 120 nmols/l) was added in duplicate. The curve was perfectly parallel to the standard curve. The assay was validated for the test species by comparing the slope of the curve produced using serial doubling dilutions of un-extracted mole-rat urine (over the range 1:1 to 1:64) against the standard curve (ANCOVA F(1,6)=4.9, P>0.05). The intra and inter assay coefficients of variation were 7 and 11% respectively. The sensitivity of the assay at 90% binding was 0.4nmols/l.
Creatinine determination
Urine concentration varies with fluid consumption; therefore creatinine is used to standardize samples. Creatinine is a breakdown product from tissue proteins and is excreted at a relatively constant rate (Schmidt-Nielsen 1997).
A modified Jaffe reaction was used to calculate the creatinine concentration for urine samples (Folin 1914).
The samples are assayed in duplicate. Ten microlitres of standard or sample were added to the wells of a micro plate, leaving two wells empty as a blank control. 200µl of picric reagent was added to all the wells, including the blanks. The picric reagent consists of saturated picric acid solution, alkaline triton solution (4.2 ml triton X-100, 12.5 ml 1N NaOH and 66.0 ml distilled, deionised water) and distilled deionised water in the proportion of 1:1:10. The alkaline triton can only be used once the product is homogenous. The microplate is then placed in the dark for a period of 1,5 hours, at room temperature, to allow colour development to occur. A standard curve (R2>0.99) was used to determine all sample values.
Chapter 4 Blood sampling
Prior to sampling, the animals were placed into a temperature regulated chamber at 36°C for 20 minutes to bring about vasodilatation, thus facilitating blood collection. Mole-rats were hand restrained while blood was taken from the saphenic vein in the foot, prior to and 20 minutes after the administration of a saline injection or a single GnRH challenge. Heparinised capillary tubes were used to collect between 300 and 400µl of whole blood, whereafter the blood was centrifuged to separate the plasma from the cellular component of the sample. Plasma was stored at -40°C until being assayed. This method has been successfully used to investigate pituitary sensitivity and secretion in naked mole-rats (Faulkes et al. 1990b, 1991), Damaraland and Mashona mole-rats (Bennett et al. 1993, 1996, 1997) and suricates (O’Riain et al. 2000).
GnRH administration
A chimaeric analogue of mammalian GnRH produced in the laboratory of R.P. Millar (Chemical Pathology, University of Cape Town), was administered to the mole-rats. The hormone was synthesized using solid phase methodology and had a purity of >98% homogeneity (Millar et al. 1989). A dose of 2µg in 100µl of sterile physiological saline was used to challenge the pituitary. Control animals were injected with 0.2ml sterile physiological saline.
LH bioassay
LH concentrations were determined using an in vitro bioassay based on the production of testosterone by dispersed mouse Leydig cells (Van Damme et al. 1974). The incubation medium (12ml Eagle’s basal medium, 2.1ml 7.5% sodium hydrogen carbonate 2ml foetal calf serum and 100ml distilled water) was placed on ice and gassed slowly under Carbogen 5 (95% O2: 5% CO2). A six week old male mouse was killed by cervical dislocation, the testes removed and decapsulated in 5ml incubation medium. The cell suspension was stirred on a magnetic stirrer for 5 minutes, filtered through fine nylon mesh and incubated under Carbogen 5 gas for 1 hour in a shaking water bath at 34ºC. Subsequently the incubated cell suspension was washed and centrifuged at 2500 r.p.m for 5 min at 4ºC. The supernatant was decanted and the cells resuspended in the incubation medium. The process was repeated after which the cell suspension was slowly stirred on the magnetic stirrer for 5 minutes. A haemacytometer was used to count the number of cells. Incubation medium was added until the number of cells counted corresponded to the final cell suspension volume (in ml). The medium was stirred for 5 minutes.
Declaration
Abstract
Acknowledgements
Glossary
Table of contents
List of figures
List of plates
List of tables
Chapter 1 – General introduction
• Reproductive regulation in mammals
• Seasonal breeding
• Cooperative breeding
• African mole-rats
• Reproductive skew in mole-rats
• Reproductive suppression in mole-rats
• Study animals
– The Cape mole-rat (Georychus capensis)
– The Natal mole-rat (Cryptomys hottentotus natalensis)
• Aims
Chapter 2 – Material and methods
• Study animals
• Chapter 3 – Gonadal hormone assays
Creatinine determination
Chapter 3 – Seasonal Gonadal hormone levels in the solitary Cape mole-rat and the social Natal mole-rat
• Abstract
• Introduction
• 3a – Cape mole-rat
• Material and methods
• Results
• Discussion
• 3b – Natal mole-rat
• Material and methods
• Results
• Discussion
Chapter 4 – Luteinising hormone responses to single doses of exogenous GnRH in the solitary Cape mole-rat (Georychus capensis) and the social Natal mole-rat (Cryptomys hottentotus natalensis).
• Abstract
• Introduction
• Material and methods
• Results
• Discussion
Chapter 5 – Neuroanatomy and neuroendocrinology of the GnRH system of Cape mole-rats and the Natal mole-rats
• Abstract
• Introduction
• Material and methods
• Discussion
Chapter 6 – Synthesis
References
GET THE COMPLETE PROJECT
