Research Group 4: Reproduction Biology

Fields of Expertise

1 Non-invasive monitoring of hormones

2 Determination of volatile substances in biological matrices

3 Qualitative and quantitative characterisation of spermatogenesis

4 Cryopreservation of sperm and oocytes of different species

5 In-vitro-maturation and -fertilisation of mammalian oocytes (IVM/ IVF)

6 Xenotransplantation of ovarian tissue

1. Non-invasive monitoring of hormones

Non-invasive monitoring means the measurement of steroid hormone metabolites in urine and faeces. Steroid hormones are rapidly and extensively metabolised by the liver and excreted with urine or faeces. As a rule hormone metabolites are chemically different from their parent hormone. All analyses are carried out by enzyme immunoassay (ELISA). This analytical principle uses an antibody directed against the native hormone which binds endogenous hormones in the biological sample. This competes with a constant amount of enzyme labelled hormone at the antibody binding site. Quantification is obtained from the relation that increasing amounts of endogenous hormone displace increasing amounts of enzyme labelled hormone from its antibody binding site. However, there are no universal methods which might allow the analyses of a distinct metabolite in all species. Moreover, species-specific differences in metabolism and gut microflora can cause these metabolites to differ between species and no chemical identity exists between hormones and their metabolites. Therefore, for each species existing methods had to be adapted or new methods had to be created for metabolite analyses. In principle, a methodological development consists of the following steps:

• Production of an antibody and label
• Scheduling an appropriate sampling regime and optimising sample preparation (storage, extraction)
• Testing different assays with the most promising antibody
• Analytical validation
• Metabolite characterisation based on HPLC-immunograms and, if possible, to carry out a radiometabolism study
• Biological validation
• Serial mesurements and data evaluation

HPLC-Immunogram: HPLC immunograms are performed to characterise the immunoreactive metabolites present in biological samples. Usually extracts from fecal and urine samples are partially purified on solid phase Sep-Pak C18 cartridges, dried down, and reconstituted in solvent prior to the injection into the HPLC column. When passing through the non-polar (reversed phase) column the metabolites are retained and separated based on differences in their polarity. A fraction collector, where each fraction is the same size and the fraction size is set in terms of time, allows fractionating of the column effluent. The immunoassay is now used as a post column detector system following separation by HPLC investigating aliquots of each fraction for immunoreactive compounds. The immunoreactivity plotted against fraction number or elution volume may reveal a pattern of an unpredictable number of peaks. The elution positions of immunoreactive peaks are compared with those of authentic standards, whose positions were verified before when known amounts of standard were injected in separate HPLC runs. In case that the antibody appears to have detected an immunoreactive peak coeluting with a standard there is a high degree of probability that the metabolite is indeed identical with the standard. In the example shown below native testosterone (T) and dihydrotestosterone (DHT) were confirmed in feces of a male white rhinocerus (Ceratotherium simum simum). In most species studied so far authentic hormones usually are represented in minor proportions.

see also: Kretzschmar P, Gansloßer U, Dehnhard M: The development of a non-invasive method to analyse the effect of season, mating behavior and fighting on gonadal activity of male white rhinoceros (Ceratotherium simum simum) in South Africa. Hormones Behav. 45, 1-9, 2004

Established analytical methods in the endocrine laboratory of the IZW

At the time, 12 enzyme immunoassays are routinely used for steroid hormone measurements in the endocrine laboratory . The applications enclosed typical measurements of steroid hormone in blood plasma. Due to the increasing demand and their wide array of applications non-invasive methods are topics of current methodological developments covering faecal and urinary metabolites. In detail the following methods are available in the endocrine lab

Gestagens
Progesterone (4-Pregnen-3,20-dione)
Pregnanediol (5b-Pregnan-3a,20a-diol)
5a-Dihydroprogesterone (5a-Pregnan-3,20-dione)
5a-Pregnan-3b-ol-20-one

Estrogens
Total estrogens
Estradiol (1,3,5(10)-Estratriene-3,17a-diol)
Estrone (1,3,5(10)-Estratriene-3a-ol-17-one)

Androgens
Testosterone (17b-Hydroxy-4-androsten-3-one)
5a-Androstan-17b-ol-3-one (DHT)
5b-Androstane-3a,11b-diol-17-one
epi-Androsterone (3b-hydroxy-5a-androstan-17-one)

Glucocorticoids
Cortisol (4-Pregnen-11b,17a,21-triol-3,20-dione)
Corticosterone (4-Pregnen-11b,21-diol-3,20-dione)
Desoxycorticosterone (4-Pregnen-21-ol-3,20-dione)

In addition we use Immulite® kits (DPC Biermann, Germany) and the Immulite automated immunoassay analyser for reproductive and stress monitoring. The apparatus is designed to analyse approximately 30 different hormones in human blood plasma. We currently investigate its applicability for the measurements of urinary and fecal hormone metabolites in different zoo animals. The endocrine lab provides analytical capacities to research institutions and zoos as a service.

If you are interested in our service:
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2. Measurement of volatiles

An alternative analytical perspective is the determination of volatile substances. This idea is based on the observation that in different species females signal oestrous to males using tactile, visual, acoustic and olfactory stimuli. Olfactory stimuli (pheromones) can trigger specific behavioural or endocrine reactions in the recipient. Behavioural effects of estrous-related pheromones (contained in urine and vaginal secretions of females during estrous) in males such as attraction, olfactory preferences or flehmen have already been described in several species. The chemical nature of the substances involved is mostly unknown. However, pheromones may represent more informative indicators of behavioural and physiological conditions than measurements of circulating or excreted hormones, which do not bear communicator functions. To identify and to analyse e.g. male directed female signals in elephants whose emission is limited to a narrow period around ovulation might improve estrous detection and thus reproductive management in elephants.

Analyses of volatiles can be performed with the solid-phase microextraction technique (SPME). Urine samples were heated and volatiles evaporated into the headspace above the urine (k1) where they were adsorbed on a specific fibre (k2). Both equilibria (k1, k2) depend on several factors, e.g. temperature and time. SPME was carried out with an CTC Combi Pal system autoinjector (see fig. below). After an adsorption time of 60 min the fibre was retracted into the needle which then was inserted into the injector of a gas chromatograph. The fibre was extended by depression of the plunger, and the analytes were desorbed at 300°C and separated by gas chromatographical analyses (GC). Detection was carried out in a mass spectra (MS) where an ionization of the substances occurred in an electromagnetic field. Each substance decomposed into characteristic ion fragments which allowed its specific detection and quantification.

In contrast to the biochemical principle of an enzyme immunoassay (EIA) the solid-phase microextraction (SPME) is based on a physical principle. Compared to an EIA, the disadvantage of the headspace SPME is the lower sensitivity and the smaller amount of samples that can be examined per day . However, the simultaneous measurement of several substances in one sample (indicating luteal activity and probably oestrous and adrenocortical activity) within 90 min after the sample arrived at the lab may offer an alternative analytical tool in the field of zoo and wildlife research.

The figure shows principle and instrumentation used to analyse volatile substances from urine. The instrument consists of three components. 1: The CTC Combi Pal system autoinjector (SPME, above): sample heating, adsorption of substances onto the fibre exposed into the headspace above the surface of the urine, injection of the fibre into the injector of the GC, 2: the gas chromatograph (GC, left part) in which the separation of the substances was performed on a 60m capillary column, 3: the mass spectra(MS, right part): ionization of the substances and separation according to the ion size, detection and data analysis.

3. Qualitative and quantitative characterisation of spermatogenesis (histology with computer-assisted image analysis, flow cytometry, electron microscopy, immunohistochemistry, molecular biology)

Spermatogenesis, the production of male germ cells, is controlled by complex regulation mechanisms at different levels of the hypothalamic-pituitary-gonadal axis. It depends on endocrine factors (gonadotrophins, testosterone) whose effects are mediated by paracrine acting growth factors. The output of spermatogenesis is the result of the quantitative relations between mitosis, meiosis, and apoptosis. These processes depends on physiological, genetical and ecological factors. These factors cause different time patterns and/or intensities of activation and inhibition, respectively, of the three components.

The qualitative characterisation of spermatogenesis is used as an prerequisite for the investigation of such factors by histological and immuno-histochemical methods. Flow cytometry is applied for the quantitative evaluation of several cell types and the mitotic and meiotic activity. The function of testis as the producer of testosterone is assessed by endocrinological tests.

4. Cryopreservation of sperm and oocytes of different species

To establish methods for sperm characterisation and preservation is an essential part of artificial insemination programs in rare species. These projects are performed in tight co- operation with research group 5 and include investigations on structural and functional integrity of sperm in dependence of season, individual and species characteristics, conditions in captivity, and used technology for freezing. Our semen bank has already been used for artificial inseminations in giant panda, elephants, rhinos, and felids. The cryopreservation of fully grown mammalian oocytes is a difficult task. We have established methods to deep-freeze ovarian slices and primordial follicles isolated from ovaries of dead animals. In felid species, approximately 3000 follicles can be gained and frozen from one single ovary.
(see also FG 5).

5. In-vitro-maturation and -fertilisation of mammalian oocytes (IVM/ IVF)

Maturation and fertilisation of oocytes in vitro is a very important tool to help childless couples and also plays a role to help "childless" rare animals "couples" to receive offspring. For IVM/IVF oocytes are punctured from ovaries by endoscopy, and then cultured in specific cell culture media to maturity. Addition of sperm to the culture dish enables fertilisation of eggs and thus development of embryos in vitro. These embryos are transferred into the mother or another recipient of the same species. We have established an IFM/IVF system for felid species and perform a project on IVM/IVF in cattle in co-operation with the Institute for Reproduction of Farm Animals, e.V., Schönow, Germany.

Furthermore, in-vitro-fertilisation of domestic cat oocytes provides an experimental tool to investigate sperm-egg-interaction within our immunocontraception project and the project on sperm competition.

6. Xenotransplantation of ovarian tissue

Xenotransplantation is the transfer of viable cells and tissue, including organs or parts of the body, between different species. Human ovarian tissue was first successfully transplanted in rabbits at the end of 19th century (KNAUER, 1896, 1898, 1900). Female germ cell transplantation was then frequently used for different research purposes, in different experiments, but to a minor for practice in therapeutic purposes.
Currently, ovarian transfer is intensely examined to help women who undergo a radio- or chemotherapy. Especially in young female cancer patient, the ovarian tissue could be removed, stored deep frozen and re-transplanted after the ending of cancer therapy in order to protect germ cells against genetic damage and maintain fertility.
All organisms have immune defense to identify and launch a rejection reaction. For organ transplants or tissue-graft patients this response is commonly suppressed with pharmaceuticals. However in-bred strains of mice and rats with a genetic failure in their cellular immune system (so called nude mouse or rat) launch no rejection reaction to foreign tissue. Therefore these animals are prime candidates for foreign tissue cultivation. There is no risk of this rejection reaction as the cultivated tissue is re-implanted into the original organism (autotransplantation). The IZW conducted first experiments concerning ovary transplantation in the early 1990 in Siberian Golden Hamsters. In this study, the strategy of a prenatal hyper sensitization was tested (HILDEBRANDT, 1993).
In the current research project at the IZW we are examining the conservation of female germ cells of highly endangered cat species after their natural death using ovarian transplantation in an alternate host. The nude rat serves the alternative host for cat ovarian tissue cultivation and preservation. These rats suffer a thymus aplasia caused by a spontaneous mutation. The thymus is physiologically responsible for the formation of T-Lymphocytes / T-cells, which provoke the rejection of foreign tissue.
All treatments were carried out in general anesthesia using a gentle anesthetic gas (Isofluran®) in combination with oxygen via a small a breathing mask. Isofluran is considered to have only less negative side effects. In all rats, the ovaries were removed, a small incision was made into the kidney capsule and small fragments of the cat ovarian cortex (ca. 2 x 2 mm) were transplanted under the kidney capsule. The capsule was closed then with a two - component - fibrin - glue (Tissucol Duo S 0.5 ml Immuno, Baxter).

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For more methodological details: see scheme of M. Fassbender

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