By Jennifer Harr, BS; Leanne Coyne, PhD; Ash Chaudhry, MS; and Robert F. Halliwell, PhD.
TJ Long School of Pharmacy & Health Sciences, University of the Pacific, Stockton, Calif.
The African clawed frog, Xenopus laevis, is widely used in developmental biology, embryology, molecular biology, and, for their oocytes, in neuro-pharmacology (Brown 2004). Academic labs and the pharmaceutical industry extensively utilize X. laevis oocytes to express recombinant nerve cell receptors and ion channels in drug studies and drug discovery efforts.
In all cases, the quantity and the quality of oocytes is critical for the collection of reliable data. Any protocol that can enhance the quality and quantity of oocytes would therefore reduce the total number of animals needed. With support from the Animal Welfare Institute, we conducted a study using electrophysiological techniques to test the hypothesis that environmental enrichment for X. laevis leads to an improved quantity and/or quality of oocytes.
Female X. laevis were housed in a large water tank, divided into two halves by a perforated Perspex sheet. One half of the tank was in a standard (unenriched) condition, and the other half was enriched by the addition of small functional items, including a plastic hollow log, a rocky cave and several plastic aquarium plants. All frogs were maintained on a 12 hour light/ 12 hour dark cycle at 21 to 23ºC and fed once every three days.
Ovulation of X. laevis was induced by injection of human chorionic gonadotropin (Sive et al., 1998). Membrane characteristics were recorded from X. laevis eggs using a two-electrode voltage-clamp technique (Halliwell et al., 1999). The data is expressed as the mean ± s.e.m of n experiments. For statistical comparisons, the Student's t-test was used.
The average membrane potential of eggs from X. laevis maintained in an enriched environment was -17 ± 1.1mV (n=89 eggs from 5 frogs) and -11 ± 0.8mV, (n=84 eggs from 5 frogs) from animals housed in the standard environment. These values are significantly different (at p? 0.001), but are much lower than the average membrane potential of -41 ± 1.3mV (n=63 cells from 3 frogs) when oocytes are removed surgically.
Membrane resistances determined for eggs obtained from X. laevis exposed to an enriched environment were 0.69 ± 0.1M?, (n = 36 eggs from 5 frogs) and 1.1 ± 0.2M? (n = 45 from 5 frogs) from frogs maintained in standard conditions. These values are not significantly different. The average membrane conductance determined for eggs obtained from animals maintained in the standard environmental condition was 2.6 ± 0.5µF (n = 45 eggs from 5 frogs) and 3.0 ± 0.4µF (n = 36 from 5 frogs) for eggs obtained from frogs housed in the enriched environment. Again, these values were not significantly different.
Although not quantified, frogs appeared to prefer having places to hide, since they were frequently observed in the cave or under the rock or plants. Frogs exposed to an enriched environment also released more eggs, with a significant increase in membrane potential compared with eggs obtained from frogs in standard laboratory conditions. However, surgically obtained oocytes are viable in vitro for up to one week longer than eggs obtained by the induction of ovulation, making oocytes more suitable for longer term (e.g. electrophysiological) studies. Nonetheless, the subtle improvement in the quantity and quality of eggs in this study suggests that it may be beneficial to incorporate environmental enrichment into experiments that utilize X. laevis.
Brown DD. A tribute to the Xenopus laevis oocyte and egg. Journal of Biological Chemistry, 279: 45291-45299 (2004).
Halliwell R.F, Thomas, P., Patten, D., James, C.H., Martinez-Torres, A., Miledi, R., & Smart, T.G. (1999) Subunit-selective modulation of GABAA receptors by the non-steroidal anti-inflammatory agent, mefenamic acid. European Journal of Neuroscience Cold Spring Harbor Laboratory Press, 11: 2897-2905.
Sive HL, Grainger RM & Harland RM (1998) Early Development of Xenopus laevis: a laboratory manual. Cold Spring Harbor Laboratory Press.