PMC:514616 / 11143-19157 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/514616","sourcedb":"PMC","sourceid":"514616","source_url":"http://www.ncbi.nlm.nih.gov/pmc/514616","text":"Results and Discussion\n\nArtificial activation\nTable 1 shows the activation effects of either mechanical stimulation or A23187 calcium ionophore exposure on the unfertilized eggs of E. coqui. This ionophore non-specifically activates the unfertilized eggs of a variety of species through stimulation of a calcium-dependent signalling cascade [45]. Ten hours after laying, 11 of 32 eggs (34%) pseudocleaved, even if left undisturbed. As unfertilized eggs do not posses a centrosome, if they are artificially activated, the mitotic apparatus cannot form properly and divisions are irregular. This well-described process is called pseudocleavage [[46]; figure 1]). Activation almost doubled to 28 of 44 (64%) when the eggs were poked with forceps. Further, 30 of 36 eggs (83%) exposed to 0.1 mM calcium ionophore pseudocleaved. These percentages include both eggs that would have auto-activated –presumably 34%- as well as eggs that were activated by the mechanical or chemical treatments.\nTable 1 Artificial activation of E. coqui eggs To test whether the pseudocleavage response was an effect of our stimuli and not a reaction tied to other uncontrolled variables, we examined eight eggs that had remained undisturbed and that had not started pseudocleavage ten hours after laying. At this time, we poked them with fine forceps, and 6 of 8 (75%) began pseudocleaving six hours later (table 1). This delay in activation as a response to a delay in the stimulus is a strong indication that our manipulation is in fact responsible for eliciting the onset of cell division.\nAn interesting observation was that one third of all eggs deposited in response to hormone treatment activated of their own accord. This may be due to the mechanical stress to which the eggs are exposed during oviposition. Clearly, mechanical stimuli are able to activate the eggs, and stress incurred in during transit from the ovisac may be sufficient to cause activation. This would presumably not affect E. coqui during natural matings because this species undergoes internal fertilization, and the eggs will have already been fertilized prior to deposition [34]. In order to examine this hypothesis, we dissected oocytes directly from a female's ovisac, circumventing the passage through the oviduct and cloaca, and attempted to activate them with calcium ionophore (table 1). Controls were also performed with and without progesterone pretreatment in order to induce maturation. Since there is no information on the stage at which E. coqui eggs are arrested or what signal takes them out of their arrest, we followed procedures used in Xenopus [43]. However, none of these oocytes activated, regardless of the treatment. This may be because the oocytes did not respond to treatment with progesterone, and so never matured. Another possibility is that oocytes need to receive a signal from the oviducts and/or be coated in jelly before they can mature.\nThe ability to initiate activation after a long delay was interesting as we suspected that E. coqui eggs might be sensitive to aging, as has been reported under certain conditions for the externally fertilizing X. laevis [47]. Because E. coqui has internal fertilization, we suspected that eggs laid unfertilized might degenerate rapidly, complicating the artificial manipulation of this species' reproduction. Consequently, we investigated the ability of eggs to be activated as a function of time. When we artificially activated eggs with the calcium ionophore at different time points and examined them six hours post treatment, the percentage that pseudocleaved was high even ten hours after being laid (Figure 2). Twenty-four hours after deposition, however, the eggs were no longer able to activate. Thus, there is an extended period in which it is possible to carry out experiments without concern for a decrease in activation potential.\nFigure 2 Artificial activation of E. coqui eggs in relation to time after deposition. Unfertilized eggs were treated with calcium ionophore to induce activation at 0 (n = 12), 1 (n = 12), 2 (n = 10), 3 (n = 12), 4 (n = 12), 5 (n = 12), 10 (n = 18), and 24 (n = 18) hours. Eggs were scored for activation by the presence of cleavage furrows at 6 hours post treatment. Note that for the 10 and 24 hour time points, six eggs at each time point had already auto-activated by 6 hours post deposition. At the 10 hour time point, nine additional eggs activated later in response to ionophore treatment, while at 24 hours, no additional eggs were observed to activate after ionophore treatment.\n\nIn vitro fertilization\nThe average fertilization efficiency for natural matings conducted in our laboratory was 72% (see table 2). As roughly one third of all unfertilized eggs laid in response to hormone treatment auto-activate (table 1), and so only approximately 66% of the eggs in a given clutch will actually be receptive to sperm. If we assume these two factors to be independent -because we hypothesize that the eggs auto-activate during laying, a problem that doesn't arise in natural matings- then only 48 of every hundred eggs will be available for IVF (100*0.66*0.72). However, despite these complications, we were able to obtain an in vitro fertilization efficiency of 27% -or, rather, 56% of all receptive eggs (27/0.48) – by simply mincing the testes and adding them directly over the eggs (see table 2). Other IVF techniques were not as successful. Using sperm diluted in SDB resulted in only a 12% total fertilization efficiency (table 3).\nTable 2 Natural mating fertilization percentages for E. coqui in captivity.\nTable 3 In vitro fertilization of E. coqui eggs Sperm concentration may play a role in fertilization efficiency as the use of diluted sperm resulted in decreased fertilization. In support of this possibility, polyspermy has been observed in this species and is apparently not deleterious to fertilization and development [40]. A second possibility is that fertilization efficiency is linked to sperm capacitation and acrosome reaction. We attempted to study this possibility by examining the acrosomes of fresh and treated sperm using Lysosensor green fluorescent dye (Molecular Probes, Eugene, OR). This dye concentrates in low pH vesicles of living cells through an unknown mechanism and was shown to accumulate and preferentially stain the acrosome in X. laevis sperm [42]. However, we were unable to observe acrosome-specific staining in E. coqui sperm using this dye (data not shown). We also examined the possibility that a component of the egg jelly coat may be important for sperm capacitance. To test this, we incubated sperm with SDB and jelly, or SDB alone, and injected this under the jelly coat of eggs. None of 12 eggs were fertilized by sperm incubated with SDB alone, while pre-incubation of sperm with an extract of the jelly coat in SDB resulted in one fertilization out of 10 eggs (10%). This is considerably less than the 27% efficiency following direct placement of minced testes over the eggs, but suggests that interactions between sperm and the jelly coat may play a role in sperm capacitance and subsequent fertilization.\nTo test the functional response of the eggs, we attempted to fertilize artificially activated eggs. As was explained above, we were able to achieve an IVF success rate of 27%. However, if we poked the eggs fifteen minutes prior to direct fertilization, none (0/36) developed (see table 3). If we assume our expected fertilization rate to be 25%, the possibility of this result being due to chance is (1-0.25)36 = 3.2 × 10-5, or less than one in ten thousand. This shows that fifteen minutes after being poked the eggs have established a block to polyspermy, one of the defining functional characteristics of activation. Hence, although the first visible indication of activation –the formation of the first cleavage furrow- will not be seen for six hours, we can conclude that the egg is undergoing the normal activation processes within minutes of being stimulated.\n\n","divisions":[{"label":"Title","span":{"begin":0,"end":22}},{"label":"Section","span":{"begin":24,"end":8013}},{"label":"Title","span":{"begin":24,"end":45}},{"label":"Table caption","span":{"begin":986,"end":1035}},{"label":"Figure caption","span":{"begin":3874,"end":4563}},{"label":"Section","span":{"begin":4563,"end":8012}},{"label":"Title","span":{"begin":4563,"end":4585}},{"label":"Table caption","span":{"begin":5519,"end":5597}},{"label":"Table caption","span":{"begin":5596,"end":5646}}],"tracks":[{"project":"2_test","denotations":[{"id":"15296510-4473722-133587970","span":{"begin":342,"end":344},"obj":"4473722"},{"id":"15296510-3874873-133587971","span":{"begin":644,"end":646},"obj":"3874873"},{"id":"15296510-6894203-133587972","span":{"begin":2134,"end":2136},"obj":"6894203"},{"id":"15296510-5567826-133587973","span":{"begin":3151,"end":3153},"obj":"5567826"},{"id":"15296510-11846477-133587974","span":{"begin":6371,"end":6373},"obj":"11846477"}],"attributes":[{"subj":"15296510-4473722-133587970","pred":"source","obj":"2_test"},{"subj":"15296510-3874873-133587971","pred":"source","obj":"2_test"},{"subj":"15296510-6894203-133587972","pred":"source","obj":"2_test"},{"subj":"15296510-5567826-133587973","pred":"source","obj":"2_test"},{"subj":"15296510-11846477-133587974","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93e3ec","default":true}]}]}}