Uncovering the mystery of hybrid animalsIn animals that reproduce by external fertilization (like sea urchins), species-specificity is vital sothat the egg of one species recognizes and fuses with the sperm of the same species.Consequently, there are not one but several steps during fertilization that require species-specific recognition for fertilization to proceed. These steps include sperm attraction, initiation ofthe acrosomal reaction at the jelly coat, and sperm-egg binding. Interestingly, in mammals thatundergo internal fertilization, there are rare examples of interbreeding between species,including mules (horse x donkey) and zonkey (zebra x donkey).Consider the following scenarioYou are a developmental biologist on vacation in Florence, Italy. Talking to a local while having agelato, you hear of an animal reserve just outside of the city where a curious animal was born.The animal has a head of a donkey, and the stripes of a zebra on its body and legs! You instantlyguess it must be a zebroid, a hybrid between a zebra and another member of the horse family.Like any curious biologist, you drag your partner to the animal reserve to check out this curiouszebroid.When you arrive at the animal reserve, you immediately spot Ippo the zebroid running aroundher pen, and you go to talk to the caretakers at the reserve. The caretakers tell you that thezebras are kept in a fenced-in pen next to an area where an endangered species of donkey fromthe Amiata region are kept. One day Martin the zebra jumped the fence and mated with Giadathe donkey. Twelve months later, Giada gave birth to Ippo!Ippo has been such a sensation at the animal reserve that ticket sales to the park have soared.Hearing that you are a biologist, the owner strides over and asks you, how likely is a hybrid eventlike this to happen again? Could they produce other combinations of animals successfully to keepprofits up? Though you are concerned about the ethical implications, your curiosity about howanimals can mate to form hybrids is too much to resist. You can’t wait to return to yourlaboratory in Middletown and study this question.Back in the lab, you begin to read about the mechanism of fertilization. You learn that Deno is aprotein expressed on the acrosomal process of mammalian sperm (deno means ‘bind’ in Greek).In their discussion section, the authors of the paper speculate that Deno may recognizeglycoproteins in the zona pellucida, though no evidence is provided in the paper. The proteinsequence of Deno is 99% identical between horse, donkey and zebra, and 70% identical betweenhorse, human, mouse and rabbit.Question 1 (21 points)Test the hypothesis that Deno is a likely candidate protein for the recognition of sperm and egg.
a) How would you design an experiment to test this hypothesis?
b) What would you predict to find from your proposed experimental design?
a) To test the hypothesis that Deno is a likely candidate protein for the recognition of sperm and egg, I would design an experiment using in vitro fertilization (IVF) techniques.
First, I would collect eggs from the donkey, horse, and zebra, and isolate them in separate dishes. Then, I would collect sperm from each species and then separate them into three groups as well.
Next, I would prepare three different treatments for each species combination:
1) Control treatment - eggs from a species (e.g., donkey) are fertilized with sperm from the same species (donkey sperm);
2) Test treatment 1 - eggs from a species (e.g., donkey) are fertilized with sperm from a different species within the same family (e.g., horse sperm);
3) Test treatment 2 - eggs from a species (e.g., donkey) are fertilized with sperm from a species outside the family (e.g., zebra sperm).
In each treatment, I would add a fluorescent dye that specifically binds to Deno protein to visualize its distribution in the sperm and egg.
After allowing fertilization to occur, I would observe the fertilized eggs under a microscope and analyze the fluorescence pattern. Specifically, I would look for the presence and distribution of Deno protein in the fertilized eggs.
b) Based on the proposed experimental design, I would predict the following outcomes:
1) In the control treatment, where eggs are fertilized with sperm from the same species, I would expect to observe a high fluorescence signal indicating the presence of Deno protein in both the sperm and egg. This would support the hypothesis that Deno is involved in sperm-egg recognition.
2) In test treatment 1, where eggs are fertilized with sperm from a different species within the same family, I would expect to observe a weaker or absent fluorescence signal in the fertilized eggs. This would suggest that the Deno protein may not recognize and bind effectively to glycoproteins in the zona pellucida from a different species within the same family.
3) In test treatment 2, where eggs are fertilized with sperm from a species outside the family, I would expect to observe a very weak or absent fluorescence signal in the fertilized eggs. This would indicate that the Deno protein may not recognize and bind effectively to glycoproteins in the zona pellucida from a species outside the family.
Overall, if the fluorescence pattern supports these predictions, it would provide evidence that Deno is indeed involved in the recognition of sperm and egg. However, further studies would be needed to fully confirm its role and the species-specificity of its binding.