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?Question 2 (36 points)

Question

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?Question 2 (36 points)

BIOL 430 FA23EXAM 2Once you prove that Deno is the recognition protein for sperm and egg, you need to prove itsfunction.a) How would you approach testing that Deno is necessary for sperm-egg recognition? Includewhat you would predict to find and how would you know you had successful results.b) How would you approach testing that Deno is sufficient for egg-sperm recognition? Includewhat you would predict to find and how would you know you had successful results.Question 3 (18 points)Based on your experimental results (above), what would you advise the animal reserve owner(use scientific reasoning in your answers)?His original questions to you were:a) How likely is a hybrid event like this to happen again?b) Could they produce other combinations of animals successfully?

Answer 1

Question 1:

a) To test the hypothesis that Deno is a likely candidate protein for the recognition of sperm and egg, I would design an experiment that involves inhibiting the function or expression of Deno in the sperm. One approach could be to use gene editing techniques, such as CRISPR-Cas9, to specifically disrupt the Deno gene in horse sperm or zebra sperm. Another approach could be to use specific antibodies or inhibitors to block the interaction between Deno and the glycoproteins in the zona pellucida during fertilization.

b) If Deno is indeed a critical protein for sperm-egg recognition, I would predict that inhibiting its function or expression in the sperm would result in a decreased ability of the sperm to bind and fertilize the egg. This would be reflected in a significantly lower fertilization rate compared to control sperm with functional Deno. Alternatively, if the antibodies or inhibitors successfully block the interaction between Deno and the glycoproteins in the zona pellucida, it would also lead to a decreased fertilization rate.

Question 2:

a) To test whether Deno is necessary for sperm-egg recognition, I would design an experiment using sperm from horses, donkeys, and zebras. I would use techniques such as gene knockdown or knockout, or CRISPR-Cas9-mediated gene editing to specifically disrupt the Deno gene in each species of sperm. The sperm with disrupted Deno would then be used in in vitro fertilization (IVF) experiments to assess their ability to fertilize eggs from the same species. If Deno is necessary for sperm-egg recognition, I would predict a significantly lower fertilization rate in the eggs fertilized by sperm with disrupted Deno compared to control sperm.

b) To test whether Deno is sufficient for egg-sperm recognition, I would design an experiment using sperm from horses, donkeys, and zebras. I would genetically engineer the sperm to express Deno protein from one species in a species that does not naturally express it (e.g., expressing horse Deno in zebra sperm or donkey Deno in horse sperm). The engineered sperm would then be used in IVF experiments with eggs from the appropriate species. If Deno is sufficient for egg-sperm recognition, I would predict successful fertilization of the eggs by the engineered sperm, resulting in a similar fertilization rate compared to control sperm.

Question 3:

Based on the experimental results, I would advise the animal reserve owner as follows:

a) The likelihood of a hybrid event like the birth of Ippo happening again would depend on the level of similarity and compatibility of the proteins involved in sperm-egg recognition between the different species. If Deno is a critical protein for species-specific recognition, as suggested by the experiments, then the likelihood of another hybrid event occurring would be lower if the donkey species from the Amiata region has significantly different proteins involved in sperm-egg recognition compared to the zebra or other horse species.

b) The ability to produce other successful combinations of animals would also depend on the similarity and compatibility of the proteins involved in sperm-egg recognition between the species. If the proteins involved in recognition are highly conserved across different species, there is a higher chance of successful hybridization. However, if the proteins involved are significantly different, the likelihood of successful hybridization would be lower. It is important to note that hybridization between different species can also result in offspring with reduced fertility or viability, so the ethical implications and long-term consequences should be considered.