In addition to A23187, design an experiment and describe how you would test the

Question

In addition to A23187, design an experiment and describe how you would test the effects of another compound on one of the other 5 factors that are necessary for capacitation (listed in the intro, do NOT pick changes in Ca2+ concentration). Your experiment should be done in mice as well as include proper test and control subjects and your expected results if the compound is successful at promoting capacitation verses not.

Factors included in the intro: (1) activation of a cAMP/protein kinase A pathway6 ; (2) loss of cholesterol7 and other lipid modifications8 ; (3) increase in intracellular pH (pHi )9 ; (4) hyperpolarization of the sperm plasma membrane potential10–12; (5) increase in intracellular Ca2+ concentration [Ca2+]

*Please make your own experiment

Explanation / Answer

A23187 is an ion carrier that would form stable complexes with the divalent cations. This is more known as calcium ionophore A23187, or calcimycin. The A23187 increases the Calcium ion concentrations . When there is insensitivity or no action of Calcium ionophores, it is due to the absence of intracellular calcium ions, and or low activity of plasma membrane calcium channels. When there is influx of calcium ions , there is induction of calcium ions via three components :

In order to check this, experimental setup can be designed such that there are two conditions-

- increase in calcium ion influx and no blockage of native calcium channels

- increase in calcium channels with blockage of the calcium channels by drugs inhibiting the same.

1) activation of a cAMP/protein kinase A pathway6 and increase in the pH

The PKa activation at the time of capacitation depends on the cAMP that is produced by the adenylyl Cyclase. This activation would lead to increase in polymerisation of actin which is essential for the hyperactivated motility and this is required for successful motility. The presence of appropriate level of HCO3- results in activation of the enzymes required for activation. Also, there is an increase in the intracellular pH . In order to prove the same, we need to inhibit the PKA that would inhibit the phosphorylation of tyrosine. PKA can be inhibited by H89 and KT 5720. Two conditions can be set, one in which the inhibitor is used and other in the normal without inhibition condition.

The pH change can be studied by varying the pH of the spe-rm solution from 5.2- 8.2. The spe-rm total motility, progressive motility (PR), hypo-osmotic swelling (HOS) rate, and sp-erm penetration. Better activity was observed at pH 7.2 and 8.2 as compared to lower pH values.

2) loss of cholesterol and other lipid modifications

The cholesterol content is essential in development of acrosomal responsiveness and in vitro fertilisation. Bovine serum albumin (BSA) and NaHCO(3) are responsible for the phosphorylation of tyrosine in the human spe-rmatozoa. This can be checked by removing cholesterol for the plasma membrane of the sper-m. Two set ups, one in which the media did not contain BSA and other in which the media contained BSA have to be grown, and in the set up with BSA, cholesterol was released which led to protein tyrosine phosphorylation.

5) increase in intracellular calcium ion concentration

There is receptor operated influx of calcium ions through native calcium channels and this is sensitive to the drugs that inhibit the receptor operated influx of calcium ions. With an increase in calcium ion concentration, there is an increase in the calcium ion influx and higher activation of sp-erm.

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