Hello Zahra.

You could make monolayer of neuroendocrine neurons first, and then add washed spermatozoa in that culture to remove many identified hydrolases present in semen or epidymal fluid due to premature acrosome reaction and the acrosome.

1) The problem is that neurons and spermatozoa require quite different components in their own media. Therefore, spermatozoa may not be happy in the medium essential to neurons vice versa.

2) You could tell any effects on sperm by the neuron within only a very narrow window before sperm deteriorate in the neuron-supporting medium.

3) Spermatozoa require specific cell viability and motility.Furthermore, sperm viability can be assesed only by sperm penetration or fertilizing ability, using zona-free hamster oocyte penetration (HOP) test or its counterpart oocyte, respectively.

4) I would suggest to make 48h-cultured neuron medium first (called conditioned medium). Spin it down to remove any cell debris and particles. Supplement with further 3~5% serum and 1/2 glucose or glutamine contents of the neuron medium to the conditioned medium to compensate the depleted nitrogenous nutrients and other unknown factors. Millipore, aliquot and store at 4C. Use them within a week. When use the conditioned medium, make sperm culture (its own medium), and then add conditioned medium 1/20~1/10 of the sperm medium volume). See the effects on sperm.

Best wishes.

Oh Thank you so much for such a detailed answer. I will definitely try following your suggestion.