When using Single GUP option, SEP clients are able to cross subnets to access the GUP. As Manish has mentioned above, this will require you open the GUP port (2967 by default) between these subnets.
With the Multiple GUP option, SEP Clients will pick from the list of GUPs only the one that is in their own subnet to update from. When using the Multiple GUP option, you are also able to define a Backup GUP. This Backup GUP is used if a SEP client cannot contact the GUP in its own subnet. The Backup GUP acts the sameway as a Single GUP, and may be used by SEP clients from other subnets.
All GUPs are meant to be able to handle upto about 10k SEP clients each (subject to hardware on the GUP).
Hopefully, this little bit of background info will help you decide how you want to proceed.