Shakespeare utilizes gender in Act IV, Scene I of Macbeth in a very curious way. Up to this point in the play, Macbeth's manhood has been criticized by his wife, Lady Macbeth. She has wondered if he had the courage to kill Duncan so that he would be able to take control of the throne. Worried about his manly ability, Lady Macbeth asks the spirits to "unsex me" so that she do things more typical of a man's behavior (like murder).
In Act IV, Scene I, Macbeth comes to the witches once again. Not satisfied with the prophecy given to Banquo (his sons will be kings), Macbeth is worried about what is next to come for him. It is in this scene where two things happen regarding gender roles.
First, Macbeth trusts the witches (women) because their earlier prophecies came true. When asked to show him more, they bring forth their "masters." While not directly stated, one could justify the apparitions as being male based upon the prophecies and warnings they provide. As every warning and prophecy is given, Macbeth fails to interpret them correctly. In the end, he is not satisfied with the witches' masters and curses them, "let this pernicious hour stand aye accursed in the calendar."
Second, Macbeth has begun to take on the true role of the male. No longer needing to be led by his wife, Macbeth plots to murder Banquo based upon the prophecy. Macbeth has finally become the man Lady Macbeth wanted him to be. No longer the weak one who "is too full o'the milk of human kindness," Macbeth is the ambitious and ruthless man she has always desired.