I can briefly address your questions, but others, such as critic Jarold Ramsey (link listed below), deal with this issue in detail. Both Macbeth and Lady Macbeth have their own interpretations of what it means to be a man: brave and ruthless in pursuing one's goals. We see Macbeth exhibit these qualities in a positive way on the battlefield as the play opens. However, in the plotting to kill Duncan, this definition becomes much more sinister. Lady Macbeth fears that Macbeth is "too full of the milk of human kindness to catch the nearest way" to the throne. In this expression, kindness is linked to femininity. When Lady Macbeth discusses the murder of Duncan with Macbeth, Macbeth is at first reluctant. Lady Macbeth accuses him of being a coward, not being man enough to do what is necessary to achieve his ambition.
Through Macduff, Shakespeare subverts this definition of manhood, to some extent. Macduff is brave. But he is also responsible, patriotic, self-sacrificing, and emotional. He cries when he is told that his wife and children have been slain. He feels responsible for their deaths because he chose his loyalty to Scotland over protection of his family. He wants to avenge their deaths, but first he will grieve: "I must feel it like a man."
An interesting minor definition of manhood, though, involves the way a man fights. It is important to receive wounds in the front, to face one's enemy in combat. Ambush, as Macbeth and the murderers do, is considered unmanly and dishonorable. Siward, for instance, is proud that his son died with his wounds in the front (facing his enemy, not running), and is clearly proud when Ross tells Siward that "like a man he died." Macduff clearly fulfills this more traditional idea of manhood in his desire to fight Macbeth "front to front." Macbeth ironically dies a man, refusing to submit when faced with his inevitable death at the hands of Macduff.
In the play Macbeth by William Shakespeare, the author starts by showing a man engaged in typical acts of male prowess - success and courage on the battle field. Yet, within a few scenes, this towering ideal of masculinity will be reduced to a jibbering wreck of emasculated maleness. In trying to manipulate Macbeth to get him to do what she wants, Lady Macbeth takes a swipe at her husband's male pride - and so, his self-esteem. She uses typically emasculating jeers and derisive taunts, even hoping that "the milk of human kindness" will leave him so that it will not stand in his way of carrying out the blood-thirsty deed. Yet she also decries femininity in herself, wishing to be unsexed so that her resolve may stay firm. "Art thou afeard" is such a predictable taunt for many characters in similar stories down through the ages.
Look at the public vs. the private roles of the Macbeths and the Macduffs.
Lady Macbeth and Lady Macduff mainly deliver their lines at home, in the private realm. Lady Macbeth convinces her husband to murder Duncan using emotion-filled intimate argument. Lady Macduff, whose husband has abandoned her, also vents her frustrations with her son and Ross, again privately. According to linguist Deborah Tannen, women tend to be "rapport talkers," consensus-builders who speak more intimately and emotionally from a domestic, private setting.
Macbeth and Macduff are all about the public realm. Both deliver speeches to other Thanes, long monologues for all to hear. They are, according to Tannen, "report-talkers. They deliver information more so than feelings. Notice what Malcolm tells Macduff after his family has been slaughtered:
The men demand action (revenge) of Macduff, instead of words. They don't expect him to use language to show feelings; instead, they expect steel to do their talking.
This is what Lady Macbeth laments in Act I:
When you durst do it, then you were a man;
And, to be more than what you were, you would
Be so much more the man. Nor time nor place
Did then adhere, and yet you would make both:
They have made themselves, and that their fitness now
Does unmake you. I have given suck, and know
How tender 'tis to love the babe that milks me:
I would, while it was smiling in my face,
Have pluck'd my nipple from his boneless gums,
And dash'd the brains out, had I so sworn as you
Have done to this.
She would trade her mother's milk for gall and dash her kids' brains out (if she had any) so that she could have the public privileges of men, such are the limitations of gender in her time.
Numerous details could go into an answer to your question concerning masculinity in Shakespeare's tragedy, Macbeth.
Lady Macbeth questions Macbeth's masculinity when he changes his mind about assassinating Duncan. Malcom tells Macduff that he must take the murder of his family like a man (get revenge) and Macduff replies that he will, but first he must "feel" it like a man. Banquo is no less brave in battle than Macbeth, and he handles the witches' predictions in a correct way, as opposed to Macbeth.
I suppose ideal masculinity in the play could be seen in the characters of Banquo and Macduff. They are loyal, brave, honest, and serve their country as best they can.
Gender roles are subverted in the play primarily by Macbeth and Lady Macbeth. Lady Macbeth takes on the stereotypical role of the aggressive male, and Macbeth is the one that has to be coaxed into the assassination. Lady Macbeth even begs the spirits she seems to believe in to "unsex me here."
Of course, the witches, or weird sisters, have beards, so gender roles are subverted by Shakespeare even before the audience sees Macbeth or his wife.