The authors investigated the mechanisms by which sex steroids regulate melanogenesis. The study contains descriptive and mechanistic studies indicating that physiologic estrogen and progesterone exert their differential effects on melanocytes by non-genomic mechanisms. Using genetic and pharmacologic approaches, the authors demonstrated that activation of nonclassical estrogen and progesterone receptors (GPER and PAQR7) are both necessary and sufficient to mediate sex hormone effects on melanocytes.

The effect of sex hormones on skin pigmentation has been observed for many years. There are relatively few studies that investigated the mechanisms that lead to such a phenomenon. A study by McLoad and his colleagues in 1994 reported no staining of ER-alpha on primary human melanocytes. Interestingly, the observation that estradiol and its 17-alpha analogue had equivalent effect to increase melanin synthesis, and the surprising similar effect of a pure anti-estrogen compound led them to speculate that the effect of estrogen on melanocytes might be mediated through a "non-classical" mechanism. (Effects of estrogens on human melanocytes in vitro. J Steroid, Biochem. Molec. Biol. 1994)

The current study is well designed, and the experiments follow a logical sequence. The manuscript is also well written. However, some of the findings contradict the previously published observations. The authors need to acknowledge those studies, and briefly discuss them. Notably, some researchers proposed that such inconsistent findings might be due to the fact that the melanocyte response to sex hormones is donor specific:

1. A few studies reported the expression of classical estrogen receptors on melanocytes: • Jee, SH et al. (1994) Effects of estrogen and estrogen receptor in normal human melanocytes. Biochem Biophys Res Commun. • Tobin, D. J., et al. (2002) Melanocytes in human scalp epidermis and hair follicles express the androgen receptor (AR) and both estrogen receptors (ER-alpha) and (ER-beta). 9th Annual European Hair Research Society Conference (poster presentation) • Im, S., et al (2002) Donor specific response of estrogen and progesterone on cultured human melanocytes. J. Korean Med. Sci. • Schmidt AN, et al. (2006). Oestrogen receptor-beta expression in melanocytic lesions. Exp Dermatol.

2. A few studies reported the effect of estradiol and progesterone on melanocyte proliferation: • Im, S., et al (2002) Donor specific response of estrogen and progesterone on cultured human melanocytes. J. Korean Med. Sci. • Wiedmann, et al (2008) Inhibitory effects of progestogens on the estrogen stimulation of melanocytes in vitro. Contraception

There are also a few clarifications that would improve the quality of the manuscript: 9, 617-629

1. The authors state that there have been no changes in melanocyte number in the organotypic human tissue. They should also clarify whether treatment of melanocytes in culture had any effect on the proliferation or not.
2. Figure 2, figure supplement 2: The authors should briefly explain why the melanin synthesis is significantly higher in Pertussis toxin alone compared to control.
3. Keratinocytes have been shown to express both classical and non-classical sex steroid receptors (Thornton, M. J. (2005) Oestrogen functions in skin and skin appendages. Expert. Opin. Ther. Targets). Since the authors discuss the therapeutic potential of topical non-classical receptor agonists/antagonist, it would be beneficial to briefly discuss their possible off-target effects. For example, is it likely that GPER agonists might increase the risk of inflammatory skin disorders or skin cancers by transactivation of EGFR on keratinocytes?