+ Article
Skin exposure to UVB light induces a skin-brain-
gonad axis and sexual behavior
Graphical abstract
Highlights
d UVB exposure increases circulating sex-steroid levels in mice
and humans
d UVB exposure enhances female attractiveness and
receptiveness toward males
d UVB exposure increases females' estrus phase, HPG axis
hormones, and follicle growth
d Skin p53 regulates UVB-induced sexual behavior and ovarian
physiological changes
Authors
Roma Parikh, Eschar Sorek,
Shivang Parikh, ..., Ruth Percik,
Aron Weller, Carmit Levy
Correspondence
carmitlevy@post.tau.ac.il
+
+ In brief
Parikh et al. find that UVB exposure
triggers a skin-brain-gonadal axis
through skin p53 activation. UVB
exposure increases in female mice sexual
responsiveness and attractiveness,
hypothalamus-pituitary-gonadal axis
hormone levels, ovary size, and estrus
duration, as well as male-female
interactions. Solar exposure in humans
enhances romantic passion and
positively correlates with male
testosterone levels.
+
+ Parikh et al., 2021, Cell Reports 36, 109579
August 24, 2021 ª 2021 The Authors.
https://doi.org/10.1016/j.celrep.2021.109579
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Article
Skin exposure to UVB light induces
a skin-brain-gonad axis and sexual behavior
Roma Parikh, 1 Eschar Sorek, 1 Shivang Parikh, 1 Keren Michael, 2 Lior Bikovski, 3,4 Sagi Tshori, 5,6 Galit Shefer, 5
Shira Mingelgreen, 5 Taiba Zornitzki, 7 Hilla Knobler, 7 Gabriel Chodick, 8,23 Mariya Mardamshina, 1 Arjan Boonman, 9
Noga Kronfeld-Schor, 9 Hadas Bar-Joseph, 10 Dalit Ben-Yosef, 11,12 Hadar Amir, 13,14 Mor Pavlovsky, 15 Hagit Matz, 15,16
Tom Ben-Dov, 1,17 Tamar Golan, 1 Eran Nizri, 15,16 Daphna Liber, 18 Yair Liel, 19 Ronen Brenner, 20 Yftach Gepner, 21
Orit Karnieli-Miller, 22 Rina Hemi, 23 Ruth Shalgi, 24 Tali Kimchi, 25 Ruth Percik, 16,23 Aron Weller, 26 and Carmit Levy 1,27, *
1 Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
2 Department of Human Services, The Max Stern Yezreel Valley Academic College, Jezreel Valley 1930600, Israel
3 The Myers Neuro-Behavioral Core Facility, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
4 School of Behavioral Sciences, Netanya Academic College, Netanya 4223587, Israel
5 Research Authority, Kaplan Medical Center, Rehovot, Israel
6 Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University, Jerusalem, Israel
7 Diabetes, Endocrinology and Metabolic Disease Institute, Kaplan Medical Center, Hadassah School of Medicine, Hebrew University in
Jerusalem, Rehovot, Israel
8 Maccabitech, Maccabi Healthcare Services, Tel Aviv, Israel
9 School of Zoology, Faculty of Life Sciences and the Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel
10 The TMCR Unit, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
11 IVF Lab & Wolfe PGD-Stem Cell Lab, Fertility Institute, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel
12 Department of Cell Biology and Development, Sackler Faculty of Medicine & Sagol School of Neuroscience, Tel Aviv University, Tel Aviv,
Israel
13 Fertility Institute, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel
14 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
15 Department of Dermatology, Tel Aviv Sourasky (Ichilov) Medical Center, Tel Aviv 6423906, Israel
16 Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
17 Department of Otolaryngology, Head and Neck surgery, Meir Medical Center, Kfar Saba 4428164, Israel
18 Faculty of Humanities, Education and Social Sciences, Ono Academic College, Kiryat Ono, Israel
19 Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
20 Institute of Pathology, E. Wolfson Medical Center, Holon 58100, Israel
21 School of Public Health, Sackler Faculty of Medicine and Sylvan Adams Sports Institute, Tel Aviv University, Tel Aviv 69978, Israel
22 Department of Medical Education, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
23 Institute of Endocrinology, Chaim Sheba Medical Center, Tel-Hashomer, Israel
24 Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
25 Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel
26 Department of Psychology and the Gonda Brain Research Center, Bar-Ilan University, Ramat Gan 5290002, Israel
27 Lead contact
*Correspondence: carmitlevy@post.tau.ac.il
https://doi.org/10.1016/j.celrep.2021.109579
SUMMARY
Ultraviolet (UV) light affects endocrinological and behavioral aspects of sexuality via an unknown mechanism.
Here we discover that ultraviolet B (UVB) exposure enhances the levels of sex-steroid hormones and sexual
behavior, which are mediated by the skin. In female mice, UVB exposure increases hypothalamus-pituitary-
gonadal axis hormone levels, resulting in larger ovaries; extends estrus days; and increases anti-Mullerian
hormone (AMH) expression. UVB exposure also enhances the sexual responsiveness and attractiveness
of females and male-female interactions. Conditional knockout of p53 specifically in skin keratinocytes abol-
ishes the effects of UVB. Thus, UVB triggers a skin-brain-gonadal axis through skin p53 activation. In humans,
solar exposure enhances romantic passion in both genders and aggressiveness in men, as seen in analysis of
individual questionaries, and positively correlates with testosterone level. Our findings suggest opportunities
for treatment of sex-steroid-related dysfunctions.
+
+ INTRODUCTION
Exposure to the ultraviolet (UV) component of solar radiation in-
creases testosterone levels in men (Myerson and Neustadt,
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+ 1939), estradiol and testosterone levels in fish (Mitchell et al.,
2014), and the attractiveness of hens to cockerels (Jones et al.,
2001). This suggests that exposure to UV plays a major role in
the regulation of sexuality on both behavioral and endocrinological
Cell Reports 36, 109579, August 24, 2021 ª 2021 The Authors. 1
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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levels. The mechanism underlying this effect remains poorly
understood.
The reproductive endocrine system includes organs such as
the hypothalamus, pituitary, thyroid, pineal and adrenal glands,
ovaries, and testes (Rawindraraj et al., 2019). The system is gov-
erned by the hypothalamus, which sends signaling mediators
such as gonadotropin-releasing hormone (GnRH) to the pituitary
gland to induce release of follicle-stimulating hormone (FSH) and
luteinizing hormone (LH). In turn, these hormones transmit sig-
nals to the male and female gonads (Rawindraraj et al., 2019),
the testicles and the ovaries, respectively, promoting sex-steroid
production and gametogenesis (Rawindraraj et al., 2019). In fe-
males, FSH and LH stimulate the production of estrogen and
progesterone, which regulate ovulation and pregnancy (Barbieri,
2014; Rosner and Sarao, 2019). In most female mammals, sexual
activity and receptivity are confined to the preovulatory period
(Wallner et al., 2019) and the estrus phase (Kim et al., 2016) of
the menstrual/estrous cycle. These cyclic hormonal changes
dictate sexual behavior. In humans, it has been shown that
men respond more favorably to a woman's scent (Kuukasja ¨rvi
et al., 2004) or facial appearance (Roberts et al., 2004) during
the preovulatory phase of the woman's cycle.
The skin, consisting of epidermal, dermal, and hypodermal
layers, is the largest body organ (Golan et al., 2015; Yousef
et al., 2020). In contrast to the vast literature on the skin as a hor-
monal target (Slominski and Wortsman, 2000; Zouboulis, 2004),
its role as a source of hormones is less understood. The skin is
capable of producing and releasing hormones including vitamin
D, various peptides derived from proopiomelanocortin (POMC),
b-endorphin (Fell et al., 2014), and corticotropin-releasing hor-
mone (CRH) (Skobowiat and Slominski, 2015), resembling the
central regulatory paradigms of the hypothalamic-pituitary-adre-
nal axis (Slominski and Wortsman, 2000; Slominski et al., 2015).
b-endorphin release into the circulation is implicated in sun-
addiction behavior (Fell et al., 2014). Furthermore, ultraviolet B
(UVB) exposure is linked to increased expression of the stress-
response hormone CRH, as well as with components of the hy-
pothalamus-pituitary-adrenal (HPA) axis, including adrenocorti-
cotropic hormone (ACTH) and corticosterone, both in the skin
and in the plasma; stimulation of corticosterone production
was seen in the absence of pituitary involvement (Skobowiat
and Slominski, 2015; Skobowiat et al., 2011, 2017; Slominski
et al., 2013, 2018). These data situate the skin as an important
component in the regulation of stress-related behaviors and
sun-addiction behavior.
Given that the furless human skin contacts the environment in
general and the sun's rays in particular, it is conceivable that the
skin plays a role in hormone-related social, sexual, and reproduc-
tive behavior, but this assumption has yet to be verified. Solar ra-
diation (bright light and radiant heat) from the sun includes infrared,
visible, and UV. UV light is further divided into UVA, UVB, and UVC
(Ho ¨lzle and Ho ¨nigsmann, 2005). Here, we report, using behavioral
tests in mice, that UVB treatment significantly enhanced the sex-
ual responsiveness of females, which in turn increases male sexual
arousal and behavior. Furthermore, UVB treatment significantly
enhanced the desire for male-female interaction and female
attractiveness to males. In terms of physiological changes, we
found that UVB treatment increased the incidence of estrus days
+
+ and enhanced ovary size and anti-Mullerian hormone (AMH)
expression in mice. Mechanistically, we demonstrated that the
UVB-induced sexual behavior and hormonal changes are medi-
ated by p53 activation in epidermal keratinocytes through a
skin-brain-gonadal axis. We also demonstrated, using question-
naires, that UVB treatment enhanced romantic passion in both
men and women and aggressiveness in men and is positively
correlated with testosterone level. This study suggests that UVB
phototherapy has potential as an ancillary treatment of sex-ste-
roid-related dysfunctions.
RESULTS
Daily UVB treatment enhances female sexual
attractiveness and receptiveness
To investigate the systemic effects of UVB radiation, we exposed
dorsally shaved mice to a single UVB dose of 800 mJ/cm 2
(Svobodova ´et al., 2012) or to 50 mJ/cm 2 daily for 8 weeks, a
sub-erythemic UVB dose that is equivalent to 20-30 min of
midday sun (Fell et al., 2014). Blood samples were collected
24 h after UVB treatment for the acute model and after 8 weeks
of UVB treatment for chronic models (Figure S1A). As expected
(Malcov-Brog et al., 2018), skin pigmentation increased in the tails
of both male and female mice upon chronic exposure compared
with mock treatment (control) (Figure S1B). Furthermore, we
found significant positive activation Z score for upstream regula-
tors b-estradiol, testosterone, and estrogen in male mice (Fig-
ure 1A, right panel) and for estrogen, androgen, b-estradiol, and
progesterone in female mice after chronic UVB treatment
compared with the controls (Figure 1A, right panel). Acute
exposure of mice did not result in significant activation of sex-ste-
roid signaling (Figure S1C), suggesting that the chronic dose of
UVB exposure is a more physiologically relevant model. Sex ste-
roids released from gonads activate the neuronal pathways
involved in sexual behavior in zebrafish (Pradhan and Olsson,
2015), and testosterone injections enhance mounting behavior
in males by priming the neural tissues mediating the mating
behavior in guinea pigs (Phoenix et al., 1959). These data suggest
that UVB treatment enhances sex-steroid signaling in both male
and female mice and thus might influence mating behavior.
Mating behavior in rodents consists of several behaviors. At-
tractivity (Beach, 1976) involves efforts to elicit a response
from the opposite sex by vocalizations and olfactory and visual
stimuli (Beach, 1976). Proceptivity includes estrus responsive-
ness and purposive vocalizations (Beach, 1976), and female
receptivity involves lordosis behavior that involves readiness to
be involved in copulation, which culminates in successful intro-
mission (Beach, 1976). To assess the effect of UVB on reproduc-
tive behavior, we conducted a mating test (Figure 1B) in which a
sexually naive female, either UVB treated or mock treated (con-
trol), was introduced into the home cage of a sexually naive male
that had been UVB treated or mock treated (control). Sexual
receptivity in rodents varies with the stage of the female estrous
cycle (Zinck and Lima, 2013). To exclude the differences in fe-
male sexual receptivity, we evaluated the estrous cycle of the fe-
male by vaginal cytology (Caligioni, 2009) and used only females
in the estrus/proestrus stage for the mating test. During the 1-h
mating test, we monitored vocalization, sniffing, self-grooming,
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+ intromission, lordosis, and ejaculation as has been done previ-
ously (Achiraman et al., 2014; Beach, 1976; Haga et al., 2010;
Kimchi et al., 2007).
To determine the effect of UVB exposure on attractivity, we as-
sessed ultrasonic vocalizations (USVs) (Costantini and D'Amato,
2006). During a male-female encounter, male mice exclusively
Figure 1. Daily UVB treatment enhances female sexual attractiveness and receptiveness
(A) Activation Z scores of predicted upstream regulators of mice upon UVB treatment for 6 weeks.
(B) Schematic representation of the mating test with male and female mice treated with UVB or control for 6 weeks.
(C-E) Total number, total time, and mean dominant frequency of USVs by control males in the presence of UVB-or control-treated females.
(F) Representative photograph of sexual behavior parameters.
(G) Total number of control male anogenital sniffing events of UVB-or control-treated females.
(H) Total time self-grooming by control males in the presence of UVB-or control-treated females.
(I) Latency intromission (left) and total number of intromissions (right) by control males on UVB-or control-treated females.
(J) UVB-or control-treated females' lordosis quotient upon control male mounting.
(K) Total number of anogenital sniffing events (left) and total time self-grooming (right) for control females in the presence of UVB-or control-treated males.
(L) Plasma testosterone levels of males upon 8 weeks of UVB or control treatment.
(M) Total number of anogenital sniffing events by UVB-or control-treated males on a control-treated female.
(N) Total time spent self-grooming by UVB-or control-treated males for a control-treated female.
(O) Latency intromission (left panel), duration of intromission (middle panel), and total number of intromissions (right panel) by a UVB-or control-treated male on a
control female.
(P) Total number of anogenital sniffing events (left) and total time self-grooming (right) by UVB-or control-treated females on a control-treated male.
Data are means ± SEM, n = 3 (A and C-E), n = 7 (G-K and M-P), n = 8 (L). For data analysis, a two-tailed, unpaired Student's t test was performed. *p < 0.05; **p <
0.01; ***p < 0.001; ns, not statistically significant.
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+ dominate the calls (frequency of 40-70 kHz), a behavior posi-
tively related to their level of sexual arousal (Kerchner, 2004).
We evaluated the effect of UVB treatment on the USVs of control
males in the presence of a UVB-treated or control female as a
stimulus. The audio recordings were then extracted into several
parameters, including total call duration and its mean dominant
frequency using UltraVox XT 3.1 software. The total number of
control male calls (Figure 1C; Figure S1D) and their total duration
(Figure 1D) were significantly higher when males were matched
with UVB-treated females than with control females. There was
no difference in frequencies of the calls with the highest energy
(mean dominant frequency) (Figure 1E), indicating that the dura-
tion and number of calls changed but the type of call did not.
These results suggest that UVB treatment of females enhances
their attractivity as indicated by the relative increase in male
vocalization parameters.
Next, we evaluated the effect of UVB treatment on female
attractiveness by measuring anogenital sniffing behavior (Clarke
and Trowill, 1971) (Figure 1F; Video S1). Males exhibited similar
sniffing behavior during the 1-h test session regardless of the
treatment the female received (Figure 1G). Self-grooming re-
flects an attraction to the opposite sex (Achiraman et al., 2014;
Haga et al., 2010), and the total duration of grooming (Figure 1F;
Videos S2A and S2B) was significantly enhanced for males in the
presence of UVB-treated females compared with control fe-
males (Figure 1H). Furthermore, we analyzed intromission, which
is a measurement of a successful mating event by the male on a
receptive female (Haga et al., 2010) (Figure 1F; Video S3). The la-
tency to intromit was significantly shorter for UVB-treated than
for control females (Figure 1I, left panel). The duration (Fig-
ure S1E) and total number of successful intromissions (Figure 1I,
right panel) were significantly greater when males were mated
with UVB-treated females than with controls. This indicated
that males are more attracted to and subsequently sexually
more successful with UVB-treated females.
During intromission, the receptiveness of the female is
measured by her lordotic response (Haga et al., 2010) (Figure 1F;
Video S3). We found a significant increase in the lordosis quo-
tient of UVB-treated females toward males compared with that
exhibited by mock-treated females (Figure 1K), suggesting that
UVB enhances female receptiveness. No change was observed
in the rearing behavior of females (Figure S1F), indicating no dif-
ference in forced intromission encounters.
Finally, male ejaculation did not significantly differ between
UVB-or mock-treated females (Figure 1F; Figure S1G; Video
S4). Similar tests were performed using UVB-treated males,
and differences between the number of ejaculations did not differ
when females were UVB or mock treated (Figure S1H). This
demonstrates that UVB treatment significantly enhances the
attractiveness and responsiveness of female mice, which in
turn increases the sexual arousal and behavior of males.
Next, we measured the effect of UVB on male attractiveness
by testing anogenital sniffing and grooming of a female in the
presence of UVB-treated or control males. Females exhibited
significantly more anogenital sniffing events toward UVB-treated
males compared with control males (Figure 1K, left panel). No
significant difference was observed in female grooming behavior
(Figure 1K, right panel). Similar tests were performed using UVB-
treated females, demonstrating same significant trend toward
the UVB-treated male compare to control male (Figure S1I), sug-
gesting an increase in male odor upon UVB exposure.
Because testosterone is involved in synthesis and secretion of
pheromones (Asaba et al., 2014), male attractivity (Mitra and
Sapolsky, 2012; Schellino et al., 2016), and social and emotional
bonds with females (van der Meij et al., 2012), we found signifi-
cantly higher plasma total testosterone levels in UVB-than
mock-treated male mice (Figure 1L). We found no change in
the level of testosterone in female mice upon UVB exposure
(Figure S1J).
Next, we tested the effect of UVB treatment on the social/sex-
ual behavior of males and females. No significant differences
were observed in anogenital sniffing, grooming behavior, latency
to intromit, duration of intromission, and number of intromissions
by control or UVB-treated male mice mated with control-treated
females (Figures 1M-1O). However, UVB treatment significantly
increased female anogenital sniffing and grooming behavior to-
ward control males (Figure 1P). This suggests that UVB treat-
ment enhances social/sexual behavior of females. Because
grooming behavior is also a known characteristic of anxiety in ro-
dents (Kalueff et al., 2016), we evaluated anxiety in an elevated
plus maze test, a classic measure of anxiety-related behavior
(Walf and Frye, 2007). Our results demonstrate that males
treated with UVB displayed a significant reduction in their anxiety
level compared with the control males (Figure S1K), as was
shown previously for male mice (Fell et al., 2014). No difference
was observed in the anxiety levels of females (Figure S1L). These
data support our conclusion that an increase in grooming
behavior might be indicative of attraction to the opposite sex ir-
respective of anxiety. Altogether, our data demonstrate that UVB
exposure enhances female attraction, the testosterone level in
males, and the social/sexual behavior of females.
UVB treatment enhances male and female sexual
behavior and female attraction
Sexual selection is based on the preference for social proximity
to an attractive partner (Puts, 2010). Given our findings that UVB
treatment significantly increases the attractiveness of males and
females, we investigated odor-triggered preferences and mate
selection through social proximity using the three-chamber test
(Yang et al., 2011), in which a subject's preference for one of
two stimuli is monitored (Figure 2A). The subject was a UVB-
or mock-treated male or female mouse, and the stimulus was
a UVB-or mock-treated male, a control female mouse, or a novel
object. The wire cages in this test setup ensure that the social
behavioral analysis is limited to the subject mouse (Yang et al.,
2011). This setup allows olfactory, auditory, and visual stimuli.
All females were in the estrus/proestrus stage.
We found that when the stimulus was a UVB-treated or a
mock-treated control female mouse, the males exhibited a clear
preference for the UVB-treated female (Figure 2B). We analyzed
our results in terms of the latency of the subject mouse to venture
into a stimulus compartment, the frequency of visits, and the visit
duration. Our analysis showed that it took significantly less time
for the male to move near the wire cage (Figure 2C, left panel) or
into the zone of the wire cage (Figure S2A, left panel) of a UVB-
treated female than a mock-treated control female. Furthermore,
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+ the subject males preferred to visit and stay near the wire cage
(Figure 2C, middle and right panels) and in the zone of the wire
cage (Figure S2A, middle and right panels) of a UVB-treated fe-
male rather than a control female. Similar trends of attraction to-
ward the UVB-treated female were observed when the subject
was a UVB-treated male (Figures S2B and S2C). These data
demonstrate a significant male preference for social proximity
to UVB-treated females, supporting our hypothesis that UVB
treatment enhances female attractiveness.
Next, we examined the effect of UVB on male attractiveness.
The subject female demonstrated no difference in the latency
to visit, the frequency of visits, or the total time spent near or in
the zone of a wire cage containing a UVB-treated or a mock-
treated male stimulus (Figures 2D and 2E; Figure S2D). Similar
trends were observed when the subject was UVB-treated female
except in a few parameters (Figures S2E and S2F), which might
be a result of an interaction between two UVB-treated animals.
Next, to check the effect of UVB on social behavior, we deter-
mined the latency to visit, the frequency of visits, and the total
time spent by UVB-treated or mock-treated males with a
mock-treated control female. We found a significant decrease
in the latency to visit and a significant increase in the total time
spent near the female by the UVB-treated male subjects
compare to the control male subjects (Figures 2F and 2G). No
difference was observed in the frequency to visit the female (Fig-
ure 2G, middle panel). This suggests that UVB treatment en-
hances male social behavior. Furthermore, we observed a signif-
icant decrease in the latency to visit and a significant increase in
the frequency of visits near a wire cage containing a male by the
UVB-treated female subjects compared with the control female
subjects (Figures 2H and 2I), although no difference was
observed in the total time spent next to a male (Figure 2I, right
panel). This suggests that UVB treatment enhances female so-
cial behavior.
To rule out the possibility that the results are due to mice
pausing in the center of the chamber, we measured the total
time spent by the subject mouse in the center of the chamber.
Notably, no difference was found between the two male subject
groups (Figure S2G) or the two female subject groups (Fig-
ure S2H). This confirmed that the observed desire for social
proximity in both male and female mice results from the UVB
treatment.
To validate that male preference for a UVB-treated female
mouse results from sexual signals, we repeated the three-cham-
ber test with a female subject, a UVB-treated female stimulus,
and a mock-treated control female stimulus (Figures S2I-S2L).
No variation in the social preference of the female subject was
observed (Figures S2I-S2L), supporting our hypothesis that
UVB treatment induces female sexual attractiveness to males.
We also performed the test with a female as a subject and a
male and a novel object (plastic block) as stimuli. The results
clearly showed that UVB-treated females preferred to visit and
stay near the male rather than near the novel object (Figures
S2I and S2M); the mock-treated control female demonstrated
no such preference (Figures S2M-S2O). These data support
our hypothesis that UVB treatment increases the social behavior
of female mice with male mice. Altogether, our observations
show that UVB treatment significantly enhances the desire for
male-female interaction and significantly increases the attrac-
tiveness of female mice.
UVB treatment induces romantic passion in humans
To conduct a controlled study of the effect of UVB treatment in
humans, we assembled a cohort of patients who were undergo-
ing phototherapy, which provides a documented dose of UVB
exposure. The patients were asked to fill an adapted Passionate
Love Scale (PLS) questionnaire (Hatfield and Sprecher, 1986)
before the first UVB treatment (time point T1) and 1 month there-
after (time point T2). Dermatosis was not expected to have
improved at the 1-month time point (Bae et al., 2017; Cameron
et al., 2002); thus, therapeutic success, or lack thereof, should
not influence the stress levels of subjects, and there should not
have been bias in our questionnaire because of therapeutic effi-
cacy. During this period, patients received a UVB dose (0.1-2.5
J/cm) two or three times a week for 10-12 UVB treatments. The
PLS, developed to measure passionate love in intimate relation-
ships, focuses on intense longing for union with the other. We
found that the male participants' scores were significantly higher
at T2 with respect to obsessive thoughts regarding their loved
ones, yearning to know everything about her, and endless desire
for affection from her (Table 1). However, they also reported
significantly less attraction to that person compared with at T1.
Female participants at T2 scored significantly higher when it
came to feeling that the person whom they loved most passion-
ately is the perfect romantic partner and experiencing a physical
response when touched by that person. Furthermore, because
we observed an increase in the level of testosterone following
UVB treatment in male mice (Figure 1L) and because testos-
terone is responsible for sexual and aggressive behavior (Muller,
2017), we asked our human cohort of patients undergoing
Figure 2. UVB treatment enhances male and female sexual behavior and female attraction
(A) Schematic representation of the three chamber test for subject and stimulus mice treated for 5 weeks with UVB or control treatment.
(B) Heatmap paths of a control male subject (n R 10) toward a UVB-or control-treated female stimulus.
(C) Latency of visit (left), frequency of visits (middle), and total time spent (right) by subject control-treated males toward the wire cage of a UVB-or control-treated
female stimulus.
(D) Heatmap paths of a control female subject toward a UVB-or control-treated male stimulus.
(E) As in (C) but for female subject movement toward a UVB-or control-treated male stimulus.
(F) Heatmap as in (B), depicting control or UVB-treated subject male movement toward control female stimuli.
(G) As in (C) but for control or UVB-treated male subject movement toward control female stimuli.
(H) Heatmap as in (B) but for UVB-or control-treated female subject movement toward control male stimuli.
(I) As in (C) but for UVB-or control-treated female subject movement toward control male stimuli.
Data are presented as means ± SEM, n R 10. For data analysis, a two-tailed, unpaired Student's t test was performed. *p < 0.05; **p < 0.01; ns, not statistically
significant.
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+ phototherapy to complete an aggression questionnaire (Buss
and Perry, 1992) at T1 and T2. Our results indicate that male par-
ticipants were significantly more verbally aggressive at T2 than at
T1 (Table S1), whereas women showed no difference. Neither
men nor women had a significant change in the level of physical
aggression between T1 and T2. Altogether, our data suggest that
in humans, UVB treatment enhances passionate love in both
genders and increases some aspects of aggressiveness in men.
UVB treatment increases estrus incidence and the
number of growing follicles
UVB treatment was found to enhance female sexual/social
behavior, receptiveness, and attractiveness in mice, so we
investigated its effect on the estrus phase, because females
are more sexually receptive and attractive during its estrus stage
(Kim et al., 2016). To this end, we followed the estrous cycle of
eight female mice for 45 days using the vaginal smear method
(Caligioni, 2009) (Figure S3A). We found a significant increase
in the percentage of estrus days from the total number of 45
tested days (Figure 3A) and in the length of the estrus phase (Fig-
ure 3B) in UVB-treated females. These data suggest that UVB
treatment modifies the estrus incidence of female mice,
increasing the number of estrus days in the cycles.
The estrous cycle is governed by the level of GnRH secretion
from the hypothalamus, which stimulates the pituitary gland to
release FSH and LH; these hormones regulate the ovarian cycle,
resulting in the production of sex-steroid hormones (Smith,
2009). We measured the levels of GnRH, FSH, and LH in the
plasma of female mice following 8 weeks of UVB or mock treat-
ment and found a significant rise in these hormones following
UVB treatment (Figure 3C). These data demonstrate that UVB
treatment induces the production of hormones involved in the
brain-gonadal axis.
FSH and LH regulate follicle growth in the ovaries, leading to
ovulation (Smith, 2009). Therefore, we surgically resected the
ovaries from UVB-treated and mock-treated control female
mice in their proestrus/estrus stage to assess the effect of the
UVB treatment on ovarian morphology. Interestingly, we found
a significant increase in the size and weight of the ovaries of
UVB-treated compared with mock-treated female mice (Fig-
ure 3D, left and middle panel), which was reflected in their histol-
ogy (Figure 3D, right panel). Moreover, there was a significant in-
crease in the expression of mRNAs encoding the progesterone
receptor (PGR), androgen receptor (AR), and estrogen receptors
(ESR1 and ESR2) in ovaries of UVB-treated females compared
with control females (Figure 3E). We also observed significant
upregulation in the enzymes involved in sex-steroid biosynthesis
in UVB-treated females compared with control females (Fig-
ure S3B). AMH suppresses the cyclic recruitment of primordial
follicles into the pool of growing follicles and inhibits FSH-depen-
dent follicle recruitment (Dewailly et al., 2016), thus playing an
important role in maintaining the ovarian reserve (Visser et al.,
2006). AMH levels are an indicator of a female's ovarian reserve,
and the number of oocytes with high AMH levels reflect a pro-
longed fertility window (Santoro, 2017). Upon UVB treatment,
we found significant upregulation of the expression of AMH
and AMHR2, which encode the AMH receptor, in the ovaries of
female mice (Figure 3F), suggesting an increase in the pool of
growing follicles. Altogether, our data indicate that UVB treat-
ment of female mice enhances their estrous cycle, gonadotropin
secretion, follicle growth, and sex-steroid synthesis.
p53 modulates UVB-mediated sexual behavior and
ovarian changes
Skin interacts with solar/UVB light and has been suggested to
result in production and release of hormones (Fell et al., 2014;
Skobowiat and Slominski, 2015). Therefore, we reasoned that
the skin plays a role in hormone-related social, sexual, and
reproductive behavior in response to solar/UVB radiation. To
identify the regulators that drive the sexual behavior and ovarian
changes induced by UVB, we determined the overlap of the up-
stream transcription regulators upregulated in mouse plasma
proteomes upon UVB treatment by Ingenuity pathway analysis
(IPA) (Table S2), with the top 10 UVB-related transcription factors
identified by GeneCards and the list of skin regulators involved in
UVB response identified by GeneCards. The overlap of these
three lists indicated that p53 is a potential regulator (Figure 4A;
Table S3). Furthermore, p53 target genes, identified by IPA,
are significantly enriched in biological processes involved in
behavior and reproduction (Figure S4A), which is in line with
Table 1. Within-group differences in passionate love
Males
Females
T1
T2
Z
T1
T2
Z
Median Range Median Range
Median Range Median Range
Obsessive thoughts on __ a
3
1-7
5
2-7
À1.63 b * 2
1-8
3
1-6
À0.65 b
Rather be with __ than anyone else
8
4-9
8
6-9
À0.96 b
6.5
1-9
7.5
1-9
À0.95 b
Yearn to know everything on __
7
4-9
8
6-9
À2.06 b * 5
1-9
5
2-9
À0.86 b
Endless appetite for affection from __
7
3-9
8
5-9
À1.71 b * 4.5
1-9
5
1-9
À1.20 b
__ is the perfect romantic partner
8
4-9
8
1-9
À0.14 b
7
2-9
8
3-9
À2.00 b *
Sense body responding when __ touches 9
3-9
8
1-9
À0.32 b
7
2-9
8.5
3-9
À1.73 b *
Possess a powerful attraction to __
8
4-9
7
1-9
À1.89 c * 7
2-9
7
2-9
À0.32 b
*p < 0.05; **p < 0.01.
a
__, name of the person whom the participant loved most passionately.
b
Based on negative ranks.
c
Based on positive ranks.
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+ our observations. Therefore, we hypothesized that the skin p53
modulates sexual behavior via a skin-brain-gonadal axis.
To test whether the UVB-induced sexual behavior changes we
observed are p53 dependent, we crossed mice that express Cre
specifically in keratinocytes (under the K14 promotor) with p53
floxed mice (Marino et al., 2000) to generate a conditional p53
knockout in epidermal keratinocytes (p53 flox/flox K14-Cre +/+ ),
referred to here as p53-KO mice (Fell et al., 2014); wild-type
p53 littermates (p53 flox/flox K14-Cre À/À ), referred to here as
p53-WT, were used as controls (Figure S4B). We treated the
p53-KO and p53-WT mice daily with UVB in a dose of 50 mJ/
cm 2 . There was a significant decrease in the levels of p53 in
the whole skin of mice, as shown at protein level (Figure S4C)
and at mRNA levels for p53 and its downstream target p21 in
the skin (Figure S4D). These results validated the efficiency of
the knockout of p53 in epidermal keratinocytes. Consistent
with the known role of p53 in the pigmentation response (Mal-
cov-Brog et al., 2018), there was no increase in skin pigmenta-
tion in the p53-KO mice after 5 weeks of UVB treatment (Fig-
ure S4E). There were no differences in body weight between
these mice before and after treatment (Figure S4F).
No increases in the circulating levels of GnRH, LH, or FSH
were detected upon UVB treatment in the p53-KO females (Fig-
ure 4B). Moreover, no changes in ovary size, weight, or histology
were noted in UVB-treated p53-KO females compared with
mock-treated p53-KO females (Figure 4C). Furthermore, there
were no differences in the levels of mRNAs encoding steroido-
genic hormone receptors (PGR, AR, ESR1, and ESR2) (Fig-
ure 4D), in the expression level of the enzymes related to each
of these receptors in the ovaries (Figure S4G) or in the expression
of AMH and AMHR (Figure 4E) when UVB-treated and mock-
treated control p53-KO females were compared. These data
support our hypothesis that skin p53 drives the changes in
the ovaries and mediates sex-steroid induction upon UVB
treatment.
To evaluate how skin p53 influences sexual behavior, UVB-
treated and mock-treated control p53-WT and p53-KO mice
were subjected to the mating test (Figure 1B). All females were
in the estrus/proestrus stage. We found no difference in the ano-
genital sniffing behavior of p53-WT and p53-KO males toward
UVB-treated or mock-treated p53-WT females (Figure 4F, left
panel). In contrast, the amount of sniffing toward the UVB-
treated p53-KO females was significantly reduced compared
with that toward the mock-treated p53-KO females and UVB-
treated p53-WT females (Figure 4F, left panel). Furthermore,
we found a significant increase in the sniffing behavior of females
(both p53-WT and p53-KO) toward UVB-treated p53-WT males
compared with mock-treated control p53-WT males, but no
enhancement of sniffing behavior was observed toward the
UVB-treated p53-KO males (Figure 4F, right panel). This sug-
gests that UVB treatment induces a male mouse odor cue that
depends on skin p53.
Both p53-WT and p53-KO males exhibited significant
enhancement of facial and genital grooming in the presence of
a UVB-treated p53-WT female compared with a control p53-
WT female, whereas this UVB effect did not occur in the pres-
ence of the p53-KO females (Figure 4G, left panel). Furthermore,
we noted an increase in the grooming behavior of females (both
p53-WT and p53-KO) in the presence of UVB-treated p53-WT
males compared with control p53-WT males, a feature not
A
D
E
F
B
C
Figure 3. Daily UVB treatment increases estrus incidence and the number of growing follicles
(A) Percentage total of the proestrus/estrus stage (estrus) and metestrus/diestrus stage (diestrus) of the control or UVB-treated female estrous cycle.
(B) Length of the estrous cycle in a control or UVB-treated female.
(C) Plasma levels of GnRH (n = 3), FSH (n = 5), and LH (n = 3) of control or UVB-treated female mice.
(D) Representative photograph of the ovaries (left), the weight of ovaries in milligrams (middle), and representative H&E staining of female mice ovaries after
8 weeks of control or UVB treatment (right; scale bar, 500 mm).
(E) Relative mRNA expression from ovary section genes involved in female steroidogenesis after an 8-week control or UVB treatment.
(F) Relative expression of AMH and AMHR from an ovary section after an 8-week control or UVB treatment.
Data are presented as means ± SEM, n = 4 (A, B, E, and F), n = 3 (D). For data analysis, a two-tailed, unpaired Student's t test was performed. *p < 0.05; **p < 0.01;
***p < 0.001; ns, not statistically significant
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+ A
C
F
H
I
J
G
D
E
B
Figure 4. p53 modulates UVB treatment-mediated sexual behavior and ovarian changes
(A) Overlap of predicted upstream regulators from mouse upon UVB treatment of 8 weeks with the top 10 UVB-related transcription factors (GeneCards) and with
skin regulators involved in UVB response (GeneCards).
(B) Plasma levels of GnRH (n = 3), FSH (n = 5), and LH (n = 3) in p53-KO females.
(C) Photograph of representative ovaries (left), weight in milligrams (mg) of ovaries (middle), and representative H&E staining (right; scale bar, 500 mm) from p53-
KO females ovaries.
(D) Relative mRNA expression levels of genes involved in steroidogenesis from p53-KO female ovaries.
(E) Relative mRNA expression levels of AMH and AMHR from p53-KO female ovaries.
(F) Total number of anogenital sniffing events by males (control-or UVB-treated p53-WT and p53-KO) toward control-or UVB-treated p53-WT or p53-KO females
(left). Total number of anogenital sniffing events by females (control-or UVB-treated p53-WT and p53-KO) toward control-or UVB-treated p53-WT or p53-KO
males (right).
(G) Total time spent self-grooming by males (control-or UVB-treated p53-WT and p53-KO) in the presence of control-or UVB-treated p53-WT or p53-KO females
(left panel). Total time spent self-grooming by females (control-or UVB-treated p53-WT and p53-KO) in the presence of control-or UVB-treated p53-WT or p53-
KO males (right panel).
(H) Total number of intromissions (left) and total duration of intromissions (right) by males (control-or UVB-treated p53-WT and p53-KO) with control-or UVB-
treated p53-WT or p53-KO females.
(I) UVB-or control-treated p53-WT or p53-KO females' lordosis quotient with control-and UVB-treated p53-WT or p53-KO males.
(legend continued on next page)
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+ observed in the presence of p53-KO males (Figure 4G, right
panel). The total number and duration of successful intromis-
sions by males (both p53-WT and p53-KO) on UVB-treated
p53-WT females were significantly higher than on control p53-
WT females, whereas no change was observed toward p53-
KO females (Figure 4H).
The lordotic response of a female was significantly higher in
UVB-treated p53-WT mice than in mock-treated control p53-
WT mice; there was no difference in p53-KO females (Figure 4I).
We also found that UVB-treated p53-WT males have signifi-
cantly higher levels of testosterone in their plasma than do
mock-treated p53-WT males and that this UVB effect was abol-
ished in p53-KO males (Figure S4H, left panel). No change was
observed in female testosterone levels upon UVB treatment (Fig-
ure S4H, right panel). These observations support our finding
that testosterone appears to be influenced by UVB-induced
skin p53. These data indicate that the enhancement of female
and male attractiveness to the opposite sex induced by UVB
treatment depends on p53 in the skin.
To further explore the mechanism by which p53 controls sex-
ual behavior upon UVB treatment, we overlapped two datasets,
p53 binding genes (identified using chromatin immunoprecipita-
tion sequencing [ChIP-seq]) (Nguyen et al., 2018) and UVB-
affected keratinocyte genes (identified using GeneCards)
(Figure 4J). To identify the downstream targets of p53 (i.e.,
UVB-induced keratinocyte genes bound by p53), we overlapped
these genes with genes known to affect the hypothalamus (Kang
et al., 2000; McCann et al., 2003; Ray et al., 1996; Schmidt et al.,
1995; Slominski et al., 2012), genes known to affect the pituitary
hypothalamus-pituitary-gonadal (HPG) and HPA axes (Rawin-
draraj et al., 2019), or genes known to affect the gonadal HPG
axis (Figure 4J). We found six overlapping genes that affect the
hypothalamus: IL6, LIF, IL-1b, NOS, Leptin, and LEP-R (Fig-
ure 4J; Table S4). IL-6 and LIF are known to increase the expres-
sion of POMC to enhance LH, FSH, and ACTH effects on the
downstream gonads and adrenal gland (Chida et al., 2005; Ray
et al., 1996). Interleukin (IL)-1b increases the expression of
CRH and GnRH from the hypothalamus (Kang et al., 2000;
Schmidt et al., 1995). NOS increases luteinizing hormone
releasing hormone (LHRH) levels (McCann et al., 2003), and
vice versa (Garrel et al., 1998). LHRH controls female lordosis
and male sexual behavior (McCann et al., 2003). Moreover, we
found one overlapping gene, CRH, that affects the pituitary.
The CRH protein is released from the skin upon UVB treatment
and acts on the local and central HPA axis (Skobowiat and Slo-
minski, 2015; Skobowiat et al., 2011).
We observed significant upregulation of IL1B, CRH, and IL6
expression in UVB-treated p53-WT males, whereas the expres-
sion of these genes remained unchanged in the UVB-treated
p53-KO males (Figure S4I, upper panel). Furthermore, we
observed significant upregulation of IL1B, IL6, LIF, and CRH,
as expected based on previous work (Skobowiat and Slominski,
2015), and of NOS1 in UVB-treated p53-WT females, but not in
UVB-treated p53-KO females, compared with mock-treated
controls (Figure S4I, lower panel). Altogether, these findings
demonstrate that p53 expressed in epidermal keratinocytes reg-
ulates sexual behavior and ovarian changes through a skin-
brain-gonadal axis.
Solar exposure enhances human sex-related steroids
To examine the relevance of mouse data to humans, we re-
cruited volunteers (n = 9 men, n = 10 women; age 18-55 years)
who were asked to avoid sun exposure for 2 days and then
spend approximately 25 min in the sun on a bright sunny midday;
this resulted in a dose of approximately 2,000 mJ/cm 2 UV radia-
tion (measured using a UVX radiometer). Blood samples were
collected on the day before sun exposure and approximately
the same time on the day of sun exposure. Similar to the mouse
proteome data (Figure 1A), we found a significant positive activa-
tion Z score for upstream regulators b-estradiol, progesterone,
testosterone, and estrogen in men (Figure 5A, left panel) and
for estrogen, progesterone, and testosterone in women following
solar exposure compared with the control day before exposure
(Figure 5A, right panel).
Furthermore, we analyzed the testosterone levels of men aged
21-25 years (n = 13,086) from the Maccabi Health Service (Cho-
dick et al., 2020) and observed a significant peak in total testos-
terone level during the summer (July), indicative of testosterone
seasonal variation (Figure 5B). This is in line with a previous
report that testosterone levels increase in men following UV radi-
ation (Myerson and Neustadt, 1939). Finally, to determine how
pigment phenotype affects these solar responses, we retrieved
testosterone-level data of men aged 20-50 years from the Clalit
Health Services data-sharing platform (Israel) and divided them
into two groups based on the amount of ultraviolet radiation
(UVR) in their country of origin. Testosterone levels were signifi-
cantly higher (n = 1,607, p = 0.004) in men originating from coun-
tries with low UVR (UV < 2,500 J/m 2 ) compared with individuals
who originated from countries with high UVR (UV R 4,500 J/m 2 )
during summer months (May-September) for all body mass in-
dex values (Figure S5, left panel). No significant differences
(n = 2,309, p = 0.499) were observed during the winter months
(October-April) (Figure S5, right panel). Because skin coloration
is strongly related to levels of UVR in a given country (Chaplin,
2004), our data support the involvement of the skin reaction to
UVR in regulation of sexual behavior. Altogether, our data sug-
gest enhancement of sex steroids upon solar exposure and
demonstrate a positive correlation between solar exposure and
testosterone levels in human males.
DISCUSSION
Fitness is defined by the individual's reproductive success (Zim-
mer et al., 2016). Conception must be timed so that offspring are
born when they have the highest chances of survival and repro-
duction. This is likely the reason for seasonality in birth rates in
(J) Overlap between p53 DNA binding-based ChIP-seq (Nguyen et al., 2018), UVB-affected keratinocyte genes (GeneCards), and genes affecting hypothalamic,
pituitary, and gonad expression (Kang et al., 2000; McCann et al., 2003; Ray et al., 1996; Schmidt et al., 1995; Slominski et al., 2012).
Data are presented as means ± SEM, n = 4 (C-E), n = 3 (F-I). For data analysis, a two-tailed, unpaired Student's t test (B-E) or two-way ANOVA (F-I) was
performed. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.
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+ humans. There is a unimodal spring-summer (end of April-May)
peak in conceptions in most of Europe and a strongly bimodal
distribution in North America, with peaks in spring and autumn
(Roenneberg, 2004). Photoperiod and temperature have been
suggested to be the major environmental factors affecting this
seasonality (Roenneberg and Aschoff, 1990a, 1990b). Because
we showed a direct response to UVB, the source is not
the endogenous circannual clock, which generates seasonal
changes in physiology and behavior in the absence of environ-
mental cues (Scanes, 2015). Therefore, UVB may serve as a
backup mechanism, ensuring optimal reproduction timing and
direct influence on fitness. It is interesting that industrialization,
which shifted work from outdoors to indoors with eternal summer
conditions, characterized by a long photoperiod and mild tem-
peratures with almost no seasonality (Stevenson et al., 2015),
happened in parallel to an amplitude reduction in peaks in human
conceptions observed in today's industrialized nations (Foster
and Roenneberg, 2008; Roenneberg, 2004). It is possible that
the reduced exposure to UVB contributed to this change.
Our data suggest skin-brain crosstalk, in which the skin acts as a
dermato-endocrine organ, releasing hormones that affect the hy-
pothalamus-pituitary-gonadal axis. The mechanism of action may
be similar to that of b-endorphins (Fell et al., 2014) and CRH (Sko-
bowiat and Slominski, 2015), which are released from the skin and
affect the opioid system and axis, respectively, and/or to nerve fi-
bers, the immune system, or as-yet-unknown regulators. Because
eyes of mice and of human volunteers were not covered, we
cannot exclude the possibility that solar/UV radiation to the eye
affected the observed sexual behavior. UVB exposure via the
eye activates the hypothalamopituitary proopiomelanocortin sys-
tem, which is upstream of the HPA and HPG axes (Hiramoto et al.,
2003). However, when we depleted p53 from skin keratinocytes,
we observed suppression of the UVB-induced sexual behavior
traits, as well as a significant decrease in the hormones of the
HPG axis, which favor our hypothesis that in addition to the
eyes, the skin has an active part in regulating sexuality.
Vitamin D synthesis is affected by UV absorption, which de-
pends on the skin tone of the individual (Webb et al., 2018; Ri-
chard et al., 2017); thus, individuals with the Fitzpatrick V (FST
V) skin type must receive a greater UV dose per unit time to syn-
thesize vitamin D compared with individuals with lighter skin
(Webb et al., 2018). In addition to the seasonality of birth rates,
conception rates, ovulation, socioeconomic status, and age
group as fertility effectors (Bobak and Gjonca, 2001; Lam
et al., 1994; Stolwijk et al., 1996), we propose that pigment
phenotype might play a role in regulating the skin-brain-gonadal
axis, thereby regulating sexual behavior.
All skin layers are innervated by sensory, sympathetic, and
parasympathetic nerve fibers that relay signals to the brain and
receive cues from it (Slominski et al., 2012). Sensory signals
from the skin to the brain include temperature, touch, pain,
stretch, itch, and vibration; they are sensed by skin receptors
that transfer the stimuli via nerve fibers directly to the brain
(Roosterman et al., 2006). Signals from the brain to the skin
include thermoregulation, sweat-gland function, blood flow,
adnexal functions (Roosterman et al., 2006), and hair graying
(Zhang et al., 2020).
Another mode of skin-brain crosstalk involves the combined
neural signals from the preoptic hypothalamus and peripheral
nerves that together trigger eccrine sweat glands (Stowers and
Liberles, 2016). In a response that is sex dependent (Stowers
and Liberles, 2016), pheromones are mainly secreted in axillary
sweat, which contains the odorous 16-androstenes (Verhaeghe
et al., 2013). Axillary secretions originate from apocrine odor
glands, eccrine sweat glands, and sebaceous glands located
in the skin (Verhaeghe et al., 2013). Pheromones are inducers
of communication and behavioral responses, including sexuality
and mating (Ferrero et al., 2013; Roberts et al., 2010; Stowers
and Liberles, 2016; Verhaeghe et al., 2013). Although the neural
triggers for pheromone synthesis and secretion are poorly un-
derstood, it has been established that eccrine sweat glands
are controlled by hypothalamus cues (Stowers and Liberles,
2016). Therefore, we cannot exclude the possibility that part of
the observed effect is mediated by pheromones: UVB radiation
may alter hypothalamus activity or directly affect axillary secre-
tion. Both possibilities should be further investigated. In line
with this, knock down of p53 has been shown to trigger the
DNA damage response in skin keratinocytes that results in
peeling of these cells (Farmer et al., 1992; Fields and Jang,
1990; Levine et al., 2006; de Pedro et al., 2018). This by itself
might be a trigger of attractiveness and should be investigated
in the future.
It is worth putting in mind that species differences play an
essential role in activating mating behavior via the circulating
sex steroids. For example, female rhesus monkeys continue to
mate with males for weeks after ovariectomy, wherein there is
withdrawal of estradiol (Baum et al., 1977). In contrast, ovariec-
tomized female mice cease to mate within few days of the
A
B
Figure 5. Solar exposure enhances human
sex-related steroids
(A) Predicted upstream regulators of the differential
blood plasma proteins from humans following a
single solar exposure (2,000 mJ/cm 2 UV) (n = 5
humans for each condition).
(B) Total testosterone levels of men aged 21-25
years (n = 13,086). Plot depicts monthly means on
a cubic spline of calendar month (January-
December; 2 degrees of freedom).
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+ procedure and resume it only upon receiving an injection of
estradiol benzoate followed by progesterone (Edwards, 1971).
Likewise, male rhesus monkeys typically continue mating for
many months after castration (Phoenix et al., 1973), whereas
most strains of male mice stop mating within 3-4 weeks after
castration (Thompson et al., 1976).
Our quantitative questionnaire results show that both sexes
have a tendency toward higher levels of passion following UV
treatment. Passion takes two forms, emotional and sexual.
UVB radiation affected different components of passion in men
than in women. UVB-treated women scored higher on questions
about physical arousal that related more to sexual passion and
idealizing the connection, whereas men scored higher on the
cognitive dimension of passion, which involves obsessive
thoughts about the partner and wanting to know more about
her. The questionnaire we used measured romantic passion,
rather than physiological/sexual passion, due to institutional re-
view board (IRB) ethical concerns regarding sensitive sexually
oriented questions. Future studies on this topic should address
physiological arousal more directly and should be geared toward
the precise identification of the different effects of UVB on the
sexual behavior of men and women.
STAR+METHODS
+
+ Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead contact
B Materials availability
B Data and code availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Mouse models and habituation
B Human cohort
B Human cohort testosterone study
B Human questionnaire
d METHOD DETAILS
B UV treatment
B Melanin intensity quantification
B Mouse blood draw
B Human cohort Solar-exposure study
B Human blood draw
B Proteolysis and mass spectrometry
B Proteomic analysis
B ELISA
B Mating test
B Ultrasonic vocalization
B Elevated plus maze test
B Three-chamber test
B Male subject and two female stimuli
B Female subject and two male stimuli
B Female subject and two female stimuli
B Female subject, a male stimulus, and a novel-object
stimulus
B Vaginal smears for estrous cycle evaluation
B RNA purification and qRT-PCT
B Histology
B Genotyping
B Immunoblotting
d QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
celrep.2021.109579.
+
+ ACKNOWLEDGMENTS
The authors thank Prof. Eli Pikarsky (The Hebrew University of Jerusalem,
Israel) for the gift of the p53-floxed mice and Prof. Itai Ben-Porath for the gift
of K14 CRE mice (The Hebrew University of Jerusalem, Israel). C.L. thanks
Prof. Yossi Yovel (Department of Zoology, Faculty of Life Sciences, Tel Aviv
University) for providing the recording instrument and analysis and Prof. Uri
Alon, Prof. Noam Sobel (Weizmann Institute of Science, Israel) for useful dis-
cussions, and Yuval and Omer Levy for infinite joy. C.L. acknowledges grant
support from the European Research Council (ERC) under the European
Union's Horizon 2020 research and innovation program (grant 726225) and
the Israel Science Foundation (ISF) (grant 2017/20). R. Parikh is the recipient
of a CBRC 2020 travel grant and 3 rd Esther and Zvi Weinstat Graduate Student
Award, 2021, and would like to thank her family for their love and support.
Research in A.W.'s lab is supported in part by the ISF (grant 1781/16), Israel
Ministry of Science and Technology (grants 3-13608 and 84/19), and EPM Inc.
+
+ AUTHOR CONTRIBUTIONS
R. Parikh, conceptualization, methodology, validation, formal analysis, inves-
tigation, writing -original draft, and visualization. E.S., conceptualization and
formal analysis. S.P., methodology and validation (human cohort study).
K.M., D.L., and O.K.-M., formal analysis (human questionnaire). L.B. and
A.W., methodology and resources. S.T., G.S., S.M., T.Z., H.K., and G.C.,
investigation and formal analysis (human testosterone data). M.M., formal
analysis (mass spectrometry). A.B., resources (ultrasonic vocalization).
N.K.-S., writing -review & editing. H.B.-J., formal analysis (ovary cross sec-
tions). D.B.-Y., H.A., and T.G., writing -review & editing. H.M. and M.P., re-
sources (human questionnaire data from phototherapy clinic). Y.L., R.B., and
E.N., visualization. Y.G., formal analysis (human epidemiological data). R.H.,
investigation. R.S., methodology and writing -review & editing (ovarian exper-
iments). T.K., visualization (sexual behavioral data). R. Percik, investigation
and writing -review & editing. C.L., conceptualization, methodology, writing -
original draft, visualization, supervision, project administration, and funding
acquisition. All authors reviewed the final draft and approved it.
DECLARATION OF INTERESTS
The authors declare no competing interests.
+
+ Received: November 9, 2020
Revised: May 12, 2021
Accepted: July 30, 2021
Published: August 24, 2021
+
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+ STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Antibodies
Anti-p53 antibody [PAb 240]
abcam
Cat #ab26; RRID:AB_303198
Anti-mouse IgG, HRP-linked
Cell Signaling Technology
Cat #7076; RRID:AB_330924
Anti-actin antibody
Sigma Aldrich
Cat #A2066; RRID:AB_476693
Anti-Rabbit IgG, HRP-linked
abcam
Cat #ab6721; RRID:AB_955447
Biological samples
Human blood plasma
Separated from blood samples in our lab
(This study)
Approval of Tel Aviv University Ethics
Committee
Mouse tissue samples
Tissues obtained after sacrifice in our lab
(This study)
IACUC permit#10-16-078
Chemicals
TRIzol
Invitrogen
Cat# 15596026
Choloroform
Bio-Lab
Cat# 3082301
2-Propanol
Sigma Aldrich
Cat #278475
Methanol
Bio-Lab
Cat #136806
Paraformaldehyde, 16%
Electron Microscopy Sciences
Cat #30525-89-4
Difco Skim Milk
Avantor Sciences
Cat #90002-594
Hematoxylin Solution, Harris Modified
Sigma-Aldrich
Cat #HHS16
Eosin Y solution
Sigma-Aldrich
Cat #HT110232
DPX Mountant for histology
Sigma-Aldrich
Cat #06522
Ketamine hydrochloride
Bremer Pharma GMBH
N/A
SEDAXYLAN
Eurovet animal health
N/A
Isoflurane, USP TerrellTM
Piramal Critical care, Inc.
N/A
Critical commercial assays
Mouse LH(Luteinizing Hormone)
Wuhan Fine Biotech Co., Ltd
Cat # EM1188
Mouse GnRH(Gonadotropin Releasing
Hormone)
Wuhan Fine Biotech Co., Ltd
Cat # EM1616-CM
Mouse FSH(Follicle-stimulating hormone)
Wuhan Fine Biotech Co., Ltd
Cat # EM1035
Testosterone
abcam
Cat # ab108666
Deposited data
Raw Mass Spectrometry Data Files
This study
The mass spectrometry proteomics data
have been deposited to the
ProteomeXchange Consortium via the
PRIDE (Perez-Riverol et al., 2019) partner
repository with the dataset identifier
Database:PXD025973
Experimental models: Organisms/strains
Mouse: C57BL/6J
Envigo
N/A
Mouse: C57BL/6J-p53flx/flx
A gift from Dr. Eli Pikarsky, The Hebrew
University of Jerusalem, Israel
N/A
Mouse: C57BL/6J-k14Cre+/À
A gift from Dr. Ittai Ben-Porath, The Hebrew
University of Jerusalem, Israel
N/A
Mouse: p53flx/flxK14CreÀ/À (Control
littermates)
+
+ Bred & genotyped in our lab (This study)
N/A
Mouse: p53flx/flxK14Cre+/+ (p53-KO
littermates)
Bred & genotyped in our lab (This study)
N/A
(Continued on next page)
e1 Cell Reports 36, 109579, August 24, 2021
Article
+
+ ll
OPEN ACCESS
+
+ RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Carmit
Levy (carmitlevy@post.tau.ac.il).
Materials availability
All in-house generated mouse strains generated for this study are available from the Lead Contact with a completed Materials Trans-
fer Agreement.
Data and code availability
d All original datasets has been deposited at the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019) partner
repository and is publicly available as of the date of publication: Database: PXD025973.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Mouse models and habituation
Unless stated otherwise, we used 5-to 6-week-old C57BL6 male and female mice (Envigo) for experiments. The p53 floxed male and
female mice were a gift from Dr. Eli Pikarsky (The Hebrew University of Jerusalem, Israel). Dr. Ittai Ben-Porath (The Hebrew University
Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Oligonucleotides
See Table S5 for RT-qPCR primers
Intergrated DNA Technologies (IDT)
N/A
See Table S5 for genotyping primers
Jackson laboratories
N/A
Software and algorithms
EthoVision XT 7
Noldus information technology
https://www.noldus.com/ethovision-xt;
RRID:SCR_000441
UltraVox XT system
Noldus information technology; version 3.1
N/A
SPSS Statistics
IBM; version 25.0
https://www.ibm.com/uk-en/analytics/
spss-statistics-software;
RRID:SCR_002865
Prism
Graphpad; version 8
http://www.graphpad.com/; RRID:
SCR_002798
Ingenuity Pathway Analysis
QIAGEN
https://digitalinsights.qiagen.com/;
RRID:SCR_008653
Proteome Discoverer 1.4
Thermo Fisher Scientific
https://www.thermofisher.com/order/
catalog/product/
IQLAAEGABSFAKJMAUH;
RRID:SCR_014477
MaxQuant
Cox and Mann, 2008; version 1.5.2.8
https://www.biochem.mpg.de/5111795/
maxquant/; MaxQuant, RRID:SCR_014485
Perseus software
Cox and Mann, 2008; version 1.6.10.43
https://www.biochem.mpg.de/5111810/
perseus
BioMart
Ensembl
https://www.ensembl.org/biomart/
martview/
9b0f3136bd9d6999b66c3a766729a6ae
Other
Virusolveâ+
Amity International
N/A
Veet hair removal cream
Reckitt
N/A
UVB Lamp (Model: XX-15MR Bench Lamp,
302 nm)
+
+ Analytik Jena US
Cat # 95-0042-15
UVX radiometer
Analytik Jena US
Cat #97-0015-02
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+ of Jerusalem, Israel) provided male and female mice in which the K14 promoter directs expression of Cre recombinase. We
confirmed the p53 knockout in keratinocytes by genotyping, qRT-PCR, and ear pigmentation analysis. Male and female mice
were habituated for at least 1 week to their new environment under a standard 12-h light/dark cycle and the conditions of constant
temperature (24 ± 1 C) and humidity (50 ± 5%) with access to food and water ad libitum prior to experimentation. The guidelines of
the Tel Aviv University Institutional Animal Care and Use Committee (IACUC) (10-16-078) were followed.
Human cohort
The human cohort of 9 men and 10 women (18-55 year of age) were recruited through convenience sampling at the Sackler School of
Medicine (Tel Aviv University, Israel). All provided written consent. The approval of the University's Ethics Committee was obtained
prior to the study.
Human cohort testosterone study
The human cohort study from Clalit was approved by Kaplan Medical Center Institutional Review Board. Total testosterone data and
related medical data were retrieved for all males aged 20 to 50 years old in Israel Central and Jerusalem districts from Clalit Health
Services using Clalit Secure Data Sharing Platform powered by MDClone (https://www.mdclone.com), and subjects with testos-
terone modifying medical conditions were excluded. Subjects were classified into four groups categorized according to average
UV radiation (UVR) in their country of origin (https://apps.who.int/gho/data/node.main.164?lang=en) by using WHO map (https://
www.who.int/gho/phe/ultraviolet_radiation/exposure/en/). Only men from countries with low UVR (UV < 2500 J/m 2 ) and high UVR
(UV R 4500 J/m 2 ) were considered for this study. Multivariate analysis of variance models was used to estimate the effect of season-
ality and country of origin on total testosterone levels. Models were assessed separately for the summer (May-September) and winter
(October-April) months and were adjusted for age and body mass index. The analysis of human cohort testosterone level data of men
aged 21-25 years old (n = 13,086) from the Maccabi Health Services, was done as previously described (Chodick et al., 2020).
Human questionnaire
For the quantitative longitudinal study, 19 subjects aged 23-73 (mean M = 45.89, SD = 15.22), 47.4% male and 52.6% female, with
skin conditions including vitiligo, eczema, and psoriasis were recruited by convenience sampling from two Israeli hospitals (Assuta
Hospital; Helsinki ethical approval 0063-17-ASMC 17 and Tel Aviv Sourasky Medical Center; Helsinki ethical approval 0151-17-TLV).
Of all the participants, 11.8% were single, 64.7% were married, and 23.5% were divorced, 35.3% had no children, and 64.7% had 1-
3 children. Data were collected through self-reported questionnaires at two time points, before exposing the participants to a UVB
treatment (T1), and approximately a month after the treatment (T2). During this period, patients were given full body (except their gen-
itals, eyes, and head) narrow band UVB exposure (0.1-2.5 J/cm) (Waldmann UV7002 UVB instrument; UV lamp (UVB) 42 x TL 01 120
W) 2-3 times a week, 10-12 UVB exposures in total. The hospitals IRB approved the study, and all participants signed informed con-
sent forms.
The PLS (Hatfield and Sprecher, 1986) was developed to measure passionate love in intimate relationships and focuses on an
intense longing for union with the other. We used a Hebrew translation of the PLS for our study. We used seven items from the short
version of the PLS relevant to our study: cognitive components of passion (intrusive thinking about the partner, idealization of the
other and the relationship); emotional components of passion (attraction, longing for reciprocity, and physiological arousal). From
the short version of the PLS, we excluded items that did not directly examine the person's passion, such as actions taken to deter-
mine the other person's feelings. We included items related to cognitive components of passion (e.g., intrusive thinking about the
other and idealization of the other and the relationship) and emotional components of passion (e.g., attraction, longing for reciprocity,
and physiological arousal). Each item was rated on a 9-point Likert-scale (1 = not at all true; 9 = definitely true), with a higher score
representing more passionate love.
The aggression questionnaire (Buss and Perry, 1992) was developed to measure four aspects of aggressiveness: physical aggres-
sion, verbal aggression, anger, and hostility. A questionnaire translated into Hebrew was used to assess two factors relevant to our
study: physical aggression (nine items; e.g., 'I get into fights a little more than the average person') and verbal aggression (five items;
e.g., 'I can't help getting into arguments when people disagree with me'). Each factor was rated on a 5-point Likert-scale (1 =
extremely uncharacteristic of me; 5 = extremely characteristic of me) and calculated by summing the answers (after re-coding
one item of physical aggression), with higher scores representing more aggression. The reliability of the scale of the original ques-
tionnaire, measured through Cronbach's alpha, was 0.85 for physical aggression and 0.72 for verbal aggression. In our study, the
Cronbach's Alpha was 0.84 in T1 and 0.77 in T2 for physical aggression (after deleting one item); and 0.81 and 0.79, respectively,
for verbal aggression.
METHOD DETAILS
UV treatment
Mice were kept in a reverse 12-h dark/light cycle (red light) and were shaved on the dorsal side in an area of approximately $60% of
the skin, excluding the ears, tail and paw regions, where hair growth is not prevalent, and were treated with depilatory cream (Veet).
Mice were exposed to daily UVB treatment of 50 mJ/cm 2 in the reverse light setting with a XX-15 stand equipped with 15-W, 302-nm
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+ UVB bulbs (Ultraviolet Products) at approximately the same time of their day (9:00 -10:00 AM) in a custom transparent plexiglass
chamber that allowed freedom of movement during the treatment. To exclude the possibility of not crawling on top of each other
and hindering the UVB skin exposure, one mouse per chamber at a time were UVB exposed (Nghiem et al., 2002). The UV emission
was measured using a UVX radiometer (Ultraviolet Products, 280 nm -320 nm) equipped with a UVB measuring head. The calibration
was done for the delivered doses of UVB emission. For the mock (control) treatment, the animals were placed in the chamber but the
UVB lamp was not turned on, which ensured that the UVB-exposed and control mice experienced the same stress conditions. After
each treatment, the container was cleaned using Virusolve (Amity International) to avoid cross-contamination of odors. Control and
UVB exposed mice used for the study were of similar age and underwent similar experimental protocols in order to exclude the pos-
sibility of hair-cycle differences as well as stress related interferences in the behavioral experiment.
Melanin intensity quantification
The reflective colorimetric measurements were performed with a DSM II Color Meter (Cortex Technology), which gives the level of
pigmentation. A white standard background (provided by the manufacturer) was used for the calibration before every measurement.
All the measurements were performed on the same background with no UV light. The tail and ear pigmentation were measured at the
end of the 8-week UVB (50 mJ/cm 2 ) or mock (control) treatment series and at the end of 5-week period for p53-KO and p53-WT an-
imals, and pigmentation intensity was scored relative to control (UV/Mock -melanin pigmentation intensity).
Mouse blood draw
Mice were anesthetized by the intraperitoneal injection of ketamine (100 mg/kg body weight; Bremer Pharma GMBH) and xylazine
(10 mg/kg body weight; Eurovet Animal Health BV), and blood was drawn from the heart with a 23G needle (KDL) at approximately the
same time of the day (between 10:00 -13:00 Israel Standard Time) for all samples. The drawn blood was transferred to EDTA-coated
microvette tubes (BD Mictrotainer) and immediately placed on ice, followed by centrifugation at 448 g for 10 min at 4 C to separate
the plasma fraction, which was then aliquoted and stored at À80 C until further use.
Human cohort Solar-exposure study
All subjects were asked to avoid or minimize their solar exposure during the 2 days prior to the experiment and were requested to
wear long-sleeved clothes on those days. On the day of the experiment (between 16:00-18:00, Israel standard time), 10 cc of intra-
venous blood was drawn by a certified physician. On the next day subjects were asked to wear short sleeves/sleeveless shirt and
shorts and be in a non-shaded area in order to expose themselves to 2000 mJ/cm 2 solar UV radiation, as measured by the UVX radi-
ometer (Ultraviolet Products, 280 nm -320 nm), between 11:00-13:00, Israel standard time. The second blood sample was then
drawn later that day, between 16:00-18:00, Israel standard time.
Human blood draw
Venous blood was drawn from the forearm, after disinfection with Alcosept (chlorhexidine gluconate 0.5% W/V and alcohol 70% V/V;
Floris) using a blood-collecting needle set (KDL). Blood was collected in Vacutainerâ tubes (BD Biosciences). The blood was allowed
to clot at room temperature for 15-30 min followed by centrifugation at 2000 g for 10 min at 4 C to separate the serum fraction, which
was then aliquoted and stored at À80 C until further use.
Proteolysis and mass spectrometry
Proteins from plasma of five human volunteers randomly selected from the cohort and from three mice were precipitated with 90%
ethanol at 90 C for 10 min, followed by centrifugation at 11,200 g for 5 min. The resulting supernatant was dried and resuspended in
9 M urea, 400 mM ammonium bicarbonate, reduced with 3 mM DTT (60 C, 30 min), modified with 12 mM iodoacetamide in 400 mM
ammonium bicarbonate (in the dark, at room temperature, 30 min), and digested in 1 M urea, 50 mM ammonium bicarbonate with modi-
fied trypsin (Promega) at a 1:50 enzyme-to-substrate ratio at 37 C for 2 h. The tryptic peptides were desalted using C18 tips (Top tip,
Glygen), dried, and re-suspended in 0.1% formic acid. The peptides were then resolved by reverse-phase chromatography on 0.075 X
180 mm fused silica capillaries (J&W Pharmalab) packed with Reprosil reversed-phase material (Dr Maisch GmbH). The peptides were
eluted with a linear 60-min gradient from 5% to 28%, then a 15-min linear gradient from 28% to 95%, followed by 25 min at 95% aceto-
nitrile with 0.1% formic acid in water, at a flow rate of 0.15 ml/min. Mass spectrometry was performed with a Q Exactive HF mass spec-
trometer (Thermo Fisher Scientific) in a positive mode, using repetitively full MS scan followed by collision induced dissociation of the 18
most dominant ions selected from the first MS scan. The mass spectrometry data from three biological repeats was analyzed using the
MaxQuant software 1.5.2.8 (Cox and Mann, 2008). The data was quantified by label-free analysis using the same software. Statistical
analysis of the identification and quantization results was done using Perseus 1.6.10.43 software (Cox and Mann, 2008).
Proteomic analysis
The proteomic dataset, which included the UniProt identifiers, were converted to gene symbols using BioMart, Ensembl. The gene sym-
bols and absolute value of the log 2 -transformed fold-change were subjected to IPA for the core analysis (QIAGEN). Matching with the
mouse Ingenuity Knowledge Database generated predicted possible upstream and transcription regulators based on the p value and
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+ activation Z-score values, which infers the activation state (increased or decreased). Fisher's right-tailed exact test was used to deter-
mine the probability of upstream analysis over-representation in the dataset. The protein network was built using the string output.
ELISA
Testosterone, LH, FSH, and GnRH levels in mouse plasma were detected and quantified after 8 weeks of UVB (50 mJ/cm 2 ) or control
treatment using the Testosterone Elisa Kit (ab108666, Abcam), LH Elisa kit (EM1188, Wuhan Fine Biotech Co., Ltd.), FSH Elisa kit
(EM1035, Wuhan Fine Biotech Co., Ltd.), and GnRH Elisa Kit (EM1616-CM, Wuhan Fine Biotech Co., Ltd.), according to the manu-
facturers' instructions.
Mating test
Procedure: This test was conducted on sexually naive female and male C57BL6, p53-WT, and p53-KO mice after UVB (50 mJ/cm 2 ) or
mock treatment. The males were individually housed for 24 h in a new cage with sawdust bedding; food and water were provided ad
libitum. Before the start of the mating test, the food and water were removed from the cage, and the females were examined for the
stage of their estrous cycle; they were mated with a male only if they were in the estrus/proestrus stage. In the mating test, a female
was introduced into the cage of the male, where she spent 1 hour, after which she was immediately returned to her cage. All tests
were conducted in a 17 3 25 cm transparent plexiglass chamber placed on a table that allowed videotaping in the ventral view with a
digital camera. The visual data were subsequently analyzed manually for respective behavior parameters. All the experiments took
place in a reverse 12-h light/dark cycle under dim, red lighting. In the case of the p53-WT and p53-KO mice, the mating test was
carried out at the end of the light phase of the standard 12-h light/dark cycle under dim, red lighting, as mating behavior was tested
during the active phase (dark phase) of the mice.
Analysis parameters and criteria: The following parameters were measured as previously described (Haga et al., 2010): number of
anogenital sniffs, grooming behavior, latency and total number of male mounting of the female, intromission latency, duration and
total number of intromissions, female lordosis response, rearing behavior, and number of ejaculations. Anogenital sniffing was
defined as actively reaching out and sniffing the genital regions of a mouse. Reaching out was defined as a mouse trying to stretch
out and sniff the other sex's genitalia. Mouse grooming behavior was scored when the mouse self-groomed its face or body.
Mounting was defined as a failed attempt of the male to climb with both forepaws on the female's back in an attempt to mate. Intro-
mission was defined as a male successfully climbing on the female with its forepaws and making pelvic thrust movements with a
stable frequency for a minimum duration of 5 s. A female's lordosis response was defined as the female standing on all four paws
grounded and elevating the hind region from the floor, creating a lordotic curve of the spine. A female was classified as receptive
only if she exhibited the lordotic posture upon mounting by a male. The lordosis analysis was performed against the total number
of mounting with pelvic thrusts by male mouse, which may or may not include penile intromission (Lordosis quotient = Total number
of female lordosis/total number of male mounts*100) in a single experimental session as previously described (Beach, 1976; Haga
et al., 2010). Rearing behavior was considered a female assuming a defensive upright posture toward a male, with both forepaws
in the air and the back straight and stretched. Ejaculation was defined as the end of the intromission period, when the male, after
ejaculating, would fall on one side and remain in that position for a couple of seconds.
Ultrasonic vocalization
Sexually naive male and female mice were subject to the above-described mating test but in a room suited for recording their ultrasonic
vocalizations. Recordings were obtained using an UltraVox XT system (Noldus Information Technology), which was capable of recording
the full spectrum of sound with a maximum frequency of 160 kHz. Detector outputs were analyzed with UltraVox XT 3.1 software (Noldus
Information Technology). The number of vocalizations, mean dominant frequency, duration of the mice vocalizing with each other was
recorded and scored. A representative spectrogram of the vocalization was extracted using the UltraVox XT 3.1 software.
Elevated plus maze test
The elevated plus maze test was performed after 5 weeks of UVB or control treatment with an apparatus measuring 90.0 cm in height
made of white plexiglass. The maze consisted of four arms in total (two open arms without walls and two enclosed arms with 15.0-cm
high walls). The mice were habituated for 30 min to the experimental room prior to the start of experiment in order to avoid the stress bias
of a new environment. Naive mice, who did not undergo any experimental protocols other than the UVB and control treatments were
used for this study thus giving us the true measure of the test unhindered from the stress due to other behavioral experiments. The con-
trol or UVB-treated mouse was placed in the center of the maze (intersection of the open and closed arms) facing the open arm and was
allowed to move for 7 min in the maze. The mouse behavior was recorded in a digital video camera mounted overhead on the ceiling and
was scored and analyzed using the Ethovision XT software (Noldus Information Technology). The test was conducted in a reverse 12-
hour light/dark condition under the dim red light setting during their active phase cycle. The females were checked for their estrous cycle
by vaginal smears prior to the experiment in order to avoid the bias of the state of the cycle influencing the anxiety parameter. Between
each trial, the maze was cleaned using Virusolve (Amity International) to avoid cross contamination of odors between gender and treat-
ments. The parameters scored included the total time spent giving the cumulative of the time spent either in the open or closed arm and
the frequency giving the number of visits by the mouse either in the open or closed arms of the elevated plus-maze.
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+ Three-chamber test
A white, rectangular, plexiglass chamber was divided into three consecutive compartments, with each of the two outer compart-
ments containing a wire cage. Small openings in the center of the two partitions facilitated movement throughout the chamber,
except into the wire cages. The wire cage limits movement of the stimulus mouse. To habituate the subject mouse to the test cham-
ber, it was placed in the center of the middle chamber with freedom of movement on either side of the compartment for 10 min the day
before the experiment; no stimuli was introduced. On the day of the experiment, the subject and stimulus mice were brought into the
experimental room and habituated to the room for 20 min. Next, the stimulus mice (or novel objects) were placed in the wire cages,
one in each cage. The subject mouse was then placed in the center of the middle compartment and allowed to move freely for 15 min.
The experiments were performed under a reverse 12-h light/dark phase under dim, red lighting. A digital camera mounted overhead
on the ceiling was used to record the mouse's behavior throughout the 15-min session, which was scored using EthoVision XT soft-
ware (Noldus Information Technology). Between each trial, the positions of the stimuli were switched, to avoid a confounding error
(preferred side of the subject mouse), and the chamber and wire cages were cleaned using Virusolve (Amity International), to avoid
cross-contamination of the odors from the subject or the stimulus mice.
The coordinates and time stamps of the subject mouse obtained from a live video feed were translated into a number of parameters
and further visualized by a heatmap generated with EthoVision XT software (Noldus) to detect the subject mouse's location and
movements. We scored the following parameters: latency toward the zone of the cage and near the cage, which are the amounts
of time taken by the subject mouse to move toward a compartment with a stimulus. The total time spent in the zone of the cage
and near the cage represents the cumulative time spent in the compartment with a stimulus. The frequency of visits to the zone
of the cage and near the cage provides the number of times the subject mouse visited a compartment with a stimulus.
In all the following schemes, mice received UVB (50 mJ/cm 2 ) or control treatment for 5 weeks. All the female mice were examined
prior to the test session for their estrous cycle stage, with only those in their estrus/proestrus stage used in the test.
Male subject and two female stimuli
The same stimulus females (one UVB-treated and one control female) were tested twice, once with a control male mouse subject and
once with a UVB-treated male mouse subject, on separate days.
Female subject and two male stimuli
The same stimulus males (one UVB-treated and one control male) were tested twice, once with a control female subject and once
with a UVB-treated female subject, on separate days.
Female subject and two female stimuli
The same stimulus females (one UVB-treated and one control female) were tested twice, once with a control female subject and once
with a UVB-treated female subject, on separate days.
Female subject, a male stimulus, and a novel-object stimulus
The same stimulus male (one set of experiments with a UVB-treated male and the other with a control male) were tested twice, once
with a control female mouse subject and once with a UVB-treated female mouse subject, on separate days. The novel object (plastic
block) was cleaned using Virusolve (Amity International) between each trial.
Vaginal smears for estrous cycle evaluation
To examine the estrous cycle, a gentle lavage technique was used to collect vaginal smears from each female mouse daily, approx-
imately at the same time, for a period of 45 days, starting when the females were 4 weeks old. In the first 2 weeks of smear collection,
females were not subject to any treatment. In the following 4 weeks, the females were subjected UVB (50 mJ/cm 2 ) or control
treatment.
The smear collection involved gently inserting a 200-ml pipette tip containing 30 ml sterile PBS X1 into the vagina to a depth of 3 mm,
and the lavage was smeared onto a plain glass slide (76 3 26 mm, Bar Naor Ltd.). The smears were then immediately viewed under a
bright field microscope (Nikon) to assess the stage of estrous cycle, as determined by examining the morphology of the cells present
in the vaginal smear, as described previously (Caligioni, 2009). The proestrus stage was characterized by the presence of nucleated
epithelial cells; the estrus stage by enucleated cornified cells; the metestrus stage by leucocytes, cornified cells, and nucleated
epithelial cells; and the diestrus stage by the predominant presence of leucocytes and lower amounts of nucleated cells. For Fig-
ure 3B, the proestrus and estrus stage were combined and the diestrus and metestrus stage were combined as previously done (Ajayi
and Akhigbe, 2020) for the subsequent analysis.
RNA purification and qRT-PCT
Flash-frozen tissues were thawed on ice, followed by homogenization with magnetic beads of the desired size (Next Advance) in a
Bullet Blender (Next Advance). Total RNA was purified using TriZol (Invitrogen) according to the manufacturer's guidelines. RNA was
quantified by measuring the OD 260 nm /OD 280 nm . For the mRNA analysis, the cDNA was prepared using the qScript cDNA synthesis kit
(Quantabio) and further subjected to qRT-PCR using PerfeCTa SYBR green FastMix (Quantabio). The data are represented as the
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+ fold changes relative to the control. All experiments were performed at least in triplicates. All the primer sequences used are pre-
sented in Table S5.
Histology
Following UVB or control treatment of female mice, the ovaries were fixed in 4% paraformaldehyde and paraffin-embedded, followed
by staining with hematoxylin (HHS16, Sigma-Aldrich) and eosin (HT110232, Sigma-Aldrich) according to the manufacturer's instruc-
tions. Sections of 5 mm were mounted using the DPX mountant (06522, Sigma-Aldrich). The images were obtained with an Aperio
Slide Scanner microscope (Leica Biosystem, USA), at 3 20 magnification.
Genotyping
Genomic DNA was extracted from the tail of a mouse using extraction buffer (25 mM NaOH, 0.2 mM disodium salt EDTA; pH 12) for
60 min, followed by incubation in neutralization buffer. PCR was performed in a 20-ml volume that included 10 ml GoTaq green master
mix ( 3 2) (Promega), 0.5 mM Cre primers (with positive control) and 0.5 mM flox primers (Integrated DNA Technologies). Reactions
were carried out in a PCR cycler (Biometra PCR Cycler) at 95 C for 3 min, followed by 35 cycles at 95 C for 30 s, a cycle at 55 C for
1 min, and an extension step at 72 C for 5 min. The PCR products were kept at 4 C until electrophoresis in 3% agarose gel. The
visualization of the PCR product was done based on its size (Cre-recombinase 100 bp, internal positive control 324 bp, flox
390 bp), and the digital images were captured in a Gel Documentation system (UVITEC Ltd.).
Immunoblotting
Whole skin tissues were dissected from mice and snap frozen in liquid nitrogen followed by homogenization in RIPA buffer with pro-
tease inhibitor (Roche) as previously described (Glaich et al., 2019). This was followed by incubation on ice for 1 hour, then the sam-
ples were centrifuged at 10,000 g for 15 mins at 4 C. The resulting clear phase protein was stored at À80 C until further use. Samples
were subjected to western blot analysis as described previously (Dror et al., 2016). Membrane was exposed overnight to antibody
targeting p53 (ab26, Abcam) and Actin (#A2066, Sigma Aldrich) and proteins were visualized with SuperSignal Chemiluminescent
Substrates (Pierce) using horseradish peroxidase-conjugated anti-mouse antibody (#7076, Cell Signaling) and horseradish peroxi-
dase-conjugated anti-Rabbit antibody (#ab6721, abcam). The p53 protein levels in each condition were normalized to actin (Q).
QUANTIFICATION AND STATISTICAL ANALYSIS
The data are shown by means and standard errors. We performed two-tailed Student's t tests for two group comparisons and
ANOVA for multiple group comparisons. For the PLS questionnaire, we used IBM SPSS (version 25.0) and conducted Wilcoxon tests
to examine within-group differences (ranks of T1 versus T2 for each gender separately). For all the tests, p values < 0.05 were consid-
ered significant. All the analyses were performed using Excel (Microsoft Corp.), SPSS (version 25.0), and GraphPad PRISM 8 soft-
ware. The statistics details and the software's used for all the experiments can be found in the resources tables and figure legends
and the Human questionnaire statistics details can be found in the STAR Methods section: Human questionnaire.
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