The essence of male scent promotes female puberty and estrus

4 5 Pheromones are chemical signals that trigger a response in another member of the same species. 6 In mice, it has been shown that exposure of females to male pheromones leads to puberty advance 7 and estrus induction 1,2,3 . In particular, these effects can be triggered by conspecific male urine, 8 suggesting that they are related to the chemical composition of this stimulus. Although these 9 phenomena were among the earliest known examples of pheromonal actions, the identities of these 10 chemical signals remain mysterious. Here we identified two small molecules in male urine, termed 11 Calin319 and Calin381 that accounted for much of the vomeronasal neuronal response to male 12 urine and were sufficient and necessary to advance juvenile female puberty and induce female 13 estrus. Besides acting as a primer pheromone, a blend of these two male compounds also acts as a 14 releaser pheromone that resulted in increased investigatory behavior by female mice. These 15 findings demonstrate that Calin319 and Calin381 are crucial male pheromones that regulate female 16 reproductive behavior in mice. This study resolves the long-standing mystery of the molecular 17 code of male urinary chemicals that control female gonadal function.

To investigate the olfactory cues that result in puberty acceleration, male urine was applied daily 44 to the external nares of juvenile females with a pipette for four continuous days (Fig. 1a). 45 Consistent with previous results 1 , females treated with male urine entered puberty three days 46 earlier (a ~12% acceleration counting from birth) than those who were treated by water (Fig. 1b). 47 To test a role for any volatiles, we delivered 30 μl fresh male urine daily to an inverted perforated 48 petri dish inside mice cages for four continuous days (Fig. 1c). Without being able to touch the 49 male urine, none of the juvenile female mice entered puberty earlier (Fig. 1d). These findings are 50 consistent with previous work showing that puberty acceleration of females requires direct contact 51 and is mediated by one or more non-volatile low molecular weight chemicals 17,18,19,20 . Therefore, 52 we focused on searching for signal chemicals for female puberty acceleration from the non-volatile 53 part of male mice urine.

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We first fractionated male urine by size exclusion column into high molecular weight (HMW > 56 3000 da) part and low molecular weight (LWM < 3000 da) part (Fig. 1e). After separately applying 57 them to the external nares of juvenile females with a pipette (Fig. 1f), we found only the LMW 58 urine fraction contained the puberty-accelerating activity (Fig. 1g). To search for chemical signals 59 underlying puberty acceleration, we first fractionated LMW urine extract by high pressure liquid 60 chromatography (HPLC). Then, we performed a forward screen for chemical compounds that 61 drive activity in vomeronasal sensory neurons (VSNs) using a combination of calcium imaging 62 and quantitative liquid chromatography-mass spectrometry (LC-MS) (Fig. 1h). This screen corre-63 lated compound abundance across fractions, as measured by negative ion nano LC-MS using high 64 mass resolving power (to enable mass accuracy of a few ppm or four decimal places) (Fig. 2a), 65 against the bioassay. We used calcium imaging, rather than behavior as in some previous stud-66 ies 10,11,12,26 , for its quantitative readout, high throughput, and possibility of circumventing the fre- interpretive context, we presented urine extracts from BALB/cJ as well as HPLC urinary fractions 71 matching its endogenous concentration in BALB/cJ male urine extract 8 (Fig. 2b-c).

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Neurons that responded to either HPLC urinary fractions or whole urine extract fell into 6 clearly-74 distinguishable groups. Three of the cell groups (clusters 1, 2, and 3) exhibited unambiguous 75 responses to the fractions, in a manner graded with concentration. All three of these fraction-76 responsive clusters were highly selective for male mouse urine, indicating that the ligand(s) 77 responsible for these neuronal activities only exist or are more concentrated in male mouse urine 78 (Fig. 2c). We performed a preliminary component-activity matching (CAM) analysis of these  (Fig. 2a, 2d).

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We then quantified these compounds in C18 urine extracts of four laboratory strains (BALB/cJ, 88 C57BL/6J, CBA, and DBA). BALB/cJ male urine had the highest abundance of both candidates 89 (Extended data Fig. 1a). Therefore, we chose BALB/cJ male urine as a staring material and purified 90 these compounds to homogeneity.

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Structural characterization of male ligands 93 Based on accurate mass measurement, Calin319 is ionized to a [M -H]with chemical formula of 94 C15H27O7 -, indicating that it is a previously unknown ligand. Following determination of its 95 chemical formula, we solved its structure by using a combination of MS-based methods, including 96 high mass resolving power, tandem MS (MS 2 and MS 3 ), and hydrogen deuterium exchange (H/DX) 97 to determine the number of hydroxyl groups (Extended data Fig. 2-6). The details of structure 98 elucidation are provided in Methods. Results of these analyses suggested that Calin319 is a 99 glucuronide with a saturated nonyl chain of undetermined isomeric structure.

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To test the validity of this structural interpretation, we performed a de novo synthesis of this 101 proposed glucuronide (Extended data Fig. 7a Fig. 8). 113 We then tested vomeronasal neuronal responses to these 16 synthetic glucuronide isomers by using  demonstrating that Calin381 is also a glucuronide (Extended data Fig. 9). After losing a C5H4 127 moiety, the structure of the remaining part of Calin381 is indistinguishable to that of Calin319 128 (Extended data Fig. 2, Extended data Fig. 9). Therefore, the structure of Calin381 includes a 129 hydrocarbon chain, a glucuronic acid and a C5H4 moiety. Thus it seems likely that Calin381 and 130 Calin319 share a common metabolic pathway. A synthetic standard of Calin381 is currently 131 unavailable and was therefore not available for direct testing against endogenous Calin381.

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Pheromonal effects of calin319 and calin381 133 To further explore the factors that control male ligands Calin319 and Calin381 expression, we 134 quantified urine from BALB/cJ juvenile male mice, BALB/cJ male mice castrated at 21 days of 135 age, and BALB/cJ female mice. Quantitative LC-MS of these samples showed that Calin319 and 136 Calin381 were about 20-fold lower in the urine of 21-day-old juvenile males, and three-fold less-137 concentrated in the urine of castrated male mice. Calin319 and Calin381 were not detected in the 138 urine of females (Extended data Fig. 1b). These results indicate that synthesis of Calin319 and

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Both male urine extract and a blend of Calin319 and Calin381 at endogenous concentrations 148 advance vaginal opening by three days (Fig. 4b). The two compounds have a synergistic effect, 149 with Calin319 having substantial activity on its own but enhanced by the simultaneous presence 150 of Calin381 (Fig. 4c). Moreover, depleting these two compounds from intact male mouse urine 151 (details provided in Methods) extracts eliminates the accelerating property of the urine extract ( Fig.   152 4d), demonstrating that these two compounds are not merely sufficient but also necessary.  To determine whether other sensory modalities might contribute to detecting Calin319 and indicating that TRPC2 function is necessary to detect and respond to pheromones that trigger 167 puberty.

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Given their similar nature, the Whitten (ovarian cycling induction) and Vandenburgh (puberty 169 induction) effects 1,2,3 have been proposed to be mediated by the same urinary pheromones 14 . To 170 determine whether our new discovered male ligands, Calin319 and Calin381, play a vital role in the induction of adult female estrus, we directly applied these two compounds to the external nares 172 of group-caged anestrus females (Extended data Fig. 10a). All of these estrous-suppressed females 173 treated by male ligands simultaneously entered estrus cycle after three days' treatment. In contrast, 174 females treated by water failed to start their estrous cycles (Extended data Fig. 10b, c). Thus, male 175 ligands Calin319 and Calin381 not only advance juvenile female puberty, but also induce adult 176 female estrus.  Calin319 or Calin381 on its own did not significantly increase the investigation time for females.

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Females investigated a blend of Calin319 and Calin381 for duration that were more than three-

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Ai38 (GCaMP3)/OMP-Cre male mice aged 8-12 weeks were used for imaging by OCPI 217 microscope 16,21, . Prior to dissection, mice were anesthetized with carbon dioxide. Sexually naive 218 three-month-old female B6D2F1 mice were used for investigatory behavior tests. 22 days old 219 BALB/cJ juvenile female mice were used for the puberty acceleration test. All of the mice except 220 for GCaMP3 mice were purchased from Jackson Laboratory.  For light sheet calcium imaging, 620 μm × 640 μm × 300 μm imaging volumes were acquired at 243 0.5 Hz using a custom OCPI microscope retrofitted with a PCO edge5.5 sCMOS camera) 16,21 .  suggesting the presence of a carboxylic group (Extended data Fig. 2-3). For further structural 305 elucidation, low-energy negative-ion collision-induced-dissociation (CID) spectra of Calin319 175, also demonstrating the present of a glucuronic acid group (Extended data Fig. 9). After losing 318 a C5H4 moiety, the structure of the remaining part of Calin381 is indistinguishable to that of 319 Calin319 (Extended data Fig. 2, Extended data Fig. 9). Therefore, the structure of Calin381 320 includes a hydrocarbon chain, a glucuronic acid and a C5H4 moiety (Extended data Fig. 9).

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Synthesis of calin319 323 We chose an enzymatic synthetic method for Calin319. Because purified or synthetic UGT is                            Fig. 2 High-resolution mass spectra (HRMS) of the product ion of Calin319 in the negative ion mode.