# Effects of minipuberty disruption on the expression of sexual behavior in female mice

Thomas Torres, Caroline Parmentier, Céline Guigon, Sakina Mhaouty-Kodja, Lydie Naulé

► To cite this version:

Thomas Torres, Caroline Parmentier, Céline Guigon, Sakina Mhaouty-Kodja, Lydie Naulé. Effects of minipuberty disruption on the expression of sexual behavior in female mice. Scientific Reports, 2024, 14, pp.31297. 10.1038/s41598-024-82653-8. hal-05377307

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1 **Title**

2 Effects of minipuberty disruption on the expression of sexual behavior in female mice

3

4 **Authors**

5 Thomas Torres¹, Caroline Parmentier¹, Céline Guigon²,³, Sakina Mhaouty-Kodja¹, Lydie Naulé¹

6 ¹Sorbonne Université, CNRS UMR8246, INSERM U1130, Neuroscience Paris Seine – Institut de
7 Biologie Paris Seine, Paris, France.

8 ²Université Paris-Cité, CNRS, Inserm, Biologie Fonctionnelle et Adaptative, F-75013, Paris, France.

9 ³Université Paris-Cité, Inserm, 3PHM, Pathophysiology and Pharmacotoxicology of the Human
10 Placenta, pre & post-natal Microbiota, Paris, F-75006, France.

11

12

13 **Corresponding author:**

14 Dr. Lydie Naulé

15 lydie.naule@sorbonne-universite.fr

16

17 **Keywords**

18 Minipuberty; Females; Sexual differentiation; Reproductive behaviors, Sex steroids
19 Abstract

20 Sex steroids influence early organization of neural structures involved in expression of sexual
21 behaviors. A critical perinatal period during which testosterone surges occur has been identified in
22 male rodents. Data are lacking for females, whose ovarian activity starts later in the postnatal period.

23 Minipuberty has been described in female as a first postnatal period of gonadotropic axis activation.

24 We transiently disrupted the minipubertal period, using daily injections of GnRH antagonist or
25 aromatase inhibitor. The results showed unchanged puberty initiation and mild disturbance of estrus
26 cyclicity. Minor modifications of some components of sexual behavior (olfactory preference, ability to
27 attract male) were observed, but had no impact on lordosis behavior. Interestingly, letrozole-treated
28 female mice showed increased anxiety state level at adulthood. This period therefore does not seem to
29 be a key organizational window for female sexual behavior but appears to participate in the
30 organization of neural structures involved in anxiety-related behavior.
31 **Introduction**

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Sexual reproduction relies on optimal display of sexually dimorphic behaviors expressed by sexual partners. These include in rodents i) the emission and sensing of olfactory and auditory cues, which inform on the hormonal and receptive state and attract the partner, and ii) the postures adopted afterward to ensure insemination, such as lordosis behavior for females, mounts, thrusts and intromissions for males¹⁻³.

Sex steroid hormones play a key role in the early organization of the neural structures involved in the expression of male and female behaviors. These processes have been extensively studied for half a century in males. However, the precise developmental period during which sex steroids act in females still needs to be documented. In rodents, it is indeed well established, since the seminal work of Phoenix et al.⁴, that testosterone released during the perinatal period by fetal and neonatal testes and its neural metabolite, estradiol, induce a permanent organization of the neural circuitry controlling male sexual behavior⁵. This consists in a potentiation of male (masculinization) and an inhibition of female (defeminization) behavioral characteristics, resulting in the expression of male-typical behaviors and the inability to adopt the female posture (lordosis).

In female rodents, there was a long-standing view that feminization of the neural circuitries regulating fertility and reproductive behaviors might occur as a default pathway. This idea stems from the fact that rodent ovaries do not produce sex steroids during the fetal and perinatal periods⁶. At these stages, the female brain is also protected from the masculinizing effects of maternal (and male sibling) estradiol through its selective binding to the α-fetoprotein⁷. In mice, ovaries start releasing estradiol around postnatal day (PND) 7 and then rise to reach a maximum at puberty. The prepubertal increase of estradiol has been shown to participate in brain feminization⁸,⁹. For instance, constitutive deletion of *cyp19a1* gene, encoding the aromatase that metabolizes testosterone into estradiol, has been shown to reduce lordosis behavior in adult mutant female mice⁸. In these mice, estradiol supplementation from
PND15 to PND25 partially restored the behavioral deficiency, while earlier administration of estradiol between PND5 and PND15 did not<sup>9</sup>. These studies suggested that the period lasting from PND15 to PND25 may represent one of the key postnatal periods involved in the feminization of the neural circuitry underlying female sexual behavior. However, these *cyp19a1* knockout mice are depleted of estrogens from the embryonic period. They also present high levels of testosterone, which could directly or indirectly interfere with neural processes in the female brain.

More recently, high levels of estradiol were detected in the serum of female mice between PND12 and PND17, resulting from a massive and transient gonadotropin surge<sup>10</sup>. This minipubertal hypothalamic-pituitary-gonadal (HPG) activity may regulate uterus and mammary gland development but its role in female reproductive function remains unknown<sup>11</sup>.

The present study aims to determine the precise role of the minipubertal period in the organization of the neural circuits controlling female reproductive function and sexual behavior. For this purpose, we used two pharmacological models to transiently disrupt the ovarian production of estrogens during minipuberty. In the GonadoSTOP model, C57BL6/J female mice were injected from PND10 to PND16 with a GnRH antagonist (Ganirelix®) to alter the gonadotropic axis. Such treatment was shown to suppress gonadotropin release and reduce estradiol levels by half after only two days of treatment<sup>10,12</sup>.

In the second model, C57BL/6J female mice were treated from PND10 to PND17 with letrozole an inhibitor of aromatase, the enzyme that metabolizes testosterone into estradiol. As aromatase blockade increases the levels of testosterone, we added two other groups treated with letrozole and flutamide (an androgen receptor antagonist) or with flutamide alone. For both models, we monitored body weight, assessed puberty initiation (age at vaginal opening and first estrus) and analyzed estrous cyclicity in adulthood. Afterwards, female mice were ovariectomized and primed with estradiol and progesterone to measure their sexual behavior under normalized hormonal levels. We performed tests for olfactory
79 preference, ability to attract the male partner and lordosis behavior. In addition, general behavior,

80 including locomotor activity and anxiety-related behavior, was assessed.
81 **Results**

82 **Effects of minipubertal treatment with Ganirelix® (Experiment 1)**

83 Pubertal onset, estrous cyclicity and other physiological parameters

84 Postnatal C57BL6/J females were injected with vehicle or Ganirelix for 7 days from PND10 to PND16 (Figure 1). The ages at vaginal opening and first estrus were monitored for both groups. The results in figure 2A-B shows that there were no differences between the control and Ganirelix-treated females for the age at vaginal opening (p = 0.84) and first estrus (p = 0.44). The duration between vaginal opening and first estrus was also comparable between the two treated groups (p = 0.15; Figure 2C).

89 In adult females, we monitored the estrous cycle for 5 weeks. One vehicle and 5 Ganirelix-treated females showed irregular cycles and were not included in the detailed analysis. A Fisher's exact test indicated no effect of the treatment on this parameter (p = 0.18). Further analysis showed that the treatment did not affect the mean cycle duration (p = 0.75; Figure 2D). Detailed analysis of the estrous cycle (Figure 2E) showed a comparable duration of the estrus and diestrus phases between controls and Ganirelix-treated females (p = 0.45, p = 0.68; respectively). In contrast, the metestrus phase was shorter by 13.6% in the Ganirelix group (p = 0.0228), while a tendency to increase was observed for the proestrus phase (p = 0.077).

97 Females from vehicle- and Ganirelix-treated mice were weekly monitored for their body weight until ovariectomy. This showed no effect of the treatment (F(1, 26) = 0.57, p = 0.45), but an effect of age (F(1.317, 34.25) = 668.6, p < 0.0001) (Figure 2F). There also was no effect on body weight at vaginal opening (p = 0.68) (Figure S3A).
The ovaries collected from controls and Ganirelix-treated adult females at the time of ovariectomy were weighed. No difference between the two groups was observed (0.024% ± 0.002 of body weight in controls versus 0.027% ± 0.001 of body weight in Ganirelix-treated females; *p* = 0.45) (Figure S3B).

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105 *Behavior*

Sexual and general behaviors were assessed in vehicle- and Ganirelix-treated females, which were ovariectomized at the end of estrous cyclicity monitoring and supplemented with estradiol and progesterone.

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109 *Sexual behavior*

The ability of females to discriminate between male and female pheromones was tested in a Y-maze with male and female stimuli, both anesthetized and placed at each end of the maze (Figure 3A). The total time spent in chemo-investigation was not different between the vehicle- and Ganirelix-treated females (48.35 ± 3.11 s versus 57.66 ± 5.88 s; *p* = 0.1751) (Figure S1A). Two-way ANOVA indicated no effect of stimulus (F<sub>(1, 48)</sub> = 0.52; *p* = 0.47) and treatment (F<sub>(1, 48)</sub> = 0.005211, *p* = 0.9428) on the number of entries into the arms (Figure S1B). The calculated discrimination index showed a preference towards male for the vehicle-treated females (*p* = 0.0363; Figure 3B). Although positive, the discrimination index calculated for Ganirelix-treated females did not reach the statistical significance (*p* = 0.0678). Comparison of the two treated groups did not show differences (*p* = 0.35).

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In mating tests, females were tested in the presence of a sexually experienced male for 30 min (Figure 3C). Males exhibited a comparable number of mounts in the presence of vehicle- or Ganirelix-treated females (*p* = 0.20) as shown in Figure 3D. The lordosis quotient did not differ between the two groups of treated females (*p* = 0.47; Figure 3E).
The ability of vehicle- and Ganirelix-treated females to attract sexually experienced males was assessed using the three-chamber test (Figure 3F). The total time spent in investigation (*p* = 0.94; Figure S1E), the number of entries into the stimulus compartments (*p* = 0.42; Figure S1F) and the percentage of time spent by males in the investigation of each stimulus (*p* = 0.12; Figure 3G) were comparable between controls and Ganirelix-treated females.

In the Y-maze test, vehicle- and Ganirelix-treated females were replaced by their urine (Figure 3H). As for the three-chamber test, the total time spent in stimulus arms (*p* = 0.78; Figure S1I), the number of entries into the two arms (*p* = 0.58; Figure S1J) and the time spent in the investigation of each stimulus (*p* = 0.39; Figure 3I) were comparable between vehicle- and Ganirelix-treated females.

## General behaviors

Locomotor activity of both treated groups was assessed in a circular corridor for 2 h. Two-way ANOVA showed an effect of time ($F_{(3,337, 83.43)}$ = 68.13; *p* < 0.0001) but not of treatment ($F_{(1, 25)}$ = 0.47, *p* = 0.50) (Figure 6A). Comparison of cumulative activity over the 2 hours of the test did not show differences between the two groups (*p* = 0.50) (Figure 6B).

Anxiety-related behavior was analyzed in an 0-maze paradigm (Figure 6E). No difference was observed between the two groups in the latency to the first entry into the open arm (*p* = 0.61; Figure 6F), or the number of entries (*p* = 0.89; Figure 6G). The time spent in the open arms was also comparable between the treated groups (*p* = 0.38; Figure 6H).

## ERα-immunoreactivity

The brains of female mice that were subjected to behavioral tests were analyzed for ERα-immunostaining. It was performed in keys hypothalamic regions controlling female sexual behavior, including the activating nuclei VMH and the inhibitory regions, ARC and POA<sup>13,14</sup>. Figure S2 shows
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a specific nuclear staining in the POA, VMH and ARC. Quantification of the number of ERα-
immunoreactive cells showed no difference between vehicle- and Ganirelix-treated females in the POA
($p$ = 0.29), VMH ($p$ = 0.78) and ARC ($p$ = 0.97) (Figure S2A-D).

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# Effects of separate and combined treatment with letrozole and flutamide during minipuberty
## (Experiment 2)

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Treatment of female mice with Ganirelix from PND10 to PND16 did not affect the expression of sexual
behavior. As Ganirelix treatment only partially suppresses the ovarian production of estradiol¹⁰, it is
possible that the remaining estradiol was sufficient to induce the organization of the neural circuitry
underlying female sexual behavior. Therefore, in Experiment 2, C57BL6/J females were daily injected
for 8 days from PND10 to PND17 with vehicle (control group), letrozole alone at 50 µg/day to inhibit
aromatase activity and thus estradiol production, flutamide alone at 0.1 mg/day, or with both
compounds (Figure 1). The anti-androgen flutamide was used to counteract the androgenic effects
potentially induced by the known elevation of testosterone levels triggered by the blockade of
aromatase and thus the metabolization of testosterone into estradiol.

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*Pubertal onset, estrous cyclicity and other physiological parameters*

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Monitoring of pubertal onset markers showed no effect of the treatment on the age at vaginal opening ($p$ = 0.54), while an effect of treatment was observed on the age at first estrus ($p$ = 0.018) (Figure 4A-B). *Post hoc* analysis indicated that females treated with both letrozole/flutamide had the first estrus 4.14 days later than those treated with flutamide ($p$ = 0.046; Figure 4B). Analysis of the interval between vaginal opening and first estrus did not show any effect of the treatment ($p$ = 0.27; Figure 4C).

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We monitored the estrous cycle for 5 weeks in adult females. One vehicle, one flutamide and 4
letrozole/flutamide-treated females showed irregular cycles and were not included in the detailed

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analysis. A Fisher's exact test indicated no effect of the treatment on this parameter (*p* = 0.072). The mean duration of the estrous cycle was unaffected by the treatment (*p* = 0.1317; Figure 4D). Detailed analysis of each cycle phase duration revealed no effect of the treatment on the duration of the proestrus and estrus and metestrus phases (*p* = 0.29, *p* = 0.99, *p* = 0.06; respectively), although the statistical significance was close to 0.05 for the metestrus phase. In contrast, an effect of treatment was observed for the diestrus phase (*p* = 0.023). Females treated with letrozole/flutamide had a longer duration compared to those treated with flutamide (+ 31.77%; *p* = 0.046) (Figure 4E).

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For body weight monitored between PND20 and PND90, two-way ANOVA showed effects of age ($F_{(1,652, 66.08)}$ = 1545, *p* < 0.0001) and treatment ($F_{(3, 40)}$ = 3.826, *p* = 0.017) (Figure 4F). *Post hoc* analysis pointed out a transient increase of body weight at PND21 for letrozole-treated females (*p* = 0.015) and letrozole/flutamide-treated groups (*p* = 0.023) compared to the vehicle-treated females, and at PND70 for the letrozole group compared to females treated with flutamide (*p* = 0.027). There also was no effect on body weight at vaginal opening (*p* = 0.095) (Figure S3C).

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The weight of ovaries, collected at the time of ovariectomy, was not different between the treated groups (*p* = 0.13) as illustrated in Figure S3D.

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## *Behavior*

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Sexual behavior was analyzed in vehicle-, flutamide-, letrozole- and letrozole/flutamide-treated mice, which were ovariectomized at the end of estrous cyclicity monitoring and hormonally primed as described above.

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## *Sexual behavior*

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In the olfactory preference test (Figure 5A), there was no effect of the treatment on the total time spent in chemoinvestigation for the four female-treated groups (*p* = 0.1689; Figure S1C). Similarly, no effect

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190 of the treatment ($F_{(3, 86)} = 0.7207; p = 0.5423$) and stimulus ($F_{(1, 86)} = 0.8275; p = 0.3655$) was observed
191 for the number of entries into the arms (Figure S1D). All females exhibited preference for the male
192 stimulus ($p = 0.007, p = 0.010, p = 0.036, p = 0.006$, for vehicle-, flutamide-, letrozole- and
193 letrozole/flutamide-treated mice; respectively). One-way ANOVA showed no effect of the treatment
194 on the discrimination index ($p = 0.98$; Figure 5B).

195 In the mating test, sexually experienced males exhibited comparable number of mounts in the presence
196 of females ($p = 0.69$), regardless of their treatment (Figure 5C-D). Furthermore, no effect of the
197 treatment was observed on the lordosis quotient ($p = 0.72$; Figure 5E).

198 In the three-chamber paradigm (Figure 5F), there were no differences in the total time spent by males
199 in the stimulus compartments ($p = 0.55, p = 0.72, p = 0.19$, for vehicle versus flutamide groups, vehicle
200 versus letrozole groups and vehicle versus letrozole/flutamide groups, respectively, Figure S1G), nor
201 in the number of entries in the compartments ($p = 0.49, p = 0.32$ and $p = 0.31$ for vehicle versus
202 flutamide groups, vehicle versus letrozole groups and vehicle versus letrozole/flutamide groups,
203 respectively) (Figure S1H). The percentage of time spent by males investigating vehicle- versus
204 flutamide-treated females ($p = 0.59$) and vehicle- versus letrozole-treated females ($p = 0.8$) was not
205 different. In contrast, males spent less time investigating letrozole/flutamide-treated mice than controls
206 (-29.37%, $p = 0.009$) as shown in (Figure 5G).

207 For urinary preference (Figure 5H), the total time spent in the stimulus arms was not different ($p =
208 0.41, p = 0.59, p = 0.59$, for vehicle versus flutamide groups, vehicle versus letrozole groups and vehicle
209 versus letrozole/flutamide groups, respectively; Figure S1K). The number of entries into each arm was
210 also comparable between the three experimental conditions ($p = 0.45, p = 0.83$ and $p = 0.68$ for vehicle
211 versus flutamide groups, vehicle versus letrozole groups and vehicle versus letrozole/flutamide groups,
212 respectively; Figure S1L). In addition, the percentage of time spent by males in investigation was not
213 different between the two stimuli for the three experimental conditions ($p = 0.18, p = 0.39, p = 0.46$)
214 for vehicle versus flutamide groups, vehicle versus letrozole groups and vehicle versus
215 letrozole/flutamide groups, respectively; Figure 5I).

216 *General Behaviors*

217 Analysis of locomotor activity by two-way ANOVA showed an effect of time ($F_{(3,225, 129.0)} = 87.56, p < 0.0001$) but not of treatment ($F_{(3, 40)} = 0.598, p = 0.62$) (Figure 6C). There was no effect of the
218 treatment on the cumulative activity over the 2 hours of the test ($p = 0.76$; Figure 6D).

220 For anxiety-related behavior, there was no effect of the treatment on the latency to the first entry into
221 the open arm ($p = 0.77$, Figure 6I), but a tendency to decrease was observed for the number of entries
222 although it did not reach the statistical significance ($p = 0.059$, Figure 6J). One-way ANOVA showed
223 an effect of treatment on the time spent into the open arms ($F_{(3, 43)} = 2.995, p = 0.041$), with reduced
224 time spent in the open arms for letrozole-treated mice compared to vehicle group (-48.51%, $p = 0.0176$)
225 (Figure 6K).
226 **Discussion**

227 In this study, using two pharmacological methods, we investigated the impact of minipubertal
228 disruption on later pubertal initiation, estrous cyclicity and female sexual behavior.

229 Disruption of the gonadotropic axis through GnRH antagonist administration (Ganirelix) during the
230 minipubertal period did not impact the age of puberty initiation, with no change in the age at vaginal
231 opening or first estrus in female mice. Adult monitoring of estrous cyclicity showed reduced duration
232 of the metestrus phase in females treated with Ganirelix, without modification of the total duration of
233 the estrous cycle. It has also been previously shown that minipubertal Ganirelix treatment induces no
234 modification in fertility at 3 months of age, suggesting that the slight treatment effect observed on the
235 duration of the metestrus phase does not appear to impact fertility in adulthood. Surprisingly, 11-
236 month-old treated females showed an increase in the percentage of mice becoming pregnant,
237 suggesting a long-term effect of the treatment on reproductive longevity (Chester et al., preprint
238 available at bioRxiv¹⁵).

239 Regarding the treatment with an aromatase inhibitor (letrozole) in the absence or presence of an anti-
240 androgen (flutamide), an effect of treatment was observed on the age at the first estrus. This difference
241 was observed only between the flutamide and letrozole/flutamide groups with no difference compared
242 with vehicles. In adulthood, an effect of treatment was observed on the duration of the diestrus phase
243 of the estrous cycle. Again, difference was noticed between the flutamide and letrozole/flutamide
244 groups. The meaning of such differences in the initiation of puberty and estrous cyclicity between these
245 two groups is not clear. Interestingly, it is documented that androgens through the androgen receptor
246 (AR) play a key role in both follicular development and female neural regulation of the gonadotropic
247 axis¹⁶⁻¹⁹. It is therefore possible that both treatments affected ovarian function and/or gonadotropic axis
248 differently, leading to the observed differences. Nevertheless, it is important to point out that the age
249 of pubertal onset and the duration of the diestrus phase of the cycle in females treated with letrozole or
250 flutamide alone or in combination were in the range of those observed for the vehicle group. In addition,
treatments with letrozole or letrozole/flutamide had slight effects on body weight at PND20, suggesting that these treatments, compared to Ganirelix, transiently affected the somatotropic axis.

At the behavioral level, analyses were performed in ovariectomized and hormonally primed females to test the direct effects of the minipubertal treatments on the neural circuitry underlying sexual behavior. Under such normalized hormonal levels, the discrimination index in the olfactory preference test was slightly affected by the treatment with Ganirelix. However, no significant difference was found with the vehicle group. Olfactory stimulation is mandatory for the activation of the other components of sexual behavior. Lordosis behavior, partner and urinary preferences were not affected by Ganirelix treatment. In agreement with these data, the number of ERα-immunoreactive cells was comparable between controls and Ganirelix-treated females in the neural circuitry underlying sexual behavior (VMH, POA and ARC).

Female mice treated during minipuberty with letrozole, flutamide or letrozole/flutamide showed comparable olfactory preference. In the three-chamber test, females of the letrozole/flutamide group attracted less the males compared to the control group. This effect was not observed in the urinary preference test, suggesting differences in the volatile cues between vehicle- and letrozole/flutamide-treated groups. Despite this reduced partner preference, mating assessed by the lordosis quotient was comparable between treatment groups.

Overall, this comprehensive behavioral analysis shows that under normalized hormonal levels, the expression of female sexual behavior was not or slightly impacted following minipubertal treatment with a GnRH antagonist or aromatase inhibitor, in the absence or presence of flutamide. This confirms a previous study showing that estradiol supplementation between PND5 and PND15 did not restore the expression of sexual behavior in aromatase knockout females⁹. In line with the classic view that neonatal estradiol masculinizes the brain structures involved in sexual behavior, this early postnatal treatment masculinized aromatase knockout females similarly to controls, which both exhibited
female-directed (male typical) mounting behavior when treated with testosterone in adulthood⁹. In the same study, estradiol treatment from PND15 to PND25 partially restored the expression of lordosis behavior of aromatase knockout adult females. These and our data strongly support that the period involved in the feminization of the neural circuits underlying female sexual behavior occurs at a later age⁹. Further research will be instrumental to delineate the precise prepubertal/pubertal periods during which neural feminization takes place, and determine the underlying mechanisms of action.

In addition to sexual behavior, we assessed general behavior. The results showed no effect of the treatments on locomotor activity. In contrast, an increased anxiety state level was observed in female mice treated during the minipubertal period with letrozole. This effect was likely induced by the transient depletion of estradiol during the minipubertal period, since hormonal levels were normalized in adult females analyzed for this behavior. No such effect was observed with Ganirelix treatment, probably explained by the incomplete reduction in estradiol levels in this model. In previous studies, we have shown that deletion of estrogen receptor (ER) ERβ or ERα in neural progenitors since embryonic day 10 resulted in increased anxiety-related behavior in adult female mice²⁰⁻²². The present data suggest that estradiol released during the minipubertal period (PND10 to PND17) participates in the organization of the neural structures involved in anxiety-related behavior. It is well known that estradiol in adulthood controls the anxiety state of females. Indeed, elevated level of estradiol during the follicular phase induce an anxiolytic effect²³. Downstream molecular pathways may imply the serotoninergic system and the expression of tryptophan hydroxylase (TPH) in the dorsal raphe nucleus²⁴. For instance, the increase in anxiety-related behavior observed in females deleted for neural ERβ was associated with a reduction in TPH2-immunoreactivity in the dorsal raphe nucleus since PND30 (unpublished observation,²²). It remains to be determined whether the estradiol released during the minipubertal period participates in the organization of this neural pathway.
299 *Perspective and significance*

300 In summary, the evaluation of the impact of minipubertal disruption on puberty initiation, estrous
cyclicity and female sexual behavior has shown that estradiol released during this period appears to
301 affect the development of female reproductive function and behavior in subtle ways but is not essential
302 for successful fertility and mating in adulthood. A transient activation of the HPG axis during
303 minipuberty, characterized by high levels of gonadotropin LH (luteinizing hormone) and FSH (follicle-
304 stimulating hormone) and elevated gonadal sex steroid production, occurs also in humans shortly after
305 birth and can last up to 3-4 years in girls²⁵. The influence of this period on women fertility remains to
306 be determined. Further studies focusing on later developmental periods (prepubertal/pubertal) will help
307 identify the critical developmental periods for brain feminization of reproductive function. Meanwhile,
308 interestingly, this study revealed that estradiol released during minipuberty may play a role in the
309 organization of anxiety-related behaviors. This is consistent with the well-established role for estradiol
310 in the regulation of women's anxiety throughout life, whether during the menstrual cycle, menopause
311 or in post-partum anxiety disorders. A better understanding of the critical periods of estradiol action
312 would enable better management of these anxiety troubles.
313

314

315 **Conclusions**

316 In this study, we showed that transient disruption of the minipubertal period, in female mice, induced
317 only minor modifications of estrous cyclicity and some components of sexual behavior (olfactory and
318 partner preferences) without impacting subsequent mating behavior. Interestingly, this study, however,
319 revealed that estradiol released during minipuberty may contribute to the organization of female
320 anxiety-related behavior. Hence, these findings support the view that estradiol acts at different periods
321 of development to organize distinct behaviors. Further research will precise the windows of action and
322 the underlying mechanisms of action.
323
323 **Methods**

324 **Animal and Treatments**

325 Female mice were obtained from local mating of male and female C57BL/6J mice (Janvier Labs).
326 Animals were maintained at a temperature of 22°C and subjected to a 12-hour light-dark cycle with
327 the dark phase beginning at 1:30 PM and ending at 1:30 AM. They had *ad libitum* access to both food
328 (A03-10; SAFE) and water.

329

330 *Experiment 1*. Female mice were subcutaneously injected each day from PND10 to PND16 with 20 µl
331 of sesame oil alone (control group) or 20 µl of sesame oil containing 10 µg of Ganirelix® (Fyremadel
332 Gé, FERRING), a GnRH antagonist (n = 12-15 per treatment, 3 different litters for control- and 4 litters
333 for Ganirelix-treated animals) (Figure 1A). Under such experimental conditions, Ganirelix treatment
334 reduced estradiol levels by 50 % in female mice¹⁰.

335

336 *Experiment 2*. Female mice were subcutaneously injected each day from PND10 to PND17 with 20 µl
337 of sesame oil and ethanol 3% for the vehicle group, or containing either 50 µg of letrozole (a reversible
338 aromatase inhibitor), 0.1 mg of flutamide (a non-steroidal anti-androgen), or both (letrozole/flutamide)
339 (n = 10-13 per treatment, 3 different litters for control- and 4 litters for letrozole-, flutamide- and
340 letrozole/flutamide-treated animals) (Figure 1B). The concentrations of letrozole and flutamide were
341 chosen based on previous studies in mice²⁶,²⁷. Administration of 50 µg per day of letrozole reduced
342 estradiol level below the limit of assay sensitivity, as in ovariectomized females²⁷. In a prenatally
343 androgenized mouse model, daily injection of the same concentration of flutamide was efficient to
344 block androgen receptor signaling and restore estrous cyclicity and GnRH neuron circuit integrity,
345 while unaffecting ovarian and adrenal gland weights²⁶.

346
347 For both Experiments 1 and 2, female mice were weaned at PND28 and identified by numbers. No
348 information on the treatment groups were available during the analyses.

349 Pubertal onset, estrous cyclicity, body and organ weights

350 The age at pubertal onset was monitored by daily examination of vaginal opening since PND20 (Figure
351 1A-B). Once vaginal opening was observed, vaginal smears, flushed with physiological saline (NaCl
352 0.9%), were collected daily to determine the age of first estrus. Vaginal smears were collected daily
353 until PND90 to examine estrous cyclicity. Microscopic examination of the vaginal smears, following
354 hematoxylin/eosin staining, allowed identification of the estrous cycle phases. The mean duration of
355 the estrous cycle and the number of days spent in each stage were quantified.

356 Body weight was assessed from PND21 to PND90. The ovaries were collected and weighed at the time
357 of ovariectomy two weeks before the beginning of behavioral assessment.

359 Behavioral tests

360 Experiments were carried out under red-light illumination, occurring 2 h after lights were turned off.
361 All sessions were recorded via videotaping for subsequent analysis. The testing sequence was
362 proceeded as following: 1st test of lordosis behavior for sexual experience, olfactory preference test,
363 locomotor activity monitoring, 2nd test of lordosis, 0-maze test, three-chamber test for partner
364 preference and Y-maze for urinary preference. Behavioral tests were separated by at least two full days
365 of complete rest or habituation to the following test. The stud males used were sexually experienced
366 three times before the beginning of behavioral tests. All devices used in these tests, except for the
367 animal home cages, were cleaned with a solution of 10% ethanol between trials.

369 *Preparation of females*
370 Female mice were ovariectomized two weeks before the beginning of behavioral tests under general anesthesia and received a 1 cm subcutaneous SILASTIC™ filled with 50 µg of estradiol benzoate (Sigma-Aldrich) diluted in 30 µL sesame oil and sealed at each end with SILASTIC™ adhesive as previously described<sup>20,28,29</sup>. Five hours before each behavioral test, females were primed with a subcutaneous injection of progesterone (1 mg/100 µL).

375

376 Lordosis

377 Each sexually-experienced female was introduced into the cage of a sexually experienced male. The 378 duration of each test was set to 30 min. The female was considered in a lordosis posture when, in 379 response to male mount, all four paws made contact with the ground, the hind section lifted while the 380 back region formed an arch. The number of male mounts, the percentage of females displaying lordosis 381 behavior and the lordosis quotient of each female were calculated. The lordosis quotient was calculated 382 as the number of lordosis postures divided by the number of male mounts as previously described<sup>20,28</sup>.

383

384 Olfactory Preference

385 Olfactory preference of female mice was tested in an enclosed plexiglass Y-maze, following a 386 previously established protocol<sup>20,30,31</sup>. The maze comprised 3 equivalent arms (25.8 cm length, 11 cm 387 width, 30.7 cm height). Female mice underwent a familiarization period over two consecutive days 388 (day 1 and 2), during which two empty perforated goal boxes were positioned at each end of two arms 389 for a duration of 10 min. On the testing day (day 3), females were placed in the empty arm and had 390 choice for 10 min between a sexually receptive female and an intact male, both placed within the goal 391 boxes in the two opposing arms. To prevent social interaction, the stimuli were anesthetized. 392 Observations included the time spent in chemo-investigation of the stimuli and the number of entries
393 into each arm of the maze. The discrimination index was calculated as the difference in time spent
394 investigating the male (M) versus the female (F), divided by the total investigation time.

395

396 *Partner Preference tests*

397 *Three-chamber test*

398 Sexually experienced males underwent a familiarization phase with the testing arena, a three-
399 chambered box (22.5 cm height, 40.5 cm width, and 62.4 cm length; 20.5 cm for the two peripheral
400 chambers and 21.4 cm for the central chamber) for 10 min over two consecutive days³⁰,³¹. During this
401 period, two empty perforated goal boxes were placed within the side chambers of the arena. On the test
402 day, each male was introduced into the neutral chamber and allowed to freely explore for 10 min. A
403 vehicle-treated female was put in a goal box, while the opposite one contained a female treated with
404 Ganirelix (Experiment 1), flutamide, letrozole or flutamide/letrozole (Experiment 2). Females were
405 randomly assigned to either the left or right chamber. The number of entries into each compartment
406 and the duration the male spent sniffing each female were analyzed.

407

408 *Y-Maze test (Urinary Preference)*

409 Sexually experienced males underwent a familiarization period with the maze for 10 min over two
410 consecutive days³⁰,³¹. On the testing day, males had choice between urine of females from the vehicle
411 group and urine from one of the treated groups, Ganirelix (Experiment 1) or flutamide, letrozole or
412 flutamide/letrozole (Experiment 2), both put inside perforated goal boxes placed at each end of the
413 maze. Urine was collected one hour prior to the test. Urine samples from all females within each
414 treatment group were combined in equal volumes, and 10 µL of this mixture was applied onto a piece
415 of filter paper. Observations were made on the time spent by males chemo-investigating each stimulus
416 and on the number of entries into each arm of the maze.
417

418 *Elevated 0-maze*

419 The elevated 0-maze consisted in a 56 cm diameter circular track (5 cm width), divided into two open and two enclosed (bordered by 17 cm-diameter walls) quadrants, elevated at 65 cm above the ground.

420 Female were placed in one of the enclosed quadrants and allowed to freely explore the maze for 9 minutes. Anxiety-like behavior was assessed by measuring the latency to the first entry into the open arms, the number of entries and the time spent in the open arms. A mouse was considered to be in an open arm when the four paws have entered.

425

426 *Locomotor activity*

427 The circular corridor used for activity measurement consisted in two concentric cylinders crossed by four diametrically opposed infrared beams²⁹,³⁰. Locomotor activity was recorded each time animals interrupted two successive beams, indicating they had traveled a quarter of the circular corridor. The activity monitored every 20 min and cumulative activity over the 120-min test were reported.

431

432 **Immunohistochemistry**

433 For experiment 1, three days after the end of behavioral assessment, females were euthanized 4 to 5 h after progesterone injection through intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10mg/kg). They were intracardiacially perfused with NaCl (0.9%) followed by 4% paraformaldehyde (PFA) in Phosphate Buffer (PB). The brains were extracted, post-fixed overnight in 4% PB-PFA, then frozen in tissue freezing medium (Leica) and stored at -20°C. The brains were then sectioned into 30 µm coronal sections using a cryostat (Leica CM 3050S).
439

For immunodetection, the sections were incubated in a solution containing 1X Phosphate Buffer Saline

440

(PBS) - 0.3% Triton with 2% normal donkey serum for 1 h to saturate non-specific binding sites.

441

Incubation with the primary antibody anti-ERα (1:400; Santa Cruz Biotechnology; rabbit) was

442

performed in 1X PBS - 0.3% Triton for 3 days at 4°C. The sections were washed then incubated with

443

chicken secondary anti-rabbit coupled with Alexa Fluor 488 (1:500; Invitrogen) in 1X PBS - 0.3%

444

Triton for 2 h at room temperature in the dark. The sections were washed, mounted on SuperFrost

445

slides, dried in the dark at room temperature, and then cover-slipped with Mowiol.

446

The slides were scanned using a Nanozoomer Hamamatsu Scanner (Hamamatsu corporation) at high

447

resolution and analyzed using NDP.view software. The regions of interest, VMH, arcuate nucleus

448

(ARC), and preoptic area (POA), were identified using the Paxinos & Franklin Mouse Brain in

449

Stereotaxic Coordinates Third Edition (2007) (plate 46, plate 43 and plate 30 respectively). After

450

delineating equivalent area zones (0.3 mm², 0.16 mm², and 0.4 mm², respectively), ERα-

451

immunoreactive neurons were counted in the left and right hemispheres.

452

453

## Statistics

454

The data are expressed as the mean ± standard error of the mean. A *p*-value < 0.05 was considered

455

significant. Normality of the data was assessed with the Shapiro-Wilks normality test. Unpaired t-test

456

and Mann-Whitney test were used to compare vehicle- and ganirelix- treated mice (Experiment 1) for

457

vaginal opening, estrous cyclicity, ovarian and uterine weights, ERα-immunoreactivity, olfactory

458

preference, lordosis and anxiety-related behaviors, as well as cumulative locomotor activity. One-way

459

ANOVA and Kruskal-Wallis tests were used to assess the same tests in Experiment 2. Paired t-test and

460

Wilcoxon test were used in both experiments to analyze partner preference in three-chamber and

461

urinary preference tests. Two-way ANOVA was used in both experiments to assess the effect of
462 treatment on body weight, number of entries in the stimulus arms for olfactory preference and 2-hour
463 monitoring of locomotor activity.

464

465 Ethical approval

466 The study was conducted according to the European legal requirements and received the approval of
467 the «Charles Darwin» Ethical committee (project number 32459). The experiments were in compliance
468 with the ARRIVE guidelines.

469

470 Data availability

471 The authors declare that the data supporting the findings of this study are available within the paper
472 and its supplementary information files.
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550 Acknowledgments

551 We thank the Institut Biologie Paris-Seine platform for taking care of the animals. This work was
552 supported by the Agence Nationale de la Recherche (ReproFun, 2020) in France (to CG and SMK).

553 Author's contribution

554 CG, SMK and LN designed the study. TT, CP and LN carried out the experiments. TT, SMK and LN
555 analyzed the data. TT, SMK and LN wrote the manuscript. All the authors corrected and edited the
556 manuscript.

557 Competing interests

558 The authors declare no competing interests.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Schematic representation of the experimental design. **A.** In Experiment 1, female mice were daily treated with the vehicle or Ganirelix® at the indicated doses from postnatal day 10 (PND10) to PND16. **B.** In Experiment 2, female mice were daily treated with the vehicle, flutamide (F), letrozole (L) or both (F/L) at the indicated doses from PND10 to PND17. For both experiments, body weight, pubertal onset and estrous cyclicity were monitored in intact females. Females were then ovariectomized and received estradiol implants (OVX+E2) and were primed with progesterone (Pg) before each behavioral test. At the end of behavioral tests, females were euthanized and tissues were collected.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Effects of minipubertal treatment with Ganirelix® on pubertal onset, estrous cyclicity and body growth. A-C. Female mice treated with the vehicle (Veh) or Ganirelix (G) from postnatal (PND)10 to PND16 were analyzed for the age at vaginal opening (A) and first estrus (B) as well as the interval between these two pubertal markers (C). D-E. The estrous cycle was monitored in adult control and Ganirelix-treated females. The duration of the estrous cycle (D) and of each stage (E) are indicated; (*p* < 0.05 for the metestrus phase). F. Body weight was followed every week from PND20 to PND86 for each female. Data are presented as means ± S.E.M. (n = 9-15 per treatment).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 3. Effects of minipubertal treatment with Ganirelix® on female sexual behavior. A-B. In the olfactory preference test, female mice treated with the vehicle (Veh) or Ganirelix (G) had choice to chemoinvestigate anesthetized male or female stimuli (A). The discrimination index, time spent in male investigation minus the time spent in female investigation divided by the total time of investigation is indicated (B). \*p < 0.05, positive index for the control group. C-E. Lordosis behavior was tested for sexually-experienced females in the presence of sexually experienced males for 30 min</figcaption></figure>
581 (C). The number of male mounts (D) and lordosis quotient (E) are indicated. F-G. In the three-chambered test, sexually experienced males had choice to investigate Veh- or G-treated females (F).

582 The percentage of time spent investigating each female is indicated (G). H-I. In the urinary preference test, sexually experienced males had choice to investigate urine from Veh- or G-treated females (H).

583 The percentage of time spent investigating each urine stimulus is indicated (I). Data are presented as means ± S.E.M. (n = 12-15 per treatment).
Pubertal onset and estrous cyclicity

<figure><img src="image_6.png" /></figure>

Body growth

<figure><img src="image_7.png" /></figure>

**Figure 4.** Effects of separate or combined treatment with flutamide and letrozole during minipuberty on pubertal onset, estrous cyclicity and body growth. **A-C.** Female mice treated with the vehicle (Veh), flutamide (F), letrozole (L) or both (F/L) from postnatal day (PND)10 to PND17 were analyzed for the age at vaginal opening (**A**), age at first estrus (**B**) and interval between the two pubertal markers (**C**). \*p < 0.05 for the indicated groups. **D-E.** Duration of the estrous cycle (D) and of
592 each stage (E) in adult females. F. Body weight was monitored weekly from PND21 to PND86, <sup>a</sup>p

593 <0.05 between Veh and L, <sup>b</sup>p < 0.05 between Veh and F/L, <sup>c</sup>p < 0.05 between F and L). Data are

594 presented as means ± S.E.M. (n = 8-13 per treatment).
<table><caption>Figure 5. Effects of separate and combined treatment with flutamide and letrozole during minipuberty on female sexual behavior. A-B. In the olfactory preference test, female mice treated with the vehicle (Veh), flutamide (F), letrozole (L) or both (F/L) had choice to chemoinvestigate anesthetized male or female stimuli (A). The discrimination index, time spent by exposed females in male investigation minus the time spent in female investigation divided by the total time of investigation (B). \*p < 0.05, positive index for the four group. C-E. Lordosis behavior was tested for</caption><tr><td>Olfactory preference</td></tr></table>

A

<figure><img src="image_8.png" /></figure>

B

<figure><img src="image_9.png" /></figure>

Lordosis behavior

C

<figure><img src="image_10.png" /></figure>

D

<figure><img src="image_11.png" /></figure>

E

<figure><img src="image_12.png" /></figure>

Partner preference

Urinary preference

F

<figure><img src="image_13.png" /></figure>

G

<figure><img src="image_14.png" /></figure>

H

<figure><img src="image_15.png" /></figure>

I

<figure><img src="image_16.png" /></figure>
601 sexually-experienced females in the presence of sexually experienced males for 30 min (C). The
602 number of male mounts (D) and lordosis quotient (E) are indicated. F-G. In the three-chamber test,
603 sexually experienced males had choice to investigate either Veh- or F- (p < 0.01 for F/L) (F). The
604 percentage of time spent investigating each female is indicated (G). H-I. In the urinary preference test,
605 sexually experienced males had choice to investigate urine either from Veh- or F-, Veh- or L-, or Veh-
606 or F/L-treated female (H). The percentage of time spent investigating each urine stimulus is indicated
607 (I). Data are presented as means ± S.E.M. (n = 10-13 per treatment).
<figure><img src="image_17.png" /><figcaption>Figure 6. Effect of minipubertal treatment on locomotor activity and anxiety-like behavior. A-D.</figcaption></figure>

Locomotor activity was monitored in a circular corridor during 2 h. The activity monitored every 20 minutes (A, C) or cumulative activity (B, D) are presented for females treated with the vehicle (Veh) and Ganirelix (G) groups in Experiment 1, and flutamide (F) alone, letrozole alone or both flutamide and letrozole (F/L) in Experiment 2. E-K. Anxiety-like behavior was assessed in a O-maze during 9
613 min (E). The latency to the first entry into the open arm (F, I), the number of entries into the open arm

614 (G, J) and the time spent into the open arm (H, K) are indicated. Data are presented as means ± S.E.M.

615 (n = 12-15 per treatment for Experiment 1; n = 10-13 per treatment for Experiment 2) *p < 0.05 for the
616 indicated groups.
1 Effects of mini-puberty disruption on the expression of sexual behavior in female mice

2 Thomas Torres, Caroline Parmentier, Céline Guigon, Sakina Mhaouty-Kodja, Lydie Naulé

3

4 Figure S1

5 Figure S2

6 Figure S3
Olfactory preference

A

<figure><img src="image_18.png" /></figure>

B

<figure><img src="image_19.png" /></figure>

C

<figure><img src="image_20.png" /></figure>

D

<figure><img src="image_21.png" /></figure>

Partner preference

E

<figure><img src="image_22.png" /></figure>

F

<figure><img src="image_23.png" /></figure>

G

<figure><img src="image_24.png" /></figure>

H

<figure><img src="image_25.png" /></figure>

Urinary preference

I

<figure><img src="image_26.png" /></figure>

J

<figure><img src="image_27.png" /></figure>

K

<figure><img src="image_28.png" /></figure>

L

<figure><img src="image_29.png" /></figure>

**Figure S1.** Effect of minipubertal treatment on female sexual behavior. A-D. In the olfactory preference test, female mice treated with vehicle (Veh), Ganirelix (G), flutamide (F), letrozole (L) or flutamide/letrozole (F/L) had choice to chemoinvestigate anesthetized male or female stimuli. The total time in investigation (A, C) and the number of entries in both stimulus compartment (B, D) were assessed. In the three-chambered tests, sexually experienced males had choice to investigate either Veh- or G-treated females (E, F) or either from Veh- or F-, Veh- or L-, or Veh- or F/L-treated females (G, H). The total time males spent in stimulus compartments (E, G) and the number of entries in
14 stimulus compartments is indicated (F-H). In the urinary preference tests, sexually experienced males
15 had choice to investigate urine from either Veh- or G-treated females (I, J) or either from Veh- or F-
16 Veh- or L-, or Veh- or F/L-treated females (K, L). The total time males spent in stimulus compartments
17 (I, K) and the number of entries in stimulus compartments is indicated (J-L). Data are presented as
18 means ± S.E.M. (n = 12-15 per treatment group for Experiment 1; n = 10-13 per treatment group for
19 Experiment 2).
<figure><img src="image_30.png" /><figcaption>Figure S2. Effect of minipubertal treatment with Ganirelix® on ERα-immunoreactivity in the neural substrates of sexual behavior. A. Representative nuclear ERα-immunolabeling in the POA, VMH and ARC of vehicle- (Veh) and Ganirelix-treated female (G). Scale bar = 100μm. B-D. Quantitative analyses of the number of ERα-immunoreactive cells in the POA (B), VMH (C) and ARC (D). Data are presented as means ± S.E.M. (n = 3-6 per treatment).</figcaption></figure>
<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

**Figure S3.** Effect of minipubertal treatment on physiological parameters. **A, C.** The body weight at vaginal opening is indicated for Experiment 1 (**A**) and Experiment 2 (**C**). **B, D.** 2 weeks before sexual behavior tests, females treated with vehicle (Veh), Ganirelix (G), flutamide (F), letrozole (L) or flutamide/letrozole (F/L) were ovariectomized and the ovaries were weighted for Experiment 1 (**B**) and Experiment 2 (**D**). Data are presented as means ± S.E.M. (n = 12-15 per treatment for Experiment 1; n = 10-13 per treatment for Experiment 2).