The role of minipuberty in female reproductive function programming – REPROFUN

Various mouse models are used in ReproFun. They all harbor a time-controlled and reversible manipulation of ovarian hormone levels (E2, T and AMH) at mini-puberty, obtained by pharmacological approaches. Model 1 displays an overall decrease in ovarian endocrine activity following GnRHR antagonist treatment. The respective roles of E2, T or AMH is specified by supplementing these mice with each of these hormones. In contrast, model 2 displays an overall increase in ovarian endocrine activity after exogenous gonadotropin (PMSG) treatment. This model mimics “excessive” mini-puberty as observed in pre-term infants. Model 3 exhibits low levels of E2 due to aromatase inhibitor (anastrozole) treatment, whereas model 4 has no actions of androgens due to androgen receptor antagonist treatment (flutamide). Models 3 and 4 complete the analysis of E2 and T's actions from Model 1.
The following methods are used on these models:
-Determination of puberty onset: daily follow-up of vaginal opening, daily vaginal smears to detect the first estrus and diestrus, and ovarian morphometric analyses to detect 1st ovulation.
- Estrous cycles: daily vaginal smears in adult females.
- Reproductive capacity (time to conceive, number and size of litters): continuous mating of females with WT males for 12 months. These parameters are completed by morphometric analyses of the ovaries to assess follicular growth and ovulation, and by measuring serum levels of different hormones attesting HPO function (LH, FSH, E2, progesterone, collaboration Dr F. Giton, IMRB, Créteil).
-Activity of the hypothalamus: analyses of the number of GnRH- and kisspeptin-immunoreactive neurons in the preoptic area and RP3V, fiber density in the ARC, and appositions of kisspeptin fibers to GnRH cell bodies in prepubertal and adult females on brain sections (immunofluorescence, confocal microscopy).
-Behaviors. For sexual behavior, naive and sexually-experienced females are analyzed for their olfactory preference, partner preference and lordosis posture, in the presence of sexually-experienced males. For maternal behavior, dams are analyzed for nursing, nest-related and self-directed behaviors. The neural circuitries involved in sexual and maternal behaviors (medial amygdala, bed nucleus of stria terminalis, preoptic area, ventromedial hypothalamus) are analyzed by immunohistochemistry for the expression of ER and its target genes (progesterone and oxytocin receptors).
-Genetic & epigenetic regulations induced by ovarian hormones during minipuberty. We will perform single nucleus RNA-seq studies coupled with ATAC seq using tissue punches of the RP3V and ARC rom prepubertal and adult mice. It will be followed by searches of histone marks and DNA methylation in signature genes, as successfully carried out in Partner 1's team.

Partner 1 has already optimized 3 out of 4 mouse models, in terms of period of treatment and drug doses, so that they are now suitable for the ReproFun project, as expected in Task 1. The characterization of the fourth model (Model 3, aromatase inhibitor) will be launched next month.
Partner 1 has analyzed the reproductive parameters on model 1, in two strains of mice (Swiss and C57BL/6). Only C57BL/6 mice were supplemented or not with ovarian hormones at the time of mini-puberty. We observed that GnRHR antagonist-treatment induces a modest delay in puberty onset in the Swiss strain (P< 0.05), but not in the C57BL/6 strain. Our complementary study by in situ ovarian analyses (PubScore) to specify the age at first ovulations, did not reveal any significant differences between groups. We observed an alteration in sexual cyclicity in treated Swiss mice (lengthening of the time spent in diestrus 2) but not in C57BL/6 mice. In line with these findings, Partner 2 observed a decrease in the number of ir-kisspeptin neurons in the AVPV at the time of preovulatory surge in Swiss mice. Analyses on C57BL/6 mice are on-going.
There was no change in fertility in the first three months of reproductive life in the two strains of mice (P>0.05, n=12-20 mice/group). This fertility study, initially planned for a period of 3 months, was continued to highlight any long-term effects. Importantly, reproductive lifespan may be extended in treated Swiss mice, since at 10 months 50% of them were pregnant, compared to 16% of control females (P<0.05; n=16-17 mice/group). These findings indicate that reproductive senescence, observed at 10 months in control females, may be postponed in treated females of the Swiss strain. The study of the «late« fertility in the C57BL/6 strain is still in progress as they were 9 months old at the time of the report. Our subsequent studies of different ovarian markers (inhibin beta A and beta B subunits, aromatase, AMH) in aging Swiss mice show that their reproductive longevity would not be due to improved ovarian function. However, we observed that aging females displayed low basal LH concentrations similar as young adult females (4 months) in the GnRHR antagonist group, while control females exhibited significantly higher LH levels. These findings suggest that the extension of reproductive life in GnRHR treated mice at the time of mini-puberty would not result from delayed ovarian aging, but rather to delayed hypothalamus aging in these mice.

Data from the ReproFun project point to a quite unexpected result: suppressing pituitary gonadotrope activity, and therefore the overall ovarian activity, specifically at the time of mini-puberty would result in longer reproductive life in female mice. Studies of the underlying mechanisms suggest that this reproductive «longevity« is not due to delayed ovarian aging, but rather to delayed hypothalamic aging. This physiological process is considered as the principal cause of reproductive senescence in rodents, but how it is triggered is not known. Our subsequent analyses will identify the involved ovarian hormone(s) and specify the hormonally-regulated genetic and epigenetic modifications occurring during mini-puberty that can impact reproductive lifespan.

1 review paper in a peer-reviewed journal:

Deciphering the roles & regulation of estradiol signaling during female mini-puberty: insights from animal models. Marie M Devillers, Sakina Mhaouty-Kodja, Céline J Guigon. Accepted 4 Nov2022, IJMS

3 scientific meetings:
1. 64ème Journées Internationales d’Endocrinologie clinique Henri-Pierre Klotz, Paris Juin 2022.
2. 22ème Congrès international de neuroendocrinology (ICN), Aout 2022, Glasgow, Ecosse
3. Journées scientifiques de la Société Française de Neuroendocrinologie (, Lyon, septembre 2022

An intriguing event in mammals is the activation of the hypothalamo-pituitary ovarian axis for a short time period during infantile life referred to as minipuberty. The levels of pituitary gonadotropins, ovarian sex steroids and anti-müllerian hormone (AMH) are then similar or even higher than in adults. Although well conserved in female mammals, its physiological role remains elusive. Our hypothesis is that this developmental phase is critical for the differentiation of neural circuitry regulating ovulation and reproductive behavior. In this context, our objectives are to seek the effect of sex steroids and AMH produced during minipuberty on female fertility. We will study reproductive performance and behavior as well as epigenetic modifications arising in underlying neural circuitry in mouse models allowing modulation of sex steroid and AMH levels specifically during mini-puberty. This project will help to understand the mechanisms of human reproduction and the origin of its dysfunctions.