Minipuberty regulates reproductive lifespan and ovarian follicular loss in a mouse model with reduced minipubertal gonadotropin levels

Abstract

Study questionWhat is the role of the physiological hypergonadotropic activity encountered at minipuberty on the implementation of female reproductive function, in a mouse model with manipulated minipubertal gonadotropin levels? Summary answerElevated minipubertal levels of gonadotropins may have long-term effects on fertility by mediating neuroendocrine aging and ovarian follicle depletion. What is known alreadyMinipuberty is characterized by the tremendous activation of the gonadotropin axis, as evidenced by elevated levels of gonadotropins regulating folliculogenesis as well as the synthesis of ovarian hormones including estradiol, testosterone, and AMH. Study design, size, durationTo determine whether hypergonadotropic activity of the gonadotropin axis at mini-puberty could impact reproductive parameters and female fertility, we used a pharmacological approach to suppress gonadotropin levels in Swiss mice by injecting daily a GnRH receptor antagonist (GnRHR) (Ganirelix, 10 g/mouse) or its vehicle between 10 and 16 postnatal days, to cover the entire duration of minipuberty. We analyzed the onset of puberty and estrous cyclicity as well as fertility in young (3 to 5 months) and middle-aged (11 months) mice from control (CTR) and antagonist-treated groups (n = 17 to 20 mice/age and treatment group). Ovaries and brains were collected, fixed and sectioned (for histology, follicle count and immunohistochemistry) or frozen (for analysis of follicular markers, aging and inflammation) from adult females, and blood was collected by cardiac puncture for hormonal assays (n = 3 to 8 mice/age and treatment group). Participants/materials, setting, methodsTo analyze the initiation of puberty, we monitored vaginal opening and performed vaginal smears to detect first estrus and diestrus 2 in control and antagonist-treated mice. We studied estrous cyclicity on vaginal smears to detect the occurrence of the different stages of the cycle at the beginning of reproductive life. Young and middle-aged mice of the two groups were mated several times with males to assess fertility rates, delay of conception and litter size. To evaluate ovarian function, we counted follicles at the primordial, primary, secondary and tertiary stages and corpora lutea by morphometric analyses, and we determined the relative intra-ovarian abundance of follicular markers (Amh, Inhba, Inhbb, Cyp19a1, Lhcgr, Fshr) by real-time RT-PCR, as well as the levels of circulating AMH and progesterone by ELISA and GC/MS, respectively. We also analyzed features of ovarian aging and inflammation (presence of oocyte-depleted follicles and multinucleated giant cells) by histology and by measuring the relative intra-ovarian abundance of Sirt1, Sod2, Tnfa and Il1b using real-time RT-PCR. To determine the impact on neuroendocrine determinants related to the control of reproduction, we analyzed circulating gonadotropin levels using Luminex assays as well as kisspeptin and GnRH immunoreactivity by immunohistochemistry in the hypothalamus, in both young and middle-aged mice. Main results and the role of chanceOur results show that the treatment had no impact on the initiation of puberty, estrous cyclicity, or fertility at the beginning of reproductive life. However, it increased reproductive lifespan, as shown by the higher percentage of antagonist-treated females than controls (33% versus 6%) still fertile at 11 months of age (P=0.0471). There were no significant differences in the number of kisspeptin and GnRH neurons, nor in the density of kisspeptin- and GnRH-immunoreactivity in the hypothalamic areas involved in reproduction between the two groups of mice studied at either 4 or 11 months. In addition, basal levels of LH and FSH were comparable between the two groups at 4 months, but not those of LH at 11 months which were much lower in females treated with antagonist than in their age-matched controls (237 {+/-} 59.60 pg/mL in antagonist-treated females versus 1027 {+/-} 226.3 pg/mL in controls, P=0.0069). Importantly, at this age, antagonist-treated mice had basal LH levels comparable to young mice (e.g., in 4-month-old controls: 294 {+/-} 71.75 pg/mL, P > 0.05), while those of control females were higher (P= 0.0091). Despite their prolonged reproductive lifespan and delayed neuroendocrine aging, antagonist-treated mice exhibited earlier depletion of their follicles, as shown by lower numbers of primordial, primary, and secondary follicles associated with lower circulating AMH levels and relative intra-ovarian abundance of Amh transcripts than control mice. However, they exhibited comparable completion of folliculogenesis, as suggested by the numbers of tertiary follicles and corpora lutea, relative intra-ovarian abundance of Cyp19a1, Inha and Inhb transcripts, and circulating progesterone levels that all remained similar to those of the control group. These observed alterations in ovarian function were not associated with increased ovarian aging or inflammation. Large scale datanone Limitations, reasons for cautionThis study was carried out on mice, which is a validated research model. However, human research is needed for further validation. Wider implications of the findingsThis study, which is the first to investigate the physiological role of minipuberty on reproductive parameters, supports the idea that high postnatal levels of gonadotropins may have long-term effects on female fertility by regulating the duration of reproductive life. Changes in gonadotropin levels during this period of life, such as those observed in infants born prematurely, may thus have profound consequences on late reproductive functions. Study funding/competing interest(s)This research was conducted with the financial support of ANR AAPG2020 (ReproFUN), CNRS, Inserm, Universite Paris Cite and Sorbonne Universite. The authors declare that they have no conflicts of interest.