Reduction in minipubertal gonadotropin levels alters reproductive lifespan and ovarian follicular loss in female mice

Abstract

Study question: What is the effect of attenuating the physiological hypergonadotropic activity encountered at minipuberty on female reproductive function in a mouse model?

Summary answer: Decreasing the surge of gonadotropins at minipuberty extended reproductive lifespan, coinciding with alterations in neuroendocrine and ovarian aging.

What is known already: Minipuberty is characterized by the tremendous activation of the gonadotrope axis, as evidenced by elevated levels of gonadotropins regulating folliculogenesis and the synthesis of ovarian hormones, but its role in fertility remains unclear.

Study design, size, duration: To determine the link between gonadotrope axis activity at minipuberty and reproductive parameters, we used a pharmacological approach to suppress gonadotropin levels in Swiss mice by injecting daily a GnRH receptor antagonist (GnRHR) (Ganirelix, 10 µg/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 (3-5 months) and middle-aged (11 months) mice from control (CTR) and antagonist-treated groups (n = 17-20 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 = 3-8 mice/age and treatment group).

Participants/materials, setting, methods: To analyze the initiation of puberty, we monitored vaginal opening and performed vaginal smears in CTR and antagonist-treated mice. We studied estrous cyclicity on vaginal smears at the beginning of reproductive life. Mice were mated several times with males to assess fertility rates, delay of conception, and litter size. To evaluate ovarian function, we counted follicles at different stages and corpora lutea, and we determined the relative intra-ovarian abundance of key follicular markers by real-time RT-PCR, as well as the levels of circulating anti-Müllerian hormone (AMH) and progesterone by ELISA and GC-MS, respectively. We also analyzed features of ovarian aging and inflammation by histology and by measuring the relative intra-ovarian abundance of some markers using real-time RT-PCR. To determine the impact on neuroendocrine determinants related to the CTR of reproduction, we analyzed circulating gonadotropin levels using Luminex assays as well as kisspeptin and GnRH immunoreactivity in the hypothalamus by immunohistochemistry.

Main results and the role of chance: Our 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 CTRs still fertile at 11 months of age (33% versus 6%; P = 0.0471). There were no significant differences in the number of kisspeptin and GnRH neurons, nor in the density of kisspeptin- and GnRH-immunoreactive neurons 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 FSH were comparable between the two groups at 4 and 11 months, but not those of LH at 11 months which were much lower in females treated with antagonist than in their age-matched CTRs (237 ± 59.6 pg/ml in antagonist-treated females versus 1027 ± 226.3 pg/ml in CTRs, P = 0.0069). Importantly, at this age, antagonist-treated mice had basal LH levels comparable to young mice (e.g. in 4-month-old CTRs: 294 ± 71.75 pg/ml, P > 0.05). 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 preantral follicles associated with lower circulating AMH levels and relative intra-ovarian abundance of Amh transcripts than CTR mice. However, they exhibited comparable completion of folliculogenesis, as suggested by the numbers of antral follicles and corpora lutea, relative intra-ovarian abundance of Cyp19a1, Inhba, and Inhbb transcripts, and circulating progesterone levels that all remained similar to those of the CTR group. These observed alterations in ovarian function were not associated with increased ovarian aging or inflammation.

Large-scale data: None.

Limitations, reasons for caution: This study was carried out on mice, which is a validated research model. However, human research is needed for further validation.

Wider implications of the findings: This study, which is the first to investigate the physiological role of minipuberty on reproductive parameters, supports the idea that suppressing the high postnatal levels of gonadotropins may have long-term effects on female fertility by extending the duration of reproductive life. Perturbations 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, Université Paris Cité, and Sorbonne Université. The authors declare that they have no conflicts of interest.

Keywords: FSH; LH; aging; hypothalamus; minipuberty; ovary; reproductive function.

Figure 1.

Determination of circulating FSH levels during the prepubertal period in Swiss mice and after treatment by the GnRHR antagonist during minipuberty. (A) Circulating levels of FSH measured by Luminex assay in wild-type mice at 8, 12, 14, 17, 21, and 28 dpn (4–12 samples per age). (B) Serum concentration of FSH measured by Luminex assay at 14, 17, and 21 dpn in CTR and GnRHR antagonist-treated mice (4–6 samples/age in CTR and antagonist-treated mice). In graphs, bars are the means ± SEM. Data were analyzed using a one-way ANOVA test (A) and a Mann–Whitney U test (B). Distinct letters indicate significant differences between groups, with P < 0.05. CTR, control.

Figure 2.

Reproductive parameters in mice with reduced minipubertal gonadotropin levels. (A) Determination of the age of vaginal opening, first estrus, and first diestrus 2, by daily morning vaginal smears (28 females/group). (B) Percentage of time spent in estrus and diestrus 2 in 3- to 4-month-old females on 21 consecutive days of vaginal smears (20 females for CTR and 17 for antagonist-treated mice). (C) Fertility rate based on the percentage of fertile mice at 3, 4, 5, and 11 months (n = 20 females/group at 3, 4, and 5 months; 17 females for CTR and 15 females for antagonist-treated mice at 11 months). (D) Litter size at 3, 4, 5, and 11 months based on the number of pups seen at birth (20 females/group at 3, 4, and 5 months; 17 females for CTR and 15 females for antagonist-treated mice at 11 months). (E) Time to conception, corresponding to the time between mating and parturition (20 CTR and 19 antagonist-treated females at 3, 4, and 5 months; 17 females for CTR and 15 females for antagonist-treated mice at 11 months). (F) Body weights at the time of vaginal opening, 4, 8 weeks, and 3–4 and 11 months (18–62 females for CTR and 16–61 for antagonist-treated mice). In graphs, bars are the means ± SEM. Data were analyzed using Student’s t-test or Mann–Whitney U test (A, B), a chi-square test (C), and a two-way ANOVA test. CTR, control.

Figure 3.

Analysis of the impact of reduced minipubertal gonadotropin levels on central determinants of reproductive function in young and middle-aged mice. (A) Serum concentrations of basal LH and FSH levels were measured by Luminex assay at 4 and 11 months in CTR and antagonist-treated mice (6–10 mice/group). (B) Representative images and quantification of the number of GnRH-immunoreactive neurons in the rostral preoptic area (rPOA) and mean density of GnRH immunoreactivity in the median eminence (ME) of 4- and 11-month-old females in diestrus (3–6 females/age and treatment group). (C) Representative images and quantification of the number of kisspeptin-immunoreactive neurons in the rostral periventricular area of the third ventricle (RP3V) composed of the anteroventral periventricular (AVPV), rostral (rPeN), and caudal (cPeN) preoptic periventricular nuclei and mean density of kisspeptin immunoreactivity in the arcuate nucleus (ARC) of 4- and 11-month-old females in diestrus (n = 3–6 females/age and treatment group). 3V = third ventricle. Scale bar = 100 µm. In graphs, bars are the means ± SEM. Data were analyzed using a one-way ANOVA test (A), Student’s t-test (4 months), or Mann–Whitney U test (11 months) (B, C). CTR, control.

Figure 4.

Impact of GnRHR antagonist treatment on follicular and corpora lutea content in middle-aged mice. (A) Histological ovarian sections of middle-aged ovaries from CTR and antagonist-treated mice, showing corpora lutea (*) and antral follicles (AF). (B–C) Morphometric analysis of the number of healthy (B) and atretic follicles (C) in each category at 11 months (6–7 ovaries from different females/group). (D) Morphometric analysis of the number of corpora lutea (CL), which were classified into three categories according to their size and the presence or not of apoptotic luteal cells (regressing: type 1; formed one or more cycles before: type 2; formed during the current cycle: type 3) (6–7 ovaries from different females/group). (E) Serum concentrations of progesterone levels were measured by GC-MS (5 samples from different females/group). In graphs, bars are the means ± SEM. Data were analyzed using Student’s t-test or Mann–Whitney U test (B–E). CTR, control; GC-MS, mass spectrometry coupled with gas chromatography.

Figure 5.

Analysis of ovarian function in middle-aged mice treated with the GnRHR antagonist at minipuberty. (A) Schematic representation of key markers of endocrine activity in the ovary. Shown are the androgen-converting enzyme Cyp19a1 aromatase, the inhibin beta subunits (Inhba, Inhbb), Amh, and Fshr, which are all expressed in granulosa cells of growing follicles, and Lhcgr (encoding the LH receptor) expressed in thecal cells. (B) Serum concentration of AMH was measured by ELISA assay at 11 months (5–6 samples from CTR and antagonist-treated mice). (C–H) Relative intra-ovarian abundance of Amh, Cyp19a1, Inhba, Inhbb, Fshr, and Lhcgr transcripts in CTR and antagonist-treated mice aged 11 months determined by quantitative real-time RT-PCR and normalized to the mRNA levels of Hprt (5–6 ovaries from different females/group). In graphs, bars are the means ± SEM. Data were analyzed using a nonparametric Student’s t-test (B–H). AMH, anti-Müllerian hormone; CTR, control.

Figure 6.

Ovarian aging and inflammation in middle-aged mice treated with the GnRHR antagonist at minipuberty. (A) Histological ovarian sections of middle-aged ovaries from CTR and antagonist-treated mice showing typical signs of aging, i.e. oocyte-depleted follicles (B) and multinucleated giant cells (C). Morphometric analyses of oocyte-depleted follicles at 11 months (6–7 ovaries from different females/group). (C) Percentage of ovaries with multinucleated giant cells at 11 months (6–7 ovaries from different females/group). (D–G) Relative intra-ovarian abundance of Sirt1, Sod2, Tnfa, and Il1b transcripts in the ovaries of 11 months for CTR and antagonist-treated mice determined by quantitative real-time RT-PCR and normalized to the mRNA levels of Hprt (6 ovaries from different females/group). In graphs, bars are the means ± SEM. Data were analyzed using Student’s t-test (B, D–G) and chi-square test (C). CTR, control.