The cyto-protective effects of LH on ovarian reserve and female fertility during exposure to gonadotoxic alkylating agents in an adult mouse model

Abstract

Study question: Does LH protect mouse oocytes and female fertility from alkylating chemotherapy?

Summary answer: LH treatment before and during chemotherapy prevents detrimental effects on follicles and reproductive lifespan.

What is known already: Chemotherapies can damage the ovary, resulting in premature ovarian failure and reduced fertility in cancer survivors. LH was recently suggested to protect prepubertal mouse follicles from chemotoxic effects of cisplatin treatment.

Study design, size, duration: This experimental study investigated LH effects on primordial follicles exposed to chemotherapy. Seven-week-old CD-1 female mice were randomly allocated to four experimental groups: Control (n = 13), chemotherapy (ChT, n = 15), ChT+LH-1x (n = 15), and ChT+LH-5x (n = 8). To induce primary ovarian insufficiency (POI), animals in the ChT and ChT+LH groups were intraperitoneally injected with 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan, while control mice received vehicle. For LH treatment, the ChT+LH-1x and ChT+LH-5x animals received a 1 or 5 IU LH dose, respectively, before chemotherapy, then a second LH injection administered with chemotherapy 24 h later. Then, two animals/group were euthanized at 12 and 24 h to investigate the early ovarian response to LH, while remaining mice were housed for 30 days to evaluate short- and long-term reproductive outcomes. The effects of LH and chemotherapy on growing-stage follicles were analyzed in a parallel experiment. Seven-week-old NOD-SCID female mice were allocated to control (n = 5), ChT (n = 5), and ChT+LH-1x (n = 6) groups. Animals were treated as described above, but maintained for 7 days before reproductive assessment.

Participants/materials, setting, methods: In the first experiment, follicular damage (phosphorylated H2AX histone (γH2AX) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay), apoptotic biomarkers (western blot), and DNA repair pathways (western blot and RT-qPCR) were assessed in ovaries collected at 12 and 24 h to determine early ovarian responses to LH. Thirty days after treatments, remaining mice were stimulated (10 IU of pregnant mare serum gonadotropin (PMSG) and 10 IU of hCG) and mated to collect ovaries, oocytes, and embryos. Histological analysis was performed on ovarian samples to investigate follicular populations and stromal status, and meiotic spindle and chromosome alignment was measured in oocytes by confocal microscopy. Long-term effects were monitored by assessing pregnancy rate and litter size during six consecutive breeding attempts. In the second experiment, mice were stimulated and mated 7 days after treatments and ovaries, oocytes, and embryos were collected. Follicular numbers, follicular protection (DNA damage and apoptosis by H2AX staining and TUNEL assay, respectively), and ovarian stroma were assessed. Oocyte quality was determined by confocal analysis.

Main results and the role of chance: LH treatment was sufficient to preserve ovarian reserve and follicular development, avoid atresia, and restore ovulation and meiotic spindle configuration in mature oocytes exposed at the primordial stage. LH improved the cumulative pregnancy rate and litter size in six consecutive breeding rounds, confirming the potential of LH treatment to preserve fertility. This protective effect appeared to be mediated by an enhanced early DNA repair response, via homologous recombination, and generation of anti-apoptotic signals in the ovary a few hours after injury with chemotherapy. This response ameliorated the chemotherapy-induced increase in DNA-damaged oocytes and apoptotic granulosa cells. LH treatment also protected growing follicles from chemotherapy. LH reversed the chemotherapy-induced depletion of primordial and primary follicular subpopulations, reduced oocyte DNA damage and granulosa cell apoptosis, restored mature oocyte cohort size, and improved meiotic spindle properties.

Large scale data: N/A.

Limitations, reasons for caution: This was a preliminary study performed with mouse ovarian samples. Therefore, preclinical research with human samples is required for validation.

Wider implications of the findings: The current study tested if LH could protect the adult mouse ovarian reserve and reproductive lifespan from alkylating chemotherapy. These findings highlight the therapeutic potential of LH as a complementary non-surgical strategy for preserving fertility in female cancer patients.

Study funding/competing interest(s): This study was supported by grants from the Regional Valencian Ministry of Education (PROMETEO/2018/137), the Spanish Ministry of Science and Innovation (CP19/00141), and the Spanish Ministry of Education, Culture and Sports (FPU16/05264). The authors declare no conflict of interest.

Keywords: DNA repair; LH; cancer; chemotherapy; fertility preservation; follicle protection; ovoprotection.

Figure 1.

LH treatment prevented follicular depletion, atresia, and stromal degeneration induced by chemotherapy. Alkylating agents were administered with or without LH and ovaries analyzed 30 days later. (A) Chemotherapy (ChT) treatment reduced the number of total follicles assessed in hematoxylin and eosin (H&E) stained sections, and LH co-administration blunted this effect. (B) All follicular subpopulations were higher in the LH-cotreated group than in the ChT group. (C) Percentages of morphologically abnormal follicles were similar in the LH and control group, but the ChT group showed a significant increase in atretic follicles. (D) Stromal degeneration index (fibrotic non-cellular or tissue absent area/total tissue area of each sample, normalized to control group index), and representative images at 2.5× (top, scale bar = 800 µm) and 10× (bottom, scale bar = 200 µm) magnification showing that LH preserves stromal morphology. Disrupted regions were identified as fibrotic areas and are indicated with black arrows in 10× images. Scatter plots show individual data and means for all groups (n = 3 in control and n = 5 in ChT and chemotherapy with LH (ChT+LH-1×) groups). Statistical significance was determined by two-tailed Mann–Whitney U test; *P-values <0.05 were considered statistically significant.

Figure 2.

LH treatment ameliorated the effects of alkylators on oocyte quantity and quality. Alkylating agents were administered with or without LH and controlled ovarian stimulation (COS) performed 30 days later to release oocytes that had been exposed to chemotherapy during their quiescent stage. (A) Number of morphologically normal metaphase II (MII) oocytes recovered after COS from all experimental groups. LH-treated mice ovulated greater numbers of MII-oocytes than ChT-treated mice (B) Number of 2-cell stage embryos. (C) Representative confocal images of meiotic spindles from MII-oocytes (n = 9 in control, and n = 12 in ChT and in ChT+LH groups). α-tubulin staining (green) (showing spindles), chromosomes (blue), and CREST-centromere proteins (red) were visualized to evaluate chromosome alignment and microtubule–chromosome attachment. The right column shows high magnification images of merged optical sections. White scale bar = 10 µm; yellow scale bar = 1.25 µm. (D) Calculated spindle areas. LH treatment reversed the chemotherapy-induced reduction in spindle area. (E) Chromosomal alignment with reference to the metaphase plate. The LH-treated group had a lower percentage of misaligned chromosomes than the ChT group. Scatter plots indicate individual data and means of all analyzed mice (n = 5 in controls, and n = 7 in ChT and ChT+LH-1× groups). Outliers are indicated by a cross. Statistical significance was determined by two-tailed Mann–Whitney U test (A and B) or linear (D) and logistic (E) regressions; *P-values <0.05 were considered statistically significant.

Figure 3.

LH co-administration improved breeding outcomes and extended reproductive lifespan. Mice were treated with alkylating agents with or without LH and 1 month later tested for breeding performance in six consecutive mating attempts. (A) ChT (black) reduced the cumulative pregnancy rate and LH treatment reversed this, with the 1× dose (green) being more effective than the 5× dose (purple). (B) ChT reduced the mean litter size and LH-1× reversed this effect. (C) ChT reduced the total number of pups and LH-1×x ameliorated this effect. (D) Representative first and last litters from all experimental groups. Graphs show means and, where indicated, SD for each experimental group (n = 4 animals/group). Statistical significance for litter size was determined by two-tailed Mann–Whitney U test; *P-values <0.05 compared with the control group were considered statistically significant.

Figure 4.

LH promoted DNA repair and cell survival and reduced the deleterious effects of chemotherapy on ovarian tissue. Alkylating agents were administered with or without LH and ovaries assessed 12 and 24 h after (A) phosphorylated H2AX histone (γH2AX) immunofluorescence (red) counterstained with DAPI (blue) of ovarian samples collected 12 h after treatments with alkylating agents with or without a low (1×) or high (5×) dose of LH (n = 3 in controls, and n = 4 in ChT, ChT+LH-1×, and ChT+LH-5× groups). Images on the left are at 20× (white scale bar = 100 µm) and on the right at 40× (yellow scale bar = 50 µm). The percentages of follicles showing γH2AX positive oocytes were quantified. The LH-treated groups had lower percentages of follicles with double-strand breaks (DSB) than the ChT group. (B) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (red) counterstained with DAPI (blue) was performed on ovarian sections 12 h after treatments. Magnifications and scale bars are as described in (A). Both LH-treated groups had a lower percentage of apoptotic follicles than the ChT group. (C) Representative western blots (WB) showing the levels of the anti-apoptotic protein B-cell lymphoma-2 (Bcl2) and the pro-apoptotic protein cleaved caspase-3 (CC3). Both doses of LH increased the Bcl2: CC3 ratio protecting the ovaries from ChT-induced cell death at 24 h. (D) Representative WB showing phosphorylated-extracellular signal-regulated Kinase 1 and 2 (pERK1/2) and ERK1/2 protein levels. ChT activated ERK1/2 signaling and LH treatments reduced this effect at 12 and 24 h. (E) Representative WB for ataxia-telangiectasia mutated kinase (ATM) and RAD51 recombinase (Rad51) proteins. LH-5×-treated ovaries expressed higher levels of ATM and Rad51 than ChT-treated ovaries at 12 and 24 h. (F) Representative WB for phosphorylated-serine/threonine-protein Kinase 1 (pAkt) and Akt proteins. Chemotherapy caused a notable increase of Akt activation at both time-points, and cotreatment with LH blocked this effect. All WBs were performed from at least two independent experiments from two pools of two ovaries each per group. (G) mRNA expression levels of the indicated DNA repair genes at 12 (left) and 24 h (right). At 12 h, the expression levels of all repair genes were higher in the ChT+LH-1× samples than in the ChT samples. At 24 h, Rad51 expression was higher in both LH groups than in the ChT group. Three independent ovarian fragments per group and time-point was analyzed, and expression levels were normalized to those of the control group. Scatter plots indicate individual data and means; bar charts display means and SDs. Statistical significance was determined by two-tailed Mann–Whitney U test; P-values <0.05 were considered statistically significant. *P < 0.05 or ^ap <0.05, ^bP <0.05 and ^cP <0.05 indicating statistical differences from the control, ChT, and ChT+LH-1× group, respectively.

Figure 5.

LH treatment protected the ovarian reserve and stromal architecture and prevented ChT-induced follicular damage in a subfertile NOD/SCID mouse model. Mice were treated with alkylating agents with or without LH and seven days later underwent COS. Ovaries were harvested and examined for follicle numbers, DNA damage, and apoptosis. (A) Total follicle numbers. (B) Follicle subpopulations. The LH group had more total follicles than the ChT group. This effect was most appreciable in the primordial and primary populations, (C) ChT depleted the quiescent population and LH blocked this effect. (D) Representative H&E stained images captured at 10× (top, scale bar = 200 µm) and magnified images of the boxed regions at 20× (bottom, scale bar = 100 µm) showing primordial (black arrows) and primary (black asterisks) follicles. (E) LH treatment was unable to reverse the ChT-induced increase in the percentage of morphologically abnormal follicles. (F) Stromal degeneration index and representative images of lesions visualized at 2.5× (top, scale bar = 800 µm) and 10× (bottom, scale bar = 200 µm). Fibrotic areas are indicated with black arrows in 10x images. LH treatment protected ovaries from ChT-induced stromal degeneration. (G) Representative images of γH2AX (red) immunofluorescence counterstained with DAPI (blue). LH treatment protects cells from ChT-induced double-strand breaks. Scale bar = 100 µm. (H) Representative images of TUNEL-staining (red) counterstained with DAPI (blue). Scale bar = 50 µm. Apoptotic follicles (≥ 20% labeled cells) were quantified. ChT treatment increased the percentage of apoptotic follicles, and LH treatment reversed this effect. Scatter plots indicate individual data and means of all analyzed mice (n = 3 in Control and ChT, and n = 4 in ChT+LH-1× groups). Statistical significance was determined by two-tailed Mann–Whitney U test; *P-values <0.05 were considered statistically significant.

Figure 6.

LH treatment ameliorated the effects of alkylators on oocyte quality in NOD/SCID mice. Mice were treated with alkylating agents with or without LH and 7 days later underwent COS to release oocytes that had been exposed to chemotherapy during their growth stage. (A) ChT reduced the number of healthy MII-oocytes ovulated after COS, and LH blunted this effect. (B) ChT decreased the number of early-cleavage stage embryos, and LH was unable to block this effect. (C) Bayesian model predicting the probability of spindle presence in MII-oocytes. LH treatment promoted spindle assembly during chemotherapy. (D) Representative images of meiotic spindles from ovulated MII-oocytes (n = 8 in Control, n = 7 in ChT, and n = 13 in ChT+LH-1× groups) and a high magnification view of the equatorial plate. α-tubulin staining (green), chromosomes (blue), and CREST-protein centromeres (red) were visualized. White scale bar = 10 µm; yellow scale bar = 1.25 µm. (E) Analysis of spindle area indicates LH treatment can ameliorate the effects of ChT. (F) Percentage of MII-oocytes with at least one misaligned chromosome as referred to the equatorial plate. ChT treatment resulted in a higher percentage of oocytes with misaligned chromosomes and LH treatment reversed this effect. Scatter plots indicated individual data and means of all analyzed mice (n = 5 in Control and ChT, and n = 6 in ChT+LH-1× groups). Statistical significance was determined by two-tailed Mann–Whitney U test (A and B), Bayesian analysis (C), linear (E) and logistic (F) regressions; *P-values <0.05 were considered statistically significant.