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.

Analysis of Secreted Proteins from Prepubertal Ovarian Tissues Exposed In Vitro to Cisplatin and LH

“…In previous papers, we reported a significant and unexpected protective effect of luteinizing hormone (LH) against the cisplatin (CIS)-induced apoptosis of the primordial follicle pool in prepubertal ovaries [ 2 ]. Moreover, we confirmed such LH protection both in prepubertal and adult animals [ 2 , 3 ]. In the in vitro system, we also found that LH treatment of the ovarian tissues generated molecular signals in the ovarian somatic cells that, through the activation of PI3K/AKT and cAMP/PKA pathways in the oocytes, prevented their apoptosis [ 2 ].…”

“…In the present work, our main aim was to characterize the extracellular in vitro microenvironment in which the oocytes within the primordial/primary follicles underwent apoptosis induced by CIS or were, for the most part, protected by concomitant administration of LH [ 2 , 3 , 6 ]. Moreover, we looked to confirm the ability of early postnatal ovaries to respond to LH stimulation.…”

“…The actions of CIS and LH were clearly reciprocally influenced when the compounds were used together offering some clues as to how the hormone influences the composition of the extracellular environment in order to protect primarily the oocytes enclosed in primordial/primary follicles from apoptosis [ 2 , 3 , 6 ]. As a matter of fact, it is likely that such LH protective effect occurs through multiple ways.…”

Deficiency of Wnt10a causes female infertility via the β-catenin/Cyp19a1 pathway in mice

“…All sections were observed with a light microscope (Olympus VS120, Olympus, Tokyo, Japan) and digitized with software VS-ASW (Olympus). The ovarian follicles were classified into 4 types as described previously 21 , 22 : primordial follicles, having one layer of flattened pre-granulosa cells surrounding the oocyte; primary follicles, showing one layer of cuboidal granulosa cells; secondary follicles, containing at least two layers of granulosa cells; and atretic follicles, exhibiting disorganized, apoptotic, and degenerative changes in the eosinophilic granulosa cells of the ooplasm, contraction and clumping of the chromatin, and wrinkling of the oocyte nuclear membrane. The endometrial thickness was measured from the luminal surface to the beginning of the circular smooth muscle layer with ImageJ software, as previously reported 2 .…”

PQQ Dietary Supplementation Prevents Alkylating Agent-Induced Ovarian Dysfunction in Mice

“…Jiang et al showed that only a single injection of BUL and CTX can cause accelerated follicles depletion in mice, mimicking the process of ovarian aging (6). This mouse model has been widely adopted as a mouse model of POI for study (9)(10)(11).…”