LAUSR

Does Luteinizing Hormone (LH) protect the follicular endowment and growth by improving oocyte-granulosa cell (GC) communication of follicles exposed to chemotherapy at the primordial stage? LH treatment protects mouse primordial follicles against alkylating agents by preventing the chemotherapy-induced follicular depletion and the impairment of oocyte-GC communication during follicular growth. Impaired folliculogenesis is one of the most common deleterious side effects of alkylating agents in ovaries. Bidirectional communication between the oocyte and surrounding GCs is crucial for oocyte development. Therefore, defective gap junctions and reduced oocyte-derived factors compromise folliculogenesis, oocyte competence and meiotic maturation. Previous findings reported a significant LH protection of follicular viability and meiotic potential of MII oocytes exposed to chemotherapy at primordial stage. Therefore, we aimed to investigate the LH effects on cell junctions and communication between oocyte and GCs in growing follicles derived from quiescent oocytes exposed to alkylating chemotherapy. Adult 6-week-old CD1 female mice were allocated to control (n = 3), chemotherapy (ChT, n = 5) and ChT+LH (n = 5) groups. Chemotherapy (120 mg/Kg of cyclophosphamide and 12 mg/Kg of busulfan) was intraperitoneally administrated to ChT and ChT+LH mice. ChT+LH animals were pretreated with 1 IU of LH, followed by a second LH dose (1 IU) along with chemotherapy 24 hours later. Control mice only received vehicle (DMSO). Mice were euthanized 30 days later to collect ovaries. Follicles were mechanically isolated by puncture with 30-gauge needles from frozen-thawed half-ovaries. Isolated follicles measuring ≥100µm were selected to represent the secondary and later developmental stages. Part of them were kept intact while others were decumulated by using narrow pipettes to obtain denuded oocytes (DOs), and GCs. Follicles, ODs and GCs were analyzed by qRT-PCR to evaluate key factors in oocyte-GC junctions (Cx37, Cx43, Cdh1, Cdh2, Tjp1) and communication (Gdf9, Bmp15, Bmpr2, Alk4, Alk5, Alk6). Chemotherapy induced a 2.1-fold reduction in the number of total isolated follicles (p = 0.036), reducing 2.7-fold the number of primordial and primary follicles (<100 µm; p = 0.034) and 1.9-fold the amount of growing follicles (≥100 µm; p = 0.036) compared to controls. LH-treated ovaries showed a 1.6-fold increase in the total follicle isolation yield when compared to ChT (p = 0.032), recovering control-like values (p=ns). This LH protection specially benefited the early-stage follicles (<100 µm), where a 1.9-fold increase in the number of isolated follicles was detected compared to ChT group (p = 0.016). Gene expression analysis of follicles (n = 168), DOs (n = 110) and GCs (from n = 153 follicles) revealed a global downregulation pattern in ChT samples for all genes, when compared to controls, with a significant fold change (FC) reduction for Gdf9 in follicles (FC: 0.36±0.16); Cx37, Cdh2 and Gdf9 in DOs (FC: 0.23±0.17, 0.09±0.03, and 0.17±0.07, respectively); and Cx37, Cx43, Gdf9 and Bmp15 in GCs (FC: 0.40±0.23, 0.17±0.07, 0.17±0.08, and 0.04±0.01, respectively). However, LH treated samples showed an overall improvement of gene expression pattern reaching control-like levels for all genes excepting for a downregulation of the Bmp15 expression in GC (FC: 0.28±0.24; p = 0.036). Animal model study performed with a reduced sample size. Therefore, these findings should be validated in further studies with human tissue samples. Wider implications of the findings: Our findings suggest that LH treatment prevents the chemotherapy-induced follicle depletion. The LH protection of primordial population seems to preserve its ability to properly establish oocyte-GC interactions during growth and development, which is required to regulate follicular maturation and oocyte competence. Not applicable