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The presence of insulin-like growth factor 2 (IGF2) mRNA has been demonstrated in human and mouse spermatozoa. However, the expression of the IGF2 protein in spermatozoa and its function is not known. To accomplish this, we searched for IGF2 protein in spermatozoa of four healthy Caucasian men by Western blot (WB) and immunofluorescence (IM). Semen samples were collected by masturbation into a sterile container after 3-4 days of sexual abstinence. The spermatozoa were separated from leucocytes and epithelial cells by swim-up. The WB demonstrated the presence of the IGF2 protein in each sample examined. IM showed that IGF2 is a cytoplasmic protein with a variable degree of expression in each spermatozoon and localized in the equatorial and post-acrosomal segments. To evaluate the effects of IGF2, porcine Sertoli cells (SCs) were isolated from neonatal Large White pigs (7-15 days) using established methods. SCs were incubated with increasing concentrations (0.33, 3.33, and 10 ng/mL) of recombinant human IGF2 (rhIGF2) for 48 hours. At the end of incubation, the following endpoints were evaluated: a) expression of the GDNF, FGF2, and SCF genes, known to be mitogens that promote gonocyte proliferation and differentiation towards their spermatogonial fate, by real-time PCR (RT-PCR); b) gene (RT-PCR) and protein (WB and IM) expression of the follicle-stimulating hormone receptor (FSHR); and c) proliferation of SCs by flow cytometry. Subsequently, the effects of IGF2 were re-evaluated by pre-treating SCs with NVP-AEW541, a not competitive inhibitor of the insulin-like growth factor 1 receptor (IGF1R), known to be activated by IGF2. For this purpose, the NVP-AEW541 was added to the culture medium at a concentration of 1 µg/mL one hour before the IGF2 and was left for the entire duration of the incubation. The results of this study showed that IGF2 significantly downregulates GDNF gene expression in a concentration-dependent manner (-40.3% at 0.33 ng/mL, -48.2% at 3.33 ng/mL, -55.5%, respectively, p Presentation: Monday, June 13, 2022 11:30 a.m. - 11:45 a.m.

Anti-Müllerian hormone (AMH) is secreted by Sertoli or granulosa cells. Recent evidence suggests that AMH may play a role in the pathogenesis of hypogonadotropic hypogonadism (HH) and that its serum levels could help to discriminate HH from delayed puberty. Moreover, the growth hormone (GH)/insulin-like growth factor 1 (IGF1) system may be involved in the function of gonadotropin-releasing hormone (GnRH) neurons, as delayed puberty is commonly found in patients with GH deficiency (GHD) or with Laron syndrome, a genetic form of GH resistance. The comprehension of the stimuli enhancing the migration and secretory activity of GnRH neurons might shed light on the causes of delay of puberty or HH. With these premises, we aimed to better clarify the role of the AMH, GH, and IGF1 on GnRH neuron migration and GnRH secretion, by taking advantage of previously established models of immature (GN11 cell line) and mature (GT1-7 cell line) GnRH neurons. Expression of Amhr, Ghr, and Igf1r genes was confirmed in both cell lines. Cells were then incubated with increasing concentrations of AMH (1.5–150 ng/mL), GH (3–1000 ng/mL), or IGF1 (1.5–150 ng/mL). All hormones were able to support GN11 cell chemomigration. AMH, GH, and IGF1 significantly stimulated GnRH secretion by GT1-7 cells after a 90-min incubation. To the best of our knowledge, this is the first study investigating the direct effects of GH and IGF1 in GnRH neuron migration and of GH in the GnRH secreting pattern. Taken together with previous basic and clinical studies, these findings may provide explanatory mechanisms for data, suggesting that AMH and the GH-IGF1 system play a role in HH or the onset of puberty.