OR18-1 The Impact of Makorin Ring Finger Protein 3 (MKRN3) Overexpression in the Arcuate Nucleus of Postpubertal Female Mice on the HPG Axis

Abstract

Abstract Background and Objective Makorin Ring Finger Protein 3 (MKRN3), an inhibitor of the KISS1 and TAC3 promoters in the hypothalamic arcuate nucleus (ARC), is an important player in age of menarche and etiology of central precocious puberty. However, its potential role in delayed puberty and/or hypogonadism is not defined. Mkrn3 expression in the ARC declines prior to the onset of puberty and remains low in adulthood, supporting its role as an inhibitor of pubertal onset. Neonatal hypothalamic overexpression of Mkrn3 leads to delayed puberty in female mice; this study aimed to explore the effects of MKRN3 overexpression in the postpubertal mouse. We hypothesized that MKRN3 overexpression in the ARC of postpubertal female mice would result in suppression of the HPG axis, manifested as hypogonadotropic hypogonadism. Methods and Results Adult female C57BL/6 mice were stereotaxically injected into the ARC with a recombinant adeno-associated virus (rAAV-Mkrn3-IRES-EGFP;n=12) to overexpress Mkrn3, or with a control virus (rAAV-EGFP;n=9). Daily vaginal smears collected six weeks post-injection showed that rAAV-Mkrn3 injected mice spent significantly more time (62.7%) in diestrus compared to controls (41.3%;P<0.01). Mean body weight did not statistically differ between groups. Eight weeks after viral injection, gonad-intact females underwent stimulation tests while in diestrus. Tail vein blood samples were collected at baseline and 20, 40 and 60 minutes after stimulation. In response to the NK3R agonist, senktide, evaluated at two doses (5 nmol and 10 nmol ip, respectively), Mkrn3-overexpressing mice had a reduced peak (i.e., T20) LH response compared to controls (P<0.001 for both dosing paradigms). In contrast, in response to kisspeptin-10 (1 nmol ip), mice responded with similar peak serum LH levels. Subsequently, tests were repeated seven days after ovariectomy and estradiol capsule replacement (OVX+E2). Peak LH levels (T20) were again significantly lower in response to senktide (5 nmol ip) in the rAAV-Mkrn3 injected females (P<0.001). Peak serum LH levels (T20) were not different in response to kisspeptin-10 (1 nmol) or GnRH (1.25 ng/g body weight). Additionally, there were no significant differences in serum LH levels between groups 7 days after OVX or OVX + E2. Conclusions In this novel mouse model, developed to investigate the mechanism of action of MKRN3, an important neuroendocrine controller of puberty initiation, these findings suggest that MKRN3 can exert inhibitory effects on the reproductive axis in female mice beyond the juvenile period. In these mice, response to kisspeptin and GnRH was preserved but the response to senktide was blunted. These findings suggest that Mkrn3 may act in ARC kisspeptin neurons to inhibit kisspeptin synthesis or secretion and/or potentially the action of neurokinin B via its receptor. Modulation of MKRN3 expression may be a future therapeutic target for reproductive disorders. Presentation: Monday, June 13, 2022 11:00 a.m. - 11:15 a.m.