Hypothalamic Overexpression of Makorin Ring Finger Protein 3 Results in Delayed Puberty in Female Mice

Abstract

Makorin ring finger protein 3 (MKRN3) is an important neuroendocrine player in the control of pubertal timing and upstream inhibitor of gonadotropin-releasing hormone secretion. In mice, expression of Mkrn3 in the hypothalamic arcuate and anteroventral periventricular nucleus is high early in life and declines before the onset of puberty. Therefore, we aimed to explore if the persistence of hypothalamic Mkrn3 expression peripubertally would result in delayed puberty. Female mice that received neonatal bilateral intracerebroventricular injections of a recombinant adeno-associated virus expressing Mkrn3 had delayed vaginal opening and first estrus compared with animals injected with control virus. Subsequent estrous cycles and fertility were normal. Interestingly, male mice treated similarly did not exhibit delayed puberty onset. Kiss1, Tac2, and Pdyn mRNA levels were increased in the mediobasal hypothalamus in females at postnatal day 28, whereas kisspeptin and neurokinin B protein levels in the arcuate nucleus were decreased, following Mkrn3 overexpression, compared to controls. Cumulatively, these data suggest that Mkrn3 may directly or indirectly target neuropeptides of Kiss1 neurons to degradation pathways. This mouse model suggests that MKRN3 may be a potential contributor to delayed onset of puberty, in addition to its well-established roles in central precocious puberty and the timing of menarche.

Keywords: MKRN3; hypothalamus; kisspeptin; neurokinin B; puberty.

Figure 1.

In vivo and in vitro validation of AAVs. (A) Cell lines (n = 3 wells for each cell line and experimental condition) derived from Kiss1 neurons from the mouse arcuate nucleus (KTaR-1) and the anteroventral periventricular nucleus (KTaV-3) demonstrate significantly increased Mkrn3 mRNA levels (P < .0001 by 1-way ANOVA, Tukey post hoc) in cells transduced with AAV-Mkrn3 (purple circles) compared with cells transduced with AAV-EGFP (green circles) or untransduced cells (black circles). Representative hypothalamic sections of (B) mediobasal hypothalamus (MBH) and (E) preoptic area at PND28 from AAV-EGFP female mice, in which Mkrn3 protein was not detected. (C, D) Mkrn3 protein detection, levels and distribution from the MBH of a representative AAV-Mkrn3 female mouse at PND28, shown at 2 different magnifications. Arrows denote examples of neuronal cell bodies demonstrating immunofluorescence. (F) Similarly, MKRN3 protein detection, levels and distribution from the POA at PND28 of a representative AAV-Mkrn3 female mouse. Scale bar 100 μm (A-E).

Figure 2.

Neonatal bilateral ICV injection of AAV-Mkrn3 leads to delayed pubertal onset in female mice. (A) Percentage of female mice with VO with increasing postnatal age is shown (left panel) for mice injected with AAV-Mkrn3 (purple circles, n = 11) or AAV-EGFP (green circles, n = 9); mean age of VO is delayed in AAV-Mkrn3 female mice (purple) compared to AAV-EGFP (green) controls (right panel; *P < .05, unpaired t-test). (B) Similarly, percentage of these 2 groups of female mice with first estrus with increasing postnatal age is shown (left panel) and mean age of first estrus is delayed in AAV-Mkrn3 female mice (n = 11) compared with AAV-EGFP controls (n = 9) (right panel; **P < .05, unpaired t-test). (C) Mean body weight of female mice measured at weaning (PND 25) to PND 60 did not differ (AAV-Mkrn3, n = 11, purple circles, AAV-EGFP n = 9, green circles, 2-way ANOVA F(1,18) = 2.986, P = .10). (D) In male mice, the mean age of preputial separation was not significantly different between AAV-Mkrn3 injected (n = 8) compared with AAV-EGFP injected (n = 9) mice (P = .76, unpaired t-test). (E) Mean body weight from PND 25 to 60 also did not differ in male mice (AAV-Mkrn3 n = 8, purple circles, AAV-EGFP, n = 9, green circles, 2-way ANOVA F(1,14) = 0.09, P = .76). Data are shown as mean ± SEM. VO; vaginal opening; PND: postnatal day.

Figure 3.

Estrous cyclicity and fertility are normal in AAV-Mkrn3 injected female mice. (A) Following puberty, estrous cycles were monitored following first estrus for 4 to 5 weeks until the fertility study was initiated at 10-12 weeks of age. Percent time spent in estrus compared with metestrus/diestrus did not differ between AAV-Mkrn3 (n = 11) and AAV-EGFP (n = 9) injected mice (P = .58, 2-way ANOVA). Fertility was assessed over a 3-month study period by mating 1:1 with a wild-type male (n = 6 per group). (D) Time to first litter, (E) mean number of litters, and (F) mean litter size did not differ significantly between the 2 groups (P = .68, P = .51, and P = .37, respectively, unpaired t-test).

Figure 4.

Hypothalamic mRNA levels in female mice on PND28 as measured by RT-qPCR. In the mediobasal hypothalamus, (A) Mkrn3, (B) Kiss1, (C) Tac2, and (D) Pdyn mRNA levels were all significantly higher in AAV-Mkrn3 mice (purple circles) than in AAV-EGFP mice (green circles). Kiss1 (E; P = .16) and Gnrh1 (F; P = .48) mRNA levels in the preoptic area were not significantly different between the AAV-Mkrn3 and AAV-EGFP mice at PND28. Data are shown as mean ± SEM (n = 5 per group), normalized to the housekeeping gene Hprt. *P < .05; **P < .01; unpaired t-tests.

Figure 5.

Immunohistochemistry shows reduced kisspeptin and neurokinin B protein levels in the arcuate nucleus at PND28 in female AAV-Mkrn3 mice compared with AAV-EGFP mice, whereas the number of GnRH cell bodies in the preoptic area were unchanged. (A) Representative photomicrograph of kisspeptin immunohistochemistry in the arcuate nucleus of AAV-EGFP (left panel) and AAV-Mkrn3 (right panel) female mice at PND28. Scale bar 50 μm. (B) Mean gray density (n = 5 per group) was significantly lower for the AAV-Mkrn3 than for the control AAV-EGFP mice (P < .001, unpaired t-test). (C) Representative photomicrograph of neurokinin B immunohistochemistry in the arcuate nucleus of AAV-EGFP (left) and AAV-Mkrn3 (right) female mice. Scale bar 50 μm. (D) Mean gray density (n = 5 per group) was significantly lower for AAV-Mkrn3 than for AAV-EGFP mice (P < .001, unpaired t-test). (E) Representative photomicrograph of GnRH immunohistochemistry in the preoptic area of AAV-EGFP (left) and AAV-Mkrn3 (right) female mice. Scale bar 50 μm. (F) Mean number of cell bodies for GnRH did not statistically differ between the 2 groups (P = .98).