The Influence of Gonadal Steroid Hormones on Immunoreactive Kisspeptin in the Preoptic Area and Arcuate Nucleus of Developing Agonadal Mice with a Genetic Disruption of Steroidogenic Factor 1

Abstract

Kisspeptin, a regulator of reproductive function and puberty in mammals, is expressed in the rostral (anteroventral) periventricular nucleus (AVPV) and arcuate nucleus (Arc), and its expression is at least partially regulated by estradiol in rodents. The aim of the present study was to determine contributions of genetic factors and gonadal steroid hormones to the sexual differentiation of kisspeptin-immunoreactive (kisspeptin-ir) cell populations in the AVPV and Arc during postnatal development using agonadal steroidogenic factor 1 (SF-1) knockout (KO) mice. To examine the effects of gonadal hormones on pubertal development of kisspeptin neurons, SF-1 KO mice were treated with estradiol benzoate (EB) from postnatal day (P)25 to P36, and their brains were examined at P36. No sex differences were observed in the SF-1 KO mice during postnatal development and after treatment with EB - which failed to increase the number of kisspeptin-ir cells at P36 to the levels found in wild-type (WT) control females. This suggests that specific time periods of estradiol actions or other factors are needed for sexual differentiation of the pattern of immunoreactive kisspeptin in the AVPV. Kisspeptin immunoreactivity in the Arc was significantly higher in gonadally intact WT and SF-1 KO females than in male mice at P36 during puberty. Further, in WT and SF-1 KO females, but not in males, adult levels were reached at P36. This suggests that maturation of the kisspeptin system in the Arc differs between sexes and is regulated by gonad-independent mechanisms.

Fig. 1

A digital inset image shows representative sections with kisspeptin-ir cells in the AVPV at P60 in WTM and WTF mice. The graph illustrates the number of kisspeptin-ir cells in the AVPV in gonadally intact wild type (WT) male (WTM) and female (WTF), gonadectomized at P21 WT male (WT/CAS), and female (WT/OVX), and agonadal SF-1 KO male (KOM) and female (KOF) mice in three different developmental stages, at P25 (before puberty), at P36 (during pubertal time) and at P60 (adult). The number of cells is presented as mean +/- SEM. *p < 0.05 (significantly different from the WTM, WT/CAS, WT/OVX, KOM and KOF); ^ap< 0.05 (significantly different from WTF at P25); ^bp< 0.05 (significantly different from WTF at P36). For the image, 3V- third ventricle, OT- optic tract, bar: 100 μm.

Fig. 2

The diagram illustrates the number of kisspeptin-ir cells in the AVPV from 36 days old gonadally intact wild type male (WTM) and female (WTF) mice (same bars as in diagram 1, included for comparison), WT male (WT/CAS) and female (WT/OVX) mice gonadectomized at P21 and SF-1 KO male (KOM) and female (KOF) mice. Gonadectomized mice and agonadal SF-1 KO mice were treated with EB from P25 to P36. Treatment restored the number of kisspeptin-ir cells in WT/OVX mice to the levels seen in WTF mice, while the number of kisspeptin-ir cells in agonadal SF-1 KO mice of both sexes was significantly lower (p< 0.05) in comparison to WTF mice even after EB treatment. The number of cells is presented as mean +/- SEM. *p < 0.05, significantly lower than EB treated WT/OVX mice and untreated WTF mice.

Fig. 3

A- D] Representative images of kisspeptin immunoreactivity in the Arc at P36 in gonadally intact WT male (A) and female (B) and agonadal SF-1 KO male (C) and female (D) mice. Gonadally intact WT and agonadal SF-1 KO female mice had significantly higher kisspeptin-ir area in the Arc during puberty at P36 than gonadally intact WT and agonadal SF-1 KO males respectively. E] The graph illustrates the kisspeptin-ir area in the Arc in gonadally intact wild type (WT) male (WTM) and female (WTF), gonadectomized at P21 WT male (WT/CAS), and female (WT/OVX), and agonadal SF-1 KO male (KOM) and female (KOF) mice in three different developmental stages, at P25 (before puberty), at P36 (during pubertal time) and at P60 (adult). The kisspeptin-ir area is presented as mean +/- SEM. *p< 0.05 (different from the WTM, WT/CAS, WT/OVX and SF-1 KO mice over all ages studied); ***p< 0.001 (higher immunoreactive area in WT than gonadectomized WT mice); ^#p< 0.05 (different from WT OVX); ^ap< 0.05 (different from WTF and WT/OVX mice); ^bp< 0.05 (different from WTF, WT/OVX and WT/CAS mice) ^cp< 0.05 (higher than KOM at P36). 3V- third ventricle, ME- median eminence, bar: 100 μm

Fig. 4

The diagram illustrates the kisspeptin-ir area in the Arc from 36 days old wild type male (WT/CAS) and female (WT/OVX) mice gonadectomized at P21 and SF-1 KO male (KOM) and female (KOF) mice. All mice were untreated or treated with EB from P25 to P36. EB treatment from P25 to P36 had no effect on the kisspeptin-ir area in both WT/CAS and WT/OVX mice when compared to untreated gonadectomized WT mice. However, EB treatment did eliminate the sex difference in agonadal SF-1 KO mice at P36 by moderately (nonsignificantly) increasing immunoexpression in males and decreasing immunoexpression in females. The kisspeptin-ir area is presented as mean +/- SEM. *p< 0.05 (different from WT/CAS).