Sexually dimorphic testosterone secretion in prenatal and neonatal mice is independent of kisspeptin-Kiss1r and GnRH signaling

Abstract

Kisspeptin, encoded by the Kiss1 gene, stimulates GnRH secretion and is therefore critical for sex steroid secretion at puberty and in adulthood. However, kisspeptin's role in regulating sex steroid secretion earlier in development is unexplored. In rodents, testosterone (T) levels are higher in prenatal and newborn males than females. We determined whether kisspeptin-Kiss1r and GnRH signaling plays a role in sexually dimorphic perinatal T secretion in mice. Our results demonstrate that 1) T levels in newborn males are elevated at 4 h but not 20 h after birth, but hypothalamic Kiss1 and neurokinin B (NKB) levels in males are not different between these time points (and both are lower than in females); 2) serum T levels in newborn Kiss1r knockout (KO) males are higher than in newborn females and similar to wild-type (WT) males; 3) perinatal hypothalamic progesterone receptor (Pgr) expression, which is dependent on circulating levels of gonadally produced T, is significantly higher in prenatal and newborn Kiss1r KO and WT males than similarly aged females; 4) multiple measures of testicular growth and function are not different between developing Kiss1r KO and WT mice until after postnatal d 5; and 5) GnRH neurons of newborn males do not exhibit high c-fos coexpression, and newborn hypogonadal (hpg) male mice (lacking GnRH) secrete elevated T, similar to newborn WT males. We conclude that, unlike in puberty and adulthood, elevated T secretion in prenatal and neonatal mice is independent of both kisspeptin and GnRH signaling, and the necessity of kisspeptin-Kiss1r signaling for testicular function is first apparent after d 5.

Fig. 1.

Serum T in PND1 C57BL/6 males (M) and females (F). Serum was isolated from blood collected from C57BL/6 pups killed between 0 and 4 h old and 16 and 20 h old, pooled within groups, and assayed for T. Males had significantly higher T levels than females at 0–4 h old (P < 0.05). There was no significant difference in T between males and females at 16–20 h old.

Fig. 2.

Gene expression of GnRH-stimulating neuropeptides in the ARC on PND1. A, Representative photomicrographs for Kiss1 and NKB mRNA expression in females (F) and males (M) that were 0–4 h old. The mean number of Kiss1 (B) and NKB (C) neurons in the arcuate, as well as the relative level of Kiss1 (B) and NKB (C) mRNA/cell (determined by grains per cell), were quantified for males and females that were ages 0–4 h old and 16–20 h old. Males had significantly fewer Kiss1 and NKB cells and lower mRNA per cell than females at either time point (P < 0.05). There were no significant differences in the number of neurons or mRNA/cell between 0 and 4 h and 16 and 20 h. 3V, Third ventricle; GPC, grains per cell.

Fig. 3.

Serum T in Kiss1r KO males and females. A. Blood serum was isolated from newborn male (M) and female (F) mice less than 4 h old and assayed for T. Both WT and Kiss1r KO males had significantly higher T levels than females of the same genotype (P < 0.05). There was no significant difference in T levels between 4-h WT and KO males. B, In contrast to neonatal mice, serum T was significantly reduced in adult Kiss1r KO males compared with adult WT male littermates (P < 0.05). C, Testis weight was also significantly lower in adult Kiss1r KO males than adult WT littermates (P < 0.05).

Fig. 4.

Pgr mRNA expression in the hypothalamus of fetal and neonatal Kiss1r KO and WT mice. Hypothalamic Pgr levels are an established indicator of fetal and neonatal androgen milieu and therefore can serve as a biomarker to recent T exposure. A, Representative photomicrographs of Pgr expression in WT and Kiss1r KO males (M) and females (F) in the AVPV and vl-VMH on E18. B and C, The mean number of Pgr neurons in the AVPV and vl-VMH on E18 and PND1 was significantly higher in males than females, regardless of age or genotype (P > 0.05).

Fig. 5.

Serum T, testis weight, and AGD were measured in WT and Kiss1r KO mice of different postnatal ages. Serum T (A), testis weight (B), and AGD (C) were not significantly different between genotypes on PND5 but were lower in KO Kiss1r males than WT males on PND 10 and PND 21 (P < 0.05). *, Significantly different from WT males. Bars labeled with different letters designate significantly different groups (P < 0.05).

Fig. 6.

A, Representative low power (left) and higher power (right) photomicrographs of GnRH (red DIG staining) and c-fos (silver grains) mRNA expression in PND1 mice. Blue arrows designate example GnRH neurons lacking c-fos expression; white arrows designate single-label c-fos cells that are not GnRH neurons; yellow arrow designates one of the few GnRH cells coexpressing c-fos. B, Quantification of the percent of total GnRH neurons double labeled with c-fos on PND1. There were no significant differences between sexes or between time points. C, Serum T levels in hpg males (M) and females (F) on the day of birth. Blood serum collected from WT and hpg mice that were killed between 0 and 4 h old was assayed for T. Both WT and hpg males had significantly higher T levels than females from the same genotype (P < 0.05). There was no significant difference in T levels between 4-h WT and hpg males.

Fig. 7.

Serum gonadotropin concentrations from PND1 WT, Kiss1r KO (A), and hpg mutant (B) mice. Individual serum samples were collected from newborn mice between 0 and 4 h after birth and assayed for LH and FSH using an LH/FSH multiplex ELISA. LH and FSH were readily detectable in newborn WT females (F) but undetectable in newborn WT males (M). LH and FSH were undetectable in Kiss1r KO and hpg mice, regardless of sex. Bars labeled with different letters designate significantly different groups (P < 0.05). The dashed line designates the lower limit of detection for the assays.

Fig. 8.

Schematic summary of differences between the adult and neonatal reproductive axis in WT and Kiss1r KO males. In adulthood, kisspeptin is a required upstream stimulant of GnRH release and subsequent secretion of gonadotropins and T. When kisspeptin signaling is disrupted in adults by a Kiss1r mutation, GnRH, gonadotropin, and T secretion decreases considerably, and there is no longer sex steroid-mediated negative feedback on the brain. In contrast to adulthood, kisspeptin-Kiss1r signaling is not required for T production on PND1. Kiss1r KO male mice are able to secrete similar elevated T as WT males, despite presumed decreased in GnRH signaling and gonadotropin production. Moreover, the presence of elevated T in PND1 Kiss1r KO males suggests that T-mediated negative feedback to the brain is still present and not impaired in newborns, unlike in older Kiss1r KO animals.