Research reportExpression of progesterone receptor, estrogen receptors α and β, and kisspeptin in the hypothalamus during perinatal development of gonad-lacking steroidogenic factor-1 knockout mice
Introduction
In mammals, the brain develops differently in males and females. In males, testes differentiate to produce testosterone as early as embryonic-day (E) 14, and the temporal increase in testosterone during the perinatal period is required for the masculinization of the brain. Sex differentiation of rodent brains is considered to be mainly influenced by estradiol (which is converted from testosterone) acting through estrogen receptors (ERs), whereas studies in primates indicate that androgens, acting through androgen receptors, have greater effects (Bakker and Brock, 2010, Lenz and McCarthy, 2010, McCarthy, 2010). In contrast, in females, ovaries differentiate to secrete estrogens after postnatal-day (P) 7 (Mannan and O'Shaughnessy, 1991). Studies using knockout (KO) mice for α-fetoprotein (AFP), a fetal plasma protein that binds estrogens with high affinity, and KO mice for aromatase (ArKO) suggest that, in rodents, masculinization and defeminization are protected in females by AFP during the prenatal period and that ovarian estrogens play a role in female-typical brain development around the second week after birth (Bakker and Brock, 2010). Thus, gonadal hormones influence brain sex differentiation at different times of development. However, the details of this mechanism remain unclear.
The preoptic area (POA) is a sexually dimorphic brain region that plays a role in the regulation of gonadotropin secretion and sexual behaviors (Simerly, 2002). Two regions of the POA, the anteroventral periventricular nucleus (AVPV) and the medial preoptic nucleus (MPN), which is located slightly caudal and lateral to the AVPV in coronal sections, are both sexually dimorphic in their structure, neurochemistry and function. Both the number of cells in and the area size of the AVPV are larger in females than in males (Davis et al., 1996), whereas those in the MPN are larger in males than in females (Gorski et al., 1978, Gorski et al., 1980, Bleier et al., 1982). The AVPV sends descending projections to the arcuate nucleus (ARC), which is more pronounced in females. In contrast, the MPN sends descending projections to the ventromedial hypothalamus (VMH), which is more pronounced in males. Formation of the sexually dimorphic POA regions involves sexually different rates of cell death (Forger, 2009, Tsukahara, 2009) and these POA regions are sensitive to gonadal hormones during development to produce sexually dimorphic expression of estrogen-responsive genes, which include progesterone receptor (PR), ERα, ERβ and kisspeptin (Lenz and McCarthy, 2010, He et al., 2013).
PR mediates progesterone’s actions on neural development during late prenatal and early postnatal periods of development. Plasma levels of progesterone, which is of maternal origin, are similar in males and females during perinatal development (Weisz, 1980). PR expression in the POA and the VMH is highly induced by estradiol (Quadros and Wagner, 2008). A study using aromatase KO (ArKO) mice indicates that estradiol converted from testosterone in the brain and estrogens secreted from the ovary contribute to up-regulation of POA PR in males during the neonatal period and in females during the prepubertal period, respectively (Brock et al., 2010). Expression of the two major ER subtypes, ERα and ERβ, in the AVPV and MPN of mice and rats is sexually dimorphic during development and in adulthood, and the expression profiles during early development differ between the two receptors (Karolczak and Beyer, 1998, Cao and Patisaul, 2011). PR expression in the neonatal rat MPN is induced by estrogens and this action is mediated via ERα but not ERβ, indicating that the two ERs may play different functional roles in sexual differentiation of the brain (Chung et al., 2006, González-Martínez et al., 2008).
Kisspeptin, a family of neuropeptides encoded by the Kiss1 gene, and its receptor have been suggested to play important roles in sexual maturation during pubertal development and in adult reproductive function (Clarkson et al., 2008, Pinilla et al., 2012, Poling and Kauffman, 2013). Kisspeptin is expressed in two brain regions, the AVPV and the ARC. Expression of Kiss1 mRNA and protein in both brain regions is sexually dimorphic during postnatal development (Clarkson and Herbison, 2006, Kauffman et al., 2007, Homma et al., 2009, Bakker and Brock, 2010, Desroziers et al., 2012, Semaan et al., 2013). Experimental approaches, including neonatal castration of males and neonatal hormone exposure of females, and studies using KO mice for genes that affect estrogen levels, such as ArKO mice (Brock et al., 2015), AFP KO mice (González-Martínez et al., 2008), and hypogonadal (hpg) mice (Gill et al., 2010), which produce very low levels of gonadal steroid hormones because of a mutation in the gonadotropin-releasing hormone 1 gene (Gnrh1), indicate that Kiss1/kisspeptin expression in the two brain regions is differentially regulated by gonadal hormones and/or non-gonadal factors during postnatal development (Pinilla et al., 2012, Brock and Bakker, 2013, Poling and Kauffman, 2013).
Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that plays important roles in the development and function of the endocrine system (Parker et al., 2002). Expression of SF-1 is detected in gonads, adrenal cortex, anterior pituitary, and the VMH of the brain during development and in adulthood (Ikeda et al., 1994). In SF-1 KO mice, gonads and adrenal glands degenerate during embryonic development before steroidogenic cells differentiate (Luo et al., 1994). SF-1 KO mice, therefore, lack gonads from the earliest time of differentiation and differ from neonatally gonadectomized animals, which lack gonadal hormones postnatally. Therefore, SF-1 KO mice represent an excellent model to study genetic and hormonal influences on sex differentiation during both embryonic and postnatal development (Parker et al., 2002, Büdefeld et al., 2012). An immunohistochemical study of ERβ in the POA in developing SF-1 KO mice suggested a role for gonadal hormones in regulating neuronal migration and positioning during development (Wolfe et al., 2005). Another study investigating expression of calbindin and neuronal nitric oxide synthase in the POA and the VMH in adult SF-1 KO mice suggested gonadal hormone-dependent and-independent mechanisms for sexually dimorphic development of these regions (Büdefeld et al., 2008). SF-1 KO mice have also been used to study the expression of Kiss1 and Kiss1 receptor in the POA and ARC during embryonic development (Knoll et al., 2013) and of kisspeptin expression during pubertal development through to adulthood (Büdefeld et al., 2016).
Sexual differences in the expression of PR, ERα, ERβ, and kisspeptin in the hypothalamic regions are well documented in adult rodents. However, only a small number of studies have focused on the patterns of sexually dimorphic expression that occur during the early postnatal period, and the results are inconsistent among studies. To precisely examine the developmental profile of these neuronal markers and to clarify the effect of gonadal hormone deficiency on sexually dimorphic brain development, we performed immunohistochemistry (IHC) and compared expression patterns between WT and SF-1 KO mice. Additionally, we treated SF-1 KO mice with diethylstilbestrol (DES), a synthetic estrogen, from P0 to P6 and from P7 to P13, and examined its effect on expression at P7 and P14, respectively.
Section snippets
PR expression in the POA of developing SF-1 KO mice
No PR-immunoreactive (+) cells were detected in the POA at E15.5. At P0 and P7, abundant PR+ cells were detected in the AVPV, which abuts the third ventricle, in WT males (Fig. 1A, E, Suppl. Fig. 1A), whereas few PR+ cells were detected in WT females and KO males and females (Fig. 1B−D, F−H). At P14 and P21, abundant PR+ cells were detected in both sexes of WT and KO mice (Fig. 1I−L, data not shown). The immunoreactivity (IR) for PR in the AVPV was quantitatively evaluated by counting PR+ cells
Discussion
Testosterone production occurs as early as E14.5, which corresponds to the time when expression of some neuronal markers, including ERα and kisspeptin, emerges in the brain (Gerlach et al., 1983, Kumar et al., 2015); therefore, sexually dimorphic events may occur at times before or around birth. However, studies using neonatally gonadectomized mice/rats, which lack gonadal hormones after birth, do not detect effects of prenatal gonadal hormones. SF-1 KO mice, which lack gonads before the onset
Animals
Heterozygous mice carrying a null mutant allele of SF-1 (SF-1+/−) on a C57BL6/J background (Luo et al., 1994) were mated to generate WT, heterozygous, and homozygous-null (SF-1 KO) offspring. The day following mating was designated as E0.5. Sexing and genotyping were performed on embryo tail biopsies by PCR (Majdic et al., 2002). The day of birth was designated as P0. SF-1 KO mice lack both adrenal glands and gonads during embryonic development and, therefore, develop female reproductive organs
Declaration of interest
None.
Acknowledgments
This research was supported by Grants-in-Aid for Scientific Research (C) 16K08476. We thank Jeremy Allen, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
Author contributions
Y.I. and T.K. designed the study. Y.I., T.K. and A.T. performed the experiments. T.K. and M.M. provided SF-1 KO mice. Y.I. analyzed the data, prepared the figures and wrote the manuscript. All authors reviewed the manuscript.
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