Development of the adreno-genital systemFemale sex determination, ovarian and adrenal gland ontogeny regulated by Wnt-4 in mice

Minna Heikkilä

Biocenter Oulu, University of Oulu
Department of Biochemistry, University of Oulu

Abstract

Although the genetic sex of an embryo is determined at conception by the presence or absence of the Y chromosome, both females and males have bipotential, undifferentiated gonads early in their development. Genes and testicular hormones direct differentiation into either testes or ovaries. The first relevant gene to be identified was the Y-linked master regulatory gene, SRY, since when several other genes have been found to be of importance for sex determination.

The primary aim here was to identify the role of Wnt-4 in the development of the gonad and adrenal gland. Wnt-4 was found to be expressed in the developing gonad, the Müllerian duct and the adrenal gland, in addition to the kidney, pituitary gland and mammary gland as observed earlier. Expression in the gonad was found to be regulated in a sex-specific manner. After sex determination Wnt-4 was downregulated in the testis, but the expression persisted until birth in the ovary. Wnt-4-deficient female mice demonstrated a partial female-to-male sex reversal and a reduction in the number of oocytes, while the M¸llerian duct was absent from both sexes. Lack of Wnt-4 in the adrenal gland led to reduced aldosterone production, indicating abnormal development of the zona glomerulosa. Flutamide administration to pregnant Wnt-4 heterozygote females was shown to partially restore the sex reversal.

The results suggest that female development is not a default pathway but needs active signalling, in which Wnt-4 plays an essential role.

Dedication

 

You can get anywhere,

take one step and

repeat it as many times as needed

 
-- (unknown) 

To Toni and Venla

Table of Contents
Acknowledgements
Abbreviations
List of original articles
1. Introduction
2. Review of the literature
2.1. Gonadal development
2.1.1. Formation of a bipotential gonad
2.1.2. Germ cell migration
2.1.3. Sex determination
2.1.4. Specific promoters of testis development
2.1.5. Specific promoters of ovarian development
2.1.6. Autosomal genes involved in early gonad formation
2.1.7. Genetic interactions in gonadal development
2.2. Adrenal gland development
2.3. The hypothalamus-pituitary-adrenal /gonadal axis (HPA/HPG)
2.3.1. Steroidogenesis
2.3.2. Mutations in biosynthetic enzymes
2.4. Wnts
2.4.1. Wnts in sex differentiation
2.4.2. Wnt-4
3. Aims of the research
4. Materials and methods
4.1. Mouse strains (I, III-IV)
4.1.1. Genotyping and sex typing of embryos
4.1.2. Tissue samples
4.2. Histology (I,III-IV), -galactosidase (I) and antibody staining (I)
4.3. Whole mount and section in situ hybridization (I-IV)
4.4. Morphometric studies (IV)
4.5. Quantitative reverse transcription-PCR (IV)
4.6. Hormone analysis (III-IV)
4.7. Flutamide treatment (III)
5. Results
5.1. Expression of Wnt-4 in the developing urogenital system (I, IV)
5.2. Lack of Wnt-4 leads to masculinization of the female reproductive system (I, III)
5.3. Steroidogenesis is initiated in the ovaries of Wnt-4 mutant females (I-IV)
5.4. Flutamide can rescue partially the Wnt-4 mutant female phenotype (III)
5.5. Wnt-4 deficiency alters the function of the adrenal cortex (IV)
5.5.1. Elevated function of the HPA-axis in Wnt-4 mutant mice (IV)
5.6. Wnt-4 may control the migration/sorting of adrenal and gonadal cells (IV)
6. Discussion
6.1. Wnt-4 expression in the gonad is regulated sex-specifically, while that in the adrenal gland is independent of sex
6.2. Lack of Wnt-4 leads to female-to-male sex reversal
6.3. Wnt-4 and the adrenal gland
6.4. Relationship of Wnt-4 to other sex-determining genes
7. Conclusions
References
List of Tables
1. Mutations in genes involved in early gonad development
2. Known phenotypes of Wnt gene knock-out mice
3. Probes used for whole mount and section in situ hybridization
4. Comparison of the gonad phenotype of Wnt-4 wild-type and mutant mice before and after the flutamide treatment
List of Figures
1. Differentiation of the gonads and the gonad-associated sex ducts. Originally both Müllerian (female specific) and Wolffian ducts (male specific) are present in both sexes. Later in the females the Müllerian duct will differentiate into the oviduct, uterus and upper part of the vagina, while Wolffian duct regresses in the absence of testosterone. In turn, in males the Müllerian duct degenerates due the action of AMH and Wolffian duct will differentiate into epididymis and vas deferens.
2. Adrenal glands and gonads share a common primordium, from which adrenal gland, testis and ovary will be formed. (Modified from Morohashi 1997).
3. Differentiation of the bipotential gonad into the ovary and the testis. (Modified from (Modified from Vaiman & Pailhoux 2000.)
4. Two-cell, two-gonadotropin model of steroidogenesis in the ovary. Theca cells produce androstenedione under the control of LH, which increases the number of LDL-receptors and hence cholesterol entry into the cells. Then androstenedione diffuse into the granulosa cells, where it will be converted to estrogen. Granulosa cells respond to FSH by inducing aromatase activity.
5. Transcription patterns in gonads of the genes involved in sex determination and differentiation. The level of the transcription is illustrated with black stripes; the more dense stripes the higher level of transcription. Gray area indicates no transcription. The ages up to 12 dpc represent the transcription in the whole gonad and thereafter only in the supporting cells. (Modified from Swain & Lovell-Badge 1999.)
6. Main gene interactions in the sex determination. In males Sry is thought to repress Dax-1, which would otherwise inhibit the activation of estrogen receptor (ER) α and . When Dax-1 is not present steroidogenic factor 1 (Sf1) is fully functional and can further participate into the activation of anti-Müllerian hormone (AMH), steroidogenic genes and Sox9. In addition, Sox9 could maintain its own expression through an autoregulatory loop. Furthermore, Sry can probably contribute also directly to the Sox9 activation. In the absence of Sry in females, the Dax-1 remains functional leading to inhibition of the genes active in male specific development.
7. Composition of the adrenal gland. In humans the adrenal gland is composed of medulla and three cortical layers; zona glomerulosa, zona fasciculate and zona reticularis. In mouse there exist no zona reticularis, but instead it is replaced with so called “X-zone”.
8. The function of hypothalamus-pituitary-adrenal (HPA) and hypothalamus-pituitary-gonadal (HPG) axes. Gonadotropin releasing hormone (GnRH) and adrenocorticotropin releasing hormone (CRH) are discharged from hypothalamic central nervous system to stimulate the function of gonadotrophs and corticotrophs in pituitary gland. In response to gonadotropins (FSH, LH) and adrenocortical tropic hormone (ACTH) the gonads and the adrenal cortex synthesize and secrete sex steroids and corticosteroids. (Modified from Morohashi 1997.)
9. Steroid hormone synthesis pathways. All steroid hormones will be synthesized from cholesterol and the end products can be classified according to their principal effects; mineralocorticoids (aldosterone), glucocorticoids (cortisol in human, corticosterone in rodents), progestins, androgens and estrogens.
10. Simplified model of Wnt/β -catenin signalling pathway. Wnt ligand activates Frizzled/LRP-5/6 receptor complex, which is followed by signalling cascade leading to accumulation of β -catenin in the cytoplasm. Then β -catenin enters the nucleus, where it modulates gene expression together with TCFs.
11. The transcription of female specific gene, follistatin is lost from the ovary of Wnt-4 deficient mouse. In situ hybridization results show that Follistatin is not transcribed in the testis (a), but strongly in the 14.5 dpc ovary of the wild-type female shown as black reaction product in unstained specimen (b), while no transcription can be detected in the one of Wnt-4 mutant female (c).
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