Genetic, endocrine and biochemical aspects of testis and ovarian differentiation and development

1. Genetic, endocrine and biochemical aspects of testis and ovarian differentiation and development Class: II M.Sc., Unit: 4 Prepared by A. Benno Susai Department of Biochemistry, SJC, Trichy

2. The establishment of chromosomal sex (46,XY or 46,XX) is considered the first step in normal sexual differentiation. The term sex determination refers to the process in which the primordial gonad is committed to development as testis or ovary. Sex differentiation refers to the development of the internal accessory duct structures and external genitalia Department of Biochemistry, SJC, Trichy

3. GONADAL DIFFERENTIATION Department of Biochemistry, SJC, Trichy BIPOTENTIAL GONAD The bipotential gonad develops from an out pouching of tissue on the posterior aspect of the celomic cavity on either side of the aorta. This structure is termed the urogenital ridge. It consists of three regions: the pronephros, the mesonephros, and the metanephros. The central region, the mesonephros, gives rise to the bipotential gonad and the first renal system. TESTIS DETERMINATION Testis determination occurs at about six weeks of gestation. The first histological evidence is the proliferation of cells from the celomic epithelium. These cells migrate into the underlying mesenchyme and give rise to Sertoli cells. Sertoli cells encircle germ cells, form the testis cords, cause mitotic arrest of germ cells, and secrete both antiMullerian hormone (AMH) and inhibin.

4. Department of Biochemistry, SJC, Trichy Leydig cells develop later than Sertoli cells and begin to secrete testosterone at seven to eight weeks of gestation. Peak serum levels of testosterone are reached by the 16th week of gestation. After testis determination, a second migration of cells from the mesonephros occurs. This migration is necessary for the development of the celomic vessel and the formation of the peritubularmyoid cells that stabilize the testis cords. Ovarian Determination Ovarian determination takes place at about seven weeks of gestation. It involves formation of follicular cells, which are thought to arise from the same progenitors as those that give rise to Sertoli cells. Follicular cells surround germ cells in a loose configuration and form primordial follicles. This process is associated with arrest of germ cells in meiotic prophase . In the absence of germ cells, the development of ovarian follicles is abnormal.

5. Genetic Control of Gonadal Differentiation Department of Biochemistry, SJC, Trichy The homeobox gene LIM1 plays an important role in formation of the gonadal ridge. The human LIM1 gene has been identified, but no mutations have been reported. A related gene termed Lhx9 is expressed in the urogenital ridge in mice, and the absence of Lhx9 causes gonadal agenesis in mice. The steroidogenic factor 1 (SF1) gene located on chromosome 9p33, is a member of the orphan nuclear receptor family. SF1 was first identified as an activator of genes involved in biosynthesis of adrenal steroids. The Wilm’s tumor suppressor (WT1) gene is located on chromosome 11p13, and encodes a transcription factor necessary for the development of the bipotential gonad and the kidney. WT1 activates transcription of sex-determining region Y (SRY), and also plays a role in testis determination.

6. Genes Involved in Testis Determination Department of Biochemistry, SJC, Trichy The SRY gene initiates the process of testis determination. SRY is located on chromosome Yp11.3, close to the pseudoautosomal boundary. It belongs to the family of high-mobility group (HMG) proteins, which contain a related DNA-binding motif termed the HMG box. The SRY gene product bends DNA and the change in chromatin results in increased transcription of target genes. SRY is expressed in cells from the celomic epithelium that become Sertoli cells, and also directs the migration of cells from the mesonephros. The SOX9 gene, located on chromosome 17q24.3–25.1 encodes an SRY-like protein expressed by Sertoli cells and cartilage. An increase in expression of SOX9 follows expression of SRY, and SOX9 may be a target gene of SRY. Overexpression of SOX9 in humans and mice results in XX sex reversal. SOX9 also interact with SF1 in regulating the expression of the AMH gene. Mutation of SOX9 results in campomelic dysplasia and 46,XY gonadal dysgenesis.

7. Genes Involved in Ovarian Determination Department of Biochemistry, SJC, Trichy Ovarian determination is still poorly understood, some progress has been made in recent years. This has come largely from the elucidation of the role of Wnt4. Wnt4 is expressed in somatic cells and induces expression of Follistatin. Together they block the migration of mesonephric cells and so antagonize male development. Lack of the Wnt4 gene expression impairs ovarian development and promotes expression of testis specific markers such as AMH and testosterone. Wnt4 and Follistatin also support survival of germ cells. The transcription factor Figa (factor in germ line a) recruits granulosa cells in the formation of follicles and promotes expression of the zone pellucida proteins in the oocyte. In addition, Foxl2 promotes appropriate differentiation of granulosa cells during folliculogenesis.

8. HORMONAL CONTROL OF SEX DIFFERENTIATION Department of Biochemistry, SJC, Trichy AntiMüllerian Hormone (AMH) AMH causes the cranial–caudal regression of Müllerian ducts during week 8 to 10 of gestation. AMH is secreted by Sertoli cells. Granulosa cells also secrete AMH, but not until the critical period of Müllerian regression has passed. AMH is a dimeric glycoprotein and a member of the transforming growth factor b (TGFb) family, which includes inhibin, activins, and other factors. The AMH receptor is a complex transmembrane serine/threoninekinase, and is a heterodimer made up of type I and type II receptors.

9. Department of Biochemistry, SJC, Trichy Testosterone Leydig cells appear between weeks 7 and 8 in the human fetus, and proliferate until the 18th week of gestation. Receptors for human chorionic gonadotropin (hCG) are present on fetal Leydig cells by at least 12 weeks of gestation and help to insure continued testosterone secretion. Biosyntheses of testosterone requires five enzymes: Steroidogenic acute regulatory protein (StAR), Side chain cleavage (P450scc), 3 β -hydroxysteroiddehydrogenase (3 β -HSD), 17-hydroxylase/17,20 desmolase (P45017), and 17βhydroxysteroiddehydrogenase (17 β -HSD).

10. Department of Biochemistry, SJC, Trichy In target tissues, testosterone is converted to DHT by steroid 5α-reductase. The conversion of testosterone to DHT is necessary for sex differentiation of male external genitalia, and there are two explanations for this. First, DHT binds to the androgen receptor (AR) with greater affinity than does testosterone. Second, DHT cannot be aromatized, and thus its action is purely androgenic. Although masculinization of prostate, urogenital sinus, and external genitalia depend on DHT, proliferation of Wolffian ducts required testosterone alone.

11. Department of Biochemistry, SJC, Trichy Estrogen The biological effects of estrogens are mediated by estrogen receptors (ERs). Estrogen diffuses freely into cells where it binds to ER in the nucleus. The ER-ligand complex dimerizes, binds to the estrogen response elements, and hence modulates transcription of estrogen-regulated genes. Two ERs, α and β, have similar affinity and specificity. The human ER β has been detected as early as 13 to 20 weeks of gestation with highest levels being found in the adrenals, and in the male reproductive tract. For example, prenatal exposure to estrogens can result in cryptorchidism, possibly by inhibiting insulin-like factor 3. Hypospadias, epididymal cysts, persistentMullerian ducts, and prostate disease have also been related to prenatal exposure to estrogen in males.

12. Basic concepts Fetal sex differentiation: • Occurs at 7-14 weeks’ fetal age only Is innately female and does not require ovaries or estrogens • As a male requires: • Sex-determining region of Y (SRY) gene • Bilateral testes producing mullerian inhibiting substance (MIS/MIF/AMH) and testosterone • 5a-reductase enzyme (external genitalia) • Testosterone and dihydrotestosterone receptor (internal and external genitalia) Department of Biochemistry, SJC, Trichy

13. Department of Biochemistry, SJC, Trichy

14. Human Gonadal Development and Differentiation Wilms tumor 1 – encodes for transcription factor essential for normal development of the urogenital system Genital ridge Wolffian D. Male int genit. Genit. Tub. Urogen. Sinus Penis Prostate Testis SOX-9 SRY Leydig cells Testosterone DHT AMH Mullerian Duct Regression SF-1 WT-1 SF-1 Sertoli cells Bipotential gonad DSS Follicular cells Theca cells Mullerian Duct Female Internal Genitalia SF-1 DOX 1,SOX9 & SRY Follicles Ovary Steroidogenic factor High mobility group- is a super family of eukaryotic DNA binding proteins Department of Biochemistry, SJC, Trichy

15. Figure 10-13 Department of Biochemistry, SJC, Trichy

16. Male genital (internal) DevelopmentSertoli cell differentiation in the medullary sex cords ~7th week Department of Biochemistry, SJC, Trichy

17. Male genital (internal) DevelopmentAnti-Mullerian hormone (AMH) secretion by Sertoli cells (reminant of cranial end of mesonephric duct) ~8-12th week Sertoli Cells Ductuli efferentes (reminant of inferior mesonephric tubules) Department of Biochemistry, SJC, Trichy

18. Male genital (internal) DevelopmentDifferentiation of the mesonephric ducts of the male ~10th-12th week Department of Biochemistry, SJC, Trichy

19. Male genital (internal) DevelopmentDifferentiation of the accessory glands of the male urethra ~10th-12th weeks Department of Biochemistry, SJC, Trichy

20. Female genital (internal) developmentAbsence of a Y chromosome Department of Biochemistry, SJC, Trichy

21. Female genital (internal) developmentAbsence of AMH in the female embryo Department of Biochemistry, SJC, Trichy

22. Female genital (internal) developmentAbsence of AMH in the female embryo Department of Biochemistry, SJC, Trichy

23. Female genital (internal) developmentAbsence of AMH in the female embryo Department of Biochemistry, SJC, Trichy

24. External genitalia developmentMale and Female external genitalia develop from the same primordia or urethral fold External genitalia is similar in male and female embryos through the 12th week. Department of Biochemistry, SJC, Trichy

25. 14 weeks 12 weeks External genitalia development.Male external genitalia. Department of Biochemistry, SJC, Trichy

26. External genitalia developmentFemale external genitalia 7th week 3rd month Department of Biochemistry, SJC, Trichy

27. The gubernaculum controls the descent of the testes and ovariesThe descent of the testes Department of Biochemistry, SJC, Trichy

28. The gubernaculum controls the descent of the testes and ovariesThe descent of the testes Department of Biochemistry, SJC, Trichy

29. The gubernaculum controls the descent of the testes and ovariesThe descent of the testes Department of Biochemistry, SJC, Trichy

30. The gubernaculum controls the descent of the testes and ovariesThe descent of the ovaries Department of Biochemistry, SJC, Trichy

31. Testicular Feminization Syndrome: the androgen receptors are abnormal or lacking, resulting in the formation of a blind-ending vagina (Male genotype, Female external phenotype, no ovaries). Since the testes are still present and AMH is produced, the paramesonephric ducts regress (Thus no superior vagina, uterus, or fallopian tubes). Abnormal development of the genital system Department of Biochemistry, SJC, Trichy

32. Abnormal development of the genital system True hermaphrodites--> have both ovarian and testicular tissue. The gonads are usually a composite ovotestes containing both seminiferous tubules and follicles. Or an individual may have an ovary on one side and a testes on the other side. Most true hermaphrodites are reared as males since a phallus is usually present at birth. Department of Biochemistry, SJC, Trichy

33. References Department of Biochemistry, SJC, Trichy Developmental Biology, Tenth Edition, Scott F. Gilbert. Chapter – 17 Devlin, 1997, Textbook of Biochemistry (with clinical correlation), John Wiley, USA. Wilson and Foster, 1992, Textbook of Endocrinology, (8thedn), W. B. Saunders, USA.