1. Class: II M.Sc., Unit: 4 Prepared by A. Benno Susai
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. Department of Biochemistry, SJC, Trichy
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
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.
Department of Biochemistry, SJC, Trichy

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 peritubular myoid 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.
Department of Biochemistry, SJC, Trichy

5. Genetic Control of Gonadal Differentiation
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.
Department of Biochemistry, SJC, Trichy

6. Genes Involved in Testis Determination
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 migra tion 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.
Department of Biochemistry, SJC, Trichy

7. Genes Involved in Ovarian Determination
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.
Department of Biochemistry, SJC, Trichy

8. HORMONAL CONTROL OF SEX DIFFERENTIATION
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/threonine kinase, and is a heterodimer made up of type I and type II receptors.
Department of Biochemistry, SJC, Trichy

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 -hydroxysteroid dehydrogenase (3 β -HSD),
17-hydroxylase/17,20 desmolase (P45017), and
17βhydroxysteroid dehydrogenase (17 β -HSD).
Department of Biochemistry, SJC, Trichy

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.
Department of Biochemistry, SJC, Trichy

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, persistent Mullerian ducts, and prostate disease have also been related to prenatal exposure to estrogen in males.
Department of Biochemistry, SJC, Trichy

12. Department of Biochemistry, SJC, Trichy
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.
Testis
Genit. Tub.
Urogen. Sinus
Penis
Prostate
SOX-9
SRY
Leydig cells
SF-1
WT-1
Testosterone DHT
AMH Mullerian Duct
Regression
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. Department of Biochemistry, SJC, Trichy
Figure 10-13
Department of Biochemistry, SJC, Trichy

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

17. Department of Biochemistry, SJC, Trichy
Male genital (internal) Development Anti-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. Department of Biochemistry, SJC, Trichy
Male genital (internal) Development Differentiation of the mesonephric ducts of the male
~10th-12th week
Department of Biochemistry, SJC, Trichy

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

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

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

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

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

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

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

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

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

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

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

31. Abnormal development of the genital system
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).
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. Department of Biochemistry, SJC, Trichy
References
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.
Department of Biochemistry, SJC, Trichy