Gonad and Sex Differentiation

Male Anatomical Structures

Female Anatomical Structures

Developmental Timing of Reproductive Organs in Humans and Lifestock

Sometimes happen even before women recognize they’re pregnant

Disruptions During Early Fetal Development Can Have Major Implications

Environmental exposures to a pregnant female can alter the development of offspring and the gametes that form the grand offspring!

• can impact not only the offspring, but bc the cells that are developing in the offsprings ovary or testes will produce the grandchildren of this individual that’s carrying the fetus, need to think about all the things that you might interact with during early stages of pregnancy that can impact not only child that’s being carried, but also the grand offspring of that individual

Sexual Differentiation Has 3 Phases (not gender, talking about biological sex)

1. Chromosomal (Genetic)

(X or Y chromosome)

2. Gonadal

(initiated by SRY expression in the bipotential embryonic gonad)

3. Phenotypic (ultimately, will differentiate into a phenotypic sex)

(induced by sex steroids)

Genetic Sex Determination (determined by DNA)

The individual is genetically MALE

There is one X and one Y chromosome

• ultimate determination of genetic sex

Chromosomes in Gametogenesis (how chromosomes are divided)

• Gametogenesis: production of germ cells (spermatozoa or oocytes)

  • Reduction of chromosomal number by half during meiosis

• Somatic cells have homologous chromosome pairs and one pair of sex chromosomes (diploid)

• Germ-cell precursors (germ-line stems cells; spermatogonia or oogonia) are mitotic and diploid

• Meiosis in germs cells consists of two special divisions where homologous chromosome pairs segregate to individual chromatids in the resulting daughter cells (haploid gamete)

  • Meiosis is the mechanism for genetic diversity through homologous recombination and independent assortment of homologous chromosomes

  • haploid containing sperm cells will either have an X or Y

• Haploid gametes (sperm and oocyte) fuse at fertilization to form a diploid embryo and becomes mitotic again

• father is going to give you a sperm cell that has either an X or a Y chromosome

Chromosomal (Genetic) Sex Determination

Sex chromosome pairs are XX (homogametic) in female, and XY (heterogametic) in males

• Meiosis in XX females always produces oocytes with an X chromosome

• Meiosis in XY males results in half of the sperm carrying an X chromosome and half carrying a Y chromosome

• At fertilization, the sex chromosome of the sperm determines the chromosomal (genetic) sex of the embryo

• from mom’s side, we can only get a single X bc woman have two X chromosomes, whereas men have an X and a Y

Genetic Sexual Determination

• Under normal physiological conditions:

  • If a Y chromosome is present, the individual is genetically and phenotypically male, no matter how many X chromosomes are present

    • when you receive a Y chromosome from your father, that individual will become a phenotypic male

  • If there is no Y chromosome, the individual is genetically and phenotypically female

• It is not uncommon for development to occur under non-normal physiological conditions

Terminology of Sex Differentiation

• Sex-determining Region of the Y-chromosome (SRY) = Testis Determining Factor (TDF)

• Anti-Mullerian Hormone (AMH) = Mullerian Inhibiting Substance (MIS) = Mullerian Inhibiting Factor (MIF)

• SRY-High Mobility Box containing gene 9 (SOX9)

• Fibroblast Growth Factor 9 (FGF9)

• Wingless-related MMTV integration site 4 (Wnt4)

• Paramesonephric ducts = Mulllerian ducts

• Mesonephric ducts = Wolffian ducts

Basis of Phenotypic Sex is Genetic (but in rare cases not chromosomal) (based on Y chromosome, not just Y itself)

• Y chromosome carries SRY, the testis determining gene

  • when SRY is present on Y chromosome (99.99%), the gonadal sex & the phenotypic sex will differentiate this early gonad into male

• SRY normally leads to male gonadal and phenotypic sexual differentiation of the bipotential indifferent gonad

  • early gonad into male

• Sex determination occurs specifically by the presence of SRY, not the Y chromosome itself

  • are instances where SRY gene gets translocated to another chromosome or the X chromosome

    • SRY gene turns on

    • even though you don’t have a Y chromosome, you can still be phenotypically male

SRY Starts the Process of Producing a Male

• The SRY gene codes for a transcription factor that binds to DNA and affects DNA function (turns gene on or off)

• SRY protein binds to specific genes in the genome

  • Male-determining genes are turned ON

• A major male-determining gene is SOX9

  • SOX9 is also a transcription factor and binds in multiple regions of the genome

  • SOX9 is a major gene that SRY turns on

  • major sex-determining gene is male fetuses

• Binding of SOX9 turns on more genes associated with maleness (male development)

  • Mutations in SOX9 can alter phenotypic sex

Sertoli Cell Differentiation Initiates Gonadal Sex Determination

• The genes turned on by SOX9 affect epithelial cells of the indifferent gonad

  • bi-potential (can either be male or female), doesn’t know yet

  • These cells differentiate to the male cell type, Sertoli cells

    • defining moment when bi-potential gonad becomes unipotential & starts going down the male pathway

• The presence of Sertoli cells induces the cells around them to develop along male lines

  • Germ cells → spermatogonia

    • primordial germ cells that had entered into bi-potential gonad

  • Mesenchymal cells → Leydig cells

    • in bi-potential gonad

• The indifferent gonad becomes a testis

  • have now developed gonadal sex

  • went from genetic sex → gonadal sex bc of this change in gene expression & the differentiation of Sertoli cells

Sertoli Cell Differentiation Initiates Gonadal Sex Determination 2

• Sertoli cells are the first cells in the bipotential gonad to differentiate in response to SRY expression

  • becomes a testis

• Sertoli cells support the development of male germ cells

  • Sertoli cells differentiate intrinsically though a genetic, SRY initiated, cascade

  • In the absence of SRY, ovarian differentiation occurs

Molecular Pathway to Sex Differentiation

• process is stepwise

• default is female

Male vs Female Sexual Differentiation from Sherwood

understand both diagrams

Gonadal Sexual Differentiation

Embryonic timing is for the mouse

Gonadal Sex Differentiation 2

• Sertoli cells express Mullerian Inhibiting Substance (MIS/AMH), which induces Leydig cell differentiation and testicular organization though paracrine signaling

• Ovarian organization occurs in the absence of SRY induced testicular development (default pathway)

How Do These “Male-determining” Genes Make a Phenotypic Male??

• Presence of a proper functioning testis, and the hormones it secretes, induces the fetus to develop into a male

Phenotypic Sexual Differentiation is Dependent on Endocrine Signaling

• Once gonadal sex is established, the Leydig cells of the testis become to produce testosterone

  • we’ve gone through genetic & gonadal sex development

  • what’s the next steps that will force us to have complete phenotypic male genitalia versus female?

  • really driven by hormone testosterone

We are all the same, until we’re not. Phenotypic Sex Differentiation (can’t tell difference b/w male & female phenotypically at these early stages)

• Reproductive tract precursors for both sexes are formed and exist simultaneously before gonadal sex differentiation

• After gonadal sex determination (formation of the testes), either the male (Wolffian), or female (Mullerian) ducts will differentiate while the other will degenerate

  • leydig cells begin to produce testosterone

    • what whole process is driven by (what ducts?)

  • phenotype will begin to change

• Hint for remembering the names: Latin word for woman: mulierem, Portuguese word for woman: mulher, Spanish word for woman: mujer

Phenotypic Sex Differentiation Relies on Hormones from Newly Developed Testes

• Sertoli cells secrete Mullerian Inhibiting Substance (MIS or AMH)

  • MIS causes degradation of Mullerian ducts

  • MIS induces Leydig differentiation

• Leydig cells produce Testosterone

  • Testosterone promotes survival of the Wolffian duct (forms internal ductal system) (in a male fashion)

• Testosterone is converted (DHT) in the target organ (in external genitalia)

  • Induces differentiation of external genitalia (penis, scrotum, etc.)

• male development is all driven by the production of testosterone & subsequently the conversion of testosterone to dihydro… will develop the phenotype of a male

Testosterone and DHT are Powerful Hormones Directing Phenotypic Sex Differentiation

• Urogenital sinus produces bladder, urethra, prostate, and bulbourethral glands

  • last 2 are male

Female is the Default Pathway for Phenotypic Sex Differentiation

• Absence of androgens (& testosterone, DHT) and MIS leads to Wolffian duct regression

• Mullerian ducts differentiate into oviducts, uterus, cervix, and upper vagina

• External genitals form clitoris and labia

• Urogenital sinus forms the bladder, urethra, and lower vagina

• Sexual differentiation is independent of female hormones such as estrogen which are high in fetal environment of the maternal uterus for both sexes

  • cannot depend on estrogen, everything would be single sex (female)

  • dependent on fetus testicle-derived testosterone

  • fetus in uterus

    • being drowned in estrogens, progesterone from mom

      • lipophilic hormones, can pass from mother to fetus easily

Sexual Differentiation Requires Proper Development of Gonads and Associated Ducts and Tissues

External Genitalia Differentiate from Common Precursors (all the same until we’re not, externally)

Before gonadal sex determination, external genital precursors are also the same in male and female embryos. External genitalia of both sexes arises from common precursors

Androgens Drive Differentiation of External Genitalia

Male sex hormones (androgens) masculinize the reproductive system

Not everything goes as planned…

Disorders of Sexual Development (DSD)

Broad range of abnormal male or female development ranging from true hermaphroditism (evidence of both sexes, have both an ovary & a testi developing at the same time) to minor hypospadias (urethral opening forms on the underside of the penis rather than the tip (problems with sex chromosomes being too many or not enough)

• Sex Chromosome DSDs (XX/XY mosaicism, XO Turners syndrome, XXY Klinefelter syndrome, SRY mutation/translocation)

  • Ovotesticular DSD (rare, ~1:20,000 births)

  • True hermaphroditism (can occur in XX or XY individuals)

• XY DSDs

  • Certain male-determining genes (Sox9, MIS, 5-alpha reductase, etc.) are non-functional

  • Ambiguous external genitalia and phenotypes ranging from simple infertility to intersexuality

• XX DSDs

  • Ovaries present but external genitalia appear male or mixed. The internal female reproductive glands are usually normal

Chromosomal Disorders of Sexual Development (DSD)

Ovotesticular DSD: True hermaphrodite

Androgenital Syndrome (an XX DSD) (fetal adrenal gland can overproduce fetal androgens)

Virilization of external genitalia only

Can be caused by maternal/environmental androgen, or fetal adrenal hyperplastic adrenomegaly

• Masculinized external genitals of XX female

  • May exhibit early puberty and short stature

Adrenal Associated Disorders of Sexual Development (have a dysfunction of one or more enzymes in pathway, this is when fetal adrenal gland can cause some problems)

Adrenogenital = Androgenital Syndrome: Masculinizing condition in XX females due to the excess fetal adrenal DHEA production

Common form is a 21-hydroxylase deficiency

• all these precursors will funnel into DHEA & you produce too much androgen

• an XX female fetus producing too much androgen will then ultimately produce too much testosterone bc this pathway is getting overloaded & it can cause an unusual phenotype

Less common DSDs due to enzymatic deficiencies:

• 3-beta hydroxysteroid dehydrogenase (HSD) deficiency = salt wasting and DSD in both sexes

• 17-beta HSD deficiency = XY DSD and amenorrhea in XX females

Disorders of Sexual Development: 5-𝛂 Reductase Deficiency (when pathways are non-functional, things happen)

• 5-𝛂 reductase deficiency in gonadal males prevents adequate conversion of testosterone to DHT

• Lack of DHT prevents fetal development of external male genitalia

  • male genitalia appear female or ambiguous at birth

  • have gonadal sex of testis

• Internal genitalia are normal male (testis, Wolffian Duct, seminal vesicles, epididymus, vas deferens)

• Female external genitalia and female phenotype: pseudohermaphroditism

• At puberty, increase in testosterone from testes causes virilization (increased growth of phallus and scrotum, male hair and muscle growth and male phenotype begins)

  • most individuals (if severe enough) get raised as females until they hit puberty

  • start to develop secondary sex characteristics of a male

• “guevedoce” = penis at 12

  • huge amount of testosterone will start to convert clitoris to looking more like a penis

Disorders of Sexual Development: Testicular Feminization Syndrome = Androgen Insensitivity Syndrome (failure of androgen receptor)

• Androgen insensitivity at the target organ tissue leads to feminization in genetic (XY) males

• Caused by an androgen receptor defect, severity of receptor mutation correlates with phenotype

• Testes are present and produce testosterone, the lack of functional receptor prevents the typical male external and internal phenotypic sexual differentiation

• fetus is male, producing testosterone, but receptor for testosterone is defective

• have a functioning testis that’s producing testosterone, but none of rest of it can be recognized

• both external & internal phenotype appear to be female

  • individuals are sterile, have testis & female reproductive parts, but this is true form of XY phenotypic female

Altered Sex Differentiation Can Be External to the Fetus (in livestock production, can happen when we have twins)

• Freemartin (condition name)

  • Most common intersex condition in cattle

    • Very rare in small ruminants

  • Dizygotic twins where one is male and the other is female

  • Anastomoses of placental vasculature

    • Androgens and AMH from male twin masculinizes the female twin

    • Creates a somatic chimera

  • About 90-95% of female calves born to a male twin will be sterile

Abnormalities in External Male Genitalia

Odd external genitalia

Abnormalities in External Female Genitalia

We still eat the, not good for repro

ICQ #1: The failure of ___ ___ to convert ___ into ___ would most likely result in altered ___ genitalia

• Leydig cells, estrogen, testosterone, external

• Target tissue, testosterone, DHT, external

• Leydig cells, testosterone, estrogen, internal

• Sertoli cells, estrogen, testosterone, internal

• Sertoli cells, estrogen, testosterone, internal

• None of the above are likely

ICQ #2: In the XY chromosome bearing fetus, genetic sex is determined by the expression of a specific gene on the Y chromosome. If this gene is present but has an inactivating mutation, what is the most likely result?

• Sox9 and other male determining genes will still activate and the offspring will appear male

• Wnt4 will activate FGF9 and other male determining genes and the offspring will appear male

• Sox9 will not be expressed properly, no activation of other male determining genes and the offspring will appear female

• Sertoli cells of the XY fetal testes will make testosterone regardless of the gene mutation and the offspring will appear male

• If the male determining genes are not expressed, 5-alpha reductase will strill produce DHT from adrenal andorgens and the offspring will appear male

Gonadal Sex is Not Yet Determined When Germ Cells Arrive at Primitive Gonad

• During primordial germ cell (PGC) generation and proliferation, phenotypic sex is still undetermined

  • Same process occurs in males and females

  • PGCs originate at a very early stage (gastrula, ~2 weeks)

Somatic Cells Play a Crucial Role in Germ Cell Development

• From an evolutionary perspective, the sole role of all somatic cells is the generation, proliferation, protection, and dissemination of germ cells

  • somatic cells help germ cells make next generation

    • eyes, ears, skin cells

  • purpose is to make sure you can deliver a gamete to next generation

  • make sure gametes are produced properly

• Somatic cells provide the microenvironment or “stem cell niche” conducive to primordial germ cell survival

  • Germ cells are responsible for generating genetic diversity through meiosis and transmitting DNA to subsequent generations

Microenvironment of the Developing Testes Transforms Primordial Germ Cells to Spermatogonia

• PGCs begin to line up on tubules & begin to differentiate into spermatogonia in the male

• microenvironment of the testis transforms a primordial germ cell

Microenvironment of the Developing Ovary Transforms Primordial Germ Cells to Oogonia

• PGCs spread out in cortex of developing ovary

• microenvironment will allow oogonia to continue mitosis & ultimately convert them into meiotic oocytes

Germ Cells of the Fetal Gonad

• Males

  • Mitotic germ cells (spermatogonia) embed in seminiferous tubule along Sertoli cells

  • Spermatogonia are then quiescent until puberty (after they form in fetus)

    • ~12-14

    • spermatogonia go to sleep after they’re developed

• Females

  • Germ cells (oogonia) continue with mitosis (continue to divide)

  • Enter meiosis during fetal development and become meiotically quiescent (oocytes are in meiotic arrest until puberty)

Primordial Germ Cells Differentiate to Oogonia in the Ovary

• Oogonia proliferate via mitosis (generate large # of oocytes)

• Enter first phase of meiosis and then become quiescent (meiotic arrest)

And now we wait until puberty…

Hypothalamus and Pituitary Control Many Endocrine Functions

Reproductive Hormones to Know

• what these hormones do, where they come from, what is their basic fxn

• understand how these things work together & how the feedback mechanisms are working in order for us to have a fxn

Reproductive Hormones to Know 2

Summary

• Gonadal development occurs early in gestation any errors or external environmental effects can have profound impacts on the offspring and the subsequent generations

• Genetic sex is determined by the Y chromosome and subsequent expression of SRY under normal conditions. In the absence of SRY expression, female is the default sex

• Sex determination requires more than SRY expression. Subsequent gene expression, enzyme and hormone activity are critical for proper development gonadal and phenotypic sex

• Abnormalities in gonadal and phenotypic sex development are common

• Primordial germ cells enter an undifferentiated gonad, but the influence of somatic cells determines their differentiation into either spermatogonia or oogonia