Chapter 1

Introduction to Molecular Regulation and Signaling

Gene expression can be regulated at several levels:
(1) different genes may be transcribed,
(2) nuclear deoxyribonucleic acid (DNA) transcribed from a gene may be selectively processed to regulate which RNAs reach the cytoplasm to become
messenger RNAs (mRNAs),
(3) mRNAs may be selectively translated, and
(4) proteins made from the mRNAs may be differentially modifi ed.
GENE TRANSCRIPTION
Genes contained in chromatin- basic unit of structure nucleosome
Each nucleosome composed of
octamer of histone proteins
140 base pairs of DNA
- linker DNA-bind with other histone proteins into clusters
- keep the DNA tightly coiled, cannot be transcribed(inactive state)
heterochromatin- chromatin appears as beads of nucleosomes on a string of DNA
For transcription- euchromatin- DNA must be uncoiled
Genes reside within the DNA strand contain regions called
a. exons, which can be translated into proteins,
b. introns, which are interspersed between exons and not transcribed into proteins
also includes the following:
c. promoter region -binds RNA polymerase for the initiation of transcription;
d. transcription initiation site
e. translation initiation site to designate the first amino acid in protein;
f. translation termination codon;
g. 3 untranslated region that includes a sequence (poly A addition site)-assists with stabilizing mRNA, to exit nucleus, and permits it to be translated
into protein. DNA is transcribed from 5 to the 3 end, and promoter region is upstream from the transcription initiation site
Promoter region
- where the RNA polymerase binds, contains sequence TATA, site is called TATA box -In order to bind
requires
additional proteins called transcription factors
transcription factors
have a specific DNA-binding domain
Transactivating domain that activates or inhibits transcription of gene whose promoter or enhancer it
has bound
activate gene expression by causing DNA nucleosome complex to unwind, by releasing polymerase so
that it can transcribe the DNA template, and by preventing new nucleosomes from forming.
Enhancers
- regulatory elements of DNA-activate utilization of promoters to control efficiency and rate of transcription
from promoter
- can reside anywhere
- bind transcription factors thru transcription factors transactivating domain
- used to regulate timing of a genes expression and its cell-specific location
- Ex. PAX6 transcription factor participates in pancreas, eye, and neural tube development, contains three
separate enhancer
- act by altering chromatin to expose promoter or by facilitating binding of RNA polymerase
silencers- inhibit transcription- activate one gene while silencing another by binding to different enhancers

DNA Methylation Represses Transcription

Methylation of cytosine bases in promoter regions Ex. X chromosome inactivation
- genes in different types of cells are repressed - Blood(DNA is highly methylated) and muscle
- for genomic imprinting- only a gene inherited from father or mother is expressed while other gene is
silenced
- 40 to 60 human genes are imprinted and methylation patterns are established during spermatogenesis
and oogenesis
- silences DNA by inhibiting binding of transcription factors or by altering histone binding resulting in
stabilization of nucleosomes and tightly coiled DNA that cannot be transcribed.

OTHER REGULATORS OF GENE EXPRESSION

Nuclear RNA (nRNA) or sometimes premessenger RNA- initial transcript of a gene

longer than mRNA- alternative splicing- introns that are removed (spliced out), exons are spliced in
different patterns, carried out by spliceosomes and proteins that recognize specific splice sites at 5 or the
3 ends of nRNA
splicing isoforms (also called splice variants or alternative splice forms)- Proteins derived from the same
gene Ex. WT1 gene have different functions in gonadal versus kidney development
post-translational modifications
- even after translation some have is cleaved to become active or phosphorylated
- 23,000 genes exist, 5 times number of genes can be synthesized

INDUCTION AND ORGAN FORMATION

A process where one group of cells or tissues causes another set of cells or tissues to change their fate
Inducer- produces a signal responder
Competence- capacity to respond to signal, requires competence factor
1. epithelial mesenchymal interactions- Many inductive interactions occur between them
epithelial- joined together in tubes or sheets
mesenchymal- are fibroblastic in appearance and dispersed
in extracellular matrices
Examples
gut endoderm and surrounding mesenchyme- produce gut-derived organs, including liver and pancreas
limb mesenchyme with overlying ectoderm (epithelium)- produce limb outgrowth and differentiation
endoderm of ureteric bud and mesenchyme from metanephric blastema- produce nephrons in kidney
2. between two epithelial tissues
Ex. induction of the lens by epithelium of optic cup
Crosstalk- between two tissues or cell types is essential for differentiation to continue

CELL SIGNALING
established by
1. paracrine interactions- proteins synthesized by one cell diffuse with with other cells
paracrine factors or growth and differentiation factors (GDFs)-diffusable proteins
2. juxtacrine interactions, do not involve diffusible proteins

Signal Transduction Pathways

Paracrine Signaling- hedgehog signaling
act by signal transduction pathways include a signaling molecule (the ligand) and a receptor
receptor spans cell membrane and has an
extracellular domain (the ligand-binding region),
transmembrane domain
cytoplasmic domain
kinase---phosphorylate other proteins using ATP as a substrate---activates a transcription factor--activates or inhibits
gene expression
Juxtacrine Signaling
3 ways it occurs
(1) protein on one cell surface interacts with a receptor on an adjacent cell in a process. Notch pathwayNotch receptor protein binds with Delta, Serrate, or Jagged proteins--- cytoplasmic side of membrane
is cleaved--- binds to transcription factor to activate gene expression
Impt in neuronal differentiation, blood vessel specifi cation, and somite segmentation
(2) Ligands in extracellular matrix secreted by one cell interact with their receptors on neighboring cells
extracellular matrix consists of large molecules secreted by cells including
collagen, proteoglycans (chondroitin sulfates, hyaluronic acid, etc.), and glycoproteins, such as
fibronectin and laminin
Ex. type IV collagen are components of basal lamina for epithelial cell attachment,
fibronectin molecules form scaffolds for cell migration.
Integrins- Receptors that link extracellular molecules such as fibronectin and laminin to cells, uses actin
can induce gene expression and regulate differentiation- chondrocytes be linked to matrix to form cartilage
3. direct transmission of signals from one cell to another by gap junctions- small molecules and ions can
pass
junctions are made of connexin proteins
other genes in the family may compensate for the loss of one of their counterparts
there is crosstalk between pathways

Paracrine Signaling Factors- GDFs

four groups/ families interacts with its own family of receptors

1. fi broblast growth factor (FGF)- stimulate the growth of fibroblasts, two dozen
produce proteins--- activate tyrosine receptor kinases called fibroblast growth factor receptors
(FGFRs)---act. Signal path
important for angiogenesis, axon growth, and mesoderm differentiation. FGF8 impt for devt of limbs and
parts of brain
2. WNT-there are 15, related to segment polarity gene, wingless in Drosophilia
frizzled family of proteins- receptors
regulating limb patterning, midbrain development, and some aspects of somite and urogenital
differentiation
3. Hedgehog
Named after pattern of bristles on leg of Drosophila resembled the shape of a hedgehog
3 hedgehog genes, Desert, Indian, and sonic hedgehog- limb patterning, neural tube induction and
patterning, somite differentiation, gut regionalization, and others
Patched- receptor--- binds with Smoothened--- transduces hedgehog signal but it is inhibited by
Patched until the hedgehog protein binds to this receptor
4. transforming growth factor-b (TGF-b) 30 members includes TGF-bs, bone morphogenetic
proteins, activin family, Mllerian inhibiting factor (MIF, anti-Mllerian hormone), and others.
Important for extracellular matrix formation and epithelial branching that occurs in lung, kidney, and
salivary gland development. BMP family induces bone formation and is involved in regulating cell
division, cell death (apoptosis), and cell migration

Other Paracrine Signaling Molecules

Neurotransmitters- serotonin and norepinephrine- act as ligands and bind to receptors
Serotonin (5HT)
- acts as a ligand for receptors- G proteincoupled receptors.
- regulates cell proliferation and migration, important for establishing laterality, gastrulation, heart
development, etc during early stages of differentiation.
Norepinephrine role in apoptosis (programmed cell death) in the interdigital spaces and in other cell types.
Chapter 2 / Gametogenesis: Conversion of Germ Cells into Male and Female
Devt begins with fertilization- union of gametes---give rise to zygote
Gametes sperm and oocyte, derived from Primordial germ cells(PGCs)
2nd week- formed in epiblast to yolk sac
4th week yolk sac to developing gonads arrive end of 5 th week
Gametogenesis- includes meiosis- reduce the number of chromosomes
Cytodifferentiation- complete maturaturation
Teratomas-PGCs that have strayed from normal migratory path

CHROMOSOME THEORY OF INHERITANCE

Humas- 23,000 genes on 46 chromosomes
linked genes- Genes on same chromosome tend to be inherited together
In somatic cells- chromosomes appear as 23 homologous pairs to form diploid number of 46
Autosomes-22 pairssex chromosomes- one pair
sex pair is XX-female XY-male
Each gamete is haploid(23 chromosomes,)
Mitosis is process- one cell divides= two daughter cells-genetically identical to parent cell
Phases
each chromosome replicates its deoxyribonucleic acid (DNA). chromosomes are extremely long
prophase- chromosomes begin to coil, contract, and condense
chromatids- two parallel subunits joined by centromere
prometaphase do chromatids become distinguishable
metaphase- chromosomes line up in the equatorial plane and doubled structure is clearly visible
Each is attached by microtubules extending from the centromere to centriole, forming mitotic spindle
Anaphase- centromere of each chromosome divides followed by migration of chromatids to opposite poles of the
spindle
Telophase- chromosomes uncoil and lengthen, nuclear envelope reforms, and cytoplasm divides
daughter cell receives half of all doubled chromosome material and thus maintains same number of
chromosomes as mother cell
Meiosis takes place in germ cells to generate male and female gametes
Meiosis requires 2 cell divisions, meiosis I and meiosis II to reduce number of chromosomes to haploid number
of 23

Same with mitosis

In Contrast
male and female germ cells (spermatocytes and
homologous chromosomes then align themselves in
primary oocytes) at beginning of meiosis I replicate
pairs, a process called synapsis
their DNA so that each of 46 chromosomes is
pairing is exact and point for point except for XY
duplicated into sister chromatids
combination
Homologous pairs then separate into 2 daughter cells, thereby reducing chromosome number from diploid to
haploid. Shortly thereafter, meiosis II separates sister chromatids. Each gamete then contains 23 chromosomes.
Crossovers- critical events in meiosis I- interchange of chromatid segments between paired homologous
chromosomes
Chiasma- X-like structure
30 to 40 crossovers (one or two per chromosome) with each meiotic I
As a result - Genetic variability, Each germ cell contains a haploid number of chromosomes, so that at
fertilization the
diploid number of 46 is restored
Polar Bodies- during meiosis(from primary oocyte becomes 1 mature gamete and 3 polar biodies
Morphological Changes During Maturation of the Gametes
Oogenesis- oogonia differentiate to mature oocytes
From PGCs arriving in Gonad---oocyte differentite in mitosis
End of 3rd month- arranged in clusters surrounded by layer of epithelial cells- the Follicular cells- originate from surface epi
of ovary
Some continue to divide in mitosis, some form Primary oocytes
5th mo-7 million germ cells maximum, at this time many becomes atretic-degenerate
7th mo- majority degenerated except near surface

primordial follicle- primary oocyte, together with its surrounding flat epithelial cells
Maturation of Oocytes Continues at Puberty
diplotene stage, a resting stage during prophase that is characterized by a lacy network of chromatin, instead
of proceeding
into metaphase
Primary oocytes remain arrested in prophase and do not finish their first meiotic division before puberty is
reached.
by oocyte maturation inhibitor (OMI)-produced by follicular cells
at birth- 600,000 to 800,000 oocytes
childhood-most become atretic-40,000
puberty- fewer than 500 will be ovulated
primary oocytes are vulnerable to damage as they age

At puberty- pool of growing follicles is established and continuously maintained

primordial follicles continous supply at puberty--- Each month, 15 to 20 follicles selected from this pool begin to
mature--- Some of these die, while others begin to accumulate fluid in a space called antrum---enter antral or
vesicular stage--- Fluid continues to accumulate--- follicles are quite swollen and are called mature vesicular
follicles or Graffian follicles
antral stage is longest
mature vesicular stage- 37 hours prior to ovulation
primary follicle- growing primordial follicle with unit of stratified epithelium of granulose cells

theca folliculi- separating Granulosa cells with ovarian connective tissue (stromal cells) theca interna, theca
externa
zona pellucid- glycoproteins on the surface of the oocyte
fi nger-like processes- follicular cells extend across the zona pellucid, for transport of materials from follicular
cells to the oocyte
antrum- Coalescence of fluid-filled spaces, follicle is termed vesicular or an antral follicle
cumulus oophorus-from Granulosa cells surrounding the oocyte that remain intact
mature vesicular (Graafi an) follicle- 25 mm or more in dameter
surrounded by theca interna composed of cells like steroid secretion, rich in blood vessels, and theca externa,
which gradually merges with ovarian connective tissue
ovarian cycle- only one follicle reaches full maturity.
When the secondary follicle is mature, a surge in luteinizing hormone (LH) induces preovulatory growth
phase
Meiosis I is completed, resulting in formation of two daughter cells of unequal size, each with 23 doublestructured chromosomes
Secondary oocyte- receives most of the cytoplasm
First polar body- receives none, lies between zona pellucida and cell membrane of the secondary oocyte in
perivitelline space, may
undergo a second division
Enters meiosis II- completed only if the oocyte is fertilized otherwise, cell degenerates approximately 24 hours
after ovulation
Metaphase- arrest 3 hours before ovulation

Spermatogenesis
Maturation of Sperm Begins at Puberty
Spermatogenesis- spermatogonia are transformed into spermatozoa
At birth- germ cells are large, pale cells surrounded by supporting cells
Supporting cells- from the surface epithelium of the testis--- become sustentacular cells, or Sertoli cellsparticipate in nutrition of spermatids and assist in release of mature spermatozoa
Puberty- sex cords become seminiferous tubules
form type A spermatogonia- marks initiation of spermatogenesis
type B spermatogonia
priamary spermatocytes-enter prolonged prophase (22 days) followed by rapid completion of meiosis I form
secondary spermatocytes
haploid spermatids
type A cells to spermatids, cytokinesis is incomplete, cell generations are joined by CYTOPLASMIC BRIDGES
Spermatogenesis is regulated by LH production by pituitary gland--- binds to receptors on Leydig cells and
stimulates testosterone production---bind to Sertoli cells to promote spermatogenesis
Follicle-stimulating hormone (FSH)- stimulates testicular fluid production and synthesis of intracellular
androgen receptor proteins
Spermiogenesis
- transformation of spermatids into spermatozoa
changes include
1. formation of acrosome- covers half of nuclear surface and contains enzymes to assist in
penetration of egg and its surrounding layers during fertilization
2. condensation of nucleus;
3. formation of neck, middle piece, and tail; and
4. shedding of most of cytoplasm as residual bodies that are phagocytized by Sertoli cells.
time required- 74 days, 300 million sperm cells daily
When fully formed- spermatozoa eneters lumen of seminiferous tubules--- epididymis
Chapter 3 / First Week of Development: Ovulation to Implantation
Ovarian Cycle

Hypothalamus release GnRH---ANTERIOR Pituitary Gland(adenohypophysis)---secrete gonadotropinsmonthly(sexual)

cycle
FSH and LH- stimulate and control change in ovary
FSH-rescue 15-20 primary stage(preantral) follicles
Only one matures, others degenerate(oocyte becomes corpus atreticum)
Also maturates follicular(granulose)s surrounding oocyte---proliferate mediated by Factor 9 (member of TFGb)
Theca interna and granulose cells secrete estrogen

Theca interna- produces androstenadione and testosterone

Granular cells-converts these hormones to estrone and B-estradiol
Result of estrogen
uterine endometrium enters follicular or proliferative stage
thinning of cervical mucus- for passage of sperm
anterior lobe of PG is stimulated to secret LH
Midcycle, LH will
elevate concentration and maturation- promoting factor, causing oocyte to complete meiosis I and initiate meiosis II
stimulate production of progesterone by following stromal cells(luteinizationb
cause follicular rupture
Ovulation
Before ovulation- Vesicula Follicle grows 25mm---become Graafian follicle (mature), abrupt inc in LH(Primary oocyte complete
Meiosis I---enter preovulatory mature vesicular stage---initiate Meiosis II(arrested in metaphase for 3 hours before

ovulation)--- stigma(bulge, avascular spot) appears

LH inc collagenase activity, digestion of collagen fibers surrounding follicle
Prostaglandin levels also inc in response to the LH surge---cause local muscular contractions in ovarian wall--extrude oocyte with its granulosa cells--- breaks free (ovulation) and floats out of ovary
cumulus oophorus cells rearrange themselves around zona pellucida to form corona radiata
After ovulation
LH causes granulosa cells remaining with theca interna develop a yellowish pigment and change into lutein
cells form corpus luteum- secrete estrogens and progesterone
Progesterone- causes uterine mucosa to enter progestational or secretory stage for implantation of embryo

Oocyte Transport
before ovulation
fimbriae sweep over surface of ovary and tube begins to contract--- oocyte w/ granulose cells carried into tube
by fimbrae and cilia--- Once in tube, cumulus cells withdraw cytoplasmic processes from zona pellucida and lose
contact with oocyte
transport regulated by endocrine status during and after ovulation
fertilized oocyte reaches uterine lumen in 3-4 days
If fertilization does not occur, corpus luteum(yellowish projection on surface of ovary) reaches maximum
development 9 days after ovulation--- shrinks because of degeneration of lutean cells (luteolysis) and forms
mass of fibrotic scar tissue, corpus albicans
progesterone production decreases--- menstrual bleeding
If oocyte is fertilized, human chorionic gonadotropin prevents degeneration of corpus luteum
HCG- hormone secreted by syncytiotrophoblast of the developing embryo
corpus luteum continues to grow and forms corpus luteum of pregnancy (corpus luteum graviditatis)
end of 3rd month-CL 1/3 to of total size of ovary, Yellowish luteal cells cont. to secrete progesterone then
regress
end of 4th month- trophoblastic component of placenta secrete progesterone to maintain pregnancy
Removal of corpus luteum of pregnancy before 4th month usually leads to abortion

FERTILIZATION- male and female gametes fuse, occurs in ampullary region of uterine tube(widest and
near ovary)
1% of sperm deposited in vagina enter cervix, survive many hours--- move by muscular contraction into uterine
tubes(30mins or as slow as 6 days)---reach isthmus become less motile and cease migration---then motile at
ovulation by chemoattractants produced by cumulus cells--- swim to the ampulla, where fertilization occurs
Spermatozoa are not able to fertilize oocyte immediately and must undergo
(1) capacitation and (2) acrosome reaction
Capacitation- period of conditioning in female reproductive tract- 7 hours
glycoprotein coat and seminal plasma proteins are removed from plasma membrane that overlies acrosomal
region of spermatozoa
acrosome reaction, occurs after binding to zona pellucida, is induced by zona proteins- release of enzymes to
penetrate zona pellucida, including acrosin- and trypsin-like substances
The phases of fertilization include
Phase 1, penetration of the corona radiata
200 to 300 million spermatozoa deposited--- 300 to 500 reach ampulla---1 fertilizes egg others aid fertilizing
sperm in penetrating the
barriers protecting female gamete

Phase 2, penetration of the zona pellucid

mediated by ligand ZP3, a zona protein, release of acrosin contacts plasma membrane of oocyte---release of
lysosomal enzymes
from cortical granules alter properties of the zona pellucida (zona reaction)--- prevent other sperm
penetration and inactivate speciesspecific receptor sites for spermatozoa
Phase 3, fusion of the oocyte and sperm cell membranes
mediated by interaction of integrins on oocyte and ligands, disintegrins, on sperm---plasma membranes of
sperm and egg fuse, actual fusion is between oocyte membrane and membrane that covers posterior region of
sperm head---head and tail of he spermatozoon
enter cytoplasm of oocyte

OOcyte responds in 3 ways

1. Cortical and zona reactions
Release of lysozomal enzymes---1. Membrane becomes impenetrable to other spermatozoa 2. Zona
pellucid prevent poly spermy(penetration of more than one sperm)
2. Resumption of 2nd meiotic division
Oocyte finishes 2nd meiotic division--- 2nd polar body(no cytoplasm) and definitive oocyte (chromosome 22
plus X) arranged in vesicular nucleus- female pronucleus
3. Metabolic activation of egg- carried by the spermatozoon, associated with early embryogenesis
Spermatozoon nucleus bulges, becomes male pronucleus---contacts female pronucleustail detaches and
degenerates---lose their nuclear envelopes---replicate DNA(synthesis)---organize in sindle for mitotic division--chromosomes split at the centromere, sister chromatids move to opposite poles---deep furrow appears dividing
cytoplasm into two parts

main results of fertilization are as follows:

Restoration of the diploid number of chromosomes, half from father and half from mother-zygote contains
a new combination of chromosomes different from both parents.
Determination of the sex of the new individual. X-carrying sperm produces a female (XX) embryo, Ycarrying sperm produces a male (XY) embryo.
Initiation of cleavage. Without fertilization, oocyte usually degenerates 24 hrs after ovulation.

CLEAVAGE
TWO-CELL STAGE-undergoes a series of mitotic divisions, smaller-blastomeres
EIGHT-CELL STAGE-form a loosely arranged clump
third cleavage-compaction- communicate extensively by gap junctions, from outer cells
16-CELL-3 days after fertilization- morula constitute inner cell mass(gives rise to tissues of embryo proper)
and outer cell mass(forms the trophoblast, later contributes to placenta)
morula enters uterine cavity---fluid begins to penetrate into intercellular spaces---blastocele, forms---embryo is
a blastocyst---inner cell
mass, now called embryoblast, outer cell mass is trophoblast-flatten and form epithelial wall of blastocyst
zona pellucida disappeared,implantation to begin
6th day- trophoblastic cells over embryoblast pole begin to penetrate bet epithelial cells of uterine mucosa

L selectin and carbohydrate receptors mediate initial attachment of the blastocyst to uterus