repro test 1

Ovarian cycle – female HPG axis

-        Hypothalamus: Releases (GnRH) in pulses.

-        Anterior Pituitary: Secretes (FSH) and (LH) in response to GnRH.

-        Ovaries: Produce sex steroids (estrogen/estradiol and progesterone) and peptide hormones (inhibin)

Oogenesis - Production of gametes in female

In utero:

-        Gamete form many primary follicles containing each one oogonium

-        Each oogonium undergoes mitosis -> each oogonium forms 2 primary oocytes

-        Primary Oocytes start meiosis and arrest in prophase 1

After birth, after puberty

-        FSH stimulates a few follicles to grow -> every menstrual cycle a few Oocytes complete meiosis I

o    Result: 2 cells = Secondary Oocyte and 1st polar body

-        Secondary Oocyte develops to metaphase II, rests in this state till after ovulation = haploid oocyte and polar body

-        Only if fertilized completion of Meiosis II (casts off second polar body)

Ovarian cycle: follicular phase

-        Day 1 of menstruation.

-        FSH and LH are released by the pituitary.

-        Follicle development: Primordial → Primary → Secondary → Mature (Graafian).

• Primordial cell contains an immature primary oocyte surrounded by granulosa cells that multiply as the follicle develops.

• The zona pellucida forms around the oocyte.

-        FSH → granulosa cells → growth + estradiol + inhibin.

-        LH → theca cells → androgens + progesterone.

-        Theca-cell androgens are used by the granulosa cells to make estradiol.

-        Increasing estradiol promotes development of the dominant follicle, which prepares for ovulation.

ovulation

-        Near the middle of the cycle

- estradiol becomes very high.

-       the high estradiol level causes positive feedback, resulting in a large LH surge.

-        LH surge → follicle wall ruptures → mature follicle is released

Lutel phase

-        Antrum contains clotted blood after ovulation -> corpus hemorrhagicum

-        Ruptured follicle turns into Corpus luteum after ovulation - Becomes a gland – secrets Progesterone + Estradiol + Inhibin

-        Corpus luteum regresses if no pregnancy occurs after 10-12 days -> scar tissue corpus albicans

- If pregnancy occurs Corpus Luteum is supported by hCG = human chorionic gonadotrophic hormone

Menstrual cycle 1.     Menstrual Phase

-        Lack of progesterone

-        Loss of blood due to breakdown of superficial layers of uterine endometrium

-        About 30 - 50 ml of mainly arterial blood.

Menstrual cycle : 2.    Proliferative phase

-        FSH grows follicles and oocytes (in ovaries) -> they produce mainly estrogen

-        Estrogen travels through blood to endometrium and thickens and builds it up

-        Uterine glands small and round

menstrual cycle 3.    Secretory phase

-        LH from ovulation and luteal phase

-        corpus luteum progesterone and estrogen -> maintains lining and enlarges glands, elongated, & coiled arteries

-        Uterine glands secrete uterine milk

4. Premenstrual phase

-        Lack of progesterone

-        Glands start breaking down

-        Blood vessels leak

-        Tissue death

Epithelium of the Cervix

-        Mucus secreting simple columnar epithelium on luminal surface

-        moist non-keratinized stratified squamous epithelium covering the external surface of the cervix

-        Transformation zone - area between columnar and squamous cells

-        cervix averages 22 mm in length and 6 mm in diameter at ovulation

Cervical mucus:

-        2 major fractions:

o    insoluble gel or mucin

o    aqueous phase containing the soluble components (lipids, fatty acids, prostaglandins, proteins, enzyme inhibitors, and immunoglobulins)

-        Physical barrier to pathogens

-        Immune barrier to pathogens

-        Sometimes more or less permeable to sperm

Changes in cervical mucus

1.     Early Follicular phase: Mucus thick, sticky

2.     Late Follicular phase

-        Mucus copious, watery, thin & alkaline → sperm can penetrate mucus

-        Dominant influence: Estrogens

3.     Ovulatory Period

-        Mucus thinnest- wet and clear at time of ovulation

-        sperm can easily penetrate through the mucus

4.     Luteal phase

-        Mucus thick, viscous & cellular

-        No ferning pattern when dried

-        Dominant influence: Progesterone

Hormonal regulation in the male (HPG-axis)

-        Hypothalamus -> GnRH -> anterior pituitary -> LH and FSH

-        LH influences Leydig cells to produce testosterone (neg feedback)

-        FSH stimulates Sertoli cells to produce ABP and inhibin (neg feedback from inhibin)

The Testes anatomy

-        Paired and contained within the scrotum

-        Septa divide testis into ~250 lobules

-        Each lobule contains 1-4 seminiferous tubules

pathway of sperm

Seminiferous tubules → Straight tubules → Rete testis → Efferent ductules → Epididymis → Vas deferens → (past seminal vesicles) ejaculatory duct → (past prostate and bulbourethral glands) urethra

Seminiferous tubules

-        Site of sperm production

-        Lined by complex stratified epithelium

Cells in the testes Seminiferous tubules:

in seminiferous tubules

-        Spermatogonia

-        FSH → Sertoli cells → ABP AND GF → nourishment & protection for developing sperm & contain receptors to bind testosterone

Interstitial space:

-       LH → Leydig cells:→ testosterone, -> testosterone diffuses into seminiferous tubules

-        Peritubular myoid cells: peristaltic movement

- Capillary cells of blood vessels

Blood-testes barrier

-        Separates the testes from the normal circulatory processes

-        Tight junctions between adjacent Sertoli cells form the blood testis barrier

o    Prevents blood and other body fluids entering the lumen of the seminiferous tubules

o    Only allows secretions from Sertoli cells to enter

o    Protects developing sperm from the body’s immune system, restricts passage of drugs

Spermatogenesis

-        Meiosis: Formation of haploid Spermatids from diploid Spermatogonia

o    Stem cells enter into spermatogenesis ~ every 16 days

1.     Mitosis: spermatogonia become primary spermatocytes

2.     Meiosis I: Reductional division Separation of homologous chromosomes

3.     Meiosis II: Equational division Separation of sister chromatids

Spermiogenesis

-        remodelling and maturing of spermatids into mature spermatozoa through differentiation

-        Round spermatids become elongated spermatids

-        Spermatids are nonmotile

-        Then spermiation = release of spermatozoa into lumen of seminiferous tubules

-        sertoli cells provide nutrients

-        4 phases

1.     Golgi phase

-        Spermatid almost spherical

• Well developed golgi apparatus

-        Small vesicles of golgi fuse -> form secretory pro- acrosomic granules

-        Vesicle fusion continues till a large acrosomal vesicle is formed

• Proximal centriole (PC) is region where tail will attach

2. cap phase

-        Golgi migrates to caudal end

• Distal Centriole (DC) forms Axoneme (AX) = flagellum

-        Acrosomic vesicle flattens and forms distinct cap:

o   Outer acrosomic membrance (OAM)

o    Inner acrosomic membrane (IAM)

-        Acrosomal contents -> enzyme

3. Acrosomal Phase

-        Nucleus elongates

-        Acrosome covers majority of anterior nucleus

-        Neck is formed

4. maturation phase

-        Mitochondria assemble in midpiece

-        Excess cytoplasm phagocytosed by Sertoli cells

Spermiogenesis after remodeling

-        Mature spermatozoa released from Sertoli cells into lumen of seminiferous tubule

-        Spermatozoa are now mature but lack motility

-        Non-motile spermatozoa transported to epididymis in testicular fluid secreted by the Sertoli cells by peristaltic contraction

Sperm anatomy and functionality

-        Head & acrosome for enzymatic penetration of the egg at fertilisation

-        Midpiece containing the mitochondrion

-        Tail for forward propulsion

-        Sperm is stored in epididymis till needed

-        During ejaculation sperm musculature in vas deferens rhythmically contracts

-        Young men produce ~300.000 sperm a minute = 400 million/day

-        Degradation of sperm which is not ejaculated

Erectile tissue in the penis

-        Corpus spongiosum: surrounds urethra

-       Corpus cavernosa: paired dorsal erectile bodies

-        During arousal

o    nerves in penis release vasoactive intestinal peptide (VIP) and nitric oxide (NO) → vasodilation

o    increased blood flow to arteries in corpus cavernosa

o    "helicine arteries“ empty into corpora cavernosa enclosed by the trabeculae engorging it with blood

o    swelling = erection & compression of veins and venules to maintain erection

Semen Accessory gland secretions

-        Seminal Vesicles: ~65% of seminal fluid, alkaline, prostaglandins, clotting proteins & fructose (ATP)

-        Prostate: ~30% of seminal fluid, contains enzymes & secrets milky, slightly acid fluid

-        Bulbourethral glands: <5% of seminal fluid, thick, clear mucus to lubricate glans penis, alkaline -> neutralizes traces of acidic urine in urethra

NORMAL SEMEN ANALYSIS

-        colour : white, opalescent

-        pH : 7.35 - 7.50

- volume : 2 to 6 ml

-        sperm count : > 40 million/ml (sterile if < 20mio/ml)

-        motility : > 60 % with good forward progression

-        morphology : > 60 % normal form

endocrine disruptors

-        Organic compounds with hormone-like activity e.g. Pesticides

- Two distinct modes of action:

o    direct impact on males after puberty

o    When substances in mother’s bloodstream can disrupt embryonic and fetal development

Meiosis I - Reductional division

-        Prophase I: chromosomes condense, spindle forms, nuclear membrane dissolves, homologous chromosomes pair and form synapsis, crossing over

-        Metaphase I: homologous chromosomes align at equator, spindle fibers attach to centromers

-        Anaphase I: homologous chromosomes separate -> drawn to opposite poles by spindle

-        Telophase I: spindle disappears, nuclear membrane forms

-        Cytokinesis: cytoplasm separates, formation of two separate cells

Meiosis II – Equational Division = Mitotic division of haploid cells produced in Meiosis I

-        Prophase II: nuclear membrane dissolves, spindle forms

-        Metaphase II: chromosomes align at equator

-        Anaphase II: Sister chromatids separate and get drawn to opposite poles by spindle

-        Telophase II: spindle disappears, nuclear membrane forms

-        Cytokinesis: cytoplasm separates, formation of two separate cells

Introduction of variation - Crossing over

-        During Prophase I: Pairing structure of homologous chromosomes: Tetrad = Synapsis = Bivalent

-        Crossing over between non-sister chromatids to exchange of genetic material

-        Several chiasmata depending chromosome length

Introduction of variation Independent assortment

-        meta-phase I: Random alignment at equator and subsequent disjunction/segregation to either pole -> distribution into the daughter cells is random

-        Anaphase I: Either of homologous chromosomes drawn to the poles

- In Anaphase II: Either of chromatids drawn to the poles

Aneuploidy

abnormal number of individual chromosomes in a cell

Monosomy X (Turner Syndrome):

Karyotype: 45, X

-        Affects 1:2000 females

-        Individuals are genetically female

-        The only viable monosomy in humans

-        No sexual maturation during puberty -> sterile

-        Short stature and normal intelligence

-        Often congenital abnormalities -> webbed skin of neck

Trisomy X – Triple X Syndrome

Karyotype: 47, XXX

-        Affects 1:1000 females

-        Individuals are genetically female

-        Healthy and fertile

Usually cannot be distinguished from normal female (phenotypically normal)

Klinefelter Syndrome:

Karyotype: 47, XXY

-        Affects 1:1000 males

-        Genetically male with male sex organs

-        Unusually small testes & sterile

-        Breast enlargement and other feminine body characteristics

-        Usually tall

-        Normal intelligence

Double Y: Karyotype: 47, XYY

-        Affects 1:1000 males

-        Genetically male

-        Nondisjunction in the second meiotic division of father

-        Normal fertility and sexual development

-        Individuals usually phenotypically normal but often taller than average

Patau Syndrome

(trisomy 13): Karyotype: 47, +13

-        Affects 1:5000 live births

-        Serious eye, brain, circulatory defects as well as cleft palate

-        Children rarely live more than a few months

Edward’s Syndrome

(trisomy 18): Karyotype: 47, +18

-        Affects 1:10,000 live births

-        Almost every organ system affected

-        Children rarely live more than a few months

Down Syndrome

(trisomy 21): Karyotype: 47, +21

-        Affects 1:800 live births

-        Altered phenotype:

-        Characteristic facial features: round face, epicanthic folds

-        Short stature

-        Congenital abnormalities of heart

-        IQ often mildly impaired

maternal age and down syndrome

-        Risk seems to be associated with delay in completion of prophase I in female gametes

-        Down Syndrome is highly correlated with age of mother (risk at 40 years ~1%)

-        Newer studies – paternal age important also: up to ¼ of Down Syndrome cases found to originate from paternal non-disjunction

Polyploidy

-        More than two complete sets of chromosomes, e.g. tetraploids (4n)

-        Often due to duplication of genome without subsequent mitosis

Sperm migration

-        Ejaculate of 1.5- to 5.0-ml of semen containing 200 - 500 million sperm -> deposited during coitus in posterior vagina

-        Rapid phase: Some sperm reaches fallopian tubes after ~5min through coordinated vaginal, cervical, and uterine contractions

-        Sustained Phase: Most sperm only reaches distal fallopian tubes after hours to days after undergoing capacitation

-        Spermatozoa undergo capacitation & acrosome reaction before fertilization can occur

-        Capacitation in isthmus of fallopian tube

Sperm activation in the female reproductive tract

1.     Capacitation

-        Acquisition of hypermobile tail

-        Sperm head changes to gain capacity to fertilize

2.     Acrosome reaction

-        Sperm head modified

-        Release of enzymes to penetrate zona pellucida

Chemical changes in sperm during capacitation

1.     Membrane modification

-        Cholesterol leaching through female tract albumin

-        Destabilizes the sperm plasma membrane -> permeable to Ca2+

2.     Modulation of enzymatic activity through

-        Increase of intracellular Ca2+ -> modulating signaling pathways

-        Increase of intracellular HCO3- -> increased intracellular pH -> activation of PKA

3.     Protein modification

-        Hypermobile flagellum

-        In sperm head so they can interact with zona pellucida

Changes of Temperature and pH, sperm pathway

-        Optimal pH for sperm viability is between 7.0 - 8.5

-        Normal vaginal pH is only 3.5 to 4.0, but seminal fluid and cervical mucus in vagina are alkaline and act as buffers

-        Vaginal pH rises to 7.0 within just seconds after ejaculation Temperature:

-        Low in testis and epididymis allowing maturation (35°C)

-        Seminal plasma 37°C

-        Increased temperature in fallopian tube (38.5°C)

Sperm survival

-        200 and 500 million sperm deposited in posterior vagina

-        Majority of sperm do not reach site of fertilization -> only ~2000-3000 spermatozoa reach oocyte

o    destroyed by vaginal acid

o    drain out of vagina

o    cannot penetrate cervical mucus

o    get destroyed by leukocytes in uterus

o    some sperm defect

o    half go in the fallopian tube not containing an oocyte

Fertilization time period

-        Secondary Oocyte has to be fertilized within 12-24h after ovulation

-        Takes ovum 72 hours to reach the uterus -> sperm has to fertilize the egg in ampulla of the fallopian tube

-        Sperm viable for up to six days

Steps of fertilization 1-3

1.     Sperm penetration of cumulus cells

2.     sperm receptors attach to zona pellucida (jelly coat) protein 3 (ZP3) triggering acrosome reaction

3.     Acrosome reaction induced by calcium and progesterone + other factors

-        Membrane surrounding the acrosome fuses with the plasma membrane of the sperm's head -> exposure of acrosome contents:

o    Digestive enzymes

o    Antigens which can bind oocyte membrane

Steps of fertilization 4-6

4. Acrosome vesicle fuses with the zona pellucida and releases digestive enzymes which soften the glycoprotein matrix of the zona pellucida -> sperm can penetrate

5. Sperm entry into perivitelline space and binding to exposed docking proteins on the oocyte membrane

6. membrane fusion between 1 sperm and ovum -> sperm nucleus enters cytoplasm (cell is now diploid)

Steps of fertilization 7-8

7. Cortical reaction: enzymes released preventing polyspermy

• A calcium-dependent exocytotic process in which the content of secretory granules is released into the perivitelline space immediately after fertilization to prevent polyspermic fertilization

• Cortical granules are specialized secretory vesicles positioned near the oocyte plasma membrane

• Fusion of sperm and oocyte membranes activates intracellular signaling pathways -> ER of oocyte releases calcium into cytoplasm

• Calcium wave triggers exocytosis of cortical granules -> enzyme content secreted into perivitelline space

  • Released enzymes modify the zona pellucida proteins -> structural change of zona pellucida = “zona hardening”

  • Formation of a specific coating on oocyte membrane with less receptors for sperm binding

• Permanent barrier to sperm entry gradually established = slow block of polyspermy in many animals

8. hardening of zona pellucida - block to polyspermy

Formation of a zygote

-        After the sperm penetrates the secondary oocyte, the oocyte completes meiosis II, forming the ovum and second polar body.

-        Sperm and ovum nuclei swell, forming pronuclei

-        The DNA in each pronucleus replicates.

-        The pronuclei approach each other and a mitotic spindle forms between them.

-        Chromosomes of the pronuclei mix and feritilisation is complete, the cell now called a zygote is ready for first cleavage

Contributions to the zygote female

-        Female Oocyte:

o    1 nucleus with 23 chromosomes and large amount of cytoplasm

o    Granules for cortical reaction to block polyspermy, mRNA and proteins for fertilization, cleavage, cell fate determination, embryo axis orientation

o    Mitochondria, nucleolus, centriole pair, ribosomes

o    Surrounded by zona pellucida = extracellular matrix – protection

o    Corona radiata – nourishment

male contributions ot zygote

o    1 nucleus with 23 chromosomes

o    Very few mitochondria and other proteins

Events after zygote formation

-        Aim: Quickly grow early embryo

-        First Cleavage: Formation of a two-cell embryo by mitosis

o    First mitotic division 24h after fertilization -> results in two cell embryo – each cell is now called a blastomere

Cleavage and cell cycle events

-        Zygote undergoes further cleavage = rapid cell division without growth

o    Cell cycle consists mainly of S phase and M phase (DNA synthesis and mitosis)

o    Very little protein synthesis (skips G1 and G2)

o    Oocyte brought lots of cytoplasm with proteins and organelles for these cleavages to occur

-        Cleavage into 4 cell embryo: 4 blastomers

-        Cleavage into 8 cell embryo = 8 blastomers

Understanding early cell fate

-        Lineage tracing of embryos developed in vivo after labeling a blastomere by photoconversion at the two-cell stage

-        Labeled embryos developed in vivo to the blastocyst stage

-        Distribution of cells not equally

Morula: Is a solid ball of cells formed after the zygote undergoes cleavage.

-        Typically occurs around 3-4 days after fertilization (~72h)

-        Consists of 16-32 blastomeres derived from the zygote, surrounded by zona pellucida

-        Blastomeres of the morula undergo compaction, flattening and develop polarity with inner and outer aspects

-        Morula then develops into the blastocyst

Blastocyst

-        Blastocyst: consists of ~100 cells

o    Inner cell mass › Becomes embryonic disc, which will form embryo and three or four extraembryonic membranes

o    Trophoblast › Display immunosuppressive factors › Participate in placenta formation

o    Blastocoel › Fluid filed cavity

-        Blastocyst enters uterine cavity ~ 4-5 days after fertilization

Hatching

-        Blastocyst sheds zonal pellucida – enables further growth

-        Hatching process typically occurs around day 5 - 6 after fertilization.

Implantation

-        Blastocyst implantation begins day 6-7

-        Blastocyst nourished by uterine secretions

-        Blastocyst Adhesion: Trophoblast cells of Blastocyst adhere to site with proper receptors and chemical signals

-        Trophoblast cells proliferate and form two distinct layers

o    Cytotrophoblast: inner layer of cells

o    Syncytiotrophoblast: › cells in outer layer lose plasma membranes, becoming multinuclear mass › Send out long protrusions that invade and digest endometrium

-        Blastocyst fully implanted in endometrium by day12 after fertilization

Hormones during Implantation of the Blastocyst

-        Implantation completed ~12 days after ovulation (~day 26-28 of menstrual cycle); about same time menstruation would occur

-        human chorionic gonadotropin (hCG) is secreted by trophoblast cells and later chorion

o    Corpus luteum is maintained by hCG to prevent menstruation

o    Corpus luteum -> becomes endocrine gland -> continues secretion of progesterone and estrogen

o    hCG levels rise until end of month 2

o    Decline as placenta begins to secrete progesterone and estrogen