• scrotum = supportive structure for the testes

• consists of loose skin and superficial fascia that hangs from the root of the penis

• externally- single pouch separated at the midline by a raphe

• internally – divided by a scrotal septum into two sacs each containing 1 testis

• the septum = dartos muscle (smooth muscle) + superficial fascia

• dartos is also found in the subcutaneous portion of the scrotal skin

• each testis is associated with a cremaster muscle – skeletal muscle that is a continuation of the internal oblique

• exterior location of the testis ensures its internal temperature is at least 2 to 3C lower than the body core

• failure of the testes to descend = cryptochidism

– 3% of full-term infants and 30% or premature infants

– untreated – results in sterility– increases the chances of testicular

cancer– 80% of those undescended testes will

descend spontaneously within the year

-testis: develop internally near the kidneys and descend through the inguinal canal during the latter half of the seventh month gestation-covered by several protection membranes

1. tunica vaginalis – serous membrane derived from the peritoneum, forms during the descent of the testes

-injury to the testes can cause an accumulation of fluid within the membrane = hydrocele-allows for easier movement of the testes within the scrotum

2. tunical albuginea – internal to the TV-extends inward to divide the testes into lobules (200-300)

-each lobule contains 1 to 3 coiled seminiferous tubules for sperm production

-lined with epithelium that produce sperm (spermatogenic cells)

• embedded among the spermatogenic cells of the seminiferous tubules – Sertoli cells– sustenacular cells– extend from the basement membrane of a seminiferous tubule to the lumen– during the Maturation phase of spermiogenesis - Sertoli cells consume the unneeded portions of

the spermatazoa.– once fully differentiated, the Sertoli cell is unable to proliferate– adjacent cells are joined together by tight junctions – blood-testis barrier– this barrier prevents an immune response against the spermatogenic cells’ surface antigens which

are recognized as they develop as being foreign– this creates a privileged immune environment– also secrete numerous cytokines and growth factors that mediate spermatogenesis

• anti-Müllerian hormone (AMH) - secreted during the early stages of fetal life. • inhibin and activins - secreted after puberty, and work together to regulate FSH secretion • androgen binding protein - faciliate spermatogenesis and sperm maturation • glial cell line-derived neurotrophic factor (GDNF) - has been demonstrated to function in promoting

undifferentiating spermatogonia - ensures stem cell self-renewal• the Ets related molecule (ERM transcription factor) - needed for maintenance of the spermatogonial

stem cell in the adult testis. • transferrin

• between adjacent seminiferous tubules are the interstitial cells or Leydig cells– for the production of testosterone (androgen)– can be a site for the development of testicular cancer - along with Sertoli cells– androgen = hormone for the development of masculine characteristics– Sertoli-Leydig tumor – type of ovarian tumor

• Arrhenoblastomas – Sertoli-Leydig cells within the ovary secrete androgens leading to virilization of the female phenotype

Medical application

• Sertoli cells & the immune system– presence of a privileged immune environment within

the testes– Sertoli cells can be transplanted into an ectopic site –

recreates this immune environment– other cells and tissues co-transplanted into these ectopic

sites are protected from the immune system• transplantation of canine pancreatic islets – production of

insulin– also secrete growth factors that improve the

engraftment, vascularization and survival of the co-transplanted tissues

Spermatogenesis• sperm development – from sperm stem cells called

spermatogonium• these spermatogonium develop in the embryonic

testes from primordial germ cells that arise from the yolk sac

• the spermatogonium remain dormant in the testes until puberty

• the maturing sperm can be found toward the lumen of the seminiferous tubule

• most mature = sperm cells or spermatozoa• takes 60-75 days to complete• 1. dissociation of some spermatogonium from the

basemement membrane of the ST• 2. differentiation of the dissociating spermatogonium

into primary spermatocytes (2n)• 3. replication of DNA within the spermatocyte and

the onset of meiosis• 4. formation of secondary spermatocytes (n) –

however despite having 23 chromosomes, these chromosomes are still comprise of two chromatids

• 5. completion of meiosis and formation of spermatids (n) – 23 chromosomes each made up of one chromatid

• 6. spermiogenesis – development of spermatids into a sperm cell

– spherical spermatids transform into elongated sperm containing an acrosome and bearing a flagellum

• as the spermatogenic cells form through meiosis they fail to undergho complete cytokinesis

– cells remain in contact throughout meiosis via cytoplasmic bridges

– accounts for the synchronization observed in the production of sperm in any given area of the seminiferous tubule

Sperm• 300 million made each day• 60 um long • major parts

– 1. head: contains the nucleus with 23 highly condensed chromosomes (one chromatid)

– 2. acrosome: covers the anterior 2/3 of the head• contains digestive enzymes to dissolve the

protective barriers of the egg (hyaluronidase and proteases)

– 3. tail or flagellum• neck - constricted region just behind the head

– contains centrioles for the production of the microtubules for the tail

• middle piece – contains mitochondria arranged in a spiral

• principal piece – longest portion of the tail• end piece – terminal portion of the tail

-release of gonadotropic releasing hormone (GnRH) from the neurosecretory cells of the hypothalamus which stimulates the gonadotrophs of the anterior pituitary gland-anterior pituitary releases gonadotropins (FSH and LH)1. Follicle stimulating hormone - stimulates spermatogenesis

-synergistic action by FSH and testosterone on the Sertoli cells – secrete androgen-binding protein into the lumen of the seminiferous tubule

-ABP binds to testosterone and keeps the concentration of this androgen high within the ST-testosterone stimulates the final stages of spermatogenesis-FSH release is inhibited by the release of inhibin by the Sertoli cells

2. Leutinizing hormone - stimulates male hormone production by the Leydig cells-testosterone synthesized from cholesterol in the testes-suppresses GnRH synthesis by negative feedback-in some targets (e.g. prostate), testosterone is converted into dihydrotestosterone (DHT)

Sertoli-Leydig cell interaction: Paracrine regulation

• Sertoli cells exert a paracrine control of the two main testicular functions, androgen secretion and spermatogenesis

• co-culture of Sertoli cells with Leydig cells – increases testosterone production by Leydig cells– causes Leydig cells to produce an increase in FSH receptors

• coculture of rat or pig Sertoli cells with rat germ cells– induces an increase in the RNA and DNA biosynthetic activities of germ

cells – increases spermatogenesis• most of the stimulatory effects of Sertoli cells seemed to be mediated

by diffusible factors like FSH (paracrine), secreted by Sertoli cells, but full

• but stimulation of germ cells seems to require cell-cell contact with Sertoli cells

Testosterone• testosterone and DHT both bind to same receptors

– receptors are found within the nuclei of the target cells• targets – bone, muscle• effects

– 1. prenatal development• stimulates the male pattern of the reproductive system

– gonads develop during the 5th week of gestation from two sets of ducts: 1) Wolffian ducts (males) and 2) Mullerian ducts (females)

– therefore the embryo has the potential to develop into either sex– BUT “maleness” determined by a gene called SRY – sex determining region of the Y chromosome– SRY protein expression induces differentiation of Sertoli cells– Sertoli cells secrete Mullerian-inhibiting Substance – apoptosis within the Mullerian ducts which inhibits the

development of female structures-in response to hCG – Leydig cells begin to synthesize testosterone-testosterone stimulates development of the epididymus, vas deferens, ejaculatory duct and seminal vesicle

• DHT stimulates development of external genitalia– development around the 8th week– from the genital tubercle (both males and females) – comprised of a urethral groove and two labiosacral swellings– elongation of part of the genital tubercle into the penis– labiosacral swellings - scrotum

• testosterone is converted in the brain to estrogens – development of certain brain regions in males– 2. development of male sexual characteristics– 3. development of sexual function

• male sexual behavior• spermatogenesis• libido in both males and females

– females – androgen release by the adrenal cortex

– 4. stimulation of anabolism• stimulate protein synthesis

Testosterone

Medical application: Anabolic steroids• a class of natural and synthetic steroid hormones• first discovered in the early 1930s • promote cell growth and division, protein sythesis (anabolism) • results in growth of several types of tissues, especially muscle and bone • increases bone remodelling and growth, increases bone marrow production of RBCs• increases size of clitoris or penis, increase vocal cord thickness, increases the libido, inhibits

spermatogenesis• different anabolic androgenic steroids have varying combinations of androgenic and anabolic

properties, and are often referred to in medical texts as AAS (anabolic/androgenic steroids)• for reversal of chronic wasting conditions including cancer and AIDS• stimulation of myogenesis

– hypertrophy of both types of muscle fibers (I and II) – mechanism of this is not completely understood – increased synthesis of muscle proteins and/or decrease degradation of muscle proteins– also – increased commitment of muscle stem cells to the myogenic lineage and inhibiting their differentiation

to the adipogenic• supraphysiological doses of testosterone in men promotes nitrogen density and increases fat free mass

(skeletal muscle mass) while at the same time decreasing fat, particularly abdominal fat.• may also play an anticatabolic role in inhibiting skeletal muscle atrophy through inhibiting

glucocorticoid action • mechanisms of action differ depending on the specific anabolic steroid• different types of anabolic steroids bind to the androgen receptor to varying degrees depending on

their chemical makeup• also associated with numerous side effects when administered in excessive doses

– increased LDL and decreased HDL, increased acne, elevated blood pressure, hepatotoxicity, and alterations in left ventricle morphology.

• e.g. methandrostenolone (Dianabol) do not react strongly with the androgen receptor – stimulates protein synthesis independently

– 1956, oral– aid to muscle growth by bodybuilders (Arnold Schwarzenegger)– continues to be produced in countries such as Mexico under the trade name Reforvit-b, Russia, Thailand, and US

black market. – relies on activity not mediated by the androgen receptor for its effects– includes dramatic increases in protein synthesis, glycogenolysis, and muscle strength – decreases the rate of cell respiration and decreases production of red blood cells - anemia– high doses (30 mg or more per day) - side effects such as gynaecomastia, high blood pressure, acne and male

pattern baldness may be seen – causes severe masculinising effects in women even at low doses– metabolized into estradiol - without the administration of inhibitors (e.g. Tamoxifen) estrogenizing effects will

appear – stacked (combined) with drugs that react strongly with the androgen receptor, such as Oxandrolone– processing of the steroid in the body decreases its ffinity for sex hormone binding globulin - protein that de-

activates steroid molecules– significantly more active than an equivalent quantity of testosterone – BUT the concomitant elevation in estrogen levels results in significant water retention. – it is often used by bodybuilders only at the start of a "steroid cycle", to facilitate rapid strength increases

• e.g. oxandrolone (Anvral, Oxandrin) binds the androgen receptor to mediate its effects• -oral, Class II steroid• -1964• -treatment of osteoporosis, alcohol hepatitis, Turner’s syndrome (XO), HIV wasting, anemia• -used frequently by bodybuilders – not easily metabolized into DHT or estrogens

Medical application: Anabolic steroids

• Testosterone (attached to various esters enanthate, cypionate, propinate or suspended in oil or water)

• Methandrostenolone / methandienone (Dianabol) • Nandrolone Decanoate (Deca-durabolin) • Nandrolone Phenylpropionate (Durabolin) • Boldenone Undecylenate (Equipoise) • Stanozolol (Winstrol) • Oxymetholone (Anadrol-50) • Oxandrolone (Anavar) • Fluoxymesterone (Halotestin) • Trenbolone (Fina) • Methenolone Enanthate (Primobolan) • 4-chlordehydromethyltestosterone (Turinabol) • Mesterolone (Proviron) • Mibolerone (Cheque Drops) • common misconceptions

– shrinks the penis – actually decreases LH and FSH which affects the size of the testes– causes cancer – no linkage– cause suicide – no linkage– “roid rage” and aggression – no linkage– http://en.wikipedia.org/wiki/Anabolic_steroids

Medical application: Anabolic steroids

-pressure generated by the Sertoli cells pushes the sperm into a series of ducts within the testes that end up as the epididymis

-within the epididymis is the ductus epididymis -also made up of a head, body and tail portion-site of sperm maturation – acquire mobility (14 days)-helps propel sperm into the:

-vas (ductus) deferens: conducting tube from testis to urethra-connects to the tail of the epididymis-connects the testes to the urethra-made up of a pseudostratified columnar epithelium witha lamina propria connective tissue plus three layers ofsmooth muscle-contractions of these muscular layers propel the sperm

Reproductive Ducts

-spermatic cord supports thevas deferens + blood vessels (testicular artery and the pampiniform venous plexus), lymphatic vessels, the cremaster muscle and autonomic nerves-passes through the inguinal canal

• ejaculatory duct – forms from the union of the seminal vesicle and the end of the vas deferens– pass through the prostate gland and terminate in the urethra

• urethra: 3 sections: • A. prostatic - runs through the prostate

• connects to ducts from the prostate and to the ejaculatory duct

• B. membranous - between prostate and penis• -through the muscles of the perineum – urogenital diaphragm

• C. spongy - through the erectile tissue of the penis

Male reproductive glands

• -glands: seminal vesicles, prostate, bulbourethral glands• -produce fluid that combine with sperm to make semen• -semen: alkaline, activates sperm cells• 1. prostate: surrounds the urethra

-forms as an outgrowth of the urethra along with the bulbourethral glands

• -secretes a thin, milky fluid that enhances sperm motility and neutralizes vaginal fluid

• 2. seminal vesicles: connect to urethra via the ejaculatory ducts• -secretes an alkaline fluid that contains sugars and

prostaglandins (stimulates uterine contractions)• 3. bulbourethral glands: 2 glands behind• the prostate• -secrete a fluid that lubricates the penis

-conveys urine and semen-body is found externally-body is comprised of two tissue types of erectile tissue surrounded by connective tissue A. corpus cavernosum - large spaces B. corpus spongiosum - smaller spaces

-surrounds the urethra-root of the penis is attached to the pelvis-corpus spongiosum enlargens at the tip - glans penis (sensory receptors)-glans penis covered with a loose fold of skin = prepuce

-ovary: production of egg-surface is covered with a germinal epithelium (simple epithelium) – does NOT give rise to the ova! -next layer is = tunica albuginea – dense irregular connective tissue capsule -outer cortex- granular tissue due to the presence of tiny ovarian follicles - inner medulla - connective tissue with blood & lymphatic vessels and nerves

Oogenesis and Follicular Development

• begins before birth• early fetal development – primordial germ cells from the yolk sac

migrate into the developing ovaries• differentiate to form oogonia (diploid stem cells)

– undergo mitosis to produce millions of germ cells• most of the germ cells degenerate by atresia• a few develop further into primary oocytes – entered prophase I of

meiosis– surrounded by a layer of follicular cells = primordial follicle– at birth – 200,000 to 2,000,000 primary follicles within the ovary– at puberty 40,000 are still present– 400 will develop further (rest undergo atresia)

-release of FSH and LH each month causes the development of one primary oocyte into a secondary oocyte-development of a few primordial follicles into primary follicles (only one will continue until ovulation)-primary follicle – primary oocyte surrounded by several layers of epithelial cells called granulosa cells

-develops a clear glycoprotein layer between the oocyte and the granulosa cells – zona pellucida-the outermost granulosa cells contact a basement membrane which begins to develop into two layers (theca layers)-now known as the secondary follicle

-secondary follicle begins to accumulate fluid in the center of the follicle (antrum)-innermost granulosa cells firmly attaches to the zona pellucida = corona radiata

-becomes larger and turns into the tertiary or mature Graafian follicle-completes meiosis I – two haploid cells-these haploid cells are uneven in size but each have 23 chromosomes (two

chromatids each - 46)-smaller cell – first polar body (discarded nuclear material)-larger cell – secondary oocyte

-receives most of the cytoplasm and has 23 chromosomes-stops at metaphase II-ovulated

• ovulation – expulsion of the secondary oocyte into the pelvic cavity with the first polar body and corona radiata

• fertilization – union of egg and sperm– penetration of the sperm into the

secondary oocyte results in the resumption of meiosis II

– the secondary oocyte splits again into two cells of unequal size (n)

– larger one is called the ovum and the smaller one is the second polar body

– combination of the ovum and the sperm results in the formation of the zygote

– the first polar body splits also into two haploid cells

– therefore meiosis of the primary oocyte produces one haploid ovum and three haploid polar bodies that degenerate

Oogenesis and Follicular Development

• uterus: receives and nourishes the embryo• -comprised of a body, a curved portion (fundus) and the cervix• -uterine wall outer perimetrium, muscular myometrium and

inner endometrium• -endometrium: mucosal layer covered with epithelium• -rich blood supply, sloughed off during menstruation• uterine tubes (Fallopian tubes): conduction of egg from ovary to

uterus• -expands at end near the ovary = infundibulum with fimbrae (fingers) for

the “catching” of the released egg• -lined with a mucosal layer and columnar epithelium with cilia

-are also cells with microvilli rather than cilia – produce a nutritive fluid for the egg

• cervix: projects into the vaginal canal

Female Reproductive Cycle

• two cycles– 1. ovarian: during and after the maturation of

the oocyte– 2. uterine: concurrent series of changes in the

endometrium of the uterus to prepare it for embryo implantation

-GnRH causes release of FSH and LH from anterior pituitary-FSH initiates follicular growth -LH stimulates the maturation of follicles-both LH and FSH stimulate the secretion of estrogens from the follicle

-LH stimulates the theca layers of the follicle to make androgens-FSH stimulates the uptake of these androgens and converts them to estrogens

-LH triggers ovulation and results in development of corpus luteum-corpus luteum produces and releases progesterone and some estrogen plus relaxin and inhibin-estrogen and progesterone regulate pregnancy, menstruation, secondary sex char’s& development of sex organs at puberty-relaxin – relaxes the uterus by inhibiting contractions of the myometrium

-important to the implantation of the embryo – produced by the placenta during pregnancy-also increases the flexibility of the pubic symphysis

-inhibin - inhibits secretion of FSH and LH

-3 major types of estrogens:1. beta-estradiol2. estrone3. estriol4. other smaller quantities-follicular estrogens:a. promote the development of the female reproductive structures, secondary sex characteristics and the mammary glandsb. increase protein anabolism, includingbone synthesisc. lower blood cholesterold. inhibit the release of GnRH, FSH and LH

1. Menstrual PhaseA. Ovarian events – FSH increase causesdevelopment of primordial follicles into primary follicles-may take several months to complete!B. Uterine events – 50-150 mL of blood,tissue fluid, mucus and epithelial cells-shed from the stratum functionalis-occurs because of declining levels ofE and P = causes the spiral arterioles to contract which kills the cells of the SFleaving the stratum basalis intact

2. Preovulatory Phase – most variable in lengthA. Ovarian events – secretion of E and inhibin from the secondary follicles-by day 6 one secondary follicle has outgrown the rest to become the dominant follicle-the dominant follicle secretes E and I which causes an inhibition of FSH and a decrease in the stimulation of other follicles-the dominant follicle develops into the Graafian follicle-forms a blister-like bulge due to an increase in fluid within the antrum of the follicle-the GF continues to increase its estrogen productionB. Uterine events – E stimulates the repair of the SF by stimulating mitosis in the SB layer-the arterioles begin to lengthen and coil within the SF (4-10mm in thickness)

• 3. Ovulation• A. Ovarian events –

rupture of the GF usually around day 14

• ovulated secondary follicle remains surrounded by its corona radiata and its zone pellucida

• triggered by a positive feedback system – high levels of E at the end of the preovulatory phase increases the secretion of GnRH, also increases the release of LH directly

• increased LH induces rupture of the GF about 9 hours after the LH peak

• basis for the at-home ovulatory tests – detect rises in LH

• B. Uterine events - none• signs of ovulation

– Increase in basal body temperature– Changes in cervical mucus– Cervix softens– Mittelschmerz---pain

• 4. Postovulatory Phase – most consistent part of the cycle (14 days)– A. Ovarian events – the mature GF collapses and bleeding from the follicle results

- the development of a blood clot results as the follicle induces bleeding – follicle is now called the corpus hemorrhagicum

• the basement membrane between the granulosa cells and the thecal layers degenerates• this mixes the granulosa and thecal cells – transformed into corpus luteum cells under

the influence of LH• luteal cells produce hormones and absorb the blood clot• if the ovum is NOT fertilized, the CL degenerates into the corpus albicans – 2 weeks• decrease in P, E and I results in the release of GnRH, FSH and LH (loss of negative

feedback)• new follicular growth begins• if fertilized – the CL persists beyond 2 weeks by the secretion of human chorionic

gonadotropin (hCG) hormone produced by the developing chorion that surrounds the embryo (8 days post-fertilization)

– B. Uterine events – P and E produced by the corpus luteum promotes the growth and vascularization of the endometrium and its thickening to about 12-18 mm

• -endometrial glands within the endometrium begin to secrete glycogen – energy for the fertilized egg

Summary

Birth Control Methods

• Surgical• Hormonal• Mechanical barriers• Periodic abstinence• Coitus interruptus• Induced abortion

Reproductive disorders

• Males– testicular cancer– prostate concer– erectile dysfunction (ED)– benign prostatic hyperplasia

• Females– PMS– PMDD– Endometriosis– Ovarian, uterine cysts– Ovarian, uterine, cervical cancer– vulvovaginal candidiasis

• Both– UTI– STDs – gonorrhea, syphillis, chlamydia, genital herpes, genital warts

Pregnancy

-fertilization in the upper third of the oviduct/fallopian tube-fertilization = union of egg and sperm-plasma membrane of the egg is surrounded by an extracellular matrix = zona pellucidaand a ring of follicular cells = corona radiata (nourishment in the follicle)-after fertilization = zygote 1. sperm penetrates corona radiata

2. several sperm enter zona pellucida-one of the glycoproteins within the ZP(ZP3) acts as a receptor for the sperm-binding causes dissolution of the acrosomeand release of digestive enzymes3. ONE sperm penetrates the plasmamembrane of the egg4. immediate change in the oocyte cellmembrane (depolarizes) -also binding results in release of intracellularcalcium which stimulates exocytosis ofsecretory vesicles whose contents inactivateZP3 and harden the zona pellucida - impervious to more sperm5. oocyte releases the zona pellucidaaway from the egg surface6. fusion of the sperm with nucleus of the egg-before fusion the secondary oocyte must completemeiosis II and form the ovum

-after sperm penetration and ovum development the nuclei of the egg and sperm undergo changes to become pronuclei-union of sperm and egg pronuclei nuclei forms the zygote-first cell division = embryo-first division takes place 24 hours post-fertilization – takes 6 hours to complete-each succeeding division takes less time-72 hr stage = 16 cells-96 hr stage = morula (embryo is the size of the original ovum, filled with cells (blastomeres)

embryonic stage:week 2 to week 8

Implantation• attaches after about 6

days• usually in the fundus or

the body of the uterus• orients its inner cell

mass toward the uterus• 7 day – the endometrium

becomes more vascularized

• 9 days – completely embedded

• following implantation, the endometrium is called the decidua– several layers with

defined functions

-day 4 – formation of morula and passage into the uterine cavity-endometrial glands release a glycogen-rich fluid = uterine milk-enters the morula through the zona pellucida and provides nourishment

-day 5 -the fluid begins to collect in the morula and reorganizes them around a fluid-filledcavity = blastocoel-embryo is now called a blastula or blastocyst (50-150 cells)

-outer layer = trophoblast - forms extraembryonic tissues (e.g. placenta,yolk sac)

-inner cell mass at one end - totipotent embryonic stem cells-by the end of day 5, the blastocyst digests a hole in the ZP and squeezes through itto undergo implantation

-second week of development - the inner cell mass flattens = embryonic disk (hypoblast and epiblast)-hypoblast = primitive endoderm-epiblast = primitive ectoderm-amniotic cavity forms between the inner cell mass and the trophoblast

-surrounded by an amniotic membrane – develops from the epiblast-fills with amniotic fluid – filtrate from maternal blood at initial stages

-formation of the yolk sac (from the hypoblast)-forms blood cells, gives rise to sex cells and the stem cells of the immune system-also forms part of the embryonic digestive tube-portion will also become part of the umbilical cord

-the outer trophoblast cells develops into two layers within the region where the blastocyst and the endometrium make contact – become part of the chorion

-these trophoblast cells will secrete digestive enzymes that allow the embryo toburrow into the decidua-also secrete hCG – rescues the corpus luteum from degeneration

• day 15: embryonic disk undergoes gastrulation to form the gastrula embryonic stage– formation of the three embryonic germ layers by differentiation of the ES cells

within the embryonic disc• epiblast form a specialized region = primitive streak

– clearly establishes a head and tail orientation– head end the streak enlargens to form the primitive node– cells from the epiblast move inward through the primitive streak– some cells displace the hypoblast and form the endoderm – other cells are retained in the area and form the mesoderm

• mesoderma forms a loose connective tissue = mesenchyme– cells remaining in the epiblast form the ectoderm

-in front of the primitive streak forms the primitive node – head and associated structures-mesodermal cells from the primitive node form a hollow tube near the future head of theembryo - become the notochord (day 22-24)(progenitor to the vertebral column)-four weeks of development - embryo forms a tubular structure-embryo begins to form definitive structures:

-neural folds of ectoderm -> nervous system (brain and spinal cord)** neurulation occurs by induction (one tissue influences the development of another)

-e.g. nervous system requires the mesodermal cells of the notochord

-portions of the mesoderm thatdo not form the notochord segment into sections calledsomites -> specific bodyregions and structures

Medical application: Stem Cells

• two broad categories of mammalian stem cells exist: embryonic stem cells, and adult stem cells

• Stem cells = primal cells that:– 1) retain the ability to renew themselves through cell division and

2) can differentiate into a wide range of specialized cell types• stem cell field grew out of findings by Canadian

scientists Ernest A. McCulloch and James E. Till in the 1960s– 1963 – self-renewing cells in the bone marrow of mice

• 1964 - single cells from a human testicular teratocarcinoma were isolated – - remained undifferentiated in culture = embryonic carcinoma

cells (EC cells)

• 1968 – bone marrow transplant was used to cure two patients of SCID

• 1978 – Hematopoietic Stem Cells (HSCs) were isolated from the bone marrow and identified

• 1981 – Embryonic stem cells (ES cells) first isolated and cultured from mouse embryos in 1981 by two independent research groups. Evans and Kaufman, Martin

• 1998 - human embryonic stem cells isolated and grown by James Thomson at the University of Wisconsin-Madison – developed a technique to isolate and grow ES cells from human

blastocysts

• stem cells can be defined by their ability to form specific cell types• potency = ability to specialize into a distinct cell type• -unipotent – ability to form only one cell type• e.g. pre-adipocyte -> adipocyte

– sometimes called progenitor cells because of their limited ability to specialize

• -bipotent – ability to form two cell types• e.g. osteochondro progenitor cells (OPCs) – bone and cartilage• -multipotent/pluripotent – ability to form many cell types

• multipotent – usually within one germ lineage (mesoderm)

• e.g. MSC & ASCs – bone, fat, cartilage and muscle• pluripotent – within more than one lineage (mesoderm & ectoderm)

• -adult stem cells????

• -totipotent – ability to form all cell types• e.g. ES cell

• adult stem cell populations – bone marrow – HSC and MSC– adipose – ASC– skin– brain/neural - NSCs– skeletal muscle– mammary– olfactory– hepatic– corneal– etc…….

• fetal stem cell populations – placental and fetal tissue derived• -placental & amniotic populations• -umbilical cord blood

– increased potentials because of their “younger” age???

• adult stem cells may have limited potency when compared to ES cells – but are still useful because of their limited ethical concerns

• in addition ES cells still may possess the ability to form cancers• -many proto-oncogenes are involved in embryonic and fetal development• -these genes turn on and off at specific times during development• -if they turn back on during adult stages = oncogenes – may cause the formation of

tumors• http://stemcells.nih.gov/

Use of adult stem cells• use of bone marrow stem cells (i.e. HSCs) leukemia and lymphoma• Potential treatments • 2.1 Brain Damage• neural stem cells • rats subjected to stroke - administration of drugs to increase the NSC division rate and may increase the survival and differentiation of

newly formed cells– within weeks, recovery of brain structure is accompanied by recovery of lost limb function

• several studies in which NSCs or ES cells are injected into damaged areas of the brain– treatment for Parkinson’s

• 2.2 Cancer • 2.3 Spinal cord injury • University of Wisconsin-Madison: differentiated human blastocyst stem cells into neural stem cells, then into the beginnings of motor

neurons, and finally into spinal motor neuron cells• University of California: injection of hES cells into paralyzed mice – limited regaining of their ability to move and walk four months

later. – stem cells regenerated neurons in addition to the myelin sheath – http://img227.imageshack.us/img227/7954/stemcellbreakthru052wl.jpg

• 2.4 Muscle damage - use in muscular dystrophy and myasthenia gravis• 2.5 Heart damage – repair of ischemic coronary arteries• 2.6 Low blood supply • 2.7 Baldness

– stem cells within hair follicles– follicle stem cells may lead to successes in treating baldness through "hair multiplication," also known as "hair cloning," as early as 2008????

• 2.8 Missing teeth • 2.9 Deafness • 2.10 Blindness and Vision Impairment• 2.11 ALS (Lou Gehrig's Disease)

– Johns Hopkins University: induced nerve damage similar to that of ALS– injection of rats with stem cells - migration to the sites of injury – regeneration of the dead nerve cells – restoration of movement– (http://www.cellmedicine.com/als.asp)

Therapeutic cloning• In somatic cell nuclear transfer (SCNT) the nucleus of a somatic cell is removed and

the rest of the cell is discarded. • the nucleus of an egg cell is removed• the nucleus of the somatic cell is then inserted into the enucleated egg cell. • the egg is stimulated to divide by an electric shock (depolarizes the egg’s plasma

membrane)• new cell begins to divide and proceedes through the various embryonic stages• SCNT is used in stem cell research - to obtain stem cells that are genetically matched to

the donor organism – e.g. potential use of genetically-customized stem cells & Parkinson's disease - stem cells

resulting from SCNT would those genes that contribute to Parkinson's disease. therefore, the disease-specific stem cell lines could be studied in order to better understand the disease

– e.g. genetically-customized stem cell lines could be generated for cell-based therapies to transplant to the patient - avoiding any complications from immune system rejection

• *** no human stem cell lines have been derived from SCNT research. In 2005, a South Korean research team led by Professor Hwang Woo-suk, published claims to have derived stem cell lines via SCNT ,but supported those claims with fabricated data

Cloning types• Molecular cloning

– procedure of isolating a DNA sequence of interest and obtaining multiple copies of it in an organism.

– frequently employed to amplify DNA fragments containing genes, an essential step in their subsequent analysis.

– cloning of any DNA sequence involves the following four steps: – fragmentation, ligation, transfection, and screening/selection.

• Genetic cloning– Cloning a cell means to derive a (clonal) population of cells from a single cell.

Asexual reproduction (also known as agamogenesis) is a form of reproduction which does not involve meiosis, gamete formation, or fertilization.

– In laymen's terms, there is only one "parent" involved. – common among simple organisms such as amoeba and other single-celled

organisms,

Horticultural-clone in horticulture means all descendants of a single plant, produced by vegetative reproduction-many horticultural plant cultivars are clones - multiplied by some process other than sexual reproduction.

e.g. some European grapes represent clones that have been propagated for over two millennia. - other examples are potato and banana.

-Grafting can be regarded as cloning, since all the shoots and branches coming from the graft are genetically a clone of a single individual-Many trees, shrubs, vines, ferns and other herbaceous perennials form clonal colonies.

Reproductive Cloning• SCNT can also be used in the reproductive cloning of animals (e.g. Dolly the sheep), and in theory could be used to

clone humans. • embyro is created by SCNT & transferred to the uterus of a female host where it continues to develop until birth• Dolly or any other animal created using nuclear transfer technology is not truly an identical clone of the donor animal.

– Only the clone's chromosomal or nuclear DNA is the same as the donor. – the mitochondrial DNA will differ– mutations will occur throughout embryonic and fetal development– so the clone is not 100% identical!

• Species cloned• Tadpole: (1952) Many scientists questioned whether cloning had actually occurred and unpublished

experiments by other labs were not able to reproduce the reported results. • Carp: (1963) In China, embryologist Tong Dizhou cloned a fish. He published the findings in an

obscure Chinese science journal which was never translated into English.[1] • Mice: (1986) first successfully cloned mammal; Soviet scientists Chaylakhyan, Veprencev,

Sviridova, Nikitin had mice "Masha" cloned.• Sheep: (1996) from early embryonic cells by Steen Willadsen. Megan and Morag cloned from

differentiated embryonic cells in June 1995 and Dolly the sheep in 1997. • Rhesus Monkey: Tetra (female, January 2000) from embryo splitting • Cattle: Alpha and Beta (males, 2001) and (2005) Brazil • Cat: CopyCat "CC" (female, late 2001), Little Nicky, 2004, first cat cloned for commercial reasons • Mule (2004): Idaho Gem, a john mule - the first horse-family clone. • (2003) Horse: Prometea• a similar process called budding has been used in cattle for decades• -an embryo is dissociated into individual cells without harm• -each cell – separate embryo• -animal is not derived from a differentiated cell but from a undifferentiated egg

HIV and AIDS

-most viral illnesses are caused by DNA viruses a. binding of virus to host cells via interaction of host and viral envelope proteins - endocytosis b. degradation of viral capsid proteins to release the naked viral DNA c. viral DNA enters host nucleus and “takes over” host machinery - d. replication, transcription and translation of viral components

e. assembly of new viral capsids and formation of new viral progenyf. release of progeny from host cell(cell death of host)

HIV• Previous names:

– human T-lymphotropic virus-III (HTLV-III)– lymphadenopathy-associated virus (LAV)– AIDS-associated retrovirus (ARV)

• Infection - transfer of blood, semen, vaginal fluid, pre-ejaculate or breast milk. • HIV is present as both free virus particles and virus within infected immune cells. three major

routes of transmission:– unprotected sexual intercourse – contaminated needles and – transmission from an infected mother to her baby HIV primarily infects vital cells of the immune system

• HIV primarily infects vital cells of the immune system – e.g. helper T cells (specifically CD4+ T cells), macrophages and dendritic cells

• infection leads to low levels of CD4+ T cells through three main mechanisms: – 1. direct viral killing of infected cells– 2. increased rates of apoptosis in infected cells– 3. killing of infected CD4+ T cells by CD8 cytotoxic lymphocytes – recognize infected cells.

• when CD4+ T cell numbers decline below a critical level – loss of cell-mediated immunity (T cell mediated immunity)

• body becomes progressively more susceptible to opportunistic infections.• about one in ten remain healthy for many years, with no noticeable symptoms

HIV timeline• AIDS epidemic was discovered June 5, 1981• U.S. Centers for Disease Control and Prevention reported a cluster of

Pneumocystis carinii pneumonia in five homosexual men in Los Angeles• originally dubbed GRID, or Gay-Related Immune Deficiency• 1982 - the CDC introduced the term AIDS• 1983 - scientists led by Luc Montagnier at the Pasteur Institute in France

discovered the HIV • originally called lymphadenopathy-associated virus (LAV). • 1984 - a team led by Robert Gallo of the US confirmed the discovery of the

virus• renamed it human T lymphotropic virus type III (HTLV-III)• President Mitterrand and President Reagan had to resolve the discovery issues• 1986 - renamed human immunodeficiency virus (HIV)

• Three of the earliest known instances of HIV-1 infection are as follows:• 1. A plasma sample taken in 1959 from an adult male living in what is now the

Democratic Republic of Congo• 2. HIV found in tissue samples from a 15 year old African-American teenager

who died in St. Louis in 1969 • 3. IV found in tissue samples from a Norwegian sailor who died around 1976

HIV classification• HIV classified as a member of the genus lentivirus - part of the family of

retroviridae Lentiviruses: responsible for long-duration illnesses, long incubation period

• transmitted as single-stranded, positive-sense, enveloped RNA virus • upon entry of the target cell, the viral RNA genome is converted to double-

stranded DNA by a virally encoded reverse transcriptase• viral DNA is then integrated into the cellular DNA by a virally encoded

integrase after cell infection - two pathways are possible: – 1. either the virus becomes latent and the infected cell continues to function– 2. the virus becomes active and replicates – produces a large number of progeny

viruse particles• Two species of HIV infect humans: HIV-1 and HIV-2. • HIV-1 is thought to have originated in southern Cameroon after jumping from

wild chimpanzees (Pan troglodytes troglodytes) to humans – HIV-1 is more virulent.– easily transmitted – is the cause of the majority of HIV infections globally.– HIV-1 is the virus that was initially discovered and termed LAV.

• HIV-2 may have originated from the Sooty Mangabey (Cercocebus atys), an Old World monkey of Guinea-Bissau, Gabon, and Cameroon

– HIV-2 is less transmittable– largely confined to West Africa

Structure and genome

• HIV is different in structure from other retroviruses. • approximately 120 nm in diameter (around 60 times smaller than a red blood cell) roughly spherical.• composed of two copies of positive single-stranded RNA• RNA codes for the virus's nine genes – but it must be converted back into DNA (reverse transcribed by an enzyme called reverse

transcriptase)• enclosed by a conical capsid composed of 2,000 copies of the viral protein p24

• single-stranded RNA is tightly bound to: nucleocapsid proteins, p7 and enzymes needed for the development of the virion such as reverse transcriptase, proteases, ribonuclease and integrase

• the capsid is surrounded by a matrix composed of the viral protein p17 – ensures the integrity of the virion particle

• matrix is surrounded by a viral envelope - two layers phospholipids taken from the membrane of a human cell upon budding of the progeny virsus from its host

• viral envelope is embedded with proteins from the host cell and about 70 copies of a complex HIV protein called Env • Env - cap made of three molecules called glycoprotein (gp) 120, and a stem consisting of three gp41 molecules (anchor the Env complex to

the viral envelope)– enables the virus to attach to and fuse with target cells– both gp120 and gp41 (especially gp120) are targets of future treatments or vaccines

• 3 genes = gag, pol, and env contain information needed to make the structural proteins for new virus particles– 1. env - codes for gp160 which is processed to form gp120 and gp41– that is broken down by a viral enzyme to form gp120 and gp41– 2. gag – codes for the capsid protein 024, the nucleocapsid proteins p6, p7 and the matrix protein p17– 3. pol – codes for the enzymes reverse transcriptase, integrase and protease

• the six remaining genes are tat, rev, nef, vif, vpr, and vpu (or vpx in the case of HIV-2) - are regulatory genes that code for proteins that control the ability of HIV to infect cells, replicate, or cause disease

– e.g. nef appears necessary for the virus to replicate efficiently, and the – e.g. vpu influences the release of new virus particles

HIV Tropism• viral tropism = cell types HIV infects. • HIV infects a variety of immune cells such as CD4+ T cells, macrophages, and microglial cells• HIV-1 entry to macrophages and CD4+ T cells is mediated through interaction of gp120 with:

– 1. the CD4 molecule – 2. with chemokine coreceptors

• Strains of HIV-1 • 1. Macrophage (M-tropic) or non-syncitia-inducing strains (NSI)

– use the β-chemokine receptor CCR5 for entry – able to replicate both in macrophages and CD4+ T cells– CCR5 receptor is used by almost all primary HIV-1 isolates regardless of viral genetic subtype. – macrophages appear to be the first cells infected by HIV– perhaps the source of HIV production when CD4+ cells become depleted in the patient– microglial cells in the CNS can be infected by the NSI strains

• 2. T-tropic isolates, or syncitia-inducing (SI) strains– replicate in primary CD4+ T cells and macrophages– use the α-chemokine receptor, CXCR4– administration of SDF-1(a ligand for CXCR4) may be able to suppress replication of T-tropic

HIV-1 isolates by down-regulating the expression of CXCR4 on the surface of these cells– HIV strains that use only the CCR5 receptor are termed R5– those that only use CXCR4 are termed X4– those that use both = X4R5– Some people are resistant to certain strains of HIV– e.g. people with a mutation in the CCR5 gene (CCR5-Δ32 mutation); these resistant to

infection with R5 virus - HIV cannot bind this coreceptor thus reducing its ability to infect target cells.

– Both X4 and R5 HIV are present in the seminal fluid which is passed from partner to partner – R5 strain seems to predominate – unknown why – but spermatozoa may selectively carry R5 HIV – they possess both CCR3 and CCR5 but not CXCR4 on their surface– genital epithelial cells preferentially infected by the X4 virus

HIV Replication• Entry to the cell• enters via adsorption of its glycoproteins on

its to receptors on the target cell• requires interactions between the envelope

protein complex Env (gp160 spike) + CD4 + either CCR5 or CXCR4 (others CCRs are known to interact!! )

• gp160 spike contains binding domains for both CD4 and chemokine receptors.

• fusion results - involves the high-affinity attachment of CD4 to gp120

• this changes the shape of gp120 and allows in now to intereact with the chemokine receptor

• a more stable two-pronged attachment, allows gp41 to penetrate the cell membrane and allows subsequent entry of the viral capsid.

• Once HIV has bound to the target cell, the HIV RNA and various enzymes, including reverse transcriptase, integrase, ribonuclease and protease, are injected into the cell

• Replication and transcription• once inside the cell reverse transcriptase liberates the single-stranded

(+)RNA from the attached viral proteins and copies it into a complementary DNA of 9 kb size

• process of reverse transcription is extremely error-prone and it is during this step that mutations may occur.

• Such mutations may cause drug resistance• reverse transcriptase then makes another complementary DNA strand

to form a double-stranded viral DNA intermediate (vDNA). • This vDNA is then transported into the cell nucleus• integration of the viral DNA into the host cell's genome is carried out

by another viral enzyme called integrase• integrated viral DNA may then lie dormant = latent stage of infection• BUT to actively produce the virus, certain HOST cellular transcription

factors need to be present• these factors are upregulated when the T cell is fighting an infection • this means that those cells most likely to be infected and killed by HIV

are in fact those currently fighting infectio• during replication - the integrated provirus is copied to mRNA which

is then spliced into smaller pieces. • these small pieces produce the regulatory proteins Tat (which

encourages new virus production) and Rev (affects processing of the mRNA to produce different viral proteins)

• as Rev accumulates it gradually starts to inhibit mRNA splicing and results in the production of the structural proteins Gag and Env from the full-length mRNA.

• the full-length RNA is actually the virus genome; it binds to the Gag protein and is packaged into new virus particles.

HIV Replication

• Assembly and release• final step of the viral cycle = assembly of new HIV-1 virions• begins at the plasma membrane of the host cell• the Env polyprotein (gp160) goes through the endoplasmic reticulum

and the Golgi complex of the host where it is processed into the two HIV envelope glycoproteins gp41 and gp120.

• these are transported to the plasma membrane of the host cell• Gag polyprotein (p55) associates with the inner surface of the plasma

membrane along with the HIV genomic RNA • during maturation, the HIV protein protease process or cleave the

proteins made by the host into individual functional HIV proteins and enzymes.

• cleavage step can be inhibited by protease inhibitors. • the various structural components then assemble to produce a mature

HIV virion

HIV Replication

• stage of infection can be determined by measuring the patient's CD4+ T cell count, and the level of HIV in the blood• initial infection occurs after transfer of body fluids • the first stage of infection = the primary, or acute infection

– a period of rapid viral replication – levels of HIV commonly approaching several million viruses per mL– accompanied by a marked drop in the numbers of circulating CD4+ T cells = acute viremia – also with the activation of CD8+ T cells - kill HIV-infected cells– antibody production– CD8+ T cell response is important in controlling virus levels– good CD8+ T cell response has been linked to slower disease progression and a better prognosis– during this period most individuals (80 to 90%) develop an influenza-like illness– often not recognized as a sign of HIV infection

• clinical latency stage = strong immune defense reduces the number of viral particles in – marks the start of this phase – vary between two weeks and 20 years – HIV is active within lymphoid organs & surrounding tissues rich in CD4+ T cells may also become infected– viral particles accumulate both in infected cells and as free virus. – Individuals are still infectious. – CD4+ T cells carry most of the proviral load– when CD4+ T cell numbers decline below a critical level, cell-mediated immunity is lost - opportunistic

infections appear. – first symptoms include: moderate and unexplained weight loss, recurring respiratory tract infections and oral

ulcerations– oral Candida species and Mycobacterium tuberculosis - increased susceptibilty to oral candidiasis (thrush) and

tuberculosis. – reactivation of latent herpes viruses causes patients to suffer from shingles from Epstein-Barr virus-induced B-

cell lymphomas, and from Kaposi's sarcoma (endothelial cell tumor) – Pneumonia caused by the fungus Pneumocystis jiroveci

• final stages of infection = AIDS– infection with cytomegalovirus (another herpes virus) or Mycobacterium avium complex

The clinical course of infection

Acute phase: CD4 T cell count drops from 800 to 500 per mm3 (normal immune fxn possible) -spike in viral load with initial infection -massive replication of CD4 T cells to try and stay ahead of viral loadChronic phase: T cell count is 200- 499 -appearance of symptoms - swollen lymph nodes, fatigue, cough, diarrheaAIDS: T cell count is below 200 -degeneration of lymph nodes -opportunistic infections - pneumonia, tuberculosis, encephalitis, Kaposi’s

sarcoma

Treatment• no vaccine (http://www.brown.edu/Courses/Bio_160/Projects1999/hiv/vacstrat.html)• prevention is avoiding exposure to the virus. • antiretroviral treatment - known as post-exposure prophylaxis reduces risk of infection

treatment for HIV infection = highly active antiretroviral therapy, or HAART• current HAART options - cocktails of least three drugs

– two types of anti-retroviral agents - nucleoside analogue reverse transcriptase inhibitors (NARTIs or NRTIs)

– plus either a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor (NNRTI).

• more aggressive treatment is recommended for children • HAART allows the stabilisation of the patient’s symptoms and viremia,• computer based study in 2006 projected that following the 2004 United States treatment

guidelines gave an average life expectancy of an HIV infected individual to be 32.1 years from the time of infection if treatment was started when the CD4 count was 350/µL

• absence of HAART - progression from HIV infection to AIDS has been observed to occur at a median of between nine to ten years and the median survival time after developing AIDS is only 9.2 months

• HAART - effective in less than fifty percent of patients- intolerance/side effects, prior ineffective antiretroviral therapy, infection with a drug-resistant strain of HIV, non-adherence and non-persistence with antiretroviral therapy (due to economics, social structure or HAART complexity)

• side effects include lipodystrophy, dyslipidaemia, insulin resistance, an increase in cardiovascular risks and birth defects

Abacavar - a nucleoside analog reverse transcriptase inhibitors (NARTIs or NRTIs)

Epidemiology• UNAIDS and the WHO estimate that AIDS has killed more than

25 million people since it was first recognized in 1981• 2005: AIDS pandemic claimed an estimated 2.8 million - of which

more than half a million (570,000) were children• Globally, between 33.4 and 46 million people currently live with HIV• 2005, between 3.4 and 6.2 million people were newly infected and

between 2.4 and 3.3 million people with AIDS died, an increase from 2004 and the highest number since 1981.

• worst affected = Sub-Saharan Africa - estimated 21.6 to 27.4 million people HIV positive– Two million [1.5–3.0 million] of them younger than 15 years of age. – More than 64% of all people living with HIV are in sub-Saharan Africa,

• Including more than three quarters of all women living with HIV– average life expectancy is 48.3 years

• South & South East Asia are second-worst affected with 15% of the total. – AIDS accounts for the deaths of 500,000 children in this region. – Two-thirds of HIV/AIDS infections in Asia occur in India (the highest

number of HIV infections in the world)• United States, the number of persons with AIDS increased from

about 35,000 in 1988 to over 220,000 in 1996