Riddle: Reproductive Microanatomy

Front 1

Structure of testis

Back 1

Covered by two-layered extension of abdominal peritoneum - tunica vaginalis
Collagenous connective tissue (tunica albuginea) - thicker posteriorly (mediastinum testis) and invaginates to form septula testis surrounding ~250 lobules
Each lobule: vascular interstitial tissue surrounding 1-4 seminiferous tubules (appear in cross section b/c highly coiled)
Each tubule 150-250 µm diameter and 80 cm long (total length 300-900 m per testis); straighten near mediastinum & connect to duct system

Front 2

Semineferous tubules and interstitial tissue

Back 2

Interstitial tissue (loose connective tissue)
-Blood vessels
-Fibroblasts
-Macrophages and mast cells
-Leydig cells – produce testosterone

Seminiferous tubules:
Thin tunica propria
-Myoid cells and/or fibroblasts
Thick seminiferous epithelium
-Sertolli cells – supporting cells
-Spermatogenic cells
--Spermatogonia – dividing mitotically
--primary (and very few secondary) Spermatocytes – undergoing meiosis (spermatogenesis)
--Spermatids – haploid germ cells; differentiating (spermiogenesis)

Front 3

Sertoli (sustentacular) cells

Back 3

Processes of Sertoli cells form junctional complexes, surround spermatogenic cells until released.

Help concentrate testosterone (~200-fold higher in seminiferous tubule than interstitial tissue) via androgen-binding protein

Contribute to blood-testis barrier
-excludes plasma proteins and antibodies from lumen – developing spermatozoa antigenically distinct
-protects developing sperm cells from chemical toxins

Front 4

Spermiogenesis

Back 4

Spermatids differentiate into structurally mature spermatozoa through several stages: condense nucleus, produce acrosomal cap with enzymes required for fertilization, generate flagellum, position mitochondria to power flagellum, shed excess cytoplasm (note: still not fully functional).

Front 5

Release of spermatids

Back 5

Through later mitotic divisions (spermatogonia) and through spermatogenesis cell division incomplete; daughter cells connected by cytoplasmic bridges
When separated from residual bodies (at end spermiogenesis) spermatozoa released as individual cells
When released appear mature structurally but are immotile and immature biochemically
Sperm production regulated regionally so appearance seminiferous epithelium heterogeneous
Takes about 74 days for spermatogonia to complete development into released spermatozoa so may take ~3 mo to see effects of treatments male infertility

Front 6

Intratesticular ducts and excurrent ducts

Back 6

Intratesticular ducts:
Derived from indifferent gonad during development
At end each seminiferous tubule get abrupt transition to the tubuli recti (straight segments), lined only by Sertoli cells.
Empty into rete testis, interconnecting channels within mediastinum lined by simple cuboidal/low columnar epithelium

Excurrent ducts:
Derived from the mesonephric duct and tubules during development
Three components
-Efferent ductules
-Epididymis
-Vas (ductus) deferens

Front 7

Efferent duct

Back 7

~20 efferent ductules anastomose and coalesce to form single ductule that connects to ductus epididymis
Lined by pseudostratified columnar epithelium with alternating clumps of short and tall cells
Endocytotic activity - fluid secreted by seminiferous epithelium resorbed by short cells of efferent duct epithelium
Thin muscular coat appears around the tubules; sperm transport is by ciliary action of columnar cells and by contraction of smooth muscle

Front 8

Epididymis

Back 8

Connective tissue capsule
Many cross-sections b/c highly coiled; 5-6 meters long
Pseudostratified epithelium produces factors that facilitate maturation spermatozoa (largely unknown mechanisms); two cells types:
-Basal cells function as stem cells
-Principal cells bear long luminal extensions (called stereocilia but immotile microvilli)
--resorb most remaining luminal fluid produced in seminiferous tubules
--phagocytose degenerate spermatozoa and remaining residual bodies
--Produce glycerophosphocholine; inhibits capacitation so full functional maturation doesn’t occur until sperrm enter female reproductive tract

Lumen often contains sperm
Little smooth muscle around mucosa along most of ductus epididymis (primarily fibroelastic tissue stroma); increases in tail as near vas deferens
Alters composition of fluid in which spermatozoa suspended
Recycling center for damaged/immature spermatozoa
Store spermatozoa and facilitate functional maturation: capacitation inhibited by glycerophosphocholine; other secretions facilitate other maturational changes; motility begins (increases when sperm cells mixed with secretions from seminal vesicle “down the line”)

Front 9

Spermatic cord and vas deferens

Back 9

Vas deferens is straight tube running behind the epididymis, within the spermatic cord
Thick muscular coat with middle circular layer and two longitudinal layers
Epithelium pseudostratified with columnar cells (with long microvilli) similar to those in epididymis (although principal cells shorter)
Other elements in spermatic cord:
-Arteries
-Veins, including pampiniform plexus (thermoregulatory function)
-Nerves
-Adipose tissue
-Smooth muscle in vas deferens
-Skeletal muscle in cremaster (thermoregulatory function)
-Loose connective tissue

Front 10

Leaving vas deferens

Back 10

Near distal end (at base of bladder) each vas deferens has a dilation called ampulla; muscle layer becomes thinner
At distal end of ampulla each vas deferens joined by a short duct from seminal vesicle
Ejaculatory duct formed by the merging of the duct from the seminal vesicle and the vas deferens distal to the ampulla (no smooth muscle in wall of the ejaculatory duct)
Right/left ejaculatory ducts run through prostate gland and open into the prostatic urethra

Front 11

Maturation of spermatozoa

Back 11

When released from seminiferous epithelium spermatozoa appear mature structurally but immotile and immature biochemically
Acquire motility and undergo other maturational factors as move through epididymis but complete maturation actively inhibited
The more distally sperm are obtained (ejaculate vs microsurgical, or percutaneous epididymal sperm aspiration versus testicular sperm aspiration/extraction) the more completely they can be evaluated and the more straightforward their use in IVF procedures (see link).
Sperm obtained through aspiration or extraction methods generally are inadequate and insufficient in number for intrauterine insemination (IUI) but may be used for intracytoplasmic injection (ICI).
-Sperm normally acquire ability to fertilize after time in female reproductive tract; capacitance requires removal/replacement of glycoconjugates on membrane so sperm head can bind to egg.
-Although only one sperm cell will penetrate ovum appears 100 or more must reach the ovum; getting through the cells that surround the oocyte appears to require teamwork.

Front 12

Seminal vesicles, prostate gland, bulbourethral glands: secretions

Back 12

Seminal vesicles produce viscous, fructose-rich fluid, 60-80% of volume of semen; pale yellow due to lipochrome

Prostate gland: conglomeration 30-50 compound tubuloalveolar glands in three concentric layers. Secretion is serous, rich in lipids, proteolytic enzymes, acid phosphatase, fibrinolysin and citric acid

Bulbourethral glands: produce thick, slippery mucus (serves to lubricate urethra)

Front 13

Seminal vesicle: structure

Back 13

Highly convoluted but unbranched tube
Essentially diverticulum of vas deferens
Epithelial tube surrounded by two layers smooth muscle (M) and external layer of fibroelastic connective tissue
Fibroelastic lamina propria thrown into folds and covered by epithelium with columnar secreting cells and round, non-specialized basal cells
Mucosal folding increases surface area for secretion; often secretions are evident within lumen

Front 14

Organization of prostate gland

Back 14

Mucosal glands (inner periurethral glands) in transition zone (TZ) empty directly into urethra

submucosal glands (outer periurethral glands) in central zone (CZ) that surrounds ejaculatory ducts (ED); more numerous and empty into urethra through ducts

Peripheral glands (main glands) in the peripheral zone (PZ) empty into urethra via long ducts opening along urethral crest.

Note fibrous stroma (St) surrounding the urethra, the capsule (Cap) and the fibrous septa (Sp) that separate the gland into lobules.

Front 15

Bulbourethral glands: structure

Back 15

Bulbourethral glands (about 5 mm diameter) produce watery, somewhat mucoid fluid containing sugars and some sialic acid
Secretions precede thicker semen along penile urethra and lubricate it
Capsule of gland contains connective tissue and smooth muscle
Glands comprise simple columnar epithelium; height varies with functional status (under control testosterone)

Front 16

General organization of female reproductive system

Back 16

Gametes develop in ovaries; also site of steroid synthesis
Ova conducted to uterus via oviducts (fallopian tubes); also site of fertilization
Uterus prepares to receive and support fertilized ovum
Lowest portion of the uterus, cervix, can dilate from lumen smaller than a pencil to a canal capable of permitting passage of a baby
Vagina is rugose fibromuscular tube, structure varies with age and hormonal activity

Front 17

Transport of the oocyte from ovary to uterus

Back 17

Following release from the ovary, ovum is “captured” by oviduct for transport to uterus.
Four tubal segments differ in proportions of muscle and epithelium and degree of convolution of mucosa
-Infundibulum bears fringe of epithelium-coated fimbriae (may adhere to ovary)
-Ampulla longest segment – normal site of fertilization
-Isthmus - narrower and thicker-walled
-Intramural segment penetrates wall of the uterus and opens into it

Front 18

Fertilization in the oviduct

Back 18

Normally occurs in ampulla
Final meiotic division of the ovum does not occur until the time of fertilization
Initial cell divisions within fertilized egg occur as transported toward uterus
Zona pellucida helps prevent polyspermy and remains intact around the fertilized egg through several cell divisions; may help prevent implantation of developing cell mass within the oviduct (must “hatch” prior to implantation)
More on the development and structure of the ovum below

Front 19

Organization of oviduct wall

Back 19

Three recognizable layers in the wall of the oviduct
Mucosa: variably folded, simple columnar epithelium with underlying lamina propria
Muscularis: two indistinct layers smooth muscle (inner circular; outer longitudinal)
Serosa: mesothelium with thin underlying layer of connective tissue

Front 20

Histology of the oviduct wall

Back 20

At higher magnification epithelium (E) and lamina propria (LP) of the mucosa are evident
Lamina propria includes blood and lymphatic vessels within typical loose connective tissue; extends into mucosal folds and may contain numerous lymphocytes.
In this section, both inner circular (MCL) and outer longitudinal layer (OLL) of the muscularis (M) are apparent between mucosa and connective tissue of the serosa (S)

Distal portion of oviduct characterized by larger diameter, thinner wall, and more complexly folded mucosa; mucosal extensions of the infundibulum (fimbria) help “capture” the ovum as it is released from the ovary
Closer to uterus oviduct is narrower with thicker, more muscular wall, smaller lumen, and less convoluted mucosa. Contraction of the smooth muscle in the wall of the oviduct helps propel the ovum toward the uterus.

Epithelium of oviduct has two cell types:
-shorter ciliated cells (CC) help propel egg toward uterus
-columnar peg cells (PC) secrete oviductal fluid that nourishes egg cell
Closer to ovary ciliated cells predominate. The light micrograph shows their cytology and also the stroma (S) of the lamina propria. The scanning electron micrograph (inset) shows cilia extending from the ciliated cells (cc) and the shorter microvilli on peg cells (aka secretory cells, sc)
Closer to the uterus (lower right) proportion of ciliated cells (CC) is greatly decreased and proportion of secretory peg cells (SC) is increased

Front 21

Summary of oviduct structure and function

Back 21

Fimbria on infundibulum help capture ovum as released from ovary (see below)
Ciliated epithelial cells help propel ovum toward uterus; become less abundant closer to uterus where there’s more smooth muscle to help with transport
Secretory peg cells help support ovum if fertilized; are more abundant closer to uterus
Sperm have to swim “upstream,” against movement of intratubal fluid; recent evidence suggests they “crawl” in the mucus environment.
Must penetrate cellular and extracellular barriers in order to fertilize ovum; usually occurs in ampulla of oviduct.

Front 22

Organization of the uterus

Back 22

Fertilized ovum delivered to uterus, pear-shaped muscular organ that comprises the body and fundus above and the cervix (which forms the roof of the vagina) below. Wall of the uterus comprises three layers:
-endometrium - mucosa with thick lamina propria
-myometrium with three layers of smooth muscle, blood vessels and lymphatics
-perimetrium - for most of uterus forms a serosa (mesometrium) continuous with the peritoneum (but with more elastic tissue); anteriorly forms adventitia

Front 23

Uterine endometrium and myometrium: histology

Back 23

Endometrium
-simple columnar epithelium
-invaginates into lamina propria to form simple, tubular uterine glands
-lamina propria well vascularized

myometrium
-Smooth muscle and lots of it
-Many blood vessels
-interstitial connective tissue

Front 24

Cyclical changes in uterine mucosa

Back 24

During reproductive years two layers recognized in endometrium:
-lower stratum basale maintained throughout ovarian cycle
-upper stratum functionale built up during part of menstrual cycle and then sloughed during menstruation

Building up of st. functionale in preparation for possible implantation includes elaboration of uterine glands and vasculature, latter includes extensive capillary beds and vascular lacunae (thin-walled venous lakes)

Pattern of vascularization critical for maintaining st. basale and regulating breakdown of st. functionale at menstruation:
-St. basale supported by straight arteries; cells of straight arteries lack steroid receptors
-St. functionale supported by spiral arteries; high density of steroid receptors on these vascular cells makes sensitive to changes levels of steroid hormones

Front 25

Changes in the uterine glands

Back 25

Uterine glands and gland cells change dramatically during ovarian cycle:

In proliferative stage glands are straight ; cells show no secretory activity

In initial secretory phase glands begin to coil; cells accumulate glycogen in basal region

In late secretory phase glands are highly coiled; cells show extensive secretory activity at apical portion

As a result uterine wall is prepared for implantation and early support of development, if fertilization occurs

Front 26

Start of the menstrual phase

Back 26

If no fertilization and implantation, decrease in estrogen and progesterone levels 10-12 days after ovulation causes cycles of contraction of spiral arteries - decreases blood flow and causes ischemia in st. functionale
Death (necrosis) of blood vessel cells and cells in st. functionale and bleeding from ruptured vessels
Portion of st. functionale detaches and rest of endometrium shrinks due to loss of interstitial fluid
By end menstrual phase endometrium reduced to thin layer

Front 27

Organization of the cervix

Back 27

Lower part of the uterus protrudes into and forms roof of vagina
Junction between uterine body and cervix is internal os, where the uterine wall and epithelium changes
Cervical canal opens into the vagina at the external os, where there is another change in the epithelium.
Cervical stroma comprises smooth muscle fibers embedded in collagen.
Endocervical epithelium extends in a slit-like fashion into lamina propria to form mucus-secreting cervical glands.
Mucus provides protective barrier to prevent bacterial movement into intrauterine cavity ; also descends into vaginal canal to provide lubrication during intercourse.

Front 28

Cervical softening

Back 28

Cervix must maintain integrity during pregnancy as uterus enlarges
Becomes distensible in preparation for labor and delivery (cervical ripening)
In contrast to body and fundus of uterus, cervix only 10-15% smooth muscle - mainly connective tissue - collagen, glycosaminoglycans and proteoglycans, also fibronectin and elastin
In late pregnancy hyaluronic acid content increases so more water trapped among collagen fibers; reduced cross-bridges among collagen fibers decreases fiber alignment and fiber strength
After cervical softening and with uterine contractions cervix passively dilates and is pulled over the presenting fetal part; elastin component helps maintain dilatation between contractions
Biochemical regulation of cervical softening involves complex interactions of several hormones and other factors
prostaglandin E2 causes dilatation small vessels in the cervix, increased collagen degradation, increased hyaluronic acid, increased chemotaxis for leukocytes (increase collagen degradation)
Nitric oxide (NO) also plays a role

Front 29

Organization of the vaginal wall

Back 29

Mucosa with stratified squamous epithelium and lamina propria that is rich in elastic fibers and thin-walled blood vessels

fibromuscular layer with ill-defined bundles of inner circular and outer longitudinal smooth muscle

adventitia with fibrocollagenous and elastic fibers, blood vessels and nerves (no mesothelium, vaginal adventitia blends with adventitia of bladder anteriorly and rectum posteriorly)

Front 30

Histological structure of the vagina

Back 30

Stratified squamous epithelium , non-keratinized
No glands in lamina propria (lubricated by secretions cervical glands and vulvovaginal glands around lower vaginal opening)
Structure varies with age and hormonal activity
-Before puberty/after menopause epithelium thin
-During reproductive years epithelium responds to estrogen: epithelial cells increase in number and size and accumulate glycogen
Glycogen content maximal at time of ovulation; breakdown by commensal bacteria produces lactic acid - lowers pH and deters invasion by bacterial pathogens and fungi

Front 31

Ovary anatomy

Back 31

Each ovary attached posterior surface of broad ligament by fold of peritoneum (mesovarium)
Superior (tubal) pole attached to pelvic wall by suspensary ligament (w/ vessels and nerves)
Inferior (uterine) pole attached to uterus by ovarian ligament
So ovary held in position close to, but not connected to, ovarian end of the oviduct

Front 32

Oogenesis

Back 32

Primordial germ cells from yolk sac migrate to developing ovary (like, way early)
Mitotic division of oogonia produces several million by time mitoses cease in second trimester
Oocytes increase in size, start meiosis I but stop in prophase; primary oocytes stay in that state for years (versus 20 days for primary spermatocytes)
By birth, about 1 million oocytes left in each ovary; by puberty about 250K left in each ovary
Only about 400 ova ultimately mature and are released, typically one in each ovarian cycle
During reproductive years each ovary contains many “resting” oocytes and smaller numbers of developing oocytes

Front 33

Ovary: organization, development

Back 33

Organization of the ovary
-Geminal epithelium outermost, covering tunica albuginea
-Cortex contains follicles & oocytes surrounded by stromal cells
-Medulla connective tissue and BVs

Follicular development & structure of primordial, primary, secondary and mature follicles
-Structure and chromosomal compliment of oocyte within each
-Changes in follicular cells through maturation and at ovulation

Process of atresia
Development, organization, function, and regression of the corpus luteum
Roles of the zona pellucida after ovulation

Front 34

Oocytes and follicular maturation

Back 34

Oogonia gone before birth –started meiosis or died trying

1º oocytes: Diploid, 4d DNA – almost all oocytes in ovary

2º oocytes: Haploid, 2d DNA –only in Graafian follicle(s)

Ovum haploid, 1d DNA – but meiosis not completed until released AND fertilized

Front 35

Ovulation

Back 35

Increased volume/pressure follicular fluid
Proteolysis of follicular wall
Deposition of glycoaminoglycans between cumulus oophorus and stratum granulosum
Cessation of blood flow in germinal epithelium over the follicle (macula pellucida or stigma)
That region ruptures and oocyte with corona radiata and cumulus oophorus is expelled
Small quantities of blood and follicular fluid pass into peritoneal cavity

Front 36

After ovulation

Back 36

Oocyte released with corona radiata, which remains with oocyte as transported into oviduct
Sperm cell must penetrate corona radiata, where last steps of capacitation occur
Change in glycoconjugates on sperm head permits binding to sperm receptors in zona pellucida
Triggers acrosome reaction, required for penetration of ovum

Front 37

Ovarian steroidogenesis

Back 37

Greatest steroid production by cells that surround follicle (theca layers) but scattered stromal cells produce significant levels of steroid hormones (probably all stromal cells have capability to produce steroid hormones under appropriate circumstances)
After ovulation corpus luteum formed from follicular wall (left after ovulation) and surrounding stromal cells; is very vascularized
Granulosa and thecal cells grow, fill with lipid, take on characteristics of steroid-secreting cells. Lipochrome provides a yellow appearance in fresh preparations
Both granulosa and theca lutein cells secrete hormones, primarily progesterone (also some estrogens)
If fertilization occurs corpus luteum will persist and increase in size to form the corpus luteum of pregnancy; otherwise it will degenerate in ~14 days

Front 38

Appearance of corpus luteum

Back 38

Section of ovary on left contains two corpora lutea (CL), each with a central blood clot (BC) surrounded by granulosa lutein cells (GL; simultaneous appearance two active corpora lutea indicates two follicles came to maturity in same cycle, potential for non-identical twins).
At higher magnification (right), the large pale-staining granulosa lutein cells (GL) can be compared to the more compact theca lutein cells (TL).

Front 39

Formation of corpus albicans

Back 39

If fertilization does not occur corpus luteum loses vascularization and cells become loaded with lipid, shrink, and undergo autolysis
Residual theca externa cells and fibroblasts produce collagen, replaces involuting lutein cells
End-result is relatively acellular, collagenous tissue called corpus albicans

Front 40

Follicular atresia

Back 40

Most oocytes never mature; disappear through atresia
Can occur any stage of development; more complex and visible for more developed follicles
For primordial/small growing follicles oocyte and surrounding follicle cells shrink and degenerate (A in a) and resorbed by surrounding stromal cells
For larger growing follicles (b), atresia probably starts in the follicle wall: granulosa cells stop dividing, neutrophils and macrophages invade, granulosa cells slough into antrum, theca interna cells hypertrophy then collapse into follicle; basement membrane of theca interna may form a hyalin layer (glassy membrane)