Male Reproduction
Sex determination & differentiation, SRY/Sox9, gonadal and phenotypic sex, PGC migration, disorders of sex development, male anatomy, spermatogenesis, testicular cooling, male endocrinology, spermatogenesis mechanics, oogenesis, fertilization, and assisted reproductive technologies (ANSC 224, Lectures 23–29)
A · Primary Germ Layers, Pituitary Development & Sex Determination Overview
- Digestive system, lungs, endocrine glands
- Muscle, skeleton, cardiovascular
- Reproductive system: gonads, uterus, cervix, part of vagina, epididymis, vas deferens, male accessory sex glands (MASG)
- Nervous system, skin, hair
- Mammary glands, hypothalamus, pituitary
- Part of vagina, penis, clitoris
Two parts derived from different tissues with different functions
- Origin: floor of the brain — infundibulum (neural ectoderm diverticulum)
- Releases ADH (vasopressin) and oxytocin
- Origin: roof of the mouth — Rathke's pouch (stomodeal ectoderm)
- Releases FSH, LH, GH, TSH, ACTH, prolactin
Reproductive tract development requires timing and coordination — errors affect ~0.5–1% of humans
Set at fertilization by the sex chromosomes: XY = male, XX = female. Y chromosome carries the SRY gene — the master switch.
SRY → testes develop (XY); no SRY → ovaries develop (XX, default). The bipotential gonad is directed by the hormonal environment.
Gonads secrete hormones (AMH, testosterone, DHT, estradiol) that drive development of internal ducts and external genitalia into male or female anatomy.
Testes → Sertoli cells (AMH) + Leydig cells (Testosterone) → male internal + external anatomy
Ovaries → no AMH → Müllerian ducts persist → uterus, oviducts, vagina. No positive hormone signal required.
B · The SRY Gene: Identifying the Master Sex Switch
In most mammals, presence of the Y chromosome → male; absence → female.
Quest timeline: researchers narrowed Y chromosome candidates from ~40,000 genes (1959) down to a single gene by 1990 — Sry.
In the presumptive testis, Sry gene is expressed in pre-Sertoli cells (somatic non-germ cells), producing Sry protein.
Sry protein → increases abundance of Sox9, another transcription factor.
Sox9 then:
- Alters transcription of many genes driving testis differentiation
- Drives production of FGF9 (testis-specific growth factor)
- Drives production of AMH (Anti-Müllerian Hormone)
- Turns off the Sry gene (Sry active only briefly, ~2 days in mice)
C · Hormones Drive Phenotypic Sex — Indifferent Gonad to Male or Female Tract
All embryos start with an indifferent gonad that has the potential to become either testis or ovary. At this stage, two duct systems co-exist:
→ In males: becomes epididymis, vas deferens, seminal vesicle, ampulla
→ In females: regresses (no testosterone)
→ In females: becomes oviduct, uterus, cervix, cranial vagina
→ In males: regresses under AMH
Also present: Urogenital sinus → becomes bladder (cranial) and vestibule/prostate (caudal)
→ Causes Müllerian duct inhibition/regression
→ Wolffian duct development: epididymis, vas deferens, seminal vesicle
(via 5α-reductase → DHT)
→ Male external genitalia: penis, scrotum, urethra
→ Brain sexual differentiation
Mesonephric tubules (future efferent ducts), mesonephric duct, paramesonephric (Müllerian) duct, undifferentiated sex cords, tunica albuginea forms
Epithelial cords → future seminiferous tubules; mesonephric tubules connect to rete testis; Müllerian duct still present but will regress
Rete testis → efferent ducts → epididymis; mesonephric duct → ductus deferens; seminiferous tubules; tunica albuginea
- PGC migration from yolk sac
- Sex cords develop in gonad; paramesonephric ducts develop
- Sex evident from gonadal structures
- Development of male ducts and testes OR female ducts and ovaries
- Formation of broad ligament (females)
- Testicular descent (species order: Bull & Ram → Boar & Human → Colt/Stallion latest)
D · Testis Descent, Hormone-Dependent Structures & Phenotype-Genotype Mismatches
The testes develop near the kidney and must migrate to the scrotum for spermatogenesis (requires cooler temperature).
- Gubernaculum — ligament from testis to scrotum; shortens and guides testis downward
- Testis passes through the inguinal ring → inguinal canal → scrotum
- Process driven by androgens (testosterone/INSL3) and differential growth
- Species timing varies (see 3rd trimester timing above)
The inguinal ring created for testis passage remains a weak point. Intestinal loops can herniate through it.
- Swine: 1/200 incidence
- Human children: 5/100 (5%)
- Can block intestinal blood flow; corrected by surgery
Epididymis, vas deferens (ductus deferens), seminal vesicle, ampulla — the Wolffian duct derivatives
Penis, scrotum, prostate, urethra (male external genitalia)
Requires: Testosterone → 5α-reductase → 5α-Dihydrotestosterone
Brain sexual differentiation: Testosterone → aromatase → Estradiol → masculinizes hypothalamic circuits
Genotype: 46;XY
Cause: 5α-reductase deficiency → little DHT in fetal life → female-appearing external genitalia at birth, undescended testes, normal internal male tract (testosterone-dependent)
At puberty: large testosterone surge overwhelms the deficiency → penis grows, testes descend → masculinization
Demonstrates distinction between T-dependent (internal) vs DHT-dependent (external) structures
SRY → Sox9 cascade establishes gonadal sex, but phenotypic sex depends on:
- Hormone production (Leydig/Sertoli cells functioning)
- Hormone receptors (androgen receptor intact)
- Enzyme activity (5α-reductase, aromatase)
Failure at any step = phenotype that doesn't match genetics
E · Primordial Germ Cell Biology, DSD & Freemartinism in Cattle
- Somatic cells — brain, liver, bone, muscle, skin, blood, etc.
- Germ cells — gametes and gamete precursors (sperm + oocytes); unique for undergoing meiosis
- Preserve genetic integrity through generations
- Generate genetic diversity (genetic recombination)
- Transmit genetic information to the next generation
- AIS (Androgen Insensitivity Syndrome) — Male pseudohermaphroditism: 46;XY, normal testes producing testosterone, but androgen receptor mutation → no response to androgens → female external phenotype, no uterus (AMH still works); 1:20,000
- Guevodoces — Male pseudohermaphroditism: 46;XY, 5α-reductase deficiency → female at birth; male at puberty
- CAH (Congenital Adrenal Hyperplasia) — Female pseudohermaphroditism: 46;XX, low glucocorticoids → adrenal overproduces androgens → masculinization of XX female
- Freemartin = female of mixed-sex (bull-heifer) twins
- Cause: shared placental vascular anastomosis (fusion of placentas) → blood exchange between twins
- AMH from male twin diffuses into female fetus → Müllerian (paramesonephric) duct inhibition → oviducts and uterus do not develop normally
- Variable reproductive tract abnormalities in the female twin
- Affects >90% of female twins co-gestated with a male
- Diagnosis: genetic test detecting Y chromosome in white blood cells (chimerism from fused blood supplies)
- Note: male co-twin is typically unaffected (testosterone dominates)
A · Male Gonads & The 5-Step Sperm Production System
Testis (singular) / Testes (plural)
Three functions:
- Gametogenesis — specifically spermatogenesis (sperm production)
- Produce male steroids — androgens (testosterone) and peptide hormones (inhibin)
- Produce fluid to move sperm through the ducts
1–25 × 10⁹ sperm/day (35,000–290,000 per second). "Plant must be air conditioned" — requires temp 2–10°C below body
Fluid absorption; membrane changes; nuclear & flagellar stabilization; motility acquisition; cytoplasmic droplet translocation (toward tail)
Stores 10–50 × 10⁹ spermatozoa; enough for 5–10 ejaculations; smooth muscle contractions move sperm at ejaculation
Add metabolic substrates (fructose, citrate), surface coatings, transport media. Glands: seminal vesicles, prostate, bulbourethral glands
Erection → Protrusion → Emission → Ejaculation
Most mammals have scrotal (extracorporeal) testes for cooling. Some species retain testes internally throughout life:
Why external? The reason is uncertain — but these internal-testis species all evolved alternate cooling mechanisms or heat-tolerant sperm.
B · Spermatic Cord Anatomy & Scrotal Cooling Adaptations
Extends from the body through the inguinal canal to each testis. Contains:
- Nerves, blood vessels, lymphatics
- Testicular artery (warm arterial supply from aorta)
- Pampiniform venous plexus (network of veins returning cool blood from testis) — surrounds the testicular artery
- Ductus deferens (vas deferens)
- Cremaster muscle (voluntary; elevates/lowers testis)
Spermatogenesis requires 2–10°C below core body temperature.
Mechanism: cool venous blood returning from the testis (≈33°C) surrounds and cools the warm testicular artery (entering at body temp ≈39°C) before it reaches the testis.
Warm incoming blood is cooled by outgoing blood — classic countercurrent exchange
A 2-lobed sac providing additional cooling adaptations beyond the pampiniform plexus
- Many sweat glands — evaporative cooling
- Very little hair — low insulation
- Very little subcutaneous fat — minimal insulation
- All features maximize heat dissipation
- Smooth muscle (involuntary)
- Has temperature sensors → trigger increased respiration rate when warm
- Cold: contracts → wrinkles scrotum → pulls testes toward body → warms
- Warm: relaxes → increases surface area → enhances cooling
- A pocket of peritoneum that descended with the testis
- Forms the vaginal cavity (fluid-filled space allowing testis to rotate freely)
- Visceral tunica vaginalis covers the testis surface directly
C · Testicular Temperature Problems & Cooling in Marine Mammals
- Bilateral: both testes retained → infertile (overheating → spermatogenesis fails) but normal testosterone and male appearance (Leydig cells still function)
- Unilateral: one testis descended → usually fertile
Many non-seasonal breeders show decreased fertility in warm months — testicular temperature rises as ambient temp increases
Testes too close to body wall → inadequate cooling → impaired spermatogenesis → subfertility
Abnormal dilation of pampiniform plexus veins → impaired countercurrent cooling → elevated testicular temperature → reduced sperm quality/fertility
Physicians suggest men with fertility problems wear boxers rather than briefs — reduces scrotal temperature by keeping testes away from body heat
Dolphins, whales, and seals have internal testes surrounded by warm body tissues and thick blubber. Yet they produce sperm. How?
Venous blood flowing through the dorsal fin and flukes is cooled by cold seawater via a periarterial venous rete (artery surrounded by ring of veins)
Cool venous blood from fins/flukes returns to juxtaposed arterial/venous plexuses near the aorta → cools the arterial blood heading to the testis
Same countercurrent heat exchange principle as the pampiniform plexus — just using fin/fluke venous return as the cooling source
The same blood plexus that cools the dolphin testis also cools the dolphin uterus — both are internal reproductive organs facing the same heat problem
A · Male Endocrinology
Releases GnRH (gonadotropin-releasing hormone) in pulses into the hypophyseal portal system. Pulse frequency and amplitude regulate FSH vs LH balance.
GnRH stimulates release of FSH and LH (gonadotropins) into systemic circulation.
FSH arm: FSH → Sertoli cells → inhibin (↓FSH feedback) + spermatogenesis support.
LH arm: LH → Leydig cells → testosterone → secondary sex characteristics + negative feedback.
Lipid-soluble; derived from cholesterol
- Examples: testosterone, estradiol, progesterone, cortisol
- Cross cell membranes freely
- Bind intracellular/nuclear receptors
- Receptor-hormone complex acts as transcription factor → gene expression
- Slow onset (hours–days) but prolonged effect
- Transported in blood bound to SSBG (sex steroid binding globulin)
Water-soluble; cannot cross membranes
- Examples: GnRH, FSH, LH, inhibin, activin, prolactin
- Bind membrane receptors (GPCRs)
- Signal via second messengers: cAMP → protein kinase A
- Fast onset (minutes) but shorter duration
- Circulate freely in blood (water-soluble)
- Produced by the liver
- Carries testosterone (and estradiol) in blood
- ~98% of testosterone is bound (inactive)
- Only free (~2%) testosterone is biologically active
- SSBG levels regulate testosterone bioavailability
All steroid hormones share this cholesterol precursor; pathway occurs mainly in Leydig cells
Testosterone is converted to more active forms in peripheral tissues
- Enzyme: 5α-reductase
- Sites: prostate, skin, hair follicles, genital skin
- DHT has greater androgen receptor affinity than testosterone
- Actions: prostate growth, male pattern baldness, external genital masculinization
- Enzyme: aromatase (CYP19)
- Sites: brain, adipose tissue, Sertoli cells
- Essential for: bone density, brain function/libido, spermatogenesis (in Sertoli cells)
- Males need some estrogen — deficiency causes osteoporosis and impaired spermatogenesis
Andropause
- Gradual decline in testosterone after age ~40
- Unlike menopause — not a complete cessation
- Symptoms: fatigue, decreased muscle mass, decreased libido, depression, decreased bone density
Testosterone Replacement Therapy (TRT)
- Forms: patches, gels, injections
- Improves symptoms of andropause
- Caution: exogenous T → negative feedback → ↓ GnRH/LH → ↓ endogenous testicular testosterone → ↓ spermatogenesis → infertility
A · Spermatogenesis & Testis Anatomy
- Seminiferous tubules — where spermatogenesis occurs
- Lined with Sertoli cells and developing germ cells
- Surrounded by contractile myoid cells
- Tubule lumen carries sperm toward rete testis → efferent ducts → epididymis
- Leydig cells — produce testosterone in response to LH
- Blood vessels (testicular artery, pampiniform veins)
- Lymphatics and nerves
- Macrophages and immune cells (regulated by BTB)
Characteristics
- Somatic cells — not germ cells
- Stop dividing at puberty → their number sets the maximum sperm production capacity
- Have FSH receptors and testosterone receptors
- Form tight junctions that create the blood-testis barrier
Functions
- Produce inhibin (↓FSH feedback) and androgen-binding protein (ABP)
- Provide nutrients, growth factors, and structural support to germ cells
- Phagocytose residual bodies after spermiogenesis
- Aromatize testosterone → estradiol (needed locally for spermatogenesis)
- Create immunologically privileged adluminal environment
- Formed by tight junctions between adjacent Sertoli cells
- Divides the tubule into two compartments:
Spermatogonia (stem cells) and early primary spermatocytes; connected to blood supply; mitosis occurs here
Meiotic cells (secondary spermatocytes) and spermatids; immunologically privileged; isolated from blood
Spermatocytogenesis (Mitosis)
- Spermatogonial stem cells (Type A) in basal compartment undergo mitosis
- Type A → Type B spermatogonia → primary spermatocytes (2n, diploid)
- Amplifies germ cell numbers; maintains stem cell pool
Meiosis (Reduction Division)
- Primary spermatocyte (2n) → Meiosis I → 2 secondary spermatocytes (n)
- Each secondary spermatocyte → Meiosis II → 2 spermatids (n)
- Net result: 1 primary spermatocyte → 4 haploid spermatids
- Occurs in adluminal compartment (above BTB)
Spermiogenesis (Differentiation — No Cell Division)
- Round spermatids → morphologically mature spermatozoa
- Acrosome formation from Golgi apparatus (enzyme cap for egg penetration)
- Flagellum develops from centrioles migrating to basal pole
- Nuclear condensation: histones replaced by protamines; DNA tightly packaged
- Mitochondria arrange into midpiece (energy for flagellar movement)
- Most cytoplasm shed as residual body → Sertoli cells phagocytose it
Oogenesis is the complete process of production of ova (mature eggs) from primordial germ cells. It has four main goals:
- Increase genetic variation (through meiotic recombination)
- Produce a haploid cell (so fertilization restores diploidy)
- Synthesize macromolecules needed for fertilization
- Produce a store of materials for the developing embryo
Four Phases of Oogenesis
- Mitotic divisions of PGCs (proliferation)
- Start of meiosis + nuclear arrest (dictyate)
- Oocyte growth (cytoplasmic phase)
- Resumption of meiosis and ovulation
PGC Migration & Proliferation
- 10–100 Primordial Germ Cells (PGCs) originate near the posterior primitive streak (hindgut region)
- They migrate to the genital ridge, attracted by Stem Cell Factor produced there
- PGCs divide mitotically to form >5,000 cells, forming primitive sex cords
- In females: PGCs → oogonia → stop mitosis prenatally → enter meiotic Prophase I → become primary oocytes
- In mice, PGCs enter the gonad at ~Day 11.5 dpc; by Day 14, they have multiplied to ~30,000
Mouse Oogenesis Timeline
PGCs form cysts → cysts break down (depends on steroids) → primordial follicles form (~Day 19–23 dpc). Unlike males, stem cell renewal ends in the fetal stage — oogonia do not renew postnatally.
Oocyte meiosis is uniquely characterized by two long-term arrests before the process completes:
Arrest #1 — Dictyate (Late Diplotene)
- Oogonia enter Prophase I triggered by local retinoic acid
- Genetic recombination occurs during Prophase I
- Oocytes arrest at late diplotene (dictyate stage)
- Maintained by high cAMP levels
- Remain arrested from prenatal stage until puberty — up to 40–50 years in women
- At puberty: LH surge re-starts meiosis; oocyte advances to Metaphase II
Arrest #2 — Metaphase II
- After LH surge: oocyte completes Meiosis I, produces 1st polar body
- Ovulated as a secondary oocyte arrested at Metaphase II
- Remains arrested at MetII until fertilization by sperm
- Fertilization triggers completion of Meiosis II → 2nd polar body
- Final result: 1 primary oocyte → 1 secondary oocyte → 1 mature ovum
Phase 3: Oocyte Growth (Cytoplasmic)
- Oocytes grow dramatically in size after birth, before puberty
- Develop the zona pellucida — a glycoprotein coat secreted by the oocyte
- Synthesize cortical granules — vesicles that will be released at fertilization
- Oocyte must reach full size before the antrum forms — only fully grown oocytes can resume meiosis
- 99.9% of oocytes become atretic and never ovulate
Ovulated Metaphase II Oocyte — Structure
The meiotically mature, fertilization-ready oocyte consists of: oocyte membrane (plasma membrane), cortical granules just inside the membrane, a zona pellucida (glycoprotein coat), a first polar body in the perivitelline space, and surrounding cumulus cells.
The oocyte does not passively wait in the follicle — it actively controls follicle development through two types of communication:
Type 1 — Paracrine Signaling
The oocyte secretes growth factors that diffuse to surrounding granulosa cells, controlling their proliferation and function.
Evidence: Booroola sheep ovulate ~2× as many oocytes due to a mutation in the receptor for a growth factor produced only by the oocyte — proving oocyte-exclusive control of this pathway.
Type 2 — Gap Junction Communication
Cumulus cell processes extend through the zona pellucida to form gap junctions with the oocyte — allowing direct exchange of ions and small nutrients.
These junctions break down at the LH surge, disconnecting the oocyte from cumulus cells as meiosis resumes.
Oocyte Numbers Over a Lifetime (Women)
| Stage | Oocyte Count | Notes |
|---|---|---|
| 20 weeks gestation | ~7 million | Peak; stem cell renewal active |
| Birth | ~1 million | Stem cell renewal has ended |
| Puberty | ~300,000 | <5% of peak number remain |
After the fetal stage, follicle/oocyte numbers decline exponentially through atresia — this is why maternal age is the major risk factor for chromosomal abnormalities. The longer oocytes sit arrested in dictyate (up to 50 years), the greater the chance of chromosome segregation errors (e.g., Trisomy 21/Down syndrome: risk rises from 0.1% at age 20 to 3.6% at age 45).
The LH surge triggers three simultaneous maturation changes in the oocyte and follicle near/at ovulation:
- Nuclear maturation — resumption of meiosis (germinal vesicle breakdown → MetII)
- Cumulus mass expansion — cumulus cells produce hyaluronic acid, pushing cells apart
- Cytoplasmic maturation — cytosol develops the ability to re-organize the sperm nucleus at fertilization
Cumulus Cell Maturation
- Immature oocyte–cumulus complexes are tightly compacted
- LH surge → cumulus cells produce hyaluronic acid → cells pushed apart (expansion)
- Sperm possess hyaluronidase to dissolve the cumulus matrix
- Many animals lose cumulus cells before fertilization is complete
Nuclear Maturation Steps (Meiotic Resumption)
- Oocyte in large follicle is activated by LH surge (sometimes 40–50 yrs after meiosis began)
- Germinal vesicle (nucleus) breaks down → meiosis I proceeds
- Oocyte completes meiosis I → enters meiosis II → arrests at Metaphase II
- Extra chromosomes deposited into 1st polar body
- Fertilization by sperm triggers completion of meiosis II → 2nd polar body
Summary: Mitosis/Meiosis During Oogenesis
- Mitosis and part of meiosis are completed before birth (stem cell proliferation ends prenatally)
- Critical meiotic recombination occurs in the fetal stage
- Eggs arrested in dictyate stage of Meiosis I until ovulation — follicle grows around them
- Oocytes help control follicle development (paracrine + gap junction communication)
- Meiosis I completed at ovulation; Meiosis II completed at fertilization
- 1 primary oocyte → 1 secondary oocyte → 1 mature ovum
Fertilization is the fusion of a sperm and egg to form a new organism. It requires the reproductive tract to bring gametes together and for each gamete to undergo final maturation. Site of fertilization: the ampulla of the oviduct.
5 Events After Semen Deposition
- Immediate transport — retrograde loss of most sperm, phagocytosis, entrance into cervix
- Cervix — privileged pathways (cervical crypts = Reservoir I); non-motile/abnormal sperm removed
- Uterus — capacitation initiated; phagocytosis of excess sperm
- Oviduct — capacitation completed; sperm develop hyperactivated motility; isthmus = Reservoir III
- Fertilization — acrosome reaction → sperm penetrates oocyte → pronuclei form
Sperm Numbers at Fertilization
Of millions deposited, fewer than 50 sperm are typically present at the ampulla at any one time. Most are lost via retrograde flow back out of the vagina, phagocytosed by macrophages, or filtered at anatomical barriers (cervical os and uterotubal junction).
Rapid Transport
Sperm arrive at the oviduct within minutes of ejaculation — but are not yet fertile. This is likely passive transport via muscle contractions.
Sustained Transport
Sperm bind to the isthmus reservoir, where they survive longer; they undergo capacitation and are slowly released to the ampulla over time — providing a steady supply of fertile sperm.
Capacitation is the final maturation of sperm in the female reproductive tract that enables fertilization ability. Ejaculated sperm cannot immediately fertilize an egg — they must first spend time in the female tract (or in appropriate in-vitro culture conditions).
Three Key Changes During Capacitation
1. Hyperactivated Motility
Sperm develop vigorous, whip-like flagellar motion needed to penetrate the zona pellucida.
2. Cholesterol Removal
Cholesterol is removed from the sperm plasma membrane, destabilizing it and priming the membrane for the acrosome reaction.
3. Coating Protein Removal
Epididymal coating proteins mask zona pellucida receptors. These are removed in the female tract, exposing ZP receptors on the sperm head.
Isthmus Reservoir & Sperm Lifespan
Sperm bind to sugar residues on isthmus epithelial cells — this binding prolongs sperm lifespan. Bound sperm capacitate slowly, then detach and are carried to the ampulla. Binding is thought to involve oviduct-specific glycoproteins.
The Zona Pellucida (ZP)
The zona pellucida is a tough glycoprotein coat surrounding the oocyte. It has three key functions:
- Sperm binding — species-specific recognition (prevents cross-species fertilization)
- Polyspermy block — modified after fertilization to prevent additional sperm from entering
- Embryo protection — physical shield during preimplantation development
ZP Structure (Mice/Mammals)
- ZP1 — structural crosslinker (no direct sperm binding)
- ZP2 — sperm receptor; degraded by cortical granule enzyme ovastacin after fertilization
- ZP3 — sperm receptor; inactivated by cortical granule enzymes after fertilization
- (ZP4 in humans)
The Acrosome Reaction
To penetrate the ZP, sperm must undergo the acrosome reaction:
- The sperm plasma membrane and outer acrosomal membrane fuse and degenerate
- This releases acrosomal enzymes (e.g., acrosin) that may help digest a path through the ZP
- The inner acrosomal membrane (IAM) is exposed — the IAM binds to the egg membrane after ZP penetration
- Hyperactivated motility provides the physical force for ZP penetration
Sperm–Egg Membrane Binding & Fusion
After ZP penetration, the acrosome-reacted sperm enters the perivitelline space. Its inner acrosomal membrane (IAM) makes contact with the egg plasma membrane. The egg has microvilli that grip the sperm; the membranes then fuse, incorporating the sperm into the egg cytoplasm.
Egg Activation at Fertilization
After sperm–egg membrane fusion, a sperm enzyme (phospholipase C ζ) is released into the egg cytosol and triggers repeated Ca²⁺ waves from intracellular stores. Ca²⁺ release activates the egg:
- Cortical granules are released (exocytosis)
- Cortical granule enzymes remove the sperm-binding site from the zona pellucida (polyspermy block)
- Egg finally completes meiosis II — expels the 2nd polar body
Slow Block to Polyspermy (Mammals)
- Mammals have a slow block at the ZP and egg membrane (engages in minutes)
- Cortical granule enzyme ovastacin cleaves ZP2 (degrades it)
- Another enzyme inactivates ZP3 (removes sperm-binding activity)
- If ovastacin is absent: sperm continue binding even after fertilization → polyspermy
Consequences of Polyspermy
- Multiple sperm introduce extra centrosomes into the egg
- Extra centrosomes cause multipolar spindles during first cleavage
- Results in abnormal chromosome numbers in daughter cells
- Cells disintegrate → embryo death
Pronuclear Formation & Syngamy
- Sperm is engulfed; sperm head swells and DNA structure relaxes → forms male pronucleus
- Sperm organelles are degraded (except the centriole, which the egg lacks)
- Egg nucleus forms the female pronucleus
- Syngamy: the nuclear envelopes interdigitate → chromosomes align on a single metaphase spindle → first mitotic division → two diploid nuclei
Preimplantation Embryo Development
- Cleavage cells = blastomeres; mitosis occurs with no overall size change
- Blastocyst differentiates into: trophoblast (→ placenta) and inner cell mass (ICM) (→ fetus)
- Embryo hatches from zona pellucida at 7–10 days to implant in uterus
A · Semen Collection, Artificial Insemination & Semen Storage
Began: 1940–1950s; now routine worldwide
Species use:
- Dairy cattle: most economically impactful use
- Turkeys: ~100% of commercial matings use AI
- Swine: >90% of commercial matings use AI
- Horses: widely used; AI accepted by many registries
Why AI is important: considered the most important technology for genetic improvement in livestock — one superior sire can produce tens of thousands of offspring
- Superior genetics: use of elite sires impossible with natural service alone
- Genetic records: accurate pedigree and mating records
- Safety: no need to keep a bull/boar on farm
- Reduced feed costs: no maintenance of live male
- Disease control: semen tested for sexually transmitted pathogens before use
- Consistent fertility: standardized processing and quality control
Collection method: artificial vagina (AV) — mimics the temperature and pressure of the female tract to induce ejaculation
Semen extender is added immediately after collection:
- Isotonic solution with buffer at neutral pH
- Energy substrate: glucose or fructose
- Antibiotics to reduce bacterial contamination
- Cryoprotectants (for freezing) or storage medium (for chilled liquid storage)
Pig sperm are stored chilled (16–18°C) for 2–5 days; most cattle semen is cryopreserved in liquid nitrogen
| Method | Deposition Site |
|---|---|
| Natural mating | Vagina / cervix |
| AI (cattle) | Uterine body |
In AI, only 0.25–0.5 mL of diluted semen is used. Depositing in the uterine body compensates for the small volume — distributing sperm closer to the oviducts. The best inseminators can successfully deposit in the uterine body ~85% of the time.
Study data: uterine body 39%, cervix 25%, left horn 13%, right horn 23% among inseminators of varying skill
Non-Penetrating Cryoprotectants
Do NOT enter the cell
- Examples: egg yolk protein, milk protein
- Mechanism: coat and stabilize the external plasma membrane, protecting it from ice damage
Penetrating Cryoprotectants
Enter the cell
- Examples: DMSO, ethylene glycol (antifreeze), glycerol
- Mechanism: enter the cell and lower the intracellular freezing point, minimizing intracellular ice crystal formation
1. Ice Crystal Formation
- Intracellular ice crystals physically break the plasma membrane
- Caused by fast cooling — water cannot exit the cell fast enough before ice nucleation occurs
- Fast freezing → more ice crystals
2. Solution Effects
- As extender freezes, pure water crystallizes first
- Remaining unfrozen liquid becomes hypertonic — salts and cryoprotectants concentrate to toxic levels
- Slow freezing → more solution effects
| Freezing Rate | Ice Crystals | Solution Effects |
|---|---|---|
| Fast | More (↑) | Less (↓) |
| Slow | Less (↓) | More (↑) |
Optimal protocols balance these two types of damage — typically a controlled-rate freezer is used for cattle semen straws.
Thawing: thaw as quickly as possible in a warm water bath at 35°C (95°F) to minimize time in the critical temperature zone where damage accumulates
Species Cryopreservation Tolerance:
- Survive freezing well: humans, horses (stallion semen freezes acceptably)
- Survive freezing poorly: swine (pig sperm very sensitive to cold shock; usually stored chilled at 16–18°C for 2–5 days), birds/poultry
Only proven commercial method: flow cytometry / cell sorting
Principle: fluorescent DNA-binding dye is applied to sperm
- Y chromosome is smaller than X chromosome → Y-bearing sperm have ~4% less total DNA than X-bearing sperm
- X-bearing sperm fluoresce brighter; Y-bearing sperm fluoresce less
- Cell sorter separates the two populations by fluorescence intensity
Accuracy: ~90%
Cost: $15–25 per straw (vs. ~$2–5 for conventional semen)
Main use: dairy cattle (selecting heifer calves)
Step-by-step procedure:
- Day 9 (midcycle): inject donor with FSH to superovulate — recruit multiple follicles
- Day 11: inject PGF2α to lyse the CL → drop progesterone → induce estrus in donor
- At estrus: breed donor via AI
- ~Day 18–19 (7 days after breeding): flush morulae/blastocysts from donor uterus by non-surgical lavage
- Transfer to recipient: deposit each embryo into the uterine horn ipsilateral (same side) as the corpus luteum on the recipient's ovary
Cycle Synchronization
The recipient surrogate must be at the same stage of the estrous cycle as the donor at the time of transfer. The uterus must be in the correct progesterone-dominated state to receive a morula/blastocyst.
Horn Placement
Embryo is deposited into the uterine horn on the same side as the CL (ipsilateral horn). The CL produces progesterone that locally prepares the horn for implantation. Heifers are preferred as recipients.
Pregnancy Rates
- Fresh embryo: ~60%
- Frozen/thawed embryo: ~40–50%
Lower rates with frozen embryos reflect damage from the freeze–thaw process
1Hormones, Steroids & Signals
| Hormone / Signal | Type | Source | Key Actions |
|---|---|---|---|
| GnRH | Peptide | Hypothalamus | Pulsatile → stimulates FSH + LH from anterior pituitary; negative feedback by T + E2 |
| FSH | Glycoprotein | Anterior pituitary | Sertoli cells → spermatogenesis support, ABP, inhibin; also follicle recruitment (Lec 27) |
| LH | Glycoprotein | Anterior pituitary | Leydig cells → testosterone; LH surge → ovulation + oocyte meiosis resumption (Lec 27) |
| Testosterone (T) | Steroid | Leydig cells (LH-stimulated) | Wolffian duct development (internal tract); spermatogenesis; secondary sex characteristics |
| 5α-DHT | Steroid | T via 5α-reductase in peripheral tissue | Male external genitalia (penis, scrotum, urethra, prostate); stronger AR affinity than T |
| Estradiol (E2) | Steroid | T via aromatase (brain, adipose, Sertoli) | Brain sex differentiation (fetal); bone density; locally essential for spermatogenesis |
| Inhibin | Peptide | Sertoli cells | Selective inhibition of FSH (negative feedback); not LH |
| AMH | Glycoprotein | Sertoli cells (fetal testis) | Müllerian duct regression; produced by pre-Sertoli cells driven by Sox9/SRY |
| SRY protein | Transcription factor | Pre-Sertoli cells (Y chromosome) | Master sex switch: upregulates Sox9 → FGF9 + AMH → testis formation |
| Sox9 | Transcription factor | Pre-Sertoli cells (SRY downstream) | Drives testis differentiation; produces AMH + FGF9; turns off SRY |
| SSBG | Glycoprotein | Liver | Carries testosterone in blood; ~98% bound (inactive); only free ~2% is bioactive |
| Retinoic acid | Retinoid | Local fetal/gonadal tissue | Triggers oogonia to enter meiotic Prophase I (Lec 27); males block this signal |
| cAMP (oocyte) | Second messenger | Granulosa → gap junctions → oocyte | Maintains meiotic arrest at dictyate stage; LH surge lowers cAMP → meiosis resumes |
| Stem Cell Factor (SCF) | Growth factor | Genital ridge | Chemoattractant for migrating PGCs; guides them from hindgut to gonad (Lec 27) |
| PLC-ζ | Enzyme (sperm-derived) | Sperm (released at fusion) | Triggers repeated Ca²⁺ oscillations in egg → egg activation at fertilization (Lec 28) |
2Key Structures
| Structure | Location / Type | Key Function |
|---|---|---|
| Hypothalamus | Brain | GnRH pulse generator — master HPG axis controller |
| Anterior pituitary | Base of brain | Secretes FSH + LH in response to GnRH |
| Bipotential (indifferent) gonad | Embryonic genital ridge | Can form testis or ovary; SRY tips it toward testis; ovary is default |
| Wolffian (mesonephric) duct | Embryonic | → Epididymis, vas deferens, seminal vesicle; requires testosterone to develop |
| Müllerian (paramesonephric) duct | Embryonic | → Oviduct, uterus, cervix (females); regresses in males under AMH from Sertoli cells |
| Testes | Scrotum (or abdomen in some species) | Spermatogenesis + testosterone + inhibin production; must be 2–10°C below body temp |
| Seminiferous tubules | Testis — tubular compartment | Site of spermatogenesis; Sertoli cells + developing germ cells; myoid cells contract |
| Sertoli cells | Lining seminiferous tubules | 'Nurse cells': BTB formation, spermatogenesis support, ABP, inhibin, AMH (fetal), phagocytose residual bodies |
| Leydig (interstitial) cells | Interstitial compartment (between tubules) | Testosterone production in response to LH; also produce small amounts of E2 |
| Blood-testis barrier (BTB) | Tight junctions between Sertoli cells | Immunologically privileged adluminal compartment; protects post-meiotic cells from immune attack |
| Epididymis — Caput (head) | Above testis | Initial sperm processing; absorbs testicular fluid |
| Epididymis — Corpus (body) | Along testis | 'Finishing shop': sperm motility acquisition, nuclear + flagellar stabilization |
| Epididymis — Cauda (tail) | Below testis | 'Warehouse': stores 10–50 × 10⁹ sperm; can supply 5–10 ejaculations |
| Spermatic cord | Inguinal canal to testis | Contains testicular artery + pampiniform plexus + vas deferens + cremaster muscle |
| Pampiniform venous plexus | Spermatic cord (around artery) | Countercurrent heat exchanger: cool venous blood cools incoming testicular artery |
| Scrotum — tunica dartos | Smooth muscle in scrotal wall | Contracts (cold → wrinkles → warms); relaxes (warm → increases surface area → cools) |
| Gubernaculum | Testis → scrotal floor | Ligament that guides testicular descent through inguinal canal; shortens under androgen/INSL3 |
| Rete testis | Inner testis (mediastinum) | Collects sperm from all seminiferous tubules → efferent ducts → epididymis |
| Vas deferens (ductus deferens) | From cauda epididymis → urethra | Sperm transport channel; smooth muscle contracts at ejaculation |
| Accessory sex glands | Seminal vesicles, prostate, bulbourethral | Add fructose, citrate, surface coatings, transport medium at ejaculation |
| Acrosome | Anterior cap of sperm head (from Golgi) | Contains enzymes (acrosin); released at acrosome reaction → ZP penetration |
| Oogonia / Primary oocyte | Fetal ovary (Lec 27) | Oogonia → enter meiotic Prophase I prenatally → arrest at dictyate as primary oocytes |
| Zona pellucida (ZP) | Glycoprotein coat around oocyte | ZP2 + ZP3 bind sperm species-specifically; modified by cortical granules → polyspermy block |
| Cumulus oophorus | Granulosa cells around oocyte (Lec 27) | Oocyte communication via gap junctions; expands (hyaluronic acid) at LH surge |
| Oviduct — Ampulla | Middle oviduct (Lec 28) | Site of fertilization; oocyte + sperm meet here |
| Oviduct — Isthmus | Proximal oviduct (Lec 28) | Sperm reservoir; capacitation completed; regulated release to ampulla |
| Cortical granules | Beneath oocyte plasma membrane | Released at fertilization → ovastacin cleaves ZP2; inactivates ZP3 → slow polyspermy block |
3Exam Traps — by Lecture
PGC Migration Route — sequence matters
Trap: The hindgut is the migration route, not the final location. PGCs are not "hindgut cells" — they use it as a highway to the gonad.
Stem Cell Factor (SCF)
Produced by the embryonic gonad / genital ridge. Acts as a chemoattractant that guides migrating PGCs from the hindgut toward the gonad. Without SCF signaling, PGCs fail to reach the gonad and die.
Duct Derivatives — know both
| Duct | Also called | Stimulus | Derivatives |
|---|---|---|---|
| Wolffian | Mesonephric | Testosterone | Epididymis, vas deferens, seminal vesicle |
| Müllerian | Paramesonephric | No AMH (default) | Oviduct, uterus, cervix, cranial vagina |
Trap: Prostate and external genitalia are not Wolffian duct derivatives — they arise from the urogenital sinus and genital tubercle (DHT-driven).
Freemartinism (Cattle)
Occurs in mixed-sex cattle twins when placental vasculature fuses early. AMH (and androgens) from the male twin travel to the female twin via shared blood supply → Müllerian duct regression + gonadal masculinization in the female. Result: infertile female co-twin. Cattle-specific — fusion occurs early enough for AMH to act; rare in other species.
Male Reproductive Tract Flow — in order
Trap: Efferent ducts come before the epididymis, not after. At ejaculation the movement is: cauda epididymis → vas deferens → ejaculatory duct → urethra.
Epididymis Regions
| Region | Droplet position | Function |
|---|---|---|
| Caput (head) | Droplet near head of sperm | Immature sperm; fluid absorption from testis |
| Corpus (body) | Droplet migrating to midpiece | Maturation; motility acquisition; membrane remodeling |
| Cauda (tail) | Droplet shed (mature) | Storage of mature, motile sperm; 10–50 × 10⁹ |
Semen Fluid Contributions
Seminal vesicles contribute the majority of semen volume (~60–70%); secrete fructose (sperm energy), prostaglandins, and coagulation proteins. Prostate contributes ~25–30% (citrate, zinc, proteases). Sperm themselves = <5% of volume.
Clinical Anatomy Traps
- Cryptorchidism → testicular temp too high → spermatogenesis fails.
- Key exam point: Bilateral cryptorchids are infertile (no sperm) but still produce testosterone — Leydig cells function normally at body temperature. Normal male secondary sex characteristics are maintained.
- Unilateral cryptorchid is usually fertile (one descended testis sufficient).
Clinical Endocrine Traps
- 5α-Reductase converts testosterone → DHT. 5α-Reductase inhibitors (finasteride, dutasteride) lower DHT → can shrink the prostate (BPH treatment) and slow male-pattern baldness; they do not eliminate testosterone itself.
- PSA (Prostate-Specific Antigen) is not cancer-specific — it is produced by normal prostate epithelium and rises with BPH, infection, or manipulation too.Trap: elevated PSA → biopsy may be unnecessary; PSA is a screening tool, not a diagnosis.
Erection Signaling Pathway
PDE5 degrades cGMP → ends erection. PDE5 inhibitors (sildenafil/Viagra) prevent cGMP breakdown → prolonged smooth-muscle relaxation → sustained erection.Trap: PDE5 inhibitors do not produce NO themselves — they require sexual stimulation to initiate the NO signal first.
Anabolic Steroid / Exogenous Testosterone Trap
Exogenous testosterone (or synthetic analogs) provides negative feedback at hypothalamus (↓ GnRH) and pituitary (↓ LH/FSH) → endogenous testicular testosterone falls → spermatogenesis impaired → testicular atrophy and infertility. Secondary sex characteristics are maintained by exogenous steroid, masking the internal damage.
Blood-Testis Barrier — exact wording
Formed by tight junctions between adjacent Sertoli cells. Creates two compartments:
Basal compartment (below BTB)
Spermatogonia + early primary spermatocytes. Connected to blood supply. Mitosis occurs here.
Adluminal compartment (above BTB)
Meiotic cells + spermatids + spermatozoa. Immune-privileged — immune cells and antibodies cannot enter.
Spermatogenesis Exam Traps
- Sequence: cell proliferation (spermatocytogenesis / mitosis) → meiosis → differentiation (spermiogenesis). Never skip or reorder.
- Duration: full process takes approximately 35–60 days depending on species (~64 days in humans).
- Trap: Secondary spermatocytes are rarely seen in histology slides because they divide very quickly through Meiosis II — their window is brief.
- Trap: The acrosome forms during spermiogenesis (differentiation phase), not during meiosis. It derives from the Golgi apparatus.
- Recombination (crossing over) occurs during Prophase I → in primary spermatocytes, not spermatogonia or spermatids.
Oogenesis — 4 Phases in Order
Oogenesis Timing Traps
- Female germ cells begin meiosis as a fetus (triggered by retinoic acid prenatally).
- Maximum oocyte number is reached in the fetal stage (~7 million at 20 wks in humans), then declines by atresia throughout life — not at puberty.
- Oocytes can remain arrested in dictyate for up to 40–50 years in humans — increasing maternal age → chromosome segregation errors.
Polar Body Traps
| State of oocyte | Polar bodies present | Meiotic stage |
|---|---|---|
| Ovulated / in oviduct (unfertilized) | 1 polar body | Metaphase II arrest |
| After fertilization (sperm entry) | 2 polar bodies | Meiosis II completed; pronuclei forming |
Trap: If a question says "2 polar bodies," fertilization has already occurred. If it says "1 polar body," the oocyte is post-LH-surge but pre-fertilization.
IVF-Ready Oocyte — What to Look For
A mature, fertilizable oocyte recovered for IVF shows: expanded cumulus mass (hyaluronic acid after LH surge) + 1 polar body visible in perivitelline space. This confirms MetII arrest — the oocyte is ready for insemination.
Sperm Storage & Fertilization Location
- Sperm storage before ovulation = oviduct isthmus (Reservoir III). Not the vagina — vagina is where sperm are deposited but they quickly move onward.
- Fertilization site = ampulla of the oviduct (middle segment, closer to ovary end).
- Only <50 sperm typically reach the ampulla despite millions deposited.
Fertilization Mechanisms — sequence & traps
- Acrosome reaction releases enzymes (acrosin) and exposes inner acrosomal membrane → allows sperm to penetrate the zona pellucida. Must occur before ZP penetration.
- Ovastacin (cortical granule enzyme) specifically cleaves and inactivates ZP2. Exam trap: ovastacin = ZP2 cleavage. Other cortical granule enzymes may inactivate ZP3, but the named enzyme for the polyspermy block question is ovastacin → ZP2.
- Sperm-oocyte membrane fusion releases PLC-ζ (phospholipase C zeta) from sperm into egg cytosol → generates IP₃ → triggers repeated Ca²⁺ waves from ER stores.
- Ca²⁺ waves cause: (1) cortical granule exocytosis → polyspermy block, (2) completion of Meiosis II → 2nd polar body expelled, (3) metabolic activation of the egg.
- Pronuclei: male pronucleus (from sperm head) + female pronucleus (egg nucleus after MII) → interdigitate → chromosomes align on single spindle → syngamy = first mitosis of the zygote.
Reproductive Technology Traps
| Topic | Key fact / trap |
|---|---|
| Embryo donor superovulation | Induced by FSH treatment (not LH) to recruit multiple follicles. |
| Cryopreservation — fast freezing | Freezing too fast → intracellular ice crystals form because water cannot leave the cell fast enough. Ice crystals lyse membranes. |
| Dolly the sheep | Surprising because she was cloned from an adult somatic cell (mammary gland) — proving adult cell nuclei can be reprogrammed to totipotency. |
| Sperm sorting (sex selection) | Uses DNA content (X sperm have ~3.5% more DNA than Y sperm) via flow cytometry / fluorescence. Not swimming speed. |
| Immotile sperm / male infertility | Best treated with ICSI (intracytoplasmic sperm injection) — single sperm injected directly into oocyte, bypassing the need for motility or zona penetration. |
| Cattle embryo transfer site | Deposit embryo in the uterine horn ipsilateral to the corpus luteum (same side as the CL/ovulation), not in the uterine body. Progesterone from the CL locally prepares that horn for implantation. |
AI Semen Deposition Site
- Natural mating in cattle: semen deposited in vagina / cervix.
- AI in cattle: semen deposited in the uterine body. Exam trap: it is NOT placed in the vagina. The small AI volume (0.25–0.5 mL) requires placement closer to the oviducts.
- AI is considered the most important technology for genetic improvement in livestock.
Cryoprotectants — Penetrating vs. Non-Penetrating
| Type | Examples | Mechanism |
|---|---|---|
| Non-penetrating | Egg yolk protein, milk protein | Coat the plasma membrane — protect it from ice damage externally |
| Penetrating | DMSO, ethylene glycol, glycerol | Enter the cell — lower intracellular freezing point, minimize ice crystal formation |
Freeze Damage — Two Mechanisms & Speed Traps
- Ice crystals: break the plasma membrane. Caused by intracellular water freezing before it can exit the cell. Fast freezing → MORE ice crystals.
- Solution effects: pure water freezes first → remaining liquid becomes hypertonic → salts and cryoprotectants reach toxic concentrations. Slow freezing → MORE solution effects.
- Thawing: always thaw as quickly as possible (35°C water bath) — do NOT thaw slowly.
- Poor cryo-tolerance species: swine and birds. Pig semen is stored chilled, not frozen.
Sperm Sexing & MOET Key Facts
- Only proven sperm sexing method: flow cytometry using fluorescent DNA-binding dye. Not swimming speed. Y sperm have ~4% less DNA (Y chromosome is smaller than X).
- Sexed semen accuracy: ~90%; cost ~$15–25/straw.
- MOET superovulation: donor is given FSH at midcycle (~Day 9). Not LH — FSH recruits multiple follicles.
- PGF2α on Day 11: lyses the CL → drops progesterone → triggers estrus in the donor.
- Embryo transfer site: deposit embryo in uterine horn ipsilateral (same side) as recipient's CL. Exam trap: NOT the contralateral horn; the CL's progesterone locally primes the ipsilateral horn.
- Cycle synchronization: recipient must be in the same stage of the estrous cycle as the donor at the time of transfer.