Male reproductive biology, spermatogenesis, and endocrinology

Journey of sperm

Seminiferous tubules > lumen > rete testis > ductuli efferentes > epididymis > vas deferens . penis

Why sperm leaving testis cannot fertilise

They lack motility and fertilisation capacity until epididymal maturation

Two compartments of testis and functions

Seminiferous tubules perform spermatogenesis
Interstitium performs steroidogenesis

Structure function of seminiferous tubules

Highly organised epithelium supports germ cell development from basal to lumen

Components of interstitium

Leydig cells - produce testosterone

Capillaries - carry released testosterone to circulation

Fibroblasts: produce ECM of interstitium and testis

Macrophages: activated in inflammation

Lymphatics: lymph drainage

Peritubular myoid cells: surround seminiferous tubules and are contractile

Mechanism of testosterone synthesis

LH binds Leydig receptors: stimulates conversion of cholesterol into testosterone and testosterone > 5-alphaDHT by 5-alpha reductase

Why DHT is important

DHT has higher androgen receptor affinity producing stronger effects

Experimental evidence for Leydig function

3beta HSD staining localises steroidogenesis to Leydig cells

EDS destroys Leydig cells leading to reduced testosterone and degeneration of spermatogenesis - Stem Leydig cells repopulate interstitium after EDS treatment

Structure of peritubular myoid cells

Smooth muscle like cells surrounding seminiferous tubules

Contract to move sperm toward the rete testis

Structure of epididymis

Highly coiled tubule with tall columnar epithelium and stereocilia

Highly coiled to provide long tubule length for sperm maturation within the space

Concentrates sperm and induces motility and fertilisation competence

Acidic to maintain sperm in inactive state for maturation

Structure function of stereocilia

Increase surface area for absorption of fluid concentrating sperm

C ros knockout experiment

Loss of C ros causes alkaline epididymal pH tail defects and infertility

the acidic environment is essential for sperm maturation

Effect of androgen receptor knockout in epididymis

Epithelial involution loss of stereocilia and impaired function

• androgens are therefore essential for epididymal maintenance

Changes to sperm in epididymis

Membrane protein lipid modification and acquisition of motility

Structure of vas deferens

Low cuboidal epithelium and thick smooth muscle tube which contracts to move sperm along narrow lumen and strong peristaltic contractions propel them during ejaculation

Stores sperm

Vasectomy mechanism

Vas deferens is cut preventing sperm entry into semen sperm are phagocytosed

Contraline ADAM mechanism

Injectable hydrogel forms physical barrier in vas deferens and can be reversed

undergoing clinical trials as a male contraceptive

Composition of semen

Sperm: 2-5%

seminal vesicles secretions: 60-75 %

prostate secretions: 25-30 %

Function of seminal vesicles

Provide fructose for sperm energy and semenogelin proteins for coagulation after ejaculation

Structure function of seminal vesicles

Secretory glands adapted to produce large volume nutrient rich fluid

dependent on androgens - as castration reduces size, while testosterone restores structure

Prostate gland function

Produces alkaline fluid with zinc and PSA

prostatic fluid is alkaline to neutralise the female reproductive tract to improve sperm survival

Function of PSA

Protease that cleaves semenogelin to liquefy semen

used as a diagnostic tool as PSA is elevated in prostate disease like BPH and cancer

Zinc mechanism in semen

Initially inhibits PSA allowing coagulation then binds semenogelin freeing PSA for liquefaction

present in semen

Importance of coagulation then liquefaction

Coagulation retains semen then liquefaction allows sperm motility

BPH mechanism

Enlargement of prostate gland due to cell proliferation

Age related

non-cancerous

Structure of penis

Contains corpus cavernosum with vascular spaces and smooth muscle

delivers sperm to reproductive path

Mechanism of erection

Anatomical: blood entering the corpus cavernosum due to smooth muscle relaxation

Sexual stimulation activates the cavernous nerve > releases Nitric oxide > stimulates guanylate cyclase to convert GT > cGMP > protein kinase decreases intracellular calcium causing smooth muscle relaxation and blood filling to the corpus cavernosum

Viagra mechanism

Inhibits PDE5, which prevents cGMP breakdown prolonging erection

Semen coagulation and liquefaction

Zinc in prostatic fluid inhibits PSA, preventing cleavage of semenogelin - this allows semenogelin to cross link and causes semen coagulation

Later, zinc binds to semenogelin instead, freeing PSA to cleabe semenogelin for liquefaction

Overall male reproduction

The seminiferous tubules produce haploid germ cells, and the interstiitum produces tesosterone for male reproduction. PTM cells move sperm out of testes by peristalsis. In the epididymis, sperm are concentrated and undergo maturation for motility and fertilisation. The vas deferens stores and transports sperm. while prostate and seminal vesicles add most of seminal fluid for alkalinity energy and coagulation of sperm for reproduction. the penis then delivers sperm to the female.

Purpose of spermatogenesis

Produce haploid and genetically diverse, motile sperm

occurs in the seminiferous epithelium from basal surface to luminal surface

Cell sequence

Spermatogonia > spermatocytes > spermatids to>spermatozoa

Phase 1 proliferation

Spermatogonia undergo mitosis at basement membrane

PCNA staining in mice marks these cells here

Stem cell niche definition

Local environment providing signals regulating self renewal and differentiation

Key factors are GDNF, FGF2, CXCL12

Key niche factors

GDNF FGF2 CXCL12

GDNF mechanism

Binds receptors on spermatogonia promoting self renewal

KO - depletion of spermatagonial stem cells and infertility

Role of retinoic acid

Triggers differentiation and entry into meiosis

occurs at puberty in males - initiates spermatogenesis

Reduced RA leads to impaired spermatogonial differentiation

thus RA is essential for differentiation

RA mechanism

genomioc RA receptor: acts as transcription factor inducing Stra8 expression

Non-genomia RA receptor: prmotes translation of KIT from existing mRNA

Phase 2: meiosis

Reduces chromosome number and generates genetic diversity

produces haploid cells from diploids

Germ cells > spermatocytes

what is diploid vs haploid

diploid: 2 homologous chromosomes in each homologous pair

Meiosis I mechanism

Homologous chromosome pairs (mother and father) separate, crossing over to exchange genetic information, and independent assortment occur

chromosome number is reduced from diploid (2n) to haploid (n)

Meiosis II mechanism

Sister chromatids separate and convert secondary spermatocytes to round spermatids - maintaining haploid chromosome

Cell progression through meiosis

Primary spermatocyte > secondary spermatocyte > spermatid

Blood testis barrier structure

Formed by Sertoli cell tight junctions that separate the basal from the apical side of the seminiferous epithelium

protects haploid germ cells from the immune system

Why barrier must open

to allow germ cells to move across the epithelium and spermatocytes to move to adluminal compartment

the tight junctions dynamically reorganise during germ cell development

Role of androgens in meiosis

Required for progression beyond meiosis into spermiogenesis

as loss of testosterone prevents formation of post meiotic cells

Phase 3: spermiogenesis

Transformation of spermatids into spermatozoa

produces structural changes needed to ensure sperm is fast, motile, and able to fertilise

phases: golgi phase > cap > acrosomal > maturation phase

Golgi phase

Round cell becomes polar - the golgi moves to the head end and centrioles move to the tail end

Cap phase

Acrosome vesicle forms a cap over nucleus and primitive flagellum develops

Acrosomal phase

Acrosome spreads around he nucleus as nucleus elongates

Maturation phase

Mitochondria arrange in midpiece and excess cytoplasm removed - the cell becomes a mature spermatazoon

DNA packaging mechanism

Histones replaced by specialised histones then by transition proteins then protamines for greater chromatin condensation

creates compact hydrodynamic sperm head

Transcription in spermiogenesis

Stops in early spermatid nucleus so proteins must be pre synthesised

Loss of BrUTP incorporation shows transcription stops in early spermatids

Phase 4: spermiation

Release of sperm from Sertoli cells into seminiferous lumen

Sertoli germ cell junctions remodel allowing release

the residual body is phagocytosed by sertoli cells

Organisation of spermatogenesis

Occurs in waves along seminiferous tubules

controlled by Pulses of retinoic acid coordinate timing

Human arrangement

Helical distribution of stages

Human spermatogenic cycle

Approximately 16 days per cycle

so total duration of spermatogenesis is 64 days

Integrated function of system

Testis produces sperm, epididymis matures, vas deferens transports, glands support, penis delivers

What is the male HPG axis?

GnRH > LH and FSH > act on testes (Leydig + Sertoli cells) to regulate testosterone production and spermatogenesis.

What is the mechanism of GnRH action?

GnRH is released in a pulsatile manner → binds GnRH receptors on pituitary gonadotrophs → stimulates LH and FSH synthesis and secretion.

What are LH and FSH?

Gonadotrophins from anterior pituitary: LH acts on Leydig cells (testosterone synthesis); FSH acts on Sertoli cells (spermatogenesis support).

Nehative feedback: testosterone inhibits GnRH and LH secretion,

inhibin B from sertoli cells selectively inhibits FSH secretion

How does negative feedback regulate the HPG axis?

Testosterone inhibits GnRH and LH secretion; Inhibin B from Sertoli cells selectively inhibits FSH secretion at pituitary level.

What experimental evidence shows testosterone negative feedback?

Castration studies: removal of testes → ↑ LH and FSH → demonstrates loss of testosterone-mediated inhibition.

Which neurons mediate steroid negative feedback?

Kisspeptin neurons in hypothalamus (especially arcuate nucleus) regulate GnRH pulse frequency.

Kisspeptin binds GPR54 receptors on GnRH neurons → modulates (reduces) GnRH pulse frequency → alters LH/FSH output.

What evidence supports role of kisspeptin neurons?

They express androgen/oestrogen receptors and mediate feedback rather than direct action on GnRH neurons.

What does LH do in the testis?

LH binds LHCGR on Leydig cells → activates cAMP pathway → stimulates testosterone synthesis.

LHCGR expression is restricted to Leydig cells (receptor localisation studies).

What happens if LH is absent?

Normal male external genitalia but delayed puberty and low testosterone → shows role in postnatal development.

What happens in LHCGR mutations?

Female external phenotype

Why can fetal development occur without LH?

hCG from placenta can activate LHCGR → substitutes for LH during fetal life.

Describe LH signalling mechanism in Leydig cells.

LH → LHCGR (GPCR) which activates adenyl cyclase to convert ATP → cAMP → PKA activation →

pKA activates StAR activation and transcription of steroidogenic enzymes which = cholesterol → pregnenolone → testosterone.

What is the role of StAR?

Transfers cholesterol from outer to inner mitochondrial membrane → rate-limiting step of steroidogenesis.

What experimental evidence supports steroidogenesis pathway?

Activation of enzymes like 3β-HSD observed in Leydig cells during testosterone synthesis.

What are Δ5 vs Δ4 pathways?

Δ5 pathway predominant in humans; Δ4 pathway predominant in rodents for testosterone synthesis.

Why is testosterone converted to DHT?

Peripheral tissues have lower testosterone → conversion to more potent androgen DHT ensures effective masculinisation.

Which enzyme converts testosterone to DHT?

5α-reductase found in skin

What is the function of DHT?

Drives external genital masculinisation and secondary sex characteristics (e.g. pubic hair, aggressiveness etc)

What is the androgen receptor mechanism?

Ligand (testosterone/DHT) binds → receptor conformational change → acts as transcription factor → regulates gene expression.

What are structural domains of androgen receptor?

DNA-binding domain + ligand-binding domain.

What is CAIS?

Complete androgen insensitivity syndrome: AR mutation → XY genotype but female phenotype.

Why are Wolffian structures absent in CAIS?

Testosterone present but no functional receptor → no maintenance of epididymis

Do germ cells express androgen receptor?

No → androgen effects on spermatogenesis are indirect.

Androgens act via Sertoli and other somatic cells

Mechanism of PTM androgen action?

Stimulates GDNF production → supports spermatogonial mitosis.

What happens in LCARKO mice?

Leydig cells undergo apoptosis in adulthood but fertility maintained.

KO mice in male androgen receptors

ARKO mice: no spermatogenesis beyond spermatogonia and failed Wolffian development. - supports androgen receptor

Sertoli-cell AR knockout → meiosis arrest + no haploid sperm + blood-testis barrier disruption - supports that androgen signalling in sertoli cells is essential for meiosis and barrier integrity

PTM-ARKO mice: reduced sperm production at all stages

LCARKO mice: leydig cells undergo apoptosis in adulthood, but fertility maintained - indicates that androgens are important for leydig cell survival but no for fertility

What are the phases of testosterone production?

Embryonic surge > neonatal surge > puberty surge > post-pubertal surge

Role of embryonic testosterone surge?

Development of Wolffian ducts + testicular descent.

What is neonatal testosterone surge?

Transient rise after birth; function unclear but may influence brain development.

What happens post-puberty?

Continuous testosterone production → spermatogenesis + secondary sexual characteristics.

Why is intratesticular testosterone important?

Levels are much higher than circulating → required for spermatogenesis.

What happens with exogenous testosterone (contraception)?

Suppresses LH → reduces intratesticular testosterone → inhibits spermatogenesis despite normal systemic androgen effects.

What does FSH do?

Stimulates Sertoli cells → supports spermatogenesis and Sertoli function.

peptide protein, so must use membrane receptors - stimulates mitosis in sertoli cells

What is ABP?

Androgen-binding protein → concentrates testosterone in seminiferous tubules.

What is inhibin B?

Hormone from Sertoli cells that selectively inhibits FSH secretion.

Heterodimer: alpha subunti (sertoli) + Beta subunit (germ cells)

What evidence suggests alternative FSH signalling?

FSH receptor KO mice still produce some sperm

What is AMH?

Anti-Müllerian hormone from Sertoli cells during embryogenesis.

regresses mullerian duct in females, keeps wollfian in males

What is 5α-reductase type II deficiency?

Mutation in Srd5a2 → impaired DHT production.

XY individuals appear female at birth bc of lack of external masculinisation

at puberty: partial masculinisation due to type I enzyme compensation

How does inhibin B regulate spermatogenesis?

Normal spermatogenesis → ↑ inhibin B → ↓ FSH → maintains homeostasis.

Overall integration of HPG axis?

GnRH → LH/FSH → Leydig (testosterone) + Sertoli (spermatogenesis + inhibin B); testosterone and inhibin B provide negative feedback to maintain balance.