PHAR303 Lecture 19 Reproductive

Testing phase for a reproductive toxicant: phase 1 fertility

• Production of gametes to implantation of the zygote.

• In vitro studies examine the effects of environmental toxins on:

  • IVF.

  • Gamete production and release.

  • Fertilization.

  • Zygote transport.

  • Zygote implantation.

phase 2 embryotoxicity, teratogenicity

• Implantation to birth, during which tissue development and epigenetic changes occur.

• Includes:

  • Implantation.

  • Embryogenesis.

  • Fetal development.

  • Parturition (birth).

pre and post natal toxicity

• Birth to puberty/adulthood

• Includes:

  • Lactation.

  • Growth and development.

  • Sexual maturation.

female reproductive system: oocyte numbers

• In early gestation, germ cells undergo mitotic division.

• Oocytes enter meiosis I and arrest in prophase I until puberty.

• All oocytes a woman will have are produced during gestation.

• Approximately 5 million oocytes are present during fetal development.

• Oocyte number decreases continuously from birth until menopause.

• Approximately 300,000 oocytes remain at puberty.

• If a woman does not become pregnant, a maximum of ~500 oocytes are ovulated during her lifetime.

• The majority of oocytes undergo apoptosis and are never ovulated.

female reproductive system:

• Oogonia are primordial follicles with 1 cell layer around them.

• They are arrested at meiosis I and remain dormant until puberty.

• At puberty, they develop into primary follicles, which have more than 1 layer of granulosa cells.

• As the number of granulosa cell layers increases, the follicle develops into secondary follicles.

• Under the presence of FSH and LH, a fluid-filled space forms in the middle of the follicle.

• This advanced secondary follicle is called an antral follicle.

• The antrum is where estrogens are produced in response to LH.

• Toxicants can accumulate in the antrum.

• The follicle continues to enlarge and becomes an ovulatory follicle.

• During the LH peak secretion, the ovulatory follicle releases the egg, which can then be fertilized.

• The remaining follicular tissue becomes the corpus luteum.

• The corpus luteum releases progesterone, which is essential for maintaining the initial phases of pregnancy.

• If the egg is not fertilized, hormone secretion from the corpus luteum stops within 14 days after ovulation, and it degenerates.

the menstrual cycle Hypothalamic Pituitary Gonadal Axis

HT releases gnrh that acts on ant put ant pit releases lh and fsh and prlactin which work on ovary to affect estra and progest prodcution lh stimulates theca cells in ovary to make andoregns then granulosa cells to convert andro to estra under fsh actrion prl maintains CL which makes progesterone

ovary releases e and p to affect vagina cervix uterus oviducy as well as feed back on HT and or ant pit

during the menstrual cycle

• FSH levels increase but remain relatively low, except for a mid-cycle peak.

• LH levels also increase and show a mid-cycle surge.

• Estradiol (estrogen) levels increase as the antrum becomes larger.

• The increase in estradiol during mid-cycle stimulates LH synthesis.

• When LH levels are high, the follicle ruptures and the egg is released.

• The mid-cycle LH surge triggers ovulation.

• The leftover follicular tissue becomes the corpus luteum (CL).

• The corpus luteum produces progesterone and some estradiol.

• If the egg is not fertilized, the corpus luteum degenerates.

• If the egg is fertilized, the corpus luteum continues producing hormones to help maintain pregnancy.

• Estrogen induces mitosis, causing proliferation (thickening) of the endometrium.

• Progesterone:

  • Suppresses estrogen receptors.

  • Slows down endometrial proliferation.

  • Decreases further endometrial thickening.

  • Prepares the endometrium for receiving and nourishing a fertilized egg.

  • This stage is called the secretory phase.

animal models of menstruation

rat cycle:

• Lasts 4–4 days.

• Is nearly identical to humans in terms of reproductive cycle patterns.

• Easy to monitor because of its short cycle length.

• Rats are specifically used in reproductive toxicity studies because they allow researchers to observe effects over many cycles.

• Multigenerational studies:

  • Used to examine effects of toxicants across multiple generations.

  • Helps determine impacts on fertility and reproductive development.

• Monkeys are most similar to humans when studying reproductive parameters, including:

  • Age at puberty

  • Cycle duration

  • Ovulation timing

  • Parturition (birth) timelines

  • Other reproductive characteristics.

potentially useful tests of female reproductive toxicity

• Weight: the first indicator of reproductive toxicity.

  • Changes in organ weight can indicate toxic effects.

• Reproductive toxicity can affect organs involved in fertility, including:

  • Ovary

  • Hypothalamus (HT)

  • Pituitary gland

  • Endocrine hormones

  • Oviduct

  • Uterus

  • Cervix

  • Vagina

• Reproductive toxicity can impact:

  • Fertility

  • Hormonal regulation

  • IVF (in vitro fertilization) outcomes

Organ endpoints

• Reproductive toxicity indicators in the ovary:

  • Organ weight

  • Ovary histology (appearance and structure of ovarian tissue)

  • Appearance of follicles

  • Number of oocytes

• Toxicants can affect:

  • Progression from one reproductive phase to another

  • Rate of follicular death

  • Follicular steroidogenesis (hormone production by follicles)

  • Follicular maturation

  • Oocyte maturation

  • Ovulation rate

  • Luteal function (corpus luteum function)

• Effects on reproduction outcomes:

  • Pre-implantation loss

  • Post-implantation loss

  • Progeny development

chemicals linked to adverse reproductive outcomes in females

• Heavy metals, cigarettes, and vapes can act as reproductive toxicants and negatively affect ovarian function.

• Anticancer drugs (chemotherapy):

  • Target cell division because cancer cells rapidly proliferate.

  • Although germ cells (oocytes) are not actively dividing, the supporting cells around the oocyte (granulosa cells) are dividing.

  • Damage to granulosa cells can lead to follicle loss and reduced fertility.

• Targeted chemotherapy:

  • Designed to target specific receptors or cell types rather than general cell proliferation.

  • May have less impact on fertility because it does not primarily affect cell division.

• Oncofertility:

  • Aims to preserve fertility before chemotherapy.

  • Can involve preserving ovarian tissue so follicles may develop later.

  • Limitations:

    • Expensive

    • Difficult

    • Low success rate

male reproductive system - spermatogenesis sperm quality

• ~1000 sperm are produced every heartbeat.

• Male reproductive toxicity is easier to assess because:

  • Sperm are continuously produced.

  • Changes in sperm production can be measured more easily.

• The hypothalamus (HT)–pituitary–testis axis controls spermatogenesis.

• The testis:

  • Produces gametes (sperm).

  • Produces steroid hormones (mainly testosterone).

• Newly produced sperm are not fully functional when they leave the testis.

• Sperm gain maturation and motility as they pass through the epididymis.

• During ejaculation:

  • The epididymis contracts to move sperm forward.

  • Sperm travel through the vas deferens.

  • Secretions from the seminal vesicles and prostate are added.

  • These fluids make up about 90% of semen volume.

male reproductive system - spermatogenesis sperm quality: hypothalamus (HT)–pituitary–testis axis

Feedback mechanism is similar to females.

• LH acts on Leydig cells to produce:

  • Testosterone (T)

  • Estrogen (E)

• FSH acts on Sertoli cells to produce:

  • Inhibin

  • Support for spermatogenesis

• Feedback regulation:

  • Testosterone (T) feeds back on the hypothalamus and anterior pituitary to regulate hormone production.

  • Inhibin feeds back on the anterior pituitary to regulate FSH release.

  • Inhibin cannot feedback on HT, cross the blood-brain barrier (BBB).

• Testosterone and inhibin are important for:

  • Spermatogenesis

  • Sex organ growth

  • Masculinization

inhibin

nhibin is a hormone produced by Sertoli cells in the testes that helps regulate sperm production.

• FSH stimulates Sertoli cells.

• Sertoli cells produce inhibin.

• Inhibin feeds back on the anterior pituitary to decrease FSH release.

• This prevents too much sperm production stimulation and helps maintain normal spermatogenesis.

Simple way to remember:

FSH → Sertoli cells → Inhibin → ↓ FSHi

sperm production and transport

• Sperm are produced in the seminiferous tubules of the testis.

• Between the seminiferous tubules is the interstitial space.

• The interstitial space contains Leydig cells, which produce steroid hormones.

• Leydig cells:

  • Produce testosterone (T).

  • Testosterone is the major male sex hormone.

  • Testosterone can be converted into other hormones.

  • The most active metabolite is dihydrotestosterone (DHT).

• Testosterone production:

  • Testosterone is made from cholesterol.

  • Several enzymes convert cholesterol → testosterone.

  • Toxicants can affect these enzymes, disrupting testosterone production.

• Transport of testosterone:

  • Microvessels carry away the lipophilic steroids produced by Leydig cells.

  • Because testosterone is lipophilic, it binds to carrier proteins:

    • Androgen-binding protein (ABP):

      • Produced by Sertoli cells.

      • Maintains a high concentration of testosterone near developing sperm.

    • Albumin:

      • Transports testosterone in the blood.

    • Sex hormone-binding globulin (SHBG):

      • Binds testosterone with high affinity and transports it in circulation.

duration of spermatogenesisw in different specues

• Humans:

  • It takes about 64 days to produce sperm (spermatogenesis).

  • It takes an additional ~10 days for sperm to pass through the epididymis and mature.

• If exposed to a toxicant:

  • Effects on sperm may not be seen immediately.

  • Changes may appear after 2–3 months because sperm production takes time.

• If exposure is reduced or stopped:

  • Recovery from toxic effects may also take 2–3 months.

  • This allows time for new, unaffected sperm to be produced.

• Toxicants can affect different stages of sperm development.

blood testis barrier

-The blood-testis barrier (BTB) is made of projections from Sertoli cells that form tight junctions.

• The BTB creates 2 compartments:

  • Basal compartment:

    • Contains spermatogonia only.

  • Adluminal compartment:

    • Contains spermatocytes and more advanced developing sperm cells.

• The blood-testis barrier creates an immune-privileged compartment:

  • Limits access of immune cells to the adluminal compartment.

  • Prevents immune cells from encountering immunogenic proteins found on developing sperm.

  • Prevents immune attacks against sperm cells.

• Sertoli cells also provide immune protection:

  • Secrete molecules that suppress immune responses.

  • Help maintain the immune-privileged environment.

• Lipophilic toxicants may still cross the blood-testis barrier and affect sperm development.

potential target sites in male reproductive toxicants

• Dopamine agonists can affect the central nervous system (CNS).

• Estrogens can affect the pituitary gland and hormone regulation.

• Antibodies can affect fertilization:

  • If the blood-testis barrier is broken, the immune system may be exposed to sperm proteins.

  • This can lead to antibody production against sperm.

  • Antibodies may interfere with sperm function and fertilization.

• Chemicals can affect:

  • Fertilization

  • Events after fertilization (early embryo development and pregnancy)

• Example: Cyclophosphamide

  • A toxicant that can affect reproduction after fertilization by damaging developing embryos/fetal development.

germ cell susceptibility

X-rays:

• Can impact mitotic and meiotic cell division.

• May damage dividing germ cells and affect sperm production.

• Cyclophosphamide:

  • Can affect early stages of spermatogenesis.

  • Damages developing sperm cells.

• Chlorambucil:

  • Affects sperm maturation.

  • Can interfere with chromatin compaction (tight packing of DNA in sperm).

• Acrylamide:

  • Can affect completed/mature sperm.

  • Its metabolites can cross the blood-testis barrier.

• Many drugs are gonadotoxic to men, especially:

  • Anticancer drugs/chemotherapy

  • Can damage sperm production and fertility.

excess heavy metals on male reproduction

• Heavy metals can affect:

  • Germ cells

  • Sertoli cells

  • Leydig cells

• Effects can occur:

  • Directly by damaging reproductive cells.

  • Indirectly by disrupting the reproductive environment.

• Lead:

  • Causes endocrine and paracrine toxicity.

  • Can disrupt hormone signaling and communication between cells.

• Cadmium:

  • Causes blood vessel constriction.

  • Leads to hypoxia (reduced oxygen supply), damaging reproductive tissues.

• Zinc toxicity:

  • Can occur due to zinc deficiency.

  • Disrupts normal reproductive function.

• Cobalt toxicity:

  • Causes effects through general hypoxia (reduced oxygen availability).

• Platinum:

  • Inhibits DNA synthesis.

  • Can affect rapidly dividing cells involved in reproduction.

sperm maturation through epididymis

• Epididymis functions:

  • Absorption

  • Secretion

  • Transport

  • Sperm maturation

  • Sperm storage

• The epididymis is less studied because it cannot be biopsied easily.

• Damage to the epididymis can cause:

  • Loss of fertility within ~1 week.

• Timing of toxicant effects on sperm depends on the stage affected:

  • Epididymis effects → seen within 1 week.

  • Late stages of spermatogenesis → effects seen after 2–3 weeks.

  • Meiosis → effects seen after 6–8 weeks.

  • Mitosis (early sperm production) → takes the longest to appear because new sperm must complete development.

semen analysis

• Semen analysis parameters used to assess sperm quality:

  • Volume → amount of semen produced.

  • Total sperm number → total number of sperm in the sample.

  • Sperm concentration → number of sperm per volume of semen.

  • Total motility → percentage of sperm that are moving.

  • Progressive motility → how well sperm move forward.

  • Sperm survival → percentage of living sperm.

  • Sperm morphology → sperm shape and structure.

• Poor sperm quality in men is thought to be related to temperature:

  • Sperm production requires the testes to be ~3–4°C cooler than body temperature.

  • Increased temperature can impair spermatogenesis.

• Abnormal sperm morphology:

  • Micro-sperm → abnormally small sperm.

  • Mega-sperm → abnormally large sperm.

  • Irregular head

  • Double head

  • Double tail

  • Curled tail around the body

• Many different sperm morphologies can be seen in normal semen; not all abnormal-looking sperm indicate infertility.

computer aided semen analysis

• Sperm motility assessment:

  • Sperm cells are placed in a chamber.

  • The chamber is placed under a microscope with a camera.

  • A computer analyzes sperm movement.

• The system measures:

  • How well sperm move

  • Duration of movement

  • Movement efficiency

  • Progressive motility (ability to move forward)

• Motility testing is:

  • More sensitive than only measuring the number of sperm produced.

  • A central tool for studying sperm quality.

• Many toxicants can affect sperm swimming ability, even if sperm numbers are not greatly reduced.

sperm chromatin

• Sperm chromatin is ~6× more densely packed than chromatin in somatic cells.

• In sperm:

  • Histones are removed and replaced by protamines.

  • Protamines allow DNA to be packed much more tightly.

• Protamines:

  • Are rich in sulfide groups.

  • Form disulfide linkages.

  • These linkages create tight DNA compaction.

• The high level of DNA compaction makes sperm DNA less accessible and less likely to be affected by some toxins.

sperm quality / chromatin biomarkers

• Chromatin analysis can be used to assess sperm quality.

• Many toxicants do not inhibit sperm production, so:

  • Sperm number may appear normal.

  • However, sperm DNA quality may still be affected.

• By examining sperm chromatin, researchers can assess:

  • DNA breaks

  • DNA crosslinks

  • Integrity of chromatin structure

  • Epigenome changes

• Chromatin analysis can detect damage that may not be seen by only measuring sperm count.

assays for sperm quality

• Comet assay:

  • Measures DNA damage in sperm.

  • The more DNA that moves away from the nucleus, the more DNA breaks are present.

  • More DNA movement = a larger “comet tail” = more damage.

• TUNEL assay:

  • Detects DNA breaks.

  • Measures the number of 3′ free hydroxyl (-OH) ends created when DNA is broken.

  • More 3′ ends = more DNA fragmentation.

• FISH (Fluorescence In Situ Hybridization):

  • Identifies chromosomal abnormalities.

  • Uses fluorescent probes viewed under a fluorescence microscope.

  • Can detect abnormal chromosome numbers or structures.

• DNA methylation analysis:

  • Used to identify epigenetic changes in sperm chromatin.

  • Uses methylation-sensitive restriction enzymes to detect changes in DNA methylation patterns.

• Epigenetic marks in sperm vs somatic cells:

  • Sperm and somatic cells have completely different epigenetic patterns.

  • Changes to sperm epigenome can affect sperm quality and potentially influence development after fertilization.

potentially useful tests of male reproductive toxicity

• Male reproductive toxicity can be assessed by examining:

  • Testis

  • Epididymis

  • Accessory glands

  • Semen

  • Endocrine function

  • Sperm motility

  • Fertility

  • Chromatin integrity

  • In vitro fertilization outcomes

• Testis measurements used to assess toxicity:

  • Testis size

  • Testis weight

  • Most of the testis weight comes from sperm-producing cells.

  • Changes in testis weight can indicate defects in spermatogenesis.

• Effects on testis weight:

  • Blocking meiosis can reduce testis weight.

  • Blocking LH → ↓ testosterone production → no/low spermatogenesis.

  • Can cause up to 90% reduction in testis weight.

• Histological analysis of the testis can measure:

  • Seminiferous tubule diameter

  • Percentage of:

    • Non-functional tubules

    • Tubules containing sperm in the lumen

  • Number and size of Leydig cells

    • Indicates effects on testosterone production

• Histology reveals the specific cellular effects of toxicants.

• Example: Two chemicals can both affect sperm production but cause different changes:

  • Methoxyacetic acid:

    • Causes spermatogonia to become disorganized.

  • m-Dinitrobenzene:

    • Cells are not shed properly into the lumen.

testicular dysgeneis syndrome