Weily Lang, Dr. Preethi RadhakrishnanDepartment of Natural SciencesLaGuardia Community College

Antioxidants Boost Male Fertility: The role of Reactive Oxygen Species (ROS) in modulating

sperm viability & fertility

Relationship between Reactive Oxygen Species and the male

reproductive system The body produces large amounts of Reactive Oxygen species

(ROS) when subjected to:

• Environmental Stressors (Smoking, Drugs, Pollution)• Systemic Pathologies (Cancer, Diabetes, Systemic infections)

This oxidative stress can be potentially harmful to male reproductive tissue, especially sperm

Sperm are very prone to lipid peroxidation under high stress (Agarwal & Esteves, 2011)

Agarwal & Esteves, 2011

Relationship between Reactive Oxygen Species and the male

reproductive system

Reactive Oxygen Species (ROS)What are ROS

- free radicals produced as byproducts of REDOX reactions

- produced during cellular stress and immune insult (Bonilla 2006)

How is ROS produced?

ATP (energy) production in the mitochondria

results in surplus of oxygen molecules

some O2 utilized, some converted into ROS radicals by mitochondrial

enzyme, Complex IV

Antioxidants

What are Antioxidants?

• Molecules which inhibit oxidation

• Scavenges free radicals

• Capable of reducing Reactive Oxygen Species (ROS)

• Examples are:

• Melatonin

• Lipoic Acid

Antioxidants used in our experiments:

Melatonin- is a lipid soluble hormone that easily

accesses cell membrane - reduces DNA destruction by

quenching free radicals (Bonilla, E. 2006).

Lipoic acid - are sulfur-containing

compounds- quench ROS by donating

electrons to ROS (Bonilla, E. 2006).

Bonilla et al., 2002 & 2006, found that dietary supplementation of Melatonin and Lipoic acid caused significant lengthening of life-span.

Interaction between Paraquat and ROS

What is the association between paraquat (PQ) and ROS?

- Organisms respond to PQ by producing a type of free radical called superoxide anions that are then converted into large quantities of ROS (Bonilla 2006).

- Therefore, we used PQ in our experiments at known doses (10mM) to artificially induce the production of ROS (Bonilla 2006).

What is paraquat (PQ)?

- Paraquat (PQ) is a nitrogen-containing herbicide - It is highly toxic to living organisms leading to poisoning and death.

FertilityWhat is fertility and how is it important?

- Fertility is a measure of number of offspring produced by an individual.

- The more offspring that are conceived the higher the chance of passing on genes to the next generation

Sexual Selection:

Pre-Copulatory sexual selection:

- Copula duration: The duration of intercourse where sperm is injected in female.

- Sperm Viability: The measure of alive sperm capable of fertilization.

- Post-Copulatory sexual selection:

- No. of offspring producedFertility is measured by the number of offspring hatched

Relationship between ROS & Fertility

ROS may directly impact fertility:

During an infection (systemic, STD)

Environmental stressors (smoking, radiation)

Pesticides (Paraquat)

Drosophila spermROS’s effect on Sperm (Lipid Peroxidation):

● Sperm membranes are high in polyunsaturated fats - making their membranes prone to ROS attacks via lipid peroxidation.

● Sperm also contain many mitochondria, which are a prime source of free radical production, again making sperm likely targets of excess ROS.

Relationship between ROS & Fertility

Agarwal & Esteves, 2011

How did we assay live/dead sperm in males?

Sperm Viability Kit (L-7011, Molecular Probes)

• Seminal Vesicles were dissected and sperm counted• SYBR 14 – is a membrane-permeable nucleic acid stain (emission max.

516 nm) • PROPIDIUM IODIDE - a dead-cell stain (emission max. 617 nm). Both

solutions stain sperm heads only• Ninety-six percent of the cells seen in light microscopy were found in the

fluorescence image.• Overall, 5% (678 out of 13 654 sperm cells) of the cells were doubly-stained

(green in centre, red at ends), and were included as dead cells. • We counted live and dead sperm were counted manually, in each frame of

the slide • Slides were counted blind of treatment

Drosophila Sperm – Sperm Viability Assay

Live sperm Dead sperm

So how can antioxidants help? Feeding Antioxidants:

Antioxidants quench and react with ROS to reduce the oxidation effect on the cell membrane and DNA. This protects sperm from damage.

Antioxidant enhanced diets, Melatonin (M), Lipoic Acid (L):

Data from these groups showed a significantly higher percentage of viable sperm and higher progeny.

Control (EtOH):

Fewer viable sperm & fewer progeny than from antioxidant-fed males.

Methodology Collect virgins

Starve males for 24 hours / separate females into different

vials

Feed males antioxidant-enhanced and control (Ethanol) diets

Paraquat assault (24 hours on filter paper)

Sperm viability assay (Propidium Iodide & SYBR Green)

Mating (males from treatments paired with females) –

observations made

Copula duration (start of mating and end of mating)

Fertility (count progeny) – Flips 1, 2 and 3.

COLLECTED VIRGINS

STARVED MALES FOR 24 HOURS ISOLATED FEMALES

MELATONIN(Antioxidant)

LIPOIC ACID(Antioxidant)

ETHANOL(Control)

PARAQUAT ASSAULT (induce ROS)MATING

& Copula Duration

SPERM VIABILITY FERTILITY (Progeny Counts)

FEED DIETS WITH TREATMENTS

Methodology

Predictions Antioxidant-enhanced feeding treatments of melatonin

and lipoic acid would positively affect male fertility.

There will be a significant decrease in the male fertility

within the control group (Ethanol Fed) due to the negative

effects of paraquat on fertility.

If we find a positive correlation between antioxidants and

fertility, we plan to focus directly on the effects of these

two antioxidants on sperm viability in the future.

Results

Results: Mating Probability

Per

cent

age

mat

ing

%

Results: Sperm Viability

Pro

port

ion

of L

ive

Spe

rm

Results: Fertility

No.

of

offs

prin

g

Conclusion

Mating probability and Copula Duration

• We found significant differences in treatments in the number of flies that mated (p = 0.002)

• We found that antioxidant fed flies had higher mating percentages than ethanol fed controls

• However, we found no significant differences in copula duration between treatments (p = 0.567)

Conclusion

Sperm viability

• Males fed Melatonin and Lipoic acid had a significantly higher proportion of live sperm (74% and 56%) than the Ethanol treatment (32%) (p = 0.0020)

• This indicated that both antioxidants Melatonin and Lipoic Acid, might protect sperm from the lipid peroxidation activity of Paraquat

Conclusion

Fertility

• Males fed Melatonin and Lipoic acid sired significantly more offspring (mean = 187 and 148) than the Ethanol fed controls (mean = 82) (p = 0.0325)

• This shows that antioxidant-fed males, passed more viable sperm to females than the Ethanol treated flies, thereby siring more offspring.

References1. Bonilla, E., et al., 2006. Paraquat –induced Oxidative stress in D. melanogaster:

Effects of Melatonin, Glutathione, Serotonin, Minocycline, Lipoic Acid and Ascorbic

Acid. Neurochem Res, 31:1425-1432

2. Bonilla, E, et al., 2002. Extension of life-span and stress resistance of D.melanogaster

by long-term supplementation with melatonin. Experimental Gerontology, 37:629-

638.

3. Dowling, D, Simmons, L.W. 2009. Reactive oxygen species as universal constraints in

life-history evolution. Proc. R. Soc. B. 276: 1737-1745.

4. Aitken, J., Roman, D, S., Antioxidant Systems and Oxidative stress in the testes. 2008.

Molecular mechanisims in Spermatogenesis.

5. Radhakrishnan, P., Fedorka, K.MF. 2011. Influence of female age, sperm senescence

and multiple mating on sperm viability in female D. melanogaster. 57: 778-83

6. Radhakrishnan, P., Taylor, P.W. 2007. Seminal fluids mediate sexual inhibition and

short copula duration in mated female Quuensland fruit flies. 53: 741-745.

Acknowledgements

Jon Rodriguez

Dr. Hendrick Delcham

NIH-Bridges R25 PAR-11-285

Dr. Thomas Onorato

Cheryl Pinzone, University of Georgia