Etiologies of Sperm Chromatin/DNA Damage Dr Ali Reza Talebi Ph.D of anatomical sciences Andrology lab

Topics 1) Unique structure of sperm chromatin 2) Mechanisms of sperm DNA damage 3) Factors affecting sperm DNA integrity

I. Unique structure of sperm chromatin Testicular phase Sperm specific histones are replaced at first by TPs and then by protamines. This complex is coiled into concentric circles “Doughnuts” and each of them attached to the sperm nuclear matrix at the Matrix Attachment Region (MAR) (Fuentes 2000, Chaitip 2001, Brewer 2002 ). Epididymal phase During epididymal transit, the thiol groups (-SH) of protamines are oxidized to create intra and inter molecular disulfide bonds, nessesary for mechanical and chemical stability of sperm DNA (Kosower 92, Golan 96 ). Ejaculation phase The chromatin stability completes by addition of seminal vesicle and prostatic zinc to the seminal fluid. One zinc per each protamine molecule (Björndahl, 2011).

Ejaculation phase: addition of zinc to the protamines

CHROMATIN / DNA ABNORMALITIES  Excessive histone  Absence or deficiency of protamines (P1, P2 )  Chromatin instability due to reduction in disulfide bonds formation  Hypostabilized chromatin due to the zinc deficiency  DNA fragmentation  DNA denaturation  Nuclear matrix anomalies

II. Mechanisms of sperm chromatin/DNA abnormalities 1 - Impairement in DNA nicking / ligation by Topoisomerase II ) Oosterhuis,2000 ) 2 - Programmed cell death ) Barroso,2000 ) 3- Genetic lesions (Vogt, 2004) 4- Oxidative stress ) Sharma 96,Agarwal 2002,Wang 2003 )

III. Etiological factors Multiple pathological factors acting at both intra- testicular and post-testicular levels may contribute to sperm DNA damage. These are categorized into intrinsic and extrinsic factors. Extrinsic factors can be classified into physical chemical biological behavioral socioeconomic.

Main etiologies of sperm DNA damage Aging Obesity Smoking Varicocele Inflammation Febrile illness Spinal cord injury Testicular cancer Environmental toxins Hormonal disturbances Testicular hyperthermia Drugs, chemotherapy and radiation

AGING Despite of men aging, the gamete production will be continued; however, the quality of generated cells and the fertility potential decreases. In fact, it is demonstrated that spermatozoa produced by men over the age of 37 show higher level of DNA damage that is three times greater than that detect in younger men. Narendra et al (2003) showed a direct relationship between sperm double-stranded DNA breaks, decrease in sperm apoptosis and age. Vagnini et al (2007) also showed a significant increase in sperm DFI with age.

The effect of obesity on sperm disorders and male infertility It has been shown that obesity can impair semen parameters like decreased total sperm count, concentration and motility and increased DNA fragmentation index (Du Plessis, 2010). The reports indicate that obesity may induce oxidative stress and sperm DNA damage as well as decreased fertilizing ability (Bakos, 2010).

Diabetic men have high rates of sperm DNA damage. It has been found that 52% of sperm from diabetic men contained fragmented DNA, which could prevent fertilization or lead to abnormalities in the offspring. The problem of diabetes is the rate of free radicals, which surge because of high blood-sugar levels. Diabetes

CIGARETTE SMOKING It has been reported that the DNA fragmentation index (% DFI) and high DNA stainability (% HDS) are significantly higher in fertile men who smoked than non-smokers. Protamine 2 concentration and the P1/P2 ratios are affected by smoking (Hammadeh, 2010) A possible explanation for these finding could be the increased leucocytes-induced oxidative stress (OS) on developing or mature sperm, and inadequate scavenging antioxidant enzymes in the seminal fluid of smoker men.

There is a higher frequency of spermatozoa with damaged DNA in the ejaculate of patients with varicocele in comparison with fertile men. RecentlyTalebi et al (2008) showed that production of spermatozoa with less condensed chromatin may be one of the explanations of infertility due to varicocele. The results also have shown that the sperm DNA damage is related to the high levels of OS found in the semen of infertile men with varicocele. Furthermore, sperm DNA integrity has been shown to improve after varicocele repair (Zini et al, 2005).

LEUKOCYTOSPERMIA AND INFECTIONS Higher amounts of DNA-damaged spermatozoa have been recognized in the semen samples from the patients with leukocytospermia in comparison with normal fertile donors. However, it is shown that post-testicular genital tract infection and inflammation result in leukocytospermia are associated with high levels of ROS and consequent sperm DNA damage It is possible that both mycoplasma and ureaplasmas can produce sperm chromatin decondensation, DNA denaturation and single- stranded DNA breaks (Evenson,2009). Finally, it should be considered that even systemic infection like an episode of influenza, may affect sperm DNA integrity. (Evenson, 2000).

The results showed that chronic SCI in rat disturbs sperm parameters as well as nuclear maturity and DNA integrity. We also demonstrated that SCI can affect the epididymal phase of chromatin condensation or disulphide bond formation which causes the chromatin hypostability (Talebi et al, 2007). One suggestion for these chromatin anomalies is that SCI may alter the epididymal autonomic innervation, so, the sperm cells which accumulate in the cauda epididymis for a long period of time, are exposed to the oxidative stresses.

The results showed that chronic SCI is associated with high rate of epididymal necrospermia in mammals such as rats. It is, therefore, recommended that an effective laboratory technique, such as Hoechst should be used for separation of live and motile sperms from necrospermic ones for assisted reproduction program.

SPERM DNA DAMAGE IN CANCER PATIENTS Many people with cancer have a serious worry regarding the high risk of occurrence of germ cells DNA damage. The data show that sterility is associated with testicular cancer even before any treatment is done. There are higher levels of sperm DNA damages in untreated cancer patients compared with healthy fertile men (Kobayashi, 2001).

ROS, reactive oxygen species; HSF1, heatshock transcription factor 1; Hsps, heat shock proteins; Stip1, stress-induced phosphoprotein 1 (Catriona Paul, 2008) HYPERTHERMIA

EXPOSURE TO XENOBIOTICS It is demonstrated that exposure to pesticides and insecticides has been associated with increased levels of DNA damages in spermatozoa (Xia et al. 2005). Exposure to xenobiotics can alter chromatin in human and animal sperm. Abnormalities such as deficiency in protamines (Cho et al., 2001) or in histone modification (Baarends et al., 2007) are related to xenobiotics. Insecticides: Pyrethroid, Carbaryl and Chlorpyrifos cause sperm DNA damage (Meeker JD, 2004 and 2008).

Metals Metals are considered as male reproductive toxicants. Workers with high blood lead levels have elevated sperm DNA damage (Hsu et al., 2009). Lead interacts with protamine 2 to decrease its binding to DNA, altering chromatin stability (Quintanilla-Vega et al., 2000). On the other hand, lead and other cations (mercury, copper) may replace zinc in chromatin structure: => Failure or delay in sperm chromatin decondensation in fertilitzation process => Susceptibility to DNA damage agents There is evidence that acute iron or cadmium cause oxidative DNA damage in sperm (Wellejus et al., 2000; Manna et al., 2008).

Radiation & sperm DNA X-irradiation can affect the fertility potential of spermatozoa and cleavage rate of embryo due to DNA damage (Hendricks, 2010) The type of DNA damage that is observed depends on the dose of irradiation and the stage of development of the exposed germ cells (reviewed in Kamiguchi and Tateno, 2002). It is likely that irradiation induces oxidative stress; (Ishii et al., 2005).

Mobile phone radiation induces reactive oxygen species production and DNA damage in human spermatozoa in vitro. (Geoffry N, 2009) Purified human spermatozoa which were exposed to electromagnetic radiation (as the same as mobile) have shown high levels of oxidative DNA damage bio-marker, 8-OH-dG, and DNA fragmentation index. Also, Aitken (2008) identified high levels of sperm DNA fragmentation following16 hours exposure to mobile.

IATROGENIC SPERM DNA DAMAGE Seminal plasma plays an important protective role against reactive oxygen species and its removal during sperm preparation may have negative effects on sperm chromatin/DNA integrity (Potts, 1999). The cryopreservation which is used widely in ART programs, is considered as another form of iatrogenic intervention that might lead to sperm DNA damage (Zini et al. 2000).

Chemotherapy A single dose of chemotherapeutic agents causes high levels of sperm DNA damages which may persist for several months after chemotherapy (Chatterjee, 2000) Many cancer chemotherapeutic agents target DNA. These damages are: Single- and double-stranded breaks Anomalies in chromatin structure Increasing aneuploidy Epigenetic changes in the DNA methylation Alterations in sperm nuclear protamine Changes in nuclear matrix proteome (Delbe` s, 2010)

Drugs like antidepressants can induce DNA damage in spermatozoa (New Scientist. 2008) The researchers gave antidepressant paroxetine to the 35 healthy men for five weeks. The men provided sperm samples before and during treatment. On first examination, the men's sperm parameters seemed healthy, but when the researchers looked at DNA fragmentation in the sperm, they found that the number of sperm exhibiting DNA damage in each sample was raised from 13% before treatment to 31% during treatment with the drug.

The results indicate that acrylamide, through effects on membrane integrity, decreased sperm vitality as well as causing abnormal sperm parameters in progressive motility and total motility.

Chronic exposure to MDMA (ecstasy) increases DNA damage in sperm This increase in sperm DNA damage could be an effect of the temperature or a genotoxic effect produced by MDMA quinone thioether metabolites (Barenys, 2009 and 2010). Cocaine can affect sperm DNA integrity The result of a study showed that cocaine exposure can increase sperm DNA strand breaks and also sperm apoptosis (Li, 1999). Acetaminophen and hydroxyurea alter spermatogenesis and sperm chromatin structure in laboratory mice It is demonstrated that high doses of acetaminophen or hydroxyurea inhibit DNA synthesis in the testis, testicular weight reduction, changes in the proportions of the various spermatid stages and an alteration in sperm chromatin structure (Wiger, 1995)

Alcohol is considered as one of the problems associated with poor semen production and sperm quality. The results of our cytochemical based study showed that ethanol consumption disturbs sperm motility, and nuclear maturity and DNA integrity of spermatozoa in rat. Therefore, ethanol abuse results in the production of spermatozoa with less condensed chromatin, and this may be one possible cause of infertility following ethanol consumption.

HORMONAL DISTORBANCES Animal studies have shown that hormonal insufficiency particularly in the axis of pituitary-testis may induce sperm DNA/chromatin defects. It has been found that FSH–receptor knockout mice in comparison with wild- type mice have higher rates of sperm nuclear protamine deficiency and lower testosterone, lower fertility potential and higher levels of sperm DNA damage (Xing, 2003). Meeker (2008), showed that estradiol and free T4 may have a protective effect against sperm DNA damage

Sperm nuclear maturation, Talebi, 2010

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