https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.2002.tb02599.x

Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques.

Menopause and risk factors for coronary heart disease

DNA damage in human spermatozoa has been associated with a range of adverse clinical outcomes, including infertility, abortion, and disease in the offspring. We have advanced a two-step hypothesis to explain this damage involving impaired chromatin remodeling during spermiogenesis followed by a free radical attack to induce DNA strand breakage. The objective of the present study was to test this hypothesis by determining whether impaired chromatin protamination is correlated with oxidative base damage and DNA fragmentation in human spermatozoa. DNA fragmentation, chromatin protamination, mitochondrial membrane potential, and formation of the oxidative base adduct, 8-hydroxy-2'-deoxyguanosine (8OHdG), were monitored by flow cytometry/fluorescence microscopy. Impairment of DNA protamination during late spermatogenesis was highly correlated (P < 0.001) with DNA damage in human spermatozoa. The disruption of chromatin remodeling also was associated with a significant elevation in the levels of 8OHdG (P < 0.001), and the latter was itself highly correlated with DNA fragmentation (P < 0.001). The significance of oxidative stress in 8OHdG formation was demonstrated experimentally using H2O2/Fe2+ and by the correlation observed between this base adduct and superoxide generation (P < 0.001). That 8OHdG formation was inversely associated with mitochondrial membrane potential (P < 0.001) suggested a possible role for these organelles in the creation of oxidative stress. These results clearly highlight the importance of oxidative stress in the induction of sperm DNA damage and carry significant implications for the clinical management of this condition.

DNA Damage in Human Spermatozoa Is Highly Correlated with the Efficiency of Chromatin Remodeling and the Formation of 8-Hydroxy-2′-Deoxyguanosine, a Marker of Oxidative Stress

For centuries, the black seed (Nigella sativa) herb and oil have been used in Asia, Middle East and Africa to promote health and fight disease. Thymoquinone (TQ), the most abundant constituent present in black seed, is a promising dietary chemopreventive agent. We investigated the effects of thymoquinone (TQ) against HCT-116 human colon cancer cells and attempted to identify its potential molecular mechanisms of action. We report that TQ inhibits the growth of colon cancer cells which was correlated with G1 phase arrest of the cell cycle. Furthermore, TUNEL staining and flow cytometry analysis indicate that TQ triggers apoptosis in a dose- and time-dependent manner. Apoptosis induction by TQ was associated with a 2.5-4.5-fold increase in mRNA expression of p53 and the downstream p53 target gene, p21WAF1. Simultaneously, we found a marked increase in p53 and p21WAF1 protein levels but a significant inhibition of anti-apoptotic Bcl-2 protein. Co-incubation with pifithrin-alpha (PFT-alpha), a specific inhibitor of p53, restored Bcl-2, p53 and p21WAF1 levels to the untreated control and suppressed TQ-induced cell cycle arrest and apoptosis. p53-null HCT-116 cells were less sensitive to TQ-induced growth arrest and apoptosis. These results indicate that TQ is antineoplastic and pro-apoptotic against colon cancer cell line HCT116. The apoptotic effects of TQ are modulated by Bcl-2 protein and are linked to and dependent on p53. Our data support the potential for using the agent TQ for the treatment of colon cancer.

Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism.

Apoptosis is a critical process for regulating both the size and the quality of the male and female germ lines. In this review, we examine the importance of this process during embryonic development in establishing the pool of spermatogonial stem cells and primordial follicles that will ultimately define male and female fertility. We also consider the importance of apoptosis in controlling the number and quality of germ cells that eventually determine reproductive success. The biochemical details of the apoptotic process as it affects germ cells in the mature gonad still await resolution, as do the stimuli that persuade these cells to commit to a pathway that leads to cell death. Our ability to understand and ultimately control the reproductive potential of male and female mammals depends upon a deeper understanding of these fundamental processes.

/pdf/apoptosis-in-the-germ-line-4bbffhxnad.pdf

Apoptosis in the germ line.

Purpose: Our purpose was to investigate the associationbetween percentage chromomycin A3 (CMA3) positivity ofspermatozoa with some sperm parameters and in vitrofertilization rate.

Methods: Spermatozoa were collected from 139 men,washed in PBS, fixed in methanol/glacial acetic acid (3:1),and then spread on slides. CMA3 positivity is expressed asthe percentage in 200 spermatozoa.

Results: Percentage of CMA3 positivity showed not only anegative correlation with fertilization rate but also asignificant difference between fertilizing and nonfertilizingpatients. Moreover, percentage of CMA3-positivespermatozoa showed a negative correlation with count and percentagemotility and a positive correlation with percentage ofabnormal morphology. Percentage of CMA3 positivity also had apositive correlation with some abnormalities of head such asamorphous and macrocephaly. Ultrastructural study showedchromatin unpackaging in high CMA3-positive semensamples in comparison with low CMA3-positive semen samples.

Conclusion: There is a close relationship among fertilizationrate, sperm parameters, and CMA3 positivity and CMA3could be considered as a useful tool for evaluation of malefertility prior to infertility treatment.

Chromomycin A3 staining as a useful tool for evaluation of male fertility.

Background: Nigella sativa has been a nutritional flavoring factor and natural treatment for many ailments for so many years in medical science. Earlier studies have been reported that thymoquinone (TQ), an active compound of its seed, contains anticancer properties. Previous studies have shown that TQ induces apoptosis in breast cancer cells but it is unclear the role of P53 in the apoptotic pathway. Hereby, this study reports the potency of TQ on expression of tumor suppressor gene P53 and apoptosis induction in breast cancer cell line Michigan Cancer Foundation-7 (MCF-7). Methods: MCF-7 cell line was cultured and treated with TQ, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out for evaluating the half-maximal inhibitory concentration (IC50) values after 24 h of treatment. The percentage of apoptotic cells was measured by flow cytometry. Real-time polymerase chain reaction (PCR) was performed to estimate the messenger RNA expression of P53 in MCF-7 cell line at different times. Results: The IC50 value for the TQ in MCF-7 cells was 25 mM that determined using MTT assay. The flow cytometry and real-time PCR results showed that TQ could induce apoptosis in MCF-7 cells, and the P53 gene expression was dramatically up-regulated by ascending time, respectively. Hence, there was significant difference in 48 and 72 h. Conclusions: Our results demonstrated that TQ could induce apoptosis in MCF-7 cells through up-regulation of P53 expression in breast cancer cell line (MCF-7) by time-dependent manner.

/pdf/effect-of-thymoquinone-on-p53-gene-expression-and-50uajpv4hr.pdf

Effect of thymoquinone on P53 gene expression and consequence apoptosis in breast cancer cell line

Cadmium (Cd) is a highly toxic element, which may cause toxicity to most organs in the body. Zinc (Zn) and magnesium (Mg) are essential minerals with probable benefits on Cd harmful effects. Finding an efficient and non-pathological treatment against Cd toxicity seems promising. Fifty adult rats were divided into ten experimental groups of five rats each. The Cd group was treated with 1 mg Cd/kg and the control group received 0.5 cm3 normal saline. The other eight groups received Zn (0.5 and 1.5 mg/kg) and Mg (0.5 and 1.5 mg/kg) either alone or in combination with 1 mg Cd/kg through IP injection for 3 weeks. Testis malondialdehyde (MDA), sperm parameters, and testis histopathology were investigated. Cd reduced sperm parameters and increased testis MDA. Moreover, Cd exposure caused a significant histological damage in testis of male rats. However, Zn or Mg treatment prevented and reversed Cd toxic alterations in testis. These findings suggest that co-administration of Zn or Mg could improve cadmium testicular toxicity in male Wistar rats.

Cadmium Testicular Toxicity in Male Wistar Rats: Protective Roles of Zinc and Magnesium

Background: Sperm DNA in human beings and most vertebrates is packed by protamines into highly compact form of chromatin. There are many staining methods to assess sperm chromatin. Three different methods of staining were used simultaneously in this study and the goal was to determine which of these sperm tests has a relation with fertilization rate in intracytoplasmic sperm injection (ICSI). Materials and Methods: Thirty couples who referred to Yamagata University Hospital (Yamagata, Japan) for ICSI were included in this study. The greater part of semen was prepared for ICSI. The remaining part was used for staining with aniline blue, acridine orange, and chromomycin A3 (CMA3). For evaluation of abnormal morphology and abnormality of head, Papanicolaou-stained smears were used. The analysis of data was done using Spearman coefficient of correlation and logistic regression model. Receiver operator characteristic (ROC) curve was used for discrimination of CMA3 staining power to identify ICSI rates. Results: Percentage of CMA3 positivity, unlike those of aniline blue and acridine orange, showed significant negative correlation with fertilization rate. Moreover, the percentage of CMA3 positivity showed a positive correlation with the percentage of abnormal morphology and abnormality of head. By dividing patients into CMA3 48% groups, the area under the curve was 0.646. Conclusions: CMA3 staining (protamine deficiency) could be considered as a useful tool for evaluation of male fertility prior to infertility treatment.

Impact of sperm chromatin evaluation on fertilization rate in intracytoplasmic sperm injection.

Background: Chromomycin A3 (CMA3) staining is one of the staining methods for detecting protamine deficiency in sperm nucleus. CMA3 is a fluorochrome that competes with protamines for binding to DNA double helix. It has been shown in our previous studies that percentage of CMA3 positive spermatozoa in semen has a close significant relationship with the fertilization rate in in vitro fertilization (IVF). The aim of this study was to examine the ultrastructural differences between sperms in patients who had high fluorescent percentages of yellow or red in CMA3 staining (protamine deficient) with patients with low fluorescent percentages. Materials and Methods: Semen samples are taken from five patients with high fluorescent percentages and five patients with low fluorescent percentages. Then the samples are passed for the different steps of preparing for electron microscopy. After the sectioning and mounting on grids, they are investigated under the transmission electron microscope. Results: Sperms in patients with low percentages of positive spermatozoa often have a normal appearance. Sperms in high fluorescent samples frequently have unpacked chromatin. Furthermore acrosomes of these sperms are thinner or disturbed. Also sometimes there are irregularities in sperm head membrane. Conclusion: Protamine deficiency in sperm nucleus can cause ultrastructural anomalies in sperm chromatin such as unpacking of it. It also is concomitant with acrosome and sperm membrane disturbances.

Farhad Golshan Iranpour

Papers

Chromomycin A3 staining as a useful tool for evaluation of male fertility.

Effect of thymoquinone on P53 gene expression and consequence apoptosis in breast cancer cell line

Cadmium Testicular Toxicity in Male Wistar Rats: Protective Roles of Zinc and Magnesium

Impact of sperm chromatin evaluation on fertilization rate in intracytoplasmic sperm injection.

The effects of protamine deficiency on ultrastructure of human sperm nucleus.