AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore

B8 

Colorectal cancer (CRC) is the second leading cause of cancer death in Australia Environmental factors play important roles in the multiple-stage process of CRC and nutritional intervention has been reported to have a major role in its prevention Particularly, vegetables and certain plant components, have been found to possess protective effects on CRC Recently, there has been a growing research interest in rosemary, a common household plant grown in many parts of the world, & its constituents The aim of this study was to evaluate and provide an overview of scientific evidence from all studies conducted over the last decade that examined effects of rosemary on cancer The Medline and Pubmed databases were searched for studies published in English, from May 1996 to July 2007, that compared rosemay/rosemary constituents to a control Preclinical evidence from animal & cell culture studies points to the anticancer properties of rosemary extract and its constituents carnosol, carnosic acid (CA), ursolic acid (UA), and rosmarinic acid (RA) No studies have reported anticancer activity of other constituents of rosemary including caffeic acid The anticarcinogenic and anti-tumorigenic properties appear to arise through dose-related changes in a number of molecular events associated with cancer It appears that the described mechanisms are not tissue specific, as evidenced by the ability of rosemary to block tumours not only in the colon, but only in the breast, liver, stomach, melanoma and leukemia cells The results described here suggested that molecular targets modulated by rosemary and its constituents are useful indicators of success in clinical cancer chemo-prevention trials

Roesmary and cancer prevention: Preclinical perspectives

The Catsper channel is a sperm-specific, Ca2+-permeable, pH-dependent, and low voltage-dependent channel that is essential for the hyperactivity of sperm flagellum, chemotaxis towards the egg, capacitation and acrosome reaction. All of these physiological events require calcium entry into sperm cells. Remarkably, Catsper genes are exclusively expressed in the testis during spermatogenesis, and are sensitive to ion channel-induced pH change, such as NHEs, Ca2+ATPase, K+ channel, Hv1 channel and HCO3
 − transporters. Furthermore, the Catsper channel is regulated by some physiological stimulants, such as progesterone, cyclic nucleotides (e.g., cAMP, cGMP), zona pellucida (ZP) glycoproteins and bovine serum albumin (BSA). All of these factors normally stimulate Ca2+ entry into sperm through the Catsper channel. In addition, the Catsper channel may be a potential target for male infertility treatment or contraception. This review will focus on the structure, functions, regulation mechanisms and medicinal targets of the Catsper channel.

The Catsper channel and its roles in male fertility: a systematic review

Dual Sensing of Physiologic pH and Calcium by EFCAB9 Regulates Sperm Motility

Lead poisoning is a stealthy threat to human physiological systems as chronic exposure can remain asymptomatic for long periods of time before symptoms manifest. We presently review the biophysical mechanisms of lead poisoning that contribute to male infertility. Environmental and occupational exposure of lead may adversely affect the hypothalamic-pituitary-testicular axis, impairing the induction of spermatogenesis. Dysfunction at the reproductive axis, namely testosterone suppression, is most susceptible and irreversible during pubertal development. Lead poisoning also appears to directly impair the process of spermatogenesis itself as well as sperm function. Spermatogenesis issues may manifest as low sperm count and stem from reproductive axis dysfunction or testicular degeneration. Generation of excessive reactive oxygen species due to lead-associated oxidative stress can potentially affect sperm viability, motility, DNA fragmentation, membrane lipid peroxidation, capacitation, hyperactivation, acrosome reaction, and chemotaxis for sperm-oocyte fusion, all of which can contribute to deter fertilization. Reproductive toxicity has been tested through cross-sectional analysis studies in humans as well as in vivo and in vitro studies in animals.

Impaired hypothalamic-pituitary-testicular axis activity, spermatogenesis, and sperm function promote infertility in males with lead poisoning.

Owing to the fact that there are more microbial than human cells in our body and that humans contain more microbial than human genes, the microbiome has huge potential to influence human physiology, both in health and in disease. The use of next-generation sequencing technologies has helped to elucidate functional, quantitative and mechanistic aspects of the complex microorganism-host interactions that underlie human physiology and pathophysiology. The microbiome of semen is a field of increasing scientific interest, although this microbial niche is currently understudied compared with other areas of microbiome research. However, emerging evidence is beginning to indicate that the seminal microbiome has important implications for the reproductive health of men, the health of the couple and even the health of offspring, owing to transfer of microorganisms to the partner and offspring. As this field expands, further carefully designed and well-powered studies are required to unravel the true nature and role of the seminal microbiome.

The seminal microbiome in health and disease.

Study question Are genetic abnormalities in CATSPER (cation channel of sperm) genes associated with idiopathic male infertility with normal semen parameters and, if so, how do they affect male fertility? Summary answer A novel copy number variation (CNV) in CATSPER2 causes idiopathic male infertility with normal semen parameters by disrupting the ability of sperm to penetrate viscous media, undergo hyperactivation and respond to progesterone. What is known already CATSPER is the principle Ca2+ channel mediating extracellular Ca2+ influx into spermatozoa. Although several case reports have suggested a causal relationship between CATSPER disruption and human male infertility, whether genetic abnormalities in CATSPER genes are associated with idiopathic male infertility with normal semen parameters remains unclear. Study design, size, duration Spermatozoa were obtained from men attending the reproductive medical center at Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi, China between January 2014 and June 2016. In total, 120 men from infertile couples and 20 healthy male donors were selected to take part in the study, based on their normal semen parameters. Participants/materials, setting, methods CATSPER and KSPER currents were assessed using the whole-cell patch-clamp technique. Whole-genome sequencing and TaqMan® CNV assays were performed to identify genetic variations. The expression levels of genes encoding the CATSPER complex were measured by quantitative real-time PCR and Western blot. Sperm motion characteristics and hyperactivation were examined with a computer-aided sperm analysis (CASA) system. Sperm responses to progesterone, assessed as increases in CATSPER current and intercellular Ca2+ concentrations ([Ca2+]i), as well as inducement of penetration ability and acrosome reaction, were examined by means of whole-cell patch-clamp technique, single-sperm [Ca2+]i imaging, penetration into methylcellulose assay and chlortetracycline staining, respectively. Main results and the role of chance An infertile man with complete disruption of CATSPER current was identified. This individual has a novel CNV which disrupts one gene copy in the region 43894500-43950000 in chromosome 15 (GRCh37.p13 Primary Assembly, nsv3067119), containing the whole DNA sequence of CATSPER2. This CNV affected the expression of CATSPER2, resulting in dramatically reduced levels of CATSPER2 proteins in the individual's spermatozoa. Although this individual exhibited normal semen parameters, his spermatozoa showed impaired penetration ability, deficient hyperactivation, and did not respond to progesterone, in terms of monovalent current potentiation, [Ca2+]i increase, penetration ability enhancement and acrosome reaction inducement, which may explain the individual's idiopathic infertility. Large scale data N/A. Limitations, reasons for caution Our novel findings require more cases to support the CATSPER2 CNV identified in this study as a common cause of idiopathic male infertility in patients with normal semen parameters. Therefore, caution must be taken when extrapolating the use of this CNV as a potential biomarker for idiopathic male infertility. Wider implications of the findings The findings from the unique human CATSPER 'knockout' model in this study not only confirm the essential roles of CATSPER in mediating progesterone response and regulating hyperactivation in human spermatozoa but also reveal that disruption of CATSPER current is a significant factor causing idiopathic male infertility. Study funding/competing interest(s) This study was funded by National Natural Science Foundation of China (81771644 and 31400996 to T.L.; 31230034 to X.Z.); National Basic Research Program of China (973 Program, 2015CB943003 to X.Z.); National Key Research and Development Program of China (2016YFC1000905 to T.L.); Natural Science Foundation of Jiangxi, China (20121BBG70021 and GJJ12015 to X.Z.; 20161BAB204167 and 20171ACB21006 to T.L.) and the open project of National Population and Family Planning Key Laboratory of Contraceptives and Devices Research (No. 2016KF07 to T.L.). The authors have no conflicts of interest to declare.

A novel copy number variation in CATSPER2 causes idiopathic male infertility with normal semen parameters.

Study question Is diethylstilbestrol (DES), a prototypical endocrine-disrupting chemical (EDC), able to induce physiological changes in human spermatozoa and affect progesterone actions? Summary answer DES promoted Ca2+ flux into human spermatozoa by activating the cation channel of sperm (CatSper) and suppressed progesterone-induced Ca2+ signaling, tyrosine phosphorylation and sperm functions. What is known already DES significantly impairs the male reproductive system both in fetal and postnatal exposure. Although various EDCs affect human spermatozoa in a non-genomic manner, the effect of DES on human spermatozoa remains unknown. Study design, size, duration Sperm samples from normozoospermic donors were exposed in vitro to a range of DES concentrations with or without progesterone at 37°C in a 5% CO2 incubator to mimic the putative exposure to this toxicant in seminal plasma and the female reproductive tract fluids. The incubation time varied according to the experimental protocols. All experiments were repeated at least five times using different individual sperm samples. Participants/materials, setting, methods Human sperm intracellular calcium concentrations ([Ca2+]i) were monitored with a multimode plate reader following sperm loading with Ca2+ indicator Fluo-4 AM, and the whole-cell patch-clamp technique was performed to record CatSper and alkalinization-activated sperm K+ channel (KSper) currents. Sperm viability and motility parameters were assessed by an eosin-nigrosin staining kit and a computer-assisted semen analysis system, respectively. The ability of sperm to penetrate into viscous media was examined by penetration into 1% methylcellulose. The sperm acrosome reaction was measured using chlortetracycline staining. The level of tyrosine phosphorylation was determined by western blot assay. Main results and the role of chance DES exposure rapidly increased human sperm [Ca2+]i dose dependently and even at an environmentally relevant concentration (100 pM). The elevation of [Ca2+]i was derived from extracellular Ca2+ influx and mainly mediated by CatSper. Although DES did not affect sperm viability, motility, penetration into viscous media, tyrosine phosphorylation or the acrosome reaction, it suppressed progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation. Consequently, DES (1-100 μM) significantly inhibited progesterone-induced human sperm penetration into viscous media and acrosome reaction. Large scale data N/A. Limitations, reasons for caution Although DES has been shown to disturb progesterone actions on human spermatozoa, this study was performed in vitro, and caution must be taken when extrapolating the results in practical applications. Wider implications of the findings The present study revealed that DES interfered with progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation, ultimately inhibited progesterone-induced human sperm functions and, thereby, might impair sperm fertility. The non-genomic manner in which DES disturbs progesterone actions may be a potential mechanism for some estrogenic endocrine disruptors to affect human sperm function. Study funding/competing interests National Natural Science Foundation of China (No. 31400996); Natural Science Foundation of Jiangxi, China (No. 20161BAB204167 and No. 20142BAB215050); open project of National Population and Family Planning Key Laboratory of Contraceptives and Devices Research (No. 2016KF07) to T. Luo; National Natural Science Foundation of China (No. 81300539) to L.P. Zheng. The authors have no conflicts of interest to declare.

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Diethylstilbestrol activates CatSper and disturbs progesterone actions in human spermatozoa

A number of culture-dependent and -independent studies have reported that the number and significance of bacterial species in semen may have been underestimated. The aim of the present study was to profile the seminal microbiome in patients with obstructive or non-obstructive azoospermia. A high-throughput sequencing method was adopted to sequence genomic DNA extracted from the semen of healthy people (C group), patients with obstructive azoospermia (OA group) and patients with non-obstructive azoospermia (NOA group). The results revealed that Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria species comprised the majority of bacteria in the C (98.14%), OA (98.26%) and NOA (90.96%) groups. Patients in the OA and NOA groups exhibited an increase in Bacteroidetes and Firmicutes, whereas the number of Proteobacteria and Actinobacteria were decreased compared with the C group. A total of 398 common operational taxonomic units were identified, of which 27 belonged to the genus Lactobacillus. Furthermore, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States analysis indicated that the pathogenic species and reduced biodiversity in the semen of patients with azoospermia may result in an increased risk of metabolic, infectious and immune diseases. In the present study, the seminal microbiome of patients with obstructive or non-obstructive azoospermia was explored, which may be useful for developing novel treatments against azoospermia as well as for its diagnosis.

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Seminal bacterial composition in patients with obstructive and non-obstructive azoospermia.

It is well known that there has been a worldwide decrease in human male fertility in recent years. One of the main factors affecting this is environmental pollution. Lead is one of the major heavy metal contaminants that threaten the health of animals and human beings in China. It preferentially accumulates in male reproductive organs and can be up to 10 µM in human seminal plasma. Lead impairs mammalian spermatogenesis and sperm quality in vivo. It also inhibits sperm functions in vitro but the underlying mechanisms remain unclear. Therefore, we aimed to investigate the in vitro toxicity of lead on human sperm functions and to elucidate the underlying mechanisms. Semen samples were collected from 20 healthy volunteers with different careers and backgrounds living in Nanchang, Jiangxi. Human sperm suspensions were treated with different concentrations of lead acetate (0, 0.5, 2.5, 10, 50, and 100 µM) and the viability, motility, capacitation and progesterone-induced acrosome reaction were examined. Treatment with 10-100 µM lead acetate dose-dependently inhibited total and progressive motility measures, capacitation and progesterone-induced acrosome reaction. It also dose-dependently decreased the intracellular concentrations of cyclic adenosine monophosphate (cAMP) and calcium ([Ca(2+)]i), and reduced the tyrosine phosphorylation of sperm proteins, all of which are thought to be key factors in the regulation of sperm function. Our findings suggest that lead inhibits human sperm functions by reducing the levels of sperm intracellular cAMP, [Ca(2+)]i and tyrosine phosphorylation of sperm proteins in vitro.

Lead Inhibits Human Sperm Functions by Reducing the Levels of Intracellular Calcium, cAMP, and Tyrosine Phosphorylation.

STUDY QUESTION Is there a role for lysine glutarylation (Kglu), a newly identified protein post-translational modification (PTM), in human sperm? SUMMARY ANSWER Kglu occurs in several proteins located in the tail of human sperm, and it was reduced in asthenozoospermic (A) men and positively correlated with progressive motility of human sperm, indicating its important role in maintaining sperm motility. WHAT IS KNOWN ALREADY Since mature sperm are almost transcriptionally silent, PTM is regarded as an important pathway in regulating sperm function. However, only phosphorylation has been extensively studied in mature sperm to date. Protein lysine modification (PLM), a hot spot of PTMs, was rarely studied except for a few reports on lysine methylation and acetylation. As a newly identified PLM, Kglu has not been well characterized, especially in mature sperm. STUDY DESIGN, SIZE, DURATION Sperm samples were obtained from normozoospermic (N) men and A men who visited the reproductive medical center between February 2016 and January 2018. In total, 61 N men and 59 A men were recruited to participate in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS Kglu was examined by immunoblotting and immunofluorescence assays using a previously qualified pan-anti-glutaryllysine antibody that recognizes glutaryllysine in a wide range of sequence contexts (both in histones and non-histone substrates) but not the structurally similar malonyllysine and succinyllysine. The immunofluorescence assay was imaged using laser scanning confocal microscopy and super-resolution structured illumination microscopy. Sperm motility parameters were examined by computer-assisted sperm analysis. MAIN RESULTS AND THE ROLE OF CHANCE Kglu occurs in several proteins (20-150 kDa) located in the tail of human sperm, especially in the middle piece and the latter part of the principal piece. Sperm Kglu was modulated by regulatory systems (enzymes and glutaryl-CoA) similar to those in HeLa cells. The mean level of sperm Kglu was significantly reduced in A men compared with N men (P < 0.001) and was positively correlated with progressive motility (P < 0.001). The sodium glutarate-induced elevation of Kglu levels in A men with lower Kglu levels in sperm significantly improved the progressive motility (P < 0.001). Furthermore, the reduced sperm Kglu levels in A men was accompanied by an increase in sperm glutaryl-CoA dehydrogenase (a regulatory enzyme of Kglu). LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Although the present study indicated the involvement of sperm Kglu in maintaining progressive motility of human sperm, the underlying mechanism needs to be investigated further. WIDER IMPLICATIONS OF THE FINDINGS The findings of this study provide an insight into the novel role of Kglu in human sperm and suggest that abnormality of sperm PLMs may be one of the causes of asthenozoospermia. STUDY FUNDING/COMPETING INTEREST(S) National Natural Science Foundation of China (81 771 644 to T.L.; 31 671 204 to X.Z. and 81 871 207 to H.C.); National Basic Research Program of China (973 Program, 2015CB943003 to X.Z.); Natural Science Foundation of Jiangxi, China (20171ACB21006 and 20161BAB204167 to T.L.; 20165BCB18001 to X.Z.). The authors have no conflicts of interest to declare.

Houyang Chen

Papers

A novel copy number variation in CATSPER2 causes idiopathic male infertility with normal semen parameters.

Diethylstilbestrol activates CatSper and disturbs progesterone actions in human spermatozoa

Seminal bacterial composition in patients with obstructive and non-obstructive azoospermia.

Lead Inhibits Human Sperm Functions by Reducing the Levels of Intracellular Calcium, cAMP, and Tyrosine Phosphorylation.

Lysine glutarylation in human sperm is associated with progressive motility.