Background: Oxidative stress is a major contributor to diabetes, which can lead to testicular damage and infertility.
Objective: This study aimed to compare the effects of metformin as a chemical drug with silymarin as an herbal agent on the sperm parameters and histopathological changes of testes in diabetic rats.
Materials and Methods: Thirty-two male Wistar rats (250-270 gr) were randomly divided into four groups: 1) control; 2) diabetic; 3) diabetic+metformin 200 mg/kg; and 4) diabetic+silymarin 100 mg/kg. Daily injections were administered intraperitoneally for 56 days. At the end of the treatment, blood sampling was performed for biochemical assessment. Then, the rats were sacrificed and their left testis and epididymis were cut for sperm analysis as well as histopathology and morphometric evaluation.
Results: Diabetes was associated with a reduced sperm count, motility, viability, maturity, and chromatin quality of sperm (p ≤ 0.001). It was also associated with a higher malondialdehide level and lower total antioxidant capacity level of serum in comparison with the control group (p ≤ 0.001). There was a significant difference in the seminiferous tubule diameter, germinal epithelium height, and testicular histopathological alterations in the diabetic rats compared with the control rats (p ≤ 0.001). Treatment with metformin and silymarin improved the above-mentioned parameters and this improvement was more substantial in silymarin-treated animals (p ≤ 0.001).
Conclusion: In diabetic rats, metformin and silymarin improved sperm parameters, sperm DNA integrity, seminiferous tubule diameter, germinal epithelium thickness, and testicular histopathological complications; this improvement was more substantial in the silymarin-treated group. So, the findings of this study suggest that silymarin is more effective than metformin in treating diabetic-induced infertility.
Key words: Diabetes, DNA damage, Metformin, Silymarin, Sperm, Testis.
Semen cryopreservation has become an essential tool for conservation efforts of the giant panda (Ailuropoda melanoleuca); however, it is severely detrimental to sperm quality. Evidence has shown that antioxidants have the potential to reverse cryopreservation-induced damage in sperm. The purpose of this study was to screen effective antioxidants that could retain sperm quality during cryopreservation and to determine the optimal dose. Seven antioxidant groups, including resveratrol (RSV = 50 μM, RSV = 100 μM, RSV = 150 μM), lycium barbarum polysaccharide (LBP = 2 mg/mL, LBP = 4 mg/mL), laminaria japonica polysaccharides (LJP = 1 mg/mL) or combination (LBP = 2 mg/mL, LJP = 1 mg/mL and RSV = 100 μM) were assessed.
RSV, LBP, LJP, or a combination of RSV, LBP, and LJP added to the freezing medium significantly improved sperm progressive motility, plasma membrane integrity, acrosome integrity, and mitochondrial activity during the cryopreservation process. Furthermore, the activities of glutathione peroxidase and superoxide dismutase were also improved. The levels of reactive oxygen species and malondialdehyde in semen were notably reduced. Hyaluronidase activity and acrosin activity were significantly increased in LBP-treated sperm. However, sperm total motility and DNA integrity were not significantly different between the groups.
RSV (50 μM) or LBP (2 mg/mL) are the best candidate antioxidants for inclusion in the freezing medium to improve the quality of giant panda spermatozoa during semen cryopreservation.
The role of dietary profiles in promoting or reducing the risk of multiple types of cancer is increasingly clear, driving the search for balanced foods and nutraceuticals. The red seaweed Grateloupia turuturu has been used as human food showing a balanced nutritional profile. This study aims to test in vivo chemopreventive effects of G. turuturu against cutaneous pre-malignant lesions in transgenic mice for the human papillomavirus type 16 (HPV16). Forty-four female HPV+/− or HPV−/− mice received a standard diet or were supplemented with 10% G. turuturu for 22 consecutive days. Cutaneous lesions (ear and chest skin) were identified histologically. Complementarily, the weights and histology of internal organs as well as blood biochemical and DNA integrity parameters were also assessed. G. turuturu consistently reduced the incidence of epidermal dysplasia induced by HPV16 on both cutaneous sites. Moreover, biochemical, DNA integrity and histological analyses confirmed G. turuturu edibility as no signs of toxicity were found. Dietary supplementation with G. turuturu is an effective and safe chemopreventive strategy in this model.
Varicocele, a condition associated with increased oxidative stress, negatively affects sperm DNA integrity and reduces pregnancy rates. However, the molecular mechanisms related to DNA integrity, damage, and repair in varicocele patients remain unclear. This study aimed to determine the role of DNA repair molecular mechanisms in varicocele-related infertility by combining an in silico proteomics approach with wet-laboratory techniques. Proteomics results previously generated from varicocele patients (n=50) and fertile controls (n=10) attending our Andrology Center were reanalyzed using bioinformatics tools, including the WEB-based Gene SeT AnaLysis Toolkit, Open Target Platform, and Ingenuity Pathway Analysis (IPA), to identify differentially expressed proteins (DEPs) involved in DNA repair. Subsequently, selected DEPs in spermatozoa were validated using western blotting in varicocele (n = 13) and fertile control (n = 5) samples. We identified 99 DEPs mainly involved in male reproductive system disease (n=66) and male infertility (n=47). IPA analysis identified five proteins [fatty acid synthase (FASN), myeloperoxidase (MPO), mitochondrial aconitate hydratase (ACO2), nucleoporin 93 (NUP93), and 26S proteasome non-ATPase regulatory subunit 14 (PSMD14)] associated with DNA repair deficiency, which showed altered expression in varicocele (P <0.03). We validated ACO2 downregulation (fold change=0.37, change%=-62.7%, P=0.0001) and FASN overexpression (fold change = 4.04, change %= 303.7%, P = 0.014) in men with varicocele compared to controls. This study combined a unique in silico approach with an in vitro validation of the molecular mechanisms that may be responsible for varicocele-associated infertility. We identified ACO2 and FASN as possible proteins involved in DNA repair, whose altered expression may contribute to DNA damage in varicocele pathophysiology.
GPCRs arguably represent the most effective current therapeutic targets for a plethora of diseases. GPCRs also possess a pivotal role in the regulation of the physiological balance between healthy and pathological conditions; thus, their importance in systems biology cannot be underestimated. The molecular diversity of GPCR signaling systems is likely to be closely associated with disease-associated changes in organismal tissue complexity and compartmentalization, thus enabling a nuanced GPCR-based capacity to interdict multiple disease pathomechanisms at a systemic level. GPCRs have been long considered as controllers of communication between tissues and cells. This communication involves the ligand-mediated control of cell surface receptors that then direct their stimuli to impact cell physiology. Given the tremendous success of GPCRs as therapeutic targets, considerable focus has been placed on the ability of these therapeutics to modulate diseases by acting at cell surface receptors. In the past decade, however, attention has focused upon how stable multiprotein GPCR superstructures, termed receptorsomes, both at the cell surface membrane and in the intracellular domain dictate and condition long-term GPCR activities associated with the regulation of protein expression patterns, cellular stress responses and DNA integrity management. The ability of these receptorsomes (often in the absence of typical cell surface ligands) to control complex cellular activities implicates them as key controllers of the functional balance between health and disease. A greater understanding of this function of GPCRs is likely to significantly augment our ability to further employ these proteins in a multitude of diseases.
Bacterial vitality after water disinfection treatment was investigated using bio-orthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM). Protein synthesis activity and DNA integrity (BONCAT–SYBR Green) was monitored in Escherichia coli monocultures and in natural marine samples after UV irradiation (from 25 to 200 mJ/cm2) and heat treatment (from 15 to 45 min at 55°C). UV irradiation of E. coli caused DNA degradation followed by the decrease in protein synthesis within a period of 24 h. Heat treatment affected both DNA integrity and protein synthesis immediately, with an increased effect over time. Results from the BONCAT method were compared with results from well-known methods such as plate counts (focusing on growth) and LIVE/DEAD™ BacLight™ (focusing on membrane permeability). The methods differed somewhat with respect to vitality levels detected in bacteria after the treatments, but the results were complementary and revealed that cells maintained metabolic activity and membrane integrity despite loss of cell division. Similarly, analysis of protein synthesis in marine bacteria with BONCAT displayed residual activity despite inability to grow or reproduce. Background controls (time zero blanks) prepared using different fixatives (formaldehyde, isopropanol, and acetic acid) and several different bacterial strains revealed that the BONCAT protocol still resulted in labeled, i.e., apparently active, cells. The reason for this is unclear and needs further investigation to be understood. Our results show that BONCAT and FCM can detect, enumerate, and differentiate bacterial cells after physical water treatments such as UV irradiation and heating. The method is reliable to enumerate and explore vitality of single cells, and a great advantage with BONCAT is that all proteins synthesized within cells are analyzed, compared to assays targeting specific elements such as enzyme activity.
Male infertility is a common health problem that can be influenced by a host of lifestyle risk factors such as environment, nutrition, smoking, stress, and endocrine disruptors. These effects have been largely demonstrated on sperm parameters (e.g., motility, numeration, vitality, DNA integrity). In addition, several studies showed the deregulation of sperm proteins in relation to some of these factors. This review inventories the literature related to the identification of sperm proteins showing abundance variations in response to the four risk factors for male infertility that are the most investigated in this context: obesity, diabetes, tobacco smoking, and exposure to bisphenol-A (BPA). First, we provide an overview of the techniques used to identify deregulated proteins. Then, we summarise the main results obtained in the different studies and provide a compiled list of deregulated proteins in relation to each risk factor. Gene ontology analysis of these deregulated proteins shows that oxidative stress and immune and inflammatory responses are common mechanisms involved in sperm alterations encountered in relation to the risk factors.
Cell-signaling pathways are essential for cells to respond and adapt to changes in their environmental conditions. The cell-wall integrity (CWI) pathway of Saccharomyces cerevisiae is activated by environmental stresses, compounds, and morphogenetic processes that compromise the cell wall, orchestrating the appropriate cellular response to cope with these adverse conditions. During cell-cycle progression, the CWI pathway is activated in periods of polarized growth, such as budding or cytokinesis, regulating cell-wall biosynthesis and the actin cytoskeleton. Importantly, accumulated evidence has indicated a reciprocal regulation of the cell-cycle regulatory system by the CWI pathway. In this paper, we describe how the CWI pathway regulates the main cell-cycle transitions in response to cell-surface perturbance to delay cell-cycle progression. In particular, it affects the Start transcriptional program and the initiation of DNA replication at the G1/S transition, and entry and progression through mitosis. We also describe the involvement of the CWI pathway in the response to genotoxic stress and its connection with the DNA integrity checkpoint, the mechanism that ensures the correct transmission of genetic material and cell survival. Thus, the CWI pathway emerges as a master brake that stops cell-cycle progression when cells are coping with distinct unfavorable conditions.
In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.