Jorge G. Farías

The Hypoxic Testicle: Physiology and Pathophysiology

Mammalian spermatogenesis is a complex biological process occurring in the seminiferous tubules in the testis. This process represents a delicate balance between cell proliferation, differentiation, and apoptosis. In most mammals, the testicles are kept in the scrotum 2 to 7°C below body core temperature, and the spermatogenic process proceeds with a blood and oxygen supply that is fairly independent of changes in other vascular beds in the body. Despite this apparently well-controlled local environment, pathologies such as varicocele or testicular torsion and environmental exposure to low oxygen (hypoxia) can result in changes in blood flow, nutrients, and oxygen supply along with an increased local temperature that may induce adverse effects on Leydig cell function and spermatogenesis. These conditions may lead to male subfertility or infertility. Our literature analyses and our own results suggest that conditions such as germ cell apoptosis and DNA damage are common features in hypoxia and varicocele and testicular torsion. Furthermore, oxidative damage seems to be present in these conditions during the initiation stages of germ cell damage and apoptosis. Other mechanisms like membrane-bound metalloproteinases and phospholipase A2 activation could also be part of the pathophysiological consequences of testicular hypoxia.

Effects of chronic hypobaric hypoxia on testis histology and round spermatid oxidative metabolism

In order to evaluate the effects of the exposition to continuous chronic hypobaric hypoxia (CCHH) and intermittent chronic hypobaric hypoxia (ICHH) on testis histology and on oxidative metabolism of spermatogenic cells (SC), male rats were exposed to a 4600‐m simulated altitude (PO2: 89.6 mmHg). After 60 days, ICHH and CCHH groups presented a significant decrease in testicular mass, an increase in interstitial space, a decrease in height of the seminiferous epithelium, depletion of cellular elements, vacuolization in epithelial cells and folding of the basal membrane. Round spermatids from animals exposed to CCHH presented a significant decrease in energy‐dependent cell shape changes. Round spermatid mitochondria of CCHH rats seem to be limited in their ability to handle reducing equivalents. These mitochondria also appear to be uncoupled under basal conditions. Round spermatids from CCHH rats evidence large oxygen consumption (QO2) insensitive to inhibition by cyanide, a process that could be partly related to lipoperoxidation. Thus, exposure of male rats to CCHH and ICHH induced evident changes in testicular morphology and loss of spermatogenic cells, in all stages of the spermatogenic cycle. This post‐meiotic spermatogenic cell loss in the testis correlated well with metabolic changes in round spermatids that evidenced a strong metabolic stress in these cells.

Cellular and molecular mechanisms in the hypoxic tissue: role of HIF-1 and ROS

Reactive oxygen species such as superoxide anion radicals (O2−) and hydrogen peroxide (H2O2) have for long time been recognized as undesirable by‐products of the oxidative mitochondrial generation of adenosine triphosphate (ATP). Recently, these highly reactive species have been associated to important signaling pathways in diverse physiological conditions such as those activated in hypoxic microenvironments. The molecular response to hypoxia requires fast‐acting mechanisms acting within a wide range of partial pressures of oxygen (O2). Intracellular O2 sensing is an evolutionary preserved feature, and the best characterized molecular responses to hypoxia are mediated through transcriptional activation. The transcription factor, hypoxia‐inducible factor 1 (HIF‐1), is a critical mediator of these adaptive responses, and its activation by hypoxia involves O2‐dependent posttranslational modifications and nuclear translocation. Through the induction of the expression of its target genes, HIF‐1 coordinately regulates tissue O2 supply and energetic metabolism. Other transcription factors such as nuclear factor κB are also redox sensitive and are activated in pro‐oxidant and hypoxic conditions. The purpose of this review is to summarize new developments in HIF‐mediated O2 sensing mechanisms and their interactions with reactive oxygen species–generating pathways in normal and abnormal physiology. Copyright

Oxidative Stress in Rat Testis and Epididymis Under Intermittent Hypobaric Hypoxia: Protective Role of Ascorbate Supplementation

Hypobaric hypoxia (HH), an environmental condition of high altitude encountered by mountaineers, miners, and observatory, rural health, border patrol, and rural education workers, jeopardizes normal physiologic functions in humans. The present study was conducted to evaluate the effects of intermittent HH (IHH; equivalent to 4600 m above mean sea level) on oxidative stress and the protective role of dietary ascorbic acid on rat testis and epididymis. Ten-week-old male Wistar rats were assigned to 1 of 6 groups: 1) normobaric (Nx), 2) Nx + physiologic solution (Nx + PS), 3) Nx + ascorbic acid (Nx + AA), 4) IHH, 5) IHH + PS, or 6) IHH + AA. Animals subjected to IHH were exposed for 96 hours followed by normobaric conditions for 96 hours for a total of 32 days. The control groups (2 and 5) were injected with doses of PS, and the treated groups (3 and 6) were injected with doses of AA (10 mg x kg(-1) body weight) at an interval of 96 hours. Rats were sacrificed on day 32 after initiation of the protocol. The testis and epididymis were collected to determine the activity and expression of glutathione reductase and the levels of lipid peroxide formation. An epididymal sperm count was also performed in each animal. The results of this study revealed that IHH induced lipid peroxidation, a reduction in glutathione reductase activity in testis and epididymis, and a significant decrease in epididymal sperm count. Treatment with AA prevented these changes. In conclusion, AA was capable of decreasing oxidative stress in testis and epididymis under IHH. This protection by AA of the IHH-induced lipid peroxidation can be explained in part by the preservation of glutathione reductase activity in these organs.

Melatonin protects the heart, lungs and kidneys from oxidative stress under intermittent hypobaric hypoxia in rats.

Melatonin (N-acetyl-5-methoxytryptamine) is the main secretory product of the pineal gland in all mammals including humans, but it is also produced in other organs. It has been previously demonstrated to be a powerful organ-protective substance under oxidative stress conditions. The aim of this study was to evaluate the protective effect of melatonin in several organs such as heart, lung, kidney, and of the reproductive system, such as testis and epididymis in animals exposed to intermittent hypobaric hypoxia and therefore exposed to oxidative stress and analyzed by lipid peroxidation. Ten-week-old male Wistar rats were divided into 6 groups for 96 hours during 32 days under: 1) Normobaric conditions, 2) plus physiologic solution, 3) plus melatonin, 4) intermittent hypobaric hypoxia, 5 plus physiologic solution and 6) plus melatonin. The animals were injected with melatonin (10 mg/kg body weight) at an interval of 96 hours during 32 days. Results indicated that melatonin decreased lipid peroxidation in heart, kidneys and lung under intermittent hypobaric hypoxia conditions. However, it did not exhibit any protective effect in liver, testis, epididymis and sperm count.

Effect of seminal plasma on Atlantic salmon (Salmo salar) sperm vitrification

This study was designed to test a vitrification method in Atlantic salmon spermatozoa and determine the capacity of seminal plasma (SP) to protect these cells from cryoinjuries. The vitrification medium consisted of a standard buffer for fish spermatozoa (Cortland medium) + 10% DMSO + 2% BSA + 0.13-M sucrose + SP at concentrations of 30% (G30), 40% (G40), or 50% (G50). Fresh sperm was used as a control. To freeze the samples, 30-μL suspensions of spermatozoa from each group were dropped directly into liquid nitrogen. The resulting spheres were placed in cryotubes for storage in liquid nitrogen. The cryotubes with the vitrified spermatozoa were thawed by placing them in a water bath at 37 °C for 45 seconds. After thawing, the following sperm quality parameters were determined by flow cytometry: DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labeling), plasma membrane integrity (SYBR-14/PI, staining technique), and mitochondrial membrane potential (JC-1 staining). An optical microscope was used to assess subjectively sperm motility, whereas fertility was determined by the presence of neurulation using five replicates per treatment in a sample of 30 eggs. Spermatozoa quality variables were preserved best when the highest concentration of SP (50%) was used (DNA fragmentation, 9.2%; plasma membrane integrity, 98.6%; mitochondrial membrane integrity, 47.2%; motility, 44.1%; and fertility, 46.2%).

Applications of recombinant Pichia pastoris in the healthcare industry

Since the 1970s, the establishment and development of the biotech industry has improved exponentially, allowing the commercial production of biopharmaceutical proteins. Nowadays, new recombinant protein production is considered a multibillion-dollar market, in which about 25% of commercial pharmaceuticals are biopharmaceuticals. But to achieve a competitive production process is not an easy task. Any production process has to be highly productive, efficient and economic. Despite that the perfect host is still not discovered, several research groups have chosen Pichia pastoris as expression system for the production of their protein because of its many features. The attempt of this review is to embrace several research lines that have adopted Pichia pastoris as their expression system to produce a protein on an industrial scale in the health care industry.

Omega 3 chronic supplementation attenuates myocardial ischaemia-reperfusion injury through reinforcement of antioxidant defense system in rats

Currently, controversial clinical data about the protective effects in the consumption of n‐3 polyunsaturated fatty acids (PUFAs) in ischaemic heart diseases exist. Improved myocardial resistance to ischaemia‐reperfusion (IR) injury results in non‐lethal myocardial infarction, which is a relevant factor in the myocardial function. We hypothesized that chronic supplementation with PUFAs reduced infarct size (IS) and induced an improvement on oxidative stress‐related parameters in IR model. Rats were supplemented with two doses of PUFAs D1 (n = 7) (0.6 g kg−1 d−1) and D2 (n = 7) (1.2 g kg−1 d−1) for 8 weeks. Control group (n = 7) received only standard diet. In ex vivo model, all rat hearts were subjected to 30 min of global ischaemia followed by 120 min of reperfusion. The IS and left ventricular function were assessed. Lipid peroxidation, reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio and antioxidant enzyme activity were measured in the whole heart. The results show a reduction in IS in a dose‐dependent manner with PUFAs D1 (30.6%) and D2 (48.5%) and higher values of left ventricular developed pressure, at the end of the reperfusion, for each dose, respectively (p < 0.05). The two PUFAs groups showed higher values of GSH/GSSG ratio and lipid peroxidation, and higher values of activity of antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase at basal condition (p < 0.05). At the end of reperfusion, the GSH/GSSG ratio and antioxidants enzyme activity did not show a significant drop in their values (p > 0.05). These findings suggested that the supplementation with PUFAs induces cardioprotection against IR injury, associated with reinforcement of the antioxidant defense system. Copyright

Acclimatization to chronic intermittent hypoxia in mine workers: a challenge to mountain medicine in Chile

In the past two decades, Chile has developed intense mining activity in the Andes mountain range, whose altitude is over 4,000 meters above sea level. It is estimated that a workforce population of over 55,000 is exposed to high altitude hypobaric hypoxia. The miners work under shift systems which vary from 4 to 20 days at the worksite followed by rest days at sea level, in a cycle repeated for several years. This Chronic Intermittent Hypoxia (CIH) constitutes an unusual condition for workers involving a series of changes at the physiological, cellular and molecular levels attempting to compensate for the decrease in the environmental partial pressure of oxygen (PO₂). The mine worker must become acclimatized to CIH, and consequently undergoes an acute acclimatization process when he reaches the worksite and an acute reverse process when he reaches sea level. We have observed that after a period of 3 to 8 years of CIH exposure workers acclimatize well, and evidence from our studies and those of others indicates that CIH induces acute and chronic multisystem adjustments which are effective in offsetting the reduced availability of oxygen at high altitudes. The aims of this review are to summarize findings of the physiological responses to CIH exposure, highlighting outstanding issues in the field.

Pharmacological models and approaches for pathophysiological conditions associated with hypoxia and oxidative stress.

Hypoxia is the failure of oxygenation at the tissue level, where the reduced oxygen delivered is not enough to satisfy tissue demands. Metabolic depression is the physiological adaptation associated with reduced oxygen consumption, which evidently does not cause any harm to organs that are exposed to acute and short hypoxic insults. Oxidative stress (OS) refers to the imbalance between the generation of reactive oxygen species (ROS) and the ability of endogenous antioxidant systems to scavenge ROS, where ROS overwhelms the antioxidant capacity. Oxidative stress plays a crucial role in the pathogenesis of diseases related to hypoxia during intrauterine development and postnatal life. Thus, excessive ROS are implicated in the irreversible damage to cell membranes, DNA, and other cellular structures by oxidizing lipids, proteins, and nucleic acids. Here, we describe several pathophysiological conditions and in vivo and ex vivo models developed for the study of hypoxic and oxidative stress injury. We reviewed existing literature on the responses to hypoxia and oxidative stress of the cardiovascular, renal, reproductive, and central nervous systems, and discussed paradigms of chronic and intermittent hypobaric hypoxia. This systematic review is a critical analysis of the advantages in the application of some experimental strategies and their contributions leading to novel pharmacological therapies.