Testicular Heat Stress Research Articles

Scrotal subcutaneous temperature is increased by scrotal insulation or whole-body heating, but not by scrotal neck insulation; however, all three heat-stress models decrease sperm quality in bulls and rams.

Ruminant testes are ~2-6°C below body temperature; increased testicular temperature reduces sperm motility and morphology. Our objective was to serially monitor scrotal subcutaneous temperatures during testicular heat stress and relate those to sperm quality. Two experiments were conducted, with temperature sensors surgically implanted in scrotal subcutaneous tissues recording temperatures every 15min and semen collected and evaluated weekly. After an initial control interval, testicular temperature was increased. In Experiment 1, in two Angus bulls, whole-scrotum insulation for 96h increased scrotal subcutaneous temperatures by ~2.0-2.5°C (P<0.05). Total and progressive motility decreased (P<0.05) and reached a nadir at Week 3 (~20 and 10%, respectively). Furthermore, morphologically normal sperm and acrosome integrity also decreased significantly, reaching nadirs at Weeks 3 (15%) and 4 (34%). In Experiment 2, 10 Dorset rams were allocated randomly into two equal groups and either: 1) exposed to 28°C ambient temperature and 30-34% RH for 8h/d for 4d; or 2) subjected to scrotal neck insulation that was applied and removed at the same time as the start and completion, respectively, of heat exposures in the other rams. Scrotal subcutaneous temperatures (monitored in three rams per group) were increased in response to whole-body heating (~0.8°C, P<0.05), but not significantly changed by scrotal neck insulation. Decreases in sperm quality were generally similar between treatments, with the most profound changes evident 4wk after heat stress, with ~10 percentage point reductions in both total and progressive motility and ~10 and 20 percentage point decreases in morphologically normal sperm in rams with whole-body heating versus scrotal neck insulation, respectively. In conclusion, scrotal subcutaneous temperature was significantly increased by scrotal insulation or whole-body heating, but not by scrotal neck insulation; however, all three heat-stress models decreased sperm motility and morphology in bulls and rams.

Adaptogenic potential of royal jelly in reproductive system of heat stress-exposed male rats.

Testicular heat stress (HS) can lead to testicular tissue destruction and spermatogenesis disturbances. Royal Jelly (RJ) has been introduced as a potent antioxidant. We investigated the effects of RJ on testicular tissue, oxidative stress and sperm apoptosis in HS-exposed rats. Compared to HS-exposed groups, RJ co-treatment could improve testosterone reduction and histopathological damages. The RJ co-administration decreased MDA level in testicular tissue, while TAC and CAT levels were remarkably increased compared to HS-exposed groups. Moreover, significant higher expression level of Bcl-2 and lower expression levels of P53 and Caspase-3 were seen following RJ co-administration compared to HS-exposed groups. Our data suggest that RJ can effectively ameliorate experimental HS-induced testiculopathies in rats through testicular antioxidant defense system restoration and germ cells apoptosis regulation.

PSII-33 Mitigation of testicular heat stress in boars with PG600

Abstract Testicular heat stress due to increasing summer temperatures negatively impacts spermatogenesis reducing semen quality in boars. Scrotal insulation (SI) can mimic summer heat stress. Our hypothesis is that PG600 can mitigate heat stress in boars. PG600 is human chorionic gonadotropin (HCG) and equine chorionic gonadotropin (ECG) which have LH- and FSH-like effects respectively. During spermatogenesis, LH impacts testosterone production by Leydig cells and FSH impacts Sertoli cells to produce androgen receptors, inhibit apoptotic signals and sustain spermatogenesis. Eight boars (8–12 months of age) were randomly assigned to two treatment groups: SI + saline or SI + PG600. The SI was applied for 48 hrs. Injections were administered 24 hrs prior to SI application and again at onset of SI. PG600 was given at a standard gilt dose to induce puberty. Semen was collected every M-W-F for two weeks prior to treatment and 44 days post-treatment. Semen was evaluated for nuclear head shape via Fourier harmonic analysis (FHA) described as Harmonic amplitudes 0–5 (HA0-5). Semen for each collection day post-treatment was compared to the average of the semen collection days pre-treatment, described as day 0. Scrotal insulation produced a 3.7°C increase in average scrotal temperature for 48 hrs. Post-SI + saline, HA0 decreased on days 21–33 (P &amp;lt; 0.05), HA1 increased on day 30 (P &amp;lt; 0.05), HA2 decreased on day 35 (P &amp;lt; 0.05), HA3 increased on days 21–33 (P &amp;lt; 0.05), HA4 increased on days 21–26 (P &amp;lt; 0.05), and HA5 increased on days 21–30 (P &amp;lt; 0.05). Post-SI + PG600, decreased the number of days post-SI when changes in harmonic amplitudes were seen (P &amp;lt; 0.05) as well as the magnitude of the response (P &amp;lt; 0.05). PG600 was for the first time shown to significantly mitigated the response of boars to SI and testicular heat stress. Increased doses of PG600 may improve results.

Acute mild heat stress alters gene expression in testes and reduces sperm quality in mice

Heat stress is a major concern in animal reproduction, as testicular temperature must be 3–5 °C below body core temperature for production of motile and fertile sperm in mammals. Although recent studies concluded that increased temperature per se was the underlying pathophysiology of testicular impairment, more studies are required to better understand the mechanisms. Therefore, our objective was to investigate the impacts of mild acute heat stress on sperm and testes, and based on mRNA, elucidate involvement of StAR, Trp53 and Trp53-dependent intrinsic and extrinsic apoptotic pathways in pathophysiology of testicular heat stress. Forty-eight C57 BCL6 elite male mice were equally allocated into six groups, anesthetized and the distal third of their body immersed in a water-bath at 40 or 30 °C (heat treatment and control, respectively) for 20 min. Intervals from heat exposure (Day 0) to euthanasia were: 8 and 24 h and 7, 14 and 21 d (plus a control group at 14 d). The epididymides were excised, minced and placed in Tyrode albumin lactate pyruvate hepes (TALPH) at 37 °C for 15 min to recover sperm. Based on computer assisted sperm analysis (CASA), heat treatment reduced total and progressive motility ∼40% (P < 0.05) on Days 14 and 21. Furthermore, percentage morphologically normal sperm was significantly decreased on Day 7, with greater reductions on Days 14 and 21, mostly due to increased midpiece defects. Acrosome integrity (FITC PSA) was decreased ∼35% at 8 h (P < 0.05) and reached a nadir on Day 14. There were decreases (P < 0.05) in seminiferous tubule diameter and testicular weight (relative to body weight) on Day 14. Testicular RNA was extracted, reverse-transcribed and cDNA used for PCR. Expression of genes Hspa1b (Hsp70) and Gpx1 had 7- and 10-fold increases (P < 0.001 for each) at 8 and 24 h, respectively, with Hspa1b remaining upregulated at 24 h, whereas StAR peaked at Day 14 (15-fold, P < 0.0001) and had returned to baseline on Day 21. Both Trp53 and Casp8 were upregulated (P < 0.05) on Day 14, whereas Bcl-2 was decreased (P < 0.05) on Days 7 and 14. In conclusion, acute mild heat stress severely reduced sperm quality and based on mRNA, there was upregulation of chaperone and antioxidant systems and Trp53-dependent intrinsic and extrinsic apoptotic pathways, with deleterious effects on sperm, spermatocytes and spermatids. These findings provided insights into the pathophysiology of heat stress and should contribute to development of evidence-based approaches to mitigate effects of testicular heating.

Heat stress effects on bovine sperm cells: a chronological approach to early findings.

Testicular heat stress affects sperm quality and fertility. However, the chronology of these effects is not yet fully understood. This study aimed to establish the early sequential effects of heat stress in bull sperm quality. Semen and blood samples of Nellore breed bulls were collected and distributed into control and testicular heat stress (scrotal bags/96h) groups. Semen samples were evaluated for sperm motility, abnormalities, plasma membrane integrity, acrosomal membrane integrity, mitochondrial membrane potential, sperm lipid peroxidation, seminal plasma lipid peroxidation, and DNA fragmentation. Blood plasma was also evaluated for lipid peroxidation. An increase in sperm abnormalities was observed 7days following heat stress. After 14days, sperm lipid peroxidation increased and mitochondrial membrane function, sperm motility, and plasma membrane integrity decreased. Heat stress effects were still observed after 21days following heat stress. An increase in sperm DNA fragmentation was observed as a late effect after 28days. Thus, the initial effects of heat stress (i.e., increasing sperm abnormalities and lipid peroxidation) suggest the presence of oxidative stress in the semen that alters mitochondrial function, sperm motility, plasma membrane integrity, and belatedly, DNA fragmentation. Although sperm abnormalities persisted and increased over time, sperm lipid peroxidation, in turn, increased only until 21days after heat stress. In this regard, these findings provide a greater understanding of the chronological effects of experimentally induced heat stress on bovine sperm, providing valuable insights about spermatogenesis during the first 28days following heat stress.

Effects of increased scrotal temperature on semen quality and seminal plasma proteins in Brahman bulls.

Environmental temperature has effects on sperm quality with differences in susceptibility between cattle subspecies and breeds, but very little is known about the seminal plasma protein (SPP) changes resulting from testicular heat stress. Scrotal insulation (SI) for 48 hr was applied to Brahman (Bos indicus) bulls. Semen was collected at 3-day intervals from before, until 74 days post-SI. The changes in sperm morphology and motility following SI were comparable to previously reported and differences were detected in measures of sperm chromatin conformation as early as 8 days post-SI. New proteins spots, in the SPP two-dimensional (2-D) gels, were apparent when comparing pre-SI with 74 days post-SI, and SPP identified as associated with mechanisms of cellular repair and protection. Similar trends between 2-D gel and Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) data was observed, with SWATH-MS able to quantify individual SPP that otherwise were not resolved on 2-D gel. The SPP assessment at peak sperm damage (21-24 days) showed a significant difference in 29 SPP (adjusted p < .05), and identified six proteins with change in abundance in the SI group. In conclusion both spermatozoa and SPP composition of bulls are susceptible to temperature change incurred by SI, and SPP markers for testicular heat insults may be detected.

Melatonin protects the mouse testis against heat-induced damage.

Spermatogenesis, an intricate process occurring in the testis, is responsible for ongoing production of spermatozoa and thus the cornerstone of lifelong male fertility. In the testis, spermatogenesis occurs optimally at a temperature 2-4°C lower than that of the core body. Increased scrotal temperature generates testicular heat stress and later causes testicular atrophy and spermatogenic arrest, resulting in a lower sperm yield and therefore impaired male fertility. Melatonin (N-acetyl-5-methoxytryptamine), a small neuro-hormone synthesized and secreted by the pineal gland and the testis, is widely known as a potent free-radical scavenger; it has been reported that melatonin protects the testis against inflammation and reactive oxygen species generation thereby playing anti-inflammatory, -oxidative and -apoptotic roles in the testis. Nevertheless, the role of melatonin in the testicular response to heat stress has not been studied. Here, by employing a mouse model of testicular hyperthermia, we systematically investigated the testicular response to heat stress as well as the occurrence of autophagy, apoptosis and oxidative stress in the testis. Importantly, we found that pre-treatment with melatonin attenuated heat-induced apoptosis and oxidative stress in the testis. Also, post-treatment with melatonin promoted recovery of the testes from heat-induced damage, probably by maintaining the integrity of the Sertoli cell tight-junction. Thus, we for the first time provide the proof of concept that melatonin can protect the testis against heat-induced damage, supporting the potential future use of melatonin as a therapeutic drug in men for sub/infertility incurred by various testicular hyperthermia factors.

Cold-induced RNA-binding protein (CIRBP) regulates the expression of Src-associated during mitosis of 68 kDa (Sam68) and extracellular signal-regulated kinases (ERK) during heat stress-induced testicular injury

In this study, the ability of cold-induced RNA-binding protein (CIRBP) to regulate the expression of Src-associated during mitosis of 68 kDa (Sam68) and extracellular signal-regulated kinases (ERK) in the mouse testis and mouse primary spermatocytes (GC-2spd cell line) before and after heat stress was examined to explore the molecular mechanism by which CIRBP decreases testicular injury. A mouse testicular hyperthermia model, a mouse primary spermatocyte hyperthermia model and a low CIRBP gene-expression cell model were constructed and their relevant parameters were analysed. The mRNA and protein levels of CIRBP and Sam68 were significantly decreased in the 3-h and 12-h testicular heat-stress groups, extracellular signal-regulated kinase 1/2 (ERK1/2) protein expression was not significantly affected but phospho-ERK1/2 protein levels were significantly decreased. GC-2spd cellular heat-stress results showed that the mRNA and protein concentrations of CIRBP and Sam68 were reduced 48h after heat stress. In the low CIRBP gene-expression cell model, CIRBP protein expression was significantly decreased. Sam68 mRNA expression was significantly decreased only at the maximum transfection concentration of 50nM and Sam68 protein expression was not significantly affected. These findings suggest that CIRBP may regulate the expression of Sam68 at the transcriptional level and the expression of phospho-ERK1/2 protein, both of which protect against heat-stress-induced testicular injury in mice.

Impact of a mild scrotal heating on sperm chromosomal abnormality, acrosin activity and seminal alpha-glucosidase in human fertile males.

The aim of this study was to observe sperm aneuploidy, DNA integrity, seminal alpha-glucosidase (NAG) and acrosin activity (AA) under testicular heat stress (SH). Spermatozoa were obtained from 30 healthy adult volunteers subjected to scrotal warming at 43°C for 30-40min on two successive days per week for 3months between February 2012 and September 2016. Aniline blue (AB), acridine orange (AO) staining, TUNEL assay and FISH analysis to evaluate sperm function, sperm DNA integrity and chromosomal abnormalities were carried on before, during and after SH. Sperm AA and NAG was measured by microplate reader. The mean parameters of sperm parameters, AA and NAG were significantly decreased. In contrast, the mean percentage of sperm DNA fragmentation and the proportion of aneuploidy of chromosomes 13, 18, 21, X and Y were significantly increased for spermatozoa collected during SH versus before SH (p<.01-.001). After stopping scrotal heating for 3months, most parameters were completely restored to pre-SH levels. Sperm parameters, sperm DNA integrity, chromosomes, AA and NAG are affected by scrotal exposure to constant SH temperatures several degrees over normal physiological temperature, and after treatment, these parameters were reversibly restored to the level before SH in adult men.

Lifestyle causes of male infertility

ObjectiveTo examine the potential effects of lifestyle factors on male reproductive health. Evidence of a global decline in human sperm quality over recent decades has been accumulating. Environmental, occupational, and modifiable lifestyle factors may contribute to this decline. This review focuses on key lifestyle factors that are associated with male infertility such as smoking cigarettes, alcohol intake, use of illicit drugs, obesity, psychological stress, advanced paternal age, dietary practices, and coffee consumption. Other factors such as testicular heat stress, intense cycling training, lack of sleep and exposure to electromagnetic radiation from mobile phone use are briefly discussed. Materials and methodA comprehensive literature search was performed to identify and synthesise all relevant information, mainly from within the last decade, on the major lifestyle factors associated with male infertility and semen quality. Database searches were limited to reports published in English only. A manual search of bibliographies of the reports retrieved was conducted to identify additional relevant articles. ResultsIn all, 1012 articles were identified from the database search and after reviewing the titles and abstract of the reports, 104 articles met the inclusion criteria. Of these, 30 reports were excluded as the full-text could not be retrieved and the abstract did not have relevant data. The remaining 74 reports were reviewed for data on association between a particular lifestyle factor and male infertility and were included in the present review. ConclusionThe major lifestyle factors discussed in the present review are amongst the multiple potential risk factors that could impair male fertility. However, their negative impact may well be mostly overcome by behaviour modification and better lifestyle choices. Greater awareness and recognition of the possible impact of these lifestyle factors are important amongst couples seeking conception.

Effect of Heat Stress on Sperm DNA: Protamine Assessment in Ram Spermatozoa and Testicle.

Sperm DNA fragmentation is considered one of the main causes of male infertility. The most accepted causes of sperm DNA damage are deleterious actions of reactive oxygen species (ROS), defects in protamination, and apoptosis. Ram sperm are highly prone to those damages due to the high susceptibility to ROS and to oxidative stress caused by heat stress. We aimed to evaluate the effects of heat stress on the chromatin of ejaculated and epididymal sperm and the activation of apoptotic pathways in different cell types in ram testis. We observed higher percentages of ejaculated sperm with increased chromatin fragmentation in the heat stress group; a fact that was unexpectedly not observed in epididymal sperm. Heat stress group presented a higher percentage of spermatozoa with DNA fragmentation and increased number of mRNA copies of transitional protein 1. Epididymal sperm presented greater gene expression of protamine 1 on the 30th day of the spermatic cycle; however, no differences in protamine protein levels were observed in ejaculated sperm and testis. Localization of proapoptotic protein BAX or BCL2 in testis was not different. In conclusion, testicular heat stress increases ram sperm DNA fragmentation without changes in protamination and apoptotic patterns.

Attenuation of heat stress-induced spermatogenesis complications by betaine in mice

High temperatures can induce oxidative stress, impairment of spermatogenesis, and reduction of sperm quality and quantity concomitant with transient periods of partial or complete infertility in male mammals. Promising beneficial effects of betaine supplementation on the epididymal spermatozoa have been reported in experimental studies; however, its effects on testicular heat stress (HS)-induced impairment have yet to be determined. In the present study, betaine (Bet) was orally administrated (250 mg/kg day) during a 14-day period, before (Bet + HS group) or after (HS + Bet group) induction of testicular HS in 7–9 week-old male mice. HS was induced by testicular immersion in water at 42 °C in stress groups. Epididymal spermatozoa and testes were collected at days 14 and 28 after HS induction in order to analyze sperm characteristics, testicular oxidative status, and histological changes. Our studies showed that HS reduced testicular weight, the quality and quantity of epididymal spermatozoa, and impaired maturation of germinal cells. The levels of MDA, catalase, SOD, and GPX were increased in the testes of HS-induced mice (P < 0.01). Although betaine treatment before and after exposure to HS enhanced antioxidant defense (P < 0.05) and accelerated germinal epithelium regeneration, its effects on the characteristics of epididymal spermatozoa were scarce. On the other hand, in the absence of heat stress, quality and quantity of epididymal spermatozoa were improved following 14 days of betaine consumption. Our study revealed the beneficial effect of betaine on HS-induced complications of spermatogenesis, as well as its potency to improve epididymal spermatozoa in intact mice.

Effect of Vitamin E and Polyunsaturated Fatty Acids on Cryopreserved Sperm Quality in Bos taurus Bulls Under Testicular Heat Stress

ABSTRACTTaurine bulls are highly susceptible to heat stress, leading to increased oxidative stress (OS) and impaired sperm viability. Polyunsaturated fatty acids (PUFAs) supplementation can be an alternative to improve semen quality, which also results in more sperm susceptibility to lipid peroxidation. Moreover, this deleterious effect can be exacerbated in animals affected by heat stress. Vitamin E is a key antioxidant that counteracts lipid peroxidation of sperm membrane caused by OS. Thus, combining PUFAs with vitamin E may improve sperm quality. In this context, this study aimed to evaluate the effect of interaction between PUFAs and vitamin E on sperm quality in Bos taurus bulls under testicular heat stress. Sixteen taurine bulls under testicular heat stress were randomly assigned in four groups: Control, Vitamin E, PUFA, and PUFA + Vitamin E. All groups lasted for 60 days. Samples were cryopreserved/thawed and analyzed for motility variables (CASA), membrane and acrosome integrity, mitochondrial activity, susceptibility to oxidative stress, DNA integrity, and sperm-binding capacity. Results showed that vitamin E had a beneficial effect on some sperm characteristics, whereas PUFA supplementation had an adverse effect when the two treatments were evaluated separately. Finally, the association between PUFAs and vitamin E did not improve sperm quality.

Recovery of normal testicular temperature after scrotal heat stress in rams assessed by infrared thermography and its effects on seminal characteristics and testosterone blood serum concentration

Reestablishment of testicular normal temperature after testicular heat stress is unknown and its effect varies widely. The aim of this study was to investigate the impact of scrotal insulation (IN) on testicular temperature and its relation to semen quality and testosterone blood serum concentration. For this, 33 rams were used; 17 submitted to IN for 72 hours (using bags involving the testes) and 16 not submitted to IN (control group). The experiment was performed between August and December 2013 in Pirassununga, Brazil (21°56″13″ South/47°28′24″ West). Seminal characteristics, testosterone blood serum concentration, rectal temperature (RT), respiratory frequency, scrotal superficies mean temperature (SSMT), and eye area mean temperature (EAMT) were analyzed 7 days before IN and 21, 35, 49, 63, and 90 days afterward. Scrotal superficies mean temperature and EAMT were measured by thermography camera FLIR T620. Testosterone was evaluated by radioimmunoassay. Analysis of variance was used to determine the main effects of treatment, time, and treatment-by-time interaction using PROC MIXED of SAS software adding command REPEAT. Pearson correlation test was used to verify correlation between SSMT, EAMT, RT, and respiratory frequency. Significant difference was considered when P ≤ 0.05. At the end of IN, SSMT was higher (P < 0.05) in insulated group (32.26 ± 0.19oC) than in control group (30.58 ± 0.18oC), and the difference between rectal and testicular (deduced from SSMT) temperatures was 1.12 °C; in the other times of the evaluation this difference was between 2.91 and 4.25 °C in IN group. Scrotal superficies mean temperature was reestablished 24 hours after IN. Rectal temperature and EAMT presented correlation (r = 0.59; P < 0.0001). There was time-by-treatment interaction for total sperm (P = 0.0038) and progressive motility (P = 0.01), abnormal spermatozoa (P < 0.0001), membranes integrity (P < 0.0001), induced thiobarbituric acid reactive substances (TBARSs; P = 0.05), and DNA integrity (P = 0.0004). These semen characteristics were negatively affected 21 days after IN, and excluding induced TBARSs and abnormalities, recovered 35 days afterward; induced TBARSs just were affected after 49 days of IN; sperm abnormalities just recovered after 63 days. Testosterone blood serum concentration was lesser in insulated rams (P = 0.03). Thus, the difference of 1.12 °C between RT and testicular temperature impacts semen quality and testosterone blood serum concentration. Moreover, this study shows that rams can recover testes temperature efficiently toward IN and that infrared thermography is an efficient tool to identify differences on SSMT.

Evaluation of Lasting Effects of Heat Stress on Sperm Profile and Oxidative Status of Ram Semen and Epididymal Sperm.

Higher temperatures lead to an increase of testicular metabolism that results in spermatic damage. Oxidative stress is the main factor responsible for testicular damage caused by heat stress. The aim of this study was to evaluate lasting effects of heat stress on ejaculated sperm and immediate or long-term effects of heat stress on epididymal sperm. We observed decrease in motility and mass motility of ejaculated sperm, as well as an increase in the percentages of sperm showing major and minor defects, damaged plasma and acrosome membranes, and a decrease in the percentage of sperm with high mitochondrial membrane potential in the treated group until one spermatic cycle. An increased enzymatic activity of glutathione peroxidase and an increase of stressed cells were observed in ejaculated sperm of the treated group. A decrease in the percentage of epididymal sperm with high mitochondrial membrane potential was observed in the treated group. However, when comparing immediate and long-term effects, we observed an increase in the percentage of sperm with low mitochondrial membrane potential. In conclusion, testicular heat stress induced oxidative stress that led to rescuable alterations after one spermatic cycle in ejaculated sperm and also after 30 days in epididymal sperm.

Testosterone production by a Leydig tumor cell line is suppressed by hyperthermia-induced endoplasmic reticulum stress in mice

AimsLeydig cells are characterized by their ability to produce testosterone. When the Leydig cells are unable to produce enough testosterone, spermatogenesis fails completely. Considering this, it is of great interest to investigate whether the expressions of steroidogenic enzymes are affected by testicular heat stress. This study aimed to demonstrate that heat induced ER-stress significantly influences steroidogenic enzyme expression and testosterone production in the Leydig cells. Main methodsC57BL/6 mice were subjected to repetitive testicular heat-treatment at 42°C for 15min per day, and heat-treated mLTC-1 cells following hCG treatment for 1h. The protein and RNA expressions were measured by Western blot, RT-PCR. The testosterone and progesterone levels were detected by EIA. The histological and pathological characteristics using hematoxylin and eosin (H&E) and antibody stains. Key findingsThe 3β-HSD expression was decreased by heat-stress and hCG treatment. While the GRP78/BiP and CHOP levels were increased by ER-stress inducers, those of the steroidogenic enzyme and progesterone were decreased. In contrast, an ER-stress inhibitor rescued the testosterone levels, even under heat-stress conditions. Moreover, the Leydig cells were randomly scattered, and severely damaged upon repetitive testicular heat-treatment. Additionally, immunohistochemical analyses revealed that cleaved caspase-3 was elevated in the testicular Leydig cells, and rescued by TUDCA. Thus, repetitive testicular heat-treatment in mice promotes excessive ER-stress, thereby leading to apoptosis of the Leydig cells and thus, decreased testosterone production. SignificanceOur findings help to provide an ER-stress mediate mechanistic explanation to the impairment of spermatogenesis upon elevation of the testicular temperature.

Unexpected requirement for a binding partner of the syntaxin family in phagocytosis by murine testicular Sertoli cells.

Testicular phagocytosis by Sertoli cells (SCs) plays an essential role in the efficient clearance of apoptotic spermatogenic cells under both physiological and pathological conditions. However, the molecular mechanism underlying this unique process is poorly understood. Herein, we report for the first time that α-taxilin protein (TXLNA), a binding partner of the syntaxin family that functions as a central player in the intracellular vesicle traffic, was dominantly expressed in SCs. Induction of apoptosis in murine meiotic spermatocytes and haploid spermatids by busulfan treatment stimulated a significant increase of TXLNA in SCs at day (d) 14 and d 24 after busulfan treatment, respectively. Consistently, TXLNA expression was steadily upregulated when SCs were co-cultured with apoptotic germ cells (GCs). Moreover, using siRNA treatment, we found that ablation of endogenous TXLNA significantly impaired the phagocytotic capacity of SCs and thereby resulted in defective spermiogenesis and reduced fertility during the late recovery after testicular heat stress. Mechanistically, upregulation of TXLNA expression by apoptotic GCs was associated with the stabilization of ATP-binding cassette transporter 1 (ABCA1), a transporter-mediated lipid efflux from SCs and influencing male fertility. TXLNA acted as an upstream suppressor of ABCA1 ubiquitination and thus promoted ABCA1 stability and accumulation following GC apoptosis. We further provide in vitro evidence that epidermal growth factor receptor (EGFR)-mediated phosphorylation regulated ABCA1 ubiquitination and was enhanced by TXLNA deficiency during testicular phagocytosis. Taken together, the TXLNA/ABCA1 cascade may serve as an important feedback mechanism to modulate the magnitude of subsequent phagocytotic process of SCs in response to testicular injury.

Effects of testicle insulation on seminal traits in rams: Preliminary study

To evaluate the effect of heat stress on sperm characteristics, as well as testicular biometrics and heat radiation, six breeds of rams: two Brazilian locally adapted breeds (Santa Ines and Bergamasca) and four exotic breeds (Dorper, Texel, Ile de France, and Hampshire Down) underwent scrotal insulation. The experiment consisted of pre-scrotal insulation (week −1), insulation (week 0), and post-insulation (week 1–11) phases. Insulation changed the scrotum and testicular measurements, the heat radiated from the gonads caused deleterious effects, both on the process of spermatogenesis and sperm maturation in the epididymis. However, all the characteristics studied showed reversibility at 11 weeks post-insulation, showing the ability of the seminiferous epithelium to react to the effects of environmental factors. The locally, adapted breeds were more resistant than exotic to testicular heat stress.

The impact of sedentary work on sperm nuclear DNA integrity

Contemporary professional jobs that often enforce a sedentary lifestyle and are frequently associated with testicular overheat, deserve special attention with respect to male fertility potential. Interestingly, the harmful effect of testicular heat stress on sperm characteristics including nuclear DNA integrity was well characterized; however, the influence of sedentary work on sperm chromatin has not yet been documented. Therefore, our research was designed to examine the potential effects of sedentary work not only on conventional semen features but also on sperm nuclear DNA status. The study was carried out on ejaculated sperm cells obtained from men who spent ≥ 50% of their time at work (≥ 17.5 h per week) in a sedentary position (n = 152) and from men who spent < 50% of their time at work in a sedentary position (n = 102). Standard semen characteristics were assessed according to the WHO 2010 recommendations, while sperm nuclear DNA fragmentation (SDF) was evaluated using the Halosperm test. There were no significant differences in the standard semen parameters between the study groups. The groups differed only in SDF parameter. The men who spent at least 50% of their work time in a sedentary position had a higher proportion of SDF than the men who spent < 50% of their time at work in a sedentary position (median value 21.00% vs. 16.50%, respectively). The incidence of low SDF levels (related to 0-15% sperm cells with abnormal DNA dispersion) was significantly lower (27.63% vs. 45.10%), the percentage of men with high SDF levels (related to > 30%) was significantly higher (30.92% vs. 16.67%) in group of men who spent at least 50% of their work time in a sedentary positon. Furthermore, these men were more than twice as likely to have not a low SDF level (OR: 0.4648) and had more than twice the risk of having a high SDF level (OR: 2.2381) than the men in less sedentary occupations. Despite lack of association between sedentary work and conventional semen characteristics our study revealed detrimental effect of seated work on sperm nuclear DNA integrity. A sedentary job doubled the risk of high levels of sperm DNA damage. The pathomechanism could be related to testicular heat stress resulting in sperm chromatin remodelling failure during spermiogenesis. Therefore, it seems reasonable to simultaneously carry out routine seminological analyses and tests assessing sperm chromatin status while diagnosing male infertility.

262 EFFECT OF SYSTEMIC ANTIOXIDANT TREATMENT IN BOS TAURUS TAURUS BULLS UNDER HEAT STRESS AND SUPPLEMENTED WITH POLYUNSATURATED FATTY ACIDS

One reason for lower fertility of European bulls in tropical regions is a higher rate of oxidative stress caused by increased production of reactive oxygen species (ROS) not compensated by antioxidant protection. In that regard, sperm are extremely susceptible to oxidative stress due to a high concentration of polyunsaturated fatty acids (PUFA) in their plasma membranes. However, the presence of these PUFA is fundamental for sperm to be fertile and resistant to cold shock. Thus, treatments that suppress oxidation may increase productivity of these animals. This study aimed to evaluate the most damaging ROS for European bulls subjected to heat stress and to determine a possible antioxidant-targeted treatment. In a second step, we sought to verify the efficiency of the interaction between a diet rich in PUFA and a targeted antioxidant treatment on the quality of ejaculated and epididymal sperm in European bulls subjected to testicular heat stress. Four Bos taurus bulls were subjected to scrotal insulation for 5 days, with semen collection (electroejaculation) 60 days after insulation. Semen from each bull was divided into 4 aliquots and subjected to 4 ROS-generating systems: superoxide anion (xanthine/xanthine oxidase), hydrogen peroxide, hydroxyl radical (ascorbate + ferrous sulfate), and malondialdehyde (MDA; lipid peroxidation product). Samples were incubated for 1 h and assessed by computerized sperm analysis (CASA); eosin/nigrosin (membrane integrity); fast-green/Bengal rose (acrosome integrity); 3,3′ diaminobenzidine (mitochondrial activity); sperm chromatin structure assay (DNA fragmentation); and thiobarbituric acid reactive substances (lipid peroxidation). Overall, MDA had the most deleterious effects on semen quality of Bos taurus bulls subjected to acute heat stress. Thereafter, 16 bulls were subjected to testicular insulation and allocated into 4 groups: control (n = 4; given mineral oil; placebo); vitamin E (n = 4, given 5 mL of Monovin® every 13 days); PUFA (n = 4; given 4 kg day–1 Megalac®); and PUFA+vitamin E (n = 4; combination of PUFA and vitamin E treatment groups). Semen was collected on the day of installation of the insulation, on the day it was removed, and 30 and 60 days later. Overall, vitamin E reduced heat stress-induced damage to sperm DNA and mitochondria, but only in samples collected from the epididymis. Similarly, the combination of vitamin E and PUFA supplementation improved sperm motility patterns. Therefore, a combined antioxidant treatment (vitamin E and PUFA) may reduce damage to sperm caused by acute heat stress in European bulls. However, this treatment may be more effective if instituted before heat stress.