Cellular responses and microRNA profiling in bovine spermatozoa under heat shock.

In brief: Elevated temperatures disturbed sperm physiology. Bovine sperm cells exposed to heat shock led to diminished mitochondrial activity, fertilizing ability, increased oxidative stress and caspase activity concomitant with a delay in embryonic developmental kinetics and modulation of sperm-borne microRNAsmiRNAs. Abstract: Sperm function is susceptible to adverse environmental conditions. It has been demonstrated that in vivo and in vitro exposure of bovine sperm to elevated temperature reduces sperm motility and fertilizing potential. However, the cascade of functional, cellular, and molecular events triggered by elevated temperature in the mature sperm cell remains not fully understood. Therefore, the aim of this study was to determine the effect of heat shock on mature sperm cells. Frozen-thawed Holstein sperm were evaluated immediately after Percoll purification (0 h non-incubation control) or after incubation at 35, 38.5, and 41°C for 4 h. Heat shock reduced sperm motility after 3-4 h at 41°C while mitochondrial activity was reduced by 38.5 and 41°C when compared to the control. Heat shock also increased sperm reactive oxygen species production and caspase activity. Heat-shocked sperm had lower fertilizing ability, which led to diminished cleavage and blastocyst rates. Preimplantation embryo developmental kinetics was also slowed and reduced by sperm heat shock. The microRNA (miR) profiling identified >300 miRs in bovine sperm. Among these, three and seven miRs were exclusively identified in sperm cells exposed to 35 and 41°C, respectively. Moreover, miR-181d was enriched in sperm cells exposed to higher temperatures. Hence, elevated temperature altered the physiology of mature sperm cells by perturbing cellular processes and the miR profile, which collectively led to lower fertilizing ability and preimplantation development.

Dissecting EPPIN protease inhibitor domains in sperm motility and fertilizing ability: repercussions for male contraceptive development.

EPPIN (epididymal protease inhibitor) is a mammalian conserved sperm-binding protein displaying an N-terminal WFDC (whey-acidic protein four-disulfide core) and a C-terminal Kunitz protease inhibitor domains. EPPIN plays a key role in regulating sperm motility after ejaculation via interaction with the seminal plasma protein SEMG1 (semenogelin-1). EPPIN ligands targeting the SEMG1 binding site in the Kunitz domain are under development as male contraceptive drugs. Nevertheless, the relative contributions of EPPIN WFDC and Kunitz domains to sperm function remain obscure. Here, we evaluated the effects of antibodies targeting specific epitopes in EPPIN's WFDC (Q20E antibody, Gln20-Glu39 epitope) and Kunitz (S21C and F21C antibodies, Ser103-Cys123 and Phe90-C110 epitopes, respectively) domains on mouse sperm motility and fertilizing ability. Computer-assisted sperm analysis showed that sperm co-incubation with S21C antibody (but not F21C antibody) lowered progressive and hyperactivated motilities and impaired kinematic parameters describing progressive (straight-line velocity; VSL, average path velocity; VAP and straightness; STR) and vigorous sperm movements (curvilinear velocity; VCL, amplitude of lateral head movement; ALH, and linearity; LIN) compared with control. Conversely, Q20E antibody-induced milder inhibition of progressive motility and kinematic parameters (VAP, VCL and ALH). Sperm co-incubation with S21C or Q20E antibodies affected in vitro fertilization as revealed by reduced cleavage rates, albeit without changes in capacitation-induced tyrosine phosphorylation. In conclusion, we show that targeting specific epitopes in EPPIN Kunitz and WFDC domains inhibits sperm motility and capacitation-associated events, which decrease their fertilizing ability; nevertheless, similar observations in vivo remain to be demonstrated. Simultaneously targeting residues in S21C and Q20E epitopes is a promising approach for the rational design of EPPIN-based ligands with spermostatic activity.

Contextualizing Autophagy during Gametogenesis and Preimplantation Embryonic Development.

Mammals face environmental stressors throughout their lifespan, which may jeopardize cellular homeostasis. Hence, these organisms have acquired mechanisms to cope with stressors by sensing, repairing the damage, and reallocating resources to increase the odds of long-term survival. Autophagy is a pro-survival lysosome-mediated cytoplasm degradation pathway for organelle and macromolecule recycling. Furthermore, autophagy efflux increases, and this pathway becomes idiosyncratic depending upon developmental and environmental contexts. Mammalian germ cells and preimplantation embryos are attractive models for dissecting autophagy due to their metastable phenotypes during differentiation and exposure to varying environmental cues. The aim of this review is to explore autophagy during mammalian gametogenesis, fertilization and preimplantation embryonic development by contemplating its physiological role during development, under key stressors, and within the scope of assisted reproduction technologies.

Applicability of Raman spectroscopy on porcine parvovirus and porcine circovirus type 2 detection.

Porcine parvovirus (PPV) is one of the major infectious causes of reproductive failure of swine. This disease is characterized by embryonic and fetal infection and death, responsible for important economic losses. PPV is also implicated as a trigger in the development of post-weaning multisystemic wasting syndrome (PMWS) caused by Porcine circovirus type 2 (PCV2). Their detection is PCR-based, which is quite sensitive and specific, but laborious, costly and time-demanding. Therefore, this study aimed to assess Raman spectroscopy (RS) as a diagnostic tool for PPV and PCV2 due to its label-free properties and unique ability to search and identify molecular fingerprints. Briefly, swine testis (ST) cells were inoculated with PPV or PCV2 and in vitro cultured (37 °C, 5% CO2) for four days. Fixed cells were then submitted to RS investigation using a 633 nm laser. A total of 225 spectra centered at 1300 cm-1 was obtained for each sample (5 spectra/cell; 15 cells/replicate; 3 replicates) of PPV-, PCV2-infected and uninfected (control) ST cells. Clear statistical discrimination between samples from both virus-infected cells was achieved with a Principal Component - Linear Discriminant Analysis (PCA-LDA) model, reaching sensitivity rates from 95.55% to 97.77%, respectively to PCV2- and PPV-infected cells. These results were then submitted to a Leave-One-Out (LOO) validation algorithm resulting in 99.97% of accuracy. Extensive band assignment was analyzed and compiled for better understanding of PPV and PCV2 virus-cell interaction, demonstrating that specific protein, lipids and DNA/RNA bands are the most important assignments related to discrimination of virus-infected from uninfected cells. In conclusion, these results represent promising bases for RS application on PCV2 and PPV detection for future diagnostic applications.

Autophagy is a pro-survival adaptive response to heat shock in bovine cumulus-oocyte complexes.

Autophagy is a physiological mechanism that can be activated under stress conditions. However, the role of autophagy during oocyte maturation has been poorly investigated. Therefore, this study characterized the role of autophagy on developmental competence and gene expression of bovine oocytes exposed to heat shock (HS). Cumulus-oocyte-complexes (COCs) were matured at Control (38.5 °C) and HS (41 °C) temperatures in the presence of 0 and 10 mM 3-methyladenine (3MA; autophagy inhibitor). Western blotting analysis revealed that HS increased autophagy marker LC3-II/LC3-I ratio in oocytes. However, there was no effect of temperature for oocytes matured with 3MA. On cumulus cells, 3MA reduced LC3-II/LC3-I ratio regardless of temperature. Inhibition of autophagy during IVM of heat-shocked oocytes (3MA-41 °C) reduced cleavage and blastocyst rates compared to standard in vitro matured heat-shocked oocytes (IVM-41 °C). Therefore, the magnitude of HS detrimental effects was greater in the presence of autophagy inhibitor. Oocyte maturation under 3MA-41 °C reduced mRNA abundance for genes related to energy metabolism (MTIF3), heat shock response (HSF1), and oocyte maturation (HAS2 and GREM1). In conclusion, autophagy is a stress response induced on heat shocked oocytes. Inhibition of autophagy modulated key functional processes rendering the oocyte more susceptible to the deleterious effects of heat shock.

Thermoprotective molecules to improve oocyte competence under elevated temperature.

Heat stress is an environmental factor that challenges livestock by disturbing animal homeostasis. Despite the broad detrimental effects of heat stress on reproductive function, the germline and the early preimplantation embryo are particularly prone. There is extensive evidence that elevated temperature reduces oocyte developmental competence through a series of cellular and molecular damages. Further research revealed that the oocyte respond to stress by activating cellular mechanisms such as heat shock response, unfolded protein response and autophagy to improve survival under heat shock. Such knowledge paved the way for the identification of thermoprotective molecules that alleviate heat-induced oocyte oxidative stress, organelle damage, and apoptosis. Therefore, this review depicts the deleterious effects of heat shock on oocyte developmental competence, heat-induced cellular and molecular changes, outlines pro-survival cellular mechanisms and explores thermoprotective molecules to improve oocyte competence.

Cellular and epigenetic changes induced by heat stress in bovine preimplantation embryos.

Exposure of the preimplantation embryo to heat stress triggers a series of cellular, molecular, and adaptive changes preventing a normal embryonic development. Heat stress disrupts the embryo cytoskeleton, intracellular calcium levels, mitochondrial function, and induces apoptosis. Moreover, heat stress can act indirectly through induction of reactive oxygen species (ROS), leading to a variety of cellular damage. Embryonic resistance to heat shock is determined by factors such as genotype, developmental stage, apoptosis, redox status, and regulatory molecules. The early embryo is very susceptible to heat stress; it acquires resistance to elevated temperature as development advances. One of the mechanisms involved in the developmental acquisition of thermotolerance is heat-induced apoptosis, which acts as a quality control mechanism to remove damaged blastomeres allowing the embryo to survive after stress. Although embryos at >8-cell stage can activate the apoptotic cascade as an adaptive response to stress, embryos at the two-cell stage are resistant to proapoptotic signals. This lack of apoptotic response has been associated to mitochondrial resistance to depolarization and epigenetic regulations, such as DNA methylation and histone deacetylation. Even though the cellular mechanisms triggered by heat stress have been studied, very little attention has been paid to the vulnerability of the epigenome to drastic temperature changes during the preimplantation period. Therefore, this review aims to characterize the effects of elevated temperature on the bovine embryo, especially addresissing developmental, cellular, and epigenetic alterations triggered in response to temperature.

Role of insulin-like growth factor 1 on cross-bred Bos indicus cattle germinal vesicle oocytes exposed to heat shock.

Germinal vesicle (GV) oocytes are susceptible to heat stress. However, neither the cellular mechanisms triggered by elevated temperature nor the thermoprotective effects of insulin-like growth factor (IGF) on GV oocytes are completely understood. Therefore, a series of experiments was conducted to determine the direct effects of IGF1 (0, 12.5, 25, 50 and 100ng mL-1) on heat-treated GV oocytes. Butyrolactone-arrested GV oocytes were cultured at 38.5°C (control) or 41°C (heat shock; HS) for 14h in the presence of different concentrations of IGF1. Exposure of GV oocytes to 41°C increased (P<0.05) the number of terminal deoxyribonucleotidyl transferase-mediated fluorescein-dUTP nick end-labelling (TUNEL)-positive oocytes. At concentrations of 12.5 and 25ng mL-1, IGF1 tended to minimise these negative effect of HS (P=0.07). However, neither HS nor IGF1 had any effect on caspase activity. HS also decreased (P<0.05) GV oocyte mitochondrial activity and developmental competence to the blastocyst stage. These deleterious effects of HS were alleviated (P<0.05) by 12.5ng mL-1 IGF1. This concentration of IGF1 did not affect cleavage rate, the percentage of TUNEL-positive blastomeres and total blastocyst cell number regardless of temperature. In conclusion, exposure of GV oocytes to HS triggered the apoptotic cascade and compromised oocyte developmental competence. Physiological concentrations of IGF1 had a beneficial effect on heat-shocked GV oocytes.

Thermoprotective effect of insulin-like growth factor 1 on in vitro matured bovine oocyte exposed to heat shock.

The role of insulin-like growth factor 1 (IGF1) on cellular function and developmental capacity of heat-shocked oocytes has not been completely understood. Therefore, the objective of this study was to determine the effect of IGF1 on apoptosis, mitochondrial activity, cytoskeletal changes, nuclear maturation, and developmental competence of bovine oocytes exposed to heat shock. Cumulus-oocyte complexes were submitted to control (38.5 °C for 22 hours) and heat shock (41 °C for 14 hours followed by 38.5 °C for 8 hours) in the presence of 0 or 100 ng/mL IGF1 during IVM. Heat shock increased the percentage of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive oocyte and reduced oocyte mitochondrial activity. However, addition of 100 ng/mL IGF1 minimized these deleterious effects of temperature. Caspase activity was affected neither by heat shock nor IGF1. Exposure of bovine oocytes to 41 °C during the first 14-hour IVM affected cortical actin localization and microtubule organization at the meiotic spindle and reduced the percentage oocytes that reached the metaphase II stage. However, in the presence of IGF1, cortical actin and percentage of metaphase II oocytes were not different between control and heat-shocked oocytes, suggesting a partial beneficial effect of IGF1. There was no effect of IGF1 on microtubule organization. Heat shock also reduced the percentage of oocytes that reached the blastocyst stage, blastocyst cell number, and increased the percentage of TUNEL-positive blastomeres. However, there was no effect of 100 ng/mL IGF1 on oocyte development to the blastocyst stage and blastocyst quality. Therefore, 100 ng/mL IGF1 prevented some heat shock-induced cellular damage in bovine oocytes but had no effect on oocyte developmental competence. In contrast, a low IGF1 concentration (25 ng/mL) had a thermoprotective effect on oocyte developmental competence to the blastocyst stage. In conclusion, IGF1 prevented part of the damage induced by heat shock on oocyte function. This effect was modulated by IGF1 concentration.

Leptin promotes meiotic progression and developmental capacity of bovine oocytes via cumulus cell-independent and -dependent mechanisms.

Leptin has been shown to exert positive effects during the maturation of bovine oocytes, influencing blastocyst development, apoptosis, and the transcript levels of developmentally important genes. The present study was conducted to characterize further the mechanisms of leptin action on oocytes and the role of cumulus cells (CCs) in this context. In the first series of experiments, cumulus-oocyte complexes (COCs) were matured in serum-free medium that contained 0, 1 or 10 ng/ml leptin or in medium that was supplemented with 10% (v/v) estrus cow serum (ECS). Leptin concentrations of 1 and 10 ng/ml stimulated the meiotic progression of oocytes. Moreover, TUNEL staining demonstrated that these leptin doses reduced the proportion of apoptotic CCs. In the second series of experiments, COCs or denuded oocytes (DOs) were matured in the presence of 0 or 10 ng/ml leptin. The percentages of COCs and DOs with extruded polar bodies were increased by leptin. In contrast, positive effects of leptin on fertilization rates and blastocyst development were only observed after treatment of COCs but not of DOs. Leptin treatment of COCs consistently enhanced blastocyst development even after parthenogenetic activation of oocytes or after the removal of CCs before fertilization. The proportion of polyspermic oocytes was not affected by leptin treatment or oocyte denudation. In the third series of experiments, COCs were matured in the presence of 0, 1 or 10 ng/ml leptin. The transcript levels of specific genes were determined by reverse transcriptase-quantitative PCR (RT-qPCR) analysis of cumulus cells and single oocytes. Leptin treatment increased the levels of FAS, FASLG, and STAT3 transcripts in oocytes, but did not affect the LEPR, BAX, and BIRC4 mRNA concentrations. In cumulus cells, leptin treatment increased the mRNA levels for LEPR, STAT3, BAX, BIRC4, and FAS, but did not alter FASLG mRNA abundance. In conclusion, leptin differentially regulates gene expression in oocytes and cumulus cells. Moreover, leptin enhances both oocyte maturation and developmental capacity via cumulus cell-independent and -dependent mechanisms.

Maturation of bovine oocytes in the presence of leptin improves development and reduces apoptosis of in vitro-produced blastocysts.

The series of events associated with oocyte growth and maturation determines the oocyte's ability to undergo successful fertilization, cleavage and embryonic development. Among the molecules involved in these events, leptin has been identified as a modulator of oocyte function. Experiments were conducted to determine whether leptin treatment of oocytes during maturation affects their developmental capacity after fertilization and whether it has long-lasting effects on apoptosis and gene expression in the resulting blastocysts. Cumulus-oocyte complexes (COCs) were matured in serum-free medium containing 0 (control), 1, 10, or 100 ng/ml leptin or in medium supplemented with 10% (v/v) estrous cow serum (ECS). Addition of leptin during oocyte maturation had no effect on cleavage rate after fertilization. However, an increased proportion of oocytes that matured in the presence of 1 or 10 ng/ml leptin developed to blastocysts, which exhibited increased cell numbers. The proportion of apoptotic cells was reduced in blastocysts originating from leptin- or ECS-treated oocytes. Transcript levels of the genes encoding leptin receptor (LEPR), signal transducer and activator of transcription (STAT3), BCL2 associated X-protein (BAX), and baculoviral inhibitor of apoptosis protein repeat-containing 4 (BIRC4, also known as XIAP), were determined by reverse transcriptase-quantitative polymerase chain reaction analysis of expanded and hatched blastocysts. Depending on the dose used, leptin treatment of oocytes resulted in increased LEPR, STAT3, and BIRC4 mRNA levels and reduced BAX mRNA levels in blastocysts. In conclusion, leptin improved the ability of the oocyte to sustain embryonic development and had long-term effects on blastocyst apoptosis and transcript abundance of LEPR, STAT3, and apoptosis-associated genes.