J. Lannett Edwards

Susceptibility of Bovine Germinal Vesicle-Stage Oocytes from Antral Follicles to Direct Effects of Heat Stress In Vitro

Abstract Delineation of maternal versus direct effects of heat stress in reducing development at the germinal vesicle (GV) stage is challenging, because oocytes spontaneously resume meiosis after removal from antral follicles. The use of S-roscovitine (inhibitor of p34cdc2/cyclin B kinase) to hold bovine oocytes at the GV stage without compromising early embryo development was previously validated in our laboratory. The objective of the present study was to assess the direct effects of an elevated temperature commonly seen in heat-stressed dairy cows on cumulus-oocyte complexes (COCs) held at the GV stage using 50 μM S-roscovitine. During roscovitine culture, GV-stage COCs (antral follicle diameter, 3–8 mm) were cultured at 38.5 or 41°C. Thereafter, oocytes were removed from roscovitine medium and allowed to undergo in vitro maturation, fertilization, and culture. Zona pellucida hardening (solubility to 0.5% pronase), nuclear stage (Hoechst 33342), cortical granule type (lens culinaris agglutinin-fluorescein isothiocyanate [FITC]), and early embryo development were evaluated. Culture of GV-stage COCs at 41°C increased the proportion that had type III cortical granules and reduced the proportion that progressed to metaphase II after in vitro maturation. Effects of 41°C on zona pellucida hardening, fertilization (penetration, sperm per oocyte, pronuclear formation, and monospermic and putative embryos), and cleavage of putative zygotes were not noted. However, culture of GV-stage COCs at 41°C for 6 h decreased the proportion of 8- to 16-cell embryos, whereas 41°C for 12 h reduced blastocyst development. In summary, antral follicle COCs are susceptible to direct effects of elevated body temperature, which may account in part for reduced fertility in heat-stressed cows.

Retinol improves bovine embryonic development in vitro

Retinoids are recognized as important regulators of vertebrate development, cell differentiation, and tissue function. Previous studies, performed both in vivo and in vitro, indicate that retinoids influence several reproductive events, including follicular development, oocyte maturation and early embryonic development. The present study evaluated in vitro effects of retinol addition to media containing maturing bovine oocytes and developing embryos in both a low oxygen atmosphere (7%) and under atmospheric oxygen conditions (20%). In the first experiment, abbatoir collected bovine oocytes were matured in the presence or absence of varying concentrations of retinol. After a 22–24 hour maturation period the oocytes were fertilized, denuded 18 hours later and cultured in a modified synthetic oviductal fluid (mSOF) in a humidified atmosphere at 38.5 degrees C, 5% CO2, 7% O2 and 88% N2. Cleavage rates did not differ among control and retinol-treated oocytes in all three experiments. Addition of 5 micromolar retinol to the maturation medium (IVM) tended (p < 0.07) to increase blastocyst formation (blastocyst/putative zygote; 26.1% +/- 2.2%) compared to the controls (21.9% +/- 1.9%). Further analysis revealed when blastocyst development rates fell below 20% in the control groups, 5 micromolar retinol treatment dramatically improved embryonic development, measured by blastocyst/putative zygote rate (14.4 +/- 2.1 vs 23.7 +/- 2.5; p < 0.02). The 5 micomolar retinol treatment also enhanced the blastocyst/cleaved rate by nearly 10% (23.7% vs 34.6%; p < 0.02). In the second and third experiments addition of 5 micromolar retinol to the embryo culture medium (IVC) under low oxygen conditions did not significantly improve cleavage or blastocyst rates, but 5 micromolar retinol significantly increased blastocyst development under 20% O2 conditions (p < 0.001). These studies demonstrate that supplementation of 5 micromolar retinol to the maturation medium may improve embryonic development of bovine oocytes indicated by their increased blastocyst rate. A significant improvement in the blastocyst development with the 5 micromolar retinol treatment under atmospheric conditions suggests a beneficial antioxidant effect during embryo culture.

Impact of heat stress on germinal vesicle breakdown and lipolytic changes during in vitro maturation of bovine oocytes

Two studies were conducted with the overarching goal of determining the extent to which lipolytic changes relate to germinal vesicle breakdown (GVBD) in bovine oocytes matured under thermoneutral or hyperthermic conditions. To this end, cumulus-oocyte complexes underwent in vitro maturation for 0, 2, 4, 6 or 24 h at 38.5 (first study) or 38.5 and 41.0 C (second study; heat stress applied up through first 12 h only, then shifted to 38.5 C). Independent of maturation temperature, triglyceride and phospholipid content decreased markedly by 2 h of in vitro maturation (hIVM; P < 0.0005). Content was lowest at 24 hIVM with no detectable impact of heat stress when exposure occurred during first 12 hIVM. Germinal vesicle breakdown occurred earlier in oocytes experiencing heat stress with effects observed as soon as 4 hIVM (P < 0.0001). Germinal vesicle breakdown was associated with lipolytic changes (R2 = 0.2123 and P = 0.0030 for triglyceride content; R2 = 0.2243 and P = 0.0026 for phospholipid content). ATP content at 24 hIVM was higher in oocytes experiencing heat stress (P = 0.0082). In summary, GVBD occurs sooner in heat-stressed oocytes. Although marked decreases in triglyceride and phospholipid content were noted as early as 2 hIVM and preceded GVBD, lipolytic changes such as these are not likely serving as an initial driver of GVBD in heat-stressed oocytes because changes occurred similarly in oocytes matured at thermoneutral conditions.

Mitochondrial-related consequences of heat stress exposure during bovine oocyte maturation persist in early embryo development

Hyperthermia during estrus has direct consequences on the maturing oocyte that carries over to the resultant embryo to compromise its ability to continue in development. Because early embryonic development is reliant upon maternal transcripts and other ooplasmic components, we examined impact of heat stress on bovine oocyte transcripts using microarray. Oocytes were matured at 38.5ºC for 24 h or 41.0ºC for the first 12 h of in vitro maturation; 38.5ºC thereafter. Transcriptome profile was performed on total (adenylated + deadenylated) RNA and polyadenylated mRNA populations. Heat stress exposure altered the abundance of several transcripts important for mitochondrial function. The extent to which transcript differences are coincident with functional changes was evaluated by examining reactive oxygen species, ATP content, and glutathione levels. Mitochondrial reactive oxygen species levels were increased by 6 h exposure to 41.0ºC while cytoplasmic levels were reduced compared to controls (P < 0.0001). Exposure to 41.0ºC for 12 h increased total and reduced glutathione levels in oocytes at 12 h but reduced them by 24 h (time × temperature P < 0.001). ATP content was higher in heat-stressed oocytes at 24 h (P < 0.0001). Heat-induced increases in ATP content of matured oocytes persisted in early cleavage-stage embryos (8- to 16-cell embryos; P < 0.05) but were no longer apparent in blastocysts (P > 0.05). Collectively, results indicate that direct exposure of maturing oocytes to heat stress may alter oocyte mitochondrial processes/function, which is inherited by the early embryo after fertilization.

Developmental consequences of supplementing with matrix metallopeptidase-9 during in vitro maturation of heat-stressed bovine oocytes.

Because latent form of matrix metallopeptidase-9 (proMMP9) levels are positively related to blastocyst development, it was hypothesized that addition during maturation may improve development of heat-stressed oocytes. To test hypothesis, 0, 30 or 300 ng/ml human proMMP9 (hMMP9) was added at 18 h of in vitro maturation (hIVM) to cumulus-oocyte complexes matured at 38.5 or 41.0ºC (first 12 h only). Heat stress decreased 24 hIVM proMMP9 levels only in 0 and 30 ng/ml groups and increased progesterone in 0 and 300 ng/ml hMMP9 groups. Heat stress decreased cleavage and blastocyst development. Independent of maturation temperature, hMMP9 at 18 hIVM decreased blastocyst development. In a second study, cumulus-oocyte complexes were matured for 24 h at 38.5 or 41.0ºC (HS first 12 h only) with 0 or 300 ng/ml hMMP9 added at 12 hIVM. Without hMMP9, heat stress decreased 24 hIVM proMMP9 levels and increased progesterone production. Addition of 300 ng/ml of hMMP9 produced equivalent levels of proMMP9 at 24 hIVM (271 vs. 279 ± 77 for 38.5ºC and 41.0ºC treated oocytes, respectively). Heat stress did not affect ability of oocytes to cleave but reduced blastocyst development. Independent of temperature, hMMP9 decreased cleavage and blastocyst development. In summary, hMMP9 supplementation during IVM did not improve development of heat-stressed oocytes even when it was added for the entire maturation period. At doses tested, hMMP9 appeared detrimental to development when supplemented during the last 12 or 6 h of oocyte maturation.

An in vivo model to assess the thermoregulatory response of lactating Holsteins to an acute heat stress event occurring after a pharmacologically-induced LH surge

Hyperthermia occurring 10-12 h after LH surge reduces quality of maturing oocyte, thereby reducing fertility. Objective was to examine consequences of an acute heat stress and the influence of certain hormones on the thermoregulatory responses of lactating cows during this critical period. Between the months of February through May, cows were transported to a facility and maintained at a temperature-humidity index (THI) of 65.9 ± 0.2 (thermoneutral) or exposed to changes in THI to simulate what may occur during an acute heat stress event (71-86 THI; heat stress); cows were rapidly cooled thereafter. Mixed model regressions with repeated measures were used to test respiration rates (RR) and rectal temperature (RT). Within 40 and 110 min of increasing THI, RR increased in a quadratic fashion (P < 0.001); RT increased by 0.04 ± 0.1 °C (P < 0.001) per unit THI. Changes in RR lagged THI and preceded rises in RT. Average THI 3-days before treatment (prior THI) influenced RR (P = 0.050) and RT (P < 0.001) changes. Increased RR was more noticeable in heat-stressed cows when prior THI was in the 40 s. Rectal temperature of heat-stressed cows was 0.8 ± 0.02 °C lower when prior THI was in the 40 s versus low 60 s. Levels of progesterone and luteinizing hormone before treatment were predictive of thermoregulatory response in heat-stressed cows. Rapid cooling decreased RR by 0.6 ± 0.1 bpm (P < 0.001) and RT by 0.02 ± 0.002 °C per min (P < 0.002). Speed and magnitude of thermoregulatory changes to an acute heat stress and after sudden cooling emphasizes importance of strategic cooling before ovulation. Efforts to do so when prior THI approaches levels expected to induce mild stress are especially important. Respiration rate is a useful indicator of the degree of hyperthermia a lactating cow is experiencing.

Heat stress impairs gap junction communication and cumulus function of bovine oocytes

The intimate association of cumulus cells with one another and with the oocyte is important for regulating oocyte meiotic arrest and resumption. The objective of this study was to determine the effects of heat stress on cumulus cell communication and functions that may be related to accelerated oocyte meiosis during early maturation. Bovine cumulus-oocyte complexes underwent in vitro maturation for up to 6 h at thermoneutral control (38.5°C) or elevated (40.0, 41.0 or 42.0°C) temperatures. Gap junction communication between the cumulus cells and the oocyte was assessed using the fluorescent dye calcein after 4 h of in vitro maturation. Dye transfer was reduced in cumulus-oocyte complexes matured at 41.0°C or 42.0°C; transfer at 40.0°C was similar to control (P < 0.0001). Subsequent staining of oocytes with Hoechst revealed that oocytes matured at 41.0 or 42.0°C contained chromatin at more advanced stages of condensation. Maturation of cumulus-oocyte complexes at elevated temperatures reduced levels of active 5’ adenosine monophosphate activated kinase (P = 0.03). Heat stress exposure had no effect on active extracellular-regulated kinase 1/2 in oocytes (P = 0.67), associated cumulus cells (P = 0.60) or intact cumulus-oocyte complexes (P = 0.44). Heat-induced increases in progesterone production by cumulus-oocyte complexes were detected during the first 6 h of maturation (P = 0.001). Heat-induced alterations in gap junction communication and other cumulus-cell functions likely cooperate to accelerate bovine oocyte meiotic progression.