Louisa A. Rispoli

Developmental competence of bovine embryos from heat-stressed ova

Because multiple ovulation embryo transfer procedures are occasionally performed in cows experiencing heat stress, the goal of this study was to assess the developmental competence of otherwise morphologically normal embryos from heat-stressed ova. To this end, the ability of compact morulae from heat-stressed and non-heat-stressed bovine ova to undergo blastocyst development after culture at 38.5 or 41.0 degrees C was examined. It was hypothesized that heat-induced perturbations in the ooplasm carry over to increase the susceptibility of the preattachment embryo to heat stress. Initially, ova were matured at 38.5 or 41.0 degrees C. The consequences of heat stress did not include altered cleavage, but did reduce the proportion of 8- to 16-cell-stage embryos (55.3 vs. 50.6%; SEM +/- 1.9). Although proportionately fewer, compact morulae from heat-stressed ova were equivalent in quality to those from non-heat-stressed ova (2.1 and 2.1; SEM = 0.04). Culture of compact morulae from non-heat-stressed ova at 41.0 degrees C did not affect blastocyst development (71.9 and 71.5%; SEM = 3.0). Furthermore, the development of compact morulae from heat-stressed ova was similar to that of non-heat-stressed ova after culture at 38.5 degrees C (68.2 vs. 71.9 and 71.5%; SEM = 3.0). However, blastocyst development was reduced when compact morulae from heat-stressed ova were cultured at 41.0 degrees C (62.3 vs. 71.9, 71.5 and 68.2; SEM = 3.1). In summary, reduced compaction rates of heat-stressed ova explained in part why fewer develop to the blastocyst stage after fertilization. The thermolability of the few embryos that develop from otherwise developmentally challenged ova emphasizes the importance of minimizing exposure to stressor(s) during oocyte maturation.

Heat stress effects on the cumulus cells surrounding the bovine oocyte during maturation: altered matrix metallopeptidase 9 and progesterone production

When the effects of heat stress are detrimental during maturation, cumulus cells are intimately associated with the oocyte. To determine the extent to which heat stress affects these cells, in this study, transcriptome profiles of the cumulus that surrounded control and heat-stressed oocytes (41 °C during the first 12 h only and then shifted back to 38.5 °C) during in vitro maturation (IVM) were compared using Affymetrix bovine microarrays. The comparison of cumulus-derived profiles revealed a number of transcripts whose levels were increased (n=11) or decreased (n=13) ≥ twofold after heat stress exposure (P<0.01), sufficient to reduce the development of blastocysts by 46.4%. In a separate study, quantitative PCR (qPCR) was used to confirm heat-induced differences in the relative abundances of the transcripts of five different genes (caveolin 1, matrix metallopeptidase 9, FSH receptor, Indian hedgehog homolog, and inducible nitric oxide synthase). Heat stress exposure resulted in >1.7-fold decrease in the protein levels of latent matrix metallopeptidase 9 (proMMP9). Heat-induced reductions in transcript levels were noted at 6 h IVM with reductions in proMMP9 protein levels at 18 h IVM (P=0.0002). Independent of temperature, proMMP9 levels at 24 h IVM were positively correlated with the development rate of blastocysts (R²=0.36; P=0.002). The production of progesterone increased during maturation; heat-induced increases were evident by 12 h IVM (P=0.002). Both MMP9 and progesterone are associated with the developmental competence of the oocyte; thus, it seems plausible for some of the negative consequences of heat stress on the cumulus-oocyte complex to be mediated through heat-induced perturbations occurring in the surrounding cumulus.

Disparate consequences of heat stress exposure during meiotic maturation: embryo development after chemical activation vs fertilization of bovine oocytes

Consequences of heat stress exposure during the first 12 h of meiotic maturation differed depending on how and when bovine oocytes were activated. If heat-stressed oocytes underwent IVF at ~24 h, blastocyst development was less than for respective controls and similar to that obtained for nonheat-stressed oocytes undergoing IVF at 30 h (i.e. slightly aged). In contrast, if heat-stressed oocytes underwent chemical activation with ionomycin/6-dimethylaminopurine at 24 h, blastocyst development was not only higher than respective controls, but also equivalent to development obtained after activation of nonheat-stressed oocytes at 30 h. Developmental differences in chemically activated vs IVF-derived embryos were not related to fertilization failure or gross alterations in cytoskeletal components. Rather, ionomycin-induced calcium release and MAP kinase activity were less in heat-stressed oocytes. While underlying mechanisms are multifactorial, ability to obtain equivalent or higher development after parthenogenetic activation demonstrates that oocytes experiencing heat stress during the first 12 h of meiotic maturation have the necessary components to develop to the blastocyst stage, but fail to do so after fertilization.

Temporal gene expression in equine corpora lutea based on serial biopsies in vivo.

A biopsy procedure was developed to enable repeated sampling of a single equine corpus luteum (CL) over the course of an estrous cycle. The tissue collected was utilized in characterizing mRNA abundance for genes involved in luteal formation, function, and regression in the cyclic mare. Serial biopsies of CL in cyclic mares (2.7 to 27.5 mg per biopsy) were collected using an ultrasound-guided transvaginal technique. Biopsies were collected from each mare on d 2 and 5 (d 0 = ovulation) of the estrous cycle, and every other day from d 12 through luteolysis. Samples were obtained from 4 mares with normal estrous cycles and 1 mare with a retained CL. The biopsy procedure did not adversely affect luteal size or function, as measured by luteal area and serum concentrations of progesterone. Real-time reverse-transcription PCR was used to quantify steady state mRNA concentrations in each tissue sample obtained. Mean abundance of steroidogenic acute regulatory protein (StAR) mRNA was not different (P = 0.102 to 0.964) on any of the sampling dates, but a trend for mRNA encoding StAR to decrease between d 12 and 14 (P = 0.10) was observed. Values for mRNA encoding StAR were positively correlated to serum concentrations of progesterone on d 5 (R = 0.95; P = 0.05) and 14 (R > 0.99; P < 0.01). Steady-state abundance of mRNA for 3β-hydroxysteroid dehydrogenase, Δ 5-Δ 4 isomerase (3β-HSD) declined between d 12 and 14 (P = 0.15). There were positive correlations between mRNA for 3β-HSD and concentrations of progesterone on d 5 (R = 0.94; P = 0.06) and 12 (R > 0.99; P = 0.05). No difference was detected in abundance of mRNA encoding cyclooxygenase-2 (cox-2; P = 0.340 to 0.840) or caspase-3 (P = 0.517 to 0.882) between any of the sampling dates. A successful luteal biopsy procedure was developed that did not negatively affect luteal function, and abundance of mRNA encoding StAR, 3β-HSD, cox-2, and caspase-3 was characterized in luteal biopsy tissue collected on d 2, 5, 12, and 14 of the estrous cycle in the mare.

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.