Biol Reprod Lalor Postdoctoral Fellowships -- Application Deadline January 15, 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print August 20, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020347
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
69/6/2068    most recent
biolreprod.103.020347v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rivera, R. M.
Right arrow Articles by Hansen, P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rivera, R. M.
Right arrow Articles by Hansen, P. J.
Agricola
Right arrow Articles by Rivera, R. M.
Right arrow Articles by Hansen, P. J.
BIOLOGY OF REPRODUCTION 69, 2068–2077 (2003)
DOI: 10.1095/biolreprod.103.020347
© 2003 by the Society for the Study of Reproduction, Inc.

Embryo

Alterations in Ultrastructural Morphology of Two-Cell Bovine Embryos Produced In Vitro and In Vivo Following a Physiologically Relevant Heat Shock1

Rocío M. Rivera3, Karen L. Kelley4, Gregory W. Erdos4, and Peter J. Hansen2,3

Department of Animal Sciences3 Interdisciplinary Center for Biotechnology Research,4 Electron Microscopy Core Laboratory, University of Florida, Gainesville, Florida 32611


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of cultured preimplantation embryos to temperatures similar to those experienced by heat-stressed cows inhibits subsequent development. In this study, the effects of heat shock on the ultrastructure of two-cell bovine embryos were examined to determine mechanisms for inhibition of development. Two-cell embryos produced in vitro were harvested at ~28 h postinsemination and cultured for 6 h at one of three temperatures: 38.5°C (cow body temperature), 41.0°C (characteristic temperature for heat-stressed cows), or 43.0°C (severe heat shock). Ultrastructural examinations revealed that both heat shocks resulted in the movement of organelles towards the center of the blastomere. In addition, heat shock increased the percentage of mitochondria exhibiting a swollen morphology. Distance between the membranes comprising the nuclear envelope was increased but only when embryos were treated at 43.0°C. To determine whether ultrastructural responses to heat shock in culture were similar for embryos produced in vitro and in vivo, two-cell embryos were collected from superovulated Angus cows 48 h postinsemination and treated ex vivo for 6 h at 38.5°C or 41.0°C. Again, heat shock caused an increase in number of swollen mitochondria and movement of organelles away from the periphery of the blastomere. Exposure of two-cell bovine embryos to physiologically relevant elevated temperatures causes disruption in ultrastructural morphology that is inimical to development. The observation that overall morphology and response to heat was similar for embryos produced in vitro and in vivo implies that the former can be a good model for understanding embryonic responses to heat shock.

embryo, environment


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Development of the cleavage-stage embryo is very susceptible to disruption by elevated temperatures [1–5]. Temperatures that have little effect on survival of most cells can be lethal to preimplantation embryos. For example, development of mouse embryos was inhibited by exposure to 39.0°C beginning at the one-cell stage [6], and development of bovine two-cell embryos was decreased by exposure to 41°C for as little as 4.5 h [7]. In contrast, temperatures in this range have no effect on survival of other cells [8]. Hypersensitivity of the preimplantation embryo to elevated temperatures is a transient phenomenon; effects of heat shock on development of cultured bovine embryos are reduced as embryos advance in development past the two- to four-cell stage [4, 7]. The susceptibility of the embryo to elevated temperature is likely one of the major causes for the decrease in fertility and increase in embryonic mortality caused by heat stress in cattle [9–11], sheep [12], rabbits [13], and mice [14].

The cellular changes caused by elevated temperature that lead to a block in embryonic development are not well understood. In other cells, heat shock can cause cytoskeletal rearrangement [15], changes in mitochondrial membrane potential and permeability [16], organelle disruption and relocalization [17, 18], increased membrane fluidity [15, 18], and membrane blebbing [19]. Most work on effects of heat shock on cultured cells involves temperatures higher than those that compromise embryonic survival or are experienced by heat-stressed females. Whereas most studies on the effects of heat shock on mammalian cells have used temperatures of 43.0–45.0°C, temperatures that would be lethal to most mammals, maternal hyperthermia of 40.0–41.0°C compromises pregnancy rate in cattle [9, 10, 20, 21]. The hypersensitivity of the early preimplantation embryo to heat shock as compared with embryos at later stages [4, 7] and with other cells implies that heat shock might be causing different types of cellular pathologies than those caused by the severe heat shocks required to disrupt most cell types.

Experiments on effects of heat shock on bovine embryos have depended largely on the in vitro-produced embryo as a model. It is not known, however, whether bovine embryos that develop in vivo respond to the deleterious effects of elevated temperatures in a manner similar to that of in vitro-produced embryos. Embryos produced in vitro differ in several ways from embryos that develop in utero. For example, they can exhibit retarded development rate [22], increased content of lipid [23], and lower survivability after freezing [24]. Thus, there is a need to verify that the in vitro-produced embryo is an acceptable model for studying effects of heat shock on embryonic development by testing whether similar types of changes are induced by heat shock in embryos that were produced in vivo.

The principal objective of this study was to characterize ultrastructural changes in two-cell embryos caused by exposure to a physiologically relevant heat shock. Another objective was to evaluate whether differences exist in the fine structure of two-cell embryos produced in vitro as compared with embryos produced in vivo and to test whether heat shock induced similar changes in both types of embryos.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

All chemicals, unless otherwise specified, were from Sigma (St. Louis, MO). Reagents for in vitro production of embryos were obtained as described elsewhere [5]. The FSH used was Folltropin-V (Vetrepharm Canada, London, ON, Canada). The GnRH used was Cystorelin (Merial, Iselin, NJ) purchased from Ag-Tech (Manhattan, KS). Percoll was from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Penicillin-streptomycin and the culture media SP-TL, Hepes-TL, and IVF-TL were from Cell and Molecular Technologies (Lavallette, NJ) and were used to prepare SP-TALP, Hepes-TALP, and IVF-TALP [25]. Potassium simplex optimized medium (KSOM; formulation MR-020-D) was also from Cell and Molecular Technologies and was used to prepare KSOM-BE1 by the addition of 3 mg/ml essentially fatty acid-free Fraction V BSA, 2.5 µg/ml gentamicin, essential amino acids (basal medium Eagle), and nonessential amino acids (minimum essential medium purchased from Sigma) as described elsewhere [26]. Bovine steer serum was from Pel Freez Biologicals (Rogers, AK). The medium CR1aa was prepared as described [27] and modified by addition of 3 mg/ml essentially fatty acid-free Fraction V BSA, 2.5 µg/ml gentamicin, and essential and nonessential amino acids. Fetal bovine serum (FBS) was from Atlanta Biologicals (Norcross, GA). The prostaglandin F2{alpha} (PGF2{alpha}) used was Lutalyse and was donated by Pfizer Animal Health (Kalamazoo, MI). Glutaraldehyde, osmium tetroxide, ethanol, acetone, EMbed-812, uranyl acetate, Reynolds lead citrate, Butvar, and copper grids were from Electron Microscopy Sciences (Fort Washington, PA).

Production of Embryos

In vitro Procedures for oocyte maturation, fertilization, and embryo culture were as described previously [5]. Ovaries were collected at a local abattoir approximately 1.5 h from the laboratory. Oocytes obtained by slashing the ovary were matured for 21 h and then inseminated with a cocktail of Percoll-purified spermatozoa from three different Angus bulls; a different group of bulls was used for each replicate. At 18–20 h after insemination, putative zygotes were denuded of cumulus cells by suspending them in Hepes-TALP medium containing 1000 units/ml of hyaluronidase type IV and vortexing in a microcentrifuge tube. Presumptive zygotes were then placed in groups of ~30 in 50-µl microdrops of KSOM-BE1 or modified CR1aa. All cultures at 38.5°C were performed in an environment of 5% CO2 in humidified air. The percentage of CO2 was adjusted in the incubators used for heat shock treatments (7% and 8% CO2 for 41.0°C and 43.0°C, respectively) to ensure that concentration of dissolved CO2 was similar between treatment and to maintain medium pH at ~7.4 [5]. The temperature of all incubators was calibrated routinely to assure accuracy of treatments.

In vivo Estrous cycles of three Angus cows were synchronized by i.m. injections of 100 µg GnRH on Day 0, 25 mg PGF2{alpha} on Day 7, and 100 µg GnRH on Day 9. On Day 19, superovulation was induced by i.m. injections of decreasing doses of FSH each morning and afternoon for 4 days (two injections of 40 mg each on the first day, two injections of 30 mg each on the second and third day, and two injections of 20 mg each on the last day). Cows received 25 and 15 mg PGF2{alpha} i.m., coincident with the sixth and seventh FSH injections, respectively. Approximately 24 h after the last FSH injection, each cow was inseminated with semen from a different Angus bull, and insemination was repeated 12 h later. Cows were killed approximately 48 h after the first insemination, and embryos were immediately flushed from the oviducts. This study was conducted with the approval of the Institutional Animal Care and Use Committee of the University of Florida.

Experiments

Inhibition of blastocyst development by heat shock at the two-cell stage Approximately 18 h postinsemination, putative zygotes were removed of their cumulus cells and placed in KSOM-BE1 or modified CR1aa medium. Two-cell embryos were collected at ~28 h postinsemination and cultured in fresh microdrops of KSOM-BE1 or modified CR1aa at one of two temperatures: 38.5°C (i.e., homoeothermic body temperature of the cow) or 41.0°C (characteristic temperature for heat-stressed cows;[5]). Embryos cultured at 41.0°C were returned to control temperature (38.5°C) after 6 h. On Day 5 after insemination, the group of embryos cultured in modified CR1aa medium were supplemented with 10% FBS (no FBS was added to the KSOM-BE1 cultured embryos). Development to the blastocyst stage was evaluated on Day 8 postinsemination. The experiment was replicated five times with a total of 60–70 two-cell embryos/treatment.

Effects of heat shock on the ultrastructure of two-cell embryos produced in vitro Two-cell embryos were collected at ~28 h postinsemination, placed in fresh microdrops of KSOM-BE1, and cultured for 6 h at one of three temperatures: 38.5°C, 41.0°C, or 43.0°C (severe heat shock). Immediately after treatment, embryos were processed for electron microscopy. The experiment was replicated four times on different days. For statistical analysis, electron micrographs of one embryo (selected at random) per replicate per treatment were used to take measurements of cellular morphology.

Effects of heat shock on the ultrastructure of two-cell embryos produced in vivo and treated ex vivo Two-cell embryos were obtained from three superovulated Angus cows. Immediately after slaughter, the oviducts of each cow were flushed with 10 ml sterile prewarmed (~39.0°C) Hepes-TALP into a sterile Petri dish. Embryos (n = 38) were immediately collected and separated by stage of development (one cell, n = 13; two cells, n = 24; four cells, n = 1). Only embryos that were at the two-cell stage were used for the experiment. Two-cell embryos from each cow were randomly divided into two groups and cultured in microdrops of KSOM-BE1 at 38.5°C or 41.0°C. After 6 h, embryos were processed for electron microscopy. For statistical analysis, electron micrographs of one or two embryos per cow per treatment were used to take measurements of cellular morphology (n = 3 for 38.5°C; n = 5 for 41.0°C).

Electron Microscopy

Embryos were fixed in 2% (v/v) glutaraldehyde in PBS (0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.2) at 4.0°C overnight (8–10 h). To ease the handling of embryos during dehydration and embedding, embryos were washed in PBS and individually placed on a glass slide, and a drop of 3% (w/v) molten low-gelling-temperature agar (Sigma) in PBS was placed on the embryo while observing it through a dissecting microscope. Once the agar was hardened, the excess was cut off to leave a square (~1 mm2) of agar containing the embedded embryo. While in agar, embryos were postfixed in 1% (w/v) buffered OsO4, dehydrated in ascending concentrations of ethanol, and stained en bloc with 2% (w/v) uranyl acetate in 75% (v/v) ethanol overnight at 4.0°C. After further dehydration to 100% ethanol followed by 100% acetone, embryos were infiltrated with EMbed-812, and ultrathin sections (~75 nm thick) were placed in Butvar-covered 75-mesh copper grids. Sections were stained with 2% (w/v) aqueous uranyl acetate and Reynolds lead citrate before viewing with a Hitachi H-7000 transmission electron microscope (Gaithersburg, MD). Semithin sections (~500 nm) were collected for light microscopic examination of whole embryos. Sections were placed on a glass slide and dried on a hot plate (~50.0°C). A drop of toluidine blue O stain (1:1 mixture of 0.2% [w/v] toluidine blue and 2% [w/v] borax) was added to the sections and incubated on the hot plate for approximately 1 min (before the slide dried out). The sections were rinsed in a stream of distilled water prior to examination.

Morphometric Analysis of Electron Micrographs

Electron micrographs of sections that contained the nucleus of at least one blastomere were used to take measurements on several aspects of cellular morphology. In addition, these micrographs were visually assessed to determine effects of heat shock on other aspects of cellular morphology. For each embryo, morphometric analysis was performed using SIS Megaview III camera and software (Soft Imaging System, Lakewood, CO). The distance from the plasma membrane to the organelle-containing region of the cytoplasm was measured at 10 random locations from each of four pictures per embryo (i.e., 40 random measurements/embryo). The distance between the two membranes that make up the nuclear envelope was measured at five random locations around the nucleus in each of four pictures per embryo. The average of these measurements was used for statistical analysis. The percentage of mitochondria exhibiting a swollen morphology was determined by counting the total number of mitochondria and the number of swollen mitochondria in each of four pictures per embryo.

Statistical Analysis

Effect of culture temperature on percentage of two-cell embryos that developed to the blastocyst stage was analyzed by least-squares ANOVA using the Proc GLM procedures of SAS (SAS for Windows, version 8e; SAS, Cary, NC). Percentage development was calculated for each replicate. Percentage data were analyzed before and after arcsine transformation. The analysis of transformed data was used to obtain probability values, whereas analysis of untransformed data was used to obtain least-squares means ± SEM.

Heat shock effects on the distance from the plasma membrane to the outermost organelles, proportion of mitochondria exhibiting a swollen morphology, and the distance between the two membranes of the nuclear envelope was also analyzed by least-squares ANOVA using Proc GLM procedures. Except for distance between the two nuclear membranes, data exhibited heteroscedasticity. Accordingly, data were transformed by log transformation before analysis. The analysis of log transformed data was used to obtain probability values, whereas analysis of untransformed data was used to obtain means ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of Embryonic Development by Heat Shock at the Two-Cell Stage

Exposure of two-cell embryos to 41.0°C for 6 h reduced the percentage of two-cell embryos that became blastocysts at Day 8 after fertilization (P < 0.001) (Fig. 1). The reduction in developmental success occurred for both media, and the temperature x medium interaction was not significant.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1. Inhibition of bovine embryonic development by heat shock at the two-cell stage. Data represent least-squares means ± SEM of five replicates with a total of 60–70 two-cell embryos/treatment.

Heat Shock-Induced Changes in Ultrastructure of Two-Cell Embryos Produced In Vitro

The cytoplasm of embryos cultured at 38.5°C was occupied primarily by vesicles and mitochondria that extended from the perinuclear region to the edge of the blastomere (Fig. 2, a and b). Vesicles appeared to be, at least in part, Golgi. Indicative of their immature status, mitochondria displayed a hooded morphology and had few cristae. Embryos contained little endoplasmic reticulum, but annulate lamellae (precursor organelles of the endoplasmic reticulum) were evident. Lysosomes were also evident, although scarce. Occasionally, a nucleolus (Fig. 4a) was apparent in the nucleus. The plasma membrane of the blastomere was invested with numerous microvilli, and clathrin-coated pits were observed occasionally.



View larger version (165K):
[in this window]
[in a new window]
  		FIG. 2. Effects of heat shock on morphology of two-cell bovine embryos produced in vitro as determined by light microscopy (A, C, and E) of 500-nm sections stained with toluidine blue O stain or by transmission electron microscopy (B, D, and F). Embryos were cultured for 6 h at 38.5°C (A and B), 41.0°C (C and D), or 43.0°C (E and F). Arrows show area of the cytoplasm devoid of organelles. ZP, Zona pellucida; PVS, perivitelline space; N, nucleus; mv, microvilli



View larger version (200K):
[in this window]
[in a new window]
  		FIG. 4. Effects of heat shock on nuclear morphology (A, C, and E) and distance between the two membranes comprising the nuclear envelope (B, D, and F). Two-cell bovine embryos produced in vitro were cultured for 6 h at 38.5°C (A and B), 41.0°C (C and D), or 43.0°C (E and F). N, Nucleus; nu, nucleolus; G, Golgi apparatus; V, vesicle. Arrows point to the nuclear envelope.

Upon heat shock, all organelles moved centrally, leaving the periphery of the blastomere devoid of organelles (Fig. 2, c–f). The mean (±SEM) distance from the plasma membrane to the organelle-containing region of the cytoplasm was increased by exposure to 41.0°C and 43.0°C (604 ± 132, 2261 ± 444, and 4463 ± 1915 nm for 38.5°C, 41.0°C, and 43.0°C, respectively; P < 0.01). In addition, numerous mitochondria were swollen in heat-shocked embryos. The percentage of mitochondria showing a swollen morphology was increased (P < 0.001; Fig. 3) by both heat shocks (0% ± 0.25%, 15% ± 4%, and 74% ± 20% for 38.5°C, 41.0°C, and 43.0°C, respectively).



View larger version (98K):
[in this window]
[in a new window]
  		FIG. 3. Heat shock-induced alterations in mitochondrial morphology. Two-cell bovine embryos produced in vitro were cultured for 6 h at 38.5°C (A), 41.0°C (B), or 43.0°C (C). MT, Mitochondrion; V, vesicle; SMT, swollen mitochondrion.

Heat shock at 43.0°C caused some ultrastructural changes that were not apparent in embryos exposed to 41.0°C. Heat shock at 43.0°C but not at 41.0°C increased (P < 0.001) the distance between the two membranes of the nuclear envelope (Fig. 4F). Distance between the membranes was 45 ± 5, 51 ± 1, and 156 ± 5 at 38.5°C, 41.0°C, and 43.0°C, respectively. After culture at 43.0°C, the organelles were more tightly redistributed towards the perinuclear region and could no longer be always recognized (Figs. 2, e and f, and 6, g–i). In addition, culture at 43.0°C resulted in vacuolization of the nucleoplasm (Fig. 4C) and cytoplasm (Figs. 2f and 5c). The cytoplasm of embryos cultured at 43.0°C for 6 h was covered with a heterogeneous dense material (Fig. 6, g–i). The nuclear chromatin underwent severe degeneration and had a spotty appearance (Fig. 4F). Moreover, embryos exhibited increased numbers of lysosomes (Fig. 6H) and withdrawal of microvilli (data not shown).



View larger version (170K):
[in this window]
[in a new window]
  		FIG. 6. Representative micrographs demonstrating appearance of cytoplasmic components in two-cell bovine embryos produced in vitro and cultured for 6 h at 38.5°C (A–C), 41.0°C (D–F), or 43.0°C (G–I). V, Vesicle; Ly, lysosomes; ER, endoplasmic reticulum; SMT, swollen mitochondrion; AL, annulate lamellae; G, Golgi apparatus; MT, mitochondrion; V, vesicle; mv, microvilli; ZP, zona pellucida; PVS, perivitelline space; mt, microtubule.



View larger version (94K):
[in this window]
[in a new window]
  		FIG. 5. Disruption of plasma membrane by heat shock. Two-cell bovine embryos produced in vitro were cultured for 6 h at 38.5°C (A), 41.0°C (B), or 43.0°C (C). mv, Microvilli; ZP, zona pellucida; PVS, perivitelline space; V, vesicle; MT, mitochondrion.

Heat Shock-Induced Changes in Ultrastructure of Two-Cell Embryos Produced In Vivo and Treated Ex Vivo

Embryos recovered from superovulated cows and treated ex vivo were ultrastructurally comparable (Figs. 7 and 8) to embryos produced in vitro (Figs. 2–6). As for embryos produced in vitro, the cytoplasm of embryos produced in vivo and cultured for 6 h was filled primarily with vesicles and mitochondria. Other organelles, including endoplasmic reticulum, Golgi apparatus, annulate lamellae, and lysosomes, were indistinguishable from those of in vitro embryos. Heat shock of 41.0°C for 6 h caused the same alterations in ultrastructural morphology as for in vitro embryos. In particular, the organelles of the embryos treated at 41.0°C for 6 h pulled away from the plasma membrane and toward the nucleus to leave an organelle-free area around the edge of the blastomere (Fig. 7). The mean distances from the edge of the plasma membrane to the organelle-containing region of the cytoplasm were 743 ± 80 and 2553 ± 518 nm for embryos at 38.5°C and 41.0°C, respectively (P < 0.05). The percentage of mitochondria that exhibited a swollen morphology was also increased by heat shock (percentage swollen: 0.7% ± 0.6% vs. 7% ± 2% for 38.5°C and 41.0°C, respectively; P = 0.07; Fig. 8). A notable finding was that lysosomes appeared with more frequency in the embryos produced in vivo and heat shocked at 41.0°C for 6 h than in the embryos produced in vitro and treated under similar conditions (data not shown).



View larger version (180K):
[in this window]
[in a new window]
  		FIG. 7. Effects of heat shock on morphology of two-cell bovine embryos produced in vivo and treated ex vivo as determined by light microscopic examination of 500-nm sections stained with toluidine blue O stain (A and C) or by transmission electron microscopy (B and D). Embryos were cultured for 6 h at 38.5°C (A and B) or 41.0°C (C and D). Arrows show area of the cytoplasm devoid of organelles. ZP, Zona pellucida; PVS, perivitelline space; N, nucleus; MT, mitochondrion; Ly, lysosome.



View larger version (142K):
[in this window]
[in a new window]
  		FIG. 8. Heat shock-induced alterations in mitochondrial morphology of two-cell bovine embryos produced in vivo. Embryos were cultured for 6 h at 38.5°C (A) or 41.0°C (B). MT, Mitochondrion; V, vesicle; SMT, swollen mitochondrion; G, Golgi apparatus.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that disruption of development caused by exposure of two-cell bovine embryos to a physiologically relevant heat shock involves alterations to the cytoskeleton and mitochondria. There were nearly no differences observed at the ultrastructural level between two-cell bovine embryos produced in vitro and those produced in vivo at 38.5°C or in their response to hyperthermia, which indicates that results based on embryos produced in vitro to study heat shock are relevant to the embryo that develops in vivo, at least with respect to ultrastructural changes.

Much is known about the cellular mechanisms whereby heat shock disrupts cell proliferation and differentiation. Heat shock can induce morphological alterations to the cytoskeleton [15] and changes to mitochondrial morphology, function, and potential [16–18, 28–30]. In addition, heat shock causes disruption and redistribution of organelles [16–18], damages the nucleus [17, 31], induces protein aggregation [32], and induces functional and ultrastructural changes in membranes [16, 19, 33]. However, most of the studies regarding cellular effects of heat shock involved temperatures >43.0°C, and the results are not relevant to effects of much milder heat shocks affecting embryo survival. Temperatures of >=43.0°C are lethal to many mammals and well above the body temperatures associated with hyperthermia-induced embryonic death in cattle (i.e., <=41.0°C [9, 10, 21]). As the results of the first experiment indicate, development of the two-cell bovine embryo is very susceptible to disruption by mild heat shock (i.e., 41.0°C for 6 h). How such a mild heat shock affects the cellular and subcellular processes orchestrating early embryonic development has not been well studied and is the topic of these experiments.

The most obvious alterations to the ultrastructure of two-cell embryos produced in vitro and in vivo caused by heat shock were the movement of organelles away from the plasma membrane and the increase in the percentage of mitochondria that exhibited a swollen morphology. The translocation of organelles toward the center of the blastomere suggests that heat shock affected the cytoskeleton because this system is involved in active transport of organelles and other components of the cell using both microtubule and actin filament systems of transport (i.e., dynein, kinesin [34, 35]). The movement of organelles in the heat-shocked two-cell embryo is reminiscent of the translocation of melanosomes in the fish melanophore. Movement of these melanized organelles via aggregation toward the perinuclear region or dispersion to the periphery of the dorsal skin cells is responsible for rapid background adaptation. Both microtubules and microfilaments are involved during aggregation and dispersion of the melanosomes [36]. It would be of interest to determine whether the aggregation of organelles following heat shock also involved similar patterns of movement of organelles on microtubules and microfilaments.

Although the present evidence suggests that heat shock fractures or alters the cytoskeleton, the specific alterations involved are not known. The specific cytoskeletal element affected by heat depends on cell type [15] and, in all likelihood, temperature. Further studies need to be conducted to verify that the movement of organelles caused by heat shock is a result of cytoskeletal rearrangement and to determine which elements are involved in this response. An additional question that arises is whether sensitivity of the cytoskeleton to 41.0°C is unique to embryos or is a general characteristic of mammalian cells.

Disruption of the cytoskeleton probably is a major cause of the inhibition of development caused by heat shock. The cytoskeleton is a cellular component that is common to the plasma membrane, nuclear membrane, and cytoplasmic organelles. The three components of the cytoskeleton (i.e., actin filaments, microtubules, and intermediate filaments) work together to enhance structural integrity, impart cell shape, provide cell motility, and position mRNA and protein [31, 34], and they play an important role in cell signaling [37], mitosis, and cytokinesis [38]. During the first three cleavage divisions, the mammalian embryo spends the majority of the cell cycle in S and M phases [39]. These cell-cycle stages are differentially affected by elevated temperatures. For instance, exposure of cells to heat shock during the S phase results in spontaneous premature chromosome condensation and micronuclear formation [40]. Heat shock during the M phase results in disassembly of the mitotic spindle, failure of cytokinesis, and polyploidy [41, 42]. Experiments to test whether heat shock of two-cell embryos results in micronuclei formation and/or alterations in the ploidy of the blastomeres would provide further evidence for cytoskeleton-mediated damage of the developmental program.

The second major effect of heat shock observed for both in vitro-produced embryos and embryos produced in vivo was swelling of mitochondria. Mitochondrial swelling occurs under several different stressful situations, such as calcium overload [43, 44], oxidative stress [44, 45], ischemia/reperfusion [46], chronic ethanol exposure [47], and hyperthermia [28]. Swelling of the mitochondrial matrix has been hypothesized to be a result of changes in the mitochondrial permeability transition caused by the opening of the high-conductance permeability transition pore on the inner mitochondrial membrane and resulting in unselective solute fluxes [43]. Under this condition, mitochondria can no longer maintain a proton gradient [48], aerobic ATP synthesis is arrested, a progressive unfolding of the inner mitochondrial cristae occurs, and several apoptosis initiator components such as cytochrome c and apoptosis-inducing factor are released [43, 49].

The estimated 15% loss in functional mitochondria caused by heat shock at the two-cell stage could cause a reduction in ATP synthesis, as has been seen in other cell types [50]. The consequences for reduced mitochondrial capacity may not immediately impact energy metabolism in the two-cell embryo because there is little metabolic activity until the 12- to 16-cell stage [51], when a shift from oxidative phosphorylation to glycolysis occurs [52, 53]. The loss of mitochondria at the two-cell stage could adversely affect later energy metabolism unless the loss is compensated for by increased mitochondrial biogenesis. In most cell types, the release of cytochrome c and apoptosis-inducing factor would trigger caspase activation and apoptosis. However, the two-cell bovine embryo is unable to undergo apoptosis after heat shock [54], and the loss of mitochondria due to heat shock would not cause cell loss through apoptosis at this stage.

The severe heat shock of 43.0°C caused a wider range of pathologies than observed at 41.0°C. These included separation of the two membranes of the nuclear envelope, development of a rough appearance on the plasma membrane that indicates loss of microvilli, increase in the number of lysosomes, appearance of electron-dense material in the nucleus and cytoplasm, and vacuolation of the cytoplasm and nucleoplasm. Some of these changes, including loss of microvilli [55], changes in cellular surface morphology and nuclear membrane [56], and concentration of organelles toward the nucleus [57], have also been seen in other cells exposed to temperatures of 43.0°C. The separation of nuclear envelope membranes and loss of microvilli could be the result of cytoskeletal rearrangement because the nuclear matrix is tightly fastened to the cytoskeletal scaffold [31]. Alternatively, a heat shock as severe as 43.0°C could affect the fluidity of membranes [33]. The electron-dense material seen in embryos exposed to 43.0°C for 6 h is aggregated protein caused by heat denaturation and exposure of hydrophobic domains [58, 59]. The increase in numbers of lysosomes in embryos exposed to 43.0°C implies that the blastomeres were undergoing autodigestion.

There were few differences in ultrastructure between two-cell embryos produced in vitro and those produced in vivo at either 38.5°C or 41.0°C. This similarity in ultrastructure agrees with results of another study comparing cleavage-stage embryos produced in vitro with those produced in vivo [60]. Later in development, differences emerge in volume density of total mitochondria, vacuoles, and nuclei [23], lipid content [23, 61], degree of compaction [60], and intracellular communication [62]. The one difference detected between embryos produced in vitro and those produced in vivo was the appearance of lysosomes after heat shock. These organelles appeared with more frequency in the embryos produced in vivo than in embryos produced in vitro. Perhaps there are subtle differences between these two types of embryos that either make the embryos produced in vivo more sensitive to the effects of heat shock (as indicated by increased number of lysosomes) or more able to recover from heat shock (because the increased number of lysosomes allow greater repair of the cell). The overall similarity in response to heat shock is indicative that at least at this early stage of development information on effects of heat shock on embryos produced in vitro is relevant to an understanding of the effects of heat shock on embryos produced in vivo. One question that remains to be answered is whether the changes in ultrastructural morphology caused by heat shock would also occur in embryos that were located in vivo at the time of heat shock, because the reproductive tract environment might mitigate some embryonic responses to heat shock.

Exposure of two-cell bovine embryos to elevated temperatures can disrupt embryonic development by causing alterations in the nucleus, cytoplasm, mitochondria, and other organelles. Most importantly, some of these changes, particularly to the cytoskeleton and mitochondria, occur at temperatures characteristic of cows experiencing hyperthermia-induced infertility.


   ACKNOWLEDGMENTS
 
The authors thank William Rembert for collecting ovaries, Jose Queijeiro for technical assistance, Jeremy Block and Heather Rosson for assistance with animals, and Larry Eubanks and the University of Florida Meat Laboratory personnel for assistance with tissue collection. The authors extend special thanks to the following for their generosity: Marshall, Adam, and Alex Chernin and employees of Central Beef Packing Co (Center Hill, FL) for providing ovaries, Scott A. Randell from Southeastern Semen Services (Wellborn, FL) for donating semen, and Pfizer Animal Health (Kalamazoo, MI) for donation of Lutalyse.


   FOOTNOTES
 
1 This research was supported in part by grants from the USDA: IFAFS 2001-52101-11318 and TSTAR 2001-34135-11150. This is Journal Series no. R-09573 of the Florida Agricultural Experiment Station. Portions of this research were presented at the 2002 Southeastern Electron Microscopy Society meeting in Athens, Georgia. Back

2 Correspondence: Peter J. Hansen, Department of Animal Sciences, University of Florida, P.O. Box 110910, Gainesville, FL 32611-0910. FAX: 352 392 5595; hansen{at}animal.ufl.edu Back

Received: 12 June 2003.

First decision: 8 July 2003.

Accepted: 14 August 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Alliston CW, Howarth B Jr, Ulberg LC. Embryonic mortality following culture in vitro of one- and two-cell rabbit eggs at elevated temperatures. J Reprod Fertil 1965 9:337-341[Abstract/Free Full Text]
  2. Ulberg LC, Burfening PJ. Embryo death resulting from adverse environment on spermatozoa or ova. J Anim Sci 1967 26:571-577
  3. Edwards JL, Hansen PJ. Elevated temperature increases heat shock protein 70 synthesis in bovine two-cell embryos and compromises function of maturing oocytes. Biol Reprod 1996 55:340-346
  4. Edwards JL, Hansen PJ. Differential responses of bovine oocytes and preimplantation embryos to heat shock. Mol Reprod Dev 1997 46:138-145[CrossRef][Medline]
  5. Rivera RM, Hansen PJ. Development of cultured bovine embryos after exposure to high temperatures in the physiological range. Reproduction 2001 121:107-115[Abstract]
  6. Gwazdauskas FC, McCaffrey C, McEvoy TG, Sreenan JM. In vitro preimplantation mouse embryo development with incubation temperatures of 37 and 39C. J Assist Reprod Genet 1992 9:149-154[CrossRef][Medline]
  7. Krininger CE, Stephens SH, Hansen PJ. Developmental changes in inhibitory effects of arsenic and heat shock on growth of pre-implantation bovine embryos. Mol Reprod Dev 2002 63:335-340[CrossRef][Medline]
  8. Rattan SI. Repeated mild heat shock delays ageing in cultured human skin fibroblasts. Biochem Mol Biol Int 1998 45:753-759[Medline]
  9. Dunlap SE, Vincent CK. Influence of post-breeding thermal stress on conception rate in beef cattle. J Anim Sci 1971 32:1216-1218
  10. Ealy AD, Drost M, Hansen PJ. Developmental changes in embryonic resistance to adverse effects of maternal heat stress in cows. J Dairy Sci 1993 76:2899-2905[Abstract/Free Full Text]
  11. Ryan DP, Prichard JF, Kopel E, Godke RA. Comparing early embryo mortality in dairy cows during hot and cool seasons of the year. Theriogenology 1993 39:719-737
  12. Dutt RH. Critical period for early embryo mortality in ewes exposed to high ambient temperature. J Anim Sci 1963 22:713-719[Abstract/Free Full Text]
  13. Shah MK. Reciprocal egg transplantations to study the embryo-uterine relationship in heat-induced failure of pregnancy in rabbits. Nature 1956 177:1134-1135
  14. Ozawa M, Hirabayashi M, Kanai Y. Developmental competence and oxidative state of mouse zygotes heat-stressed maternally or in vitro. Reproduction 2002 124:683-689[Abstract]
  15. Coss RA, Linnemans WAM. The effects of hyperthermia on the cytoskeleton. Int J Hyperthermia 1996 12:173-196[Medline]
  16. Funk RHW, Nagel F, Wonka F, Krinke HE, Golfert F, Hofer A. Effects of heat shock on the functional morphology of cell organelles observed by video-enhanced microscopy. Anat Rec 1999 255:458-464[CrossRef][Medline]
  17. Welch WJ, Suhuan JP. Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment. J Cell Biol 1985 101:1198-1211[Abstract/Free Full Text]
  18. Wang TT, Chiang AS, Chu JJ, Cheng TJ, Chen TM, Lai YK. Concomitant alterations in distribution of 70 kDa heat shock proteins, cytoskeleton and organelles in heat shocked 9L cells. Int J Biochem Cell Biol 1998 30:745-759[CrossRef][Medline]
  19. Jewel SA, Bellomo G, Thor H, Orrenius S, Smith MT. Bleb formation in hepatocytes during drug metabolism is caused by disturbances in thiol and calcium ion homeostasis. Science 1982 217:1257-1259[Abstract/Free Full Text]
  20. Turner HG. Genetic variation of rectal temperature in cows and its relationship to fertility. Anim Prod 1982 35:401-412
  21. Putney DJ, Drost M, Thatcher WW. Embryonic development in superovulated dairy cattle exposed to elevated ambient temperature between Days 1 to 7 post insemination. Theriogenology 1988 30:195-209
  22. Holm P, Booth PJ, Callesen H. Kinetics of early in vitro development of bovine in vivo- and in vitro-derived zygotes produced and/or cultured in chemically defined or serum-containing media. Reproduction 2002 123:553-565[Abstract]
  23. Crosier AE, Farin PW, Dykstra MJ, Alexander JE, Farin CE. Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro. Biol Reprod 2001 64:1375-1385[Abstract/Free Full Text]
  24. Enright BP, Lonergan P, Dinnyes A, Fair T, Ward FA, Yang X, Boland MP. Culture of in vitro produced bovine zygotes in vitro vs in vivo: implications for early embryo development and quality. Theriogenology 2000 54:659-673[CrossRef][Medline]
  25. Parrish JJ, Susko-Parrish JL, Crister ES, Eyestone WH, First NL. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 1986 25:591-600[CrossRef][Medline]
  26. Soto P, Natzke RP, Hansen PJ. Identification of possible mediators of embryonic mortality caused by mastitis: actions of lipopolysaccharide, prostaglandin F2{alpha}, and the nitric oxide generator, sodium nitroprusside dihydrate, on oocyte maturation and embryonic development in cattle. Am J Reprod Immunol 2003 50:263-272
  27. Rosenkrans CF, Zeng CQ, McNamara GT, Schoff PK, First NL. Development of bovine embryos in vitro as affected by energy substrates. Biol Reprod 1993 49:459-462[Abstract]
  28. Cole A, Armour EP. Ultrastructural study of mitochondrial damage in CHO cells exposed to hyperthermia. Radiat Res 1988 115:421-435[CrossRef][Medline]
  29. Macouillard-Poulletier de Ganes F, Leducq N, Diolez P, Belloc F, Merle M, Canioni P, Voisin PJ. Mitochondrial impairment and recovery after heat shock treatment in a human microglial cell line. Neurochem Int 2000 36:233-241[CrossRef][Medline]
  30. Willis WT, Jackman MR, Bizeau ME, Pagliassotti MJ, Hazel JR. Hyperthermia impairs liver mitochondrial function in vitro. Am J Physiol 2000 278:R1240-R1246
  31. Gniadecki R, Olszewska H, Gajkowska B. Changes in the ultrastructure of cytoskeleton and nuclear matrix during HaCaT keratinocyte differentiation. Exp Dermatol 2001 10:71-79[CrossRef][Medline]
  32. Wachsberger PR, Coss RA. Alterations in nuclear matrix ultrastructure of G1 cells following heat shock: resinless section electron microscopy, biochemical, and immunofluorescence studies. J Cell Physiol 1993 155:615-634[CrossRef][Medline]
  33. Lepock JR. Involvement of membranes in cellular responses to hyperthermia. Radiat Res 1982 92:433-438[CrossRef][Medline]
  34. Capco DG. Specializations in cytoskeletal function during early development. In: Hesketh JE, Pryme IF (eds.), The Cytoskeleton, Role in Cell Physiology, vol. 2. Greenwich, CT: Jai Press; 1996:59–112
  35. Stebbings H. Microtubule-based intracellular transport of organelles. In: Hesketh JE, Pryme IF (eds.), The Cytoskeleton, Role in Cell Physiology, vol. 2. Greenwich, CT: Jai Press; 1996:113–140
  36. Skold HN, Norstrom E, Wallin M. Regulatory control of both microtubule- and actin-dependent fish melanosome movement. Pigment Cell Res 2002 15:357-366[CrossRef][Medline]
  37. Huang SH, Yang KJ, Wu JC, Chang KJ, Wang SM. Effects of hyperthermia on the cytoskeleton and focal adhesion proteins in a human thyroid carcinoma cell line. J Cell Biochem 1999 75:327-337[CrossRef][Medline]
  38. Lodish H, Berk A, Zipursky SL, Baltimore D, Darnell J. Molecular and Cell Biology. New York: WH Freeman and Co.; 2000: 795–847.
  39. Barnes FL, Eyestone WH. Early cleavage and the maternal to zygotic transition in bovine embryos. Theriogenology 1990 33:141-152
  40. Swanson PE, Carroll SB, Zhang XF, Mackey MA. Spontaneous premature chromosome condensation, micronucleus formation, and non-apoptotic cell death in heated HeLa S3 cells. Am J Pathol 1995 146:963-971[Abstract]
  41. Coss RA, Dewey WC, Bamburg JR. Effects of hyperthermia on dividing Chinese hamster ovary cells and microtubule in vitro. Cancer Res 1982 42:1059-1071[Abstract/Free Full Text]
  42. Vidair CA, Doxsey SJ, Dewey WC. Heat shock alters centrosome organization leading to mitotic dysfunction and cell death. J Cell Physiol 1993 154:443-455[CrossRef][Medline]
  43. Ichas F, Mazat JP. From calcium to cell death: two conformations of the mitochondrial permeability transition pore. Switching from low- to high-conductance state. Biochim Biophys Acta 1998 1366:33-50[Medline]
  44. Halestrap AP, McStay GP, Clarke SJ. The permeability transition pore complex: another view. Biochimie 2002 84:153-166[Medline]
  45. Lemasters JJ. The mitochondrial permeability transition and the calcium, oxygen and pH paradoxes: one paradox after another. Cardiovasc Res 1999 44:470-473[Free Full Text]
  46. Ooie T, Takahashi N, Saikawa T, Nawata T, Arikawa M, Yamanaka K, Hara M, Shimada T, Sakata T. Single dose of geranylgeranylacetone induces heat-shock protein 72 and renders protection against ischemia/reperfusion injury in rat heart. Circulation 2001 104:1837-1843[Abstract/Free Full Text]
  47. Li HS, Zhang JY, Thompson BS, Deng XY, Ford ME, Wood PG, Stolz DB, Eagon PK, Whitcomb DC. Rat mitochondrial ATP synthase ATP5G3: cloning and upregulation in pancreas after chronic ethanol feeding. Physiol Genomics 2001 6:91-98[Abstract/Free Full Text]
  48. Willis WT, Jackman MR, Bizeau ME, Pagliassotti MJ, Hazel JR. Hyperthermia impairs liver mitochondrial function in vitro. Am J Physiol 2000 278:R1240-R1246
  49. Susin SA, Zamzami N, Kroemer G. Mitochondria as regulators of apoptosis: doubt no more. Biochim Biophys Acta 1998 1366:151-165[Medline]
  50. Calderwood SK. Role of energy in cellular responses to heat. In: Bowler K, Fuller BJ (eds.), Temperature and Animal Cells. Cambridge, MA: Company of Biologists; 1987:213–233
  51. Khurana NK, Niemann H. Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo. Biol Reprod 2000 62:847-856[Abstract/Free Full Text]
  52. Thompson JG, Partridge RJ, Houghton FD. Oxygen uptake and carbohydrate metabolism by in vitro derived bovine embryos. J Reprod Fertil 1996 106:299-306[Abstract/Free Full Text]
  53. Cummins JM. Mitochondria: potential roles in embryogenesis and nucleocytoplasmic transfer. Hum Reprod Update 2001 7:217-228[Abstract/Free Full Text]
  54. Paula-Lopes FF, Hansen PJ. Heat-shock induced apoptosis in bovine preimplantation embryos is a developmentally-regulated phenomenon. Biol Reprod 2002 66:1169-1177[Abstract/Free Full Text]
  55. Leyko W, Bartosz G. Membrane effects of ionizing radiation and hyperthermia. Int J Radiat Biol Relat Stud Phys Chem Med 1986 49:743-770[Medline]
  56. Marcocci L, Mondovi B. Biochemical and ultrastructural changes in the hyperthermic treatment of tumor cells: an outline. In: Bicher HI, McLaren JR, Pigliucci GM (eds.), Consensus on Hyperthermia for the 1990's. New York: Plenum; 1990:99–120
  57. Coakley WT. Hyperthermia effects on the cytoskeleton and on cell morphology. Symp Soc Exp Biol 1987 41:187-211[Medline]
  58. Lepock JR, Frey HE, Heynen ML, Senisterra GA, Warters RL. The nuclear matrix is a thermolabile cellular structure. Cell Stress Chaperones 2001 6:136-147[CrossRef][Medline]
  59. Wachsberger PR, Coss RA. Alterations in nuclear matrix ultrastructure of G1 mammalian cells following heat shock: resinless section electron microscopy, biochemical, and immunofluorescence studies. J Cell Physiol 1993 155:615-634
  60. Plante L, King WA. Light and electron microscopic analysis of bovine embryos derived by in vitro and in vivo fertilization. J Assist Reprod Genet 1994 11:515-529[CrossRef][Medline]
  61. Rizos D, Fair T, Papadopoulos S, Boland P, Lonergan P. Developmental, qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo and in vitro. Mol Reprod Dev 2002 62:320-327[CrossRef][Medline]
  62. Boni R, Tosti E, Roviello S, Dale B. Intercellular communication in in vivo- and in vitro-produced bovine embryos. Biol Reprod 1999 61:1050-1055[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 ReproductionHome page
A. M Brad, K. E M Hendricks, and P. J Hansen
The block to apoptosis in bovine two-cell embryos involves inhibition of caspase-9 activation and caspase-mediated DNA damage
Reproduction, December 1, 2007; 134(6): 789 - 797.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
B. Loureiro, A. M. Brad, and P. J. Hansen
Heat shock and tumor necrosis factor-{alpha} induce apoptosis in bovine preimplantation embryos through a caspase-9-dependent mechanism
Reproduction, June 1, 2007; 133(6): 1129 - 1137.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
R. J. Collier, C. M. Stiening, B. C. Pollard, M. J. VanBaale, L. H. Baumgard, P. C. Gentry, and P. M. Coussens
Use of gene expression microarrays for evaluating environmental stress tolerance at the cellular level in cattle
J Anim Sci, April 1, 2006; 84(13_suppl): E1 - E.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
R. M. Rivera, G. M Dahlgren, L. A. de Castro e Paula, R. T Kennedy, and P. J Hansen
Actions of thermal stress in two-cell bovine embryos: oxygen metabolism, glutathione and ATP content, and the time-course of development
Reproduction, July 1, 2004; 128(1): 33 - 42.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
R. M. Rivera, K. L. Kelley, G. W. Erdos, and P. J. Hansen
Reorganization of Microfilaments and Microtubules by Thermal Stress in Two-Cell Bovine Embryos
Biol Reprod, June 1, 2004; 70(6): 1852 - 1862.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
69/6/2068    most recent
biolreprod.103.020347v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rivera, R. M.
Right arrow Articles by Hansen, P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rivera, R. M.
Right arrow Articles by Hansen, P. J.
Agricola
Right arrow Articles by Rivera, R. M.
Right arrow Articles by Hansen, P. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH