Using a food web model to predict the effects of Hazardous and Noxious Substances (HNS) accidental spills on deep-sea hydrothermal vents from the Mid-Atlantic Ridge (MAR) region.

Deep-sea hydrothermal vents host unique ecosystems but face risks of incidents with Hazardous and Noxious Substances (HNS) along busy shipping lanes such as the transatlantic route. We developed an Ecopath with Ecosim (EwE) model of the Menez Gwen (MG) vent field (MG-EwE) (Mid-Atlantic Ridge) to simulate ecosystem effects of potential accidental spills of four different HNS, using a semi-Lagrangian Dispersion Model (sLDM) coupled with the Regional Ocean Modelling System (ROMS) calibrated for the study area. Food web modelling revealed a simplified trophic structure with low energy efficiency. The MG ecosystem was vulnerable to disruptions caused by all tested HNS, yet it revealed some long-term resilience. Understanding these impacts is vital for enhancing Spill Prevention, Control, and Countermeasure plans (SPCC) in remote marine areas and developing tools to assess stressors effects on these invaluable habitats.

Author Correction: Removal of deep-sea sponges by bottom trawling in the Flemish Cap area: conservation, ecology and economic assessment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Removal of deep-sea sponges by bottom trawling in the Flemish Cap area: conservation, ecology and economic assessment.

Deep-sea sponge grounds are vulnerable marine ecosystems, which through their benthic-pelagic coupling of nutrients, are of functional relevance to the deep-sea realm. The impact of fishing bycatch is here evaluated for the first time at a bathyal, sponge-dominated ecosystem in the high seas managed by the Northwest Atlantic Fisheries Organization. Sponge biomass surfaces created from research survey data using both random forest modeling and a gridded surface revealed 231,140 t of sponges in the area. About 65% of that biomass was protected by current fisheries closures. However, projections of trawling tracks estimated that the sponge biomass within them would be wiped out in just 1 year by the current level of fishing activity if directed on the sponges. Because these sponges filter 56,143 ± 15,047 million litres of seawater daily, consume 63.11 ± 11.83 t of organic carbon through respiration, and affect the turnover of several nitrogen nutrients, their removal would likely affect the delicate ecological equilibrium of the deep-sea benthic ecosystem. We estimated that, on Flemish Cap, the economic value associated with seawater filtration by the sponges is nearly double the market value of the fish catch. Hence, fishery closures are essential to reach sponge conservation goals as economic drivers cannot be relied upon.

NPC1 regulates ER contacts with endocytic organelles to mediate cholesterol egress.

Transport of dietary cholesterol from endocytic organelles to the endoplasmic reticulum (ER) is essential for cholesterol homoeostasis, but the mechanism and regulation of this transport remains poorly defined. Membrane contact sites (MCS), microdomains of close membrane apposition, are gaining attention as important platforms for non-vesicular, inter-organellar communication. Here we investigate the impact of ER-endocytic organelle MCS on cholesterol transport. We report a role for Niemann-Pick type C protein 1 (NPC1) in tethering ER-endocytic organelle MCS where it interacts with the ER-localised sterol transport protein Gramd1b to regulate cholesterol egress. We show that artificially tethering MCS rescues the cholesterol accumulation that characterises NPC1-deficient cells, consistent with direct lysosome to ER cholesterol transport across MCS. Finally, we identify an expanded population of lysosome-mitochondria MCS in cells depleted of NPC1 or Gramd1b that is dependent on the late endosomal sterol-binding protein STARD3, likely underlying the mitochondrial cholesterol accumulation in NPC1-deficient cells.

Trihalomethanes in liver pathology: Mitochondrial dysfunction and oxidative stress in the mouse.

Trihalomethanes (THMs) are disinfection byproducts found in chlorinated water, and are associated with several different kinds of cancer in human populations and experimental animal models. Metabolism of THMs proceeds through enzymes such as GSTT1 and CYP2E1 and gives rise to reactive intermediates, which form the basis for their toxic activities. The aim of this study was to assess the mitochondrial dysfunction caused by THMs at low levels, and the resulting hepatic histological and biochemical changes in the mouse. Male ICR mice were administered with two THMs: dibromochloromethane (DBCM) and bromodichloromethane (BDCM); once daily, by gavage, to a total of four administrations. Animals were sacrificed four weeks after DBCM and BDCM administrations. Blood biochemistry was performed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TB), albumin (Alb), total protein (TP), creatinine, and urea. Animals exposed to DBCM and BDCM showed elevated ALT and TB levels (p < 0.05) as compared with controls. Histological analysis confirmed the presence of vacuolar degenerescence and a multifocal necrotizing hepatitis in 33% of animals (n = 2). Mitochondrial analysis showed that THMs reduced mitochondrial bioenergetic activity (succinate dehydrogenase (SQR), cytochrome c oxidase (COX), and ATP synthase) and increased oxidative stress (glutathione S-transferase (GST)) in hepatic tissues (p < 0.05). These results add detail to the current understanding of the mechanisms underlying THM-induced toxicity, supporting the role of mitochondrial dysfunction and oxidative stress in liver toxicity caused by DBCM and BDCM. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1009-1016, 2016.

E-cadherin and ß-catenin expression during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl) nitrosamine in mice.

BACKGROUND: E-cadherin and ß-catenin are adhesion molecules that promote integrity and stability of the urothelium. A decrease in their expression is associated with more aggressive tumour phenotypes with the ability to invade and metastasize. MATERIAL AND METHODS: 45 ICR male mice were used, of which 25 received N-butyl-N-(4-hydroxybutyl)nitrosamine (0.05%) in drinking water for a period of 12 weeks. Immunohistochemical expression was evaluated in all urinary bladder preparations for E-cadherin and for ß-catenin. RESULTS: Preneoplastic lesions showed staining patterns similar to normal urothelium. In simple and nodular hyperplasia, membrane staining was dominant (66.7-78.6 and 50-100%, respectively). In dysplasia a cytoplasmic pattern was prevalent (86.7-100%). Neoplastic lesions exhibit an abnormal staining pattern (100%) with heterogeneous staining (cytoplasmic, nuclear and membrane staining). A strong correlation was observed between both adhesion molecule staining patterns (r = 0.83; p = 0.039). CONCLUSIONS: In mice, as in humans, E-cadherin and ß-catenin are valuable tools to investigate cellular adhesion status of urothelium and can be considered as indicators of tumour aggressiveness and evolution.

Review: BBN as an urothelial carcinogen.

Experimental urinary bladder tumours have been proposed as a useful model for the study of urinary bladder carcinogenesis, as well as for evaluating new therapeutic strategies. Consequently, the administration of chemical carcinogens is one of the most commonly used methods for inducing urinary bladder tumours. N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) is, undoubtedly, the most-used urothelial chemical carcinogen. BBN belongs to the nitrosamine family, a wide group of alkylating agents that are able to induce bladder tumours in laboratory animals. Depending on the animal species, the spectrum of urothelial lesions induced by BBN varies, but is similar to those observed in humans. BBN has a high propensity to induce mutations affecting the expression of genes such as p53, RAS and H19 among others. The aim of this study was to review BBN as a urothelial tumour inducer, taking into consideration its chemical characteristics, properties and spectrum of lesions induced, as well as its possible applications.

Experimental study of the anticancer effect of gemcitabine combined with sirolimus on chemically induced urothelial lesions.

BACKGROUND: The purpose of this study was to determine the efficacy of a combination of gemcitabine and sirolimus in a mouse model of invasive bladder cancer. MATERIALS AND METHODS: Gemcitabine (50 mg/kg) and sirolimus (1.5 mg/kg) were administered to animals previously exposed to N-butyl-N-4(hydroxybutyl)nitrosamine in drinking water. Tumour development was determined by histopathological evaluation. RESULTS: Both drugs were well tolerated by animals. The incidence of lesions in mice treated with gemcitabine was lower in comparison to those not treated, however this result was not statistically significant. The incidence of invasive bladder cancer in animals treated with sirolimus was statistically lower (20%) than in animals not treated (54%) (p=0.008). The results indicate that this drug combination has no statistical significance on the development of pre-neoplastic urothelial lesions and had only a minor impact on invasive bladder cancer incidence in mice. CONCLUSION: The combination of gemcitabine and sirolimus had only a marginal impact on invasive bladder cancer in a mouse model.

High doses of olive leaf extract induce liver changes in mice.

Virtually ever since it was first commercialized in 1995, there have been several studies focusing on the use of olive leaf extract (OLE) as a natural therapy and its medical properties. The aim of this study was to investigate the effects of three different concentrations of OLE on the function of mice livers over the course of 14 weeks. Female ICR mice were divided into four groups, depending on OLE concentration used: 0%, 0.25%, 0.5%, and 0.75%. Alanine aminotransferase, alkaline phosphatase, total bilirubin and albumin serum concentrations were all measured. Histopathological changes of the liver were observed after haematoxylin and eosin, reticulin, and Masson's trichrome staining was carried out while liver mitochondrial bioenergetics were also evaluated. Alanine aminotransferase and alkaline phosphatase serum enzyme activities increased significantly in the groups in which 0.5% and 0.75% OLE concentrations were used. Histologically, all the groups exposed to OLE exhibited hyperplasia of the bile ducts, cholestasis, hepatocyte necrosis and inflammatory infiltrated. Hepatic fibrosis was observed in the groups featuring 0.5% and 0.75% OLE concentrations. The mitochondrial membrane potential, respiratory control ratio and ADP/O of samples from animals fed the higher OLE concentration was significantly decreased when compared to the control group.

Temporal change in deep-sea benthic ecosystems: a review of the evidence from recent time-series studies.

Societal concerns over the potential impacts of recent global change have prompted renewed interest in the long-term ecological monitoring of large ecosystems. The deep sea is the largest ecosystem on the planet, the least accessible, and perhaps the least understood. Nevertheless, deep-sea data collected over the last few decades are now being synthesised with a view to both measuring global change and predicting the future impacts of further rises in atmospheric carbon dioxide concentrations. For many years, it was assumed by many that the deep sea is a stable habitat, buffered from short-term changes in the atmosphere or upper ocean. However, recent studies suggest that deep-seafloor ecosystems may respond relatively quickly to seasonal, inter-annual and decadal-scale shifts in upper-ocean variables. In this review, we assess the evidence for these long-term (i.e. inter-annual to decadal-scale) changes both in biologically driven, sedimented, deep-sea ecosystems (e.g. abyssal plains) and in chemosynthetic ecosystems that are partially geologically driven, such as hydrothermal vents and cold seeps. We have identified 11 deep-sea sedimented ecosystems for which published analyses of long-term biological data exist. At three of these, we have found evidence for a progressive trend that could be potentially linked to recent climate change, although the evidence is not conclusive. At the other sites, we have concluded that the changes were either not significant, or were stochastically variable without being clearly linked to climate change or climate variability indices. For chemosynthetic ecosystems, we have identified 14 sites for which there are some published long-term data. Data for temporal changes at chemosynthetic ecosystems are scarce, with few sites being subjected to repeated visits. However, the limited evidence from hydrothermal vents suggests that at fast-spreading centres such as the East Pacific Rise, vent communities are impacted on decadal scales by stochastic events such as volcanic eruptions, with associated fauna showing complex patterns of community succession. For the slow-spreading centres such as the Mid-Atlantic Ridge, vent sites appear to be stable over the time periods measured, with no discernable long-term trend. At cold seeps, inferences based on spatial studies in the Gulf of Mexico, and data on organism longevity, suggest that these sites are stable over many hundreds of years. However, at the Haakon Mosby mud volcano, a large, well-studied seep in the Barents Sea, periodic mud slides associated with gas and fluid venting may disrupt benthic communities, leading to successional sequences over time. For chemosynthetic ecosystems of biogenic origin (e.g. whale-falls), it is likely that the longevity of the habitat depends mainly on the size of the carcass and the ecological setting, with large remains persisting as a distinct seafloor habitat for up to 100 years. Studies of shallow-water analogs of deep-sea ecosystems such as marine caves may also yield insights into temporal processes. Although it is obvious from the geological record that past climate change has impacted deep-sea faunas, the evidence that recent climate change or climate variability has altered deep-sea benthic communities is extremely limited. This mainly reflects the lack of remote sensing of this vast seafloor habitat. Current and future advances in deep-ocean benthic science involve new remote observing technologies that combine a high temporal resolution (e.g. cabled observatories) with spatial capabilities (e.g. autonomous vehicles undertaking image surveys of the seabed).

DNA cytometry and kinetics of rat urothelial lesions during chemical carcinogenesis.

The aims of this study were to evaluate the DNA content of chemically-induced rat urothelial lesions and their relationship to the proliferation index and histological patterns. Sixty female Fisher 344 rats were divided randomly into six groups, four groups were exposed to N-butyl-N-(4-hydroxybutyl) nitrosamine for a period of 10 and 20 weeks, and two groups of ten rats were used as control animals. Paraffin sections were Feulgen stained and analyzed using DNA image cytometry analysis; histograms were classified as either diploid or aneuploid. Ki-67 immunoreactivity was determined by means of the streptavidin-biotin-complex immunoperoxidase method. All normal urothelium from the control groups were found to have diploid DNA content. The same histogram pattern was found in the simple hyperplasia group. As regards the other histological lesions, the frequency of the aneuploidy varied depending on the lesion type: 20% of aneuploidy were nodular hyperplasia, 32% of aneuploidy were dysplasias, 25% of aneuploidy were papilloma, 44% of aneuploidy were papillary neoplasm of low malignant potential, 22% of aneuploidy were low-grade papillary carcinoma, 100% of aneuploidy were high-grade papillary carcinoma and 100% of the aneuploidy were invasive carcinoma. Our results revealed the existence of a statistically significant relationship between DNA ploidy and histological pattern lesions (r=0.3, p<0.023). The Ki-67 proliferation index was significantly higher in aneuploid lesions than in diploid (r=0.56, p=0.01). There was also a statistically significant difference in the Ki-67 proliferation index in relation to the histopathological pattern (r=0.751, p<0.01). DNA content was associated with the Ki-67 proliferation index and histopathological grade. DNA content and prolife-ration index have critical roles to play during urothelial carcinogenesis.

Cell proliferation and DNA content in rat urothelial lesions after repeat intravesical instillations of mitomycin C and bacillus Calmette-Guérin.

AIM: To study cell proliferation and DNA content in urothelial lesions identified after repeated intravesical instillations of mitomycin C (MMC) and bacillus Calmette-Guérin (BCG) in normal rat urothelium. MATERIAL AND METHODS: A total of 45 rats were divided into nine equal groups: those intravesically instilled with MMC; those receiving BCG intravesically, and a control group intravesically instilled with a physiological solution of sodium chloride (SF). Animals were killed 1, 4 or 8 weeks after the last intravesical instillation. An immunohistochemical streptavidin-biotin-peroxidase technique was performed to investigate Ki-67 expression and the DNA ploidy status was measured using static cytometry. RESULTS: In urothelium exposed to MMC lesions such as simple hyperplasia, dysplasia, carcinoma in situ(CIS), and squamous cell metaplasia were identified. The proliferation index presented values of 11.73, 22.43 and 31.46% in hyperplasias, dysplasias, and CIS, respectively (p < 0.05). The frequency of abnormal DNA content amongst those animals exhibiting simple hyperplasias 25% were aneuploid, in the dysplasia 85.2% were aneuploid (p = 0.041). CIS were all multiploid, and squamous cell metaplasias were all diploid. Animals treated with BCG and SF presented no urothelial lesions and a diploid DNA content. CONCLUSIONS: The aneuploid and multiploid DNA content and proliferation index observed in urothelial lesions identified after repeated intravesical instillations of MMC reflect a high degree of genomic instability in such lesions which in itself may lead to rapid regeneration of new phenotypes.

A phyllodes tumor of the urinary bladder in a rat.

AIM: The aim of this short communication was to describe a case of phyllodes tumor of the urinary bladder discovered in a female Fisher 344 rat that died during an experimental protocol to induce and study urothelial lesions by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). METHODOLOGY: From a group of several female rats exposed to BBN via drinking water over the course of 20 weeks, one animal died. At necropsy, a solid mass was identified in the urinary bladder lumen, with a diameter of 0.8 x 0.7 cm. This tumor was processed for histopathological examination and Feulgen coloration. RESULTS: Microscopically, the mass in the bladder was observed to be a phyllodes tumor. DNA content measured by image analysis of a Feulgen-stained section of the tumor and stroma cells displayed diploid DNA content in both components of the tumor. CONCLUSION: This is the first reported phyllodes tumor in a rat's urinary bladder. The exact prognosis and histogenesis of phyllodes tumors of the urinary bladder remains to be determined by the accumulation of data from additional cases.

E-cadherin expression during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl) nitrosamine in rats.

An alteration in the expression of E-cadherin has been observed in many epithelial neoplasms. No data exist, however, for the expression of this protein in an animal model for urinary bladder cancer. The present study investigated the expression of E-cadherin in rat urothelial preneoplastic lesions and tumours induced by oral administration of N-butyl-N-(4-hydroxybutyl) nitrosamine, during 10, 15 and 20 weeks. Simple hyperplasia and squamous metaplasia showed a similar E-cadherin pattern when compared with normal urothelium, with its expression confined to cell membrane. Thirty eight percent of the nodular hyperplasia, 41.4% of the dysplasia and 100% of the papillomas showed a weak E-cadherin expression. All papillary neoplasm of low malignant potential, low-and high-grade papillary carcinoma, and invasive carcinoma revealed an abnormal staining pattern with an increase in cytoplasm reactivity and discontinuous cell membrane positivity. The loss of expression for low-grade papillary carcinoma versus simple hyperplasia, nodular hyperplasia and dysplasia was statistically significant (p = 0.0001, p = 0.007 and p=0.008, respectively). There was a similar decrease in E-cadherin expression for papillary neoplasm of low malignant potential versus simple hyperplasia, nodular hyperplasia and dysplasia (p = 0.0001; p = 0.001 and p=0.0001, respectively). These results suggest that alteration in the expression of this adhesion molecule in rat may be indicative of tumour progression in N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder cancer.

Bioaccumulation of Hg, Cu, and Zn in the Azores triple junction hydrothermal vent fields food web.

In this work, mercury (Hg), copper (Cu) and zinc (Zn) concentrations and tissue distribution are determined in seven benthic invertebrates species (the key species) from the Mid Atlantic Ridge (MAR) hydrothermal vent fields. The samples were collected from three hydrothermal vent fields--Menez Gwen, 840 m; Lucky Strike, 1700 m and Rainbow, 2300 m--near the Azores Triple Junction. These fields are characterized by different depths, geological context and chemical composition of the hydrothermal fluid, particularly the metal content, which is reflected by the metal concentrations in the organisms. Indeed, our results show that organisms from Menez Gwen presented the highest Hg concentrations, while those from Lucky Strike and Rainbow were richer in Cu and Zn. The potential transfer of these metals through two trophic links are also evaluated and include (1) the mussel Bathymodiolus azoricus and the commensal worm Branchipolynoe seepensis, and (2) three different species of shrimps and the crab Segonzacia mesatlantica. No evidence of Hg biomagnification in either of the vent food chains is clearly observed but an increase in Hg accumulation from prey to predator in the crustacean food chain. The same pattern was observed for Cu and Zn, even though these metals are not known to be generally biomagnified in food chains.

Experimental bladder carcinogenesis-rodent models.

Several rodent models of bladder cancer development have been established. The aim of this review article is to provide a critical assessment of different animal models available for the study of bladder carcinogenesis, its chemoprevention and therapy. All, except for transgenic and knockout animals, require 8-12 months experimental periods in order to generate a high yield of neoplasias. Spontaneous bladder tumor models are extremely rare. The significance of the results from animal experiments is dependent upon the selection of a suitable animal model. There are no rules regarding the choice of a model, it is however very useful to have knowledge of relevant comparative medical aspects concerning this subject. We describe chemical carcinogens most commonly used to induce bladder cancer, pellet implantation and urinary calculi, agents that promote bladder cancer, and irradiation. We also evaluated other tools such as cell cultures, tumor implantation and transgenic models for bladder cancer, that have been developed to study the process. The review considers how several imaging techniques can be applied to study rodent bladder carcinogenesis.

Estudo estereológico comparativo de complexos cumulus-ovócito aspirados de folículos durante o ciclo estral em bovinos / Comparative stereological study of cumulus-oocyte complexes aspirated from follicles during the estrous cycle in bovine

Realizou-se uma análise estereológica comparativa de complexos cumulus-ovócito (COCs) de bovino da raça Holtein-Friesian aspirados de folículos antrais pequenos (com diâmetro de 1-4mm) e médios (com diâmetro de 4-8mm) durante as fases de metaestro, diestro e de proestro. Foram estimados o volume médio dos COCs, dos ovócitos (com e sem zona pelúcida), dos núcleos dos ovócitos e das células foliculares e seus respectivos núcleos. Estimou-se a espessura da zona pelúcida e calculou-se a percentagem relativa da freqüência dos diferentes tipos de células foliculares encontradas no cumulus. Os folículos pequenos apresentaram crescimento acelerado e sem sincronia entre o volume do citoplasma e o do núcleo. No folículo médio ocorreu expansão harmoniosa núcleo-citoplasmática. Identificaram-se três populações de células foliculares (C1, C2 e C3), cuja distribuição na massa do cumulus é independente de sua posição relativamente ao ovócito. Durante o ciclo estral, as células C1 foram progressivamente substituídas por C2 e estas, por C3.

A stereological study of medium antral follicles during the bovine estrous cycle.

Stereological methods were applied to bovine cumulus-oocyte complexes (COCs) in order to characterize them quantitatively during the estrous cycle. COCs from medium (4-8mm) antral follicles with a compact and complete cumulus mass, and with an uniform or a non-visible ooplasm were aspirated from ovaries of Holstein-Friesian cows, fixed in glutaraldehyde, randomly embedded in glycol-methacrylate, and sectioned at 20 microm. The unbiased nucleator principle was used for estimating the mean volumes of complexes, oocytes, cumulus cells, and nuclei of oocytes and cumulus cells. The thickness of the zona pellucida and the relative numerical percentages of the several morphological types (C1-C3) of cumulus cells were also evaluated. The optical disector procedure was used for cumulus cell sampling. Quantitative data show that COCs appear heterogeneous for all studied parameters. From metestrus to proestrus the volumes of COCs and oocytes remained constant, the volumes of oocytes and oocyte nuclei were correlated, the thickness of the outer zona pellucida decreased, and the relative numerical frequency of follicular type C3 cells increased. Results suggest that COCs from distinct estrus stages are structurally different, with type C3 follicular cells gradually differentiating from cell types C1 and C2.

Stereological characterization of bovine (Bos taurus) cumulus-oocyte complexes aspirated from small antral follicles during the metestrous and proestrous phases.

Bovine cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries are used for in vitro maturation and fertilization after selection on the basis of morphological appearance of the cumulus and ooplasm. In this context, a quantitative characterization of COCs could provide additional criteria for selecting the most competent complexes. Bovine COCs from small (1-4mm) antral follicles were aspirated from metestrous and proestrous stage ovaries of Holstein-Friesian cows, fixed in glutaraldehyde, randomly embedded in glycol-methacrylate, and sectioned at 20 microm. The unbiased nucleator principle of stereology was used for estimating the mean volumes of complexes, oocytes, cumulus cells, and nuclei of oocytes and cumulus cells. The thickness of the zona pellucida and the relative numerical percentages of the several morphological types (C1, C2 and C3) of cumulus cells were also evaluated. The optical dissector procedure was used for cumulus cell sampling. Quantitative data show that the variability among complexes is generally high, especially for the volume of COCs. There were no linear correlations between the studied parameters, except between the volume of the oocyte and nucleus at metestrus. At proestrus, the volumes of COCs, oocytes and nuclei of oocytes, the volume of follicular cells and the thickness of the inner zona pellucida, were significantly higher than at metestrus. The relative numerical frequency of follicular type C1 cells was lower whereas that of type C3 cells was higher at proestrus than at metestrus. In conclusion, small antral follicles had larger COCs and oocytes at proestrus compared to metestrus and the COCs also had a higher percentage of follicular type C3 cells. Results suggest that for the same type of follicle size there may exist different functional populations of COCs at distinct stages of the bovine ovarian cycle.

Stereologic characterization of bovine (Bos taurus) cumulus-oocyte complexes aspirated from small antral follicles during the diestrous phase.

Bovine ovarian cumulus-oocyte complexes (COCs) are used for in vitro maturation and fertilization after selection by size and morphology, but their developmental potential remains low. Stereology could provide more objective criteria for selecting the most competent complexes, but its application is lacking in cattle. COCs from small (1-4 mm) antral follicles were aspirated from diestrous ovaries of Holstein-Friesian cows, fixed in glutaraldehyde, randomly embedded in glycol-methacrylate, and sectioned at 20 microm. The unbiased nucleator principle was used for estimating the mean volumes of complexes, oocytes, cumulus cells, and nuclei of oocytes and cumulus cells. The thickness of the zona pellucida and the relative numerical percentages of the several morphologic types of cumulus cells were also evaluated. The optical disector procedure was used for cumulus cell sampling. Volume estimation based on a real physical unique point did not differ from those based on a particular point among many or on a virtual central point, and the mean cumulus cell volume was estimated by using the single section bearing the unique reference point. Quantitative data showed that COCs appear heterogeneous for all studied parameters and that the cumulus mass contains three different cell populations.