Mammalian reproduction hinges upon the timely ovulation of a fully differentiated oocyte. This event is the culmination of a complex and dynamic developmental relationship between the oocyte and the antral follicle housing it; the antral follicle constitutes a specialized microenvironment or niche, uniquely suited to the needs of the oocyte as it approaches ovulation. During this time, the oocyte must complete its final growth, capacitation, and nuclear and cytoplasmic maturation. Its microenvironment-the antral follicle-is in turn responsible for the integrity of these processes and the production of a high quality oocyte. Components of the antral follicle, including three distinct somatic cell types (theca, granulosa and cumulus), the basal lamina, and follicular fluid, each have active and regulatory roles in oocyte differentiation. Several milestones in antral folliculogenesis also have an influence on oocyte development. This review will discuss the antral follicle microenvironment with specific attention to its importance in oocyte differentiation. As assisted reproductive technologies (ART) often require stages of oocyte differentiation to occur in vitro rather than in vivo, current knowledge of the antral follicle microenvironment will also be discussed with respect to its clinical applications.

In this study, we evaluated the ability of the equine clinical treatments N-acetylcysteine, EDTA, and hydrogen peroxide to disrupt in vitro biofilms and kill equine reproductive pathogens (Escherichia coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae) isolated from clinical cases. N-acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms of E. coli isolates. The CFU of recoverable P. aeruginosa and K. pneumoniae isolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CFU of E. coli isolates, K. pneumoniae isolates were observed to have a reduction in CFU, and minimal effects were observed for P. aeruginosa isolates. Chelating agents (EDTA formulations) reduced E. coli CFU but were ineffective at disrupting preformed biofilms or decreasing the CFU of P. aeruginosa and K. pneumoniae within a biofilm. No single nonantibiotic treatment commonly used in equine veterinary practice was able to reduce the CFU and biofilm biomass of all three Gram-negative species of bacteria evaluated. An in vivo equine model of infectious endometritis was also developed to monitor biofilm formation, utilizing bioluminescence imaging with equine P. aeruginosa isolates from this study. Following infection, the endometrial surface contained focal areas of bacterial growth encased in a strongly adherent "biofilm-like" matrix, suggesting that biofilms are present during clinical cases of infectious equine endometritis. Our results indicate that Gram-negative bacteria isolated from the equine uterus are capable of producing a biofilm in vitro, and P. aeruginosa is capable of producing biofilm-like material in vivo. Bacterial endometritis is an important cause of subfertility in mares (1). Endometrial infections are reported in 25 to 60% of mares who fail to become pregnant following breeding (1), contributing to a major economic loss for the equine industry (1, 2). The most common species of bacteria identified during the clinical diagnosis of bacterial endometritis include Streptococcus equi subsp. zooepidemicus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (3, 4). The isolation of one of these species of bacteria is considered to be clinically relevant due to observed reductions in pregnancy rates after identification (2). The detection of these bacteria in the uterus would result in treatment with uterine lavage and broad-spectrum antibiotics to reduce bacterial load and eradicate the remaining bacteria (5, 6).Bacterial endometritis that is refractory to traditional antimicrobial treatment is a significant challenge in the equine breeding industry (4). One possible explanation often cited for the failure of antibiotic treatment is the growth of bacterial pathogens in a biofilm (1, 5). Uterine isolates of E. coli, P. aeruginosa, and K. pneumoniae have been proposed to most likely be associated with a biofilm, due to the observation of repeated antibiotic treatment failures in equine reproducti...

Bacteria in a biofilm community have increased tolerance to antimicrobial therapy. To characterize the role of biofilms in equine endometritis, six mares were inoculated with lux-engineered Pseudomonas aeruginosa strains isolated from equine uterine infections. Following establishment of infection, the horses were euthanized and the endometrial surfaces were imaged for luminescence to localize adherent lux-labeled bacteria. Samples from the endometrium were collected for cytology, histopathology, carbohydrate analysis, and expression of inflammatory cytokine genes. Tissue-adherent bacteria were present in focal areas between endometrial folds (6/6 mares). The Pel exopolysaccharide (biofilm matrix component) and cyclic di-GMP (biofilm-regulatory molecule) were detected in 6/6 mares and 5/6 mares, respectively, from endometrial samples with tissue-adherent bacteria (P < 0.05). A greater incidence (P < 0.05) of Pel exopolysaccharide was present in samples fixed with Bouin's solution (18/18) than in buffered formalin (0/18), indicating that Bouin's solution is more appropriate for detecting bacteria adherent to the endometrium. There were no differences (P > 0.05) in the number of inflammatory cells in the endometrium between areas with and without tissue-adherent bacteria. Neutrophils were decreased (P < 0.05) in areas surrounding tissue-adherent bacteria compared to those in areas free of adherent bacteria. Gene expression of interleukin-10, an immune-modulatory cytokine, was significantly (P < 0.05) increased in areas of tissue-adherent bacteria compared to that in endometrium absent of biofilm. These findings indicate that P. aeruginosa produces a biofilm in the uterus and that the host immune response is modulated focally around areas with biofilm, but inflammation within the tissue is similar in areas with and without biofilm matrix. Future studies will focus on therapeutic options for elimination of bacterial biofilm in the equine uterus.

These data provide clear evidence that the events of antral folliculogenesis are relevant to ROS dynamics in vivo. Furthermore, such studies will help to optimize in vitro conditions for oocyte culture protocols, particularly when combined with a comparison of oocyte quality with respect to source follicle characteristics.

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