Follicular fluid is a unique body fluid containing a large number of biochemical components, some in extremely high concentrations, whilst in the middle of this relatively isolated biological compartment lies the arrested oocyte awaiting the signals to resume meiosis and progress towards timely ovulation. Delineation of these signals poses a challenging task. While there have been numerous reports on the concentrations of various steroids and peptides in follicular fluid after both natural and stimulated follicular growth and, similarly, the relationship between various components of oocyte maturation and the perceived signal has been studied in depth, the precise chronology of the endocrine changes relative to the sequence of oocyte maturation has not been systematically evaluated. Ideally the changing endocrine profile of the late preovulatory follicle would be best investigated by repeated frequent sampling (say every 2 h) of a single follicle. Unfortunately in the human such a longitudinal study would be difficult and so as an alternative approach we have considered the pattern of simultaneous endocrine changes in a cross-sectional group of follicles timed with respect to a common event (the start of the endogenous LH surge). The study of human follicular fluid is relatively simple because of the large volumes (2-8 ml) present in a preovulatory follicle but precise timing of the endogenous LH surge is more difficult due to the problems of frequent blood sampling. An alternative although slightly less precise method is to measure LH in frequent urine collections.
E. A. Lenton, H. King, E. J. Thomas, S. K. Smith, R. I. McLachlan, S. MacNeil and I. D. Cooke
J A McGlothlin, G D Lester, P J Hansen, M Thomas, L Pablo, D L Hawkins and M M LeBlanc
An experimental model of ascending placentitis was developed in the mare to characterize the uterine myoelectrical pattern in late gestation and determine how ascending placentitis altered this pattern. In experiment 1, myometrial electrical activity was analyzed during the early morning, late morning and evening hours in four mares in the last 15 days of gestation to identify patterns of activity. In experiment 2, nine mares received intra-cervical inoculations of Streptococcus equi subspecies zooepidemicus. Myoelectrical activity in the early morning and evening hours in these mares was compared with four control mares. In experiment 1, the number of spike burst clusters >30 s was greater in the evening than in the late morning hours (P < 0.04). Spike burst activity (number × duration) of mares in experiment 1 was similar during day and night recordings until the last 6 days of gestation when it gradually increased each evening until parturition (P < 0.05). In experiment 2, control mares experienced a gradual increase in the number of small spike burst clusters in the last 6 days (P = 0.008) and an increase in large and small spike burst clusters in the evening hours in the last 4 days of gestation (P = 0.03). Mares with experimentally induced placentitis never exhibited a rise in spike burst clusters but had an increase in the mean duration and activity index of large spike burst clusters in the 4 days before parturition (P < 0.04). In conclusion, control mares had a progressive, reversible rise in myoelectrical activity at night in the week preceding parturition. This was not observed in mares with experimentally induced placentitis. They exhibited an increase in the intensity and duration of large spike burst clusters possibly in response to local inflammation.
Robert C Burghardt, James R Burghardt, James D Taylor II, Adele T Reeder, Bar T Nguen, Thomas E Spencer, Kayla J Bayless and Greg A Johnson
The integrity of the fetal–maternal interface is critical for proper fetal nourishment during pregnancy. Integrins are important adhesion molecules present at the interface during implantation; however, in vivo evidence for integrin activation and focal adhesion formation at the maternal–conceptus interface is limited. We hypothesized that focal adhesion assembly in uterine luminal epithelium (LE) and conceptus trophectoderm (Tr) results from integrin binding of extracellular matrix (ECM) at this interface to provide increased tensile forces and signaling to coordinate utero-placental development. An ovine model of unilateral pregnancy was used to evaluate mechanotransduction events leading to focal adhesion assembly at the maternal–conceptus interface and within the uterine wall. Animals were hysterectomized on days 40, 80, or 120 of pregnancy, and uteri immunostained for integrins (ITGAV, ITGA4, ITGA5, ITGB1, ITGB3, and ITGB5), ECM proteins (SPP1, LGALS15, fibronectin (FN), and vitronectin (VTN)), cytoskeletal molecules (ACTN and TLN1), and a signal generator (PTK2). Focal adhesion assembly in myometrium and stroma was also studied to provide a frame of reference for mechanical stretch of the uterine wall. Large focal adhesions containing aggregates of ITGAV, ITGA4, ITGA5, ITGB1, ITGB5, ACTN, and PTK2 were detected in interplacentomal uterine LE and Tr of gravid but not non-gravid uterine horns and increased during pregnancy. SPP1 and LGALS15, but not FN or VTN, were present along LE and Tr interfaces in both uterine horns. These data support the idea that focal adhesion assembly at the maternal–conceptus interface reflects adaptation to increasing forces caused by the growing fetus. Cooperative binding of multiple integrins to SPP1 deposited at the maternal–conceptus interface forms an adhesive mosaic to maintain a tight connection between uterine and placental surfaces along regions of epitheliochorial placentation in sheep.
Benjamin B Parrott, John A Bowden, Satomi Kohno, Jessica A Cloy-McCoy, Matthew D Hale, Jacqueline T Bangma, Thomas R Rainwater, Phillip M Wilkinson, John R Kucklick and Louis J Guillette Jr
Epigenetic modifications are key mediators of the interactions between the environment and an organism's genome. DNA methylation represents the best-studied epigenetic modification to date and is known to play key roles in regulating transcriptional activity and promoting chromosome stability. Our laboratory has previously demonstrated the utility of the American alligator (Alligator mississippiensis) as a sentinel species to investigate the persistent effects of environmental contaminant exposure on reproductive health. Here, we incorporate a liquid chromatography–tandem mass spectrometry method to directly measure the total (global) proportion of 5-methyl-2′-deoxycytidine (5mdC) in ovarian and whole blood DNA from alligators. Global DNA methylation in ovaries was significantly elevated in comparison with that of whole blood. However, DNA methylation appeared similar in juvenile alligators reared under controlled laboratory conditions but originating from three sites with dissimilar environmental qualities, indicating an absence of detectable site-of-origin effects on persistent levels of global 5mdC content. Analyses of tissues across individuals revealed a surprising lack of correlation between global methylation levels in blood and ovary. In addition, global DNA methylation in blood samples from juvenile alligators was elevated compared with those from adults, suggesting that age, as observed in mammals, may negatively influence global DNA methylation levels in alligators. To our knowledge, this is the first study examining global levels of DNA methylation in the American alligator and provides a reference point for future studies examining the interplay of epigenetics and environmental factors in a long-lived sentinel species.
Frankie J White, Robert C Burghardt, Jianbo Hu, Margaret M Joyce, Thomas E Spencer and Greg A Johnson
Secreted phosphoprotein 1 (SPP1, osteopontin) is the most highly upregulated extracellular matrix/adhesion molecule/cytokine in the receptive phase human uterus, and Spp1 null mice manifest decreased pregnancy rates during mid-gestation as compared with wild-type counterparts. We hypothesize that Spp1 is required for proliferation, migration, survival, adhesion, and remodeling of cells at the conceptus–maternal interface. Our objective was to define the temporal/spatial distribution and steroid regulation of Spp1 in mouse uterus during estrous cycle and early gestation. In situ hybridization localized Spp1 to luminal epithelium (LE) and immune cells. LE expression was prominent at proestrus, decreased by estrus, and was nearly undetectable at diestrus. During pregnancy, Spp1 mRNA was not detected in LE until day 4.5 (day 1 = vaginal plug). Spp1-expressing immune cells were scattered within the endometrial stroma throughout the estrous cycle and early pregnancy. Immunoreactive Spp1 was prominent at the apical LE surface by day 4.5 of pregnancy and Spp1 protein was also co-localized with subsets of CD45-positive (leukocytes) and F4/80-positive (macrophages) cells. In ovariectomized mice, estrogen, but not progesterone, induced Spp1 mRNA, whereas estrogen plus progesterone did not induce Spp1 in LE. These results establish that estrogen regulates Spp1 in mouse LE and are the first to identify macrophages that produce Spp1 within the peri-implantation endometrium of any species. We suggest that Spp1 at the apical surface of LE provides a mechanism to bridge conceptus to LE during implantation, and that Spp1-positive macrophages within the stroma may be involved in uterine remodeling for conceptus invasion.