Jennifer L. Marcinkiewicz

Tumor Necrosis Factor α Enhances Oocyte/Follicle Apoptosis in the Neonatal Rat Ovary1

Abstract Tumor necrosis factor α (TNFα) is a multifunctional cytokine present in oocytes and macrophages in the neonatal rat ovary. The presence of both TNFα and its receptors in the neonatal rat ovary suggests a potential role for it in follicle assembly or oocyte atresia. Previous studies have provided support for effects of TNFα on isolated granulosa and theca cells and intact follicles; however, to our knowledge, this is the first study to investigate the effects of TNFα on the earliest stages of follicular development. Effects of TNFα on oocyte/follicle number and apoptosis were investigated using an ovarian organ-culture system that supported assembly of primordial follicles in vitro. Ovaries were collected on the day of birth and treated with TNFα (0, 0.1, 1.0, 10, or 50 ng/ml), a function-blocking TNFα antibody (5 μg/ml), or control immunoglobulin (Ig) G. At 1 ng/ml, TNFα decreased follicle and oocyte numbers during 3 days of culture, whereas higher (10 and 50 ng/ml) or lower (0.1 ng/ml) doses had no effect. Treatment with TNFα antibodies increased the number of oocytes and follicles compared to nonspecific IgG control. To determine whether the decreased oocyte/follicle numbers were due to an apoptotic effect of TNFα, apoptosis was examined by DNA laddering. At 1 ng/ml, TNFα increased apoptotic DNA laddering twofold, with no significant effect from lower or higher doses. The cells undergoing apoptosis, as determined by in situ end-labeling, were oocytes, interstitial cells, and granulosa cells. These findings suggest that TNFα may be involved in oocyte atresia that normally occurs during the perinatal period.

Cortical dopaminergic innervation among humans, chimpanzees, and macaque monkeys: A comparative study

In this study, we assessed the possibility that humans differ from other primate species in the supply of dopamine to the frontal cortex. To this end, quantitative comparative analyses were performed among humans, chimpanzees, and macaques using immunohistochemical methods to visualize tyrosine hydroxylase-immunoreactive axons within the cerebral cortex. Axon densities and neuron densities were quantified using computer-assisted stereology. Prefrontal areas 9 and 32 were chosen for evaluation due to their roles in higher-order executive functions and theory of mind, respectively. Primary motor cortex (area 4) was also evaluated because it is not directly associated with cognition. We did not find an overt quantitative increase in cortical dopaminergic innervation in humans relative to the other primates examined. However, several differences in cortical dopaminergic innervation were observed among species which may have functional implications. Specifically, humans exhibited a sublaminar pattern of innervation in layer I of areas 9 and 32 that differed from that of macaques and chimpanzees. Analysis of axon length density to neuron density among species revealed that humans and chimpanzees together deviated from macaques in having increased dopaminergic afferents in layers III and V/VI of areas 9 and 32, but there were no phylogenetic differences in area 4. Finally, morphological specializations of axon coils that may be indicative of cortical plasticity events were observed in humans and chimpanzees, but not macaques. Our findings suggest significant modifications of dopamines role in cortical organization occurred in the evolution of the apes, with further changes in the descent of humans.

Cholinergic innervation of the frontal cortex: differences among humans, chimpanzees, and macaque monkeys.

Cholinergic innervation of the frontal cortex is important in higher cognitive functions and may have been altered in humans relative to other species to support human‐specific intellectual capacities. To evaluate this hypothesis we conducted quantitative comparative analyses of choline acetyltransferase‐immunoreactive axons in cortical areas 9, 32, and 4 among humans, chimpanzees, and macaque monkeys. Area 9 of the dorsolateral prefrontal cortex is involved in inductive reasoning and specific components of working memory processes, while area 32 of the medial prefrontal cortex has been implicated in theory of mind. Area 4 (primary motor cortex) was also evaluated because it is not directly associated with higher cognitive functions. The findings revealed no quantitative species differences in the three cortical areas examined, indicating that human cognitive specializations are not related to a quantitative increase in cortical cholinergic input. However, species‐specific morphological specializations were observed. Clusters of cholinergic fibers that may be indicative of cortical plasticity events were present in chimpanzees and humans, but not in macaques. The other significant morphology noted was the common and distinctive oval or ovoid perisomatic staining in macaque cortices. This feature was also sporadically observed in chimpanzee cortex. Our findings suggest a potential alteration of cortical cholinergic afferents within the prefrontal cortex of humans and chimpanzees, to the exclusion of macaque monkeys. J. Comp. Neurol. 506:409–424, 2008.

In Utero and Lactational Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin and 2,3,4,7,8-Pentachlorodibenzofuran Reduces Growth and Disrupts Reproductive Parameters in Female Rats

Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) are widespread environmental pollutants. TCDD is well known for its adverse effects on female reproduction when administered acutely to immature or adult rats. It is also known that fetal/neonatal exposure to this compound alters reproductive parameters. It is unknown whether exposure to PCDF causes similar adverse effects in offspring. The objectives of the study were to investigate the effects of in utero and lactational (IUL) exposure to TCDD and PCDF on subsequent growth, estrous cycles, and ovulation. Additionally a gonadotropin-primed immature rat model was used to investigate possible direct effects on the ovary after IUL exposure to TCDD (2.5 μg/kg) by evaluating 1) ovarian morphometrics and 2) serum estradiol concentrations. Body weights were reduced in animals with IUL exposure to TCDD and PCDF relative to those in controls at 10 days of age (P < 0.05 for each), and this difference was maintained until termination of the experiment at 125–165 days of age (P < 0.05). Exposure to TCDD or PCDF also disrupted regular estrous cycles and inhibited ovulation rate. On Day 23 (before eCG stimulation), ovaries from animals exposed to TCDD contained the same number of primordial, primary, secondary, preantral, and antral follicles as ovaries from control animals. On Day 25 (48 h after eCG stimulation), ovaries from TCDD-exposed rats had significantly fewer large preovulatory follicles when compared with ovaries from controls. The numbers of smaller follicles (both antral and small antral) were not different. Serum estradiol was significantly lower in TCDD-exposed animals 48 h after eCG stimulation.

Expression of 14-3-3 protein isoforms in mouse oocytes, eggs and ovarian follicular development

BackgroundThe 14-3-3 (YWHA) proteins are a highly conserved, ubiquitously expressed family of proteins. Seven mammalian isoforms of 14-3-3 are known (β, γ, ε, ζ, η, τ and, σ). These proteins associate with many intracellular proteins involved in a variety of cellular processes including regulation of the cell cycle, metabolism and protein trafficking. We are particularly interested in the role of 14-3-3 in meiosis in mammalian eggs and the role 14-3-3 proteins may play in ovarian function. Therefore, we examined the expression of 14-3-3 proteins in mouse oocyte and egg extracts by Western blotting after polyacrylamide gel electrophoresis, viewed fixed cells by indirect immunofluorescence, and examined mouse ovarian cells by immunohistochemical staining to study the expression of the different 14-3-3 isoforms.ResultsWe have determined that all of the mammalian 14-3-3 isoforms are expressed in mouse eggs and ovarian follicular cells including oocytes. Immunofluorescence confocal microscopy of isolated oocytes and eggs confirmed the presence of all of the isoforms with characteristic differences in some of their intracellular localizations. For example, some isoforms (β, ε, γ, and ζ) are expressed more prominently in peripheral cytoplasm compared to the germinal vesicles in oocytes, but are uniformly dispersed within eggs. On the other hand, 14-3-3η is diffusely dispersed in the oocyte, but attains a uniform punctate distribution in the egg with marked accumulation in the region of the meiotic spindle apparatus. Immunohistochemical staining detected all isoforms within ovarian follicles, with some similarities as well as notable differences in relative amounts, localizations and patterns of expression in multiple cell types at various stages of follicular development.ConclusionsWe found that mouse oocytes, eggs and follicular cells within the ovary express all seven isoforms of the 14-3-3 protein. Examination of the differential expression of these 14-3-3 isoforms in female germ cells and ovarian follicles provides the foundation for further investigating 14-3-3 isoform-specific interactions with key proteins involved in ovarian development, meiosis and oocyte maturation. This will lead to a better understanding of the individual functional roles of the 14-3-3 protein isoforms in mammalian oogenesis and female reproductive development.

The immunological and metabolic responses to exercise of varying intensities in normoxic and hypoxic environments.

Blegen, M, Cheatham, C, Caine-Bish, N, Woolverton, C, Marcinkiewicz, J, and Glickman, E. The immunological and metabolic responses to exercise of varying intensities in normoxic and hypoxic environments. J Strength Cond Res 22(5): 1638-1644, 2008-The purpose of this study was to determine the effects of varying intensities of exercise in normoxic and hypoxic environments on selected immune regulation and metabolic responses. Using a within-subjects design, subjects performed maximal tests on a cycle ergometer in both normoxic (PiO2 = 20.94%) and hypoxic (PiO2 = 14.65%) environments to determine &OV0312;O2max. On separate occasions, subjects then performed four randomly assigned, 1-hour exercise bouts on a cycle ergometer (two each in normoxic and hypoxic environments). The hypoxic environment was created by reducing the O2 concentration of inspired air using a commercially available hypoxic chamber. The intensities for the exercise bouts were predetermined as 40 and 60% of their normoxic &OV0312;O2max for the normoxic exercise bouts and as 40 and 60% of their hypoxic &OV0312;O2max for the hypoxic exercise bouts. Blood samples were collected preexercise, postexercise, 15 minutes postexercise, 2 hours postexercise, and 24 hours postexercise for the determination of interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), glucose, glycerol, free fatty acids, epinephrine, norepinephrine, and cortisol. There were no significant differences (p < 0.05) between condition or intensity for IL-1 or TNF-α. Significant differences (p < 0.05) between intensities were demonstrated for epinephrine, norepinephrine, and cortisol (p < 0.05). A significant difference was identified between normoxic and hypoxic environments with respect to nonesterifed fatty acids (0.45 ± 0.37 vs. 0.58 ± 0.31 mEq·L−1, respectively; p = 0.012). During prolonged exercise at 40 and 60% of their respective &OV0312;O2max values, hypoxia did not seem to dramatically alter the response of the selected immune system or metabolic markers. Exercise training that uses acute hypoxic environments does not adversely affect immune regulation system status and may be beneficial for those individuals looking to increase endurance performance.

The effects of gender and menstrual phase on carbohydrate utilization during acute cold exposure.

OBJECTIVE The purpose of this study was to evaluate the effects of gender and menstrual cycle on the percent of carbohydrate (CHO) utilized during cold water immersion (20 degrees C). Previous research has suggested that males and females utilize CHO differently during submaximal exercise. This study examined whether this differential response is replicated during a submaximal elevation in metabolism, as demonstrated during thermogenesis (i.e., shivering during acute cold exposure). METHODS Male and female subjects between the ages of 18 and 30 years were recruited for this study. Female subjects underwent the experimental trial once during the follicular phase and once during the luteal phase of their menstrual cycle. Subjects were immersed to the first thoracic vertebra until esophageal temperature reached 36.5 degrees C or for a maximum preocclusion period of 40 minutes. Peripheral temperature homeostasis via cuff occlusion (right arm and left leg) took place for 10 minutes, after which the pressure cuffs were released (postocclusion) and the subjects remained in the water for an additional 10 minutes. The following variables were measured: respiratory exchange ratio, percent of CHO utilization, and oxygen consumption (Vo2). RESULTS Analysis of variance demonstrated no significant difference between genders or phases of the menstrual cycle in respiratory exchange ratio, percent CHO utilization, or Vo2 during cold water immersion. A significant difference was observed between men and women for absolute Vo2. CONCLUSIONS These data suggest that although men and women differ with respect to absolute aerobic metabolism, this distinction does not cause a differential response with respect to substrate utilization during acute cold exposure.

The Effect of Cold Exposure on the Hormonal and Metabolic Responses to Sleep Deprivation

Abstract Objective.—The purpose of this study was to determine the effect of 12°C cold exposure for 180-minutes on the hormonal responses of sleep-deprived individuals. Methods.—Participants underwent 2 cold-air trials: 1 after a normal night of sleep (ie, 6–8 hours) and 1 after 33 hours of sleep deprivation (SDEP). A venous blood sample was taken at baseline and then at 90-and 180-minute cold-exposure time points. Repeated-measures analysis of variance was used to determine significance between a normal night of sleep and SDEP for norepinephrine, epinephrine, cortisol, insulin, thyroid hormones triiodothyronine and thyroxine, glucose, and non-esterified fatty acids. Results.—There was no significant main effect for time, trial, or interaction for insulin, thyroid hormones, epinephrine, cortisol, and glucose (P ≤ .05). A significant main effect for time for norepinephrine and non-esterified fatty acids was demonstrated (P < .001). Discussion.—The lack of significant differences in the hormonal and metabolic responses to cold exposure combined with SDEP may have been because of an ability of the individual to continue to respond despite the environmental stressor or the physiological effect elicited from cold exposure, thereby possibly masking physiological responses of SDEP. Conclusions.—On the basis of these data, SDEP combined with protracted cold exposure apparently was not a great enough stressor to cause a differential response in the hormonal and metabolic parameters.

The Effects of Nicotine on the Metabolic and Hormonal Responses During Acute Cold Exposure

Abstract Objective.—To examine the effects of nicotine on the metabolic and hormonal responses during acute cold exposure. Methods.—Participants in this study included 6 men and 5 women between the ages of 19 and 25 years. Each subject performed 2 cold-air trials (CATs) consisting of a 30-minute baseline (BASE) period and a 120-minute exposure to 10°C air. One CAT was performed after a nicotine (NIC) dosing using a 21-mg transdermal patch, whereas the other CAT was performed after a placebo (PL) treatment. Blood samples for metabolic and hormonal measurements were obtained at the end of BASE and immediately after the cold exposure. Results.—When examining the sexes separately, there was no difference in norepinephrine between PL and NIC (P = .066). There was also no difference in epinephrine between PL and NIC in either sex (P = .634). From BASE to 120 minutes of the CAT, there was a significant decrease in cortisol (P = .036), but this response was similar between the 2 treatments (P = .077). Glucose and glycerol concentrations were not different between the PL and NIC treatments. At BASE, nonesterified fatty acid (NEFA) concentration was lower during PL compared with NIC (P = .021); however, at 120 minutes of the CAT, NEFA was greater during PL compared with NIC (P = .035). Conclusions.—During 120 minutes of cold exposure, NIC resulted in alterations in the responses in NEFA, whereas the other blood measurements were not significantly different between the 2 groups.