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

BACKGROUND: The 14-3-3 (YWHA) proteins are a highly conserved, ubiquitously expressed family of proteins. Seven mammalian isoforms of 14-3-3 are known (ß, 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. RESULTS: We 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 (ß, 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. CONCLUSIONS: We 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.

Differences in cortical serotonergic innervation among humans, chimpanzees, and macaque monkeys: a comparative study.

In this study, we assess the possibility that the evolution of human intellectual capacities was supported by changes in the supply of serotonin to the frontal cortex. To this end, quantitative comparative analyses were performed among humans, chimpanzees, and macaques. Immunohistochemical methods were used to visualize serotonin transporter-immunoreactive (SERT-ir) axons within the cerebral cortex. Areas 9 and 32 were chosen for evaluation due to their roles in working memory and theory of mind, respectively. Primary motor cortex was also evaluated because it is not associated with higher cognitive functions. The findings revealed that humans do not display a quantitative increase in serotonin innervation. However, the results indicated region- and layer-specific differences among species in serotonergic innervation pattern. Compared with macaques, humans and chimpanzees together displayed a greater density of SERT-ir axons relative to neuron density in layers V/VI. This change was detected in cortical areas 9 and 32, but not in primary motor cortex. Further, morphological specializations, coils of axons, were observed in humans and chimpanzees that were absent in macaques. These features may represent a greater capacity for cortical plasticity exclusive to hominoids. Taken together, these results indicate a significant reorganization of cortical serotonergic transmission in humans and chimpanzees.

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.

The effects of nicotine on the metabolic and hormonal responses during acute cold exposure.

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 degree 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.

The involvement of tumor necrosis factor-alpha (TNF) as an intraovarian regulator of oocyte apoptosis in the neonatal rat .

Tumor necrosis factor-alpha (TNF) is a cytokine produced not only by various cells of the immune system, but also by various cells in the reproductive system. We have demonstrated that oocytes are an important source of TNF and that the onset of oocytic TNF expression occurs around birth. TNF receptors are localized on oocytes, granulosa cells and interstitial cells allowing for the possibility of autocrine or paracrine actions of TNF. The late fetal/early neonatal period represents a time during which several key events occur, including formation of the primordial follicle and extensive oocyte apoptosis. We have utilized an ovary culture system to examine the involvement of TNF in early ovarian function. This culture system allows both primordial follicle function and apoptosis to occur in vitro. Our results show that TNF can decrease oocyte and primordial follicle number through stimulation of oocyte apoptosis in vitro. TNF thus may serve as an important intraovarian factor involved in the determination of the size of the primordial follicle pool.

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.

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 microg/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.

Tumor necrosis factor alpha enhances oocyte/follicle apoptosis in the neonatal rat ovary.

Tumor necrosis factor alpha (TNFalpha) is a multifunctional cytokine present in oocytes and macrophages in the neonatal rat ovary. The presence of both TNFalpha 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 TNFalpha on isolated granulosa and theca cells and intact follicles; however, to our knowledge, this is the first study to investigate the effects of TNFalpha on the earliest stages of follicular development. Effects of TNFalpha 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 TNFalpha (0, 0.1, 1.0, 10, or 50 ng/ml), a function-blocking TNFalpha antibody (5 microg/ml), or control immunoglobulin (Ig) G. At 1 ng/ml, TNFalpha 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 TNFalpha 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 TNFalpha, apoptosis was examined by DNA laddering. At 1 ng/ml, TNFalpha 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 TNFalpha may be involved in oocyte atresia that normally occurs during the perinatal period.