Cholesterol side-chain cleavage cytochrome P450 and 3beta-hydroxysteroid dehydrogenase expression and the concentrations of steroid hormones in the follicular fluids of different phenotypes of healthy and atretic bovine ovarian follicles

Bovine ovarian antral follicles exhibit either one or the other of two patterns of granulosa cell death in atresia. Death can commence either from the antrum and progress toward the basal lamina (antral atresia) or the converse (basal atresia). In basal atresia, the remaining live antrally situated cells appeared to continue maturing. Beyond that, little is known about these distinct patterns of atresia. Healthy (nonatretic) follicles also exhibit either one or the other of two patterns of granulosa cell shape, follicular basal lamina ultrastructure or location of younger cells within the membrana granulosa. To examine these different phenotypes, the expression of the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in granulosa cells and concentrations of steroid hormones in follicular fluid were measured in individual histologically classified bovine antral follicles. Healthy follicles first expressed SCC and 3beta-HSD in granulosa cells only when the follicles reached an approximate threshold of 10 mm in diameter. The pattern of expression in antral atretic follicles was the same as healthy follicles. Basal atretic follicles were all <5 mm. In these, the surviving antral granulosa cells expressed SCC and 3beta-HSD. In examining follicles of 3-5 mm, basal atretic follicles were found to have substantially elevated progesterone (P < 0.001) and decreased androstenedione and testosterone compared to healthy and antral atretic follicles. Estradiol was highest in the large healthy follicles, lower in the small healthy follicles, lower still in the antral atretic follicles, and lowest in the basal atretic follicles. Our findings have two major implications. First, the traditional method of identifying atretic follicles by measurement of steroid hormone concentrations may be less valid with small bovine follicles. Second, features of the two forms of follicular atresia are so different as to imply different mechanisms of initiation and regulation.     

Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17β-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In contrast, reduction of S1P and the S1P/CER in the antral follicle could trigger cellular death and atresia.     

Effects of follicular size and FSH on granulosa cell apoptosis and atresia in porcine antral follicles

The purpose of this study was to establish a culture model for isolated intact porcine antral follicles and investigate the relationship between granulosa cell apoptosis and follicular atresia. Small (<3 mm), medium (3–5 mm) and large (>5 mm) healthy porcine follicles were isolated and cultured in serum‐free TCM199 with or without follicular stimulating hormone (FSH). Microscopic identification of healthy follicles was confirmed by histology. A spontaneous onset of apoptotic cell death in granulosa cells was observed from cultured antral follicles. The apoptotic rate of granulosa cells from small follicles cultured for 24 hr was higher than those of large and medium follicles, accompanied with high FasL mRNA abundance in granulosa cells. Supplementation with 3 or 5 IU/ml FSH significantly inhibited the percentage of granulosa cells that became apoptotic. FSH did not significantly alter estradiol secretion from cultured follicles. Progesterone secretion significantly decreased after culture for 48 hr, coinciding with the morphological changes observed. FasL and Fas mRNA were expressed in the healthy, early atretic, and progressed atretic porcine follicles regardless of follicular size. However, FasL but not Fas mRNA levels increased during follicular atresia. Addition of FSH significantly decreased FasL rather than Fas mRNA levels in granulosa cells and could attenuate apoptosis. Small follicles seemed to be more susceptible to atresia as compared to medium and large follicles. Mol. Reprod. Dev. 77: 670–678, 2010. © 2010 Wiley‐Liss, Inc.     

Gene expression of endothelin-1 in the porcine ovary: follicular development

We have investigated which follicular compartment and stage of follicular development are associated with endothelin-1 (ET-1) gene expression in the porcine ovary. The localization of mature ET-1 peptide and of its mRNA was determined by immunohistochemistry and by in situ hybridization. Stage of follicular development associated with ET-1 expression was investigated in terms of follicular class and occurrence of atresia. The latter was investigated by determining the occurrence of DNA fragmentation in apoptotic cells on adjacent sections to those used for ET-1 gene expression. Fifteen ovaries from 10 prepubertal pigs stimulated with gonadotropin were collected; a total of 1050 follicles were examined. Specific ET-1 immunoreactivity was restricted to the ovarian vasculature and to the granulosa cell compartment of antral follicles. The pattern of ET-1 mRNA expression was similar to that found for ET-1 immunoreactivity. Primordial, primary, and most secondary follicles did not express ET-1. The theca cell layer did not express ET-1 regardless of follicle developmental stage. ET-1 expression occurred with a significantly greater probability (P < 0.001 by the likelihood ratio test) in the granulosa cell compartment of antral follicles than in any other follicle class. Furthermore, in antral follicles, ET-1 expression occurred with a greater likelihood in large antral follicles than in small antral follicles (P < 0.001 by the likelihood ratio test). In small antral follicles, only 16.8% expressed ET-1; in contrast, 66.7% of large antral follicles exhibited ET-1 expression. It is interesting that in follicles in which ovulation had already occurred, intense ET-1 expression was found only in the prominent developing vasculature, the other cells present in the luteinized follicle did not display any ET-1 expression. The pattern of ET-1 gene expression observed in this study would be in agreement with our previous suggestion of a plausible physiological role for ET-1 in preventing premature progesterone production by granulosa cells of an antral follicle. The occurrence of atresia and expression of ET-1 in the same follicle was rare. Small and large antral follicles constituted 5.1% and 5.6%, respectively, of the examined follicles in this category. The majority of atretic follicles did not express ET-1 and, conversely, follicles that expressed ET-1 were not atretic. To the best of our knowledge, this is the first report in which large, nonatretic follicles are clearly identified as the population of follicles expressing ET-1. The results of this study delineate the follicular developmental stage and the compartment of when and where ET-1 may be physiologically meaningful.     

Distribution and Y397 phosphorylation of focal adhesion kinase on follicular development in the mouse ovary

Several protein tyrosine kinases (PTKs) are identified as follicle survival factors that suppress apoptosis in granulosa cells. Focal adhesion kinase (FAK/PTK2) interacts with numerous signaling partners and is important for cell adhesion, survival and other vital processes in which FAK autophosphorylation at Y397 (pY397 FAK) is critical for activating signaling pathways. Despite its important roles in apoptosis, the expression and function of FAK in the ovaries remain unknown. Here, we describe FAK expression, including pY397 FAK, in normal healthy mouse ovaries and its association with follicular development and/or atresia. Normal healthy mouse ovaries were used for western blot (n > 60) and immunohistochemical (n > 180) analyses. Western blot results in immature and mature mice revealed that total FAK and pY397 FAK were highly expressed in the ovary and immunohistochemistry results in 3-week-old mice showed they were localized to granulosa cells of ovarian follicles, especially preantral follicles. In 3-week-old mice treated with 5 IU pregnant mare serum gonadotropin (for obtaining homogenous populations of growing or atretic follicles), western blotting revealed that follicular atresia progression involved decreased phosphorylation of Y397 at 72 and 96 h after treatment, particularly in granulosa cells of atretic follicles, as shown by immunohistochemistry results at 72 h after treatment. Moreover, immunostaining patterns of FAK and cleaved caspase-3 were negatively correlated in serial sections of 3-week-old mouse ovaries. These results suggest that FAK is most active in ovarian follicle granulosa cells and that its phosphorylation at Y397 is histologically meaningful in follicular development in normal healthy ovaries.     

Local regulation of granulosa cell maturation

Fluid from small antral follicles inhibits several functions of porcine granulosa cells from 3-10-mm follicles in vitro, whereas fluid from large follicles stimulates cells from small follicles. Local factors may be needed in vivo to enable granulosa cells to fully respond to gonadotrophins. Only those follicles containing local stimulators may develop while those containing inhibitors may become arrested in development or become atretic. We have compared the actions of GnRH analogs and chondroitin sulfate (CS) on porcine granulosa cell steroidogenesis with actions of follicular fluids. GnRH agonist mimicked follicular fluid inhibition of progesterone secretion but GnRH antagonist did not antagonize follicular fluid's inhibitory actions. GnRH antagonist mimicked follicular fluid enhancement of basal and LH-stimulated progesterone secretion, but did not mimic follicular fluid enhancement of FSH action or stimulation of estrogen secretion. GnRH agonist blocked the enhancement of LH-stimulated progesterone secretion by both GnRH antagonist and stimulatory follicular fluid. CS inhibited basal and LH-stimulated progesterone secretion but did not inhibit pregnenolone utilization, aromatase activity or estrogen secretion. GnRH-like molecules and CS may be partially responsible for follicular fluid actions on granulosa cells. The actions of other molecules are needed to explain the total effects of follicular fluids on granulosa cells.     

IGF-Ⅰ及其受体、IGF结合蛋白-2和LH受体mRNA在卵泡中的表达

利用原位杂交和原位DNA－3’末端标记的方法研究了胰岛素样生长因子河（IG－I）、IGF－I受体、IGF结合蛋白－2、和促性腺激素受体的信使核糖核酸（mRNA）在不同生长与闭锁阶段的大鼠卵巢卵泡中表达的变化。结果表明：IGF－I主要在正常生长的初级卵泡、窦前卵泡和小窦状卵泡中表达。在各生长与成熟阶段的卵泡中都检测到IGF－I受体mRNA,闭锁卵泡的IGF－I受体表达降低。窦前与窦状的生长和闭锁卵泡均表达IGFBP－2。促卵泡激素（FSH）受体在窦前和小窦状卵泡的表达水平比其在大卵泡中的高。窦前与小窦状卵泡仅在膜细胞中表达黄体生成素（LH）受体mRNA,大卵泡的膜细胞与颗粒细胞均表达LH受体,在闭锁卵泡中仅在膜细胞中观察到LH受体的信号。综上结果,提示IGF－I,IGF－I受体和FSH受体在窦前和小窦状卵泡中的协同表达对卵泡的早期发育有重要作用。LH受体mRNA特异地在大卵泡的颗粒细胞中表达可能与优势卵泡选择相关。     

Theca interna: the other side of bovine follicular atresia

Currently, histological classifications of ovarian follicular atresia are almost exclusively based on the morphology of the membrana granulosa without reference to the theca interna. Atresia in the bovine small antral ovarian follicle has been redefined into antral or basal atresia where cell death commences initially within antral or basal regions of the membrana granulosa, respectively. To examine cell death in the theca interna in the two types of atretic follicles, bovine ovaries were collected and processed for immunohistochemistry and light microscopy. Follicles were classified as healthy, antral atretic, or basal atretic. Follicle diameter was recorded and sections stained with lectin from Bandeiraea simplicifolia to identify endothelial cells or with an antibody to cytochrome P450 cholesterol side-chain cleavage to identify steroidogenic cells and combined with TUNEL labeling to identify dead cells. The numerical density of steroidogenic cells within the theca interna was significantly reduced (P < 0.001) in basal atretic follicles in comparison with other follicles. Cell death was greater in both endothelial cells (P < 0.05) and steroidogenic cells (P < 0.01) of the theca interna of basal atretic follicles compared with healthy and antral atretic follicles. Thus, we conclude that the theca interna is susceptible to cell death early in atresia, particularly in basal atretic follicles.     

Relationships Between Respiratory Function Disorders and Serum Copper Levels in Copper Mineworkers

The aim of this study was to investigate the respiratory function disorders that could be related to dust exposure during the production of copper mine in copper mineworkers (CMWs). The study included 75 male CMWs (mean age, 32.0 ± 7.1 years, 58.6% smokers) and 75 male age- and smoking status-matched healthy control subjects. Serum Cu level was significantly higher in the CMW group (0.80 ± 0.62 μg/ml) than the control group (0.60 ± 0.39 μg/ml) (p = 0.017). Significant negative correlations were found between serum Cu level and forced expiratory volume in first second (r = −0.600; p < 0.001) and between serum Cu level and forced vital capacity (r = −0.593; p = <0.001) in CMWs. Serum Cu level was significantly higher in the restrictive type pulmonary function disorders group (1.36 ± 0.62 μg/ml) than obstructive type (0.90 ± 0.55 μg/ml) and normal pulmonary function pattern group (0.53 ± 0.43 μg/ml) (p < 0.001). Patients with radiological parenchymal abnormalities had significantly higher serum copper levels than those without abnormalities (1.53 ± 0.52 vs. 0.71 ± 0.52 μg/ml, respectively; p = 0.002). In conclusion, result of the study has shown a negative association between pulmonary functions disorders and radiological abnormalities and serum Cu levels in CMWs.     

Multiparametric study of atresia in ewe antral follicles: histology, flow cytometry, internucleosomal DNA fragmentation, and lysosomal enzyme activities in granulosa cells and follicular fluid

The differential quantitative participation of apoptosis and necrosis in ewe antral follicles of two different sizes, separated in four stages of atresia using macroscopic, histologic, and esteroid quantification methods was assessed. Annexin V binding and propidium iodide (PI) uptake was used to detect healthy live cells (Annexin V negative/PI negative), early apoptotic cells (Annexin V+/PI-), and necrotic or late apoptotic cells (PI+). Additionally we used internucleosomal DNA fragmentation as a quantitative estimate of apoptosis. Presence and distribution of lysosomal enzymes in follicular fluid and granulosa cells was used as a measure of necrotic cell death. DNA flow cytometry and gel electrophoresis were positively correlated with the progression of atresia, small atretic follicles tend to have higher percentages of internucleosomal cleaved DNA than follicles >6 mm. Annexin/PI binding also indicates that apoptosis and necrosis increase with atresia progression, generally apoptosis outweighs necrosis in small follicles. Acid phosphatase and glucosaminidase in follicular fluid of 3-6 mm follicles showed no significant modifications between healthy and initially atretic follicles, and only a small, but significant increase in activity in advancedly atretic follicles. On the contrary, lysosomal enzyme activity in follicles >6 mm showed positive correlation between atresia stages and the activities of acid phosphatase and glucosaminidase in follicular fluid. A similar size-differential behavior was found in free or membrane-bound lysosomal enzyme activity of granulosa cells. Necrosis, but principally apoptosis, were present during all stages of follicular maturation indicating that growth and maturation of ovarian follicles involves a continuous renewal of granulosa cells, regulated by apoptosis. Mechanisms regulating this equilibrium may participate in the final destiny, whether ovulation or atresia of ovarian follicles.     

mRNA expression pattern of gonadotropin receptors in bovine follicular cysts

Follicular growth and steroidogenesis are dependent on gonadotropin binding to their receptors in granulosa and theca cells of ovarian follicles. The aim of the present study was to evaluate the expression patterns of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) in ovarian follicular structures from cows with cystic ovarian disease (COD) as compared with those of regularly cycling cows. Relative real-time RT-PCR analysis showed that the expression of FSHR mRNA in granulosa cells was highest in small antral follicles, then decreased significantly as follicles increased in size, and was lowest in cysts. FSHR mRNA was not detected in the theca cells of any follicular category, including cysts. LHCGR mRNA expression in granulosa cells was significantly higher in large antral follicles than in cysts, and not detected in granulosa cells of small and medium antral follicles. In theca cells, the expression level of LHCGR mRNA in medium antral follicles was higher than in small and large antral follicles, whereas that in follicular cysts it was similar to those in small and medium antral follicles, but higher than that in large antral follicles. Our findings provide evidence that there is an altered gonadotropin receptor expression in bovine cystic follicles, and suggest that in conditions characterized by altered ovulation, such as COD, changes in the signaling system of gonadotropins may play a fundamental role in their pathogenesis.     

Serum Levels of Cu,Se, and Zn in Adult Rural/Urban Residents in Ghana: Paradigm Shift?

Deficiencies in Cu, Se, and Zn impair one or more biochemical functions, and excess are associated with toxicity. Baseline studies on the Ghanaian population are scanty. The study was undertaken to determine whether significant rural/urban differences in the serum levels of Cu, Se, and Zn did exist. Forty males/60 females from rural and 50 males/50 females from urban Ghanaian communities were sampled. Serum Cu, Se, and Zn were determined using flame atomic absorption spectrometry. Cu level for rural and urban subjects was 997 ± 333 and 979 ± 290 μg/L, respectively (p = 0.68). However, Cu levels were significantly higher in the rural females (1,063 ± 367 μg/L) than the rural males (898 ± 249 μg/L; p = 0.0085). Se levels for rural/urban subjects were 97 ± 36 and 87 ± 31 μg/L, respectively (p = 0.03). Zn levels in the rural/urban subjects were 312 ± 218 and 150 ± 102 μg/L, respectively (p = 0.002). Additionally, Zn was significantly higher in rural females (428 ± 204 μg/L) than the urban females (166 ± 103 μg/L; p = 0.0002). Finally, Zn was significantly higher in rural females (428 ± 204 μg/L) than males (172 ± 116 μg/L; p = 0.0028). In conclusion, Cu, Se, and Zn were higher in the rural group compared to the urban group, and the generally low Zn levels were confirmed in another cohort follow-up study.     

DNA degradation in mural granulosa cells of non- and slightly atretic follicles of fresh and cold-stored domestic cat ovaries

Apoptosis of granulosa cells is associated with follicular atresia and may occur before atresia becomes morphologically evident. Detection of DNA fragmentation by in situ end-labeling (ISEL) with terminal transferase allows the histological assessment of apoptotic cells on conventional histological sections. Degradation of DNA also may occur after prolonged cold storage of ovaries caused by the release of lysosomal enzymes. The objectives of this study were to assess follicle atresia and the impact of cold storage for 8, 12, 24, and 48 hr after ovarian excision by assessing DNA degradation in mural granulosa cells of cat ovaries. Follicles were distinguished by morphological criteria as nonatretic (NA), slightly atretic (SA), or atretic, and the mean number (±SEM) of granulosa cells labeled by ISEL was determined. About 50% of follicles showed some sign of atresia independent from the stage of the reproductive cycle of the ovarian donor. Number of ISEL-stained granulosa cells for NA and SA, freshly collected follicles was 7.5 ± 0.6 and 9.3 ± 0.8 cells/field, respectively, compared to 16.2 ± 0.8 cells/field in the wall of atretic follicles (P < 0.001). Fresh NA follicles from luteal phase ovaries had more (P < 0.05) labeled granulosa cells (9.2 ± 0.7 cells/field) than measured in follicles of cats in a follicular phase (5.7 ± 0.7). During cold storage, DNA degradation began within 12 hr (NA, 12.2 ± 0.7 cells/field; SA, 13.3 ± 0.5), both values being different (P < 0.05) from fresh controls. By 24 hr, DNA degradation was at the level of a positive control subjected to DNAse treatment. In summary, results reveal that granulosa cell DNA degeneration precedes the loss of developmental capacity of cat oocytes during atresia and postexcision storage. Finding irreversible changes in granulosa cell DNA after storage of cat ovaries for >12 hr may be important for developing oocyte rescue protocols for rare felids in cases where prolonged storage and transport may be required. Mol. Reprod. Dev. 48:350–355, 1997. © 1997 Wiley-Liss, Inc.     

Germinal vesicle configurations and patterns of polypeptide synthesis of procine oocytes from antral follicles of different size, as related to their competency for spontaneous maturation

The cytogenetic configurations of germinal vesicle (gv) chromatin were analyzed for pools of porcine oocytes harvested from small (1.0-2.0 mm), medium (3.0-5.0 mm), and large (6.0-10.0 mm) antral follicles. Groups of oocytes from these follicular classes also were examined by high-resolution, two-dimensional, polyacrylamide gel electrophoresis to compare their patterns of polypeptide synthesis. The results show a high incidence of gross and cytogenetic degeneration among oocytes from small antral follicles as compared with those from medium or lage follicles. Pools of oocytes could be separated, on the basis of gross morphology and integrity of adherent granulosa cells, into two classes: "Type A" which appeared normal, and "type B" which appeared to be atretic. Among selected "type A" oocytes a particular chromatin configuration, termed "fibrous" characterizes the gv of oocytes from small follicles; whereas a different configuration, termed "diffuse," characterizes the gv of oocytes from large follicles. The patterns of polypeptide synthesis were markedly different for samples of "type A" oocytes of the three follicular classes; and the patterns for oocytes from medium and large follicles were more similar to each other than to patterns for oocytes from slall follicles. The incidences of maturational development beyond the gv stage in vitro were similar for "type A" oocytes from the three follicular classes (i.e., 66% to 82% maturation); although "type B" oocytes underwent maturation beyond the gv at a significantly reduced incidence (i.e., 20% to 29% maturation). "Type A" oocytes from large follicles completed maturation in vitro (i.e., underwent the first meiotic division) at a significantly higher incidence (55%) than did oocytes from small (11% to 20%) or medium (16%) follicles. Our findings are consistent with the hypotheses that a high proportion of oocytes from small antral follicles are atretic, and that a developmental program controls the molecular and cytogenetic changes occurring in porcine oocytes during follicular growth. These changes appear to be highly correlated with the acquisition of competency to complete maturation in vitro, and possibly also are required for normal fertilization and embryogenesis.     

Mechanism of granulosa cell death during follicular atresia depends on follicular size

Changes in granulosa cell lysosomal and mitochondrial functions in relation to follicular size and to the stage of atresia were studied by fluorescent emission spectra and intensity using flow cytometry. Antral follicles were grouped by size in two groups: small, 3-6 mm and large, >6mm in diameter, and classified into three stages of atresia: non-atretic, initially atretic and advanced atretic. Differences in Rhodamine 123 (Rh123) and Acridine Orange (AO) fluorescent intensity indicated that changes in mitochondrial function are the primary mechanism of granulosa cell death in atretic follicles 3-6 mm in diameter, while its role in granulosa cell death in >6 mm atretic follicles seemed to be less important. However, modifications in lysosomal function (shown by a decrease in fluorometric intensity of AO incubated granulosa cells) were mainly associated with cell death in large atretic follicles. Our results support the hypothesis that the pathway of granulosa cell death during follicular atresia depends on the state of energy metabolism or on the production of hypoxic conditions related to follicular size. Changes in mitochondrial membrane potential and production of permeability transition pores were the main changes found in small follicles, while lysosomal function destabilization seemed to be the major cause of granulosa cell death during atresia in large follicles.     

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

Regeneration of plants from <Emphasis Type="Italic">Fraxinus americana</Emphasis> hypocotyls and cotyledons

A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5, respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting (78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to the emerald ash borer.     

Comparison of metabolite levels in callus of <Emphasis Type="Italic">Tecoma stans</Emphasis> (L.) Juss. ex Kunth. cultured in photoperiod and darkness

Tecoma stans is a tropical plant from the Americas. Antioxidant activity and both phenolic compound and flavonoid total content were determined for callus tissue of T. stans cultured in either a set photoperiod or in darkness. Callus lines from three explant types (hypocotyls, stem, and leaf) were established on B5 culture medium supplemented with 0.5 μM 2,4-D and 5.0 μM kinetin. While leaf-derived callus grew slower under a 16-h photoperiod (specific growth rate, μ = 0.179 d⁻¹, t _D = 3.9 d) than in darkness (μ = 0.236 d⁻¹, t _D = 2.9 d), it accumulated the highest amount (p < 0.05) of both phenolics (86.6 ± 0.01 mg gallic acid equivalents/g) and flavonoids (339.6 ± 0.06 mg catechin equivalents/g). Similarly, antioxidant activity was significantly higher (p < 0.05) when callus was cultured in period light than when grown in extended darkness. Antioxidant activity measured with a 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS)-based assay was 350.5 ± 15.8 mmol Trolox/g extract for callus cultured under a defined photoperiod compared to 129.1 ± 7.5 mmol Trolox/g extract from callus cultured in darkness. Content of phenolic compounds and flavonoids was in agreement with a better antioxidant power (EC₅₀ = 450 μg extract/mg 1,1-diphenyl-2-picrylhydrazyl) and antiradical efficiency. Results of the present study show that calli of T. stans are a source of compounds with antioxidant activity that is favored by culture under a set photoperiod.     

Blood Selenium Status in Normal Punjabi Population of Pakistan

Selenium concentrations in the blood of 112 (56 females and 56 males) normal subjects, from different regions of the Punjab (Pakistan), have been determined using the technique of inductively coupled plasma-mass spectrometry. The whole blood selenium concentrations were found to be 452 ± 12 ppb (parts per billion or nano-gram of Se per gram freeze-dried blood or 96 ± 3 μg/L ), with 470 ± 16 ppb (or 100 ± 4 μg/L) in female and 435 ± 16 ppb (or 92 ± 4 μg/L) in male population. Compared with other populations of the world, these levels are amongst the lowest.