Ultrastructural epithelial zonation of the primate endometrium (rhesus monkey)

The uterine endometrium of menstruating primates (rhesus monkey, human) consists of a germinal basalis that regenerates a transient functionalis during each menstrual cycle. The endometrium is further subdivided into 4 zones that differ histologically and in epithelial mitotic rate along the longitudinal axes of the uterine glands and microvasculature (Bartelmez et al: Contrib. Embryol. Carnegie Inst., 34:99-146, 1951; Bartelmez: Am. J. Obstet. Gynecol., 74:931-955, 1957; Padykula et al.: Biol. Reprod., 32:1103-1118, 1118, 1984; Biol. Reprod., in press, 1988). The zones are defined as follows: functionalis I, luminal epithelium; functionalis II (upper straight gland segments); basalis III (middle gland segments), and basalis IV (bottoms of the glands). The surrounding stroma and microvasculature also differ zonally. Ultrastructural epithelial differences are evident among the 4 zones during 3 distinct functional states during natural menstrual cycles and after ovariectomy: 1) basal level after ovariectomy and 2) estrogen dominance and 3) progesterone dominance. Zonal structural differences persist at a minimal level of differentiation after ovariectomy and thus zonation is an inherent property. During estrogen dominance, distinctive ultrastructural differences are evident among the 4 zones, such as epithelial cell heterogeneity in functionalis I and homogeneity in functionalis II. Also a distinctive glandular cell type occurs in basalis III and IV that is recognized by a highly irregular cisternal rough endoplasmic reticulum that permeates the cytoplasm. During progesterone dominance, ultrastructural differences exist among the 4 zones except for similarity between the epithelial cells of functionalis II and basalis III. Postovulatory epithelial cells of functionalis I and II and basalis III become postmitotic via progesterone inhibition but intracellular differentiation continues progressively. Postovulatory epithelial mitotic activity in basalis IV escapes progesterone inhibition as the [3H]thymidine labeling index continues to increase from 1 to 12% during the menstrual cycle (Padykula et al.: Reprod., 30(Suppl.1):92 (Abstr. 123), 1984). This post-ovulatory proliferation coupled with progressive differentiation in basalis IV may represent a stem-progenitor set of cells for postmenstrual endometrial regeneration or alternatively for creation of the maternal placenta.     

Selective enrichment and biochemical characterization of seven human skin fibroblasts cell types in vitro

The mitotic and postmitotic populations of the human skin fibroblast cell line HH-8 are heterogeneous when studied in vitro. There are reproducible changes in the frequencies of the mitotic fibroblasts (MF), MF I, MF II, MF III, and the postmitotic fibroblasts (PMF), PMF IV, PMF V, PMF VI, and PMF VII. For biochemical characterization, methods for selective enrichment of homogeneous populations of these seven fibroblast cell types have been established. Clonal populations with 95% purity for the mitotic fibroblasts MF I, MF II, and MF III can be raised in uniform clone types of fibroblasts (CTF) CTF I, CTF II, and CTF III. Pure clonal subpopulations of MF I type cells are present in mass populations in the range of 1-20 cumulative population doublings (CPD). Populations of mitotic fibroblasts represent nearly homogeneous populations of MF II (75-85% purity) in the range of 28-34 CPD and MF III (73-86% purity) in the range of 48-53 CPD. These populations can be easily expanded to up to 10(7)-10(8) cells. The spontaneous transition of MF III to PMF VI takes 140-180 days. In order to shorten this period and increase the proportion of distinct postmitotic types, mitotic fibroblast mass populations (CPD 30-32, MF II: 75-85% purity) have been induced by uv-irradiation to differentiate to nearly homogeneous populations of PMF IV, PMF V, PMF VI, and PMF VII within 4 to 36 days of culture. Using this method, 10(7) cells of one differentiation stage can be obtained. Spontaneously arising and experimentally selected or induced homogeneous clonal and mass populations of MF I, MF II, MF III, PMF IV, PMF V, PMF VI, and PMF VII express an identical differentiation-dependent and cell-type-specific [35S]methionine-labeled polypeptide pattern.     

Clonogenicity of human endometrial epithelial and stromal cells

The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single- cell suspensions of epithelial and stromal cells were cultured at cloning density (300-500/cm(2)) in serum medium or in serum- free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF- I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGF alpha, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF- I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except alpha(6)-integrin. All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified. This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.     

Progesterone-dependent expression of keratinocyte growth factor mRNA in stromal cells of the primate endometrium: keratinocyte growth factor as a progestomedin

In vitro studies have shown that keratinocyte growth factor (KGF, also known as FGF-7) is secreted by fibroblasts and is mitogenic specifically for epithelial cells. Therefore, KGF may be an important paracrine mediator of epithelial cell proliferation in vivo. Because stromal cells are thought to influence glandular proliferation in the primate endometrium, we investigated the hormonal regulation and cellular localization of KGF mRNA expression in the rhesus monkey uterus. Tissues were obtained both from naturally cycling monkeys in the follicular and luteal phases of the cycle, and from spayed monkeys that were either untreated or treated with estradiol (E2) alone, E2 followed by progesterone (P), E2 plus P, or E2 plus P plus an antiprogestin (RU 486). Northern blot analysis of total RNA with 32P- labeled probes revealed that the level of KGF mRNA in the endometrium was 70-100-fold greater in the luteal phase or after P treatment than in untreated, E2-treated, or follicular phase animals. Northern analysis also showed that KGF mRNA was present in the myometrium but was unaffected by hormonal state. RU 486 treatment prevented the P- induced elevation of endometrial KGF mRNA. P-dependent elevation of endometrial KGF expression was confirmed by measurement of KGF protein in tissue extracts using a two-site enzyme-linked immunosorbent assay. In situ hybridization with nonradioactive digoxigenin-labeled cDNA probes revealed that the KGF mRNA signal, which was present only in stromal and smooth muscle cells, was substantially increased by P primarily in the stromal cells located in the basalis region. Smooth muscle cells in the myometrium and the walls of the spiral arteries also expressed KGF mRNA, but the degree of this expression did not differ with hormonal state. P treatment led to increased proliferation in the glandular epithelium of the basalis region and to extensive growth of the spiral arteries. We conclude that the P-dependent increase in endometrial KGF resulted from a dual action of P: (a) a P- dependent induction of KGF expression in stromal cells, especially those in the basalis (zones III and IV), and (b) a P-dependent increase in the number of KGF-positive vascular smooth muscle cells caused by the proliferation of the spiral arteries. KGF is one of the first examples in primates of a P-induced, stromally derived growth factor that might function as a progestomedin.     

利用NOAA NDVI和MSAVI遥感监测中国北方不同纬度带植被生长季变化

将中国北方13个省按纬向划分为5个区域：32°～36°N（区域Ⅰ）、36°～40°N（区域Ⅱ）、40°～44°N（区域Ⅲ）、44°～48°N（区域Ⅳ）和48°～52°N（区域Ⅴ）,然后利用Savitzky-Golay滤波算法平滑了1982～1999年NOAA/AVHRR NDVI和MSAVI时间序列影像,基于经验正交函数（EOF）分析提取了不同区域植被NDVI和MSAVI主分量,估测了1982～1999年中国北方不同纬度带的植被生长季开始、结束和长度,最后对1982～1999年不同区域的生长季参数进行了线性拟合,分析了不同区域的植被生长季变化趋势.研究表明,不同纬度带的植被生长季开始日期均表现出不同程度的提前趋势,区域Ⅳ的植被生长季开始提前趋势最大;生长季结束日期呈现推迟的趋势,区域Ⅱ的植被活动结束日期的推迟趋势最大,而区域Ⅲ最小.整个生长季长度呈延长趋势,延长日期在10 d以上.     

The progesterone antagonist onapristone retards the advanced endometrial transformation after gonadotropin stimulation in rabbits

Ovarian stimulation with gonadotropins (GN) during human in vitro fertilization and embryo transfer (IVF/ET) therapy alters the ovarian steroid output, especially that of progesterone. As a consequence, endometrial transformation is advanced, and embryo implantation is hampered. This study used the rabbit model to determine if the application of the progesterone antagonist (PA) onapristone (ONA) could retard endometrial development after GN-stimulation. Rabbits were GN-stimulated twice daily with 5 IU FSH and 5 IU LH on 3 consecutive days with a) hMG (n = 10) or b) with a mixture of recombinant FSH and LH (n = 10). The animals were then mated, and hCG was injected i.v. to ensure ovulation. This day is designated as day 0 post coitum (d 0 p.c.). On day 2 p.c., five animals of each group were treated with 20 mg ONA/kg body weight and five with vehicle for control. On d 5 p.c. endometrial transformation was analyzed by morphology, uteroglobin (Ugl)-mRNA expression, and proliferation. Embryos were flushed from the uteri. Their number and morphology was evaluated. The endometrium of GN-stimulated control animals demonstrated very long endometrial glands and narrow stromal septa. Ugl-mRNA expression was restricted to the cells at the bottom of the gland. 17.0 +/- 4.6% (mean +/- SD) of glandular cells and 6.0 +/- 5.3% of luminal epithelial cells proliferated. In ONA-treated animals, endometrial glands were significantly shorter, and the pattern of arborization was less pronounced. Endometrial gland cells and luminal epithelial cells expressed Ugl-mRNA. Furthermore, glandular and luminal cells proliferated with high intensity (38.6 +/- 6.8% and 36.4 +/- 9.3%, respectively). These results indicate that the status of endometrial differentiation was retarded after ONA-treatment. Nevertheless, the embryos of these ONA-treated animals were well developed. In conclusion, after GN-stimulation, ONA treatment retarded the advanced endometrial transformation in rabbits. Therefore, postovulatory administration of a PA might be a possible strategy to modulate the advanced endometrial development in IVF-cycles.     

Cell population dynamics of periodontal ligament stimulated with parathyroid extract.

Young adult rats were injected with parathyroid extract (PTE). Periodontal ligament (PDL) adjacent to a previously resorbing alveolar bone surface was divided into four zones number I to IV, from bone to cementum. Zones I and IV were within 25 mmu of the bone and cementum surfaces, respectively, while a line bisecting the center of the PDL separated Zones II and III. A net increase of about 16 total nuclei in all zones was observed during the first five hours after PTE administration. Since local mitosis accounted for no more than two nuclei, approximately 14 cells apparently migrated into the area. Over the first five hours Zones I and II combined showed a 21-cell increase, being apparently the sole recipients of cells migrating into the field (14) plus approximately seven more from Zone III, which lost cells during the time period. The concurrent histological observation in Zone II, of increased intravascular monocytes and perivascular macrophages during the first five hours, suggests cells are migrating into Zone II via vascular channels. The data suggest two sources for increased PDL cellularity associated with stimulated osteoclast histogenesis: (1) local PDL cellular proliferation and (2) influx of migrating cells (probably through vascular channels) during first five hours after PTE.     

Sanitary factors and mycotoxin contamination in the argentinian wheat crop 1993/94

In Argentina, due to climatic conditions, Fusarium head blight (FHB) caused by Fusarium graminearum, affected the 1993/94 wheat crop. To evaluate the severity of this disease, samples of wheat where gathered from four zones of the wheat area. Sanitary conditions and mycotoxin contamination were determined. One zone (IIN) was intensely affected by FHB with 90% of samples in grade III (bad quality). No samples were grade I (good quality). The other zones were less affected falling into grade I or II (moderate quality). In all samples tested F. graminearum was the most prevalent species singly or in combination with others. Zone II N, with a DON mean level of I1.26 ppm, did not fulfil aceptability limits, whereas zones IIS, III and IV with overall means of 2.12, 1.57 and 1.0 ppm, respectively, did. Statistical analysis showed a close relation between percentage FHB and DON contamination (r:-0.71, p<0.01) in infected samples.     

Dopamine production by isolated glomeruli and tubules from rat kidneys

To locate the sites of dopamine (D) production in rat renal cortex, we separated glomeruli and proximal tubules by sieving or centrifugation in Percoll after collagenase digestion. After centrifugation layer I contained 60-80% glomeruli and 20-40% tubule fragments, half of which did not stain with alkaline phosphatase, layer II contained 0-5% glomeruli, 10-25% tubule fragments other than proximal tubules, and 70-85% proximal tubule fragments. Layer IV contained 85-95% proximal tubules. Gluconeogenic rates were (micromoles per hour per gram wet weight) as follows: I, 4 +/- 1; II, 7 +/- 2; and IV, 16 +/- 1. Norepinephrine (NE) content was (picomoles per gram wet weight) I, 310 +/- 30; II, 540 +/- 40; IV, 195 +/- 60. D content was (picomoles per gram wet weight) I, 26 +/- 6; II, 46 +/- 13; IV, 33 +/- 7. Surgical denervation 4-6 days previously reduced the norepinephrine content of layers I and II to 35 +/- 10 (p less than 0.001) and of IV to 60 +/- 20 (p less than 0.05) and the D content of layers I and II to 13 +/- 6 and 6 +/- 6 pmol/g, respectively (p less than 0.01); D content of layer IV was unchanged. D production from 10(-7) M 3,4-dihydroxyphenylalanine (DOPA) was (nanomoles per gram per minute) I, 0.2 +/- 0.03; II, 0.7 +/- 0.1; IV, 1.0 +/- 0.04. DOPA consumption was (nanomoles per gram per minute) I, 0.6 +/- 0.1; II, 1.4 +/- 0.3; and IV, 1.8 +/- 0.2. Denervation did not change D production or DOPA consumption.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental biology of uterine glands.

All mammalian uteri contain endometrial glands that synthesize or transport and secrete substances essential for survival and development of the conceptus (embryo/fetus and associated extraembryonic membranes). In rodents, uterine secretory products of the endometrial glands are unequivocally required for establishment of uterine receptivity and conceptus implantation. Analyses of the ovine uterine gland knockout model support a primary role for endometrial glands and, by default, their secretions in peri-implantation conceptus survival and development. Uterine adenogenesis is the process whereby endometrial glands develop. In humans, this process begins in the fetus, continues postnatally, and is completed during puberty. In contrast, endometrial adenogenesis is primarily a postnatal event in sheep, pigs, and rodents. Typically, endometrial adenogenesis involves differentiation and budding of glandular epithelium from luminal epithelium, followed by invagination and extensive tubular coiling and branching morphogenesis throughout the uterine stroma to the myometrium. This process requires site-specific alterations in cell proliferation and extracellular matrix (ECM) remodeling as well as paracrine cell-cell and cell-ECM interactions that support the actions of specific hormones and growth factors. Studies of uterine development in neonatal ungulates implicate prolactin, estradiol-17 beta, and their receptors in mechanisms regulating endometrial adenogenesis. These same hormones appear to regulate endometrial gland morphogenesis in menstruating primates and humans during reconstruction of the functionalis from the basalis endometrium after menses. In sheep and pigs, extensive endometrial gland hyperplasia and hypertrophy occur during gestation, presumably to provide increasing histotrophic support for conceptus growth and development. In the rabbit, sheep, and pig, a servomechanism is proposed to regulate endometrial gland development and differentiated function during pregnancy that involves sequential actions of ovarian steroid hormones, pregnancy recognition signals, and lactogenic hormones from the pituitary or placenta. That disruption of uterine development during critical organizational periods can alter the functional capacity and embryotrophic potential of the adult uterus reinforces the importance of understanding the developmental biology of uterine glands. Unexplained high rates of peri-implantation embryonic loss in humans and livestock may reflect defects in endometrial gland morphogenesis due to genetic errors, epigenetic influences of endocrine disruptors, and pathological lesions.     

Proliferation of type II pneumonocytes after X-irradiation

This paper reports preliminary data on the proliferative response of type II cells in the mouse lung over a five-month period after external thoracic doses of 2, 5, 10 and 12 Gy of X-rays. The DNA labelling index (LI) of control (0 Gy) mice was at all times exceedingly low (0.3-0.4 per cent). The LI after 2 and 5 Gy showed a slight though transient fall below controls during the first week post-irradiation, and thereafter the LIs were similar to the controls for the 5 months of the experiment. The LI after 10 and 12 Gy again showed a significant depression during the first week, but this was followed by a significant increase (P = 0.01) in LI which peaked at 4 weeks after irradiation. The LI returned to control values at 3-4 months and again rose significantly (P = 0.05) at 5 months. The first wave of proliferation corresponds to data showing an increase in surfactant in alveolar fluids within 2-6 weeks of 10-15 Gy of X-rays; and the second wave coincides with the pneumonitic phase and is consistent with a delay before the alveolar epithelial continuity is sufficiently compromised by the low rates of type I cell loss to trigger a compensatory wave of type II cell divisions. This relatively chronic radiation response is discussed and contrasted with the dramatic and immediate hyperplastic responses which many toxic irritants produce in type II epithelial cells.     

Distribution of dopa-oxidase isozymes among mosquito vectors of viral encephalitis

Distribution of DOPA-oxidase isozymes in larval Culex pipiens pipiens, Culex pipiens quinquefasciatus and their hybrids was determined by agarose gel electrophoresis. Three zones of enzyme activity were detected in the subspecific and hybrid populations. Zone I was cathodic whereas Zones II and III were anodic. Electrophoretic variability was evident only in Zone II which was dimorphic for enzyme activity. Zones I and III were monomorphic. The subspecific and hybrid populations were distinguishable from one another only in Zone II. In all three populations, melanization was most intense in Zone II, less in Zone I, and least in Zone III.     

The ratio of oxidative phosphorylation complexes I-V in bovine heart mitochondria and the composition of respiratory chain supercomplexes

The ratios of the oxidative phosphorylation complexes NADH:ubiquinone reductase (complex I), succinate:ubiquinone reductase (complex II), ubiquinol:cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and F1F0-ATP synthase (complex V) from bovine heart mitochondria were determined by applying three novel and independent approaches that gave consistent results: 1) a spectrophotometric-enzymatic assay making use of differential solubilization of complexes II and III and parallel assays of spectra and catalytic activities in the samples before and after ultracentrifugation were used for the determination of the ratios of complexes II, III, and IV; 2) an electrophoretic-densitometric approach using two-dimensional electrophoresis (blue native-polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis) and Coomassie blue-staining indices of subunits of complexes was used for determining the ratios of complexes I, III, IV, and V; and 3) two electrophoretic-densitometric approaches that are independent of the use of staining indices were used for determining the ratio of complexes I and III. For complexes I, II, III, IV, and V in bovine heart mitochondria, a ratio 1.1 +/- 0.2:1.3 +/- 0.1:3:6.7 +/- 0.8:3.5 +/- 0.2 was determined.     

Role of prostaglandins in the cortical distribution of renal blood flow following reductions in renal perfusion pressure

The purpose of this study was to examine the role of prostaglandins in the redistribution of renal cortical blood flow that occurs following reductions in renal perfusion pressure. The distribution of blood flow to the renal cortex was examined using radio-labeled microspheres (15 +/- 1 micron). It was found that in animals not treated with a prostaglandin synthesis inhibitor a decrease in renal perfusion pressure to the limit of renal blood flow autoregulation was associated with a decrease in fractional flow to the outer cortex (Zone I) and an increase in fractional flow to the inner cortex (Zones III and IV). A further decrease in renal perfusion pressure below the limit of autoregulation produced a further decrease in the fractional flow to Zone I and a further increase in fractional flow to Zones III and IV. In contrast, in animals treated with the prostaglandin synthesis inhibitor meclofenamate (5 mg/kg, i.v. bolus) a reduction in renal perfusion pressure to the limit of renal blood flow autoregulation produced no change in fractional blood flow to any of the 4 cortical zones. A further decrease in renal perfusion pressure, however, did produce a fall in fractional blood flow to Zone I and an increase in fractional flow to Zones III and IV. In conclusion, the results of this study indicate that within, but not below, the limit of renal blood flow autoregulation prostaglandin synthesis is an important factor in the regulation of renal cortical blood flow distribution.     

Composition of long-chain bases in sphingomyelin of the guinea pig Harderian gland

Sphingomyelin from the guinea pig Harderian gland was isolated and characterized. The purified sphingomyelin gave a broad spot on thin-layer chromatography. The fatty acid composition of the whole sphingomyelin was 71% nonhydroxy acids and 29% 2-hydroxy acids. Methyl-branched fatty acids were only 2% of the total acids. The long-chain bases were composed of straight-chain sphingenines (50%) and sphinganines (6%). Methyl-branched long-chain bases were 44% of the bases. The sphingomyelin was further separated into four fractions (I, II, III, IV) by high-performance liquid chromatography. The ratio of fractions I, II, III, and IV was approximately 2:5:2:1, respectively. The fatty acids of fractions I and II consisted of nonhydroxy acids and those of fractions III and IV were 2-hydroxy acids. The long-chain bases of fractions I and III were sphinganines including 10-, 9-, and 8-methylsphinganines and anteiso-sphinganines. These methyl-branched bases occupied about 70% of the total sphinganines. The long-chain bases of fractions II and IV consisted of sphingenines. The methyl-branched unsaturated bases were only 30% of the total sphingenines, all in the anteiso-form. Thus, the sphingomyelin obtained from guinea pig Harderian gland had complex compositions of fatty acids and long-chain bases, and half the number of long-chain bases had methyl branches. The methyl-branched fatty acids were only a minor component. These characteristics are similar to those of cerebrosides isolated from the same source.     

Efficient in vitro production of cat embryos in modified synthetic oviduct fluid medium: effects of season and ovarian status.

The effects of season (January-March = I; April-June = II; July-September = III; October-December = IV) and ovarian status (freshly ovulated, follicular, luteal, intermediate, or inactive) on the efficiency of the in vitro production of domestic cat embryos were evaluated. Ovaries and testes from cats with access to daylight were collected at local veterinary clinics. A total of 6843 cumulus-oocyte complexes (COCs) were recovered from 363 pairs of ovaries, matured in TCM 199 supplemented with BSA and gonadotropins (IVM), fertilized with epididymal sperm in a medium based on Tyrode albumin lactate pyruvate (IVF), and cultured in synthetic oviduct fluid (SOF) medium supplemented with 10% estrous cow serum (ECS) and essential and nonessential amino acids. The proportion of freshly ovulated, follicular, or luteal ovaries was higher (P < 0.05) in seasons II (64.4%) and III (60.5%) than in seasons I (42.0%) and IV (30.6%). The average number of COCs recovered per donor was not influenced by season. After IVM/IVF, cleavage rates (Day 2) were significantly higher (P < 0.05) in seasons II (mean +/- SEM: 53.1% +/- 1.9%) and III (54.6% +/- 2.8%) than in seasons I (48.4% +/- 1.4%) and IV (44.9% +/- 3.0%). Blastocyst rates on Day 6 were similar in seasons I (25.3% +/- 1.3%), II (28.2% +/- 1.5%), and III (29.6% +/- 2.3%) but were significantly lower (P < 0.01) in season IV (18.6% +/- 2.4%). The corresponding blastocyst rates on Day 8 were 28.9% +/- 1.3%, 33.7% +/- 1.6%, 37.9% +/- 2.3%, and 23.6% +/- 2.6%. In addition, we found a significant effect (P < 0.05) of ovary type; luteal, follicular, and intermediate ovaries yielding a higher proportion of developmentally competent oocytes than did freshly ovulated and inactive ovaries. These data show that the culture system used in our study supports development of IVM/IVF cat oocytes to blastocysts at a higher rate than those obtained with other methods. Although embryos could be produced throughout the year, the efficiency was significantly affected by season and ovary type.     

Overexpression of GRF encapsulated in PLGA microspheres in animal skeletal muscle induces body weight gain

Biodegradable nanospheres or microspheres have been widely used as a sustained release system for the delivery of bioagents. In the present study, injectable sustained-release growth hormone-releasing factor (GRF) (1-32) microspheres were prepared by a double emulsion-in liquid evaporation process using biodegradable polylactic-co-glycolic acid (PLGA) as the carrier. The entrapment efficiency was 89.79% and the mean particle size was 4.41 mum. The microspheres were injected into mouse tibialis muscle. After 30 days, mice injected with GRF (1-32) microspheres (group I) gained significantly more weight than any other treatment group, including mice injected with the naked plasmid (group II) (10.26 +/- 0.13 vs. 9.09 +/- 0.56; P < 0.05), a mixture of microspheres and plasmid (group III) (10.26 +/- 0.13 vs. 8.57 +/- 0.02; P < 0.05), or saline (IV) (10.26 +/- 0.13 vs. 6.47 +/- 0.26; P < 0.05). In addition, mice treated with the GRF (1-32) microspheres exhibited the highest expression levels of GRF as detected by PCR, RT-PCR, and ELISA (mean 2.56 +/- 0.40, P < 0.05, overall comparison of treatment with groups II, III, and IV). Additionally, rabbits were injected in the tibialis muscle with the same treatments described above. After 30 days, the group treated with GRF (1-32) microspheres gained the most weight. At day 30 postinjection, weight gain in group I was 63.93% higher than group II (plasmid) (877.10 +/- 24.42 vs. 535.05 +/- 26.38; P < 0.05), 108.59% higher than group III (blank MS) (877.10 +/- 24.42 vs. 420.50 +/- 19.39; P < 0.05), and 93.94% higher than group IV (saline) (877.10 +/- 24.42 vs. 452.25 +/- 27.38; P < 0.05). Furthermore, IGF-1 levels in the serum from GRF microsphere-treated group were elevated relative to all other groups. The present results suggest that encapsulation of GRF with PLGA increases GRF gene expression in muscle after local plasmid delivery, and stimulates significantly more weight gain than delivery of the naked plasmid alone.     

Parameters of dietary selenium and vitamin E deficiency in growing rabbits.

4 x 5 growing female rabbits (New Zealand White) with an initial live weight of 610 +/- 62 g were fed a torula yeast based semisynthetic diet low in selenium (<0.03 mg/kg diet) and containing <2 mg alpha-tocopherol per kg (group I). Group II received a vitamin E supplementation of 150 mg alpha-tocopherylacetate per kg diet, whereas for group III 0.40 mg Se as Na-selenite and for group IV both supplements were added. Selenium status and parameters of tissue damage were analyzed after 10 weeks on experiment (live weight 2,355 +/- 145 g). Selenium depletion of the Se deficient rabbits (groups I and II) was indicated by a significantly lower plasma Se content (group I: 38.3 +/- 6.23 microg Se/mL plasma, group II: 42.6 +/- 9.77, group III: 149 +/- 33.4, group IV: 126 +/- 6.45) and a significantly lower liver Se content (group I: 89.4 +/- 18.2 microg/kg fresh matter, group II: 111 +/- 26.2) as compared to the Se supplemented groups III (983 +/- 204) and IV (926 +/- 73.9). After 5 weeks on the experimental diets differences in the development of plasma glutathione peroxidase were observed. As compared to the initial status group (45.2 +/- 4.50) pGPx activity in mU/mg protein was decreased in group I (19.1 +/- 7.08), remained almost stable in the vitamin E supplemented group II (46.3 +/- 11.2) whereas an elevated enzyme activity was measured in the Se supplemented groups III (62.4 +/- 23.9) and IV (106 +/- 19.9). In the rabbit organs investigated 10 weeks of Se deficiency caused a significant loss of Se dependent cellular glutathione peroxidase activity (GPx1) of 94% (liver), 80% (kidney), 50% (heart muscle) and 60% (musculus longissimus dorsi) in comparison to Se supplemented control animals. Damage of cellular lipids and proteins in the liver was due to either Se or vitamin E deficiency. However damage was most severe under conditions of a combined Se and vitamin E deficiency. It can be concluded that the activity of plasma glutathione peroxidase is a sensitive indicator of Se deficiency in rabbits. The loss of GPx1 activity indicates the selenium depletion in various rabbit organs. Both selenium and vitamin E are essential and highly efficient antioxidants which protect rabbits against lipid and protein oxidation.