Large Cytoplasmic Lipid Droplets Research Articles

Evaluation of a serum-free culture medium for the enhanced vitrification cryosurvival of bovine in vitro-derived embryos

This is a feasibility study aimed to determine the effectiveness of a culture media designed to enhance embryonic cryopreservation based on laboratory-accessed quality phenotypes of in vitro produced bovine embryos. Recovered bovine oocytes were matured and fertilized in vitro and the presumptive zygotes that were obtained were randomly distributed into three culture media: (i) Control (C): modified synthetic oviduct fluid (mSOF) culture medium supplemented with 5 mg/mL BSA and 2.5% fetal calf serum; (ii) Medium A (MA): serum-free altered mSOF medium A; and iii) Medium B (MB): serum-free altered mSOF medium B. Embryos cultured in MA and MB presented a better viability after cryopreservation (P < 0.05) without affecting the production rates of blastocysts (P > 0.05, MB only) when compared to C. The MB-derived blastocysts had the highest (P < 0.05) hatching rate (26.1%, 53/203) and higher (P < 0.05) transferable embryos rate (92.1%, 187/203) compared to control-derived blastocysts (85.8%, 175/204). The total cell number in the embryos after vitrification/warming process was greater (P < 0.05) in the MA (144.9 ± 5.9) and MB (151.3 ± 7.1) compared to that in the control (121.4 ± 6.1). The number of large and giant cytoplasmic lipid droplets was reduced (P < 0.05) in MB-derived embryos compared to control-derived embryos. Additionally, the lipid profiles of MB-derived blastocysts were closely related to those observed in the in vivo-derived blastocysts. We observed different transcriptional profiles abundance of in vitro produced bovine embryos derived from control, MA and MB groups. Therefore, the novel MB culture medium increased embryonic cryosurvival and favored the production of bovine embryos in vitro with improved cellular and molecular laboratory-accessed quality phenotypes.

Triacylglycerol synthesis directed by glycerol-3-phosphate acyltransferases −3 and −4 is required for lipid droplet formation and the modulation of the inflammatory response during macrophage to foam cell transition

Background and aimsThe transition of macrophage to foam cells is a major hallmark of early stage atherosclerotic lesions. This process is characterized by the accumulation of large cytoplasmic lipid droplets containing large quantities of cholesterol esters (CE), triacylglycerol (TAG) and phospholipid (PL). Although cholesterol and CE metabolism during foam cell formation has been broadly studied, little is known about the role of the glycerolipids (TAG and PL) in this context. Here we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). MethodsWe used RAW 264.7 cells and bone marrow derived macrophages (BMDM) treated with oxidized LDL (oxLDL). ResultsWe showed that TAG synthesis is induced during the macrophage to foam cell transition. The expression and activity of GPAT3 and GPAT4 also increased during this process, and these two isoforms were required for the accumulation of cell TAG and PL. Compared to cells from wildtype mice after macrophage to foam cell transition, Gpat4−/− BMDM released more pro-inflammatory cytokines and chemokines, suggesting that the activity of GPAT4 could be associated with a decrease in the inflammatory response, probably by sequestering signaling precursors into lipid droplets. ConclusionsOur results provide evidence that TAG synthesis directed by GPAT3 and GPAT4 is required for lipid droplet formation and the modulation of the inflammatory response during the macrophage-foam cell transition.

Ageing is associated with brown adipose tissue remodelling and loss of white fat browning in female C57BL/6 mice.

Fat storage changes throughout life and affects body metabolism. Ageing impact on brown (BAT) and white adipose tissue (WAT) deserves attention, especially in females, because they are less prone to age-induced weight gain. While in male mice the impact of ageing on adipose tissue remodelling is well characterized, the effects in female mice remain largely unclear. Thus, we investigated BAT and WAT remodelling during ageing in female C57BL/6 mice. At 3months, body weight was 24±0.3g (mean±SD), and it increased from 6 to 9months of age (+20%, P<0.0001). Oral glucose tolerance test showed no disturbance of glucose metabolism. All WAT depots became heavier, and white adipocytes hypertrophied. The subcutaneous and visceral WAT had clusters of beige cells in younger mice, but they were progressively lost by ageing, indicating loss of WAT browning. Older mice had hypertrophied classic brown adipocytes that had larger cytoplasmic lipid droplets than younger mice. Pearson's correlation showed that WAT mass has a weak correlate with BAT mass, although white adipocyte diameter has a strong correlation with classic brown adipocyte size. In conclusion, our results indicate that female C57BL/6 mice have a progressive age-dependent loss of subcutaneous and visceral WAT browning, and this process runs in parallel with BAT morphological changes towards a fat storer phenotype, independent of cycling or disturbances in glucose metabolism.

Zinc Finger Homeodomain Factor Zfhx3 Is Essential for Mammary Lactogenic Differentiation by Maintaining Prolactin Signaling Activity

The zinc finger homeobox 3 (ZFHX3, also named ATBF1 for AT motif binding factor 1) is a transcription factor that suppresses prostatic carcinogenesis and induces neuronal differentiation. It also interacts with estrogen receptor α to inhibit cell proliferation and regulate pubertal mammary gland development in mice. In the present study, we examined whether and how Zfhx3 regulates lactogenic differentiation in mouse mammary glands. At different stages of mammary gland development, Zfhx3 protein was expressed at varying levels, with the highest level at lactation. In the HC11 mouse mammary epithelial cell line, an in vitro model of lactogenesis, knockdown of Zfhx3 attenuated prolactin-induced β-casein expression and morphological changes, indicators of lactogenic differentiation. In mouse mammary tissue, knock-out of Zfhx3 interrupted lactogenesis, resulting in underdeveloped glands with much smaller and fewer alveoli, reduced β-casein expression, accumulation of large cytoplasmic lipid droplets in luminal cells after parturition, and failure in lactation. Mechanistically, Zfhx3 maintained the expression of Prlr (prolactin receptor) and Prlr-Jak2-Stat5 signaling activity, whereas knockdown and knock-out of Zfhx3 in HC11 cells and mammary tissues, respectively, decreased Prlr expression, Stat5 phosphorylation, and the expression of Prlr-Jak2-Stat5 target genes. These findings indicate that Zfhx3 plays an essential role in proper lactogenic development in mammary glands, at least in part by maintaining Prlr expression and Prlr-Jak2-Stat5 signaling activity.

Characterization and manipulation of a DGAT2 from the diatom Thalassiosira pseudonana: Improved TAG accumulation without detriment to growth, and implications for chloroplast TAG accumulation

Improving the ability of microalgae cells to accumulate triacylglycerol (TAG) without a negative effect on growth or biomass accumulation is desirable for economically-viable biofuels production. Metabolic engineering is one method to increase TAG yields, provided that appropriate gene target(s) are identified. Microalgal lipid and TAG synthesis is incompletely characterized, but it is clear that it differs in fundamental ways from plants and other eukaryotes. We characterize a DGAT2 homolog from the diatom Thalassiosira pseudonana, and show that it shifts intracellular location from the chloroplast during exponential growth, to the endoplasmic reticulum in stationary phase when large cytoplasmic lipid droplets accumulate. This is the first direct localization of a chloroplast DGAT2, which confirms the ability of these organelles to accumulate TAG in evolutionarily-diverse classes of microalgae. The enzyme is also not directly localized to cytoplasmic lipid droplets, suggesting that TAG synthesis and lipid droplet formation are separate processes. Overexpression of the enzyme resulted in significantly increased TAG accumulation without a negative effect on growth or biomass accumulation. Overexpression lines had a distinct shift in fatty acid composition relative to wild-type. The results shed light on fundamental processes of algal lipid metabolism and a practical benefit of improved productivity in transgenic lines.

Actin filament-associated protein 1 is required for cSrc activity and secretory activation in the lactating mammary gland

Actin filament-associated protein 1 (AFAP1) is an adaptor protein of cSrc that binds to filamentous actin and regulates the activity of this tyrosine kinase to affect changes to the organization of the actin cytoskeleton. In breast and prostate cancer cells, AFAP1 has been shown to regulate cellular responses requiring actin cytoskeletal changes such as adhesion, invadopodia formation and invasion. However, a normal physiological role for AFAP1 has remained elusive. In this study, we generated an AFAP1 knockout mouse model that establishes a novel physiological role for AFAP1 in lactation. Specifically, these animals displayed a defect in lactation that resulted in an inability to efficiently nurse. Histologically, the mammary glands of the lactating knockout mice were distinguished by the accumulation of large cytoplasmic lipid droplets in the alveolar epithelial cells. There was a reduction in lipid synthesis and the expression of lipogenic genes without a corresponding reduction in the production of beta-casein, a milk protein. Furthermore, these defects were associated with histological and biochemical signs of precocious involution. This study also demonstrated that AFAP1 responds to prolactin, a lactogenic hormone, by forming a complex with cSrc and becoming tyrosine phosphorylated. Together, these observations pointed to a defect in secretory activation. Certain characteristics of this phenotype mirrored the defect in secretory activation in the cSrc knockout mouse, but most importantly, the activity of cSrc in the mammary gland was reduced during early lactation in the AFAP1 null mouse and the localization of active cSrc at the apical surface of luminal epithelial cells during lactation was selectively lost in the absence of AFAP1. These data define, for the first time, the requirement of AFAP1 for the spatial and temporal regulation of cSrc activity in the normal breast, specifically for milk production.

Lactation Defect with Impaired Secretory Activation in AEBP1-Null Mice

Adipocyte enhancer binding protein 1 (AEBP1) is a multifunctional protein that negatively regulates the tumor suppressor PTEN and IκBα, the inhibitor of NF-κB, through protein-protein interaction, thereby promoting cell survival and inflammation. Mice homozygous for a disrupted AEBP1 gene developed to term but showed defects in growth after birth. AEBP1 −/− females display lactation defect, which results in the death of 100% of the litters nursed by AEBP1 −/− dams. Mammary gland development during pregnancy appears normal in AEBP1 −/− dams; however these mice exhibit expansion of the luminal space and the appearance of large cytoplasmic lipid droplets (CLDs) in the mammary epithelial cells at late pregnancy and parturition, which is a clear sign of failed secretory activation, and accumulation of milk proteins in the mammary gland, presumably reflecting milk stasis following failed secretory activation. Eventually, AEBP1 −/− mammary gland rapidly undergoes involution at postpartum. Stromal restoration of AEBP1 expression by transplanting wild-type bone marrow (BM) cells is sufficient to rescue the mammary gland defect. Our studies suggest that AEBP1 is critical in the maintenance of normal tissue architecture and function of the mammary gland tissue and controls stromal-epithelial crosstalk in mammary gland development.

Adipophilin regulates maturation of cytoplasmic lipid droplets and alveolae in differentiating mammary glands

Milk lipids originate by secretion of triglyceride-rich cytoplasmic lipid droplets (CLDs) from mammary epithelial cells. Adipophilin (ADPH)/Plin2, a member of the perilipin family of CLD binding proteins, is hypothesized to regulate CLD production in these cells during differentiation of the mammary gland into a secretory organ. We tested this hypothesis by comparing CLD accumulation in differentiating mammary glands of wild-type and ADPH-deficient mice. ADPH deficiency did not prevent CLD formation; however, it disrupted the increase in CLD size that normally occurs in differentiating mammary epithelial cells. Failure to form large CLDs in ADPH-deficient mice correlated with localization of adipose triglyceride lipase (ATGL) to the CLD surface, suggesting that ADPH promotes CLD growth by inhibiting lipolytic activity. Significantly, mammary alveoli also failed to mature in ADPH-deficient mice, and pups born to these mice failed to survive. The possibility that CLD accumulation and alveolar maturation defects in ADPH-deficient mice are functionally related was tested by in vivo rescue experiments. Transduction of mammary glands of pregnant ADPH-deficient mice with adenovirus encoding ADPH as an N-terminal GFP fusion protein prevented ATGL from localizing to CLDs and rescued CLD size and alveolar maturation defects. Collectively, these data provide direct in vivo evidence that ADPH inhibition of ATGL-dependent lipolysis is required for normal CLD accumulation and alveolar maturation during mammary gland differentiation. We speculate that impairing CLD accumulation interferes with alveolar maturation and lactation by disrupting triglyceride homeostasis in mammary epithelial cells.

Nocturnin Regulates Circadian Trafficking of Dietary Lipid in Intestinal Enterocytes

Efficient metabolic function in mammals depends on the circadian clock, which drives temporal regulation of metabolic processes. Nocturnin is a clock-regulated deadenylase that controls its target mRNA expression posttranscriptionally through poly(A) tail removal. Mice lacking nocturnin (Noc(-/-) mice) are resistant to diet-induced obesity and hepatic steatosis yet are not hyperactive or hypophagic. Here we show that nocturnin is expressed rhythmically in the small intestine and is induced by olive oil gavage and that the Noc(-/-) mice have reduced chylomicron transit into the plasma following the ingestion of dietary lipids. Genes involved in triglyceride synthesis and storage and chylomicron formation have altered expression, and large cytoplasmic lipid droplets accumulate in the apical domains of the Noc(-/-) enterocytes. The physiological significance of this deficit in absorption is clear because maintenance of Noc(-/-) mice on diets that challenge the chylomicron synthesis pathway result in significant reductions in body weight, whereas diets that bypass this pathway do not. Therefore, we propose that nocturnin plays an important role in the trafficking of dietary lipid in the intestinal enterocytes by optimizing efficient absorption of lipids.

Lactation Defect in a Widely Used MMTV-Cre Transgenic Line of Mice

BackgroundMMTV-Cre mouse lines have played important roles in our understanding about the functions of numerous genes in mouse mammary epithelial cells during mammary gland development and tumorigenesis. However, numerous studies have not included MMTV-Cre mice as controls, and many investigators have not indicated which of the different MMTV-Cre founder lines were used in their studies. Here, we describe a lactation defect that severely limits the use of one of the most commonly used MMTV-Cre founder lines.Methodology/Principal FindingsTo explore the role of protein tyrosine phosphatase Shp1 in mammary gland development, mice bearing the floxed Shp1 gene were crossed with MMTV-Cre mice and mammary gland development was examined by histological and biochemical techniques, while lactation competency was assessed by monitoring pup growth. Surprisingly, both the Shp1fl/+;MMTV-Cre and MMTV-Cre female mice displayed a severe lactation defect when compared to the Shp1 fl/+ control mice. Histological and biochemical analyses reveal that female mice expressing the MMTV-Cre transgene, either alone or in combination with floxed genes, exhibit defects in lobuloalveolar expansion, presence of large cytoplasmic lipid droplets in luminal alveolar epithelial cells postpartum, and precocious induction of involution. Using a PCR-based genotyping method, the three different founder lines can be distinguished, and we determined that the MMTV-Cre line A, the most widely used MMTV-Cre founder line, exhibits a profound lactation defect that limits its use in studies on mammary gland development.Conclusions/SignificanceThe identification of a lactation defect in the MMTV-Cre line A mice indicates that investigators must use MMTV-Cre alone mice as control in studies that utilize Cre recombinase to excise genes of interest from mammary epithelial cells. Our results also suggest that previous results obtained in studies using the MMTV-Cre line A line should be re-evaluated if the controls did not include mice expressing only Cre recombinase.

Chronic Ethanol Consumption in Mice Alters Hepatocyte Lipid Droplet Properties

Hepatosteatosis is a common pathological feature of impaired hepatic metabolism following chronic alcohol consumption. Although often benign and reversible, it is widely believed that steatosis is a risk factor for development of advanced liver pathologies, including steatohepatitis and fibrosis. The hepatocyte alterations accompanying the initiation of steatosis are not yet clearly defined. Induction of hepatosteatosis by chronic ethanol consumption was investigated using the Lieber-DeCarli (LD) high fat diet model. Effects were assessed by immunohistochemistry and blood and tissue enzymatic assays. Cell culture models were employed for mechanistic studies. Pair feeding mice ethanol (LD-Et) or isocaloric control (LD-Co) diets for 6 weeks progressively increased hepatocyte triglyceride accumulation in morphological, biochemical, and zonally distinct cytoplasmic lipid droplets (CLD). The LD-Et diet induced zone 2-specific triglyceride accumulation in large CLD coated with perilipin, adipophilin (ADPH), and TIP47. In LD-Co-fed mice, CLD were significantly smaller than those in LD-Et-fed mice and lacked perilipin. A direct role of perilipin in formation of large CLD was further suggested by cell culture studies showing that perilipin-coated CLD were significantly larger than those coated with ADPH or TIP47. LD-Co- and LD-Et-fed animals also differed in hepatic metabolic stress responses. In LD-Et but not LD-Co-fed mice, inductions were observed in the following: microsomal ethanol-oxidizing system [cytochrome P-4502E1 (CYP2E1)], hypoxia response pathway (hypoxia-inducible factor 1 alpha, HIF1α), endoplasmic reticulum stress pathway (calreticulin), and synthesis of lipid peroxidation products [4-hydroxynonenal (4-HNE)]. CYP2E1 and HIF1 α immunostaining localized to zone 3 and did not correlate with accumulation of large CLD. In contrast, calreticulin and 4-HNE immunostaining closely correlated with large CLD accumulation. Importantly, 4-HNE staining significantly colocalized with ADPH and perilipin on the CLD surface. These data suggest that ethanol contributes to macrosteatosis by both altering CLD protein composition and inducing lipid peroxide adduction of CLD-associated proteins.

118 ARE LIPID CONTENTS AND APOPTOSIS DETERMINANTS OF IN VITRO-PRODUCED BOVINE EMBRYO SUSCEPTIBILITY TO VITRIFICATION?

Supplementation of fetal calf serum (FCS) during culture of bovine in vitro-produced embryos (IVPE) has been correlated with lipid accumulation, representing a limiting factor on embryo cryotolerance. In the present experiment we studied lipid content and apoptosis in Nelore IVPE cultured in different concentrations of FCS. The experimental design was a 4 × 2 × 2 factorial, in which embryos, exposed or not to the metabolic regulator, phenazine ethosulfate (PES), were cultured in 4 concentrations of FCS. In 16 replicates, 560 IVPE were vitrified after cultured in SOFaa supplemented with 0, 2.5, 5 or 10% FCS. On Day 2.5 of culture, one-half of the embryos in each group were treated with 0.3 μM of PES and then returned to standard culture conditions (5% O2, 5% CO2 and 90% N2 atmosphere at 38.5°C). All embryos were vitrified on Day 7 (Campos-Chillòn LF et al. 2006 Theriogenology 65, 1200-1214). After warming, embryos were placed into culture for 12 h under standard conditions. A sample of fresh embryos was stained with Sudan Black B for quantification of small (&lt;2 μm), medium (2-6 μm), and large (&gt;6 μm) cytoplasmic lipid droplets. Apoptosis was analyzed, before and after vitrification, using TUNEL. As a positive control (CV), in vivo-produced embryos were collected from a superovulated Nelore cow. Data were analyzed using ANOVA followed by Tukey’s test; and Pearson’s linear correlation (P &lt; 0.05). The results indicate that FCS concentration and PES treatment had no influence (P &gt; 0.05) on cleavage and blastocyst formation rates (mean of 85.7 ± 2 and 36.6 ± 5, respectively). The total number of cells in fresh embryos was also similar (P &gt; 0.05) among groups (mean of 134.1 ± 28). Additional results are shown in Table 1. Elevated concentrations of FCS increased lipid accumulation (r = 0.9) and the percentage of apoptotic cells in fresh and vitrified embryos, reducing the re-expansion rate after warming. Moreover, the number of apoptotic cells observed in fresh embryos was strongly correlated with the apoptosis observed after vitrification (r = 0.95). Although PES was able to reduce lipid accumulation, it was not efficient at preventing apoptosis in vitrified embryos. In summary, the results of this experiment indicate that the concentration of FCS during culture did not influence cleavage and blastocyst formation in bovine IVPE. We conclude that the apoptosis rate in fresh embryos is a key factor affecting survival after vitrification. Table 1.Lipid droplets, re-expansion, apoptosis of in vivo and IVPE Acknowledgements: FAPESP (07/57766-4).

Lipoblastic Nerve Sheath Tumors

Benign nerve sheath tumors of soft tissue can occasionally adopt unusual or unfamiliar morphologic appearances that may introduce difficulties for diagnosis, such as multinucleation, bizarre nuclei, intranuclear vacuoles, and other degenerative changes. Tumor cells adopting a signet-ring or lipoblast-like configuration, however, are mostly associated with epithelial malignancies, liposarcoma and melanoma, and have been only rarely observed in spindle cell tumors of soft tissue. We report 5 cases of benign nerve sheath neoplasms that displayed prominent signet-ring cells with lipoblast-like features. The cases presented as solitary soft tissue masses in the groin, thigh, retroperitoneum, and shoulder in 4 men and 1 woman between the ages of 31 to 57 years. Four tumors predominantly showed features of schwannoma and one of neurofibroma; however, intimately admixed with the spindle cell population, there were also numerous scattered mature adipocytes as well as lipoblast-like cells displaying a signet-ring cell appearance. Immunohistochemical studies showed strong S-100 protein positivity in the spindle cells as well as in the signet-ring lipoblast-like cells and the mature adipocytes. The signet-ring cells were negative for mucin stains, cytokeratin, EMA, CEA, and several other differentiation markers. Ultrastructural examination was performed in 2 cases. The signet-ring cells contained large cytoplasmic lipid droplets that displaced the nuclei to the periphery, consistent with lipoblastic differentiation, whereas complex, interdigitating cytoplasmic processes covered by basal lamina material characteristic of nerve sheath differentiation could be identified in the spindle cells. Four patients for whom follow-up was available were alive and well with no evidence of recurrence over a period of 28 to 116 months (median follow-up, 50 months). The presence of mature fat and signet-ring lipoblast-like cells within a nerve sheath neoplasm is quite rare and may signify a process of aberrant differentiation. Neurogenic tumors should be added in the differential diagnosis of spindle cell tumors capable of displaying prominent signet-ring cell features.

Blocking BRE expression in Leydig cells inhibits steroidogenesis by down-regulating 3β-hydroxysteroid dehydrogenase

Conversion of cholesterol to biologically active steroids is a multi-step enzymatic process. Along with some important enzymes, like cholesterol side-chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD), several proteins play key role in steroidogenesis. The role of steroidogenic acute regulatory (StAR) protein is well established. A novel protein, BRE, found mainly in brain, adrenals and gonads, was highly expressed in hyperplastic rat adrenals with impaired steroidogenesis, suggesting its regulation by pituitary hormones. To further elucidate its role in steroidogenic tissues, mouse Leydig tumor cells (mLTC-1) were transfected with BRE antisense probes. Morphologically the BRE antisense cells exhibited large cytoplasmic lipid droplets and failed to shrink in response to human chorionic gonadotropin. Although cAMP production, along with StAR and P450scc mRNA expression, was unaffected in BRE antisense clones, progesterone and testosterone yields were significantly decreased, while pregnenolone was increased in response to human chorionic gonadotropin stimulation or in the presence of 22(R)OH-cholesterol. Furthermore, whereas exogenous progesterone was readily converted to testosterone, pregnenolone was not, suggesting impairment of pregnenolone-to-progesterone conversion, a step metabolized by 3beta-HSD. That steroidogenesis was compromised at the 3beta-HSD step was further confirmed by the reduced expression of 3beta-HSD type I (3ss-HSDI) mRNA in BRE antisense cells compared with controls. Our results suggest that BRE influences steroidogenesis through its effects on 3beta-HSD action, probably affecting its transcription.

Characterization of large luteal cells and their secretory granules during the estrous cycle of the cow.

Large steroidogenic cells of the bovine corpora lutea were evaluated for morphological changes on Days 3, 7, 11, 14, 17, and 19 of the estrous cycle. Large cells were readily identified by size (25-50 microns diameter), numerous mitochondria, and the presence of dense secretory granules (150-300 nm in diameter). These granules were found in a discrete cluster and were not dispersed throughout the cytoplasm. Only 3% of the large cells contained a cluster of granules on Day 3. The percentage was highest during midcycle (Day 7, 84%; Day 11, 64%), dropped on Day 14 (26%), and was lowest on Days 17 (16%) and 19 (8%). Electron microscopic immunocytochemistry showed that oxytocin and neurophysin were co-localized in these granules on all days evaluated. As early as Day 14, large cells were observed with characteristics typical of regressing corpora lutea, i.e., a reduction in cells with secretory granules, large cytoplasmic lipid droplets, and swollen mitochondria with dense inclusions. However, since this was a time of the cycle when plasma concentrations of progesterone were very high, this corpus luteum is referred to as involutive rather than regressive. Our results may be summarized as follows: 1) from Day 7 to Day 14 there was a 69% decline in the number of large cells containing oxytocin-laden secretory granules. This occurred prior to the rise in uterine oxytocin receptors and the large luteolytic pulses of prostaglandin that reportedly occur after Day 14. The role of this apparent early release of oxytocin is not known. 2) Large steroidogenic luteal cells of the estrous cycle have morphological characteristics similar to those of large luteal cells during pregnancy. However, large luteal cells of the estrous cycle contain oxytocin whereas those of pregnancy are devoid of oxytocin.

Ultrastructural changes in the mouse liver induced by hepatotoxin from the freshwater cyanobacterium Microcystis aeruginosa strain 7820

The time-course of ultrastructural changes was studied in mouse liver hepatocytes after i.p. injection of lethal (100 μg/kg) and sublethal (10 μg/kg) doses of the heptapeptide hepatotoxin from Microcystis aeruginisa strain 7820, a freshwater blue-green alga (cyanobacterium). At both dose levels the hepatocytes show progressive intracellular changes over time periods of 10, 20, 30, and 60 min. The changes resulting from a lethal dose were more prominent and rapid compared to those of the sublethal dose. The most common responses to lethal and sublethal doses were vesiculation of rough endoplasmic reticulum, swollen mitochondria and degranulation (partial or total loss of ribosomes from vesicles). These vesicles appear to have formed from the dilated parts of rough endoplasmic reticulumby fragmentation or separation. At the lethal dose an increased amount of whorl shaped rough endoplasmic reticulum along with large membrane-bound vacuoles were observed in the cytoplasm. With the sublethal dose an increase in the amount of small and large cytoplasmic lipid droplets occurred. These ultrastructural changes parallel the pathological events which lead to animal death by hemmorrhagic shock.

Glial and axonal development in optic nerve of myelin deficient rat mutant

Development of glial cell lines and axons is reported for the optic nerve of the myelin deficient rat mutant, md, 3–46 days postnatally. In mutants, optic nerves do not increase in area after 16 days of age whereas, in normal rats, they enlarge through 46 days of postnatal life. The density of glial cells, determined in cross-sections, is similar in md and normal littermates through 19 days postnatally. Thereafter, glial densities are greater in the mutant. Nonetheless, total glial counts are reduced in md as compared to the normal, because cross-sectional areas and lengths of mutant nerve 30–46 days after birth are smaller than those of age-matched, normal littermates. Differential counts of glial cells, made by ultrastructural criteria, show that md optic nerves contain abnormal, vacuolated, immature oligodendroglia from the third postnatal day. Furthermore, oligodendrocytes are reduced in number in older mutants; they constitute 1% of optic nerve neuroglia at 46 days. Astrocytic numbers are increased in relative, not in absolute, terms from 19 days, and microglial numbers are greater than normal in the oldest mutants. Reactive microglia, containing large cytoplasmic lipid droplets, constitute 4–8% of the glia of md nerve 19–46 days postnatally. Mean axonal areas are similar in normal rats and mutants at 19 and 43–46 days of age. However, mitochondrial density is greater in md axons 19 days after birth and mean areas of axonal mitochondria are significantly larger in 43–46 day mutants than in age-matched, normal littermates. Additionally, the percent area of axoplasm occupied by mitochondria is increased in md at both 19 and 43–46 days of age. The myelination defect in md appears to be due primarily to an oligodendroglial abnormality which precedes the normal age of onset of myelination dAstrocytic and microglial changes are secondary. Axonal enlargement proceeds normally over 46 days of postnatal life. Overall, the data do not provide definitive support for an axonal basis for the myelination defect, although measurable differences in axonal mitochondria between mutants and normals are demonstrable and qualitative abnormalities do occur in the axons of the mutant.

Ultrastructure of the central nervous system in a myelin deficient rat.

Myelin deficiency (md) is a new mutant in the Wistar rat caused by an X-linked recessive lethal gene. One-half of the male offspring develop tremor and ataxia at 10-12 days of age and seizures at 16-21 days. Usually, the animals die 24-28 days postnatally unless survival is prolonged by anticonvulsants. Light microscopic examination of the C.N.S. shows a complete lack of myelin. The P.N.S. is normally myelinated, however. Frontal cortex, corpus callosum, optic nerves, cerebellum and spinal cord were studied routinely in affected animals aged 3-46 days. Abnormal males were identified three days after birth by the absence of myelinated axons from the ventral funiculus of the cervical cord. In mutants aged 3-16 days, axons had the usual ultrastructural features but were either entirely non-myelinated or, rarely, were invested by poorly organized, non-compacted, myelin-like loops of membranes, 2 to 4 in number. In mutants aged 17-20 days, axonal swellings appeared. These increased in number with longer survival times and contained large numbers of microtubules, neurofilaments, mitochondria and dense bodies. Normal C.N.S. myelin was not observed at any age. Two types of abnormal glial cell occur in md. The first, present in white matter at three days of age, is an abnormal oligodendrocyte. The cytoplasm contains dilatation of the rough-surfaced endoplasmic reticulum and the nuclear envelope is widened. A second cell-type, conspicuous by 10 days, has an electron-dense nucleus with prominently clumped chromatin and large cytoplasmic lipid droplets. This second cell type is believed to be a microgliacyte. The number of cytologically-normal oligodendrocytes decreases as mutants age while hypertrophied, filament-rich astrocytes occur in increasing numbers. The myelin defect in md C.N.S. is probably due to an abnormality of oligodendrocytes. Axonal alterations are probably secondary. Myelin deficiency resembles the murine mutant, Jimpy (jp), although ultrastructural changes in oligodendrocytes appear to be dis-similar and md, in contrast to jp, contains no normal-appearing C.N.S. myelin.

An ultrastructural study of the natural secretory cycle of the parathyroid gland of the bat.

AbstractCyclical ultrastructural changes in chief cells of the bat parathyroid during hibernation are described. The morphology indicates that the cells increase in activity at the start of hibernation and decrease in activity at the end. During early hibernation, the main alterations are an increase in granular endoplasmic reticulum, an increase in the number of prosecretory granules associated with the Golgi apparatus, an increase in the number of mature secretory granules, and the appearance of numerous, large cytoplasmic lipid droplets. An unusual finding is that the lipid droplets are preferentially associated with mitochondria and glycogen deposits. At this time there is also an increase in the size of intercellular spaces and the concentration of secretory granules near the plasma membrane. By late hibernation, however, there is a marked reduction in the amount of granular endoplasmic reticulum, fewer mitochondria, inconspicuous Golgi apparatuses, and a reduction in the size of intercellular spaces. In addition, lipid droplets are absent. These observations are discussed in relation to the cyclic secretory changes which occur in bat parafollicular cells and in bone during hibernation.

Lipid absorption, transport and hepatic assimilation with electron microscopy.

An electron microscopic study of intestine and liver in rats ingesting a high-lipid diet or receiving corn oil by stomach tube was carried out. Studies of the intestinal phase of lipid absorption in rats showed what appears to be the direct transfer of particles across the cell membrane confirming the postulate that some of the process of lipid absorption involves triglyceride particles. Lipid droplets, representing chylomicrons were identified in hepatic sinusoids. They were traced into the pericellular space of Disse, which they had entered through the wide pore spaces in the sinusoidal endothelial cytoplasm. Lipid particles were also observed in different stages of direct transfer across the hepatic parenchymal cell membrane, and their formation of larger cytoplasmic lipid droplets within the cytoplasm of liver cells. This mechanism is proposed as a major device of the body in dispersing the lipemia which occurs after the ingestion of a fat meal.