Messenger RNA synthesis in synchronized Chinese hamster ovary cells

Chinese hamster ovary cells were synchronized without inhibitors by mitotic selection and labelled in G1, S or G2 phase by incubation for 90 min with [3H]- OR [14C]uridine. Purified polyribosomes were extracted with phenol and the polyadenylated mRNA prepared by poly(U)-Sepharose chromatography. Poly-adenylated [3H]uridine-labelled mRNA from the G1 phase of the cell cycle was compared by exponential polyacrylamide gel electrophoresis in formamide with [14C] uridine-labelled polyadenylated nRNA from the S or G2 phase. The electrophoretic patterns obtained correspond to the size range expected for mRNA (7-28 S). No prominent differences were detected between mRNAs synthesized in different phases of the cell cycle. From these data we conclude that the major size classes of polyribosomal poly(A)-containing mRNA are synthesized in equal ratios throughout the cell cycle.     

RNA and protein synthesis in the earwig ovary

Cytochemical and autoradiographic studies on the ovaries of Labidura riparia indicate that the oöcyte is supplied with RNA synthesized in the nurse cell nucleus. There is no indication that the oöcyte synthesizes its own RNA. Neither DNA nor protein can be shown to enter the oöcyte from external sources. Although largely cytoplasmic, the incorporation of lysine into protein also localizes in the nucleolus of older oöcytes.     

Histological study of follicular atresia in the ovary of the domestic hen (Gallus domesticus)

Semi-serial (1 in 20) sections of ovaries were studied and only two types of atresia were identified--non-bursting and bursting. Smaller, non-yolky follicles (less than 1 mm diameter) showed non-bursting atresia. Atresia in follicles greater than 1 mm diameter was invariably of the bursting type which involved the rupture of the follicular wall, and the extrusion of yolk and cellular debris through the rupture site into the stroma. However, this rupture site was small and consequently was not visible in every section but it could always be seen when the follicle was followed in semi-serial sections. The mitotic index of granulosa cells in bursting atretic follicles was much lower than that for normal follicles. The most common criteria for distinguishing non-bursting atretic follicles were the extremely shrunken, irregularly shaped oocytes and the separation of the granulosa from the theca. In bursting atretic follicles, reliable indications were the presence in the ooplasm of some cells or cellular debris, and disorganization of the yolk and granulosa tissue. The presence of pycnotic nuclei in the granulosa cells was not a consistent feature of all atretic follicles of the hen.     

Messenger RNA sequence and expression of rat pancreatitis-associated protein, a lectin-related protein overexpressed during acute experimental pancreatitis.

Rat pancreatitis-associated protein (PAP) is an additional protein appearing in pancreatic juice after induction of prancreatic inflammation. Its messenger RNA was cloned and sequenced from pancreas. The deduced amino acid sequence revealed that PAP was synthetized as a preprotein with, in its mature form, a predicted molecular weight of 16,630. A search in protein data bases revealed a marked homology with the carbohydrate binding region of animal lectins; no hemagglutination activity could be shown for PAP, but the protein induced extensive bacterial aggregation. In healthy rats, the very low level of PAP expression in pancreas could be increased up to 4-fold by physiological stimuli such as chronic hormonal or cholinergic stimulation of pancreatic secretion and adaptation of rats to a carbohydrate-rich diet. By contrast, induction of acute experimental pancreatitis by retrograde injection of sodium taurocholate resulted in dramatic overexpression. Pancreatic concentration of PAP mRNA increased more than 300 x within 12 h whereas concentrations of mRNAs encoding major secretory proteins such as amylase decreased. PAP overexpression persisted during the 2 days of the acute phase and then returned to the control level during pancreatic recovery. PAP mRNA could not be evidenced in liver, stomach, salivary glands, brain, kidney, or testis. Its pattern of expression during severe pancreatic aggression suggests that it might be a stress protein involved in the control of bacterial proliferation.     

Blood flow in the reproductive tract of the domestic hen following treatment with a pituitary gonadotropic inhibitor.

The effect of pituitary gonadotropic inhibitor--methallibure on the weight and blood flow through reproductive tract was studied in immature hens. It was found that methallibure lowers the weight of the reproductive tract and simultaneously inhibits (about 2--3 times) the blood flow through the ovary and oviductal segments. However, the action of methallibure is of short duration and it is limited to the time of its administration only.     

Messenger RNA in the cytoskeletal framework: analysis by in situ hybridization

The possibility of an association of mRNA with the cytoskeletal framework (CF) of ascidian (Styela plicata) follicle cells was examined in this study. The approach was to extract the follicle cells with Triton X-100 and determine whether mRNA persisted in the insoluble residue by two methods, in situ hybridization with poly(U) and actin DNA probes and the incorporation of radioactive isotopes into RNA. Triton X-100 extraction of follicle cells yielded a filamentous CF containing approximately 70% of the total poly (A) but only 9% of the total lipid, 23% of the total protein, and 28% of the total RNA. In situ hybridization with a poly (U) probe indicated that approximately 70% of the poly (A) was associated with the CF. In situ hybridization with a cloned actin DNA probe indicated that approximately 60% of the actin mRNA was associated with the CF. Autoradiography of detergent- extracted follicle cells, which had been labeled with [3H]uridine or [3H]adenosine, indicated that greater than 90% of the newly synthesized poly (A)+RNA was preserved in the CF. Thus more newly synthesized mRNA than steady-state mRNA may be present in the Triton X-100 insoluble fraction. It is concluded that a significant proportion of the mRNA complement of ascidian follicle cells is associated with the CF.     

The hyperstriatum and nesting behaviour in the domestic hen

Bilateral ablation of the ventral hyperstriatum resulted in hens ceasing to use trap-nests which they had formerly used. The appetitive component of their pre-laying behaviour was normal in form but was not directed towards the trap-nests, as it was in control birds. In the absence of trap-nests in a pen control birds laid in corners and were conservative in choice of site but both these characteristics were abolished by these ablations. Other bilateral ablations involving the Wulst had less drastic effects on pre-laying behaviour while bilateral ablations in the posterior telencephalon had no effect on this behaviour pattern.     

Immunocytochemical analysis of the expression of gap junction protein connexin 43 in the rat ovary

The present immunocytochemical study examines in the rat ovary the pattern of expression of connexin 43 (Cx43), a subunit of gap junctions. Using a well-characterized specific antiserum against rat Cx43, immunoreactivity was not detected in the fetal ovary, i.e., prior to follicular formation. However, in the ovary of 20-day-old, 35-day-old, and adult rats, strong Cx43-immunore-activity was associated with the cell borders of the follicular epithelium/granulosa cells of all developmental stages (primordial follicles, preantral and antral secondary follicles). In general, immunoreactivity of the granulosa cells of large antral follicles appeared more intense than the one of smaller follicles. Staining was also seen in oocytes (cytoplasmic staining). Theca cells of large antral follicles, but not of small follicles were immunoreactive. Immunoreactive interstitial cells were not seen in ovaries of 20- and 35-day-old animals, but staining in these cells was present in adult rats. In large follicles with signs of atresia, granulosa cells lacked Cx43-immunoreactivity, whereas Cx43-immunoreactivity in their theca interna strikingly increased. Corpora lutea in the cyclic adult rats were heterogeneously stained, with either no detectable immunoreactivity, staining of cell borders of most luteal cells, or with conspicuous staining of only a few cells. In the pregnant animals on gestation days (GD) 12, 14, and 17, all luteal cells stained strongly for Cx43 at the cell surface. Shortly before delivery (GD 21), however, the staining pattern vanished and only few, presumably luteal cells remained immunoreactive. In Western blots (using homogenates of whole ovaries), the Cx43 antiserum recognized a major band of approximate Mr 43 × 10³, together with minor bands, which may reflect the presence of several differently phosphorylated Cx43 forms. This is indicated by treatment with alkaline phosphatase, which reduced the banding pattern to one single band. In summary, the gap junction molecule Cx43 is abundantly expressed in all endocrine compartments of the rat ovary. The staining pattern obtained in the present study indicates that Cx43 and presumably gap-junctional communication are associated with follicular development, atresia, and the development of the interstitial gland, as well as with the development and regression of the corpus luteum. The heterogeneous staining within the ovary furthermore hints to a contribution of the local intraovarian factors in the regulation of Cx43 expression. © 1995 Wiley-Liss, Inc.     

Messenger RNA structure participating in the initiation of synthesis of cucumber mosaic virus coat protein

The sequence of the 5'-terminal 106 nucleotides of cucumber mosaic virus (strain Y) RNA 4, the mRNA coding for viral coat protein, has been determined. The first AUG was located at 77 nucleotides from the 5'-terminus and was confirmed to be an initiation codon by analysis of the N-terminal amino acid sequence of the protein. The nucleotide sequence (positions 77-106) beyond the AUG codon predicted the sequence of ten amino acids corresponding to the N-terminal region of the protein, which exactly matched the determined amino acid sequence containing an acetyl methionine as the N-terminal amino acid. The distance of the initiation codon AUG from the cap structure was 76 nucleotides and the longest among the mRNAs for coat protein of plant viruses so far reported (9-36 nucleotides). This noncoding region is rich in U residues (40%) and the number of G residues (21 nucleotides) is the largest among these mRNAs (usually 1 or 2 residues). A possible secondary structure is postulated for the region, which might be implicated in efficient translation of the RNA 4 in vivo.     

Distribution of follicular growth, atresia and ovulation in the ovary of the domestic hen (Gallus domesticus) at different ages

Ovaries of laying hens of 14, 18, 30, 45 and 86 weeks of age were divided transversely into two or more distinct regions. Differences in follicular populations between these regions were observed. The central segments of the ovary in birds of 14 weeks of age produced most of the visible (greater than 0.5 mg) developing follicles (P less than 0.01). This was a transient effect during ovarian maturation. In birds of 18 weeks of age, but not yet in lay, more follicles of greater than 8 mm in diameter were observed in the anterior part of the ovary than the posterior part (P less than 0.05). Follicles of this size are almost certain to ovulate. The posterior segment of the ovary of birds of 30, 45 and 86 weeks of age contained more follicles beginning the rapid growth phase, as measured by follicles of 1.4-1.8 mm in diameter, than did the anterior segment (P less than 0.01). Higher levels of atresia in the posterior segment (P less than 0.001) resulted in fewer follicles of greater than 8 mm (P less than 0.001) and fewer post-ovulatory follicles than in the anterior segment. We conclude that most of the eggs produced during the hen's laying year must be from ovulations from the anterior part of the ovary.     

The vocal expression of feeding motivation and frustration in the domestic laying hen, Gallus gallus domesticus

Thwarting of feeding behaviour in the laying hen results in an increase in stereotyped pacing, displacement preening, and the gakel-call. These behaviours therefore reflect the frustration arousal caused by the thwarting of feeding behaviour. This raises the question whether the level of frustration also varies with the intensity of the motivation to perform the thwarted behaviour. This study investigated the relationship between the intensity of the motivation and level of frustration on the one hand and the gakel-call on the other hand. In Experiment 1, the strength of the motivation to feed was varied by thwarting hens in their feeding behaviour in an operant procedure after different durations of food deprivation (0, 8, 23 and 47 h). Trend analysis showed that with increasing hunger state, an increasing number of gakel-calls was given. No effect of treatments on temporal characteristics of the gakel-call was found. In Experiment 2, the level of frustration was varied by reducing or increasing the duration of access to food for food-deprived hens compared to the duration of access during training. It was assumed that a shorter duration of access to food compared to training would elicit frustration, which in turn would affect the performance of behaviours indicative of thwarting. However, we found neither a relation between the number of gakel-calls nor the temporal features of the gakel-call and the duration of access to food. Possibly, the differences between treatments were not large enough to induce differences in frustration level. Also, other factors that might have influenced the motivation are discussed.