In situ hybridization for the detection of feline interleukin 1 alpha mRNA on the paraffin-embedded section using biotin-labeled probes.

In situ hybridization (ISH) technique with a biotin-labeled probe was established for detecting feline interleukin 1 (IL-1) alpha mRNA in necropsied specimens. Homology between human IL-1 alpha cDNA used as a probe and feline IL-1 alpha mRNA was confirmed by means of dot blot hybridization using the biotin-labeled probe. Hence, we tried by this biotinylated probe to detect mRNA of IL-1 alpha in paraffin-embedded sections. The following results were obtained for the routine procedures: 1) coating slides with poly-L-lysine and/or heating at 60 degrees C at least for 6 hours gave an excellent result for the adhesion of the tissue sections, 2) 10 micrograms/ml solution of proteinase K treatment for 30 minutes or 50 to 100 micrograms/ml solution of proteinase K treatment for 10 to 30 minutes at 37 degrees C gave the good results in the detection of ISH signal, 3) suitable denaturation time of probes at 70 to 90 degrees C was 5 to 15 minutes, and 4) effective hybridization was obtained by incubation for 24 hours at 4 degrees C, for 18 to 24 hours at 25 degrees C or for 5 to 24 hours at 37 degrees C.     

Control of ovarian follicles activity in the ewe.

During the ovine estrous cycles, three waves of follicular growth, closely associated with the FSH secretion pattern, were observed. The parameters of these follicular waves and the ability of follicles to produce steroids in vitro were studied in various conditions. In vivo, the follicular events were similar between the breeding season and the anestrus, except for the lack of ovulation; but at the end of the breeding season and in anestrus, the follicles lose a big part of their aromatization ability. In ewes carrying the Booroola fecundity gene or Cambridge fecundity gene, the reduction in follicular atresia seems to be one of the main follicular features implicated in the control of high ovulation rate. In vitro, the most relevant difference is an early acquisition of estrogen production ability of small follicles in Booroola fecundity gene barring ewes. Fluoro-gestone-acetate (FGA) pessaries reduced the number of growing follicles; despite this effect disappearing after the sponge withdrawal, the ovulation rate is significantly reduced. But an equine chorionic gonadotrophin (eCG) treatment restores the ovulation rate (OR) by reducing the atresia rate of pre-ovulatory follicles. In similar conditions, a pretreatment of the ewes with melatonin again reduced the atresia rate of large follicles and resulted in an increased ovulation rate. In vitro, FGA blocked aromatization ability, and melatonin inhibited both androstenedione and estradiol production, but a further treatment with eCG partly restores the steroid secretion. Immunization against androstenedione leads to a higher OR, owning to a reduced atresia of large follicles. Daily growth hormone injections for a hole cycle resulted in an increased follicular population and ovulation rate, while FSH plasma levels decreased and the follicle sensitivity to gonadotrophins was reduced.     

Maintenance of the corpus luteum of early pregnancy in the ewe. IV. Changes in luteal sensitivity to prostaglandin F2 alpha throughout early pregnancy

Two experiments were conducted to examine the temporal aspects of luteal resistance to the luteolytic effect of prostaglandin (PG) F2 alpha during early pregnancy. In Exp. 1, 14 pregnant and 12 nonpregnant ewes were treated with PGF2 alpha either on d 10 or 13 post-estrus. Jugular venous blood samples were collected at -30 min, 0, 6, 12, 18, 24, 30 and 36 h post-injection for quantification of progesterone. The difference (delta P) between pre-treatment and post-treatment concentrations of progesterone was calculated for each ewe. There was a significant interaction between pregnancy status and day of treatment on delta P (P less than .05). Pregnant and nonpregnant ewes treated on d 10 showed a large delta P. A large delta P also was observed in nonpregnant ewes treated on d 13 post-estrus. However, delta P in pregnant ewes treated on d 13 was smaller than in the other three groups (P less than .05). The temporal patterns of concentrations of progesterone in serum were different among treatment groups (P less than .05). A suppression in the concentration of progesterone was observed by 24 h post-injection in all four treatment groups. Progesterone returned to pre-treatment levels only in pregnant ewes treated on d 13. In Exp. 2, 47 pregnant ewes were treated with PGF2 alpha on d 10, 13, 16, 19, 22, 26 or 30 postestrus. Blood samples were collected and data were analyzed as described for Exp. 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

原位杂交法检测hsp mRNA在猪组织细胞中的分布

根据hsp70和hsp90 mRNA基因序列,设计并合成引物,将PCR扩增的基因片段克隆到pGEM-T载体,重组质粒经筛选、鉴定,利用体外转录系统,制作地高辛标记的RNA探针,建立检测hsps mRNA原位杂交方法。利用自建的原位杂交方法,对hsp70、hsp90 mRNA进行组织细胞定位研究。结果显示:hsp70 mRNA和hsp90 mRNA广泛分布于各组织细胞中,尤其在细胞核中相对集中,在肾小管上皮细胞胞浆中也呈强阳性着色。     

Characterization of gonadotropic and ovarian steroid hormones during the periovulatory period in high ovulating select and control line gilts

Cyclic gilts from Control (C, randomly selected, n = 11) and Relax Select (RS, nine generations of selection for increased ovulation rate followed by seven generations of relaxed or random selection, n = 9) lines of the University of Nebraska Gene Pool population (derived from 14 different breeds) were utilized to characterize differences in gonadotropic and ovarian steroid hormones during preovulatory and postovulatory phases of the estrous cycle. Blood samples were collected during four periods (0500, 1100, 1700 and 2300) daily beginning 2 d prior to anticipated estrus (d -2, d 18 of a 20-d estrous cycle), and continuing through d 4 postestrus (d 0 = 1st of standing estrus). Sampling within a period consisted of five blood samples at 15-min intervals. All plasma samples were analyzed for concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH). Neither mean LH nor peak concentration of LH during the preovulatory surge differed between genetic lines (P greater than .10). Concentrations of FSH increased faster (line X period, P less than .05) and tended (P less than .1) to peak at a higher concentration in RS (.88 ng/ml) than in C (.54 ng/ml) gilts (P less than .05) during the 12 h preceding the FSH and LH preovulatory peaks. The second FSH surge began approximately 24 h after the preovulatory FSH peak. Peak FSH concentrations were observed at 42 h in both lines (1.46 vs 1.74 ng/ml for C and RS gilts, respectively). The higher FSH concentration in RS gilts established during the preovulatory surge was maintained through the second FSH surge (P less than .01). No line differences were detected in plasma concentrations of estradiol-17 beta and progesterone.     

Cell-specific localization of oestrogen receptor beta (ESR2) mRNA within various bovine ovarian cell types using in situ hybridization

The localization of oestrogen receptor beta (ESR2) mRNA, in this article denominated as (ERbeta) mRNA, was examined using in situ hybridization in the ovaries of randomly selected cows, irrespective of the cycle stage of the animals. A 602-bp fragment of ERbeta mRNA was cloned, sequenced and digoxigenin (DIG)-labelled. Semi-quantitative evaluation showed that the scores for ERbeta mRNA were moderate to high in the follicle cells of both primordial and primary follicles, but lower in granulosa cells of secondary follicles. In vital tertiary follicles, the total ERbeta mRNA expression was low but varied between the different animals. In both obliterative and cystic atretic follicles, high to moderate ERbeta mRNA scores were noticed in the granulosa cells. The stroma cells surrounding primordial and primary follicles and the theca cells of secondary follicles showed moderate ERbeta mRNA levels, whereas the ERbeta mRNA score in theca interna and theca externa cells of vital tertiary follicles was distinctly higher. In the theca cells of atretic follicles the score was even higher. Cells of corpora hemorrhagica and corpora lutea had moderate ERbeta mRNA scores, while higher scores were seen in cells of corpora albicantia. Cells of the surface epithelium had a moderate score for ERbeta mRNA, whereas cells of the tunica albuginea and deep stroma showed high ERbeta mRNA scores. The present findings have clearly established a cell-specific localization of ERbeta mRNA in several cell types in the bovine ovary.     

In situ hybridization for the detection and localization of swine Chlamydia trachomatis

Gnotobiotic piglets were inoculated intralaryngeally with swine Chlamydia trachomatis strain R33 or orally with swine C. trachmatis strain R27. Archived formalin-fixed, paraffin-embedded tissues from piglets euthanatized 4-7 days postinoculation were examined by in situ hybridization for C. trachomatis nucleic acid using a nonradioactive digoxigenin-labeled DNA probes that targeted specific ribosomal RNA or omp1 mRNA molecules of the swine C. trachomatis strains. Positive hybridization signals were detected in bronchial epithelial cells, bronchiolar epithelial cells, pneumocytes, alveolar and interstitial macrophages, and jejunal and ileal enterocytes. Chlamydia-infected cells had a strong signal that was confined to the intracytoplasmic inclusions. Positive hybridization signals were not detected in tissue sections from an uninfected control piglet or in C. psittaci-infected sheep placenta. The morphology of host cells was preserved despite the relatively high temperature required in parts of the incubation procedure. The data indicate that in situ hybridization can be used to detect swine C. trachomatis in formalin-fixed, paraffin-embedded tissue specimens.     

Endocrinology of the postpartum period in the Pelibuey ewe

The postpartum (PP) period in the Pelibuey ewe was studied. Laparotomies were performed on 14 ewes in the first year at d 10, 20 and 30 PP, and at d 10 and 20 PP in the second year on 17 ewes. Progesterone concentrations were determined in serum taken daily, from 4 to 7 d after parturition until estrus. Temporal fluctuation of luteinizing hormone (LH) was determined in samples taken at 30-min intervals for 4 h weekly. The mean interval from lambing to first ovulation was longer (P less than .001) in 1980 (59 +/- 4.9 d) than 1979 (26 +/- 3.1 d), the mean interval from lambing to first estrus was also longer (P less than .001) in 1980 (91 +/- 5.6 d) than 1979 (51 +/- 5.5 d). Follicles were present on the ovaries of the majority of the ewes at d 10. The mean diameter of the largest follicles on each ovary was reduced (P less than .025) in ewes in 1980 (6 mm) compared with 1979 (7.7 mm). Corpora lutea (CL) occurred in 67 and 75% of the ewes by d 20 and 30, respectively in 1979; no CL were found by d 20 in 1980. Progesterone profiles suggested that the PP period was composed of a period of anestrus, and a period of cyclic ovarian activity with one, two or three ovulations without behavioral estrus. In some ewes, the first cycle was of shorter duration, and its CL secreted less progesterone (P less than .05) relative to CL of silent and regular estrous cycles. Luteinizing hormone peaks were recorded as early as 6 d PP. When progesterone concentrations were elevated to luteal phase levels, the frequency, but not magnitude, of LH peaks per 4-h bleeding period was reduced (P less than .05) relative to anestrus. It is concluded that there are periods of anestrus and of silent cycles, which precede the first postpartum estrus in Pelibuey ewes.     

Effect on the ewe and lamb of low zinc intake throughout pregnancy

Throughout pregnancy, 30 primiparous Finn cross ewes were given a low Zn (less than or equal to 1 ppm) semi-purified diet. A 100-g hay supplement was fed three to seven times/week. Supplemental Zn (20 ppm) was provided in the drinking water of 14 ewes. At parturition, lambs were removed from ewes before suckling. Viable lambs not taken for tissue analysis were given 200 ml cow colostrum and raised on an artificial feeder. Throughout gestation, unsupplemented (-Zn) ewes gained less weight and had lower plasma Zn levels than Zn-supplemented (+Zn) ewes. One -Zn ewe was not pregnant, three aborted, one resorbed, one delivered mummified twins at term and two delivered malformed lambs. Average weight of lambs born to -Zn ewes d 136 or later (excluding mummified twins and one weighing less than 20% as much as its twin) was 1.8 +/- .6 (SE) kg. Only three lambs born to -Zn ewes were vigorous enough to put on the artificial feeder; none survived. One +Zn ewe was not pregnant. Of 23 lambs born to the remaining +Zn ewes, five were used for tissue analysis, two lambs of triplets were born dead, twins born d 138 died at birth. One twin died 6 d after birth. The 14 remaining lambs were weaned in good health. Average birth weight of +Zn lambs was 3.3 +/- 1.0 kg. Increased salivation was seen in -Zn ewes after 6 wk of low Zn intake.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ hybridization method for studies of cell wall deficient M. paratuberculosis in tissue samples

Cell wall deficient forms of mycobacteria may be important in the pathogenesis of Crohn's disease and sarcoidosis. However, no method has been available to localize this type of organisms in tissue sections. We developed an in situ hybridization method for the demonstration of Mycobacterium paratuberculosis spheroplasts (cell wall deficient forms) in paraffin embedded tissue sections.M. paratuberculosis spheroplasts were prepared by treatment with glycine and lysozyme. Pieces of beef were injected with the prepared spheroplasts. The samples were fixed in buffered formalin and paraffin embedded. A M. paratuberculosis-specific probe derived from the IS900 gene was used. Specificity was controlled by using an irrelevant probe and by hybridizing sections with spheroplasts from other bacteria.Beef samples injected with M. paratuberculosis spheroplasts were the only samples that hybridized with the probe. Beef samples containing acid-fast or spheroplast forms of M. smegmatis and M. tuberculosis as well as the acid-fast forms of M. paratuberculosis did not hybridize with the probe. Unrelated bacterial controls, i.e. Helicobacter pylori and Escherichia coli were also negative in the assay. In situ hybridization with the IS900 probe provides a specific way to localize M. paratuberculosis spheroplasts in tissue sections and may be useful for studies of the connection between M. paratuberculosis and Crohn's disease and sarcoidosis. The assay may also be valuable for studies on Johne's diseased animals.     

In situ hybridization for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods.

Archived formalin-fixed, paraffin-embedded tissues from 25 pigs naturally infected with transmissible gastroenteritis virus (TGEV) were examined by in situ hybridization for TGEV nucleic acid using a nonradioactive digoxigenin-labeled cDNA probe that targeted the nucleocapsid sequence of TGEV strains. The results of in situ hybridization for the detection of TGEV were compared with virus isolation (VI), a fluorescent antibody test (FAT), and transmission electron microscopy (TEM). VI, FAT, and TEM were tested over a course of time before the in situ hybridization was performed. Positive hybridization signals were detected in duodenal, jejunal, and ileal enterocytes from 21 pigs. Hybridization signals were confined to the cytoplasm. Intestinal specimens from 25 piglets were evaluated by 4 tests. Twenty-one of 25 were positive by in situ hybridization. Of these 21 samples, 5 (24%) were positive for TGEV by all 4 tests, 15 (71%) were positive by FAT, 14 (67%) were positive by VI, and 6 (29%) were positive by TEM. In situ hybridization for the detection of TGEV in formalin-fixed, paraffin-embedded tissues provides a rapid means of confirmation of a histopathological diagnosis of TGEV without virus isolation, or when only formalin-fixed intestinal specimens were available.     

In situ hybridization for the demonstration of bovine leukemia virus transcripts in lymphosarcoma cells using biotinylated probes.

Bovine leukemia virus (BLV) RNA was demonstrated in peripheral blood lymphocytes treated with concanavalin A or phytohemagglutinin from a BLV-infected ox, and in lymphosarcoma cells cultured without mitogen from an enzootic bovine leukosis cow by in situ hybridization using biotinylated DNA probes.     

实验性禽脑脊髓炎中MIP-1β和IL-8的mRNA原位杂交实验

禽脑脊髓炎病毒内蒙古地方毒株NH937株经脑内注射感染.于攻毒后3,5,10,14,20,25,30 d,取攻毒组4只,对照组2只.取材大脑、小脑于95%乙醇中固定,经原位杂交染色,结果:着染部位主要在大脑皮层锥体细胞及皮质下各核团的较大神经元的胞浆内,呈现蓝紫色连成片或单独数个较大颗粒,小脑蒲肯野细胞胞浆内也较多见;此外小胶质细胞胞浆内也发现很多呈阳性.在病情严重时的10 d开始增多,直到病情恢复的30 d时,仍然很普遍.相对于未攻毒的阴性对照组明显增多.说明禽脑脊髓炎过程中脑组织细胞内MIP-1β和IL-8的mRNA大量上调,主要与趋化T淋巴细胞到血管外形成管套,再从管套弥散向周围神经组织;同时趋化小胶质细胞到病毒感染的靶细胞形成卫星现象和噬神经元现象等有关.     

The effect of inhibition of prostaglandin F2 alpha synthesis on placental expulsion in the ewe.

Five ewes were injected with two doses of a nonsteroidal anti-inflammatory drug (NSAI), lysine acetyl salicylate, at birth of their first lamb and one hour later, and five others were injected once only, at birth of their first lamb. A control group of six animals was constituted. The times needed for fetal expulsion and placental release were recorded. The peripheral plasma PgF2 alpha (as PGFM) levels were measured prepartum during the seven last days of gestation, at parturition, then 1 h, 2 h and 12 h after lambing. The results were compared among and within treatment groups. They indicate that the physiological increase in peripheral PGFM levels starts two days before lambing and that the level peaks at lambing. The normal decrease after parturition is emphasized by NSAI injections as detected 1 h and 2 h posttreatment (p < 0.01). The NSAI drug is short-acting as revealed by the lower PGFM levels in twice-treated animals 2 h after birth compared to once treated animals and the similar low levels in all three groups 12 h after birth. The fetal membranes were expelled normally in all treated and nontreated animals, but the time needed for placental expulsion in ewes injected with two doses of NSAI was longer than in controls (p < 0.05). A negative correlation (p < 0.05) was found between plasma PGFM levels measured two hours after lambing and the time needed for fetal membrane expulsion. PgF2 alpha appears to have a role in placental release in the ewe.