Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.21.1 (DNase)
 7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have recently purified a novel pituitary polypeptide, designated 7B2. Subsequently, we developed a sensitive and specific radioimmunoassay (RIA) for this novel polypeptide. Our aim in the present study was to investigate the release of 7B2 from rat pituitary induced by various hypothalamic factors [luteinizing hormone-releasing factor (LH-RH), corticotropin-releasing factor (CRF), and growth hormone-releasing factor (GRF)]. The anterior pituitaries were removed from rats and immediately dispersed enzymatically (a mixture of collagenase/dispase/deoxyribonuclease/chicken serum) and plated on collagen-coated multiwell plates in culture medium containing 10% fetal bovine serum. After 2 days of attachment period, the medium was replaced with fresh medium every 24 h. The primary cell culture was incubated with various concentrations of LH-RH, CRF or GRF. Subsequently, the concentrations of IR-7B2, IR-LH, IR-FSH, and IR-ACTH released into the medium were quantified by specific RIA. LH-RH, at a concentration as low as 7.5 ng/ml (6 X 10(9) M: dose range 7.5-60 ng/ml) stimulated the release of IR-7B2, IR-LH, and IR-FSH, by 2- to 3-fold, 17- to 18-fold, and 3-fold, respectively, over basal levels. No significant increase of IR-7B2 was observed when stimulated by CRF or GRF at doses as high as 100 ng/ml. In addition, K+ (50 mM) stimulated the release of all the peptides measured. In conclusion, our studies suggest that the novel peptide 7B2 is under LH-RH control and indirectly confirm the immunohistochemical results of its cellular co-localization in FSH and LH cells.
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PMID:Specific release of a novel pituitary polypeptide, 7B2, from rat anterior pituitary cells in vitro by luteinizing hormone-releasing hormone. 310 Sep 76

The effects of growing of Sertoli cells isolated from rat seminiferous epithelium by a modified procedure, and responses of these cells to dBcAMP or FSH stimulation was estimated using morphological methods. The modified isolation procedure included repeated mechanical rinsing of tubule pieces with a modified EDTA containing Hanks medium. Moreover, streptornase instead DNase was added to the trypsine containing medium and this resulted in a better dispersion of tubule components. The culture conditions remained unmodified. A high degree homogeneity of the cultured cell population and an evident reactivity of these cells to dBcAMP and FSH was achieved. Furthermore, an observation of giant cells, visible in monolayer among the typical Sertoli cells, is discussed in this paper. Contrary to small number of myoid cells, being survived in the culture, these giant cells did not show positive reaction to alkaline phosphatase.
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PMID:Culture of rat Sertoli cells isolated with a modified procedure. Morphological identification of cell population and cell reactivity. 627 74

A cell culture system was developed to study the function of porcine granulosa cells from primary and secondary follicles. Primary follicles were isolated from 1- to 3-day-old pigs. Secondary follicles were isolated from 50- to 60-day-old pigs. Follicles were isolated after a digestion for 15 min with 0.25% trypsin followed by 15 min with 1000 U DNAase. Follicles were plated at 100 primary follicles or 30 secondary follicles per well in 48-well plates and cultured in media containing 10% fetal bovine serum (FBS). During initial plating, follicles attached to the plate and cells spread from the point of attachment. This resulted in monolayer cultures of granulosa cells from primary or secondary follicles. On day 4 of culture, media were replaced with 0.5 ml media containing one of the following treatments: control (media only); 10% FBS; 100 ng FSH; 2 mmol 8-bromo-cAMP l-1 or 50 ng epidermal growth factor (EGF). Media and cells were harvested on day 6, after 2 days of treatment. FBS and EGF increased DNA in granulosa cell cultures from primary or secondary follicles (P < 0.01). Treatment with 8-bromo-cAMP increased DNA in granulosa cell cultures from primary but not from secondary follicles (P < 0.05). Conversely, treatment with FSH increased DNA in granulosa cell cultures from secondary but not from primary follicles (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Response of porcine granulosa cells isolated from primary and secondary follicles to FSH, 8-bromo-cAMP and epidermal growth factor in vitro. 810 42

To study the potential intragonadal role of inhibin and inhibin-related proteins in the developing gonad, a method was developed to culture testicular cells of chicken embryos. A single-step collagenase/DNase digestion was used to disperse the cells. Except for the primordial germ cells and the erythrocytes, the cells attached well to plastic culture dishes. Moreover, they could easily be grown in the absence of serum or other additives. Inhibin secretion was measured using a heterologous radioimmunoassay validated for use in this species. The fetal testicular cells secreted high amounts of immunoactive inhibin and remained responsive to gonadotrophins. Two different cell populations could be recognized in monolayers of testicular cells: the first population had a fibroblast-like stromal appearance, resembling interstitial cells; the second had an epitheloid appearance and contained large numbers of refractile vacuoles, resembling Sertoli cells. Both cell populations were enriched using a Percoll density gradient. The epitheloid cells displayed a higher capacity to secrete immunoactive inhibin, while the stromal cells were responsible for the bulk of androgen secretion. FSH, but also LH, stimulated inhibin secretion in the epitheloid cells. Although ovine LH was the most potent stimulus for androgen secretion by the stromal cells, ovine FSH was also capable of increasing androgen output in stromal cells and to a lesser extent in epitheloid cells. As the two enriched cell populations were still contaminated by other cell types, this may indicate, as in other species, that FSH-induced paracrine factors are involved in the regulation of androgen secretion in the developing gonad.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Gonadotrophin-regulated production of immunoactive inhibin and androgen by cultured testicular cells from chicken embryos. 853 24

Initial studies in our laboratory demonstrated that a large proportion of domestic dog advanced preantral (APAN) and early antral (EAN) follicles contained grown oocytes that had acquired the dense cytoplasmic lipid characteristic of preovulatory oocytes. The objective of this study was to assess nuclear maturation of those oocytes after in vitro culture. Both APAN and EAN follicles (152 to 886 microns in diameter) were isolated from ovaries by treatment with collagenase and DNase. The follicles were cultured in Dulbecco's Modified Eagle's medium/nutrient mixture F-12 Ham culture medium supplemented with 20% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 1% (v/v) antibiotic-antimycotic, 1 microgram FSH/ml, 10 IU hCG/ml and 1 microgram estradiol/ml. Within each group (APAN or EAN), control follicles were not cultured (0 h), and 2 to 12 follicles per well were incubated under a humidified atmosphere of 5% CO2 in air at 37 degrees C for 24, 48 or 72 h. After 24 h of culture, significantly more (5.3%, 20/374; P < 0.05) oocytes from APAN follicles reached the metaphase I to metaphase II stages (MI to MII) than the percentage of control follicles observed at 0 h (0.9%, 3/318). Continued culture resulted in a further increase (P < 0.05) in the percentage of oocytes reaching MI to MII by 48 h (11.5%, 47/407), which remained unchanged at 72 h (9.9%, 40/404). The percentage of oocytes from EAN follicles reaching MI to MII did not significantly increase after 24 h of culture. However, there was an increase (P < 0.05) by 48 h of culture (8.7%, 11/126), which remained unchanged at 72 h (7.5%, 8/106). These results show that dog oocytes cultured within advanced preantral and early antral follicles in vitro are competent to resume meiosis to the metaphase stage.
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PMID:In vitro maturation of domestic dog oocytes cultured in advanced preantral and early antral follicles. 1073 1

The in vitro growth and developmental pattern of caprine preantral follicles cultured in agar gel was observed. Preantral follicles 50 to 150 microm in diameter were isolated from prepuberal goat ovaries by treatment with collagenase and DNase. The isolated preantral follicles were cultured in agar gel for up to 14 days. A group of 10 follicles in different developmental stages was cultured in a culture well coated with 0.6% agar gel and filled with DMEM medium supplemented with FCS (10%), hypoxanthine (2 mmol/mL), dbcAMP (2 mmol/mL), FSH (100 ng/mL), insulin-transferrin-selenium (ITS) (50 ng/mL), IGF-1 (50 ng/mL), hydrocortisone (40 ng/mL) and antibiotics. Follicle viability was determined under an inverted phase-contrast microscope according to morphological and histological criteria, and follicle growth was assessed by their size and appearance. The results showed that the three-dimensional structures and forms of follicles were basically maintained intact during culture. Primary follicles developed into secondary follicles and a few of them into antral follicles. A large portion of secondary follicles entered the antral stage, and oocytes also acquired growth. The formation of theca lamina and zona pellucida was observed. The survival capacity of secondary follicles was greater than primary follicles. The survival rates for primary and secondary follicles were 11.36% (5/44) and 71.16% (53/74), respectively. During in vitro development the follicles demonstrated dominance. This experiment revealed the preliminary characteristics of the in vitro development of caprine preantral follicles.
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PMID:In vitro development of caprine ovarian preantral follicles. 1107 Nov 38

Activin has been previously demonstrated to directly stimulate the synthesis of GnRH receptors and to increase FSH secretion in non-human pituitary cell cultures (PCC). Several results in Macaque monkeys failed to support an unequivocal role for Inhibin in FSH suppression. Whereas the bioactivity of Inhibin and Activin has been demonstrated in rat PCC, no data exist on human pituitary response to these peptides. We studied, therefore, the secretion of FSH and LH by dispersed human fetal PCC from > 140 midtrimester abortions in response to recombinant human (rh-) Activin-A, Inhibin, and other secretagogues. After mechanical and enzymatic dispersion, using collagenase and deoxyribonuclease, the human fetal pituitary cells were cullured on extracellular matrix (ECM) like material coaled 24 well plate (Primaria, Falcon) in fetal calf serum-containing medium. After 3 days incubation in serum-containing medium, the PCC were washed and preincubated for 90 mins in serum-free medium and incubated with rh-ActivinA, Inhibin, TGF-p, Follistatin, sex steroids, and GnRH in quadruplicate wells. The EC50 of rh-Activin-A for FSH secretion was ~ 10 ng/mL. rh- Activin-A was a more potent secretagogue for FSH secretion than GnRH. On the contrary, GnRH (20 ng/mL) was more potent than rh-Activin A for LH secretion. Nevertheless, a significant increase in LH secretion into the medium was brought about by rh-Activin-A. Inhibin decreased FSH secretion but LH response to Inhibin was inconsistent. GnRH opposed the inhibitory effect of Inhibin on both gonadotropins. In dynamic, short term, repetitive exposure of fetal pituitary fragments to rh-Activin-A (superfusionl we could not receive -a similar increase in LH & FSH as in static incubations, as opposed to a short GnRH exposure. Melatonin did not inhibit LH secretion in human PCC as opposed to rodents. In addition to their endocrine, paracrine, and sutocrine effects and to their role as possible markers, the TGF-b superfamily members may atiect embryogenesis and possibly immunomodulation of the fetus. In contrast to others, who could detect Inhibin-B only in male but not in female fetuses sera, we have measured Inhibin-B in both male and female midtrimester fetal sera, challenging the previous assumption that the fetal origin is only Sertoli cells. Human fetal PCC express the previously reported physiologic responses to Activin and Inhibin generated in non-human experiments on gonadotropin secretion in-vitro, and may serve as a physiologic model for studying human gonadotrope responses to the TGF-b family of peptides. Our preliminary data may provide the first unequivocal evidence for the validity of the Activin/Inhibin hypothesis in human.
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PMID:Response of human fetal pituitary cells to activin, inhibin, hypophysiotropic and neuroregulatory factors in vitro. 1175 7

The number of female germ cells in pig fetuses decreases by 70% between day 50 after mating and day 300 after birth. Approximately 55% of antral follicles undergo degeneration (atresia) except during the 3 days before oestrus, when only 15% of the follicles survive to ovulate. Apoptosis, a form of programmed cell death, is recognized as the mechanism of germ cell death and follicle atresia at all stages of folliculogenesis. The internucleosomal cleavage of genomic DNA caused by caspase-induced deoxyribonuclease activity was measured in pig granulosa cells by DNA fluorescence flow cytometry, densitometry of fluorescently labelled internucleosomal DNA fragments and immunohistochemical analysis of the 3' end labelling of deoxyribonuclease-nicked DNA on frozen tissue sections. Follicular atresia during the 3 days before oestrus is associated with a 60-70% decrease in the secretion of FSH. In granulosa cells, apoptosis is associated with decreased cell proliferation and reduced production of oestradiol and inhibin. In cultured pig granulosa cells, FSH and IGF-I are anti-apoptotic and a caspase inhibitor blocked apoptosis, thereby providing evidence of caspase activity. Oocytes in most follicles have resumed meiotic maturation; therefore, one role for apoptosis and follicle atresia may be to act as a barrier to ovulation of oocytes that have not remained in meiotic arrest.
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PMID:Apoptosis during folliculogenesis in pigs. 1198 Jan 88

1. To correlate the morphological observations with the known gonadotropic activity of FSH in the turtle testis, studies of the binding of iodinated FSH were conducted. 2. These demonstrated the presence of gonadotropin-binding sites of high affinity (apparent Kd = 10(-10) M) for [125I]rFSH in turtle testicular membrane preparations. 3. Although these sites did not bind iodinated human LH or avian LH, these hormones, as well as PMSG and FSH, were effective competitive inhibitors of the binding of the radioligand. 4. Binding of the radioligand to the testis was influenced by duration of incubation and temperature. 5. Binding activity was lost after incubation with proteolytic enzymes (trypsin, pronase) but not with DNAase, lipase, collagenase and neuraminidase. 6. The binding exhibited target organ specificity (no binding observed in brain, epididymis, lung, muscle and pancreas). 7. In addition, the number of binding sites varied according to the stage spermatogenesis, being highest when the tubules contained spermatocytes and spermatids, intermediate when the tubules consisted to Sertoli cells and spermatogonia and lowest st spermiation.
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PMID:Characterization of the testicular binding site for iodinated rat FSH in the turtle, Chrysemys picta. 1457 99

Expression of the FSH receptor (Fshr) is restricted to testicular Sertoli cells and ovarian granulosa cells, thereby limiting the direct targets of FSH action to these somatic cells of the gonads. Earlier studies indicate that transcription of Fshr in the gonads requires elements outside the gene's immediate 5' flanking sequence. To help uncover candidate regulatory sequences, comparative genomics and deoxyribonuclease I hypersensitivity mapping were employed. A total of 156 evolutionarily conserved sequences were found, and partial deoxyribonuclease I hypersensitivity mapping across 45 kb of 5' flanking sequence and the first intron identified four hypersensitive sites, DHS1-4. Notably, DHS1 and DHS2 localized to conserved sites in the promoter region and exon 1 and correlated with the active state of the gene. DHS3 also corresponded to a conserved site (site 7) but was more pronounced in nonexpressing myoid cells, suggesting a role in gene silencing. Transient transfection analysis of DHS3 confirmed its role in gene silencing, a function that was promoter, cell type, and position dependent. Protein-DNA binding studies on DHS3 revealed that octamer transcription factor 1 (OCT-1) and GATA-4 bound site 7, in vitro, and transient transfection analysis showed that their binding sites were required for silencing activity. Furthermore, chromatin immunoprecipitation revealed that OCT-1 bound to site 7 in the endogenous gene, but only in myoid cells. In contrast, GATA-1 bound site 7 predominantly in Sertoli cells, suggesting that it attenuates silencer activity. The findings reveal that OCT-1 binds within DHS3 to silence Fshr transcription and implicate members of the GATA family in the modulation of this activity.
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PMID:Silencing of Fshr occurs through a conserved, hypersensitive site in the first intron. 1581 54


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