54 IMMUNOLOCALIZATION OF STEROID SULFATASE AND ESTROGEN-SPECIFIC SULFOTRANSFERASE IN BOVINE FOLLICLES

2016 ◽  
Vol 28 (2) ◽  
pp. 157 ◽  
Author(s):  
C. Blaschka ◽  
G. Schuler ◽  
C. Wrenzycki
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Protein Level ◽  
Organic Anion ◽  
Steroid Sulfatase ◽  
Oestrus Cycle ◽  
Polyclonal Rabbit ◽  
Anion Transporter ◽  
Morphological Criteria ◽  
Sulfated Steroids

Steroid hormones are regulators in the fine-tuned mechanism of follicular development in cattle. Their concentration and property can be modulated via different processes. Sulfoconjugation via sulfotransferases (SULT) changes them from being hydrophobic to hydrophilic molecules, thereby preventing them from diffusing freely across the lipid bilayer and necessitating a transport system like the sodium-dependent organic anion transporter (SOAT; SLC10A6). In addition, sulfated steroids can no longer bind to their nuclear receptors, rendering them biologically inactive. Steroid sulfatase (STS) removes the sulfate moiety from conjugated steroids, transforming them to the free active forms. Data about the concentration of sulfated steroids in follicular fluid and the expression of the related enzymes are limited in horses and cattle. Recently, transcripts of the corresponding enzymes have been detected in cumulus cells of immature bovine cumulus-oocyte complex. Furthermore, it has been shown that small amounts of sulphated steroids are present in bovine follicular fluid. The objective of the study was to investigate the steroid metabolizing enzymes at the protein level via immunohistochemistry (IHC). Bovine ovaries collected at a slaughterhouse were categorized according to their oestrus cycle stage. Three pairs of ovaries of each cycle stage (proestrus, oestrus, postestrus, interestrus) were collected. Morphological criteria (ovaries: presence/absence of follicles and/or CL, size and number of follicles; closure of the cervix, amount of mucus) were employed to categorize them. Samples for IHC were fixed in formalin. After washing and dehydration, the samples were finally embedded in paraffin and mounted onto blocks. Indirect immunoperoxidase staining methods were applied using the streptavidin-biotin technique for signal enhancement following standard procedures. Tissue sections of 3 µm were mounted onto glass slides, and a polyclonal rabbit anti-human STS and a polyclonal rabbit anti-bovine SULT1E1 primary antibody were used to detect STS or SULT1E1, respectively. Negative controls were included using nonspecific rabbit IgG. The evaluation of the staining was descriptive in relation to a positive control (bovine placenta). Immunostaining for STS was detected in granulosa cells of antral and secondary follicles and in the endothelium of blood vessels, irrespective of the stage of the oestrus cycle. Moreover, staining for SULT1E1 was restricted to granulosa cells in antral follicles, again irrespective of the stage of the oestrus cycle. However, staining intensity for STS and SULT1E1 was only weak. These data indicate for the first time the presence of the steroid metabolising enzymes STS and SULT1E1 in bovine ovaries at the protein level. However, due to the low expression level, further studies are needed to clarify the function. We gratefully acknowledge the financial support of the German Research Foundation (DFG; FOR 1369, WR 154/3–1).

275 TEMPORAL PATTERN OF STEROID HORMONE CONCENTRATIONS DURING IN VIVO AND IN VITRO MATURATION OF BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 226 ◽  
Author(s):  
C. Blaschka ◽  
H. Stinshoff ◽  
F. Poppicht ◽  
C. Wrenzycki
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Steroid Hormone ◽  
Organic Anion ◽  
Hormone Concentration ◽  
Cholesterol Sulfate ◽  
Anion Transporter ◽  
Sulfated Steroids

Steroid hormone concentration and property can be modulated via different processes. Sulfoconjugation via sulfotransferases (SULT) changes steroids from hydrophobic to hydrophilic, necessitating a transport system such as the sodium-dependent organic anion transporter (SOAT; SLC10A6). Steroid sulfatase (STS) removes the sulfate moiety from conjugated steroids, transforming them to the free active ones. Moreover, present in vitro maturation systems do not completely mimic the in vivo situation resulting in oocytes of reduced quality. The present study investigates the local effects of sulfated steroids during follicular and oocyte development in vivo and in vitro. Follicles of bovine abattoir-derived ovaries were categorized according to their size (3 to 5, 6 to 8, 9 to 14, and >15 mm) after dissection and measurement via a caliper. Only nonatretic follicles were used (Kruip and Dieleman, 1982). Follicular fluid was collected via aspiration and analysed for the presence of steroids and their sulfated counterparts via LC-MS/MS. Moreover, oocytes were in vitro maturated with a standard protocol. The medium was measured via radioimmunoassay after 0, 4, 8, 12, 16, 20, and 24 h to detect 17β-oestradiol (E2) and progesterone (P4). Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey's test. A P-value of ≤0.05 was considered significant. It was possible to detect 17β-oestradiol, progesterone, testosterone, 17β-oestradiol sulfate, estrone sulfate, pregnenolone sulfate, cholesterol sulfate (Table 1), and furthermore androstendione, estrone, androsterone, and 17OH-pregnenolone. During IVM, P4 significantly increased in the medium (4 h: 3.3 ± 1.0 ng mL–1; 24 h: 9.8 ± 1.7 ng mL–1), whereas the E2 concentration did not change (4 h: 52.8 ± 12.1 pg mL–1; 12 h: 68.4 ± 3.7 pg mL–1; 24 h: 66.9 ± 19.7 pg mL–1). In addition, preliminary data suggest that transcripts of the steroid metabolizing and transporting enzymes (SULT1E1, STS, SLC10A6) were present in cumulus cells from immature bovine COC. These results indicate for the first time that only small amounts of sulfated steroids are present in bovine follicular fluid. However, the related enzymes are present at the mRNA level. Further studies are underway to analyse the protein level. Table 1.Steroid hormone concentration in follicular fluid We thank Prof. Dr Wudy and A. Sánchez Guijo for the LC-MS/MS analysis. Furthermore, we gratefully acknowledge the financial support of the German Research Foundation (DFG; FOR 1369).

Characterization of inducible nature of MRP3 in rat liver

2000 ◽  
Vol 278 (3) ◽  
pp. G438-G446 ◽  
Author(s):  
Kotaro Ogawa ◽  
Hiroshi Suzuki ◽  
Tomoko Hirohashi ◽  
Toshihisa Ishikawa ◽  
Peter J. Meier ◽  
...  
Keyword(s):  
Bile Duct ◽  
Common Bile Duct ◽  
Protein Level ◽  
Bile Duct Ligation ◽  
Organic Anion ◽  
Mrna Level ◽  
Common Bile Duct Ligation ◽  
Sd Rats ◽  
Anion Transporter ◽  
Duct Ligation

We found previously that expression of multidrug resistance-associated protein (MRP) 3 is induced in a mutant rat strain (Eisai hyperbilirubinemic rats) whose canalicular multispecific organic anion transporter (cMOAT/MRP2) function is hereditarily defective and in normal Sprague-Dawley (SD) rats after ligation of the common bile duct. In the present study, the inducible nature of MRP3 was examined, using Northern and Western blot analyses, in comparison with that of other secondary active [Na+-taurocholic acid cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (oatp1), and organic cation transporter (OCT1)] and primary active [P-glycoprotein (P-gp), cMOAT/MRP2, and MRP6] transporters. α-Naphthylisothiocyanate treatment and common bile duct ligation induced expression of P-gp and MRP3, whereas expression of Ntcp, oatp1, and OCT1 was reduced by the same treatment. Although expression of MRP3 was also induced by administration of phenobarbital, that of cMOAT/MRP2, MRP1, and MRP6 was not affected by any of these treatments. Moreover, the mRNA level of MRP3, but not that of P-gp, was increased in SD rats after administration of bilirubin and in Gunn rats whose hepatic bilirubin concentration is elevated because of a defect in the expression of UDP-glucuronosyl transferase. However, the MRP3 protein level was not affected by bilirubin administration. Although the increased MRP3 mRNA level was associated with the increased concentration of bilirubin and/or its glucuronides in mutant rats and in SD rats that had undergone common bile duct ligation or α-naphthylisothiocyanate treatment, we must assume that factor(s) other than these physiological substances are also involved in the increased protein level of MRP3.

scholarly journals The effect of cellular and physiological indicators on gender determination

2021 ◽  
Vol 67 (3) ◽  
pp. 69-75
Author(s):  
Milat Ismail Haje
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Protein Level ◽  
Physiological Indicators ◽  
Gender Determination ◽  
Significant Difference ◽  
Special Value ◽  
Follicle Size ◽  
Infertile Patients

Gender determination, in addition to having special value to parents, has particular importance in sex-linked diseases. This study aimed to investigate the cellular indicators (i.e. BMP-6 protein and PPAR? protein expression levels in granulosa cells) and the physiological indicators on gender determination. For this purpose, on 68 infertile patients referred to the clinic, ovarian stimulation was performed by different protocols and then ruptured by different HCG. Follow-up of patients was performed after they became pregnant after five months. U/S was done for knowing the gender of the baby then after labor rechecked another time. Also, granulosa-luteal cells (GLCs) were isolated from the follicular fluid of 68 women participating in the study. BMP-6 protein and PPAR? protein were measured using Western blotting. Results showed that the total number of delivered babies was 68, 41 males (60.3%) and 27 females (39.7%). About physiological indicators results, there was no significant association between the age of the mother and sex of the baby (P=0.934). No significant association was detected between the month during which the conception occurred and the sex of the baby (P=0.734). The same result was obtained for the follicle side (P=0.236), and follicle size (P=0.659), there was no significant association between the sex of the baby with the following factors: protocol of treatment (P=0.417), IVF after HCG (P=0.237), HCG type (P=0.572), parity (P=0.282), and type of infertility (P=0.376). The cellular indicators results showed that the BMP-6 protein level in granulosa cells of mothers with daughters was almost twice as high as mothers with sons (P=0.043). But there was no significant difference between mothers with daughters and mothers with sons in PPAR? protein level (P=0.12). It can be concluded that except for BMP-6 protein level, none of the cellular and physiological indicators affects gender determination. Therefore, this cell indicator can probably be evaluated as an effective indicator in determining gender.

Butyryl- and acetylcholine-esterases in human IVF-derived follicular fluid and luteinizing granulosa cells

2013 ◽  
Vol 121 (03) ◽  
Author(s):  
J Blohberger ◽  
D Einwang ◽  
D Berg ◽  
U Berg ◽  
S Hecht ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Human Ivf

Characterization of hepatobiliary organic anion transporter regulation in the Zucker rat

2004 ◽  
Vol 42 (08) ◽  
Author(s):  
A Geier ◽  
CG Dietrich ◽  
C Gartung ◽  
F Lammert ◽  
HE Wasmuth ◽  
...  
Keyword(s):  
Organic Anion ◽  
Organic Anion Transporter ◽  
Zucker Rat ◽  
Anion Transporter

МОДЕЛИРОВАНИЕ СИСТЕМ ЭКСТРАКОРПОРАЛЬНОГО СОЗРЕВАНИЯ ООЦИТОВ КРУПНОГО РОГАТОГО СКОТА С ИСПОЛЬЗОВАНИЕМ СТРУКТУРНЫХ КОМПОНЕНТОВ ОВАРИАЛЬНЫХ ФОЛЛИКУЛОВ *

2019 ◽  
pp. 20-22
Author(s):  
T.I. KUZMINA ◽  
I.V. CHISTYAKOVA
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Bovine Serum ◽  
Egg Quality ◽  
Fetal Bovine Serum ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cultural Systems ◽  
Fetal Bovine

Создание эффективной унифицированной системы дозревания донорских ооцитов обеспечит повышение результативности инновационных клеточных репродуктивных технологий. В исследовании проведен сравнительный мониторинг показателеймейотического созревания ооцитов коров, созревших в различных системах, дополненных структурными компонентами фолликулов (СКФ стенки фолликулов, клетки гранулезы, белки) и фолликулярной жидкостью,а также потенций к развитию из них доимплантационных эмбрионов. Анализу подверглись ооциты, прокультивированные в следующих системах:среда ТС199 с добавлением 10 фетальной бычьей сыворотки (ФБС), 50 мкг/мл эстрадиола, 10 мкг/мл лютеинизирующего гормона (ЛГ), 10 мкг/мл фолликулостимулирующего гормона (ФСГ) среда ТС199 с 10 эстральной сывороткой коров среда ТС199 с 50 жидкости из фолликулов диаметром 9 мм среда ТС199 с добавлением белков фолликулярной жидкости молекулярной массой 65 кДасреда ТС199 с 10 ФБС и 1106 клеток гранулезы среда ТС199 с 10 ФБС и тканью фолликула. В культуральные среды ко всем исследованным группам ооцитов добавляли антибиотики. Использование CКФ обеспечило значительное снижение доли ооцитов с дегенерированным хроматином, что способствовало увеличению уровня доимпланационных эмбрионов на стадии бластоцисты. Так, доля бластоцист, развившихся из ооцитов, созревших в среде со стенками фолликулов,составила43,5. В этой же группе выявлен минимальный уровень дегенерированных зародышей (6,45). Полученные данные предлагается использовать при моделировании систем дозревания ооцитов коров с целью повышения качества яйцеклеток.The creation of an effective unified maturation system of donor oocytes provides an increase in the efficiency of innovative cellular reproductive technologies. The comparative analysis of the meiotic maturation indicators of bovine oocytes, which were matured in different cultural systems modified by follicular structural components (FSC follicular walls, granulosa cells, proteins) and follicular fluid, as well as the potential for preimplantation embryonic development were evaluated in this study. Oocytes matured in following cultural systems: medium TC199 supplemented with 10 fetal bovine serum and 50 g/ml of estradiol, 10 g/ml of luteinizing hormone (LH), 10 g/ml of folliclestimulating hormone (FSH) medium TC199 with 10 estrous cow serum medium TC199 with 50 liquid from follicles with a diameter of 9 mm medium TC199 supplemented with the follicular fluid proteins with molecular weight 65 kDa medium TC199 with 10 fetal bovine serum and 1106 granulosa cells medium TC199 with the addition of 10 fetal bovine serum and follicle tissues were analyzed. Antibiotics were added to cultural media of all experimental groups of oocytes. The usage of FSC ensured the decrease in the proportion of oocytes with degenerated chromatin, which contribute the rise of the level of preimplantation embryos at the blastocyst stage. Thus, the proportion of blastocysts developed from oocytes matured in medium supplemented with follicular walls was 43.5. In the same experimental group, the number of degenerated embryos was 6.45. The obtained data are supposed to be used for modeling the cultural systems of cow oocytes in order to improve the egg quality.

PCOS follicular fluid derived exosomal miR-424-5p induces granulosa cells senescence by targeting CDCA4 expression

2021 ◽  
pp. 110030
Author(s):  
Dong Yuan ◽  
Jing Luo ◽  
Yixuan Sun ◽  
Lijuan Hao ◽  
Jing Zheng ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid
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