Reciprocal epithelial:endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation

The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by >90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation.     

Inhibition of protein synthesis in a polyribosome-dependent cell-free system by a specific ribonuclease prepared from the follicles of two different stages of development of the silkmothAntherea pernyi

Summary Crude, cell-free protein-synthesizing systems were prepared from follicles of two different stages of development in the ovariole of the silkmothAntherea pernyi. The efficiency of the translation of natural and synthetic mRNAs in these systems was compared with that in a cell-free wheat germ system. A postmitochondrial extract (S-30) from the follicles almost completely inhibited protein synthesis in a polyribosome-dependent, cell-free systems. A specific ribonuclease, obtained from the post mitochondrial extract by ammonium sulphate precipitation, heat denaturation and DEAE-cellulose chromatography, inhibited polyribosome-dependent protein synthesis. The effect of this specific ribonuclease on the structural integrity of radioactive RNAs and ribosomal subunits, which were isolated from Ehrlich ascites tumor cells, was also studied.     

超声成像在扬子鳄卵巢等器官形态检测中的应用

扬子鳄(Alligator sinensis)是中国特有的珍稀物种,该物种的生长、繁殖状况一直备受关注,分别在2014年3月(冬眠期)和6月(繁殖期)对17条成年雌性扬子鳄腹部应用VIVIDⅠ型彩色多普勒超声(B型超声)便携诊断仪进行检测,并对3月份卵巢卵泡大小用SPSS 19.0回归分析。扬子鳄心、肝、肠等内脏器官形态在B型超声诊断仪下清晰可见。冬眠期17条扬子鳄两侧卵巢中共检测到41枚卵泡,繁殖期共42枚。冬眠期卵泡处于低回声暗区或无回声暗区的未成熟状态,繁殖期多数卵泡的发育状态与3月相同,少数卵泡呈高回声暗区,此时已形成卵黄颗粒,也有呈高回声亮光区形成卵黄膜的卵泡。对3月份检测的17条扬子鳄具有卵泡的19个卵巢大小与卵泡大小进行回归分析,卵巢随卵泡发育而增大。本研究探讨了B超这种低损伤方法对扬子鳄检测的有效性,同时评估卵泡发育状况,提高繁殖鳄的筛选正确率。     

Antioxidant capacity of follicular fluid in relation to follicular size and stage of estrous cycle in buffaloes

The present study was designed to evaluate the changes in the concentrations of different antioxidants, such as glutathione (GSH), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT), in the follicular fluid collected from different follicular size categories in relation to stage of estrous cycle in buffaloes. In addition, malondialdehyde (MDA) as an indicator for lipid peroxidation was also estimated. Fifty pairs of buffalo ovaries were collected from a local slaughterhouse. Based on ovarian structures, the cycle was divided into follicular and luteal phase. The follicles on each pair were classified into three groups; small (≤3 mm), medium (4-9 mm) and large (≥10 mm). The concentrations of SOD, CAT, GSH, and GR in the follicular fluid of each group as well as MDA were estimated. Results indicated that there was a significant decrease (P < 0.05) in the average numbers of small follicles obtained at the follicular phase than those obtained at the luteal phase of the cycle. However, the mean numbers of the large sized follicles was significantly increased (P < 0.05) in the follicular phase than in the luteal phase. Large follicles obtained at the luteal phase had a significantly higher (P < 0.05) concentration of GSH than that obtained from small ones. A significant (P < 0.05) effect of follicular size on GR concentrations was observed. The concentration of SOD tended to be higher in large follicles obtained at the follicular phase than that collected at the luteal phase (56.7 ± 3.7 vs. 28.1 ± 6.7 U/mL, respectively). On the contrary, a significantly higher concentration (P < 0.05) of SOD was recorded in small follicles as compared with medium and large follicles collected at the luteal phase. CAT concentrations did not significantly differ among different follicular sizes between follicular and luteal phases as well as within each phase. Malondialdehyde concentration was significantly decreased (P < 0.05) in the follicular fluid obtained from small follicles collected at the follicular phase compared with those obtained at the luteal phase. In conclusion, the present study showed that the concentrations of enzymatic antioxidants except for CAT vary according to the follicle size and the stage of the estrous cycle suggesting their possible role in the process of follicular development during estrous cycle in buffaloes.     

Cryopreservation of human ovarian tissue: comparison of rapid and conventional freezing

Cryopreservation, which is the most important procedure in ovarian tissue banking, can be divided into two methods: conventional freezing and rapid freezing. In previous study, the higher effectiveness of rapid freezing in comparison with the conventional freezing for human oocytes and embryos was shown. Data on comparison of these two methods for human ovarian tissue are limited. The aim of this study was to compare conventional freezing and rapid freezing for human ovarian tissue. Ovarian tissue fragments from 14 patients were transported to the laboratory within 22–25 h in a special, isolated transport box, which can maintain a stable temperature of between 5 and 8 °C for 36 h. Small pieces of ovarian tissue (1 × 1–1.5 × 0.7–1 mm) were randomly distributed into four groups: Group 1: control, fresh pieces immediately after receiving transport box, Groups 2 and 3: experimental pieces after rapid freezing/warming, and Group 4: experimental pieces after conventional freezing/thawing. All pieces were cultured in vitro for 14 days. The viability of the tissue by in vitro production of hormones and development of follicles after culture was evaluated. The level of estradiol 17-β and progesterone was measured using heterogeneous competitive magnetic separation immunoassay. For histological analysis, the number of viable and damaged follicles was counted. After culture of fresh tissue pieces (Group 1), rapidly frozen/warmed pieces (Groups 2 and 3), and conventionally frozen/thawed pieces (Group 4), the supernatants showed estradiol 17-β concentrations of 358, 275, 331, and 345 pg/ml, respectively, and progesterone concentrations of 3.02, 1.77, 1.99, and 2.01 ng/ml, respectively. It was detected that 96%, 36%, 39%, and 84% follicles for Groups 1, 2, 3, and 4, respectively, were normal. For cryopreservation of human ovarian tissue, conventional freezing is more promising than rapid freezing.     

Osmotic tolerance and freezability of isolated caprine early-staged follicles

Isolated caprine early-staged follicles were submitted to osmotic tolerance tests in the presence of sucrose, ethylene glycol (EG), or NaCl solutions and were exposed to and cryopreserved (by slow or rapid cooling) in MEM alone or MEM supplemented with sucrose, EG (1.0 or 4.0 M), or both. When follicles were exposed to 1.5 M NaCl, only 2% of the follicles were viable, whereas 87% of the follicles were viable after exposure to 4.0 M EG. Regarding exposure time, the highest percentage of viable follicles was obtained when follicles were exposed for 10 min to 1.0 M EG + 0.5 M sucrose; exposure for 60 s to 4.0 M EG + 0.5 M sucrose also maintained high percentage viability in follicles. Slow cooling in the presence of 1.0 M EG + 0.5 M sucrose (75%) or rapid cooling in the presence of 4.0 M EG + 0.5 M sucrose (71%) resulted in a significantly higher proportion of viable follicles than all other treatments (P < 0.05). A 24-h culture of frozen-thawed follicles was used to assess survival; only slow-frozen follicles showed viability rates similar to control follicles (64% vs. 69% respectively; P > 0.05). Interestingly, the percentage of viable rapid-cooled follicles (59%) was similar to that obtained after in vitro culture of conventional slow-cooled follicles but was significantly lower than that in controls. Thus, in addition to determining improved procedures for the exposure of follicles to EG and sucrose before and after freezing of caprine early-staged follicles, we report the development of rapid- and slow-cooling protocols.     

Synchronization of ovulation and fertility in weaned sows treated with intravaginal triptorelin is influenced by timing of administration and follicle size

A 100 μg dose of triptorelin was tested for synchronizing ovulation in sows. In Experiment 1, conducted in April through June, sows (n = 125) were assigned to Control (untreated), TG-96 (Triptorelin Gel (TG) given intravaginally at 96 h post-weaning), or TG-E (given intravaginally at estrus). To optimize AI timing, sows were inseminated at 2 and 26 h after estrus for Control and TG-E and at 8 and 32 h following TG-96. Ovulation by 48 h post-treatment tended to be affected by treatment (P = 0.08) and more (P < 0.05) TG-96 sows ovulated (57.9%) compared to Controls (34.2%), but TG-E (45.1%) did not differ (P > 0.10). Duration of estrus was reduced (P < 0.005) in TG-96 (51 h) and TG-E (58 h) compared to Controls (65 h). There was no treatment effect on farrowing rate (71%) or total born (10.4). Average follicle size <6.5 mm at 96 h after weaning was associated with reduced (P < 0.01) estrus, ovulation and farrowing rate. Experiment 2 was conducted in August through September using 503 weaned sows. The TG-96 treatment reduced duration of estrus (P = 0.03), but treatment did not affect estrus expression, farrowing rate or total pigs born. In conclusion, use of a 100 μg dose of triptorelin intravaginally at 96 h or at estrus advanced ovulation and when used with timed insemination, resulted in similar farrowing rates and litter sizes comparable to sows mated based on estrus. However, ovulation induction and timed AI success may benefit from an approach that ensures sows have adequate follicle development at time of treatment.     

Occurrence of colloid-containing follicles in the pars distalis of pituitary glands from aging guinea pigs

Summary Colloid-containing follicles in the pars distalis of pituitary glands from guinea pigs at various ages ranging from 5 days to 36 months were examined by the periodic acid-Schiff (PAS) reaction, immunohistochemistry, and electron microscopy. The follicles storing PAS-positive colloid were first detected in 6-month-old animals, in which only a few follicles were present and mean diameters of colloid deposits were small: 4.3±1.0 m in males and 4.1±0.4 m in females. Thereafter, the follicles gradually increased in number and size with age. The largest number of follicles was observed in the senile groups: 410.5±92.3 in males, 454.7±84.7 in females. Mean diameters of colloid masses in the senile groups were more than 2 times larger than those in 6-month-old animals: 10.0±0.1 m in males, 9.7±0.1 m in females. These findings suggest that the formation of colloidcontaining follicles in the guinea-pig pars distalis is an aging phenomenon. The follicular lumina were mainly surrounded by thin cytoplasmic processes or cell bodies of folliculo-stellate cells immunoreactive for S-100 protein. The lining folliculo-stellate cells showed aggregations of intermediate-sized filaments, numerous lysosomes and colloid-like inclusions. Granulated cells in contact with colloid were occasionally encountered. Intracellular cavities storing colloid-like and fibrous materials were detected in the syncytial formation of GH cells.     

The involvement of hypophyseal‐gonadal and hypophyseal‐adrenal axes in arsenic‐mediated ovarian and uterine toxicity: Modulation by hCG

This study evaluated the involvement of hypophyseal‐gonadal and hypophyseal‐adrenal axes as a possible mechanism of sodium arsenite toxicity in ovary and uterus by the coadministration of hCG. Subchronic treatment of 0.4 ppm of sodium arsenite/(100 g body weight day) via drinking water for seven estrous cycles significantly suppressed the plasma levels of leutinizing hormone, follicle‐stimulating hormone, and estradiol along with sluggish ovarian activities of Δ⁵,3β‐hydroxysteroid dehydrogenase and 17β‐hydroxysteroid dehydrogenase followed by a reduction in gonadal tissue peroxidase activities in mature female rats at diestrous phase. Noticeable weight loss of the ovary and uterus along with prolonged diestrous phase and increased deposition of arsenic in the plasma and in these reproductive organs were also demonstrated following the ingestion of arsenic. Follicular atresia and thinning of the uterine luminal diameter were evident after sodium arsenite treatment. Effective protection of gonadal weight loss, suppressed ovarian steroidogenesis, and altered ovarian and uterine peroxidase activities were noticed when 1.0 IU hCG/(100 g body weight day) is given in arsenic‐intoxicated rats. Normal estrous cyclicity was restored toward the control level after hCG coadministration, though the elimination of elementary arsenic from the plasma and gonadal tissues was impossible. A significant recovery in the restoration of ovarian and uterine histoarchitecture was prominent after hCG treatment. Adrenal hypertrophy and steroidogenic arrest of the adrenal gland along with altered level of brain monoamines in the midbrain and diencephalons following arsenic intoxication were also ameliorated after hCG coadministration. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:29–41, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20309