In vitro maturation and early developmental capacity of bovine oocytes cultured in pure follicular fluid and supplementation with follicular wall.

Mammalian oocytes mature in follicular fluid (FF), surrounded by follicular cells. In the present study, in vitro maturation of bovine oocytes cultured in FF from dominant follicles 15-17mm in diameter (with various forms of heat pretreatment) and supplementation with follicular wall from follicles 3-5mm in diameter (FW1) were examined. Heat pretreatment of FF was as follows: (1) no treatment (FF1); (2) 56 degrees C for 30min (FF2); and (3) 100 degrees C for 20s (FF3). After IVM in FF1, oocytes underwent IVF and IVC and embryo development was assessed (up to the morula stage). The rate of oocyte maturation was decreased in pure FF1 versus control (44.5% versus 62.8%, P<0.001). In the control medium, FW1 did not significantly affect nuclear maturation. By contrast, the addition of FW1 to FF1 increased the rate of matured oocytes approximately two-fold (85.9% versus 45.6%, P<0.001). Furthermore, the maturation rate in the FF+FW1 system declined (from 85.9 to 71.0%, P<0.001), whereas that in the FF system increased (from 45.6 to 71.6%, P<0.001) with increased temperature of the FF treatment. Supplementation of the control medium with FW1 increased the yield of morulae (42.6% versus 13.7%, P<0.001). However, the stimulatory effect of FW1 on the morula rate was much higher in pure FF1 (72.5% versus 31.7%, P<0.001). These findings indicated, for the first time, the stimulatory impact of FW1 on in vitro maturation and early developmental capacity of bovine oocytes cultured in pure FF from dominant follicles. We also inferred that bovine FF constituents affecting bovine oocyte maturation and the meiosis-promoting ability of the FW were heat-labile.

Ovulatory cycle-related alterations in the thecal growth and membrane protein content of thecal tissue of hen preovulatory follicles.

In the hen ovary, each preovulatory follicle in the hierarchy, irrespective of its size and the level of its maturity is exposed to the preovulatory LH surge in each ovulatory cycle of an egg laying sequence. In the present study, the thecal weight and membrane protein content of theca layers at different stages of hen ovulatory cycle were assessed. Hens were killed 2 h (stage I), 9 h (stage II), 16 h (stage III), and 23 h (stage IV) after oviposition. The first (F1), second (F2), third (F3), fourth (F4) and fifth (F5) largest yellow follicles were utilized. In all follicles except F1, the thecal weight rose considerably between stages I and III (P < 0.05) followed by a slight cessation of the thecal growth at stage IV. The mean content of the theca membrane protein in F1-F5 follicles was lowest at stage III, increasing at stage IV (P < 0.05), although, in the case of individual follicles the difference was significant (P < 0.05) in F3 follicles only. Estradiol-17beta levels in the plasma were lowest (but not significant) at stage III, and a fourfold increase in the plasma progesterone concentration occurred at stage IV. These findings demonstrate for the first time the ovulatory cycle-related alterations in the thecal weight and membrane protein content in the hen preovulatory follicles. Data suggest that the preovulatory rise in ovarian steroid hormones is probably involved in transient termination of the growth and induction of differentiation of the theca in preovulatory follicles as they pass from one category to the next.

Characterization of growth hormone binding sites in granulosa and theca layers at different stages of follicular maturation and ovulatory cycle in the domestic hen.

The currently available evidence points to a possible influence of growth hormone (GH) on avian folliculogenesis, which can be mediated by both hepatic- and ovarian-derived IGF-I. Therefore, the purpose of the present study was to reveal GH-binding sites in granulosa and theca layers of preovulatory follicles and to determine the binding characteristics depending on the degree of follicular maturation and the stage of the ovulatory cycle in the hen. Hens were killed 2 h (stage I), 9 h (stage II), 16 h (stage III), and 23 h (stage IV) after oviposition, and the five largest yellow follicles (from F1 to F5) were removed. GH-binding sites in granulosa and theca layers from F1 to F5 follicles were characterized using a radioreceptor assay. Equilibrium dissociation constants (K(d)) and binding capacities (B(max)) were determined by Scatchard analysis of saturation curves, which revealed a single class of high-affinity GH-binding sites in both theca tissue and granulosa cells. In F1, F2, and F5 follicles, B(max) and K(d) for GH-binding sites in the granulosa layer changed during the ovulatory cycle, decreasing between stages I and III, to increase again at stage IV, with alterations in K(d) being less profound. No significant differences in binding capacities and affinities of GH-binding sites in the theca layer were found between various stages of the cycle. Furthermore, the concentration of GH-binding sites in the granulosa layer rose, whereas that in the theca layer fell with follicular enlargement. These findings indicate the presence of high-affinity GH-binding sites in both granulosa and theca layers of hen preovulatory follicles. Data also demonstrate that GH-binding sites in these tissues are regulated in a tissue-specific manner. Furthermore, the regulation of binding capacity of GH binding in granulosa cells by hormonal factors associated with ovulatory cycle is apparently not dependent on the state of follicular maturation.