Paul V. Holmes

Significance of the Number of Embryonic Cells and the State of the Zona Pellucida for Hatching of Mouse Blastocysts In Vitro Versus In Vivo

Abstract We investigated the course of mouse blastocyst hatching in vitro after experimental modulation of the hatching process by growth hormone or by laser treatment and compared it to embryos grown in vivo. When embryos were grown in vitro, successful hatching was dependent on blastocyst expansion and was based on a minimum number of embryonic cells. Embryos grown in the presence of growth hormone were more advanced in their development and hatched earlier. When an artificial opening was laser-drilled into the zona pellucida, hatching occurred at lower numbers of embryonic cells. In vivo, escape from the zona pellucida occurred earlier and independent of blastocyst expansion. However, when we isolated in vivo-grown blastocysts with intact zonae that had developed in vivo and then cultured them in vitro, blastocysts started to expand and hatched the following day when a sufficiently high number of embryonic cells was present. Our data show that successful hatching in vitro is dependent on a sufficiently high number of embryonic cells, which enables blastocyst expansion and zona shedding. In vivo, the lower number of embryonic cells detected in zona-free blastocysts indicates that the underlying mechanism of zona escape is different, does not depend on blastocyst expansion, and presumably involves lytic factors from the uterus.

Aspects concerning the role of prostaglandins for ovarian function.

The present review briefly summarizes the current view of the mechanisms whereby prostaglandins (PGs) may act to modulate ovarian function. Particular concern is devoted to the aspects of ovarian function which are under current investigation by the authors, namely follicular maturation and rupture, granulosa cell and oocyte maturation, and the formation and maintenance as well as the regression of the corpus luteum (CL). PGs are formed in the granulosa cells of the preovulatory follicle in response to gonadotropin action and are at their highest levels around the time of ovulation. PGs are believed to be obligatory for follicular rupture, but their mechanism of action is still unknown. In the process of oocyte maturation PGs can mimic the stimulatory effects induced by LH or hCG, but are probably not obligate mediators of these effects. The importance of PGs for CL formation and maintenance is so far unsettled, and PGs may be regarded more as modulators than as inducers for these processes. The most well-documented effect of PGs is the induction of CL regression. Evidence is presented that PGs may have this effect also in the human.

Studies on the Mechanism of Ovulation Using the Model of the Isolated Ovary

Using the isolated perfused rabbit and rat ovaries as experimental models, we have studied various biochemical aspects of the ovulatory process. In rabbits, ovulations were induced by injecting hCG prior to the perfusion or by adding LH directly to the medium. In PMSG-treated rats, ovulations were induced by adding LH to the perfusion system. Steroids and other metabolites were analyzed in the perfusate and in follicular fluid. Steroid levels in follicular fluid were high early in the preovulatory development, but declined to very low levels 4 hours after LH stimulation. Levels of prostaglandins E and F rose as ovulation approached. In both perfusion models, indomethacin blocked ovulation without affecting steroid release or oocyte maturation. In the rabbit, PGF2 alpha reversed the indomethacin-induced inhibition and was able to induce follicular rupture by itself. Manipulations of the follicular fluid content of progesterone and estradiol to supraphysiological levels did not affect follicular rupture or oocyte maturation in the rabbit model. When the initial increase in LH-induced steroidogenesis was blocked by a 3 beta-ol-dehydrogenase inhibitor, ovulation was not affected. In rats, inhibition of estradiol production by an aromatase blocker did not affect the ovulatory process. When the endogenous formation of cyclic AMP is increased by pretreatment with a phosphodiesterase inhibitor, the LH-induced ovulation frequency increases in rabbits. Furthermore, forskolin, which increased the adenylate cyclase activity, stimulated steroidogenesis and induced follicular rupture. Recent experiments in the rat indicate that cyclic AMP acts on the ovulatory process via an effect on prostaglandin synthesis.

Ovulation in the isolated perfused rat ovary as documented by intravital microscopy

Surface cell changes at the apices of preovulatory follicles and ovulations were documented in isolated perfused ovaries from immature rats treated with pregnant mare serum gonadotropin (20 IU) and 48 h later with human chorionic gonadotropin (hCG) (10 IU). A video camera coupled to an inverted microscope and a video recorder captured the preovulatory and ovulatory events at a cellular level. At around 8 h post-hCG, the follicular apex changed from a smooth and optically homogeneous appearance into a rough surface with bleb formation and extrusions of single cells through minute perforations (early stigma formation). At approximately 10 h, a sticky material formed a basketlike structure with trapped cells (late stigma formation). At 12 to 15 h, ovulation took place at a constant speed and with no contractions of the follicular wall. This indicates that ovulation can occur with no visible circumfollicular muscular activity. Furthermore, the observations of a leakage of cells over an extended period of time indicates that the follicular wall is partly digested several hours before ovulation occurs.

The Preovulatory Decline in Follicular Oestradiol Is Not Required for Ovulation in the Rabbit

Using a method of in vitro perfusion of the rabbit ovary with a chemically defined medium in a recirculation system, normal appearing follicular ruptures occurred following exposure of the ovaries to hCG in vivo (100 IU) or following addition of LH (0.25 microgram/ml of NIH B9) to the perfusate. The addition of oestradiol-17 beta (10 micrograms/ml) to the perfusate did not inhibit these follicular ruptures, although the follicular fluid oestradiol contents were increased more than 100-fold as compared to the control side not receiving the addition of oestradiol. These data suggest that the physiological decline of follicular oestrogen, normally observed in vivo prior to ovulation in the rabbit, is not required as part of the mechanism of ovulation and that normal appearing ovulations can occur even though follicular oestrogen levels are kept artificially elevated.