No abstract

No abstract

Summary. The

No abstract

Summary. Mouse oocytes exposed to 1 \ g=m\ g Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10\g=m\g/ml),obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 \g=m\gH-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 pg H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 \g=m\g H\ x=r eq-\ 33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.

Proteinases are likely effectors of endometrial menstrual breakdown. We have investigated proteinase production by human endometrial stromal cells subjected in vitro to progesterone (P) withdrawal, the physiologic stimulus for menstruation. Culture media of cells exposed to estradiol, P, or estradiol plus P had low levels of proteolytic activity similar to cultures maintained in the absence of steroids. P withdrawal, or addition of RU486 to P-treated cultures, stimulated proteinase secretion. The stromal cell proteinase was characterized by gelatin zymography, inhibitor profile, and organomercurial activation, as a metalloproteinase present mostly as a 66-kD proenzyme with lower levels of a 62-kD active form. The P withdrawal-induced metalloproteinase was identified as matrix metalloproteinase-2 (MMP-2) by Western blotting. The increase of MMP-2 induced by P withdrawal was associated with the metalloproteinasedependent breakdown of stromal cultures, involving dissolution of extracellular matrix and dissociation of stromal cells. Northern analysis showed the differential expression of MMP-2 mRNA in late secretory phase endometrium. These findings are consistent with the involvement of stromal cell-derived MMP-2 in the proteolysis of extracellular matrix promoting cyclic endometrial breakdown and the onset of menstrual bleeding. ( J. Clin. Invest. 1996 . 97:438-447.)

Arginine, cystine, histidine, leucine and threonine were needed for outgrowth of the mouse blastocyst in vitro. Omission of lysine, methionine, phenylalanine, tryptophane and tyrosine from the culture medium markedly reduced blastocyst outgrowth, h u t did not inhibit it completely; while omission of isoleucine and valine reduced the extent of outgrowth only slightly. Blastocysts kept for seven days i n a free-floating condition by omitting arginine and leucine from the medium, grew out when these amino acids were added. Such behavior may be analogous to delayed implantation in utero and suggests that the free amino acid content of the u t c p s could be a n important factor i n the control of implantation. Blastocysts delayed from implanting i n the uterus by ovariectomy were activated to outgrowth in a complete medium, but the intracellular changes associated with outgrowth occurred more slowly than i n undelayed blastocysts.

No abstract