Luz Ma. Ballesteros

Metalloproteinase activity during growth, maturation and atresia in the ovarian follicles of the goat

Metalloproteinases are an important group of hydrolytic enzymes which participate in interstitial matrix degradation during tissue remodelling processes and therefore may be required during follicular growth and maturation. The activity of metalloproteinases (collagenases, gelatinase, and Pz-peptidase), was measured during growth, maturation and atresia of goat antral follicles. These follicles (n = 67) were separated by size and also classified into four groups: non-atretic (Group I); early atretic (Stage I) (Group II); moderately atretic (Stage II) (Group IIIa); and, late atretic (Stage III) (Group IIIb). Pz-peptidase was greater in granulosa than in thecal cells, and almost absent in follicular fluid. In non-atretic follicles, activity in granulosa cells increased with increasing follicle size, whereas activity peaked in 3-6 mm follicles in thecal cells. Atresia was associated with declining activity in thecal cells from follicles in the 3-6 mm range and in granulosa cells from the > 6 mm range. Interstitial collagenase activity was significant and similar in granulosa and thecal cell extracts and low in follicular fluid from non-atretic follicles. Activity increased significantly in thecal cells, but decreased significantly in granulosa cells from large (> 6 mm) non-atretic follicles. Atresia was associated with declining activity in both types cells and increasing activity in follicular fluid. Gelatinase activity was some times associated with five regions corresponding to molecular weights of 22.1, 30.7, 39.6, 63.8 and 71.4 kDa, and rarely at 91.3 and 81.2 kDa. Overall activity declined with atresia in thecal cells from follicles in the 3-6 mm range, but not in those > 6 mm. In granulosa cells from follicles 3-6 mm, activity varied widely with stage of atresia, while in cells from follicles > 6 mm, activity was greatly increased in atretic follicles.

Subcellular distribution of trace metals in the normal and in the copper treated human secretory endometrium.

Abstract The subcellular distribution of some metals has been studied in the normal human secretory endometrium and in the secretory endometrium of women wearing a copper-T intrauterine device. The sustained release of copper inside the uterine cavity induces some significant changes in the metal composition of this tissue; Mg, Ca and Cu increase while Zn decreases. Neither Na nor K concentrations show any significant change. Under these conditions, endometrial nuclei concentrations seem to be able to passively come to equilibrium with the tissue concentrations of the elements studied, showing only a significant retention of Ca. On the contrary, mitochondria show differential concentrations of all metals. Ca is retained, but all the other elements remain at low levels. Copper is significantly increased in all fractions, but the relative increase is particularly noticeable in the microsomes (6 x). Zinc tends to decrease in all fractions but reaches statistical significance only in the microsomes and the supernatant. These results are analyzed in relation to the mechanism of action of the Cu-T.

Effect of Steroid Hormones on Membrane Sugar Transport in Human Spermatozoa

Using the model of exchange transport, we found that glucose transport through the human spermatozoa membrane (447 +/- 54 pmoles/min/10(8) cells) is probably the rate-limiting step in sugar utilization. Sugar transport was more efficient for glucose than for fructose (182 +/- 32 pmoles/min/10(8) cells) and depends on a highly asymmetric carrier with at least two transporting sites. Transport was drastically dependent on pH with an optimal pH of 7.4, showing a decrease of more than 60% with a change of 1 pH unit. Testosterone and 17-B estradiol increased the amount of transported sugar (619 +/- 73 and 922 +/- 110 pmoles/min/10(8) cells, respectively), while progesterone has no effect.

RNA metabolism of the normal and the copper treated human endometrium

Ribonucleic acid (RNA) metabolism of the normal and copper-treated ( Cu-T200 IUD) human endometrium was investigated. The relative concentration of total, messenger, ribosomal and transfer RNA was measured in normal and Cu-treated endometrium using the technique of affinity chromatography in polysepharose. The transition from the proli ferative to the secretory endometrium in normal women was accompanied by significant increases (p less than .05) in total RNA, messenger RNA and in ribosomal RNA. The relative proportions of bound and free messenger RNA were also modified by endometrial maturation changing from 70% bound messenger RNA in the proliferative to 83% in the secretory phase. Cu-T200 Cu release appeared to particularly affect RNA metabolism in the secretory phase. During the proliferative phase only the concentration of transfer RNA and the proportion of bound to free messenger RNA were modified by the Cu-T200. The Cu-T200 induced significant decreases (p less than .01 and p less than .05) in all RNA parameters, with the exception of the RNA/deoxyribonucleic acid ratio.

Copper as a Dissociating Agent of Liver and Endometrial Polysomes**Supported in part by a grant from The Ford Foundation.

In an effort to explain the mechanism of the increased efficacy of the copper-releasing IUD, the in vitro action of copper ions on the polysome patterns of rabbit endometrium and liver was studied. The uterine horns were extracted from female rabbits with prior sexual experience. The horns were sliced longitudinally, and the endometrium carefully scraped off with a curette. For each experiment the scrapings from 4 to 6 cornua were pooled, suspended in buffer A (.14 M sucrose containing .05 M Tris-hydrochloride, .05 M potassium chloride, .005 M magnesium chloride, 200 mcg/ml heparin and brought to pH 7.6), and divided into 5 equal fractions. To 4 of the fractions, copper (as copper chloride) was added to reach final concentrations of .15, 13, 1, and 1.5 mM, respectively. The fifth fraction served as a control with no copper added. The liver was extracted, homognized in buffer A, and treated in the same way as the endometrium. The homogenates were further adjusted, treated, centrifuged, and analyzed. Results showed that the in vitro addition of copper in concentrations as low as .15 mM produced a significant decrease in the amount of polysome aggregates that could be recovered from endometrium. Liver required concentrations of at least .3 mM to show a similar effect. For concentrations of 1.5 mM copper, the recoverable amount of the polysome fraction from liver and endometrium was 40% and 25%, respectively, of that recoverable without added copper. When the obtained polysomes were analyzed in a sucrose gradient, additions of increasing concentrations of copper induced a progressived decrease of the heavy components of the polysome patterns with a concomitant increase in the lighter components. The results which show copper as a possible dissociating agent of liver polysomes are important, but it is doubtful that liver concentrations of those required to dissociate polysomes (.3 mM) will ever be reached by the use of these IUDs. It is postulated that the impairment in polysome aggregation is part of the mechanism of the copper IUD, and it may modify both the endometrial characteristics and the blastocyst viability required for successful embryo development.