Holger Gips

Bovine adrenal cortex adenylate cyclase: Properties of the particulate enzyme and effects of guanyl nucleotides

SummaryThe preparation of a partially purified plasma membrane fraction from bovine adrenal cortex is described. Adenylate cyclase in this particulate preparation retained high sensitivity to ACTH and is also stimulated by 5′-guanylyl-imidodiphosphate [Gpp(NH)p]. GTP, in contrast to Gpp(NH)p, had very little intrinsic activity to stimulate adenylate cyclase. GTP could however, with high affinity, inhibit the Gpp(NH)p effects on adenylate cyclase. When the concentration of creatine phosphate, a component of the ATP-regenerating system in the adenylate cyclase assay mixture, was lowered from 20 to 2 mM (at 0.1 mM ATP, 5 mM Mg2+) GTP, dGTP and other nucleotides like ITP and much less UTP or CTP gained considerable intrinsic activity in the presence of ACTH to stimulate adenylate cyclase. The apparent affinities of the nucleotides for ACTH-stimulated adenylate cyclase from bovine adrenal cortex (at 2 mM creatine phosphate) were, GTP=dGTP>Gpp(NH)p>Gpp(CH2)p (5′-guanylyl-β, γ-methylene-diphosphonate) >ITP>UTP>CTP. These findings indicate that regulatory nucleotide binding sites exist for bovine adrenal cortex adenylate cyclase. Their specificity is similar to the nucleotide sites modulating angiotensin binding in bovine adrenal cortex plasma membranes (Glossmann et al., 1974a). The regulatory nucleotide binding sites for the adrenal cortex adenylate cyclase complex can also be identified under conditions where only Gpp(NH)p has high intrinsic activity (e.g. at 20 mM creatine phosphate) but other nucleotides like GTP act as antagonists. Both stimulants, ACTH and Gpp(NH)p, appear to remain firmly bound to the particulate membrane preparation, as suggested by preincubation experiments.

Sperm Selection Methods for Intracytoplasmic Sperm Injection (ICSI) in Andrological Patients

AbstractPurpose: To improve the chances of successful in vitro fertilization, spermatozoa have to be separated from semen before insemination. Therefore, sperm preparation methods are of great importance.nMethods: To obtain sufficient numbers of spermatozoa from patients with cryptozoospermia or severe OAT syndrome, only Minipercoll centrifugation and migration-sedimentation (MS) are practicable methods. The present study was performed to compare these two methods with regard to sperm concentration, motility, vitality, morphology, and chromatin condensation. The number of spermatozoa obtained after minipercoll was higher than that after MS, but sperm quality in all parameters examined was clearly better after MS than after Minipercoll. In the second stage of this study, the MS method was used for preparation of the spermatozoa for intracytoplasmic sperm injection (ICSI).nResults: Over a period of 13 months, 159 cycles were treated by ICSI. Of 1045 aspirated oocytes, 790 were injected. The fertilization rate was 70.4% of injected oocytes (556 oocytes with clearly visible pronuclei). In 146 cases, embryonic transfer was achieved; 58 patients became pregnant (39.7% per transfer and 36.5% per cycle).nConclusions: Although the abortion rate was very high (18 women lost their embryos), the results demonstrate that the microinjection method can be successfully used in combination with a MS method for preparation of spermatozoa.

Influence of polarization effects in ooplasma and pronuclei on embryo quality and implantation in an IVF program.

AbstractPurpose: The presence of a clear half-moon-like zone of cytoplasm in oocytes is called “halo effect.” The prognostic value of this effect is not yet determined. Aligned nucleoli in pronuclei (PN) represent a further polarization phenomenon and a marker for implantation potential. Aim of the prospective study was to evaluate the influence of the halo effect on IVF outcome and to compare the results with observed polarization in PN.nMethods: A total of 374 cycles with embryonic transfer were analyzed regarding halo effect and pattern of nucleoli. The oocytes were single-cultured to observe the following embryo quality of each PN stage.nResults: Cycles with halo-positive oocytes showed a significant higher pregnancy rate (44.0% vs. 31.1%; p < 0.05). Furthermore, higher pregnancy rates in cycles with polarized nucleoli were observed. Polarized PN resulted in a significant lower fragmentation and higher cleavage rate of embryos. The fragmentation rate was significantly lower in halo+ oocytes, but the cleavage rate was not influenced.nConclusions: The results indicate that the presence of a polarized zone of human fertilized oocytes can be a useful indicator for good oocyte quality. Since the origin of ooplasmic polarization seems to be a different process compared with the alignment of nucleoli, the observation will give additional predictive information about the implantation potential.

The preparation of brush border membranes from rat kidney using an aqueous two-phase polymer system.

SummaryAn aqueous two-phase polymer system (dextran — polyethylene glycol) has been employed to prepare a brush border membrane fraction from rat kidney. The procedure is rapid, simple and can be performed without the use of an ultracentrifuge. Brush borders isolated with this method are well preserved and of reasonable purity. The marker enzymes maltase and 5′-nucleotidase were enriched between 7 and 15 fold.

Placental steroid metabolism in a case of placental sulfatase deficiency

The importance of the placental 3 β-steroid sulfatase for placental biosynthesis of estrogens is demonstrated by a case report of a placental sulfatase deficiency. The absolute deficiency of this enzyme in our case is demonstrated in vivo by the intravenous dehydroepiandroster-rone sulfate loading test and in vitro by placental enzyme tests. Steroid concentrations in serum after injection of dehydroepiandrosterone sulfate (DHEA-S) are compared with those in a group of pregnant women without placental enzyme defects and in a group of nonpregnant women. Placental in vitro tests demonstrate the intact 3 /3-hydroxysteroid dehydrogenase-Δ4,5 isomerase-aromatase-system in the placenta with sulfatase deficiency. The lack of placental hydrolysis results in low concentrations of estrone and estradiol-17 β in the maternal serum, which are only 5.9% and 12.5%, respectively, of the mean values of the control group. This indicates that more than 90% of estrone and more than 85% of estradiol-17 β measured in the maternal serum aref rom DHEA-S as precursor. The remaining concentrations are converted mainly from dehydroepiandrosterone (DHEA), which needs no hydrolysis. Maternal serum concentrations of estriol are under the detection limit of the radioimmunoassay. This is due to the absence of the so called “neutral pathway” and the “phenolic pathway” with 16 or-hydroxydehydroepiandrosterone sulfate (16 α -OH-DHEA-S) and DHEA-S respectively as precursors. The insignificance of the “placental pathway” for the total biosynthesis of estriol is demonstrated. The placental sulfatase seems to have no great importance for the biosynthesis of the C21-steroids.

Different cumulative pregnancy rates in patients with repeated IVF- or ICSI cycles: possible influence of a male factor.

The low rate of ongoing pregnancies in IVF cycles leads to a high number of repeated cycles in couples with previously failed attempts. Therefore it would be helpful to have a prediction about the chance of becoming pregnant in a repeated cycle. In a retrospective study the data of about 4246 cycles were analysed. Because the pregnancy rates in IVF‐ and ICSI cycles are generally different, these two groups were distinguished between and the outcome in patients with one, two or more attempts was anlaysed. The rate of ongoing pregnancies per patient was lower after IVF (24.9%) than after ICSI (32.9%), but was similar or even slightly increased in patients with more than one attempt. On the other hand, there was a high pregnancy rate with ICSI in the first two cycles (35.9%), but patients with more than two ICSI cycles had a significantly lower chance of becoming pregnant (20.7%). Factors that are known to influence the pregnancy rate, such as stimulation protocol, oocyte quality or number of transferred embryos, were similar in all groups. However, significantly reduced embryo quality with successive cycles was only observed in ICSI patients. There might be a negative selection of patients with poor embryo quality and previously failed attempts after ICSI, possibly due to an andrological factor. The differences between IVF‐ and ICSI patients are based on treatment indications, and andrological diseases are the predominant indication for ICSI. Although no correlation was found between changes in conventional sperm parameters and number of treated cycles, there might be a subgroup of andrological patients selected by repeatedly failed ICSI cycles. Chromosomal or genetic disturbances in spermatozoa of this subgroup could be the reason for failure.