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The development of ovarian tissue cryopreservation will expand the range of clinical applications in reproductive medicine. This emerging technology may have beneficial opportunities for patients, particularly in oncology, as well as for the process of oocyte donation. However, it will also lead to new moral problems requiring critical reflection concerning the criteria for tissue banking and future clinical applications. Because cryopreservation of ovarian tissue nowadays is the focus of experimental research, technology assessment is currently appropriate, anticipating introduction into clinical practice. Specific guidelines, developed by the medical profession in cooperation with ethicists and lawyers can contribute to prudent clinical use.

The neuropeptide oxytocin (OT) has been detected in testis and epididymis of several mammals. The peptide affects steroidogenesis and sperm transport in vivo. Effects of OT on sperm motility in vitro seems to be contradictory. As no data are available on the presence of OT in human semen and on the relationship of OT with sperm characteristics, we assessed OT level in semen samples in 3 groups of patients: (I) normozoospermic, (II) astheno-/oligo-/teratozoospermic, and (III) azoospermic subjects. Furthermore, we studied the relationship between the concentration of OT in semen and the sperm characteristics. OT was measured in seminal plasma by radioimmunoassay after extraction. OT semen levels did not differ in control patients (I: 1.72 +/- 0.78 pg/mL; n = 10), patients with poor semen quality (II: 1.66 +/- 0.91 pg/mL; n = 11), or in vasectomized patients (III: 1.28 +/- 0.65 pg/mL; n = 11). No statistically significant relationships between the OT levels and sperm characteristics (density: 0.0693; total sperm count: 0.0845; percentage of motility: 0.1341; morphology: 0.3478) have been found. The neurosecretory peptide oxytocin is present in human seminal plasma of normal as well as of vasectomized subjects. OT is not only derived from the testis; OT levels in poor semen samples are not different from controls. No relationship was found between OT seminal plasma levels and sperm characteristics.

In a human in-vitro fertilization (IVF) programme, the effect of co-culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.

The study was designed to clarify the physiological role of FSH in maintaining the meiotic arrest in vitro of oocytes derived from intact and from hypophysectomized hamsters. The sensitivity to FSH in maintaining the meiotic arrest of oocytes during incubation of pre-antral follicles of adult golden hamsters was estimated: log dose-response curves were characterized by an ED50 of about 0.2 pg FSH/ml (= 1 microU). After hypophysectomy oocytes of the pre-antral follicles were screened for the presence of a germinal vesicle (GV). The breakdown of the GV (GVBD) rose from 0 on day 0 to 48.8 +/- 13.8% on day 1, to 55.9 +/- 6.8% on day 2 and to 64.3 +/- 7.4 on day 6 (p < 0.001 vs day 0). Incubation without FSH of the pre-antral follicles obtained from the same hypophysectomized animals showed an almost complete disappearance of the GV at the same time intervals. Incubation with 2 pg FSH/ml (= 10 microU) still demonstrated the inhibiting effect of FSH on the meiotic resumption: 13.4 +/- 4.3% (day 0), 36.9 +/- 16.3% (day 1), 38.7 +/- 21.0% (day 2) and of 38.1 +/- 12.6% (p > 0.1) on day 6. These results demonstrate that very low doses of FSH are able to maintain the meiotic arrest in oocytes of pre-antral follicles of the golden hamster and that deprivation of FSH by hypophysectomy involves a disappearance of GV oocytes to a certain extent.

Objective: To study the levels of glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 in human preovulatory ovarian follicular fluid (FF) and pooled granulosa and cumulus cells., Design: The relation of glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 with P and 17 beta-E2 concentrations were studied., Setting: The Department of Obstetrics and Gynecology, the Department of Gastroenterology, and the Laboratory of Endocrinology and Reproduction of the University Hospital Nijmegen in Nijmegen, the Netherlands., Patient(s): Infertile women participating in an IVF program., Result(s): Detectable amounts of glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 were found in ovarian FF and pooled cumulus and granulosa cells. Concentrations of glutathione S-transferase Alpha 1-1 were always much higher than those of glutathione S-transferase Pi 1-1. Both ovarian FF concentrations of glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 did not correlate with ovarian FF concentrations of 17 beta-E2 and P., Conclusion(s): The high FF concentrations of glutathione S-transferase Pi 1-1 and especially of glutathione S-transferase Alpha 1-1 suggest that these enzymes may play an important role in the detoxification processes in the follicles. The lack of correlation between follicular P and 17 beta-E2 and glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 indicates that both enzymes presumably are not present as a result of the high steroid levels.

The aim of this study was to create reliable models to predict the probability of achieving an ongoing pregnancy during in-vitro fertilization (IVF) treatment: model A, at the start of the first treatment, model B at the time of embryo transfer, and model C, during the second treatment at the end of the first IVF treatment. Prognostic models were created using data from the University Hospital Nijmegen (n = 757) and applied to the data from the Catharina Hospital Eindhoven (n = 432), The Netherlands, to test their predictive performance. The predictions of model B (made at time of embryo transfer) were fairly good (c = 0.672 in the test population). For instance, 93% of the patients who had a predicted probability of achieving an ongoing pregnancy of < 10% did not achieve an ongoing pregnancy. However, the predictions of the other two models (A and C) for Eindhoven were less reliable. The predictive value of model C was fairly high in Nijmegen (c = 0.673). Its poor performance in the test population may be explained partly by differences in effectiveness of the ovulation stimulation protocols and the decision about when to discontinue the cycle. Thus, before using prognostic models at an IVF centre, their reliability at that specific centre should be tested.

The effects of gonadotropin stimulation on mouse embryo uptake and incorporation of 35S-methionine were studied. We found that the uptake of 35S-methionine was reduced in embryos of stimulated females in both the two-cell and the blastocyst developmental stage. The incorporation of 35S-methionine into protein was not statistically significantly different between the embryos of stimulated and those of unstimulated females. Qualitatively, protein synthesis was equal in both groups as determined with one-dimensional SDS-PAGE. The results are discussed and we conclude that mouse embryo viability in vivo is decreased by ovarian stimulation.

Objective: To study the contribution of smoking and alcohol consumption to semen quality., Design: Retrospective analysis., Setting: University-based fertility clinic., Patients and Methods: Smoking and alcohol consumption were investigated in a control group (68) and in a group of 47 subjects with defined poor semen quality (PSQ). The control group was composed of subjects whose semen showed a greater than 60% morphological normality, a greater than 60% motility with a linear progression, and a density of greater than 20 million spermatozoa/mL. The group with PSQ was composed of subjects whose semen showed a less than 30% morphological normality, less than 60% motility, characterized by slow, weak motility, and a density of less than 20 million spermatozoa/mL. Medical dossiers were studied regarding the life style of the subjects., Results: The distribution of heavy smokers and light smokers did not differ statistically between the groups. There appeared to be a higher, but statistically insignificant, proportion of heavy smokers in the PSQ group (50%) compared to the control group (32.3%; P < .1); nor were significant differences found between cases and controls with respect to alcohol consumption pattern. In the PSQ group, a comparison of the semen characteristics of the daily drinkers with those of all the other subfertile patients showed no statistical difference concerning semen volume (4.1 +/- 1.9 vs. 3.3 +/- 1.3 mL; P > .1), sperm density (10.6 +/- 7.8 vs. 8.9 +/- 5.8 million spermatozoa/mL; P > or = .1), and percentage of motile spermatozoa (27.0 +/- 15.1 vs. 25.5 +/- 16.1%; P > .1). However, a lower percentage of normal sperm morphology was observed in the daily-drinker group (17.6 +/- 7.2% vs. 23.0 +/- 6.5% for the other subfertile patients; P < .05)., Conclusion: Factors such as smoking and alcohol consumption do not seem to play a pivotal role in the etiology of poor semen quality, but a pattern of excessive alcohol consumption may decrease further an already low percentage of sperm with normal morphology.

This study was undertaken to evaluate the effects of human skin fibroblast monolayers on human sperm function and fertilization in vitro. Sperm function was evaluated using the hamster oocyte penetration assay (HOPA) and zona binding assay (ZBA) in medium alone and in co-culture with human skin fibroblast monolayers and suspensions. The ZBA was also studied in fibroblast conditioned medium and in bovine oviduct cell monolayers and suspensions. Fertilization was measured both in in-vitro fertilization (IVF) couples with a normal semen analysis (first study; randomized) and in IVF couples with subnormal semen analysis (second study; each patient served as its own control). The HOPA results were not significantly different with or without fibroblasts. In all co-culture situations and in conditioned medium the ZBA scored significantly lower than medium alone. No significant differences with respect to IVF were observed between the co-culture and the control group in either study. The mean fertilization rate per patient was approximately 60% in the group with normal semen analysis and approximately 25% in the group with abnormal semen analysis. From this study we conclude that although co-culture with human skin fibroblasts and epithelial cells influences the results of some sperm function tests, it does not influence fertilization in vitro.

Objective: To test the effects of steroid-containing media such as follicular fluid (FF) and serum and of steroid-free but human protein-rich medium on hyperactivation (HA) of human spermatozoa with and without a preincubation period., Design: Analysis of HA using a computer-aided sperm analysis system., Setting: Academic hospital and andrologic laboratory., Patients: Twenty-six sperm samples were obtained from 26 different IVF patients., Interventions: None., Results: After a preincubation period of 3 hours, FF and serum enhanced the HA after 1 hour. A human plasma protein-rich solution did not. Without preincubation, no stimulation of HA was observed., Conclusion: Steroids may be involved in HA, and a preincubation period may be required for an appropriate FF-induced HA.

Unlabelled: Isolation of meiotic arrested oocytes from mammalian ovaries involves a resumption of the meiosis. Studying the effect of FSH on this process it was found that doses up to of 0.1 IU/ml did not maintain the meiotic arrest in hamster oocytes in pre-antral follicles during static incubation conditions. However, when the FSH medium was replaced at 120, 60 and 30 min the resumption of the meiosis gradually decreased. Using a replacement frequency of 30 min a complete log dose-curve was constructed (ED50: 0.5 mU/ml; minimum effective dose: 0.1 mU/ml). The lack of a FSH response during static incubation was not caused by exhaustion of its bioactivity: mixing of the FSH containing medium, obtained from a 4 hours' static incubation, resulted in an inhibition similar to the inhibition that occurred during the replacement procedure., In Conclusion: FSH is able to maintain the meiotic arrest in pre-antral follicle enclosed oocytes at low doses only when the medium is in circulation.

A pool of human pituitaries obtained from allegedly healthy subjects (traffic victims) and plasma samples from patients with Nelson's syndrome were analyzed by high performance liquid chromatography, and the corticosteroidogenic bioactivity and ACTH immunoreactivity were measured. Three bioactive forms of ACTH were detected in plasma samples and pituitary extract. The major form (peak III) coeluted with human ACTH-(1-39), showed a bioactive to immunoreactive ratio (B/I ratio) of about 1, and represented about 80% of the total bioactivity in both the plasma samples and the pituitary extract. Peak I, with a B/I ratio greater than 1, represented about 5%, and peak II, with a highly variable B/I ratio, represented about 7% of the bioactivity in both the plasma and pituitary extracts. A fraction with a very low B/I ratio was found to coelute with corticotropin-like intermediate lobe peptide. These data suggest that in Nelson's syndrome, ACTH secretion by the pituitary gland does not differ from that in normal subjects, at least qualitatively.

Oocyte-cumulus cell complexes (OCCC) were aspirated from medium-sized (3-5 mm phi) porcine follicles to study quantitatively the process of forming monolayers. A monolayer index (MLI) was therefore developed. This index was evaluated by addition of graded quantities of fetal bovine serum. Furthermore, we observed that addition of prostaglandin E2 accelerated the monolayer formation (P < 0.001 at 20 hours versus 40 hours). The effect of PGE2 regarding both monolayer formation and cumulus cell expansion has been discussed.

The contribution of the positive charge of lysine in position 11 of ACTH to the corticosteroidogenicity was tested in a purified isolated rat adrenal cell assay. ACTH analogues with Lys11 or Arg11 had the same bioactivity. Replacement of lysine by norleucine or protection of its positive charge diminished the potency by about a factor of 80. The extent of this reduction has been discussed in relation to the importance of the basic amino acid sequence 15-18 of the peptide. The result suggests a ten times higher contribution of Lys11 to the bioactivity of ACTH than the surmised potency of the individual amino acids present in the sequence 15-18.

To study the effects of sperm density on the results of computer-assisted semen analysis (CASA), 10 washed semen samples were diluted and measured with the CellTrak/S CASA system in a concentration range of 10-180 x 10(6) spermatozoa/ml. All sperm motility parameters were influenced to some extent by sperm density. The motility percentage was influenced significantly in 5 samples (P < 0.005), the straight line velocity in all samples (P < 0.0005 in 7 samples), the curvilinear velocity in 3 samples (P < 0.005), the linearity in 9 samples (P < 0.0005 in 6 samples) and the lateral head displacement in 9 samples (P < 0.005 in 6 samples). In general, the CellTrak/S data are influenced significantly if sperm density exceeds 50 x 10(6) spermatozoa/ml. The influence of sperm density on the motility parameters can be explained both by the accuracy of the CASA system and by actual changes in the motility of the spermatozoa. In the light of other published studies, it is concluded that sperm motility measurements with CASA systems should be assessed using 25-50 x 10(6) spermatozoa/ml, especially in studies concerning lateral head displacement and the linearity, as in sperm hyperactivation studies.

The relationship between bioactive and immunoreactive adrenocorticotrophin (ACTH) was studied under various conditions. Rats were stressed by ether and the immunoreactive (I-ACTH) and bioactive (B-ACTH) ACTH levels were compared to the normal values obtained from "handled" rats. The bioactivity was determined by measuring the corticosteroid production in an isolated rat adrenal cell system. Stress exposure enhanced both the I-ACTH and B-ACTH levels. I-ACTH rose from 86.9 +/- 37.9 to 363 +/- 148.7 pg/ml, whereas the B-ACTH levels rose from 34.2 +/- 11.9 to 424.2 +/- 170.9 pg/ml. The bioactive to immunoreactive ratio (B/I ratio) was calculated as 0.47 +/- 0.13 in the control group and rose to the significantly higher value of 1.10 +/- 0.22 in the stressed rats. The conclusion is that under normal circumstances only about 50% of plasma immunoreactive ACTH shows corticosteroidogenic activity and that the B/I ACTH ratio might be a useful tool to estimate the physiological phase of the pituitary releasing activity.

The purpose of this study was to investigate the influence of antisperm antibodies in the male, the female, or both partners on the outcome of in vitro fertilization treatment. The results in terms of ongoing pregnancies in the male and female antibody-positive group were the same as in the antibody-negative group. In the double antibody-positive group two of the three patients became pregnant. When high levels of antisperm antibodies were present on the spermatozoa, the fertilization rate was significantly reduced. In the female positive group no clear relationship between the antibody titer and the fertilization percentage could be detected. Abnormal semen quality was responsible for a much lower fertilization rate than the presence of antibodies. The conclusion of this study is that in vitro fertilization provides an equal change of conception in couples with antisperm antibodies in comparison with couples with no antibodies if the other semen parameters are normal.

A new system for co-culture in human in-vitro fertilization (IVF), using human skin fibroblasts, is described and tested pre-clinically. The first test involved the development of 1-cell mouse embryos which exhibit the 2-cell developmental block in vitro. Passage through this block (pb1-ratio) was determined by the ratio of compacted morula stages on day 4 of incubation. For nine human skin cell lines tested (fetal, neonatal and adult), the pb1-ratio was approximately 0.45 (0.07 in culture medium alone; P less than 0.0005). At the compacted morula stage, a second developmental block was observed. The ratio of passing this block (pb2-ratio) was 0.70 +/- 0.09 on skin fibroblasts obtained from fetal or neonatal tissue. On fibroblasts from adult patients the pb2-ratio was 0.30 +/- 0.04 (P less than 0.0005). The second test examined the influence of skin fibroblasts from fetal or neonatal tissue on human sperm motility. After 24 h of incubation, all skin cell lines had a positive influence (P less than 0.01) on the percentage motility compared to culture medium alone. The curvilinear velocity was not significantly increased. From the results we conclude that (i) human skin fibroblasts (especially from fetal tissue) have a positive influence on the development of mouse embryos in vitro, (ii) there is a positive influence of human skin fibroblasts on the percentage motility of human spermatozoa, and (iii) a clinical trial of co-culture with human skin fibroblasts can be justified.

Objective: To study the reaction pattern of acrosome reaction in human semen and correlate it to the results of in vitro fertilization (IVF)., Design: The percentage of acrosome-reacted spermatozoa of 41 IVF semen samples was determined after 0, 2, 4, and 24 hours of incubation in human tubal fluid medium supplemented with 10% human pool serum., Setting: St. Radboud Hospital, Catholic University of Nijmegen, The Netherlands., Patients: Forty-one IVF couples., Interventions: None., Main Outcome Measure: Acrosome reaction was determined using fluorescein isothiocyanate conjugated concanavalin A lectin. To avoid false-positive signals from dead spermatozoa, the sperm viability was determined., Results: Three kinetic patterns of acrosome reaction could be distinguished: (1) normal reacting pattern (percentage of acrosome-reacted spermatozoa less than 10% at 2 hours and greater than 5% at 4 hours; 75% fertilization in IVF); (2) a quickly reacting pattern (percentage of acrosome-reacted spermatozoa greater than 10% at 2 hours; 22% fertilization in IVF); and (3) a nonreacting pattern (percentage of acrosome-reacted spermatozoa less than 5% at all time intervals studied; 15% fertilization in IVF)., Conclusions: The timing of acrosome reaction and the percentage of acrosome-reacted spermatozoa are very important parameters in IVF.

Objective: To study human sperm motility in a coculture system for in vitro fertilization (IVF)., Design: We studied the viability and motility (percentage and curvilinear velocity) of human spermatozoa after incubation in: (1) medium 199 supplemented with fetal calf serum (M199/FCS) together with Vero cells; (2) Vero cell conditioned M199/FCS; (3) M199/FCS supplemented with Vero cell extract; and (4) some control media. In a second experiment, FCS was substituted by sera from different IVF patients., Setting: Semen samples were obtained from the fertility laboratory of the St. Radbound Hospital, Nijmegen, The Netherlands., Patients: Twelve men of couples with fertility problems., Interventions: None., Main Outcome Measure: The motility parameters were determined with a computerized motility-analyzing system, after 24 hours' incubation at 37 degrees C and 5% CO2. Viability was determined after eosin Y staining., Results: Sperm viability was the same in all media. In the coincubation system, the Vero cell-conditioned medium, and the experiment with human sera, the sperm motility parameters were higher (P less than 0.005) than in the control media. Vero cell extract did not have this positive effect., Conclusions: Coincubation has a positive effect on sperm motility and may be beneficial to IVF, intrauterine insemination, and artificial insemination.

The corticosteroidogenicity of ACTH-derived peptides was tested in a purified isolated rat adrenal cell system. The activity was related to the activity of standard ACTH (synthetic hACTH; potency fixed at 100). The peptides ACTH NH2 and ACTH showed steroidogenicity at pharmacological doses (potencies: 0.00067 and 0.00032, respectively). None of the peptides tested inhibited or potentiated the ACTH-induced steroidogenesis at low doses (0.5-50 000 pg). The results suggest the presence of a second center within the ACTH molecule capable of inducing steroidogenesis.

The functioning of the hypothalamo-pituitary-adrenal axis was assessed in 10 adult women with idiopathic hirsutism treated for 2 weeks with the anti-androgen cyproterone acetate in a dose of 50 mg b. d. daily and in 4 patients treated for at least 3 months. Basal plasma ACTH and cortisol levels and the cortisol response to 8 h ACTH infusion were comparable before and during short-term treatment. The plasma ACTH and cortisol responses to insulin induced hypoglycaemia before and during anti-androgen therapy also were of the same order of magnitude. In the 4 patients treated for at least 3 months also no suppressive effect of the anti-androgen on basal plasma cortisol levels was observed. From these data the conclusion seems warranted that short-term cyproterone acetate treatment in the given dose not significantly influences pituitary-adrenal function in adult women with idiopathic hirsutism.

The effects of the intravenous anesthetic etomidate have been investigated on ACTH-induced steroidogenesis in vitro, using purified isolated rat adrenal cells. It was found that etomidate almost completely blocked corticosterone production induced by physiological concentrations of ACTH at doses of 200 ng or greater. The mean inhibitory etomidate concentration resulting in 50% inhibition approximated 1.5 X 10(-7)M which is in the range of concentrations measured after clinical doses of etomidate.

In 23 consecutive patients with Cushing's disease and 52 control subjects, the responses of ACTH and cortisol to TRH and LRH were investigated. From the pattern of cortisol levels after the administration of the releasing hormone in the controls, a criterion for paradoxical responsiveness could be derived (maximum cortisol increase, greater than 6.0 micrograms/100 ml). According to this criterion, 9 patients with Cushing's disease showed a paradoxical responsiveness to one or both releasing hormones (3 to both TRH and LRH, 3 to TRH alone, and 3 to LRH alone; group I). In all patients tested, paradoxical responses of cortisol were preceded by paradoxical increments in ACTH. The remaining 15 patients showed no paradoxical increments in ACTH or cortisol after TRH or LRH (group II). ACTH levels in group I (89 +/- 28 pg/ml) were significantly lower than those in group II (185 +/- 164 pg/ml; P less than 0.02). Nevertheless, in both groups, a similar plasma cortisol level was found, suggesting a relatively higher bioactivity of ACTH in group I. A second difference between both groups was a lower amplitude of cortisol variability during the day in group I. The 2 groups did not differ in clinical data, such as age, sex distribution, sellar volume, and duration of disease, or dexamethasone suppressibility, bromocriptine sensitivity, and basal PRL levels. These latter findings do not favor an intermediate lobe origin of Cushing's disease in patients with paradoxical responses to TRH/LRH. To conclude, TRH/LRH responsiveness of ACTH/cortisol discloses two subsets of patients with Cushing's disease.

In order to investigate variations in the microenvironment of oocytes within a cohort of maturing follicles the follicular volumes as well as the intrafollicular concentrations of oestradiol (E2) and progesterone (P) were measured in the golden hamster. At 10 h before ovulation the follicular volumes varied from 0.009 to 0.037 mm3 (mean +/- SD: 0.0187 +/- 0.0071 mm3; n = 36). Large follicles (greater than 0.025 mm3; n = 8) contained statistically significantly lower E2 and P levels (30.1 +/- 10.4 and 517 +/- 113 mumol/l, respectively) than the medium sized group (less than 0.025 and greater than 0.015 mm3; n =20): 46.9 +/- 16.0 (P less than 0.02) and 919 +/- 264 (P less than 0.0001) mumol/l, respectively. Small follicles (less than 0.015 mm3) showed the highest steroid levels: 97.0 +/- 33.3 and 1590 +/- 517 mumol/l for E2 and P (P less than 0.001 versus the medium sized group values). Correlation coefficients for the steroid concentrations and the follicular volumes appeared to be -0.674 for E2 and -0.612 for P (P less than 0.001). At the time studied a positive correlation between E2 and P concentrations in the follicles was found: r = 0.655 (P less than 0.001). The mean ratios of intrafollicular over serum steroid concentrations appeared to be approx 36 x 10(3) in the case of E2 and about 17 x 10(3) in the case of P. These results clearly show that there is an inverse relationship between follicular volume and intrafollicular steroid concentrations. The presence of a fine regulatory mechanism for a collective maturation of follicles is hypothesized.

A highly sensitive bioassay, using preincubated purified isolated rat adrenal cells, has been developed for measuring plasma ACTH. This bioassay enables detection of ACTH in plasma of about 0.9 pmol/l. Bioactive ACTH (B-ACTH) plasma levels were determined during insulin-induced hypoglycaemia in 12 female subjects and the values were compared with immunoreactive ACTH (I-ACTH) levels. The mean (+/- SD) basal B-ACTH level amounted to 1.7 +/- 0.8 pmol/l, the mean I-ACTH to 2.9 +/- 1.4 pmol/l. The highest mean B-ACTH plasma value was found 30 min after insulin injection: 14.7 +/- 15.7 pmol/l (I-ACTH: 12.8 +/- 9.9 pmol/l). By 90 min the B-ACTH level had returned to baseline (1.6 +/- 0.8 pmol/l), whereas the I-ACTH level was still significantly higher (5.4 +/- 2.7 pmol/l) than at time zero. Remarkably, the B-ACTH to I-ACTH ratio (B/I ratio) showed a biphasic profile during the insulin tolerance test, the ratio increasing from 0.60 +/- 0.77 at time zero to 1.08 +/- 0.35 at the ACTH peak, and decreasing after that to a lower value of 0.33 +/- 0.11 at 90 min. From these results it is concluded: (1) in the morning hours a considerable amount of circulating I-ACTH has no steroidogenic activity; (2) the B/I ratio temporarily increases immediately after insulin injection but gradually decreases afterwards to values half the baseline level at 90 min. Whereas this decrease at 90 min can be explained by differences in disappearance rates, the increase of B-ACTH relative to I-ACTH at 30 min indicates that estimations of immunoreactive ACTH reflect the biological activity of newly released ACTH with greater precision than at steady state level. Thus, the B/I-ACTH ratio can be used as a tool for measuring the state of the pituitary releasing activity.

The ability of two pro-opiomelanocortin-derived peptides, the melanotropin potentiating factor (MPF) and beta-endorphin (beta EP), to affect corticosteroid production was studied in purified isolated rat adrenal cells. Addition of MPF or beta EP, in doses from 5 pg to 5 micrograms, alone did not result in a corticosterone production. Furthermore, no effect of MPF or beta EP in doses from 5 pg to as high as 5 micrograms for both peptides upon the ACTH or alpha-MSH-induced corticosteroidogenesis was observed (p greater than 0.1). It is concluded that both MPF and beta EP do not influence the steroidogenic activity in the adrenal gland. Use of these peptides for discrimination of the ACTH/alpha-MSH receptor interactions is suggested.

To determine the optimal conditions for in vitro spermatozoa vitality, human and hamster spermatozoa were incubated at 37 degrees C in T6 medium supplemented with different biologic fluids (10% v/v). The fluids tested were human serum (HUS), hamster serum (HAS), and human follicular fluid (HUF). After incubation the spermatozoa were investigated for their qualitative and quantitative motility. Human spermatozoa maintained a good vitality in all fluids tested (approximately 25% motility after 18-h incubation). The hamster spermatozoa had after an incubation of 4 h a motility of 28.4% in HUS, 14.2% in HAS, and 2.2% in HUF. The quality of the motility was also extremely low in HUF, whereas it was adequate in HUS and in HAS. The presence of species-specific substances in mammalian follicular fluid is discussed.