Our results indicate that isolated 46,XY and 46,XX DSD can be assigned to two separate regulatory regions, XYSR and XXSR, far upstream of SOX9. The 1.9 kb SRY-responsive subfragment from the XYSR might constitute the core of the Sertoli-cell enhancer of human SOX9, representing the so far missing link in the genetic cascade of male sex determination.

We determined the concentrations of tumour necrosis factor (TNF)-alpha, interleukins (IL)-1 beta, -6, -8 and -1-receptor antagonist (IL-1-ra) and of oestradiol and progesterone in the follicular fluid of 111 women undergoing in-vitro fertilization (IVF) and of six women with ovarian cysts in order to elucidate mid-cycle mechanisms causing dissociation of the follicle wall and local rupture of the ovarian tissue complex. Four stimulation protocols were administered: gonadotrophin releasing hormone agonist/human menopausal gonadotrophin (GnRHa/HMG), clomiphene citrate/HMG (CC/HMG), HMG and follicle-stimulating hormone (FSH). Concentrations of TNF alpha and IL-1 beta were below 15 and 3 pg/ml respectively. IL-6 (median 4.1, 3.5-4.4 pg/ml, 95% CI) was higher after stimulation with FSH (5.6 pg/ml) than with HMG (3.2 pg/ml, P < 0.05) or GnRHa/HMG (3.7 pg/ml, P < 0.05), and after stimulation with CC/HMG (5.5 pg/ml) than with HMG (P < 0.01) or GnRHa/HMG (P < 0.001). IL-8 ranged from 32 to 1241 pg/ml (147, 117-178 pg/ml) and IL-1-ra from < 31 to > 10,000 pg/ml (156, 109-192 pg/ml). Cytokine levels did not correlate to oestradiol or progesterone concentrations. The ovarian cysts contained similar IL-8 (14-540 pg/ml) and IL-1 beta (< 30 pg/ml), but higher IL-6 (13.6-> 500 pg/ml) and lower IL-1-ra concentrations. We assume that IL-6, IL-8 and IL-1-ra are involved in peri-ovulatory cellular interactions. Thus, ovulation appears to be a cytokine-regulated process of an 'inflammation' (IL-6 and IL-8) followed by 'anti-inflammatory' reactions (IL-1-ra).

All oocytes from a patient who had undergone four unsuccessful in-vitro fertilization attempts showed neither a polar body nor pronuclei when examined for fertilization. In 19 inseminated oocytes that were spread for karyotypic analysis, one haploid set of metaphase II chromosomes and a remarkable condensed structure were found. Hormonal and morphological criteria implied that the oocytes had been mature at the time of retrieval. Since non-inseminated oocytes contained only one set of metaphase II chromosomes, the condensed structure appeared to represent the sperm chromatin in the state of premature chromosome condensation due to a block in oocyte maturation. Since the first and second polar body, as well as their chromatin, were undetectable in all the patient's oocytes, a rapid maturation to metaphase II before retrieval and prolonged arrest in this state before fertilization, accompanied by degeneration of the first polar body, appear to be responsible for the condition. In accordance with this notion, degenerate spindles (typical of post-ovulatory aged oocytes) and separating chromosomes (probably representing presegregating chromatids) were observed by antitubulin immunofluorescence.

Absence of polar body formation, or premature chromatin condensation (PCC) in human oocytes can cause infertility. We studied in-vitro maturing mouse oocytes in order to identify risk factors for such conditions, and for the precocious segregation of homologues or chromatids. Treatment with the actin-binding drug cytochalasin D (10 micrograms/ml) arrested oocytes in metaphase I. Upon exposure to Ca(2+)-ionophores, anaphase I was triggered in the absence of cytokinesis. Chiasmata resolved and homologues separated instantaneously. In some oocytes predivision of all chromatids occurred. Homologues or chromatids never separated even after exposure to Ca(2+)-ionophores when microtubules were depolymerized, although bivalents could eventually decondense. Thus, in meiosis I checkpoints exist which ensure that homologue separation only takes place when a metaphase I spindle is present but cytokinesis and anaphase progression can be uncoupled. Cycloheximide induced a sequential separation of homologues in oocytes with intact metaphase I spindle, resulting in metaphase II chromosomes and bivalents in individual cells as also found in some human oocytes of aged females. In oocytes which progressed to metaphase II but failed to extrude a first polar body, the two sets of chromosomes eventually aligned on a common spindle ('diploid' metaphase II). PCC of one set was never observed. Ageing in vitro of cytochalasin D-blocked metaphase I oocytes had no pronounced effect on chromosome segregation.

A cytogenetic-cytological study was performed on unfertilized human oocytes (first polar body visible) after intracytoplasmic sperm injection (ICSI) with respect to the rate of prematurely condensed sperm chromosomes (G1-PCC). Out of 163 prepared oocytes derived from 41 ICSI cycles, 133 (approximately 82%) could be analysed successfully. a total of 60 oocytes (45.1%) showed metaphase II chromosomes in the haploid range along with an intact sperm head and 27 oocytes (20.3%) were missing the sperm head, but two of them showed an approximately diploid set of chromosomes; 38 oocytes (28.4%) exhibited the maternal metaphase II chromosomes as well as G1-PCC of the sperm nucleus showing a remarkable variation in the degree of condensation. Ten ICSI cycles (each followed by an embryo transfer) were characterized each by 2-3 oocytes demonstrating G1-PCC. It is concluded that the main cause of failed fertilization after ICSI is the failure of oocyte activation. When the sperm nucleus is able to act with the chromosome condensing factors and the oocyte does not become activated, this will lead to the induction of PCC. Absence of the sperm head might be due to injection or ejection of the spermatozoon in the perivitelline space except for two cases in which fertilization might have occurred. Finally, the observation of both a single chromatin region (n = 6) or two chromatin regions (n = 2) indicated oocyte activation which, however, was followed by developmental arrest.