The sterol 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS [follicular-fluid meiosis-activating sterol]) from human follicular fluid has recently been identified as a compound that induces the resumption of meiosis. FF-MAS and various oxysterols have been reported to transactivate the orphan receptor LXRalpha. The objective was to determine the biological activity of synthetic FF-MAS on the resumption of meiosis and final maturation of mouse oocytes in vitro. In order to evaluate whether LXRalpha might mediate FF-MAS action on the oocyte, we compared the capability of various compounds to activate LXRalpha-dependent transcription and to induce resumption of meiosis in the oocyte assay. Ovaries were isolated from immature mice primed with FSH 48 h before collection. Naked oocytes (NkO) and cumulus enclosed oocytes (CEO) were isolated from follicles. The oocytes were cultured in two groups, NkO and CEO, respectively, in media containing either 3 mM hypoxanthine, 5 microM IBMX, or 0.100 mM dbcAMP to maintain the oocytes in the germinal vesicle stage. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown (GVBD) after 24 h of in vitro culture. FF-MAS overcame the meiotic inhibition by hypoxanthine in both the NkO group and CEO group in a dose-dependent manner within the concentration range 0.07-7 microM. FF-MAS displayed similar potency in all inhibitory agents used. Also, FF-MAS significantly increased the formation of polar bodies in both the CEO and NkO group. The oxysterols 22(R)-hydroxycholesterol (a potent ligand for the LXRalpha receptor), 16-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, as well as cholesterol, were tested without any significant effect on maturation compared to that of controls. Oxysterols and FF-MAS were observed to activate LXRalpha. In conclusion, the results reported here clearly demonstrate that synthetic FF-MAS exclusively is capable of mediating resumption of meiosis in vitro in both NkO and CEO irrespective of the inhibitory substance used. In contrast, the oxysterols and cholesterol had no significant biological activity on this oocyte function, and consequently we found no correlation between LXRalpha activation and meiosis stimulation.

Synthetically produced meiosis-activating sterol, a sterol originally derived from follicular fluid (FF-MAS), induces meiotic maturation of mouse oocytes in vitro. We therefore compared FF-MAS-induced maturation of naked mouse oocytes arrested in prophase I by either hypoxanthine (Hx) or forskolin (Fo) with spontaneous maturation of naked oocytes. FF-MAS-treated oocytes overcame the meiotic block by Hx or Fo, although germinal vesicle breakdown was delayed by 11 h and 7 h, respectively. We also investigated the influence of FF-MAS on chromosome, microtubule, and ultrastructural dynamics in Hx-cultured oocytes by immunocytochemistry and electron microscopy. Similarly to spontaneously matured oocytes, chromosomes became aligned, a barrel-shaped spindle formed, and overall organelle distribution was normal in FF-MAS-matured oocytes. The number of small cytoplasmic asters was elevated in FF-MAS-treated oocytes. Although the number of cortical granules (CGs) was similar to that in spontaneously matured oocytes, the overall distance between CGs and oolemma was increased in the FF-MAS group. These observations suggest that the initiation of meiotic maturation in FF-MAS-treated oocytes in the presence of high cAMP levels leads to a delayed but otherwise normal nuclear maturation. FF-MAS appears to improve oocyte quality by supporting microtubule assembly and by delaying CG release, which is known to contribute to reduced fertilization.

Transgenic mice with tissue-specific expression of the human insulin gene in the beta cells of the pancreas do not produce insulin-specific antibodies when injected with human insulin. Tolerant transgenic mice injected with human or porcine insulin reflect the clinical situation. When injected with bovine insulin the transgenic mice produce antibodies. The potential immunogenicity of 12 recombinant human insulin analogues has been tested in this transgenic model. The analogues were designed either to prevent hexamer formation or to improve chemical stability or both. The analogues have amino acid substitutions or deletions at residue 8, 10 and 21 in the A-chain and residue 3, 9, 27 and 28 in the B-chain. The results show that substitution of single amino acids in the A-chain loop of human insulin for the corresponding amino acids in bovine insulin at residues A8 or A10 is sufficient to elicit an antibody response in responder mice. Only human insulin analogues with substitutions at residues 8 or 10 in the A-chain elicit antibody formation in the transgenic mice, whereas non-transgenic control groups respond to insulin and all analogues. Antibodies developed against the human insulin analogues are cross reactive with recombinant human insulin. Antibodies developed against an immunogenic analogue could therefore neutralize both the analogue and the native insulin and thereby aggravate the patient's condition. This transgenic mouse immunogenicity model should be useful as an in vivo model to map immunogenic areas of recombinant proteins.

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The sterol 4,4-dimethyl-5-cholesta-8,14,24-trien-3-ol (follicular fluid meiosis-activating sterol [FF-MAS]) isolated from human follicular fluid induces resumption of meiosis in mouse oocytes cultured in vitro. The purpose of this study was to examine the hypothesis that differential signal transduction mechanisms exist for FF-MAS-induced and spontaneous in vitro resumption of meiosis in mouse oocytes. Mouse oocytes were dissected from ovaries originating from mice primed with FSH 48 h before oocyte collection. Mechanically denuded germinal vesicle (GV) oocytes were in vitro matured in medium supplemented with hypoxanthine and FF-MAS or allowed to mature spontaneously; both groups were exposed to individual compounds known to inhibit specific targets in the cell. After 20-22 h of in vitro maturation, resumption of meiosis was assessed as the frequency of oocytes in GV breakdown (GVBD) stage. Pertussis toxin (2.5 microg/ml) did not influence resumption of meiosis in either group. Dibutyryl cyclic GMP (320 microM) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD, whereas the subtype 5 phosphodiesterase-inhibitor zaprinast (50 microM) inhibited GVBD in both groups. Microinjection of the catalytic subunit of cAMP-dependent protein kinase into oocytes inhibited spontaneous GVBD, but not FF-MAS-induced GVBD. An inhibitor of cytoplasmic polyadenylation, cordycepin (80 microM), inhibited or retarded spontaneous GVBD to a further extent than it did FF-MAS-induced GVBD. Spontaneous GVBD was more sensitive to the histone H1 kinase-inhibitor olomoucine (250 microM) than was FF-MAS-induced GVBD. Addition of the mitogen-activated protein kinase (MAPK)-inhibitor PD 98059 (50 microM), phospholipase C-inhibitor U-73122 (10 microM), p21(ras)-inhibitor lovastatine (250 microM), and the src-like kinase inhibitor PP2 (20 microg/ml) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD. Both MAPKs, extracellular regulated kinase (ERK) 1 and ERK2, were phosphorylated under FF-MAS-induced meiotic resumption, in contrast to spontaneous meiotic resumption, in which ERK1 and ERK2 phosphorylation occurred 2 h after GVBD. In the present study, we show that FF-MAS acts through an MAPK-dependent pathway, and we suggest that src-like kinase, p21(ras), and phosphoinositide signaling lie upstream of MAPK in the FF-MAS-activated signaling pathway. Clearly, striking pathway differences are present between spontaneous versus FF-MAS-induced meiotic resumption.

In-vitro studies in mouse oocytes have shown that the C-29 endogenously occurring sterol FF-MAS (follicular fluid meiosis-activating sterol) is a potent inducer of meiotic maturation leading to increased fertilization rates. We have used synthetic FF-MAS to induce meiotic maturation in immature human oocytes aspirated from polycystic ovarian syndrome patients. The patients were asked to give written consent to donate half of their aspirated oocytes to investigate the influence of culture conditions on maturation kinetics. The oocytes were aspirated from follicles 8-12 mm in diameter under ultrasound guidance after initial treatment with a gonadotrophin-releasing hormone agonist and s.c. injections of recombinant FSH for 3 days. The other half of the oocytes remained outside this present study. They were reserved for the patients' benefit and were fertilized with appropriate embryo stages being transferred. Fertilization and transfer were not attempted for the study oocytes. Synthetic sterol FF-MAS was added to the culture media at a concentration of 20 micromol/l and nuclear maturation was compared to a control group of oocytes cultured in media only supplemented with vehicle (TCM-199 supplemented with 0.2% ethanol v/v); thus no additional hormones, growth factors, serum or follicle fluid were added. In 31 cycles, oocytes were randomly allocated to one of seven treatment groups: fixed immediately upon aspiration (0 h group) or after in-vitro maturation culture in the presence or absence of FF-MAS for 22, 30 or 40 h respectively. A total of 81 oocytes were processed for light microscopy. The optimal timing of maturation was observed following 30 h of in-vitro culture, when 67% of FF-MAS-treated oocytes had completed nuclear maturation to the metaphase-II stage compared to 29% in the control group. The maturation time of 30 h appeared significantly superior to both 22 and 40 h, but only in the presence of FF-MAS. Cumulus expansion was most profound in the FF-MAS group after 30 h whereas all oocytes had shed the cumulus investment after 40 h. Our observations indicate that FF-MAS positively influences the absolute frequency and the kinetics of human oocytes undergoing nuclear maturation.

To explore the possible signaling pathways of meiosis-activating sterol (MAS)-induced oocyte maturation and to elucidate whether the MAS pathway involves transcription or translation, arrested immature mouse oocytes were cultured with either the protein synthesis inhibitor cycloheximide or the heteronuclear RNA inhibitors alpha-amanitin or actinomycin D, respectively. Moreover, the possible involvement of a G protein-coupled receptor mechanism in MAS-mediated oocyte maturation was explored by influencing oocyte maturation with cholera toxin (CT). MAS-induced oocyte maturation was completely blocked by the addition of 50 microg/ml cycloheximide 4 h before the addition of MAS. Simultaneous addition of MAS and the protein synthesis inhibitor also significantly reduced the meiotic resumption compared to that in MAS-treated controls. In contrast, neither of the treatment regimens to inhibit transcription of DNA to RNA was observed to have any effect on the MAS-induced resumption of meiosis. CT was observed to inhibit MAS-induced, but not spontaneous, oocyte maturation in vitro, suggesting a putative involvement of G protein-coupled receptor mechanism in the MAS mode of action. In conclusion, protein synthesis was found to be an essential requirement for maintaining the oocytes' responsiveness to MAS-induced resumption of meiosis, in contrast to transcription.

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