Christian Grøndahl

Meiosis-Activating Sterol Promotes the Metaphase I to Metaphase II Transition and Preimplantation Developmental Competence of Mouse Oocytes Maturing in Vitro

Abstract The objective of this study was to determine the effects of a sterol found in ovarian follicular fluid, known as meiosis-activating sterol (FF-MAS), on the maturation of mouse oocytes in vitro. Possible effects of FF-MAS in promoting the metaphase I (MI) to metaphase II (MII) transition (nuclear maturation) and the competence of oocytes to complete preimplantation embryo development to the blastocyst stage (cytoplasmic maturation) were assessed. Cumulus cell-enclosed oocytes that were compromised in their ability to undergo nuclear maturation and subsequent development because of the age or genotype of the female were isolated at the germinal vesicle stage and matured in vitro using media supplemented with 0 to 20 μM FF-MAS. Oocytes that progressed to MII were inseminated in vitro, and the percentages developing to the 2-cell and blastocyst stages were determined. The sterol was omitted from the media used for oocyte insemination or preimplantation development. FF-MAS promoted a significantly higher percentage of oocytes in all groups to progress to MII in vitro. Moreover, FF-MAS treatment of oocytes maturing in vitro dramatically increased the competence of all but one of the groups of oocytes to complete preimplantation development. Therefore, FF-MAS improved mouse oocyte quality by promoting both nuclear and cytoplasmic maturation in vitro.

Treatment of infertility

J Sùnksen*, DA Ohl, H Momose, FT Rocha, TEP Barros and F Biering-Sùrensen Department of Urology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark, Department of Surgery, Section of Urology, University of Michigan, Ann Arbor, Michigan, USA; Department of Urology, Hoshigaoka Koseinenkin Hospital, Osaka, Japan; Spinal Injury Unit, University of SaÄo Paulo, SaÄo Paulo, Brazil; Centre for Spinal Cord Injured, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

Activation of meiotic maturation in rat oocytes after treatment with follicular fluid meiosis-activating sterol in vitro and ex vivo

Abstract Meiosis-activating sterols (MAS) have been found to induce meiotic maturation in mouse oocytes in vitro. In the present study we have extended these observations by investigating the effects of follicular fluid MAS (FF-MAS) on rat oocyte maturation in vitro and ex vivo. Rat oocytes freed from their follicles were cultured with FF-MAS (0 μM, 1 μM, 3 μM, 10 μM, 30 μM) for 22 h in a medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 250 μM). A dose-dependent significant increase in germinal vesicle breakdown (GVB) was observed after adding FF-MAS to the culture medium in both cumulus-enclosed (CEO) and denuded (DO) oocytes. A time course study (0, 3, 8, 14, and 22 h) showed a significant increase in GVB after 14 h when DO and CEO were cultured in the presence of 10 μM FF-MAS + 250 μM IBMX. Furthermore immature rats were primed with eCG (20 IU) and 48 h later perfused ex vivo for 12 h in a recirculating system with either FF-MAS (0 μM, 10 μM, 30 μM, 60 μM), cholesterol (60 μM), or LH (0.2 μg/ml) in the presence of 200 μM IBMX, respectively. In addition, ovarian perfusion was carried out with FF-MAS (30 μM, 60 μM) or 0.2 μg/ml LH in the absence of IBMX. After 12 h, oocytes were freed from the ovaries and checked for GVB. By using the ex vivo perfused rat ovary, we found that FF-MAS, starting at 30 μM, was dose-dependently able to overcome IBMX-induced meiotic arrest leading to a comparable increase in GVB as was observed for LH. Furthermore, it was found that FF-MAS in the absence of IBMX was also able to induce meiotic maturation. Our data are consistent with the notion that the maturation-inducing effects of FF-MAS are mediated by different mechanisms compared to spontaneous maturation.

Resumption of Meiosis Induced by Meiosis-Activating Sterol Has a Different Signal Transduction Pathway than Spontaneous Resumption of Meiosis in Denuded Mouse Oocytes Cultured In Vitro

Abstract 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 μg/ml) did not influence resumption of meiosis in either group. Dibutyryl cyclic GMP (320 μM) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD, whereas the subtype 5 phosphodiesterase-inhibitor zaprinast (50 μM) 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 μM), 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 μM) than was FF-MAS-induced GVBD. Addition of the mitogen-activated protein kinase (MAPK)-inhibitor PD 98059 (50 μM), phospholipase C-inhibitor U-73122 (10 μM), p21ras-inhibitor lovastatine (250 μM), and the src-like kinase inhibitor PP2 (20 μg/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, p21ras, 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.

Autoradiographic Localization of Specific Binding of Meiosis-Activating Sterol to Cumulus-Oocyte Complexes from Marmoset, Cow, and Mouse

Abstract The sterol, 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol (FF-MAS), isolated from human follicular fluid, can induce resumption of meiosis in denuded and cumulus-enclosed mouse oocytes inhibited by hypoxanthine, IBMX, or dibutyric cyclic adenosine monophosphate. In this study the distribution of FF-MAS binding sites in denuded oocytes and in cumulus-oocyte complexes (COCs) was studied using light microscopic (LM) and transmission electron microscopic (TEM) autoradiography in marmoset, cow, and mouse oocytes. Denuded (n = 39) and cumulus-enclosed (n = 28) marmoset, cow, and mouse oocytes were cultured in the presence of [3H]FF-MAS with and without excess of unlabeled FF-MAS, respectively. In denuded oocytes LM autoradiography demonstrated specific binding to the oolemma and zona pellucida and, to some extent, the cytoplasm. In the nucleus, no specific binding of [3H]FF-MAS was demonstrated. In some COCs the labeling was dispersed throughout the zona pellucida, the oolemma, and the cytoplasm as well as the cumulus cells; whereas in others, only the outer part of the cumulus cells were labeled. TEM autoradiograms of denuded cow oocytes (n = 6) demonstrated that specific [3H]FF-MAS binding was closely related to the oolemma and that a low level of [3H]FF-MAS binding to cumulus cell remnants was present. In conclusion, specific binding of FF-MAS is predominant at the oolemma of denuded oocytes, suggesting the existence of a plasma membrane-associated molecule with affinity for FF-MAS (i.e., a putative FF-MAS receptor).

The effect of FF-MAS on porcine cumulus–oocyte complex maturation, fertilization and pronucleus formation in vitro

Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.

Action of hypoxanthine and meiosis-activating sterol on oocyte maturation in the mouse is strain specific

Follicular fluid meiosis-activating sterol (FF-MAS) is regarded as an important compound relevant to meiotic resumption in mammalian oocytes. The objective of this study was to investigate the influence of FF-MAS on germinal vesicle breakdown (GVBD) and first polar body (PBI) extrusion with regard to culture conditions, state of the oocyte and mouse strain. Denuded oocytes (DO) and cumulus-enclosed oocytes (CEO) were retrieved from PMSG-primed Quackenbush or C57BL/6J x DBA/2 (C57) mice and cultured for 20 h in alpha-MEM medium under the following conditions: (i) 250 micromol/l dibutyryl cAMP (dbcAMP) +/- EGF, 1 ng/ml or FF-MAS, 20 micromol/l; (ii) 4 mmol/l hypoxanthine (HX) +/- EGF or FF-MAS; (iii) HX + EGF + FF-MAS; and (iv) HX + FF-MAS 5 h priming and subsequent culture with HX + EGF. Oocyte GVBD and PBI emission were recorded and stained with Hoechst 33342. Very limited meiotic inhibition was observed in Quackenbush mice in comparison with C57 mice. FF-MAS promoted maturation in C57 DO and CEO and Quackenbush DO. In Quackenbush DO and CEO and C57 DO a significant increase in atypical PBI extrusion occurred, but not in C57 CEO as well as in EGF-treated Quackenbush CEO primed or co-cultured with FF-MAS. These results support a meiosis resumption function for FF-MAS and suggest that in its presence, the quality of the MII oocytes retrieved appears to be influenced by the strain of the mice, the state of the oocyte and the presence or absence of growth factors in the culture medium.

Meiosis activating sterols derived from diosgenin

Continuing research based on the meiosis activation properties of the endogenous sterol FF-MAS is reported. The synthesis and SAR of 16- and 26-substituted sterols are described, utilising diosgenin as starting material. Selected sterols were tested for their ability to induce oocyte maturation in hypoxanthine arrested mouse oocytes in vitro.

Meioseaktivierende Sterole verbessern die Perspektive für die Eizellreifung in vitro

Zwei meioseaktivierende Sterole (MAS) werden physiologischerweise in der Follikelflussigkeit des Ovars sowie im Hodengewebe von verschiedenen Saugetieren, einschlieslich Mensch, angereichert. Es handelt sich in der Follikelflussigkeit um FF-MAS (4,4-Dimethyl-5α-cholesta-8,14,24-triene-3β-ol), im Hoden um T-MAS (4,4-Dimethyl-5α-cholesta-8,24-diene-3β-ol). Beide Substanzen sind Zwischenprodukte in der Cholesterinbiosynthese aus Lanosterin. Sie werden im Ovar mit groser Wahrscheinlichkeit in den Kumuluszellen, im Hoden in den postmeiotischen Spermatogenesestadien gebildet. Durch Gonadotropine, insbesondere durch luteinisierendes Hormon (LH), wird die Produktion von FF-MAS im Ovar deutlich gesteigert. Es kann angenommen werden, dass in vivo LH uber die gesteigerte Produktion von FF-MAS in den Kumuluszellen der Eizelle das Signal fur die Wiederaufnahme der Meiose uber Kommunikationskanale wie„gap junctions“ vermittelt. FF-MAS, aber auch T-MAS, stimuliert die Wiederaufnahme der meiotischen Eizellreifung in Maus und Ratte sowohl in vitro wie auch im ex vivo perfundierten Ovar in der Anwesenheit von Meioseinhibitoren, wie z. B. Hypoxanthin. Die durch die FF-MAS Kultur verbesserte Synchronisierung der meiotischen sowie zytoplasmatischen Reifung von Mauseeizellen geht einher mit einer verbesserten Befruchtungsrate in vitro. FF-MAS stellt somit ein neues interessantes Target fur die Behandlung der Infertilitat in vitro dar. Inwieweit die Verbesserung der Befruchtungsrate einhergeht mit einer verbesserten Praimplantationsentwicklung von Embryonen sowie einer gesteigerten Implantationsrate, bleibt weiteren Untersuchungen vorbehalten.