Jan Lund Ottesen

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 impact of tail tip amputation and ink tattoo on C57BL/6JBomTac mice

Genetic material for polymerase chain reaction (PCR) and Southern blot analysis on transgenic mice is normally obtained by tail biopsy. Additionally, it may be necessary to tattoo the mice, as it is essential to have a good and permanent identification. The aim of this study was to evaluate the effects of amputating the tip of the tail to obtain a biopsy for genetic analysis and of ink tattooing on welfare in C57BL/6J mice, a strain often used as genetic background for transgenes. The behaviour of the animals, fluctuating asymmetry (FA, a measure of developmental instability) and the level of restitution in the remaining part of the tail were evaluated and used for an assessment of the impact of these procedures on the welfare of the animals. One group of mice was marked by tail tattooing at various ages. Another group of mice were tail amputated at 12 or 20 days of age. Body weight and FA were followed, and at the end of the experiment, the level of fear/anxiety was assessed using a light–dark box. In the group of tail-amputated animals observation of climbing behaviour and a beam walking test for balance was performed. Seven weeks after tail amputation, the animals were euthanized. The remaining part of the tail was evaluated histopathologically. Body weight, behaviour in the light–dark box and balance test results were not influenced by tail amputation or tattooing. FA was only transiently increased by tattooing. Climbing behaviour was reduced just after tail amputation at 20 days of age. No signs of neuromas were found in the amputated tails, but seven weeks after amputation a significant number of mice did not have fully regenerated glandular tissue and hair follicles in the tail. It is concluded that both tail amputation and tail tattooing seem to have minor short-term negative effects on welfare and that the tissues on the tail probably do not regenerate fully after amputation.

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.

Eye, body or tail? Thermography as a measure of stress in mice

Infrared thermography has been suggested as a non-invasive, objective tool to evaluate animal welfare. In this study, we investigated: 1) how body temperature, measured through thermal imaging, is affected by different mild stressors frequently experienced by laboratory mice; 2) which methodology to use for assessing temperature variations with infrared thermography; 3) whether the chosen stressors cause anxiety in mice. Eighty C57BL/6 male mice were included in the study. The mice were allocated to either a control group or one of three groups being subjected to a mild stressor once daily for 4 days: 1) anaesthesia with isoflurane for 10 min; 2) handling by scruffing; 3) intraperitoneal injection of 0.2 ml 0.9% saline. On all four intervention days, thermal images were obtained in all groups and all animals were assessed for fur status and body weight. On day five, all animals were tested in the elevated-plus-maze for 5 min. From the thermal images, the maximum eye temperature, the maximum tail base temperature and the average body temperature were obtained. Ten minutes of anaesthesia with isoflurane led to a decrease in maximum eye temperature, average body temperature and maximum tail base temperature. The animals recovered from this drop in temperature within 10 min. No drop in temperature was seen after scruffing or intraperitoneal injection of saline. Based on the number of missing values, intra-rater and inter-rater agreement, the average body temperature was found most ideal for measuring body temperature variations in mice. Finally, the elevated plus maze did not reveal any differences in anxiety between the groups and the body weight did not decrease at any time point during the study.