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Regular Article |
a Laboratory of Reproductive Biology, The Rigshospital, DK-2100 Copenhagen Ø, Denmark
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, gamete biology, granulosa cells, human chorionic gonadotropin, meiosis, oocyte development, ovary, ovulation, pituitary hormones, progesterone, steroid hormones
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-cholest-8,14,24-triene-3ß-ol, originally isolated from human ovarian follicular fluid (FF-MAS), and 4,4-dimethyl-5-cholest-8,24-diene-3ß-ol, isolated from bull testes (T-MAS). The MAS reside in the postsqualene terminus of the cholesterol biosynthesis pathway and constitute the last two metabolic intermediates of, altogether, three 4,4-dimethylsterols, the shared precursor of which is the meiotically inactive lanosterol (Fig. 1). Culture media formulated with MAS trigger the resumption of meiosis in isolated mouse and human oocytes [1–7].
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In mammals, meiosis is initiated early during life in the female gonads, and oocytes then embark on the diplotene stage of the prophase. Meiosis is arrested in the oocyte until its corresponding follicle enters the gonadotropin-dependent growth phase. In vivo, the dominant follicles respond to the midcyclic gonadotropin surge, which leads to the resumption of oocyte meiosis and ovulation [8]. Oocytes devoid of their cumulus cell lining do not respond to gonadotropins, whereas the cumulus-oocyte complexes (COC) respond by resuming oocyte meiosis when stimulated with FSH in vitro [9]. The murine theca and mural granulosa cells of preovulatory follicles display a high affinity to hCG [10, 11] and FSH [12]. The preovulatory gonadotropin surge therefore signals meiotic resumption in the oocyte through somatic cells. Data support a scenario whereby gonadotropins generate a positive stimulus against a background of meiotic suppression [13]. Also, the meiotic triggering action of FSH in mice requires a syncytial coupling between the oocyte and the cumulus cells by gap junctions [14]. However, culture media conditioned by FSH-stimulated COC stimulate the resumption of oocyte meiosis, an effect suggested to result from MAS secretion by the COC [9]. The signaling between the granulosa cells and the oocyte may therefore be transmitted through an intracellular pathway, via a paracrine (i.e., extracellular) pathway, or both.
A number of reports deal with the action of FF-MAS in particular during oocyte maturation. In vitro, FF-MAS stimulate meiotic maturation [1–3, 5, 6] and improve cytoplasmic maturation [4]. Concentrations needed to overcome physiological levels of the natural meiosis-inhibiting purines for mouse oocytes are approximately 3–4 µg/ml [1–3]. However, this rather operational level may remain largely dependent on the protocol for introduction of the extremely lipophilic molecule into the culture medium, but to our knowledge, no data regarding actual concentrations in the resulting media have yet been reported. The physiological concentration of FF-MAS plus T-MAS in human follicular fluid is approximately 1 µg/ml [15], but so far, no data are available regarding mouse ovaries or mouse follicular fluid.
In vivo, the activity of the FF-MAS-producing P45014DM enzyme (Fig. 1) was induced in rat ovaries by pregnant mare serum gonadotropins [16], but whereas hCG treatment of the intact rat increased the P45014DM transcript in cultured follicles, eCG had no effect in vivo [17]. However, the accumulation of MAS above any critical or control level has not been verified. Due to the multifunctional regulation of the P45014DM enzyme in cholesterol biosynthesis [18, 19], a relation between gene transcription and sterol accumulation may not be straightforward. Nevertheless, FF-MAS accumulates in follicular stimulated prepubertal mouse ovaries in vivo following an ovulatory stimulation in mice [20]. The ability of MAS to trigger meiotic resumption under in vitro conditions in denuded oocytes, combined with its abundance in FF, has prompted the notion that MAS may be the physiological trigger of meiotic resumption following the midcyclic gonadotropin surge [21]. In any case, an endo- or paracrine signaling process that links the gonadotropin surge and meiotic resumption should comprise a diffusible molecule that works on oocytes released from their somatic cell lining and be modulated by gonadotropins in a timed fashion that is compatible with the meiotic processes. The former quality has been ascribed to MAS by several groups, as referenced above, whereas its regulation by gonadotropins is the subject of this study.
The aim of the present study was to examine the induction and timing of the accumulation of 4,4-dimethylsterols comprising meiotically inactive lanosterol and meiotically active MAS [1] in mouse ovaries after stimulation with gonadotropins. Prepubertal mice were primed with serum gonadotropins and subsequently stimulated with hCG. Ovarian sterols were extracted and assayed at predetermined time points following the ovulatory stimulation. The ovaries were examined histologically to follow the timing of the meiotic progression. The data reveal that the intraovarian accumulation of the 4,4-dimethylsterols is highly controlled by gonadotropins in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immature female B6D2 F1 hybrids of C57 black x DBA 2 mice (weight, 12–15 g) were used in all experiments.
Ovarian Stimulation and Tissue Preparation for In Vivo Studies
Female mice were primed with varying doses of i.p. injections of Suigonan (Intervet, Skovlunde, Denmark), that is, a mixture of two-thirds eCG and one-third hCG in terms of gonadotropin activity, to stimulate follicular growth. The priming was given between 1100 h and 1500 h. The animals were then left for 46 h, after which they were given an ovulatory stimulation with hCG (Profasi; Serono, Copanhagen, Denmark) or PBS (control). All hormones were diluted in PBS, and all animals received a similar volume of injection at all times.
Mice weighing 13–17 g at 0–46 h after the hCG stimulation were killed by cervical dislocation. The tissue samples containing whole ovaries were subsequently excised and placed in a plastic vial containing PBS. Uterine remnants, tuba ovarica, and bursa ovarica were cut away under a preparation microscope. Cleansed ovaries were dried briefly on a paper tissue, placed in a glass vial, weighed, and freeze-dried. After drying, the ovaries were weighed again and subjected to organic extraction according to a previously reported method [15]. Dry weight:wet weight ratios were fairly constant (±5%) for similarly treated ovaries. Extracts were dried and reconstituted in 120–130 µl of mobile phase for high-performance liquid chromatography (HPLC; see below). Re-extraction revealed that <5% of sterol analytes remained in the samples after extraction.
Sterol Analysis
The ovarian 4,4-dimethylsterol content was analyzed by HPLC using a previously described, two-step chromatographic method [15]. In brief, freeze-dried ovaries (two or four per sample) were extracted in 1.0 ml of 75% n-heptane:25% isopropanol (v/v). The organic extract was treated for HPLC straight-phase (SP) separation (ChromSpherSi, 5 µm, 250- x 4.6-mm HPLC column, running in 99.5% n-heptane:0.5% isopropanol [v/v] at 1.00 ml/min). The 4,4-dimethylsterols were collected and subsequently resolved by reverse-phase (RP) separation (LiChrospher RP8-EC, 5 µm, 250- x 4.6-mm HPLC column, running in 92.5% acetonitrile:7.5% water [v/v] at 1.00 ml/min and 40°C). The FF-MAS, T-MAS, and lanosterol eluted as single peaks as determined by ultraviolet absorption between 200–300 nm. Before the SP sample analysis, a standard mixture containing 4,4-dimethylsterols, cholesterol, and progesterone (P4) was run three times to tabulate the time window for 4,4-dimethylsterol elution and to confirm the stability of response factors for standards. Lanosterol (L5768; Sigma Chemical Co., St. Louis, MO), cholesterol (C6760; Steraloids, Newport, RI), and P4 (P-0130; Sigma) were obtained commercially, whereas FF-MAS and T-MAS standards were produced in our laboratory as described earlier [15]. All standard curves were linear in the range of all sample values. Recovery during RP separation was assessed by running the standard mixture in SP separations (internal sample set standards) and applying this mixture to RP separations as for unknown samples. These "carrying-over" standards were compared to "direct" RP standards. Chromatographic recoveries for the 4,4-dimethylsterols were assessed by comparing the amount measured in carrying-over standards during RP analysis with the amount injected in SP. Data were excluded for a particular analyte if the carrying-over standard displayed <80% or >120% recovery. The limits of detection (LODs) for individual analytes are given in absolute amounts in Table 1. The actual LODs in terms of milligrams per gram of tissue or parts per million (ppm) were dependent on the weight of the extracted ovaries. The limit of quantification (LOQ) was taken as threefold the LOD. To obtain as many values above the LOQ as possible, SP windows from two samples were sometimes pooled and run together during RP. This operation reduced n to one-half the value for SP samples, but always 
2.
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Tissue Sectioning and Examination
Ovaries from gonadotropin-stimulated mice killed 3, 5, or 7 h after the hCG challenge were isolated as described above, immersed in Bouin fixative for 2 h, and processed for paraffin embedding. Ovaries were serially sectioned at 5 or 30 µm and stained with periodic acid-Schiff and hematoxylin. The sections were examined at 400x magnification, and oocytes of all preovulatory follicles that displayed an intact corona radiata were counted. The morphological characteristics of the oocytes comprised spherical germinal vesicle (GV), GV ondulation (o-GV), chromosome condensation (condensed), GV breakdown (GVB), and chromosomal metaphase I arrangement (MI). The numbers of oocytes with GV, o-GV or condensed chromosomes, and GVB or MI were assessed.
Statistics
Samples with analyte values between the LOQ and LOD or below the LOD were given the value of (LOQ + LOD)/2 or LOD/2, respectively, for graphical purposes. Several sterol levels for control animals were below the LOQ, and the effect of hCG in vivo was therefore evaluated by the nonparametric Mann-Whitney U test. Gonadotropin dose-response with respect to sterol and P4 accumulation was analyzed by a multiple-range test. Free versus total sterols were analyzed by a paired t-test, and the effect of hCG on the amount of total sterols was analyzed by a t-test. All tests were carried out with Statgraphics for Windows Plus 2.1 (Statistical Graphics Corp., Rockville, MD). A P value 
0.05 was considered to be indicative of statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After challenging the gonadotropin-primed mice with either hCG or vehicle, the 4,4-dimethylsterols increased in hCG-treated mice compared to the controls. The sterols reached an intraovarian maximum after 10–22 h (Fig. 2). After 3 h, the levels of the 4,4-dimethylsterols in hCG-treated animals were significantly higher than in control animals (Table 2), and they remained so for at least 46 h. No qualitative or major quantitative differences were observed in the response patterns of the 4,4-dimethylsterols following stimulation with hCG. The control levels of lanosterol tended to be higher than MAS levels, in that lanosterol was detected in 7 of 20 control samples whereas FF-MAS and T-MAS (data not shown) were detected in 2 of 20 and 0 of 20 control samples, respectively, despite very similar LODs for lanosterol and T-MAS and far lower LODs for FF-MAS (Table 1). Progesterone displayed maximum intraovarian appearance 1–3 h after hCG stimulation (i.e., before significant 4,4-dimethylsterol accumulation), and the level of P4 was higher in controls at 10 h after saline injection compared to initial (i.e., 0 h) levels. In general, P4 was increased in hCG-treated animals compared to controls, except for a single time point: After 10 h, P4 in the controls reached a level comparable to the 1–3-h peak for hCG-treated animals.
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Timing of Meiotic Resumption in Gonadotropin-Primed Mouse Ovaries Following hCG Stimulation
Mice were primed with gonadotropins and given hCG as described above. Clear signs of relief from the diplotene arrest, as determined by o-GV/chromosome condensation, were identified 3 h after hCG (Fig. 3). More than 70% of oocytes residing in the preovulatory follicles had resumed meiosis, as determined by o-GV/chromosome condensation at this time, and more than 70% of the oocytes were GVB/MI at 5 h after hCG stimulation (Fig. 4).
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Gonadotropin Dose-Response for Accumulation of FF-MAS and P4
The hCG stimulation was given in doses that varied between 0 and 10 IU to study the response pattern of sterol accumulation and the sensitivity of the mouse ovaries. Due to the qualitative and quantitative equality among the 4,4-dimethylsterols, only FF-MAS were assayed. Mice were killed 5 h after hCG stimulation. Qualitatively, P4 and FF-MAS displayed the same pattern: a bell-shaped response pattern, with maximum accumulation at 2 IU of hCG per animal (Fig. 5). The lack of FF-MAS detection but low P4 detection in the control animals was consistent with the time-response pattern described above. The FF-MAS displayed a sharper increase around the maximum stimulation, as did P4. No difference was found in the amounts of free cholesterol in the ovaries. Ovarian weights differed only slightly at 5 h after hCG. A maximum of 45% increase in wet weight relative to no hCG stimulation was measured when 2 IU of hCG were used.
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In another setup, the gonadotropin priming dose was increased (0–60 IU), whereas the ovulatory stimulation given after 46 h was kept constant (10 IU hCG). Ovarian P4 and FF-MAS accumulation were measured 3 h later. The average ovarian weight increased dramatically with increasing doses of gonadotropins, and it reached a maximum of 112% above the level for unstimulated (i.e., 0 IU) control animals (Fig. 6). The sterol and P4 values were normalized to the ovarian weights to express analyte densities in the tissue (i.e., µg of analyte per g of tissue, or ppm). Progesterone increased with increasing gonadotropin stimulation in the low window (2.4 IU of gonadotropins), when FF-MAS was not induced to accumulate to any significant extent, whereas FF-MAS increased with increasing gonadotropin stimulation in the high window (12 IU gonadotropins), when P4 decreased. Free cholesterol was significantly lower in the unstimulated and the highly stimulated animals compared to the intermediate groups. However, most of the gain in ovarian weight by gonadotropin-stimulated mice may be attributed to the edematous effect of the gonadotropins, which in turn reduces the cholesterol density in the tissue. Progesterone was produced as a response to hCG despite the lack of follicular stimulation (0 IU of gonadotropins), but it declined significantly at >2.4 IU of gonadotropins. The FF-MAS did not accumulate above detectable levels until the priming dose of gonadotropins exceeded 0.49 IU, and it sustained its maximum accumulation between 12 and 60 IU.
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Ovarian FF-MAS Accumulation Is Not Caused by De-Esterification of Sterylester Stores
To investigate the extent of sterol esterification and whether the appearance of gonadal sterol might stem from de-esterification of sterylester stores in hCG-responsive cells, gonadotropin-primed mice were stimulated with hCG or saline as before. The mice were killed 7 h after hCG/saline, and their ovaries were prepared and extracted as described above. One-half of each extract was saponified to hydrolyze steryl esters, and the other half was analyzed without saponification. If FF-MAS was stored as fatty acid esters in the ovaries, saponification of the ovarian extracts from control animals would yield FF-MAS in amounts comparable to those found in extracts of ovaries from hCG-treated animals. The amounts of cholesterol increased after hydrolysis, whereas the amounts of FF-MAS did not (Fig. 7). No difference was found in amount of cholesterol in nonsaponified samples from hCG-treated and control animals, but the amount of total (i.e., free + esterified) cholesterol was significantly lower in the hCG-treated animals compared to control animals. The lack of FF-MAS in controls was not due to MAS degradation during saponification, in that a full FF-MAS recovery was obtained in saponified samples from hCG-stimulated animals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been proposed that MAS acts as the mediator of meiotic resumption after the preovulatory surge of gonadotropins in mammals [9, 21]. Culture media conditioned by FSH-stimulated COC trigger naive, naked oocytes to resume meiosis [9], and inhibitors of P45014DM that are proposed confer the accumulation of MAS sterols induce meiotic resumption in vitro [6, 32]. Operationally, the critical level of FF-MAS in culture is approximately 3–4 µg/ml (or ppm). The total level of MAS in hCG-stimulated mice was significantly higher than the respective control levels after 3 h, at which time they had a tissue density slightly <3 ppm. Assuming an almost even distribution of MAS in the hCG-stimulated ovary due to the antral follicles occupying most of the ovarian volume at this time, these values are theoretically comparable to the levels required to confer an action in vitro. However, MAS peaked between 10 and 22 h after hCG stimulation in vivo and reached at least 15 ppm after 15 h, whereas oocyte prophase/metaphase transitions were apparent in the preovulatory follicles after 5 h. Moreover, GV morphology characteristic of meiotic resumption appeared after 3 h in most preovulatory follicles. Meiosis was therefore resumed in >70% of the preovulatory oocytes at a time when intraovarian MAS had reached <20% of the peak value measured 15 h after hCG, and most MAS was synthesized by the time meiotic resumption had been triggered in the preovulatory follicles. Moreover, no major qualitative or quantitative differences were observed between the accumulatory patterns for inactive lanosterol and MAS. Also, in vitro stimulation of mouse oocytes with FF-MAS result in GVB only after 10–18 h [4]. This is approximately twice the time required for FSH-induced meiotic resumption in vitro, which takes approximately 7 h [13]. Considering the present data and the referred kinetics of FSH and MAS action, the importance of MAS for meiotic progression remains questionable. The postmeiotic MAS accumulation presented here appears inconsistent with gonadotropin-induced meiotic resumption mediated by MAS in vivo.
The accumulation of 4,4-dimethylsterols in the ovaries following stimulation was transient. In mice, induced ovulation takes place approximately 12 h after hCG stimulation [33], which approximately coincides with the intraovarian MAS peak. During ovulation, follicular fluid is extruded into the tuba and peritoneal lumen and is thereby lost from the ovary. Because MAS has been localized to the follicular fluid [1, 15], the decreasing intraovarian content of MAS after 10–22 h may be due to loss of follicular fluid from the ovary as opposed to metabolism by cholesterogenic enzymes.
The production of P4 took place in vivo despite the lack of an exogenous ovulatory stimulation, but it was delayed by 5–14 h compared to exogenously stimulated mice. This delayed P4 production may be a response to an endogenous LH surge. In any case, such an LH surge was not sufficient to stimulate the accumulation of 4,4-dimethylsterols, which corresponds with the lower sensitivity of P4 to hCG stimulation compared to FF-MAS production. It was also demonstrated that the unstimulated, prepubertal mouse ovary was capable of responding to hCG by synthesizing P4, paralleling the production of ovarian P4 by preantral follicles in the rat [34]. However, the 4,4-dimethylsterols did not accumulate in the same unstimulated ovaries, which demonstrated yet another qualitative divergence between the two responses.
The 4,4-dimethylsterol accumulation may result from a gonadotropin-dependent stimulation of enzymes in line of precholesterol biosynthesis, an inhibition of the terminal cholesterogenic enzyme steps, or both. The FF-MAS-producing P45014DM enzyme activity was increased at least six times above control level by eCG in rat ovaries [16]. However, hCG treatment of the intact rat only caused a small increase in the P45014DM transcript in cultured follicles, and eCG had no effect in vivo [17]. The CYP51 gene encodes P45014DM and is a ubiquitously expressed housekeeping gene that is controlled by well-known cholesterol homeostasis factors [18]. However, regulation of the P45014DM enzyme is mainly conferred by a cAMP/cAMP-responsive element modulator in the testis, which is opposed to its regulation by sterol regulatory element-binding proteins in the liver [19, 35]. The upregulation of P45014DM in gonads may therefore result from both lowered cholesterol availability and events that are unrelated to cholesterol availability, of which the former most likely does not result in the accumulation of FF-MAS.
High concentrations of P4 inhibit the multidrug resistance P-glycoprotein-mediated transfer of metabolites in membranes of the endoplasmic reticulum, which causes the accumulation of intermediate steps in cholesterol biosynthesis [36]. Although the intraovarian increase in P4 precedes the accumulation of 4,4-dimethylsterols, accumulation of P4 was temporally delayed in the unstimulated ovaries but occurred without a concomitant accumulation of 4,4-dimethylsterols. The latter could be due to the shorter period during which the putative endogenous LH can act compared to the long-lived hCG in circulation, resulting in P4 accumulation only. This is consistent with the increased hCG sensitivity of this response compared to the sterol response. The results herein therefore neither support nor refute the action of a steroid-dependent accumulation of the precholesterol sterols, but they appear to indicate two prerequisites for ovarian 4,4-dimethylsterol accumulation in mice: an execution of follicular growth, which in the present context was achieved by follicular priming of prepubertal mice with gonadotropins, followed by a massive ovulatory stimulation, which in this case was conferred by exogenous hCG. The ovulatory stimulation initiates metabolic rearrangements that result in 4,4-dimethylsterol accumulation in the ovary. The response may be modulated by, or even dependent on, the accumulation of P4.
The male germ cell plasma membrane contains significant amounts of precholesterol sterols, which may facilitate motility [37, 38]. Moreover, the occurrence of desmosterol in monkey testis correlated to the onset of spermatogenesis and testosterone production [39] and thereby also correlated to LH stimulation of the Leydig cells. Similar relations may exist for the cellular environment of the female germ cell, which proved to be true for hCG-induced sterol accumulation in the ovary during the present study. An altered sterol composition could influence the sterol production and/or the physical qualities of the plasma membrane. The latter may in turn facilitate maturation, as evidenced by increased peripheral granules in FF-MAS-treated versus control oocytes [4]. Nevertheless, the functional significance, if any, of sterol accumulation in the female gonads has yet to be determined.
In conclusion, mouse ovaries synthesize 4,4-dimethylsterols from a prelanosterol precursor in response to hCG stimulation in a time- and dose-dependent manner. The sterol synthesis is dependent on previous follicular recruitment by gonadotropins. The synthesis does not appear to be coordinated with cholesterol production for steroidogenesis, as determined by the uncoordinated accumulation of the 4,4-dimethylsterols and P4 during different stimulatory regimes in the prepubertal mouse. However, the timing of MAS accumulation in the mouse ovaries of follicular-stimulated mice appears to be inconsistent with the concept of MAS as the physiological mediator of gonadotropin-stimulated meiotic resumption but, rather, to be correlated with ovulation. The biological importance of MAS remains puzzling, and a verification of the importance of MAS in oocyte maturation in vivo requires the ability to inhibit cholesterol biosynthesis de novo in ovaries without provoking side effects on other biosynthetic pathways (e.g., steroidogenesis).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Danish Research Council grants 9400824, 9601015, and 9700832. 
2 Correspondence: Mogens Baltsen, Laboratory of Reproductive Biology, The Juliane Marie Center, The Rigshospital, Blegdamsvej 9, section 5712, Copenhagen University Hospital, DK-2100 Copenhagen Ø, Denmark. FAX: 45 35455824; mogens.lrb{at}rh.dk
Accepted: August 6, 2001.
Received: February 26, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-demethylase during the preovulatory period. Biol Reprod 2001; 64:299-309-demethylase, a cytochrome P450 whose expression is regulated by oxysterols. Arch Biochem Biophys 1996; 329:73-81[CrossRef][Medline]-demethylase (CYP51)—a cholesterol biosynthetic enzyme involved in production of meiosis activating sterols in oocytes and testis. Pflueg Arch 2000; 439: (suppl 3): R56-R57[CrossRef][Medline]-demethylase (CYP51) in spermatids. Mol Endocrinol 1999; 13:1951-1962This article has been cited by other articles:
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  S. Cukurcam, I. Betzendahl, G. Michel, E. Vogt, C. Hegele-Hartung, B. Lindenthal, and U. Eichenlaub-Ritter Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes Hum. Reprod., March 1, 2007; 22(3): 815 - 828. [Abstract] [Full Text] [PDF]
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C.L. Marin Bivens, B. Lindenthal, M.J. O'Brien, K. Wigglesworth, T. Blume, C. Grondahl, and J.J. Eppig A synthetic analogue of meiosis-activating sterol (FF-MAS) is a potent agonist promoting meiotic maturation and preimplantation development of mouse oocytes maturing in vitro Hum. Reprod., October 1, 2004; 19(10): 2340 - 2344. [Abstract] [Full Text] [PDF]
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C. Bergh, A. Loft, K. Lundin, S. Ziebe, L. Nilsson, M. Wikland, C. Grondahl, J.-C. Arce, and for the CEMAS II Study Group Chromosomal abnormality rate in human pre-embryos derived from in vitro fertilization cycles cultured in the presence of Follicular-Fluid Meiosis Activating Sterol (FF-MAS) Hum. Reprod., September 1, 2004; 19(9): 2109 - 2117. [Abstract] [Full Text] [PDF]
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C. L. Marin Bivens, C. Grondahl, A. Murray, T. Blume, Y.-Q. Su, and J. J. Eppig Meiosis-Activating Sterol Promotes the Metaphase I to Metaphase II Transition and Preimplantation Developmental Competence of Mouse Oocytes Maturing in Vitro Biol Reprod, May 1, 2004; 70(5): 1458 - 1464. [Abstract] [Full Text] [PDF]
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M. Cotman, D. Jezek, K. F. Tacer, R. Frangez, and D. Rozman A Functional Cytochrome P450 Lanosterol 14{alpha}-Demethylase CYP51 Enzyme in the Acrosome: Transport through the Golgi and Synthesis of Meiosis-Activating Sterols Endocrinology, March 1, 2004; 145(3): 1419 - 1426. [Abstract] [Full Text] [PDF]
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total sterols > free sterols (paired t-test)