Content of meiosis activating sterols in equine follicular fluids: correlation to follicular size and dominance

ELSEVIER

CONTENT OF MEIOSIS ACTIVATING STEROLS IN EQUINE FOLLICULAR FLUIDS: CORRELATION TO FOLLICULAR SIZE AND DOMINANCE

M. Baltsen, 1 I.B. Bogh2 and A.G. Byskov 1 i Laboratory of Reproductive Biology The Rigshospital, DK-2100 Copenhagen, Denmark 2 Department of Clinical Studies, Royal Veterinary and Agricultural University DK- 1870 Frederiksberg C, Denmark Received for publication: Accepted:

December 1, 2000 February 14, 2001

ABSTRACT Meiosis activating sterols (MAS) are pre-cholesterol sterols that can be isolated from follicular fluid (FF-MAS) or testes (T-MAS). Meiosis activating sterols trigger the resumption of meiosis in cultured meiotically competent oocytes. In the present work MAS, cholesterol and progesterone were assayed by HPLC in follicular fluids collected from pony mares at fixed days after the last ovulation. Follicles were divided into two groups according to whether they were aspirated before or after Day 17 after the last ovulation. The latter group was further divided according to whether the follicle diameter was _<22 mm or > 27 mm. Both FF-MAS and T-MAS were detected in almost all samples. Overall, the total amount of MAS in the follicular fluids increased with the size of the follicles but was accompanied by a decrease in the amount of free cholesterol. The amounts of MAS and progesterone in > 27 mm follicles aspirated after Day 17 were significantly higher as compared to the other groups. A transversal cohort analysis showed that the largest follicle at the time of aspiration had the highest level of MAS after day 17 of the cycle, which was not always true for follicle samples aspirated before Day 17 of the cycle. The study demonstrates that the content of MAS in equine follicular fluids increased during follicular maturation concomitant with a decrease in the concentration of free cholesterol. Moreover, MAS concentration is higher in dominant follicles than in subordinate follicles. The MAS may therefore play an as yet unknown physiological role during pre-ovulatory maturation. © 2001 by ElsevierScience Inc.

Key words: meiosis activating sterols (MAS), equine follicular fluid, cholesterol, progesterone, dominance Acknowledgments We are grateful to T. Roed for technical assistance during HPLC assays and to Bente Synnestvedt and Army Pedersen for technical assistance during follicular aspirations. This work was supported by The Danish Medical Research Council (grant 9400824 and 9700832), The Danish Biotechnological Research and Development Programme (grant 9502022), The Danish Environmental Programme and The Danish Agricultural And Veterinary Research Council (grant 9307568). Theriogenology 56:133-145, 2001 ©2001 Elsevier Science Inc.

O093-691X/O1/$-see front matter PII: S0093-691X(01)00549-0

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~TRODUCTION Antral follicles respond to systemic and local factors which collectively select the dominant follicles (1). During the approximately 23-day-long estrous cycle in mares, major and minor waves of follicles develop, where only the major waves give rise to a dominant follicle (22). A major wave may be either primary or secondary. Dominant follicles of primary waves ovulate in estrus. Secondary waves emerge during late estrus or early diestrus and produce dominant follicles that often regress, although some occasionally ovulate during diestrus (9,31). Primary wave follicles are recruited 12 to 14 days before ovulation from the pool of follicles smaller than 6 mm (17). In a model involving follicular ablation, it was found that the time for deviation occurs approximately 6 days after the emergence of follicles larger than 6 mm in a wave (20). Moreover, after Day 17 of the estrous cycle dominance was appointed to follicles that exceeded approximately 27 mm in diameter, whereas follicles smaller than 22 mm appeared to be either subordinate or pre-deviated follicles. On average, follicles between 22 and 27 mm in diameter may be either large subordinate or dominant (9,20), or large follicles from minor waves (22). By such rationale, only follicles that reach a size larger than 27 mm in diameter may be judged as dominant follicles of primary waves in the mare. Mechanisms that confer the selection of the dominant follicle are as yet unknown. In other mammals FSH-receptor expression and estradiol concentration are highest in the dominant follicles after deviation (18,23), but no biochemical signal has been shown to partition follicles in this respect before deviation. Meiosis activating sterols (MAS), originally found in extracts from human preovulatory follicular fluid and bull testes (13), have been proposed as local messengers that trigger the resumption of oocyte meiosis (1). Isolated MAS induced the resumption of meiosis in murine ooeytes cultured with physiological concentrations of hypoxanthine (8,13,29), which is the most probable natural inhibitor of meiosis in mice (16). Two species of MAS were isolated: 4,4dimethyl-5c~-cholesta-8,14,24-triene-313-ol from human follicular fluid (designated FF-MAS), and 4,4-dimethyl-5c~-cholesta-8,24-diene-3[~-ol from bull testes (designated T-MAS). Individual samples of equine follicular fluid also contain MAS (5,10). Both FF-MAS and T-MAS are intermediates in the cholesterol biosynthesis pathway. The FF-MAS is the C14-demethylated product of lanosterol and T-MAS is the C14-reduced product of FF-MAS (2) (Figure 1). The gene that encodes the P45014DM enzyme responsible for the C14-demethylation of lanosterol (Figure 1) has been cloned in rats (34). The P45014DM activity can be induced by gonadotropins in rat ovaries, where it reaches expression levels comparable to those for liver tissue (39). Although MAS-species induce oocyte maturation in vitro, their roles in vivo have not yet been established. It seems, however, that excess of MAS to cultured oocytes not only promotes maturation but also improves cytoplasmic maturation and oocyte survival in vitro (15,30). The objective of the present study was to measure the amount of MAS, their biosynthetic precursor lanosterol, free (underivatized) cholesterol and progesterone in equine follicles at different stages of the follicular phase, using a previously validated chromatographic assay (4). The aim was to test whether the accumulation of MAS differed from groups of dominant and subdominant follicles or follicles aspirated before deviation, and to determine whether MAS concentrations varied according to the follicles of the same cohort.

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P45014DM acetate ~

squalene ~ - - ~ lanosterol -+ FF-MAS ~ T-MAS ~

cholesterol - - - ~ progesterone

Figure 1. Biosynthetic pathway of MAS. A single arrow indicates a single enzymatic conversion step. Underlined substances trigger resumption of meiosis. The P45014DM enzyme indicated converts inactive lanosterol to active FF-MAS. MATERIALS AND METHODS From March 1994 to November 1997 ovarian follicular fluid samples were collected from 5 Norwegian pony mares and one Thoroughbred mare weighing between 350 and 450 kg. The mares were kept indoors in stables and exposed to a 12 h + 12 h light/darkness cycle. During estrus, ovaries and uteri of the mares were examined daily by ultrasonography (Aloka SSD 500, 5 MHz linear transducer, UST 588U-5, Aloka Co Ltd., Tokyo, Japan). Ovulation was considered imminent based on the following characteristics: the largest follicle measured >40 mm and felt soft on palpation per rectum; ultrasonically, the shape of the preovulatory follicle attained a nonspherical form; ultrasonically the outer appearence of the follicle increased in thickness; the endometrial edema was maximal or declined. The day of ovulation was termed Day 0. Follicular Aspirations Follicles > 6 mm in diameter were evacuated by transvaginal ultrasound-guided aspiration, and the follicular content was aspirated into a syringe attached to the aspiration needle, as described earlier (11). The volume of each follicular fluid sample was measured and the diameter of each follicle was calculated by assuming that it was spherical. The follicular samples were centrifuged at 4°C for 10 min at 1000 x g, decanted into cryopreservation tubes (cat. 366524, Life Technologies, T~strup, Denmark) and stored at -18°C for later analysis. A first round of follicular aspirations included the following experimental groups: 1) follicular fluid of the largest follicle from untreated mares (N = 4) on Day 18, 2) follicular fluid from untreated estrous mares (N = 5) when ovulation was considered imminent, and 3) follicular fluid from estrous mares (N = 3) treated intramuscularly with 15 mg Luprostiol (Prosolvin, Intervet Scandinavia A/S, Skovlunde, Denmark) 7 to 8 days before aspiration to induce luteolysis. Aspirations from 2) and 3) were considered Day 23 follicles. In a second round of follicular aspirations, 4 Norwegan Pony mares were assigned to follicular fluid collection on Days 14, 16, 18 and 20. All mares had at least one natural ovulation between follicular aspirations. HPLC Assay Assays of 4,4-dimethylsterols (lanosterol, FF-MAS and T-MAS), cholesterol and progesterone were run as described for human FF (4). In brief: FF was thawed at 37°C and centrifuged for 5 min at 2000 x g. We mixed 1.00 mL thoroughly with 50 p.L 0.3 M NaH2PO4 and 2.50 mL 75% n-heptane:25% isopropanol (v/v) for 2 hours and centrifuged for 5 min at 2000 x g. The organic phase was dried under reduced pressure and reconstituted in mobil phase for HPLC straight phase (SP) assay. Samples were loaded onto a ChromSpherSi, 5 p.m, 250x4.6 mm HPLC column running in (v/v) 99.5% n-heptane:0.5% isopropanol at 1.00 mL/min. Cholesterol

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and progesterone was read from SP chromatograms. The 4,4-dimethylsterol elution window was collected and subjected to reversed phase (P_P) separation by reconstitution in acetonitrile and loading onto a LiChrospher RP-8, 5 gm, 250x4.6 mm HPLC column running in (v/v) 92.5% acetonitrile: 7.5% water at 1.00 mL/min, 40°C. The FF-MAS (8'50"), T-MAS (10'50") and lanosterol (11'40") were eluted as single peaks as determined by ultraviolet absorption between 200 and 300 nm. The limits of quantification (LOQ) were 7 ng/mL for FF-MAS, 23 ng/mL for T-MAS and lanosterol and 3 ng/mL for progesterone. The limits of detection (LOD) were one third of LOQs. Analyte concentrations below LOQ were assigned (LOQ+LOD)/2 and analyte concentrations below LOD were assigned LOD/2 for plotting purposes. Samples were analyzed in duplicate in different assays. Measurements occasionally exceeded the a priori stated limit of 20% inter-assay standard deviation for duplicates. Only 7 of 344 analyte measurements had an SD > 50%. Lanosterol and possibly other 4,4 dimethylsterols may be subject to some degradation in tissue samples when stored over months, even at temperatures below freezing (33). However, extracts from samples were stable when stored at 4°C, in that immediate rechromatography and rechromatography after weeks of storing produced very similar results (data not shown). Size Categories Before analysis, the aspirated follicles were grouped in a priori defined size categories ranging from 12 to 15 mm in diameter (volume 1 to 1.9 mL; Category I), 16 to 20 mm (2 to 4.6 mL; Category II), 21 to 30 mm (4.7 to 14.5 mL; Category III) and > 30 mm (> 14.6 mL; Category IV). Follicles _< 12 mm were not included because of a prerequisite equality of LOQ during HPLC assay, where 1 mL follicular fluid was analyzed. Follicles were randomly chosen for analysis by picking from a group of FFs one FF per follicle size category, if possible, in each aspiration sample. The largest follicle in an aspiration sample was always chosen for analysis. If there were no samples in one of the size categories, an additional sample was chosen from the larger size category. Chronological Arrangement and Cohort Analysis of Follicles From Mares Follicles were arranged according to the day of aspiration in the estrous cycle. Moreover, for each day follicles < 22 mm and follicles > 27 mm were grouped. For cohort analysis, the size of each aspirated follicle from four mares and its content of MAS was plotted from every day of aspiration. The follicle that contained the highest amount of total MAS among the follicles on each aspiration day was marked. Preovulatory follicles (ovulation considered imminent) were regarded as Day 23 follicles in all cases. Statistical Analysis All data are expressed as means +/- standard error of mean (SEM). Often, the data did not meet the requirements for distribution and size that enabled parametrical tests. Therefore, the non-parametrical Kruskal-Wallis method was applied in order to compare group medians (Statgraphics Plus 2.1, Statistical Graphics Corporation, MD). A significance level of P < 5 % was chosen.

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RESULTS Levels of 4-Methylsterols The concentration of total MAS (FF-MAS + T-MAS) in the aspirated follicles ranged from 14.5 to 220 ng/mL. In 53 of the 61 samples in which both MAS species could be quantified, the concentration of T-MAS was higher than that of FF-MAS. Lanosterol levels ranged up to 250 ng/mL and were below LOQ in 2 samples. Progesterone ranged up to 354 ng/mL, whereas 3 samples were below LOQ. Free cholesterol measured between 9.5 and 46.1 ~tg/mL with a mean concentration of 26.2 p.g/mE In only 4 samples the level of FF-MAS was below LOQ and in 6 samples the level was below LOQ for T-MAS (Table 1). Table 1. Range of concentrations of metabolites in equine follicular fluid Analyte

Limit of quantification (ng/mL)

Lanosterol FF-MAS T-MAS Free cholesterol Progesterone a

23 7 23 3

N Numbers below LOQ Range (ng/mL) a 69 69 69 70 66

2 4 6 0 3

0 - 250 0 - 110 0 - 181 9.5 - 46.1 a 0 - 354

Cholesterol values are ~tg/mL

Size Categories By dividing the follicles into the previously defined size categories, the median of lanosterol and total MAS concentrations were significantly higher in the largest category (> 30 mm; > 14.6 mL; Category IV) as compared to the median of the smaller ones (Categories I, II, III) (Figure 2). The concentration of total MAS was 57 ng/mL in Category I and 144 ng/mL in Category IV. The T-MAS increased 198% and contributed 84% of the absolute increase in total MAS between Categories I, II, III follicles and Category IV follicles whereas FF-MAS increased by only 55%. Concomitantly, the amount of free cholesterol decreased significantly from above 30 ~tg/mL in Category I to below 20 p.g/mL in Category IV (Figure 2). Progesterone had the largest relative increase among the analytes and the mean concentration increased 480% from about 20 ng/mL in the smaller size categories (I-III) to just above 100 ng/mL in the largest category (IV). Chronological Arrangement Based on studies on follicular dynamics in mares we divided follicles of each aspiration into a group up to 22 mm in diameter (nondeviated) and a group above 27 mm in diameter (dominant), respectively.

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150 I

E

100

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I Lanoste¢ol~~ FF-MAS ToMAS ~ Total MAS

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)<

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Progesterone

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x x xx × × × × × x xx × x

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I (12-15 mm)

ii

II (16-20 ram) III (20-30 ram)

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x IV (> 30 rnm)

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Figure 2. Influence of follicular size on the concentration of MAS, lanosterol and progesterone (bars, left axis) and free cholesterol (line, right axis) in equine FF. Values are represented by bars with SEM indicated by error bars. Differences in letter indices designate statistical differences for a particular analyte between size categories. The amounts of total MAS were higher in follicles larger than 27 mm in samples taken on Day 18 or later in the cycle as compared to follicles below 22 mm in diameter (Figure 3a). Moreover, concentrations of total MAS in the groups of large follicles from Day 18 or later were higher than all groups sampled on Days 14 or 16. Progesterone displayed a similar but slightly delayed pattern, in that only large, Day 23 follicles seemed different from other day/size groups (Figure 3a). Three groups were compared statistically: 1) follicles aspirated before Day 17, 2) follicles aspirated after Day 17 and up to 22 mm in diameter and 3) follicles aspirated after Day 17 and larger than 27 mm. This arrangement revealed that the large follicles aspirated after deviation had a significantly higher level of MAS and progesterone than the other two groups (Figure 3b). Moreover, there were no significant differences between large follicles before deviation and small follicles after deviation with respect to total MAS or progesterone.

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A 200

F2~2~ Total MAS, < 22 mm Total MAS, > 27 mm Progesterone, < 22 mm Progesterone, > 27 mm

._1

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0 < Day 17; all

> Day 17; < 22 mm > Day 17; > 27mm

Figure 3. Relation between cycle day for aspiration of the follicle and the concentration of total MAS and progesterone (3A) in equine FF. The SEMs are indicated by error bars. The number above the bars indicates analyzed follicles in the different groups. Figure 3b is a statistical comparison of follicles aspirated before and after deviation and grouped into small and large follicles. Differences in letter indices designate statistical differences for a particular analyte between size categories.

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Cohort Analysis For individual mares, the content of total MAS were depicted for individual follicles at each day of aspiration. Figure 4 is a plot of MAS versus aspiration day and follicular size, the latter represented by a circle with a diameter linearly correlated to the follicular diameter. We found that in all four mares the largest follicle on Day 18 and 20 of the cycle also had the highest level of total MAS. However, in only 1 mare onDay 14 and 2 mares on Day 16 did the largest follicles also have the highest MAS content among the cohort of follicles. DISCUSSION This study shows that lanosterol and MAS are present in the sub-micromolar range in equine FF and that their concentrations depend on the size of the follicle and possibly also on selection for dominance. Follicles aspirated as early as Day 14 in the estrous cycle contain detectable levels of MAS, although the average value of total MAS in the preovulatory equine follicles was three times higher (144 ng/mL). In preovulatory FF from women treated with hCG 36 hours before aspiration, total MAS is around 900 ng/mL (4). It is likely that the higher concentrations in the preovulatory FF of these women, apart from possible species differences, reflect differences in LH/hCG tonus during the preovulatory maturation, i.e., large doses of administered exogeneous hCG in IVF women versus the endogeneous LH in the mare. In fact, rat ovaries respond to hCG by elevating P45014DM-activity and thereby increasing the conversion of lanosterol to FF-MAS (2). Moreover, FF-MAS increased in mouse ovaries after an hCG challenge in gonadotropin-primed immature mice (12). In mares, granulosa cells of the dominant follicle also contain the highest density of LH/CG receptors whereas there are no differences in FSH receptor content (19). The data from our cohort study imply that dominance is accompanied by an elevated level of MAS later in the follicular phase compared to other follicles of the same cohort. This may therefore result from increased sensitivity to LH, which corresponds to the induction of MAS by the midcycle LH surge. The finding that MAS is present as early as Day 14 of the mare estrous cycle and increases up to ovulation, is consistent with the LH profile in mares, where LH increases from the nadir several days before ovulation (32,38). The question is whether MAS accumulation is a phenomenon secondary to cholesterol biosynthesis by the granulosa cells during preovulatory follicular maturation, or, whether MAS actually exert a unique effect on their own. The concentration of MAS in FF may result from two processes: one invoked to increase MAS in order to promote or enhance ooeyte maturation and the other to increase the delivery of substrates for steroidogenesis by increasing cholesterol biosynthesis. Although we find a reversed relationship between free cholesterol and MAS in equine FF during follicular maturation, elevated levels of MAS was only apparent in large (> 30 mm) follicles as compared to smaller follicles, whereas free cholesterol also decreased between the smaller follicles. This phenomenon may reflect a delayed response to cholesterol demand or a mechanism devoted to increase signalling molecules for meiosis, or both. In general, cholesterol biosynthesis de novo during steroidogenesis is modulated by accessibility of exogenous cholesterol (35). However, lipoproteins rather than cholesterol synthesis de novo contribute to most of the cholesterol used as substrate for steroid synthesis in ovaries (27). This conforms with the finding that human granulosa cells increase messenger RNA encoding the

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141 Cohort analysis, Individual2

Cohort analysis, Individual1 Total MAS (ng/mL)

Total MAS (ng/mL)

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(3 100 50

0 14

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Cohort analysis, Individual 3

Cohort analysis, Individual 4

Total MAS (ng/mL)

Total MAS (ng/mL)

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V

150

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Day of cycle

ii

oo

0 12

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Figure 4. Transversal cohort analysis of MAS in follicles of four. The concentration of MAS in FF is plotted as a function of follicular size and day of aspiration. The diameter of individual bubbles is linearly related to the calculated diameter of the respective follicle. The largest follicle on each day of aspiration is indicated by a dark color. Day 23 follicles are represented by more than two dark follicles for Individual 1 and 3, indicating that two preovulatory follicles has been measured in different cycles. LDL-receptor as a response to hCG (24). Nevertheless, in rats the midcyclic LH-surge also stimulates HMG-CoA reductase, the key regulatory step in de novo cholesterol biosynthesis (3,35). In addition, porcine granulosa cells increase cholesterol synthesis de novo when stimulated with FSH (6). However, hCG stimulates cholesterol biosynthesis de novo to some

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extent independent of cholesterol availability (3). Moreover, studies from the cow indicate that steroidogenesis by granulosa cells is only dependent on de novo cholesterol synthesis when exogeneous cholesterol is unavailable, as exemplified in the cow (36). Consequently, the increased MAS content in equine FF during preovulatory maturation most likely represents a phenomenon unrelated to steroidogenesis. The amount of MAS that bathes the oocyte during follicular phase maturation could mark qualities related to maturation, ovulation or fertilization (14). This study showed that the concentration of MAS in mare follicles increased approximately 3-fold, from about 50 ng/mL in small antral follicles to above 150 ng/mL in the large preovulatory follicles. The percentage of mature mare oocytes retrieved from follicles during the follicular phase increases with increasing follicular size (25), a relationship also seen in other species. It has been shown that adding FFMAS to cultured mouse oocytes supports microtubule assembly and delays the release of cortical granula (30). Also, a recent study revealed that human oocytes displayed improved meiotic maturation and survival during culture after culture with FF-MAS (15). It is thus possible that MAS in the follicular fluid might improve cytoplasmic factors during oocyte maturation in vivo. The progesterone values measured in the present work are comparable to earlier reported values for mare follicles from untreated animals in the early and late dominant phase and are in accordance with investigations based on immunological methods (21,28,37). Although progesterone production like MAS, is induced by LH, this study indicates that progesterone increases later than MAS and that progesterone levels in the dominant follicle have reached a level above subordinate and pre-deviation follicles only at the preovulatory stage. Belin et al. (7) also found increased progesterone levels in late stage dominant follicles only when follicular growth was ended. Future studies may reveal whether MAS and progesterone differ in response patterns and magnitudes to LH/CG during follicular maturation. In conclusion, this study reveals that in equine FF the concentration of 4,4-dimethylsterols (lanosterol and MAS) increase with follicular size. The concentration of MAS is higher in follicles > 27 mm in diameter as compared to follicles _< 22 a m and follicles aspirated before Day 17 of the estrous cycle. In these respects MAS resembles the pattern observed for progesterone. Moreover, the amount of free cholesterol decreases during follicular maturation at the same time as the 4,4-dimethylsterols increase. Future studies may determine whether accumulation in FF correlate to oocyte morphology, maturation and/or fertilization. REFERENCES 1. 2.

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