Assessment of the need for follicle stimulating hormone in early preantral mouse follicle culture in vitro

Human Reproduction vol.12 no.4 pp.759–768, 1997

Assessment of the need for follicle stimulating hormonein early preantral mouse follicle culture in vitro

R.Cortvrindt, J.Smitz 1 and A.C.Van Steirteghem and the technique for in-vitro culture of carefully characterizedindividually cultured early preantral follicles now permit

Centre for Reproductive Medicine, University Hospital and Medicalassessment of the exact role of each factor added to the medium.School, Dutch-speaking Brussels Free University, LaarbeeklaanUntil now, most authors have recorded mouse follicle in-vitro101, B-1090 Brussels, Belgiummaturation starting from mid-preantral follicles (diameters of1To whom correspondence should be addressed130–340µm) in 5- to 6-day cultures (Nayudu and Osborn,

In two consecutive controlled experiments 160 early pre- 1992; Bolandet al., 1994a,b; Hartshorneet al., 1994; Spearsantral follicles were cultured in order to evaluate effects et al., 1994). Gonadotrophins were invariably added to theof recombinant follicle stimulating hormone (r-FSH) on culture medium. Long-term (10 days) in-vitro culture withoutsurvival, differentiation, oestradiol and inhibin secretion, FSH after isolation of murine cumulus–oocyte complexescumulus mucification and cumulus–corona–oocyte detach- from preantral follicles in groups was shown to be possiblement by human chorionic gonadotrophin (HCG) stimula- when meiosis-inhibiting substances were added to the mediumtion. Nuclear maturation in oocytes was also assessed (Daniel et al., 1989; Eppig and Schroeder, 1989). In morefollowing addition of HCG. A histological analysis of recent experiments with ovarian organ culture from newborncultured follicles was carried out on semi-thin sections at mice, followed by culture of groups of the isolated granulosa–various culture stages. Addition of r-FSH was essential for oocyte complexes, Eppig and O’Brien (1996) used purifiedfollicle survival for 16 days: without r-FSH only 11% of FSH and obtained metaphase-II oocytes in only 6% of thethe follicles survived for 12 days (with r-FSH: 79%) and initially cultured follicles. Since recombinant FSH (r-FSH)none of these mucified after the HCG stimulus. r-FSH became available, it has been used in in-vivo and in-vitropromoted granulosa cell proliferation and antral-like cavity studies in the murine model (Galwayet al., 1990; Mannaertsformation. Without r-FSH, histology of the cultures demon- et al., 1991). Lately, it has been used in short-term cultures (3strated degeneration and reduced granulosa cell prolifera- days) of isolated late-preantral (diameter 180–200µm) mousetion; oestradiol and inhibin production were reduced. This follicles (Almahbobiet al., 1995).study illustrates the essential role of FSH in promoting the In this study we investigated the effect of r-FSH on thein-vitro growth of early preantral mouse ovarian follicles growth, differentiation and secretory capacity of singly culturedand in maintaining the oocyte under meiotic arrest. early preantral follicles (diameter 95–142µm), isolated fromKey words:in-vitro/mouse/ovary/preantral follicles/recombin- 14-day-old prepuberal mice, over a 16 day period (Cortvrindtant FSH et al., 1996). With this culture system we were not aiming to

keep an intact three-dimensional follicular structure, but weaimed rather at a simplified system that could adequatelysupport the oocyte’s needs for growth from the preantral stage

Introduction (50–55 µm) up to a preovulatory size (70–75µm). Folliclematuration was assessed by observing the potential of an HCGIn-vitro maturation of preantral follicle-enclosed oocytes and

of oocyte–granulosa–cell complexes from preantral follicles stimulus to mucify and detach the cumulus–oocyte complexesand to stimulate oocyte nuclear maturation to metaphase II.from mouse ovaries has been achieved successfully by many

researchers (Eppig and Schroeder, 1989; Qvistet al., 1990;Nayudu and Osborn, 1992; Bolandet al. 1994a,b; Hartshorne Materials and methodset al., 1994; Spearset al., 1994; Eppig and O’Brien, 1996).

AnimalsThe maturation media were essentially composed of a richF1 hybrid female mice (C57Bl/6J3CBAca; 14-day-old) werestandard medium, a serum factor and more-or-less purifiedused for dissection of ovarian follicles. These prepuberal micehormones and/or meiosis-inhibiting factors.were killed by cervical dislocation and the ovaries were asepticallyAs was shown nearly 25 years ago by in-vitro experimentsremoved from the body and briefly incubated in L15 Leibovitzon ovaries from infantile mice (Ryle, 1972), growth of one-medium (Life Technologies, Merelbeke, Belgium) supplemented with

and two-cell layer follicles was stimulated by follicle stimulat- 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin anding hormone (FSH), while luteinizing hormone (LH) appeared100 mg/ml streptomycin (all purchased from Life Technologies).to stimulate mitosis in the theca cells. Ryle’s observationsMechanical dissection of the ovaries was done using fine insulinwere made on whole ovaries in in-vitro explants and usingneedles (26 G 1/2; Becton Dickinson, Erembodegem, Belgium).gonadotrophin preparations which at the time contained manyThis technique yielded 30–40 good-quality preantral follicles per

ovary.impurities. The availability of recombinant gonadotrophins

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Culture conditions and media percentage of follicles with at least some theca cells visible on theouter site of the basal membrane. The oocyte–granulosa cell appositionFollicles were cultured individually in 20µl droplets ofα-minimalwas scored as the percentage of follicles with an intact contactessential medium (MEM; glutamax) enriched with 5% FCS,between the granulosa cells and the oocyte. Attachment to the culture10 µg/ml transferrin and 5µg/ml insulin (Boehringer Mannheim,plate was evaluated as the percentage of follicles showing anMannheim, Germany) in 60 mm tissue-culture-treated dishesoutgrowth of fibroblast-like cells from the follicle over the bottom of(Falcon®; Becton Dickinson) covered with paraffin oil (Sigma,the dish that were starting to establish a monolayer around the follicle.Bornem, Belgium). r-FSH (Org 32489; batch IP 190/0824), used in

The survival of intact follicle units was recorded, as well as thea dose of 100 mIU/ml (final concentration), was kindly donated byfrequency of denuded oocytes released during culture at the differentOrganon (Oss, The Netherlands). The FSH activity of the r-FSH wastime intervals of our observations. Follicles were regarded as survivingcalibrated by the manufacturer in the in-vivo Steelman–Pohley ratthe culture conditions as long as the oocyte was surrounded byassay, against an International Standard Preparation (Steelman andgranulosa cells and the complex remained attached to the culturePohley, 1953). The in-vivo bioactivity per ampoule was 166 IUdish. The morphological appearance of growth and differentiation of(relative to I.S. 70/45). The dose of 100 mIU/ml was selected on thethe follicles during culture was classified as follows: (i) ‘follicular’basis of previously published papers showing a maximally stimulatedfollicles, which had kept their spherical structure, i.e. oocyte coveredin-vitro follicular growth at this dose (Nayudu and Osborn, 1992;by granulosa cells surrounded by a basal membrane; (ii) ‘outgrowing’Rose and Kloosterboer, 1995). The final stimulus for progression offollicles, which showed pronounced outgrowth of the granulosa cellsmeiotic maturation was administered by using 1.5 IU/ml HCGthrough the basal membrane. Granulosa cells migrated around the(Chorulon; Intervet, Turnhout, Belgium). The culture dishes werebasal membrane and over the established monolayer of fibroblast-incubated at 37°C, at 100% humidity and 5% CO2 in air (Jouan,like cells covering the original follicular structure; (iii) ‘antral-like’Saint Herblain, France).follicles, which showed translucent areas within the granulosa-cellThe standard culture system (containing r-FSH) can be brieflymass around the oocyte, indicating the formation of antral-like cavities.described as follows. The follicles which contain one or two layers

At the end of each culture (after 12 or 16 days), final oocyteof granulosa cells, an intact basal membrane and a normal centrallymaturity was evaluated by measuring the oocyte’s diameter andlocated oocyte (not collapsed), as judged under the dissecting micro-assessing its ability to undergo GV breakdown and first polar bodyscope, are plated singly in 10µl medium droplets under oil afterextrusion after HCG addition. A histological study of the culturesthree washing steps. After 24 h (day 1), permitting the follicles towas undertaken at 4, 6, 8, 10, 12 and 14 days on semi-thinadapt, a careful characterization of the follicles was done under theplastic sections.inverted microscope equipped with a Hoffman contrast-modulation

system at magnification3400 (Figure 1A). On day 2, 10µl ofHistological analysis of the folliclesmedium was added to each droplet. Subsequent replenishments wereFollicles were grown singly on 13 mm Thermanox glasses (ICNdone every 2 days by changing half (10µl) of the medium.Biomedicals, Asse, Belgium) in 4-well culture dishes in similarThe morphological appearance of the follicles was recorded beforeconditions to those of the culture plates. Follicles were fixed in 2.5%replenishment. In this system we allowed the follicles to plate downglutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) and embedded induring culture (Cortvrindtet al., 1996).Epon as described previously (Cortvrindtet al., 1996). Serial 1µm

Experimental design sections were cut parallel to the bottom of the culture dish and stainedwith Toluidine Blue. The semi-thin sections were observed under aThe effects of r-FSH were studied in long-term culture of earlylight-transmission microscope.preantral follicles. In the first experiment, 80 early preantral follicles

were randomly divided over four culture dishes in individualSampling of culture fluid for oestradiol and inhibin assessment indroplets of medium (20 droplets per dish). Two dishes had 100 mIU/12-day culturesml r-FSH added to the medium, the two others contained no r-FSH

activity. The aim was to observe the cultures for a 16 day period.Culture medium was replenished every 48 h by retrieving and addingDuring a second repeat experiment, a similar number of follicles10 µl of medium. The 10µl samples of retrieved medium from(n 5 80) was involved, and 1.5 IU/ml HCG was added on day 12surviving follicles only were pooled per culture condition. We decidedof culture. to monitor the effect of r-FSH by measuring two important secretory

At the end of each culture period, HCG was added to a finalproducts and regulators of follicular growth: the steroid oestradiolconcentration of 1.5 IU/ml. The response to HCG was assessedand the protein inhibin. Oestradiol was measured using a com-14–16 h later on the basis of mucification of the oocyte–cumulusmercially available radioimmunoassay from Clinical Assays (Sorincomplex, detachment of mucified oocyte–cumulus complex fromFueter, Brussels, Belgium) after an appropriate dilution withthe follicle and the nuclear stage of the oocytes. Under thesteroid-free serum. The oestradiol assay has a measurement range ofinverted microscope the mucification was clearly seen by the expan-4–500 ng/l (ng/l: 272.45 nM/l), a sensitivity of 4 ng/l and a totalsion of the cumulus, which became a geliform mass. After denudationprecision,10% (% CV). The antibody is highly specific for 17β-of cumulus and corona cells, the oocyte was observed and classifiedoestradiol. Inhibin was measured with an enzyme-linked immunosorb-as a germinal vesicle (GV) oocyte when the GV was seen. Disappear-ent assay from Medgenix (Fleurus, Belgium). The antibodies used inance of the GV was notified as GV breakdown, and MII (metaphasethis sandwich assay recognized distinct epitopes of theα-subunit ofII) meant the presence of a polar body in the perivitelline space. human inhibin. Antibodies for inhibin cross-reacted with 32 and 57

kDa inhibin and other precursors, freeα subunits and its precursors.Observation of follicle growth and differentiation The inhibin assay has a total precision,12% (% CV) and a sensitivity

of 0.1 MU/ml. Concentrations of inhibin were expressed in theThe morphological changes during in-vitro culture were observedevery other day under the inverted microscope equipped with a manufacturers’ own units (Medgenix Units). One MU corresponds

to 400 pg of recombinantα-βA human inhibin from Genentech orHoffman contrast-modulation system (Nikon, Tokyo, Japan). Thefollicle diameter was measured between the basal membrane with an to 143 units porc inhibin MRC 86/690. Inhibin was measured in

conditioned medium after appropriate dilution in inhibin-free serum.ocular micrometer. Presence of theca cells was recorded as the

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Figure 1. Specific features of the culture system with and without recombinant follicle stimulating hormone (r-FSH) using invertedmicroscopy with a Hoffman contrast-modulation system. (A) Preantral follicle after mechanical isolation surrounded by two layers ofcuboidal granulosa cells (G), a basal membrane with some attached theca and/or interstitial cells (arrow) (scale bar5 40 µm). (B) Follicleculture on day 4: at the bottom of the plate a monolayer of fusiform cells (F) has developed. This monolayer is overgrown by granulosacells (G) that have proliferated out of the original follicle. The oocyte remains covered by several granulosa-cell layers (arrow) (scalebar 5 80 µm). (C) The follicle culture on day 12 shows a demarcated structure of granulosa cells grown on a monolayer. At the central partof the mound, antral-like cavities became confluent (arrow). The oocyte is covered by a dense cumulus mass (scale bar5 240 µm). (D) Arepresentative example of a day 8 culture containing r-FSH. Antral-like cavities are being formed (arrow) (scale bar5 240 µm).(E) A representative picture of a day 8 follicle from a culture without r-FSH. The monolayer has proliferated on the bottom (arrow). Thegranulosa cells have not proliferated and hence the structure remains ‘follicular’ (scale bar5 40 µm). (F) A representative example of a‘non-surviving’ follicle-unit on day 6 of culture without r-FSH. The oocyte is released from its surrounding granulosa cells which remainattached to the monolayer, which consists clearly of several cell types (arrows) (scale bar5 80 µm).

Dilutions were made in serum provided by the manufacturers, as theResultsimmunoassays were designed for measurements in a serum matrix.

Observations of follicle culturesThe immunoassays used in these experiments were validated in the

After mechanical isolation of the follicles from the 14-day-mouse model and showed linear dilution curves parallel to theold mouse ovaries, those to be cultured were selected understandard curve.

In a side-experiment, the net effect of r-FSH on steroidogenesisthe stereo microscope according to their intactness of structureand inhibin production by the follicles was determined. Standardand depending on whether a clear centrally located oocyte wasprotein supplement of 5% FCS was replaced by 0.1% bovine serumpresent. All follicles necessary for one experiment were pooledalbumin (BSA, fraction V; Sigma) to eliminate any possible sourceand then randomly divided over the different culture conditions.of exogenous LH or androgen-precursor molecules. The mean6 SD oocyte diameter in the selected early

preantral follicles was 556 3.6 µm as judged in a separateStatistical analysisexperiment, where the oocytes were denuded of the granulosaThe effect of r-FSH on follicle survival and differentiation in the twocells immediately after mechanical isolation.experiments was assessed byχ2 analysis for trend. The comparison

Extensive characterization of the early preantral folliclesof the effect of r-FSH on oocyte nuclear maturation and degenerationput into culture was done on day 1 under the invertedwas done on the absolute numbers, with contingency table analysismicroscope with a Hoffman contrast modulation systemfollowed by χ2 analysis. Comparisons between follicle and oocyte

diameters were done using Student’st-test. (Figure 1A). The results are summarized in Table I.

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Table I. Characterization of isolated follicles in culture with or without recombinant follicle stimulatinghormone (r-FSH)

Day 0 Day 1 Day 2No. folliclesat starta Follicle diameter (µm) Percentage of follicles presenting No. attached

to dishMean6 SD Range Presence of Close granulosa cell/

theca cellsb oocyte apposition

16-day culture1 r-FSH 40 1186 14.3 100–142 58 75 48– r-FSH 40 1126 13.2 95–135 72 73 6212-day culture1 r-FSH 40 1096 11.1 88–142 77 85 70– r-FSH 40 1106 10.6 90–142 80 82 82

aFollicle diameter at start was measured 12–24 h after isolation, to allow recovery from manipulation stressand the resumption of a normal spherical structure.bThecal material was not quantified, as the cells were required for later culture. Their presence wasestablished by observation under an inverted microscope equipped with Hoffman modulation atmagnification3400.

In both experiments (16 days and 12 days of cultures) the mIU/ml r-FSH, 80% of follicles reached day 16 of culture andin 58% of the follicles antral-like cavities were observedearly preantral follicles (day 1) had a similar average diameter

(t-test, not significant), i.e. all follicular diameters were between (Figure 1C and Figure 2C).Differentiation and growth during culture progression are88 and 142µm. For most follicles there were at least some

theca or interstitial cells attached to the basal membrane. In depicted by absolute numbers (Figure 2). Addition of r-FSHsignificantly (P , 0.01) enhanced the outgrowth of granulosaat least 58% of the follicles, a clearly visible theca cell layer

could be seen. Attention was paid to the apposition of the cells in all culture dishes (Figure 2B and E). Outgrowth ofgranulosa cells through the basal membrane occurred in almostcoronal granulosa-cell-layer and the oocyte. In 73–85% of the

follicles the apposition was intact. In the remaining follicles, half of the follicles on day 4 of culture. On day 8, mostfollicles had completely lost their follicular shape (Figure 1D).some of the connections between oocyte and granulosa cells

were damaged on a localized side of the oocyte. However, Antral-like cavities were visualized at the earliest on day 8and were progressively formed (Figure 2C and F). On day 12,this slight damage restricted to a part of the oocyte’s surface

did not influence in-vitro growth or differentiation of the 30% of the follicles had cavities and on day 16 almost 60% did.Without r-FSH addition to the medium, granulosa cellfollicle, with growth and maturation of the oocyte.

On day 2 of culture, 48–82% of the follicles showed proliferation was remarkably reduced in all follicles (Figure1E). The oocyte became denuded and was released into theoutgrowth of the cells from the follicle’s surface on the culture

dish, attaching the follicle to the plate. The presence of r-FSH culture droplet (Figure 1F). The remaining follicular structurewas subsequently lost by granulosa-cell dispersion. The forma-in the culture medium had no influence on the formation of

the monolayer of large fibroblast-like cells around the follicle. tion of antral-like cavities was never observed in FSH-freecultures (Figure 2C and F).These fusiform fibroblast-like theca–interstitial cells were the

first to form a monolayer around the follicle on day 4 (FigureHistological observations on serially sectioned follicles1B ). From then on, follicles were observed with the stereo

microscope at each replenishment of the medium and a All follicles described in this section were those which survivedthe culture condition without r-FSH reasonably well. Thecomparison of the cultures with and without r-FSH was made.

Every 2 days, the changes in the follicle cultures were recorded embedding technique used here did not permit retention ofdegenerating follicles, as they did not firmly adhere to theand the surviving follicles were classified either as ‘follicular’,

‘outgrowing or diffuse’, or ‘antral-like’ as described above. Thermanox coverglass and were lost during embedding.Analysis of semi-thin sections (1µm) from follicles culturedResults of the survival and of the evolution of follicle

growth for each of the two experiments are summarized in for 4 days revealed that the original follicle structure remainedwell conserved. No obvious differences were observed betweenFigure 2A and D for each of the two culture plates with and

without r-FSH. After culture day 8, the survival of the follicles follicle culture with or without r-FSH. Within the enclosedoocytes a GV was recognized and the cytoplasm showed ain FSH-free medium showed a dramatic reduction (P , 0.01).

Only 35 and 32% of the follicles survived day 10 in the two homogeneous structure.The 6 day cultures with r-FSH were structurally comparableexperiments. Only an average of 11% of the follicles were

able to survive to day 12 of culture. These follicles were still to the 4-day culture except that granulosa cells had proliferated(Figure 3A). The basal membrane was interrupted in somefollicle-shaped and did not increase obviously in diameter,

indicating a poor granulosa cell proliferation, while only the parts, permitting the outgrowth of granulosa cells which hadcolonized the outer side of the basal membrane. Within thecells (theca–interstitial cells) forming a monolayer in the

bottom of the plate proliferated. In the presence of 100 borders of the basal membrane the granulosa cells remained

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Figure 2. Results are shown per culture day, per plate and per recombinant follicle stimulating hormone (r-FSH) condition. The left panelsdescribe the survival (A) and the differentiation (B andC) of the 16-day cultures whilst the right panels show survival (D) anddifferentiation (E andF) of the 12-day cultures. InA andD, the small numbers at the top of the bars indicate numbers of follicles; shadedbars, r-FSH supplemented cultures; hatched bars, without r-FSH. In the differentiation graphs (B andE) the number of follicles with‘outgrowth’ decreased in time as they differentiated further into structures showing antral-like cavities, which were then classified as antral(C andF).

demarcated, and between cells small lacunae were visible (onset observed within those layers of granulosa cells that weredistant from the oocyte.of antral-like cavity formation?). Without r-FSH, granulosa-cell

outgrowth was only very discrete; most cells remained within Follicles cultured without r-FSH were markedly smaller(Figure 3D). The granulosa-cell mass was still mostly locatedthe basal membrane and seemed rather compact (Figure 3B).

By day 8, follicles cultured with r-FSH had a much more within the basal membrane and was very compact. Thegranulosa cells consisted mainly of a large nucleus, andprominent cellular mass than did those cultured without (Figure

3C). The original follicle was still recognizable as a structure secretory vesicles were never present. Oocytes in these follicleswere very often located eccentrically and sometimes migratedbordered by the remainder of the basal membrane and contained

3–4 loosely interconnected granulosa-cell layers. A large mass through the basal membrane, following the movement of thegranulosa cells.of granulosa cells was polarized on the basal membrane, but

on its outer side. These oval cells showed long protrusions In the follicles cultured for 12 days with r-FSH, oocyteswere still at the GV stage and were surrounded by multiple(sometimes from five layers away) onto the basal membrane.

These cells also contained more cytoplasm than the granulosa layers of granulosa cells (Figure 3E). Most follicles did notshow the original follicular structure and the basal membranecells within the edges of the original follicle. The nucleus was

located at the apical region, and secretory granules were had almost completely disappeared. The granulosa cells were

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Figure 3. Histological characteristics of semi-thin sections. The left panels illustrate representative features of the cultures withrecombinant follicle stimulating hormone (r-FSH), and the right panels those without r-FSH. (A) Follicle culture for 6 days with r-FSH.Granulosa cells have proliferated within the basal membrane and have colonized the basal membrane on its outer side (scale bar5 20 µm).(B) A representative example of a follicle cultured for 6 days without r-FSH. An outgrowth of granulosa cells through the basal membranehas not taken place owing to reduced proliferation of the granulosa cells (scale bar5 30 µm). (C) Follicle of 8 days with r-FSH. Granulosacells have broken through the basal membrane. Note the polarization of granulosa cells on the outer side of the basal membrane (BM)which has not expanded parallel to follicle growth. Granulosa cells are connected to the outer side of the BM through long protrusions(arrow) (scale bar5 40 µm). (D) Follicle cultured for 8 days without r-FSH: oocyte protruding through the basal membrane (arrow), butremaining in contact with granulosa cells (scale bar5 30 µm). (E) Follicle grown for 12 days with r-FSH: the oocyte remains surroundedby 3–4 layers of granulosa cells (cumulus cells); no pyknotic cells are visible (scale bar5 40 µm). (F) Cultures without r-FSH for 10 daysshowed granulosa-cell proliferation mostly within the borders of the basal membrane. Patches of necrosis are visible. The oocyte has agerminal vesicle and cellular organelles are condensed around the germinal vesicle in the cytoplasm, which is a sign of degeneration (scalebar 5 30 µm).

loosely interconnected but clearly separate. The cytoplasm of only sporadically at the border of the antral-like cavities wheresome granulosa cells had become detached and pyknotic.the granulosa cells from the layers most distant from the

oocyte contained many secretory vesicles. In between the Within the follicular cell mass no pyknosis was observed.Without r-FSH, the original follicular structure was stillgranulosa cells, several lacunae had formed; these were

organized radially to the oocyte. Pyknotic cells were found recognizable (Figure 3F). The many granulosa cells were

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densely packed within the basal membrane. The oocyte provided this stimulus was given on day 12. Only 3% of alloocytes remained blocked in the GV stage on day 12. Progres-remaining within the follicle contained a clear GV. Many

granulosa cells were pyknotic. Areas of necrosis were visible sion to metaphase II was achieved by 41% of oocytes; themajority (53%) of the oocytes were blocked at metaphase I; aand antral-like cavity formation was never observed.

Day 12 cultures with r-FSH showed signs of further few oocytes degenerated.differentiation within the granulosa-cell mass. The oocyte wassurrounded by 4–5 layers of tightly packed granulosa cellswhich sent protrusions towards the zona pellucida: thisgranulosa-cell mass formed the cumulus oophorus. This struc-ture most frequently capped the uppermost level of a cylindricalstructure whose wall consisted of 10–12 granulosa-cell layerssurrounding the antral-like cavity. Within the antral-like cavity,detached and degenerating granulosa cells were recognizable.With addition of r-FSH to the culture medium, cellular degen-eration within the granulosa-cell mass was seldom observed.On day 12, all of the enclosed oocytes were still meioticallyarrested at the GV stage. As culture with FSH progressed,the follicles revealed no new characteristics. Without r-FSH,follicles surviving until day 12 showed features similar tothose cultured for 10 days.

Evaluation of oestradiol and inhibin secretion in the condi-tioned medium of 12-day cultures supplemented with eitherFCS (5%) or BSA (0.1%) in relation to r-FSH addition

The measurements of oestradiol and inhibin were done onpooled samples per culture condition, from individual culturedroplets containing surviving follicles. Although the resultsshown in Figure 4 are therefore purely descriptive, they areinformative regarding the large differences observed betweenconditions with and without r-FSH. During the entire cultureperiod with 5% FCS, the follicles without additional r-FSHsecreted small amounts of oestradiol which remained aroundthe level of sensitivity of the radioimmunoassay. With r-FSH,an exponential increase was observed every 2 days. With r-FSH in the medium, inhibin production became measurablein the culture fluids on day 4; inhibin concentrations in mediawithout r-FSH never exceeded 50% of controls. Thus, toexclude a possible source of androgens or LH activity viaFigure 4. The upper panel gives the concentration of oestradiolFCS, follicle cultures were set up in addition using 0.1% BSA(61 SD) in serum-based cultures with (shaded bars) or without

(black bars) r-FSH and of cultures supplemented with 0.1% bovineas protein source. The oestradiol measurements revealed anserum albumin with (horizontal striping) or without (verticalequivalent oestrogen production in serum-free culture supple-striping) r-FSH. The lower panel gives inhibin concentrations (61

mented with r-FSH. Serum-free cultures without r-FSHSD).supplements also produced little oestradiol. The inhibinproduction in serum-free conditions was decreased from day8 onwards; without r-FSH, inhibin production remained lowerTable II. Fate of the oocytes and follicles cultured with recombinant follicle

stimulating hormone (r-FSH) for 16 days and 12 days after administration ofthan in r-FSH-supplemented cultures.human chorionic gonadotrophin (HCG). In both cultures the nuclearmaturation stage of the oocytes was assessed if the oocytes could be denuded.Response of cultured follicles to addition of HCGResults are absolute numbers and percentages (in parentheses)

Early preantral follicles cultured without r-FSH showed poorDuration of No. of follicles Stages post-HCGdevelopment and did not respond to the HCG stimulus byculture

mucification. In the cultures supplemented with r-FSH, the(days) At start At HCG GV MI MII COC Degstimulationresponse of the follicles to the HCG stimulus was significantly

(χ2: 12,6; P , 0.001, contingency table analysis) influenced16 40 31 – 10 (31) 7 (23) 7 (23) 7 (23)

by culture duration (Table II). 12 40 32 1 (3) 17 (53) 13 (41) – 1 (3)In all, 95% of the follicles cultured with r-FSH for 12 days

GV 5 germinal vesicle; MI5 metaphase I oocytes; MII5 metaphase IIdemonstrated mucification in response to HCG. In almost alloocytes; COC5 cumulus–oocyte complexes non-responsive to HCG and for

follicles cultured with 100 mIU/ml r-FSH, the oocyte had the which nuclear staging was not be possible; Deg5 degenerated oocytes innon-responsive follicles.potential to resume meiosis upon addition of the HCG stimulus

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In day 16 cultures, only 54% of follicles showed mucification brane might have been compromised (Chunet al., 1994;Hsuehet al., 1994). Furthermore, an insufficient production16 h after addition of HCG (this was significantly less than in

day 12 cultures;P , 0.001). When mucification took place, of oestrogens within the follicle could have initiated theprocess leading to granulosa-cell death. It has been shown inoocytes within the cumulus complexes were easily freed from

the surrounding cumulus cells. In those follicles from day 16 hypophysectomized rats that oestrogens prevent DNA frag-mentation within ovarian follicles (Billiget al., 1993). Thewhich mucified, enclosed oocytes showed progression to

metaphase II at a frequency (54% mucified follicles; out of cultures lacking r-FSH also more frequently showed an ‘escape’of the oocyte from its original follicular structure. This oocyte-these 42% oocytes progressed to metaphase II) similar to that

in 12-day cultures. On day 16, half of the 46% of the follicles release phenomenon might be explained by a reduced contactbetween oocyte and granulosa cells, which is established bythat had failed to mucify could be dissected only after vigorous

in and out aspiration of the diffuse follicles with a fine glass gap junctions, sites of tenacious adherence between granulosacells and oocyte through the zona pellucida (Anderson andpipette, and degenerated oocytes were found. In the other half,

the oocytes were impossible to retrieve owing to advanced Albertini, 1976). It has been shown that the number ofgap junctions can be increased by administration of FSHdegeneration of the cumulus–oocyte complex. Signs of follicle

and oocyte degeneration (granular oocyte cytoplasm, disruption (Amsterdam and Rotmensch, 1987; Hsuehet al., 1994) aswell as by oestrogens, which are also lacking in the FSH-freeof contacts with the oocyte, GV breakdown, darkening of the

granulosa-cell mass) were observed more frequently in cultures (Merket al., 1972). The release of the oocyte fromthe follicular structure in the absence of r-FSH suggests that16-day cultures (P , 0.01 on oocyte degeneration rate). This

observation suggests that day 16 follicles had already surpassed the specialized contacts are not formed as they would benormally in a growing follicle.the optimal moment of maturity in terms of their response to

the HCG stimulus. In the absence of r-FSH, the differentiation within thefollicle was compromised; antral-like cavity formation wasnever observed. When cultured with 100 mIU/ml r-FSH, the

Discussion formation of small antral-like cavities became visible on day8; these evolved into a large central cavity in 60% of allIn this study, early preantral follicles (stages 3b and 4 from

Pedersen and Peters, 1968) from prepuberal mice aged 14 follicles by day 16. This effect of FSH on differentiation in amurine follicle in-vitro culture model has also been reporteddays were subjected to one of two culture conditions: either

with or without r-FSH. The main point clarified by these previously by others (Gore-Langton and Daniel, 1990; Nayuduand Osborn, 1992; Bolandet al., 1993, 1994a). Moreover,experiments was that addition of FSH was essential for the

development of early preantral folliclesin vitro up to a oestrogens (whose production was impaired in the r-FSH-freecultures) and the presence of an oocyte also play importantfunctional ‘preovulatory’ stage. Only when r-FSH was added

to the culture medium was follicle survival ascertained with a roles in the process of antrum formation (Gore-Langton andArmstrong, 1988).reproducibly high yield and formation of large antral-like

structures became possible. Most follicles cultured without r-FSH became atretic. Onday 12, follicles were still only sporadically alive. However,Opting to isolate the early preantral follicles by mechanical

dissection instead of using dissociating enzymes was based on these follicles were not fully differentiated as no mucificationof the cumulus–oocyte complex response could be induced bya desire to conserve all cell types and receptor systems of the

ovarian follicle for culture. At the age of 14 days, the ovary the HCG stimulus.These results are corroborated by histological observations.of the mouse strain used showed a very large percentage of

follicles containing one or two cuboidal granulosa-cell layers, For the correct interpretation of the observations from histo-logical preparations, it is important to realize that we couldwhich made dissection easy as only very few antral follicles

and no corpora lutea were yet present. Previous studies analyse only those follicles that performed relatively wellwithout FSH. Indeed, the embedding technique was onlyprovided good evidence that these follicles are primed by the

postnatal gonadotrophin rise, which peaks around day 10 of effective on follicles firmly attached to the glass coverslip.The FSH-stimulated follicles showed a greater granulosa-celllife (Stiff et al., 1974). This gonadotrophin surge could make

the follicles sensitive to FSH through the expression of FSH mass with an active or passive disruption of the basal mem-brane, which apparently did not continue to grow further inreceptors on granulosa cells in early-stage follicles (Eshkol

et al., 1970). conjunction with the increased follicular volume expansion bythe proliferating granulosa cells during culture. On the semi-In our in-vitro culture system the sensitivity of the early

preantral follicle stages to r-FSH was clearly observed on day thin sections, differences in follicular differentiation, apartfrom antral-like cavity formation, between the two culture6 of culture by the increased oestradiol and inhibin secretion

in the condition with r-FSH in comparison to r-FSH-free conditions were revealed by the many secretory vesiclesobserved in the profuse cytoplasm of the granulosa-cell layersmedium. Omission of r-FSH from the culture medium resulted

in cell death from day 8 onwards at several foci within the located distally within r-FSH supplemented cultures, whereasno such vesicles were ever seen in semi-thin sections fromfollicle. The vitality of granulosa cells seemed compromised

in the absence of r-FSH. Apart from the role of FSH in r-FSH-free conditions.In contrast to previously published studies on in-vitro follicleavoiding apoptosis in the ovary, the diffusion of several

essential chemical or physical factors through the basal mem- culture in rodents (Gore-Langton and Daniel, 1990; Qvist

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FSH in preantral follicle culture

et al., 1990; Bolandet al., 1994b), no LH activity was present early follicular stages for at least 16 days. Although it hasbeen provenin vivo (Galwayet al., 1990) andin vitro (Eppigin culture medium in this study, as we used r-FSH and no

immunoreactive gonadotrophins could be assayed in the FCS and Schroeder, 1989) that FSH alone can be used to provokeovulation, in our culture system HCG was chosen to inducepreparation used. All FSH preparations used by other authors

had some degree of LH contamination (except Almahbobi in-vitro ovulation, as this permitted us to evaluate indirectlythe presence of the LH receptors and their ability to respondet al., 1995, who cultured larger follicle stages only briefly)

which could have supported LH-dependent actions during to stimulation by HCG. On day 16 of in-vitro culture, theresponse to a stimulatory dose of 1.5 IU HCG/ml resulted inculture. To be completely certain that there were no gonadotro-

phins or androgen contaminants in the culture medium, we full nuclear maturation (MII) in only 23% of the oocytes,while in as many as 46% of the follicles the oocyte had alreadychanged the serum (FCS) supplement to a pure BSA (0.1%)

preparation and got a similar output of oestradiol in the degenerated and was no longer responsive to the stimulus.This observation suggested to us that we had surpassed theconditioned medium. Our long-term cultures were supple-

mented with r-FSH alone in the total absence of LH. In the optimal culture time for obtaining fertilizable oocytes. Thesubsequent culture period of 12 days, chosen to study the fullr-FSH-supplemented cultures, the amount of oestradiol pro-

duced was comparable to previously published data from meiotic capacity of oocytes grownin vitro, was found to bemore suitable in terms of obtaining a higher number ofindividually cultured follicles (Bolandet al., 1994b). As

already suggested by others (Rose and Kloosterboer, 1995), metaphase II oocytes (40%) that could normally lead tofertilization and in-vitro embryo development up to the hatchedthe final concentration of 100 mIU/ml of r-FSH was sufficient

to cover the needs in our culture system (Nayudu and blastocyst stage, as previously reported (Cortvrindtet al.,1996).Osborn, 1992).

Several hypotheses could be postulated to explain the normal In conclusion, the long-term in-vitro culture of early preantralfollicle stages from the mouse requires FSH if full growth andoestradiol production rate in an LH- and androgen supply-free

culture medium. The basal androgen production could have meiotic competence of oocytes are to be obtained. r-FSH isan essential component in this culture system: it stimulatesbeen sufficient during the first culture days to support the

substrate needs for oestradiol. Possibly, other paracrine factors granulosa cell proliferation and differentiation, prevents pre-mature release of the oocyte from the follicle and maintainsproduced by the granulosa cells under the influence of FSH

acting upon theca, such as insulin-like growth factor (IGF)-I it under meiotic arrest. Without addition of any LH activityduring 12-day culture, oocytes are able to reach a diameter ofor inhibin, could have stimulated androgen production (Zachov

and Magoffin, 1995). Inhibin production in our FSH-supple- between 70 and 75µm and meiotic competence. Oocytematurity was better when HCG was administered on day 12mented cultures increased exponentially over time and might

have stimulated thecal androgen production. Furthermore, of culture instead of day 16, with 16 days of culture resultingin an increased rate of oocyte degeneration.granulosa cells in culture could have been transformed during

outgrowth, expressing steroidogenic enzymes not found in theintact follicle, e.g.∆5–3β-, 3α-, 17β- and 20α-hydroxysteroid

Acknowledgementsdehydrogenases or 17α-hydroxylase expression could have

Katy Billooye is thanked for her technical help making the semi-thinbeen amplified (Bjersing and Carstensen, 1967; Fischer andsections through the follicles and Nadia Fenners for typing thisKahn, 1972; Bjersing, 1977). manuscript. Bernadette Mannaerts, PhD from Organon BV (Oss, The

The dependency of our culture system on FSH is differentNetherlands) is thanked for providing the recombinant FSH (Org32489) preparation. Fritz Vanhoef is sincerely thanked for thefrom that of other systems reported (Eppig, 1977; Danielet al.,artwork. This study was financially supported by the Belgian Fund1989; Bolandet al., 1994a,b). Addition of FSH in Boland’sfor Medical Research. J.S. holds a fellowship from the Belgianculture set-up did not reveal any difference in cell number orFund for Medical Research (grant no. G.3C01.93N).

follicle diameter as compared to an FSH-free medium. Thisdifference in FSH requirements between our system and

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