2014

http://informahealthcare.com/gyeISSN: 0951-3590 (print), 1473-0766 (electronic)

Gynecol Endocrinol, 2014; 30(5): 388–391! 2014 Informa UK Ltd. DOI: 10.3109/09513590.2014.888411

PREANTRAL FOLLICLES

Recombinant activin A enhances the growth and survival of isolatedpreantral follicles cultured three-dimensionally in extracellularbasement matrix protein (matrigel) under serum-free conditions

Yilmaz Guzel1, Gizem Nur Sahin2, Mujde Sekeroglu1, and Alparslan Deniz1

1Department of Obstetrics and Gynecology, Okmeydani Training and Research Hospital, Istanbul, Turkey and 2American Hospital Women’s Health

Center, Assisted Reproduction Unit, Nisantasi, Istanbul, Turkey

Abstract

Development of in vitro technologies that will allow the culture of early stage follicles beforeantral stage is an essential part of research in reproductive biology in order to understand theovarian folliculogenesis better. Current evidence suggests that oocyte and somatic cells-derived growth factors interacting with each other and extracellular matrix proteins atparacrine level are involved in this early, gonadotrophin-independent phase of follicle growth.Basement membrane matrix protein (Matrigel�) is a soluble gel rich in extracellular matrixproteins and growth factors. Activin A promotes preantral follicle growth in vivo by inducingthe proliferation of granulosa cells and by upregulating the expression of FSH receptor andaromatase enzyme. We hypothesized that activin A and matrigel may provide a better in vitroenvironment for early stage preantral follicles. Preantral follicles isolated from 14–21 day oldBALB/c mice were cultured in matrigel ± activin A for four days. The growth (119.4% versus45.4%, p50.05; respectively) and survival rates (76.3% versus 43.7%, p50.05; respectively) ofthe follicles treated with activin A were significantly higher compared to those without activinA. These results suggest that Activin A and matrigel provide a better in vitro milieu for thegrowth of isolated ovarian follicles.

Keywords

3-dimensional culture, Activin A, growth,matrigel, preantral follicle

History

Received 1 December 2013Revised 9 December 2013Accepted 24 January 2014Published online 25 March 2014

Introduction

Understanding folliculogenesis thoroughly is of crucial import-ance for the success of assisted reproductive technologies. Therecruitment of a cohort of antral follicles for growth andthe selection of dominant follicle for ovulation are mediated bythe hormones of the hypothalamic-pituitary-ovarian axis atendocrine level. By contrast, the growth of early stages offolliculogenesis before antral stage is mediated by paracrine-autocrine signals. Current evidence suggests that oocyte andsomatic cell-derived growth factors interacting with each other andextracellular matrix proteins are involved in this early, gonado-trophin-independent phase of follicle growth. To gain informationabout folliculogenesis, several culture methods were developed inthe past to culture isolated follicles at early stages of development,namely primary, preantral and early antral follicles. Underconventional culture conditions on standard plate, the follicleslose their integrity and spherical structure with abolishment ofcross-talk between the oocyte and surrounding somatic cell layers.To provide a better in-vitro environment to enhance the growth andviability of follicles in culture, several culture systems and modelswere introduced. Matrigel� matrix is a reconstituted basementmembrane preparation that is extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellularmatrix proteins. This material, once isolated, is approximately 60%

laminin, 30% collagen IV, and 8% entactin. Entactin is a bridgingmolecule that interacts with laminin and collagen IV, andcontributes to the structural organization of these extracellularmatrix molecules. Matrigel matrix also contains heparan sulfateproteoglycan (perlecan), TGF-b, epidermal growth factor, insulin-like growth factor, fibroblast growth factor, tissue plasminogenactivator and other growth factors which occur naturally in theEHS tumor. Recently, 3-dimensional culture of follicles embeddedin extracellular matrix proteins or matrigel to mimic in vivoenvironment has been reported with success in human, mouse andother species [1–5]. While three-dimensional culture of preantralfollicles in such matrices allowed longer culture period, thestructure and integrity of growing preantral follicles in matrigelresemble those cultured on standard culture plate under serum-freeconditions supplemented with only recombinant FSH and bovineserum albumin. The growth pattern of preantral follicles culturedin matrigel and standard plate are quite similar; characterized byslowly expanding and disorganized granulosa cell layers sur-rounded by dispersed cells in the neighboring zones. Activin A is amember of transforming growth factors superfamily and wasshown to maintain follicle structure and promote follicle growth byinducing the proliferation of granulosa cells and by upregulatingthe expression of FSH receptor and aromatase enzyme [6].Recently it has been shown that activin A increases the survival ofpreantral follicles in the cultured whole ovaries from postnatalmice and promotes follicular integrity in the cultured preantralbovine follicles [7,8]. We therefore investigated in this study if theaddition of activin A to a defined serum-free culture formulationmay help sustain the growth and survival of preantral folliclescultured in matrigel.

Address for correspondence: Dr.Yilmaz Guzel, MD, Department ofObstetrics and Gynecology, Okmeydani Training and Research Hospital,Darulaceze Cad. No:25, Okmeydani, Sisli, Istanbul, Turkey. Tel:+90(532)7479027. E-mail: yguzel47@gmail.com

Gyn

ecol

End

ocri

nol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Lav

al o

n 07

/10/

14Fo

r pe

rson

al u

se o

nly.

Materials and methods

Reagents

Dulbecco’s modified eagle’s medium–F12 (DMEM-F12) waspurchased from Gibco (Invitrogen, Carlsbad, CA). Bovine serumalbumin (BSA), collagenase (from clostridium histolyticum typeIA; cat no. 9891), Dnase-I (from bovine pancreas; cat no. D4263)were purchased from Sigma (St Louis, MO). Recombinant FSHwas obtained from Serono (Serono, Turkey). Growth factor-reduced (GFR) matrigel (cat no. 356230) was purchased fromBD Bioscience (San Jose, CA). Human recombinant activin A(cat no. 114700) was from Calbiochem (EMD Biosciences,San Diego, CA).

Animals and follicle Isolations

Immature 14-day-old BALB/c mice were used in all experiments.The local Institutional Animal Care and Use Committee of ourhospital approved the protocol. The ovaries were removed aftereuthanasia and minced into two or three pieces in pre-equilibratedand HEPES-buffered DMEM-F12 culture medium.Then thepieces were digested with collagenase type IA, Dnase-I inDMEM-F12 supplemented with 5% BSA for 30 minutes at 37 �Cas described previously [1]. Preantral follicle were classified assecondary follicles with multilayer granulosa cells; folliclediameter5200 micron diameter, no antrum formation. Thefollicles were mechanically isolated using 28–30 gauge needlesunder stereomicroscope (Olympus SZX12, Japan) and those withintact basal membrane and theca cells were chosen for culture.

Culture Medium

A defined serum-free culture medium was prepared usingDMEM-F12 supplemented with 100 mIU/mL recombinant FSH,3 mg/mL BSA and 100 U/mL penicillin-G, 100mg/mL strepto-mycin, 0.25 mg/mL amphotericin-B at 37 �C, and 5% CO2 in airwithout or with (30 ng/mL) activin A. Half of the culture mediawas replaced every day.

Preparation of matrigel and follicle culture

First, GFR matrigel was allowed to liquefy at 4 �C and was thendiluted with the culture medium in a 1:1 ratio (100 mL from each)and placed in eight-well-format chamber slides. Isolated follicleswere transferred into matrigel using pipets, then the chamberslides were put in the incubator to allow the matrigel topolymerize for 30 minutes; subsequently, 100mL of culturemedia was added on the top of the solidified matrigel.

Assessment of follicle growth and survival

Follicles were imaged every other day using Olympus IX 71inverted microscope with digital imager under 300 magnifications.Follicle diameter was measured as micrometer (mm) usingOlympus DP2-BSW software (Japan). Follicles with disruptedbasal lamina or somatic cell layers; extruded, damaged or

misshapen oocytes; and those that did not attach to culture platesin the first 24 hours of the culture period were excluded from theanalysis.

Statistical analysis

Follicle diameters were expressed as micrometer [themean ± (SEM)]. The growth of follicles between the groupswere compared using Student’s t-test. Chi-square test was used tocompare the percentages of the growth and survival rates betweenthe groups. The survival curves were created using the Kaplan–Meier method and compared using Log-rank (Mantel Cox) test,p50.05 was considered signficant.

Results

Preantral follicles cultured in matrigel with activin A grew morerapidly and reached significantly larger diameters after four dayculture period. The mean follicle diameters during culture andthe percentages of growth at the end of the culture are shown inTable 1. Even though the mean diameters of the follicles culturedwith and without activin A were comparable at baseline (Day0;101.9 ± 6 versus 93.6 ± 15, p40.05; respectively), the meanfollicular diameter on day 4 was significantly larger in activin Atreated ones compared to those cultured without activin A(223.7 ± 20 versus 136.3 ± 35, p50.05; respectively) on day 4 ofthe culture. The mean diameter of the follicles treated with activinA increased by 119.4% after four days of culture, which wassignificantly higher than 45.4% growth rate of those withoutactivin A (p50.05) (Table 1) (Figure 1).

The growth curves of the follicles treated with and withoutactivin A are shown in Figure 2. Both groups have comparablegrowth curves on the day 1 and 2 of culture. But on the followingdays, 2–4, the follicles grew more rapidly in the presence ofactivin A, and therefore the differences in the mean folliclediameters on these time points were significantly different due toaccelerated growth of preantral follicles induced by activin A.

We also compared the survival curves of the follicles treatedwith and without activin A. As shown in Figure 3, starting as earlyas on day 1 of culture, follicular atresia was observed when activinA was not present. On the following days of culture, folliculardisintegration continued in the activin A (�) group with resultant64.3% of the follicles lost to atresia compared to 23.7 % of activinA-treated group (p50.01). Consequently, the survival curveswere significantly different, showing the survival promotingeffects of activin A on the Log-rank analysis (Hazard Ratio: 5405;95% CI of ratio 1.636 – 17.86).

Discussion

We conducted this study to investigate if activin A improves thegrowth and survival of isolated mouse preantral follicles in 3Dculture with matrigel. Our results showed beneficial effects ofactivin A on these parameters. The follicles maintained theirstructural integrity and, grew and survived better when culturemedia was supplemented with activin A. The follicles had a well-organized structure.

Table 1. The follicle diameters expressed as the mean ± [SEM] micrometer (mm) and the mean diameters of the follicles on each day of culture andtheir percentage of growth.

Culture days

Groups N Day 0 Day 1 Day 2 Day 3 Day 4 % Growth

Activin A (�) 12 93.6 ± 15* 103.1 ± 16y 116.4 ± 19z 124.4 ± 18� 136.3 ± 35x 45.5#Activin A (+) 10 101.9 ± 6* 121.9 ± 6y 157.5 ± 7z 187.7 ± 11� 223.7 ± 20x 119.4#

*,y: p40.05; z,�,x: p50.05 (compared to the control in the same time point; t-test analysis).#: p50.05 (Contingency table analysis, chi-square test).

DOI: 10.3109/09513590.2014.888411 3-D follicle culture activin 389

Gyn

ecol

End

ocri

nol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Lav

al o

n 07

/10/

14Fo

r pe

rson

al u

se o

nly.

Early stages of follicle growth up to antral stage ischaracterized by an activin-rich environment in contrast to inhibindominant growth phase of antral stage follicle, which are recruitedas a cohort for further growth and selected for dominance and

ovulation [6]. Small follicles produce more activin A relative toinhibin A, whereas larger selected antral follicles secrete moreinhibin A [9]. Therefore activin A is required for the growth ofsmall follicles. Accordingly, activin A was shown to promote FSHreceptor expression in immature granulosa cells and preantralfollicles in culture by RT-PCR [8,9], and supress the growth ofprimary follicles while inducing follicular growth at later stages[1,10,11]. Activin A is also involved in the regulation ofaromatase activity, estrogen synthesis, LH receptor expressionand oocyte maturation [12].

Oktem and Oktay showed that activin A promoted thegrowth and survival isolated mouse preantral follicles inmatrigel in a seven-day culture period [1], but the formulationof culture media was different from ours. During this process,the authors also added ITS + 3 (insulin-transferrin-selenite) totheir culture medium. Cossigyn et al. has recently showed thatactivin A alone decreased the proportion of atretic follicles inthe primary and preantral classes, whereas the combinedtreatment of Activin A + FSH increased the proportion ofatretic preantral oocytes when postnatal day 4 mouse ovarieswere treated with activin A (50 ng/ml), with or without FSH(100 ng/ml) after 10 days of culture _ [8]. Their results conflictwith ours since we did not observe any increase in the atreticproportion of preantral follicles after combined treatment with

Figure 2. The growth curves of the folliclescultured with and without activin A. Thefollicles treated with activin A grew fasterand reached the sizes significantly higherthan those cultured without activin A at theindicated time points.

Figure1. Isolated preantral follicles cultured in matrigel with activin A maintained their structure and integrity, and grew faster with the unifromformation of multilayers of granulosa cell in contrast to those which grew slower with defective formation of granulosa cell layers with many dispersedcells surrounding the follicle in the absence of activin A. Scale bar: 100 microns.

Figure 3. The survival analysis and curves of the follicles. Activin Apromoted the survival of preantral follicles in matrigel. At the end of fourday culture period, 64.3% of the follicles underwent atresia withoutactivin A compared to only 23.7% of the activin A treated follicles(p50.01).

390 Y. Guzel et al. Gynecol Endocrinol, 2014; 30(5): 388–391

Gyn

ecol

End

ocri

nol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Lav

al o

n 07

/10/

14Fo

r pe

rson

al u

se o

nly.

activin A + FSH. A possible explanation for this could be thedifferences in the culture model and the age of mice (four dayold), since the authors used postnatal ovarian tissue culture,not isolated follicle culture, making it impossible to evaluatethe pure effects of activin A on isolated follicles due to thepossible interactions of activin A and FSH with the growthfactors originating from different compartments (i.e. theca cells,oocyte and stroma) in the whole ovary.

A novel finding in this study is that the growth pattern ofthe preantral follicles treated with activin A is different fromthose cultured without activin A. Rapidly expanding granulosacell layers were well-organized with a few scattered cellsoutside the follicles, suggesting a beneficial effect of activin Aon the maintenance of follicular structure. This is particularlyimportant since even partial disintegration or disorganizedgrowth of the follicle may interfere with cross-talk between theoocyte and somatic cell compartments leading to follicle atresiaor insufficient growth in vitro. Indeed, this is commonlyobserved when isolated follicles are cultured 2-dimensionallyon standard culture plates. Follicles lose their spherical featureand turn into sessile like flattened structures resulting in theabolishment of the communication between the oocyte andgranulosa cell layers [13].

In conclusion, culturing early stage ovarian follicles inmatrigel provides a 3-D environment rich in extracellular matrixproteins and growth factors resembling physiological in vivomilieu may help promote follicular growth and survival whensupplemented with recombinant activin A.

Declaration of interest

The authours have nothing to declare and no conflict ofinterest.

References

1. Oktem O, Oktay K. The role of extracellular matrix and activin-A inin vitro growth and survival of murine preantral follicles. Reprod Sci2007;14:358–66.

2. Sharma GT, Dubey PK, Meur SK. Survival and developmentalcompetence of buffalo preantral follicles using three-dimensionalcollagen gel culture system. Anim Reprod Sci 2009;114:115–24.

3. Amorim CA, Van Langendonckt A, David A, et al. Survival ofhuman pre-antral follicles after cryopreservation of ovarian tissue,follicular isolation and in vitro culture in a calcium alginate matrix.Hum Reprod 2009;24:92–9.

4. Hovatta O, Silye R, Abir R, et al. Extracellular matrix improvessurvival of both stored and fresh human primordial and primaryovarian follicles in long-term culture. Hum Reprod 1997;12:1032–6.

5. Belli M, Vigone G, Merico V, et al. Towards a 3D culture of mouseovarian follicles. Int J Dev Biol 2012;56:931–7.

6. Oktem O, Urman B. Understanding follicle growth in vivo. HumReprod 2010;25:2944–54.

7. McLaughlin M, Bromfield JJ, Albertini DF, et al. Activin promotesfollicular integrity and oogenesis in cultured pre-antral bovinefollicles. Mol Hum Reprod 2010;16:644–53.

8. Cossigny DA, Findlay JK, Drummond AE. The effects of FSH andactivin A on follicle development in vitro. Reprod 2012;143:221–9.

9. Yamoto M, Minami S, Nakano R, et al. Immunohistochemicallocalization of inhibin/activin subunits in human ovarian folliclesduring the menstrual cycle. J Clin Endocrinol Metab 1992;74:989–93.

10. Liu X, Andoh K, Yokota H, et al. Effects of growth hormone, activinand follistatin on the development of preantral follicle fromimmature female mice. Endocrinol 1998;139:2342–7.

11. Mizunuma H, Liu X, Andoh K, et al. Activin from secondaryfollicles causes small preantral follicles to remain dormant at theresting stage. Endocrinol 1999;140:37–42.

12. Findlay JK. An update on the roles of inhibin, activin and follistatinas local regulators of folliculogenesis. Biol Reprod 1993;48:15–23.

13. Smitz JE, Cortvrindt RG. The earliest stages of folliculogenesisin vitro. Reprod2002;123:185–202.

DOI: 10.3109/09513590.2014.888411 3-D follicle culture activin 391

Gyn

ecol

End

ocri

nol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Lav

al o

n 07

/10/

14Fo

r pe

rson

al u

se o

nly.