This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Fibroblast growth factor-10 maintains the survival and promotesthe growth of cultured goat preantral follicles

R.N. Chavesa,*, I.B. Lima-Verdeb, J.J.H. Celestinoa, A.B.G. Duartea, A.M.C.V. Alvesa,M.H.T. Matosc, C.C. Campelloa, K.P.O. Named, S.N. Báod, J. Buratini Jre,

J.R. Figueiredoa

a Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA), Faculty of Veterinary Medicine, State University of Ceará,Av. Paranjana 1700, Campus Itaperi, Fortaleza, 60740-903, CE, Brazil

b Technology and Research Institute, Tiradentes University, Aracaju, 49032-490, SE, Brazilc Nucleus of Biotechnology Applied to Ovarian Follicle Development, Federal University of São Francisco Valley, Petrolina, 48902-300, PE,

Brazild Laboratory of Electron Microscopy, Department of Cell Biology, University of Brasilia, Campus Darcy Ribeiro, Brasília, 70910-900, DF,

Brazile Department of Physiology, Institute of Biosciences, State University Paulista, Botucatu, 18618-000, SP, Brazil

Abstract

The aim of the present study was to investigate the effects of fibroblast growth factor-10 (FGF-10) on the survival, activation(transition from primordial to primary follicles), and growth of goat preantral follicles cultured in vitro. Pieces of ovarian cortexwere cultured for 1 and 7 d in the absence or presence of FGF-10 (0, 1, 10, 50, 100, and 200 ng/mL). Noncultured and culturedtissues were processed and analyzed by histology, transmission electron microscopy, and viability testing. Results showed that after7 d, a greater percentage (79.9%) of morphologically normal follicles (containing an oocyte with regular shape and uniform cytoplasm,and organized layers of granulosa cells without a pyknotic nucleus) was observed when cultured with 50 ng/mL of FGF-10 whencompared with other concentrations of FGF-10 (0 ng/mL, 67.3%; 1 ng/mL, 68.2%; 10 ng/mL, 63.3%; 100 ng/mL, 64.4%; 200 ng/mL,52.7%). Ultrastructural analyses and viability testing using fluorescent markers confirmed the follicular integrity of FGF-10 (50ng/mL)-treated fragments after 7 d of culture. After 7 d, all FGF-10 concentrations reduced the percentage of primordial follicles andincreased the percentage of developing follicles. In the presence of 50 ng/mL of FGF-10, follicles increased in diameter after 7 d ofculture when compared with other concentrations tested. In conclusion, this study demonstrates that FGF-10 maintains the morpho-logical integrity of goat preantral follicles and stimulates the growth of activated follicles in culture. The culture conditions identifiedhere contribute to the understanding of the factors involved in goat early follicular development.© 2010 Elsevier Inc. All rights reserved.

Keywords: Goat; early follicles; viability; ovary; FGF-10

1. Introduction

The fibroblast growth factor (FGF) family consti-tutes one of the most important groups of paracrinefactors that act during cell development [1] and consistsof at least 23 different signaling polypeptide members[2]. Several studies have demonstrated that this family

* Corresponding author. Tel.: �55.85.3101.9852; fax: �55.85.3101.9840.

E-mail address: rncvet@gmail.com (R. N. Chaves).

Available online at www.sciencedirect.com

Domestic Animal Endocrinology 39 (2010) 249–258www.domesticanimalendo.com

0739-7240/10/$ – see front matter © 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.domaniend.2010.06.006

Author's personal copy

plays an important role in maintaining homeostasis inmany tissues and organs [3]. Fibroblast growth factorsare involved in various cellular processes, includingchemotaxis, cell migration, differentiation, cell sur-vival, apoptosis, embryonic development, angiogene-sis, wound healing, and oncogenesis [4,5]. In addition,experimental studies have shown the critical involve-ment of the FGF family and its receptors in the regu-lation of reproductive processes, particularly in ovarianfolliculogenesis [6–8]. Some FGFs are involved inregulation of preantral and antral follicular develop-ment, angiogenesis, survival, and proliferation of gran-ulosa cells [9,10]. However, some FGFs inhibit steroi-dogenesis in both theca and granulosa cells in thebovine [7]. These biological activities are mediatedthrough 1 of the 4 FGF receptors (FGFR 1–4), a com-plex family of transmembrane receptor tyrosine kinases[11].

Among the members of the FGF family, fibroblastgrowth factor-10 (FGF-10), also known as keratinocytegrowth factor-2 (KGF-2), is a 13.9-kD heparin-bindingprotein. Fibroblast growth factor-10 promotes epithelialcell motility, differentiation, migration, and woundhealing [12]. In mice, FGF-10 is essential for the nor-mal development of lungs, limbs, teeth, skin, mammarybuds, the stomach, and the heart [13–18]. Expression ofFGF-10 has also been found in the adult ovine uterus[19] and the bovine corpus luteum [20], and FGF-10mRNA has been detected in human normal ovariantheca, stroma, and endometrial stromal cells [21]. In thebovine ovary, FGF-10 mRNA was detected in thecacells, oocytes of antral follicles, and in primordial,primary, and secondary follicles [7]. Immunohisto-chemistry revealed the presence of FGF-10 protein inoocytes of bovine preantral and antral follicles and intheca and granulosa cell layers of antral follicles. Also,thecal expression of FGF-10 mRNA changes with fol-licle viability [7].

Although FGF-10 expression was detected in allclasses of preantral follicles, it is unknown whether itplays a role in the mechanisms controlling preantralfolliculogenesis. Therefore, the hypothesis testedwas that FGF-10 may affect the in vitro survival anddevelopment of goat preantral follicles. Consideringthe ovarian FGF-10 expression pattern in ruminantsand its role in cell differentiation in other organs, thisstudy aimed to determine if FGF-10 affects the invitro survival, activation (transition from primordialto primary follicles), and growth of early goat pre-antral follicles.

2. Materials and methods

2.1. Source of ovaries

Ovaries (n � 12) from 6 adult, nonpregnant, mixed-breed goats (Capra hircus) that were 1–3 y of age werecollected from a local slaughterhouse. All animals werecyclic and in good body condition. Immediately postmor-tem, the ovaries were washed in 70% ethanol for 10 s andthen washed again in minimum essential medium (MEM)supplemented with 100 �g/mL penicillin and 100 �g/mLstreptomycin. The pairs of ovaries were transported to thelaboratory in MEM at 4 °C within 1 h postmortem [22].Unless otherwise mentioned, the chemicals used in thepresent study were purchased from Sigma Chemical Co.(St. Louis, MO, USA).

2.2. Experimental protocol

In the laboratory, the ovaries were stripped of sur-rounding fat and fibrous tissue, and the ovarian cortexwas recovered and divided into 13 pieces approxi-mately 3 mm � 3 mm (1 mm thick) in size using aneedle and scalpel under sterile conditions. For eachanimal, 1 slice of tissue was randomly selected andimmediately fixed by immersion for 12 h in Carnoy,dehydrated in ethanol, and embedded in paraffin (Ve-tec, Rio de Janeiro, Brazil). Sections of 7 �m werestained with periodic acid-Schiff–hematoxylin (PASstaining system; Sigma) for histological analysis (freshcontrol, day 0), whereas a smaller fragment (1 mm3)was cut from the previous one and subsequently fixedfor ultrastructural examination. The remaining slicesof ovarian cortex were cultured individually in 1 mLof culture medium in 24-well culture dishes; theculture conditions were 39 °C in an atmosphere of5% CO2 in air. The basic control medium, referred toas �-MEM�, consisted of �-MEM (pH 7.2–7.4) sup-plemented with insulin-transferrin-selenium (10�g/mL insulin, 5.5 �g/mL transferrin, 5.0 ng/mLsodium selenite), 0.23 mM pyruvate, 2 mM glu-tamine, 2 mM hypoxanthine, and 1.25 mg/mL bovineserum albumin. The medium was supplemented withrecombinant human FGF-10 (PeproTech, Inc., RockyHill, NJ, USA) at different concentrations (0, 1, 10,50, 100, or 200 ng/mL). Ovarian slices from eachanimal were cultured for 1 or 7 d in medium supple-mented with different concentrations of FGF-10, andeach treatment was repeated 6 times, thus using theovaries of 6 different animals. The culture mediumwas stabilized at 39 °C for 4 h prior to use and wasreplenished every second day. The concentrations ofFGF-10 used in this experiment were based on data

250 R.N. Chaves et al. / Domestic Animal Endocrinology 39 (2010) 249–258

Author's personal copy

previously published in several species (human [23],bovine [7], goat [24], and mouse [25]).

2.3. Morphological analysis and assessment of invitro follicular growth

Fresh control and cultured tissues were fixed inCarnoy for 12 h and then dehydrated in increasingconcentrations of ethanol. After paraffin embedding(Vetec, Rio de Janeiro, Brazil), the wax blocks contain-ing the goat tissues were completely and serially sec-tioned at a thickness of 7 �m, and every fifth section wasmounted on an individual glass slide and stained using thePAS staining system. The stage of follicular development(eg, primordial, primary, and secondary follicles) of seri-ally sectioned ovarian tissues and their survival were as-sessed microscopically. Coded anonymized slides wereexamined using a Nikon microscope (Japan) under 400�magnification. Care was taken to count each follicle onlyonce, as performed in our earlier studies [26]. To avoiddouble counting, each follicle was examined in everysection and matched with the same follicle on adjacentserial sections, which ensures that each follicle wascounted only once, regardless of its size.

The developmental stages of preantral follicles havebeen previously defined [27] as primordial (1 layer offlattened granulosa cells around the oocyte) or devel-oping follicles. Developing follicles can be subdividedinto intermediate (both flattened and cuboidal granu-losa cells around the oocyte), primary (a single layer ofcuboidal granulosa cells around the oocyte), and second-ary (2 or more layers of cuboidal granulosa cells aroundthe oocyte and no sign of antrum formation) categories. Inthe current study, follicles were individually classified asmorphologically normal when an intact oocyte waspresent and when the follicle was surrounded by granu-losa cells that were well organized into 1 or more layersand had no pyknotic nucleus. Atretic (degenerated) folli-cles were defined as having a retracted oocyte possiblydisplaying a pyknotic nucleus, and/or disorganized gran-ulosa cells. Overall, 180 preantral follicles were evaluatedfor each treatment (30 follicles per animal � 6 repeti-tions), and the percentages of healthy primordial and de-veloping follicles were calculated on day 0 (fresh control)and after culture in each treatment.

To evaluate follicular activation (transition from pri-mordial to primary follicles, when surrounding squa-mous pregranulosa cells become cuboidal and begin toproliferate) and growth, only morphologically normalfollicles with a visible oocyte nucleus (equatorial sec-tion) were recorded, and the proportion of primordialand growing follicles was calculated at day 0 (fresh

control) and after 1 or 7 d of culture with treatment. Inaddition, from the basement membrane, major and minoraxes of each oocyte and follicle were measured using alight microscope fitted with an eyepiece micrometer(Zeiss, Cologne, Germany) under 400� magnification.The average of these 2 measurements was used to deter-mine the diameters of both the oocyte and the follicle.

2.4. Ultrastructural analysis of goat preantral follicles

For more in-depth evaluation of follicular morphol-ogy after histological analysis, ultrastructural studieswere performed on fragments of fresh control and treat-ment groups that maintained follicular morphology andpromoted growth. Briefly, 1-mm3 pieces of goat ovar-ian tissues were fixed in 2% paraformaldehyde and2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer(pH 7.2) for 4 h at room temperature (approximately25 °C). After fixation, fragments were postfixed in 1%osmium tetroxide, 0.8% potassium ferricyanide and 5mM calcium chloride in 0.1 M sodium cacodylatebuffer for 1 h. Subsequently, the samples were dehy-drated through an acetone gradient, and the tissues wereembedded in Spurr’s resin. For light microscopy stud-ies, 3-�m-thick sections were cut on an ultramic-rotome (Reichert Supernova, Heidelberg, Germany)and stained with toluidine blue. Ultrathin sections of 60to 70 nm thickness were contrasted with uranyl acetateand lead citrate and examined under a Jeol 1011 (Jeol,Tokyo, Japan) transmission electron microscope. Thedensity and integrity of ooplasmic and granulosa cellorganelles, as well as vacuolization and basementmembrane integrity, were evaluated. For ultrastructuralanalysis, 3–5 follicles were examined per group.

2.5. Assessment of preantral follicle viability byfluorescence microscopy

Based on the results of the morphological and ultra-structural analyses, the viability of follicles culturedwith the concentration of FGF-10 that maintained fol-licular morphology and ultrastructural integrity wasfurther analyzed using fluorescent probes.

Additional pairs of goat ovaries (n � 2) were col-lected from a slaughterhouse, and then cut into frag-ments at the laboratory. One of these fragments wasimmediately processed for follicle isolation (fresh con-trol), and the remaining fragments were cultured for 7 dwith FGF-10 (50 ng/mL), as described above. After theculture period, fragments were processed for mechan-ical isolation using the method described by Lucci et al[28]. Briefly, using a tissue chopper (The Mickle Lab-oratory Engineering Co., Gomshal, Surrey, UK) ad-

251R.N. Chaves et al. / Domestic Animal Endocrinology 39 (2010) 249–258

Author's personal copy

justed for a sectioning interval of 75 �m, samples werecut into small pieces. Next, the fragments were placedin MEM, resuspended 40 times using a large Pasteurpipette (approximate diameter 1600 �m), and resus-pended again 40 times with a smaller Pasteur pipette(approximate diameter 600 �m) to dissociate preantralfollicles from the stroma. The material obtained waspassed through 100-�m nylon mesh filters, resulting ina suspension containing preantral follicles less than 100�m in diameter. This procedure was carried out at roomtemperature within a 10-min time frame.

Isolated preantral follicles were analyzed using a2-color fluorescence cell viability assay based on thesimultaneous detection of live and dead cells with cal-cein-AM and ethidium homodimer-1, respectively. Thefirst probe detects intracellular esterase activity in via-ble cells, whereas the latter labels nucleic acids innonviable cells with plasma membrane disruption. Thetest was performed by adding 4 �M calcein-AM and 2�M ethidium homodimer-1 (Molecular Probes, Invitro-gen, Karlsruhe, Germany) to a suspension of isolatedfollicles and incubating at 37 °C for 15 min. After beinglabeled, follicles were washed once by centrifugation at100 � g for 5 min and resuspended in MEM. Follicleswere then mounted on glass microscope slides in 5 �Lof antifading medium (DABCO, Sigma, Deisenhofen,Germany) to prevent photobleaching, and they werethen examined using a DMLB fluorescence microscope(Nikon, Eclipse 80i, Tokyo, Japan). The emitted fluo-rescence signals of calcein-AM and ethidium ho-modimer-1 were collected at 488 and 568 nm, respec-tively. Oocytes and granulosa cells were consideredviable if their cytoplasm stained positively with cal-cein-AM (green) and their chromatin was not labeledwith ethidium homodimer-1 (red).

2.6. Statistical analysis

The mean number of surviving follicles at all stages(primordial and developing), obtained after 1 or 7 d inthe various culture conditions, were initially submittedto Kolmogorov-Smirnov and Bartlett’s tests to confirmnormal distribution and homogeneity of variance, re-spectively. Analysis of variance was then performedusing the GLM procedure of SAS, version 8 (SASInstitute, Inc., Cary, NC, 1999), and Dunnett’s test wasapplied to compare FGF-10-treated groups against the 0ng/mL of FGF-10 group. The Student-Neuman-Keulstest was used for comparisons among FGF-10 concen-trations and numbers of surviving follicles between 1and 7 d of culture [29]. Data for follicular viability,assessed through fluorescence microscopy, were ana-

lyzed as dispersion of frequencies using the chi-squaretest. Differences were considered to be significant whenP � 0.05, and results were expressed as the mean �standard error of means (SEM).

3. Results

3.1. Effects of FGF-10 on follicular survival

In the present study, 180 follicles were analyzed byclassical histology in each treatment, totaling 2340 pre-antral follicles (1104 primordial, 825 intermediate, 384primary, and 27 secondary). Fig. 1A-E shows morpho-logically normal follicles after 0 (control), 1, and 7 d ofculture with FGF-10 at 0 ng/mL and 50 ng/mL; Fig. 1Fshows a degenerated follicle after 7 d of culture withFGF-10 at 100 ng/mL. In degenerated follicles, weobserved retracted oocytes, pyknotic nuclei, and disor-ganized granulosa cells.

The percentage of morphologically normal folliclesin the fresh control (noncultured tissue) and after 1 or7 d of culture, in the absence or presence of FGF-10,are shown in Fig. 2. In all treatments, after 1- or 7-dculture, a reduction (P � 0.05) in the percentage ofmorphologically normal follicles occurred compared tothe fresh control, except in the treatment with FGF-10at 50 ng/mL after 1 d of culture (P � 0.05). In addition,a significantly greater percentage of morphologicallynormal follicles was observed in tissues cultured with50 ng/mL of FGF-10 than with 100 and 200 ng/mL(P � 0.05). Moreover, treatment with 200 ng/mL ofFGF-10 caused a significant reduction in the percentageof morphologically normal follicles when compared to0 ng/mL of FGF-10 dose or the other concentrations ofFGF-10 (P � 0.05). After 7 d, a greater percentage(P � 0.05) of normal follicles in tissues cultured with50 ng/mL of FGF-10 was observed compared to allother treatments with FGF-10. Similar to the resultsafter 1 d of culture, the greatest concentration ofFGF-10 (200 ng/mL) caused the smallest percentage ofmorphologically normal preantral follicles (P � 0.05)compared with 0 ng/mL of FGF-10 dose and all otherconcentrations of FGF-10. With the progression of theculture period from 1–7 d, a decrease (P � 0.05) in thepercentage of morphologically normal follicles in alltreatments was observed (P � 0.05).

3.2. Follicular activation during in vitro culture ofcortical fragments

The percentage of morphologically normal primor-dial and developing follicles (intermediate, primary,

252 R.N. Chaves et al. / Domestic Animal Endocrinology 39 (2010) 249–258

Author's personal copy

and secondary) in fresh tissue or in tissues cultured for1 or 7 d with different treatments is shown in Fig. 3.Fresh ovarian tissues contain predominantly primordial

follicles (74.5%) and a smaller percentage of develop-ing follicles (25.5%). After 7 d of culture, in all treat-ments, the percentage of primordial follicles (Fig. 3A;P � 0.05) was reduced, concomitant with a significantincrease in the percentage of developing follicles (Fig.3B; P � 0.05) compared to the fresh control. Moreover,on day 1, the addition of FGF-10 (200 ng/mL) to theculture medium significantly increased follicular tran-sition from primordial compared to the fresh control, 0ng/mL of FGF-10 dose, and other groups cultured withFGF-10, except when 100 ng/mL of FGF-10 was used.

With the progression of the culture period from 1–7d, the percentage of developing follicles in all treat-ments increased (P � 0.05) in contrast to nonculturedcontrol tissues. The mean distribution of developingfollicles after 7 d of culture in all cultures combinedwas 64.0%, 33.5% and 2.5% for intermediate, primary,and secondary follicles, respectively.

3.3. Evaluation of oocyte and follicle diameter infresh and cultured tissue

Table 1 shows the follicle and oocyte diametersbefore and after in vitro culture. After comparing frag-

Fig. 1. Representative image of goat ovarian fragments on day 0 (control, A) and after 1- or 7-day culture in the presence of 0 ng/mL (B-C) and50 ng/mL (D-E) of FGF-10 or after 7-day culture with 100 ng/mL (F) of FGF-10. Degenerated preantral follicles often displayed oocyte retraction(F, arrow) and disorganization of granulosa cell layers. Scale bars represent 50 �m (E) and 20 �m (A-D; F). O, oocyte; Nu, oocyte nucleus; GC,granulosa cells. �400, periodic acid-Schiff-hematoxylin.

Fig. 2. Percentage (mean � SEM) of morphologically normal pre-antral follicles in the fresh control (noncultured) and after 1 or 7 dculture with varying concentrations of FGF-10. * Differs significantlyfrom the fresh control (P � 0.05). a,b,c Differs significantly amongconcentrations within each day of culture (P � 0.05). � Differssignificantly with the progression of the culture period from days 1 to7 in the same treatment (P � 0.05).

253R.N. Chaves et al. / Domestic Animal Endocrinology 39 (2010) 249–258

Author's personal copy

ments cultured for 1 d with noncultured fragments, nosignificant differences were observed regarding folliclediameter. However, after 7 d, follicular diameter wassignificantly greater in ovarian tissues cultured with 50ng/mL of FGF-10 (P � 0.05) than those cultured withthe same concentration at day 1 and the other treat-

ments after 7 d. In addition, no significant influence ofFGF-10 on oocyte diameter was observed after 1 and7 d of culture (P � 0.05).

3.4. Ultrastructural features of cultured follicles

Ultrastructural features of follicles evaluated in thefresh control (Fig. 4A) and in the group treated with 50ng/mL of FGF-10 (Fig. 4B) were similar. These folli-cles exhibited sparse vesicles spread throughout theooplasm and had an intact oocyte, nuclear and base-ment membranes, as well as a large oocyte nucleus withdecondensed chromatin and a visible nucleolus. In ad-dition, the organelles—such as rounded mitochondriawith peripheral cristae and continuous mitochondrialmembranes and both smooth and rough endoplasmicreticulum—were uniformly distributed in the homoge-neous ooplasm. Granulosa cells were ultrastructurallynormal and well organized around the oocyte, showingan elongated and large nucleus with irregular mem-brane and a high nucleus:cytoplasm ratio.

3.5. Assessment of follicle viability after culture

Preantral follicles (n � 30) were analyzed after 7 dof culture in �-MEM� supplemented with 50 ng/mLFGF-10. After this quantitative analysis, all follicles(100%; P � 0.05) remained viable and were stainedgreen by calcein-AM assays (Fig. 5), confirming thatthis treatment was able to maintain follicular viability.In addition, the percentage of viable follicles was sim-ilar to that seen in the fresh control group (P � 0.05).

4. Discussion

The present study reports the influence of FGF-10on the development of goat preantral follicles in vitro,

Fig. 3. Percentage (mean � SEM) of (A) primordial and (B) devel-oping follicles (intermediate, primary, and secondary) in noncultured(fresh control) and tissues cultured for 1 or 7 d in the presence ofvarying concentrations of FGF-10. * Differs significantly from thefresh control (P � 0.05). a,b Differs significantly among concentra-tions within each day of culture (P � 0.05). � Differs significantlywith the progression of the culture period from days 1 to 7 in the sametreatment (P � 0.05).

Table 1Oocyte and follicle diameters (mean � SEM) in noncultured ovarian fragments (fresh control) and in ovarian fragments cultured for 1 or 7 dtreated with varying concentrations of FGF-10. The doses of FGF-10 were in ng/mL.

Non-culturedCultured

Follicle diameter (�m) Oocyte diameter (�m)

69.22 � 1.56 49.59 � 1.41

Day 1 Day 7 Day 1 Day 7

FGF-10 (0) 70.92 � 2.00 72.27 � 2.17b 52.68 � 1.79 50.68 � 1.85FGF-10 (1) 72.15 � 2.00 73.39 � 1.32b 51.29 � 1.58 54.69 � 1.40FGF-10 (10) 71.84 � 1.65 69.83 � 2.11b 51.91 � 1.18 49.44 � 2.44FGF-10 (50) 74.16 � 2.16d 80.50 � 2.20a,c 52.22 � 1.31 55.00 � 2.04FGF-10 (100) 69.83 � 2.29 69.99 � 2.05b 48.20 � 1.43 49.90 � 1.92FGF-10 (200) 71.69 � 1.53 68.29 � 1.69b 49.13 � 1.55 49.29 � 1.68

a,b Differs among concentrations within a column (P � 0.05).c Differs from the fresh (noncultured) control (P � 0.05).d Differs with the progression of the culture period from days 1–7 in the same treatment within a row (P � 0.05).

254 R.N. Chaves et al. / Domestic Animal Endocrinology 39 (2010) 249–258

Author's personal copy

demonstrating its importance in the maintenance ofviability and further follicular growth. Maintenance offollicular viability is the prerequisite for culture ofpreantral follicles. The current study shows that addi-tion of 50 ng/mL of FGF-10 was effective in maintain-ing a high percentage of morphologically follicles incortical tissue cultured for 7 d. Thus, this FGF-10concentration acts as a survival factor and/or atresiainhibitor, which progressively occurs with time in cul-ture. This result is in agreement with the results of

Goldfarb [30], who demonstrated that FGF familymembers have a critical role in cell survival. Moreover,Kwabi-Addo et al [31] demonstrated that FGFs caninhibit cell death in several tissues by preventing DNAinjury induced by oxygen reactive species, which re-duces the formation of H2O2 [32].

Alternatively, in our study, extreme FGF-10 concen-trations (ie, 0, 1, 10, 100, or 200 ng/mL) caused adecrease in follicular viability after 7 d of culture. Weinfer that small concentrations of FGF-10 may be in-

Fig. 4. Electron micrographs of follicles before and after 7 d of culture. (A) Morphologically normal preantral follicle from noncultured ovarian fragments(control; original magnification 3000�); (B) Morphologically normal preantral follicles after culture with 50 ng/mL FGF-10 (original magnification3000�). O, oocyte; nu, nucleolus; GC, granulosa cell; ne, nuclear envelope; m, mitochondria; bm, basement membrane. Scale bars � 10 �m.

Fig. 5. Viability assessment of goat preantral follicles using fluorescent probes. (A) An isolated preantral follicle after culture with 50 ng/mLFGF-10 that was classified as viable (B) because cells were labeled by calcein-AM (green fluorescence). Scale bars � 50 �m.

255R.N. Chaves et al. / Domestic Animal Endocrinology 39 (2010) 249–258

Author's personal copy

sufficient to promote the maintenance of goat follicularsurvival. Nevertheless, high concentrations of FGF-10may have a detrimental effect on follicular health, prob-ably increasing follicular apoptosis via the activation ofthe mitogen-activated protein kinases (MAPK) path-way [23], which is involved in both cell survival andapoptosis, with the specific cell response depending onthe cellular context [33].

This study demonstrates a decrease in the number ofprimordial follicles of goat ovarian cortical tissue and aconcomitant increase in the number of developing fol-licles in all treatments compared to the fresh controlduring in vitro culture for 7 d in a defined medium.Fibroblast growth factor-10 did not have an additionalbeneficial effect, except on day 1 of culture, in whichthe concentration of 200 ng/mL of FGF-10 promoted agreater percentage of developing follicles than 0 ng/mLof FGF-10. Some studies using several growth fac-tors and hormones have been performed with goatspecies, showing similar results of follicular activa-tion [23,24,34–37]. Moreover, another study hasshown that 10 ng/mL of FGF-2, another member of theFGF family, did not influence bovine primordial folli-cle activation [38]. According to other authors [39,40],the transition from primordial to developing folliclesduring in vitro culture occurs spontaneously, that is, with-out the addition of growth factors or hormones. In vitroculture conditions may support the development of folli-cles enclosed in ovarian fragments, possibly through therelease of stimulatory factors in the oocytes and the gran-ulosa, theca, and stromal cells [36]. Another hypothesis isthat in vitro culture appears to induce activation of pri-mordial follicles because the media are richer in nutrientsand/or oxygen than the ovarian cortex in vivo, because thecortical region of the ovary is less vascularized than themedullary region [40].

An increase in follicular diameter was observed onlyafter culture of ovarian tissue for 7 d with 50 ng/mL ofFGF-10 compared to the fresh control and otherFGF-10 concentrations. However, FGF-10 did not haveany influence on oocyte diameter. The FGF family isemerging as a group of factors that are potentiallyimportant for follicle growth [6]. Others have shownthat FGF-10 is a mitogenic factor for epithelial cells[31,41,42] and has a high affinity for the FGFR-2breceptor [42]. Granulosa cells express the FGFR-2breceptor [10], and FGF-10 derived from the oocyte andtheca cells most likely acts as a paracrine signal amongoocytes and theca and granulosa cells. Most likely, 50ng/mL of FGF-10 was a concentration sufficient tostimulate the growth of numerous early unilaminar fol-

licles, namely, primordial and intermediate, but it wasnot enough to promote further primary follicle growth.Moreover, in the present study, it was not possible tovisualize any significant increase in oocyte growth,because primordial and intermediate follicles had sim-ilar diameters [43].

Our results on follicular survival after classical his-tology were confirmed by ultrastructural analysis. Us-ing transmission electron microscopy, it was observedthat important cellular structures such as mitochondria,the endoplasmic reticulum, granulosa cells, and thebasement and nuclear membranes were preserved after7 d of culture in the presence of 50 ng/mL of FGF-10.Therefore, this technique is an important tool to detectearly morphological changes after follicular culture invitro. In addition, according to Lopes et al [44], follic-ular atresia changes occur first at the ultrastructurallevel, before they can be identified by light microscopy.

In addition to ultrastructural analysis, preantral fol-licles cultured for 7 d with 50 ng/mL of FGF-10 werefurther analyzed using a more accurate method basedon fluorescent probes, which confirmed the previousresults obtained with light and electron microscopyregarding follicular survival. Thus, viability assessmentappears be a reliable, practical, and fast method toanalyze follicular viability. Recently, this method hasalso been used successfully to evaluate preantral folli-cle viability in goats [45,46].

In conclusion, this study demonstrates that FGF-10acts in a concentration (50 ng/mL)-dependent mannerto maintain the morphological integrity of goat prean-tral follicles, and to stimulate the growth of activatedgoat preantral follicles in culture. These results providea basis for future studies of the appropriated cultureconditions required to support complete preantral fol-licle growth and subsequent oocyte maturation in vitro.

Acknowledgments

This research was supported by grants from CNPQ(RENORBIO: grant number 554812/2006-1) andFINEP–Brazil. R. N. Chaves is a recipient of a grantfrom CNPq (Brazil). The authors declare that there isno potential conflict of interest that can be perceived asprejudicing the impartiality of the research reported.

References

[1] Powers CJ, Mcleskey SW, Wellstein A. Fibroblast growth fac-tors, their receptors and signaling. Endoc Relat Cancer 2000;7:165–97.

256 R.N. Chaves et al. / Domestic Animal Endocrinology 39 (2010) 249–258

Author's personal copy

[2] Ornitz DM, Itoh N. Fibroblast growth factors. Genome Biol2001;2:1–12.

[3] Baird A, Hsueh AJ. Fibroblast growth factor as an intraovarianhormone: differential regulation of steroidogenesis by an angio-genic factor. Regul Pept 1986;16:243–50.

[4] Basilico C, Moscatelli D. The FGF family of growth factors andoncogenes. Adv Cancer Res 1992;59:115–65.

[5] Bottcher RT, Niehrs C. Fibroblast growth factor signaling dur-ing early vertebrate development. Endocr Rev 2005;26:63–77.

[6] Buratini J, Teixeira AB, Costa IB, Glapinski VF, Pinto MGL,Giometti I, Barros CM, Cao M, Nicola ES, Price CA. Expres-sion of fibroblast growth factor-8 and regulation of cognatereceptors, fibroblast growth receptor-3c and -4, in bovine antralfollicles. Reproduction 2005;130:343–50.

[7] Buratini J, Pinto MGL, Castilho AC, Amorim RL, Giometti IC,Portela VM, Nicola ES, Price CA. Expression and function offibroblast growth factor 10 and its receptor, fibroblast growthfactor receptor 2B, in bovine follicles. Biol Reprod 2007;77:743–50.

[8] Machado MF, Portela MF, Price CA, Costa IB, Ripamonte P,Amorim RL, Buratini Jr J. Regulation and action of fibroblastgrowth factor 17 in bovine follicles. J Endocrinol 2009;202:347–53.

[9] Sordoillet C, Savona C, Chauvin MA, De Peretti E, Feige JJ,Morera AM, Benahmed M. Basic fibroblast growth factor en-hances testosterone secretion in cultured porcine Leydig cells:site(s) of action. Mol Cell Endocrinol 1992;89:163–71.

[10] Berisha B, Sinowatz F, Schams D. Expression and localizationof fibroblast growth factor (FGF) family members during thefinal growth of bovine ovarian follicles. Mol Reprod Dev 2004;67:162–71.

[11] Berisha B, Steffl M, Amselgruber W, Schams D. Changes infibroblast growth factor 2 and its receptors in bovine folliclesbefore and after GnRH application and after ovulation. ReprodRes 2006;131:319–29.

[12] Ware LB, Matthay MA. Keratinocyte and hepatocyte growthfactors in the lung: roles in lung development, inflammation andrepair. Am J Physiol 2002;282:924–40.

[13] Sekine K, Ohuchi H, Fujiwara M, Yamasaki M, Yoshizawa T,Sato T, Yagishita N, Matsui D, Koga Y, Itoh N, Kato S. FGF10is essential for limb and lung formation. Nat Genet 1999;21:138–41.

[14] Komi-Kuramochi A, Kawano M, Oda Y, Asada M, Suzuki M,Oki J, Imamura T. Expression of fibroblast growth factors andtheir receptors during full-thickness skin wound healing inyoung and aged mice. J Endocrinol 2005;186:273–89.

[15] Marguerie A, Bajolle F, Zaffran S, Brown NA, Dickson C,Buckingham ME, Kelly RG. Congenital heart defects inFGFR2-IIIb and FGF10 mutant mice. Cardiovasc Res 2006;71:50–60.

[16] Spencer-Dene B, Sala FG, Bellusci S, Gschmeissner S, StampG, Dickson C. Stomach development is dependent on fibroblastgrowth factor 10/fibroblast growth factor receptor 2b mediatedsignaling. Gastroenterology 2006;130:1233–44.

[17] Veltmaat JM, Relaix F, Le LT, Kratochwil K, Sala FG, VanVeelen W, Rice R, Spencer-Dene B, Mailleux AA, Rice DP,Thiery JP, Bellusci S. Gli3-mediated somitic Fgf10 expressiongradients are required for the induction and patterning of mam-mary epithelium along the embryonic axes. Development 2006;133:2325–35.

[18] Yokohama-Tamaki T, Ohshima H, Fujiwara N, Takada Y, Ichi-mori Y, Wakisaka S, Ohuchi H, Harada H. Cessation of FGF10

signaling, resulting in a defective dental epithelial stem cellcompartment, leads to the transition from crown to rootformation. Development 2006;133:1359–66.

[19] Chen C, Spencer TE, Bazer FW. Fibroblast growth factor-10: astromal mediator of epithelial function in the ovine uterus. BiolReprod 2000;63:959–66.

[20] Castilho AC, Giometti IC, Berisha B, Schams D, Price CA,Amorim RL, Papa PC, Buratini JR. Expression of FibroblastGrowth Factor 10 and Its Receptor, Fibroblast Growth FactorReceptor 2B, in the Bovine Corpus Luteum. Mol Reprod Dev2008;75:940–45.

[21] Taniguchi F, Harada T, Iwabe T, Ohama Y, Takenaka Y,Terakawa N. Aberrant expression of keratinocyte growth factorreceptor in ovarian surface epithelial cells of endometrioma.Fertil Steril 2008;89:478–80.

[22] Chaves RN, Martins FS, Saraiva MVA, Celestino JJH, LopesCAP, Correia JC, Lima-Verde IB, Matos MHT, Báo SN, NameKPO, Campello CC, Silva JRV, Figueiredo JR. Chilling ovarianfragments during transportation improves viability and growthof goat preantral follicles cultured in vitro. Reprod Fertil Dev2008;20:640–7.

[23] Taniguchi F, Harada T, Sakamoto Y, Yamauchi N, Yoshida S,Iwabe T, Terakawa N. Activation of mitogen-activated proteinkinase pathway by keratinocyte growth factor or fibroblastgrowth factor-10 promotes cell proliferation in human endome-trial carcinoma cells. Endocrinol Metab 2003;88:773–80.

[24] Zhou H, Zhang Y. Regulation of in vitro growth of preantralfollicles by growth factors in goats Domest Anim Endocrinol2005;28:235–42.

[25] Harada H, Toyono T, Toyoshima K, Yamasaki M, Itoh N, KatoS, Sekine K, Ohuchi H. FGF10 maintains stem cell compart-ment in developing mouse incisors. Development 2002;129:1533–41.

[26] Matos MHT, Lima-Verde IB, Luque MCA, Maia JE, Jr SilvaJRV, Celestino JJH, Martins FS, Báo SN, Lucci CM, FigueiredoJR. Essential role of follicle stimulating hormone in the main-tenance of caprine preantral follicle viability in vitro. Zygote2007;15:173–82.

[27] Silva JRV, Hurk VD, Costa SHF, Andrade ER, Nunes APA,Ferreira FVA, Lôbo RNB, Figueiredo JR. Survival and growthof goat primordial follicles after in vitro culture of ovariancortical slices in media containing coconut water. Anim ReprodSci 2004;81:273–86.

[28] Lucci CM, Amorim CA, Báo SN, Figueiredo JR, RodriguesAPR, Silva JR, Gonçalves PBD. Effect of the interval of serialsections of ovarian in the tissue chopper on the number ofisolated caprine preantral follicles. Anim Reprod Sci 1999;56:39–49.

[29] Steel RGD, Torrie JH, Dickey DA. Principles and Proceduresof Statistics: A Biometrical Approach. 3rd ed. New York:McGraw-Hill; 1997.

[30] Goldfarb M. Functions of fibroblast growth factors in vertebratedevelopment. Cytokine Growth Factor Rev 1996;7:311–25.

[31] Kwabi-Addo B, Ozen M, Ittmann M. The role of fibroblastgrowth factors and their receptors in prostate câncer. EndocrRelat Cancer 2004;11:709–24.

[32] Upadhyay D, Bundesmann M, Panduri V, Correa-Meyer E,Kamp DW. Fibroblast growth factor-10 attenuates H2O2-in-duced alveolar epithelial cell DNA damage. Am J Respir CellMol Biol 2004;31:107–13.

[33] Lin A. Activation of the JNK signaling pathway: breaking thebrake on apoptosis. Bioassays 2002;25:17–24.

257R.N. Chaves et al. / Domestic Animal Endocrinology 39 (2010) 249–258

Author's personal copy

[34] Martins FS, Van Den Hurk R, Santos RR, Silva JRV, MatosMHT, Celestino JJH, Rodrigues APR, Pessoa C, Ferreira FVA,Figueiredo JR. Development of goat primordial follicles after invitro culture of ovarian tissue in Minimal Essential Mediumsupplemented with coconut water. Anim Reprod 2005;2:106–13.

[35] Silva JRV, Tharasanit T, Taverne MAM, Van Der Weijden GC,Santos RR, Figueiredo JR, Van Den Hurk R. The activin-follistatin system and in vitro early follicle development ingoats. J Endocrinol 2006;189:113–25.

[36] Bruno JB, Lima-Verde IB, Martins FS, Matos MHT, LopesCAP, Maia-Jr JE, Báo SN, Nobre Junior HV, Maia FD, PessoaC, Moraes MO, Silva JRV, Figueiredo JR, Rodrigues APR.Característica histológica, ultra-estrutural e produção de ni-trito de folículos pré-antrais caprinos cultivados in vitro naausência ou presença de soro. Arq Bras Med Vet Zootec2008;60:1329 –37.

[37] Lima-Verde IB, Matos MHT, Bruno JB, Martins FS, SantosRR, Báo SN, Luque MCA, Vieira GAB, Silveira ER, RodriguesAPR, Figueiredo JR, Oliveira MAL, Lima PF. Effects of �-to-copherol and ternatin antioxidants on morphology and activa-tion of goat preantral follicles in vitro cultured. Arq Bras MedVet Zootec 2009;61:57–65.

[38] Derrar N, Price CA, Sirard MA. Effect of growth factors andco-culture with ovarian medulla on the activation of primordialfollicles in explants of bovine ovarian cortex. Theriogenology2000;54:587–98.

[39] Fortune JE, Kito S, Wandji SA, Srsen V. Activation of bovineand baboon primordial follicles in vitro. Theriogenology 1998;49:441–9.

[40] Cushman RA, Wahl CM, Fortune JE. Bovine ovarian corticalpieces grafted to chick embryonic membranes: A model for

studies on the activation of primordial follicles. Hum Reprod2002;174:48–54.

[41] Spencer TE, Stagg AG, Joyce MM, Jenster G, Wood CG, BazerFW, Wiley AA, Bartol FF. Discovery and characterization ofendometrial epithelial messenger ribonucleic acids using theovine uterine gland knockout model. Endocrinology 1999;140:4070–80.

[42] Ohuchi H, Ohchi H, Hori Y, Yamasaki M, Harada H, Sekine K,Kato S, Itoh N. FGF10 acts as a major ligand for FGF receptor2 IIIb in mouse multi-organ development. Biochem Bioph ResComm 2000;277:643–9.

[43] Gougeon A. Regulation of ovarian follicular development inprimates: facts and hypotheses. Endocr Rev 1996;17:121–155.

[44] Lopes CAP, Santos RR, Celestino, JJH, Melo MA, Chaves RN,Campello CC, Silva JR, Báo SN, Jewgenow K, Figueiredo JR.Short-term preservation of canine preantral follicles: Effects oftemperature, medium and time. Anim Reprod Sci 2009;115:201–14.

[45] Bruno JB, Celestino JJH, Lima-Verde IB, Lima LF, MatosMHT, Araújo VR, Saraiva MVA, Martins FS, Name KPO,Campello CC, Báo SN, Silva JRV, Figueiredo JR. Expression ofvascular endothelial growth factor (VEGF) receptor in goatovaries and improvement of in vitro caprine preantral folliclesurvival and growth with VEGF. Reprod Fertil Dev 2009;21:679–87.

[46] Rosseto R, Lima-Verde IB, Matos MHT, Saraiva MVA, Mar-tins FS, Faustino LS, Araújo VR, Silva CM, Name KP, Báo SN,Campello CC, Figueiredo JR, Blume H. Interaction betweenascorbic acid and follicle-stimulating hormone maintains follic-ular viability after long-term in vitro culture of caprine preantralfollicles. Domest Anim Endocrinol 2009;37:112–23.

258 R.N. Chaves et al. / Domestic Animal Endocrinology 39 (2010) 249–258