Canine preantral follicles cultured with various concentrations of follicle-stimulating hormone (FSH)

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Theriogenology 74 (2010) 749 –755 www.theriojournal.com

Canine preantral follicles cultured with various concentrations of follicle-stimulating hormone (FSH) Michelle Karen Brasil Serafima,*, Valdevane Rocha Araújob, Gerlane Modesto Silvab, Ana Beatriz Graça Duarteb, Anderson Pinto Almeidab, Roberta Nogueira Chavesb, Cláudio Cabral Campellob, Cláudio Afonso Pinho Lopesb, José Ricardo de Figueiredob, Lúcia Daniel Machado da Silvaa b

a Faculty of Veterinary Medicine, Laboratory of Carnivore Reproduction (LRC), State University of Ceara, Fortaleza, CE, Brazil Faculty of Veterinary Medicine, Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA), PGCV, State University of Ceara, Fortaleza, CE, Brazil

Received 20 October 2009; received in revised form 14 February 2010; accepted 26 March 2010

Abstract The objective was to evaluate the effects of various concentrations of exogenous FSH during in vitro culture of isolated canine preantral follicles. Preantral secondary follicles (⬎200 ␮m) were isolated by microdissection and cultured for 18 d in supplemented ␣-Minimum Essential Medium (␣-MEM). There were three treatment groups: 1) absence of FSH (control medium); 2) FSH100 (fixed concentration of 100 ng/mL throughout the entire culture period); and 3) sequential FSH (FSHSeq – 100, 500, and 1,000 ng/mL were added sequentially). Following culture, all follicles from all treatments were still viable (marked green by calcein-AM). The initial (D0) average follicle diameter for the control, FSH100, and FSHSeq was (mean ⫾ SEM) 298.96 ⫾ 7.02, 286.00 ⫾ 5.87, and 275.39 ⫾ 174 6.55 um, respectively (P ⬎ 0.05). Mean diameter of follicles treated with FSHSeq on Day 18 (D18-439.80 ⫾ 14.08 ␮m) was greater than those of the other treatments (P ⬍ 0.05). Daily follicular growth rate (␮m/d) of follicles in the FSHSeq treatment (6.47 ⫾ 0.55) was significantly faster than for both the control (3.67 ⫾ 0.32) and FSH100 (4.47 ⫾ 0.38) treatments. Furthermore, FSH100 and FSHSeq treatments had a significantly higher rate of antrum formation than the control group on D12 of culture, whereas after D12, FSH100 had a significantly higher rate of extrusion compared to the control (P ⬍ 0.05). In conclusion, the sequential addition of FSH to the culture medium maintained the survival of isolated canine preantral follicles and promoted an increased rate of follicular growth and antrum formation. © 2010 Elsevier Inc. All rights reserved. Keywords: Preantral follicles; Ovarian follicular development; In vitro maturation; FSH; Dog

1. Introduction Research into reproductive technologies, e.g., in vitro oocyte maturation (IVM), fertilization (IVF), and

* Corresponding author. Tel.: ⫹55.85.3101.9859; fax: ⫹55.85. 3101.9840. E-mail address: [email protected] (Michelle K.B. Serafim). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.03.028

embryo culture (IVC), are conducted in the bitch in an attempt to elucidate the reproductive biology of dogs, as well as expand knowledge regarding conservation of endangered species [1]. However, the greatest obstacle to the application and development of these biotechnologies is differences between dogs and other mammalian species in reproductive physiology; the major gap has been the low efficiency of IVM for canine oocytes [2].

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In most mammals, oocyte maturation occurs within the ovarian follicle, with one or more mature oocytes being ovulated. In dogs, the female gamete has peculiar characteristics [3], e.g., the oocyte is immature when ovulated and matures 3–5 d after ovulation. Furthermore, the canine oocyte has a high lipid content, which results in a dark uniform cytoplasm, with extremely compact cumulus and corona radiata cells [4]. Since the yield of IVM canine oocytes is low, the applicability of this biotechnology is limited. However, this could be overcome with in vitro culture of oocytes derived from preantral follicles [5]. Consequently, the manipulation of oocytes enclosed in preantral follicles (MOEPF) becomes a viable alternative, since preantral follicles constitute a massive (thousands to millions, depending on the species) reserve pool of female gametes. The main objective of MOEPF is to recover preantral follicles for subsequent in vitro culture to maturation, and to prevent follicular atresia that occurs naturally in vivo [6]. Development of an in vitro culture system that supports the growth of the preantral follicle in domestic species is an ambitious goal. This growth is supported with a culture medium enriched with inorganic salts, vitamins, energetic substrates, and amino acids, among other substances [5], similar to media used for other species [7]. Several substances that influence follicular growth can be added to the culture medium. Among these substances, noteworthy substances are bovine serum albumin (BSA; used as a protein source), energy substrates, antioxidants (e.g., ascorbic acid), and the combination of insulin, transferring, and selenium (ITS). Moreover, the effects of adding growth factors, hormones (including FSH), or both, to the culture medium has been reported [8]. It is well established that FSH promotes follicular growth, as well as antrum formation in mice [9,10], humans [11], cattle [12], sheep [13,14], swine [15], and goats [16]. The effects of FSH, human chorionic gonadotropin (hCG) and estradiol on in vitro maturation of canine oocytes enclosed in advanced preantral follicles and early antral follicles has been reported [17]. In that study, only meiotic progression of oocytes was assessed, whereas follicular growth and development during prolonged in vitro culture were not evaluated. Consequently, additional studies are needed to elucidate the mechanisms of early folliculogenesis in the bitch, especially since an in vitro culture system for canine preantral follicles has never been tested. Moreover, more in-depth studies regarding the effects of exogenous FSH during in vitro culture of canine preantral follicle are needed. Therefore, the objective of this

study was to evaluate the effect of adding FSH at various concentrations and for various intervals during in vitro culture of isolated canine preantral follicles. 2. Materials and methods 2.1. Collection and transport of ovaries Ovaries (n ⫽ 60) from bitches (undefined crossbreds) in various stages of the estrous cycle, were collected immediately after euthanasia in the Center for Zoonosis Control in Fortaleza, CE, Brazil. After collection, ovaries were washed once in 70% alcohol for 10 s and twice in HEPES-buffered Minimum Essential Medium (MEM) plus penicillin (100 ␮g/mL) and streptomycin (100 ␮g/mL) and transported to the laboratory in ⬍1 h at 4 °C [18]. 2.2. Isolation of canine advanced preantral follicles In the laboratory, after removing the ovarian bursa and other adjacent tissues and ligaments, the ovarian cortex was sliced (approximately 1 mm thick). Then, secondary preantral follicles ⬎200 ␮m in diameter were manually isolated by microdissection, using 26 G needles attached to 1-mL syringes [15] under a stereomicroscope dissecting microscope (SMZ 645 Nikon, Tokyo, Japan). After isolation, follicles were immersed in drops of culture medium and were washed three times before being transferred to culture plates. Only follicles considered morphologically normal, i.e., with a centrally located spherical and dark oocyte, surrounded by two or three compact layers of granulosa cells, and with no apparent damage to the basal membrane, were used [19]. 2.3. In vitro culture Follicles were cultured individually in 100 ␮L drops of medium under mineral oil, using 60-mm diameter Petri dishes (Corning Inc., Lowell, MA, USA, catalog no. 430166). The culture medium was ␣-MEM supplemented with BSA (3 mg/mL), glutamine (2 mM), hypoxanthine (2 mM), ITS (insulin 6.25 ␮g/mL, transferrin 6.25 ␮g/mL, and selenium 6.25 ng/mL), ascorbic acid (50 ␮g/mL), and various concentrations of recombinant FSH (FSHrec). In this study, the following groups were tested: control (basic medium); FSH100 (fixed concentration of 100 ng/mL throughout the entire culture period); and sequential FSH (FSHSeq – added sequentially – 100, 500, and 1,000 ng/mL, starting 0, 6, and 12 d after the onset of culture). Follicles were cultured at 39 °C with 5% CO2, and the medium was changed every 2 d.

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Before, during, and after culture, viability, diameter, follicular growth rate, and rate of antrum formation were evaluated. To evaluate growth, follicles were measured every 6 d using an ocular micrometer attached to a stereomicroscope (SMZ 645 Nikon, Tokyo, Japan), using the average of two measurements (vertical ⫹ horizontal/2). Oocyte diameter was also measured after in vitro follicle culture. To evaluate the rate of antrum formation, the emergence of a translucent cavity filled with follicular fluid was observed visually while changing the culture medium, and the diameter of the follicle was measured.

centages. Differences were considered to be significant when P ⬍ 0.05.

2.4. Analysis of follicular survival

Following 18 d of culture, all follicles were subjected to a viability analysis with the fluorescent markers calcein-AM and ethidium homodimer-1 for live and dead cell staining, respectively. Remarkably, on D18, all follicles (100%) were stained green by calcein-AM, confirming that all of the treatments were able to maintain follicular viability until the end of culture (Fig. 1).

To verify follicular viability at the end of the culture period, a two-color fluorescence cell viability assay based on the simultaneous determination of live and dead cells by calcein-AM and ethidium homodimer-1, respectively, was used. The first probe detected intracellular esterase activity of viable cells, whereas the latter labeled nucleic acids of non-viable cells with plasma membrane disruption. The test was performed by adding 4 ␮M calcein-AM and 2 ␮M ethidium homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe, Germany) to the cells, followed by incubation at 37 °C for 15 min. Afterwards, follicles were washed three times in HEPES-buffered TCM199, mounted between a slide and a coverslip, and evaluated (100⫻) with an epifluorescence microscope (Leica, Germany). The emitted fluorescent signals of calcein-AM and ethidium homodimer were collected at 488 and 568 nm, respectively. Oocytes and granulosa cells were considered live if the cytoplasm was stained positively with calcein-AM (green) and chromatin was not labeled with ethidium homodimer (red). Similar methodology was previously used to evaluate the viability of preantral follicles of dogs [18] and goats [16]. 2.5. Statistical analysis Data were initially submitted to Shapiro-Wilk and Bartlett tests to confirm normal distribution and homogeneity of variances, respectively. Follicular diameters met both criteria, and were submitted to ANOVA, with means compared by the Least Significant Difference (LSD), as described [20]. Growth rates (mm/s) did not have homogeneity of variances, even after transformation of data, and therefore were analyzed with a Kruskal-Wallis non-parametric test. Data were expressed as mean ⫾ SEM. Rates of follicle viability, extrusion, and antrum formation were compared with a Chi-square test, and the results were expressed as per-

3. Results The rate of recovery of canine preantral follicles with a diameter ⬎200 ␮m was satisfactory; more than 100 follicles from each set of ovaries were obtained. Overall, 306 preantral follicles were randomly allocated among treatments. 3.1. Follicular viability

3.2. Follicle and oocyte diameter and follicular growth rate The average diameter of follicles pre-culture (D0) were 289.96 ⫾ 7.02, 286.00 ⫾ 5.87, and 275.39 ⫾ 6.55 ␮m for control, FSH100, and FSHSeq was respectively (P ⬎ 0.05). As culture progressed, follicles in each treatment group increased in diameter (P ⬍ 0.05; Fig. 2). Furthermore, on D12, follicle diameter was greater in FSHSeq than control (398.07 ⫾ 12.06 vs. 343.11 ⫾ 8.61 ␮m, P ⬍ 0.05). There was no significant difference between FSHSeq and FSH100 on D0, D6, and D12, although on D18, FSHSeq (439.80 ⫾ 14.08 ␮m) was larger (P ⬍ 0.05) than the other two treatments. The daily rate of follicular growth was higher (P ⬍ 0.05) in follicles on FSHSeq treatment (6.47 ⫾ 0.55 ␮m/d) than that of the control (3.67 ⫾ 0.32 ␮m/d) and FSH100 (4.47 ⫾ 0.38 ␮m/d); the latter did were not significantly different from each other. Granulosa cell proliferation was not specifically characterized, but the number of granulose cell layers apparently increased during culture in all groups. Following culture, oocyte diameters were 110.46 ⫾ 11.28, 114.80 ⫾ 19.09 and 112.41 ⫾ 18.52 ␮m in the groups control, FSH100 and FSHSeq, respectively (P ⬎ 0.05). 3.3. Antrum formation Immediately after isolation, preantral follicles were spherical, with an oocyte surrounded by at least two layers of granulosa cells (without an antrum), and with an intact basal membrane. After the first 6 d of culture,

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Fig. 1. Viable canine ovarian follicles (stained green with calcein-AM) cultured in vitro for 18 d in the absence of FSH (Control, A, and B), presence of FSH at a fixed concentration (FSH100, C, and D) or FSH added sequentially (FSHSeq, E, and F). The scale bars represent 100 ␮m.

the rate of antrum formation was 40.8, 46.7, and 44.9% for the control, FSH100, and FSHSeq, respectively (Fig. 3). Moreover, the latter two treatments had a significantly higher rate of antrum formation than the control group on D12 of culture. On D18, there was no significant difference among treatments in the percentage of follicles with an antrum (overall mean 87.6%). 3.4. Follicular extrusion All follicles used had an intact basal membrane on D0. However, the rate of follicular extrusion significantly increased from D0 to D6 in all treatments (Fig. 4). When

the treatments were compared with each other beginning at D12, FSH100 had a significantly higher extrusion rate than the control. 4. Discussion This is apparently the first study to evaluate the in vitro development of canine preantral follicles by examining the effect of various concentrations of FSH and varying culture periods. We chose to use the method of mechanical isolation by microdissection to recover canine preantral follicles.

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Fig. 2. Mean ⫾ SEM of the follicle diameter after in vitro culture, in the presence or absence of various concentrations of FSH (100 ng/mL or sequential) for 18 d.

Fig. 4. Mean extrusion rate of canine preantral follicles cultured in vitro in the presence or absence of various concentrations of FSH (100 ng/mL or sequential) for 18 d.

AB

Within a day, treatments without a common superscript differed (P ⬍ 0.05).

AB Within a day, treatments without a common superscript differed (P ⬍ 0.05).

a-d Within a treatment, days without a common superscript differed (P ⬍ 0.05).

a,b Within a treatment, days without a common superscript differed (P ⬍ 0.05).

The mechanical isolation method has been used before for other animals, including wild cats [21], swine [15], mice [22], humans [23], goats [16], buffalo [24], sheep [25], and cattle [12]. Furthermore, Durrant et al [19] also recovered preantral follicles from female dogs using an enzymatic isolation method and, despite having obtained follicles at various stages of development, there were high rates of degeneration. In further investigation of the enzymatic isolation method with murine preantral follicles, there was more disruption of the

Fig. 3. Mean percentage of canine preantral follicles forming an antrum, following in vitro culture in the presence or absence of various concentrations of FSH (100 ng/mL or sequential) for 18 d. AB Within a day, treatments without a common superscript differed (P ⬍ 0.05). a-c Within a treatment, days without a common superscript differed (P ⬍ 0.05).

basal membrane, in addition to greater degeneration [26,21]. The medium used for the culture of canine preantral follicles in this study was supplemented ␣-MEM. This medium was used successfully by Silva et al. [16] to culture caprine preantral follicles, with the addition of a fixed concentration of FSH (1,000 ng/mL), which enhanced survival, growth, and development of follicles. Perhaps that concentration of FSH caused an increase in the rate of follicular extrusion, characterized by rupture of the basal membrane. Similar results were found in this study when canine preantral follicles were cultured in medium supplemented with FSH (FSH100 and FSHSeq), promoting a high extrusion rate (relative to the control) on Day 6 of culture. The presence of this hormone may have overstimulated follicular growth, as well as the increase of the antral cavity, without the concomitant remodeling of the basal membrane. Conversely, the use of ascorbic acid in the composition of the basic medium favored the integrity of the basal membrane of the follicle, which resulted in remodeling, and, consequently, promoted maintenance of follicular viability for long culture periods. In previous studies, canine follicles were cultured for 3 d [17,27]. However, in the present study, the initiation of antrum formation with the accumulation of follicular fluid between the oocyte and the granulosa cells was observed after the sixth day. Consequently, 3 d was an insufficient interval for follicular development with antrum formation and adequate growth. In this experiment, reasonable growth of canine preantral follicles was achieved, which has apparently never

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been previously reported. Therefore, this methodology has potential for additional studies, with the objective of obtaining viable oocytes destined to mature in vitro. It was noteworthy that, in a previous study [28], the rate of degeneration of canine oocytes that had matured in vitro was very high, and there was limited success with the rates of maturation to metaphase II (only 20% in some cases [2]). In this study, follicles were cultured individually in 100 ␮L drops of medium under mineral oil, directly on plastic petri dishes. In other studies, canine follicles were cultured in groups of 5–10/mL/well [27], or 2–12/ 750 ␮L/well [17], using 24-well plates. However, Hartshorne et al [8] concluded that the method of culture of individual follicles was more appropriate to evaluate follicular metabolism, steroidogenesis, oocyte development, or even the hormonal influence on the culture of follicles. Furthermore, when murine follicles were cultured individually, they achieved excellent growth, normal antrum formation, and subsequent development [29]. This may be valid for canine follicles, but a comparison between individual and group culture has not yet been reported. With respect to oocyte size after in vitro follicle culture, oocytes derived from all treatments had mean diameters apparently greater than the minimum value (110 um) previously reported for canine oocytes to acquire meiotic competence [30,31]. In the present study, the group treated with FSHSeq had a significantly larger diameter than the control (439.80 ⫾ 14.08 vs. 373.33 ⫾ 10.39 ␮m) after 18 d of culture. The presence of FSH in the culture medium facilitated the use of glucose and production of estradiol, and it can also stimulate the follicles to grow by causing proliferation of granulosa cells [32]. In that regard, FSH stimulated ovarian follicular growth and maintained granulosa cell integrity in swine [33], sheep [13], humans [34], and cattle [35]. This result may be due to an increase in the number of FSH receptors as the follicle develops, which becomes increasingly sensitive to this stimulus [36]. Therefore, the increasing concentrations of FSH used in this study (FSHSeq) mimiced what occurs in vivo, considering that there may be an increase in the number of receptors that will support the higher concentrations during the final phases of follicular growth. Antral formation may be affected by FSH [31], since this was observed when preantral follicles from swine [33], sheep [13], goats [16,37], humans [34], and cattle [12,38] were cultured in vitro. Similarly, in the present study, the addition of FSH had a significantly greater

rate of antrum formation than that of the control group, after 12 d of culture. Antral cavity formation consists of progressive accumulation of fluid among granulosa cells and is hormonally regulated (possibly mediated by FSH [39]). Canine oocytes reached their maximum diameter at the onset of antrum expansion [40], which occurred when mean follicle diameter was ⬃350 um [39]. After the follicle has entered the antral stage, granulosa cells differentiate into two types: mural cells, which form the antrum wall, and cumulus cells, which continue to provide nutrition and regulating substances to the oocyte [41]. Similarly, the oocyte synthesized compounds that regulated activities in surrounding cells [42]. In conclusion, the sequential addition of FSH to the culture medium maintained the survival of isolated canine preantral follicles. Furthermore, FSH increased the rates of ovarian follicular growth and antrum formation during prolonged culture. The culture system described in this study was an important step in the production of potentially viable oocytes that could be used for maturation, fertilization, and subsequent in vitro production of embryos. Acknowledgements Michelle Karen Brasil received financial support from Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP; masters scholarship). Lúcia Daniel Machado da Silva and José Ricardo de Figueiredo were recipients of a grant from CNPq. We express our appreciation to the Center for Zoonosis Control (CCZ) of Fortaleza Municipality (Brazil). References [1] Songsasen N, Wildt DE. Oocyte biology and challenges in developing in vitro maturation systems in the domestic dog. Anim Reprod Sci 2007;98:2–22. [2] Farstad W. Assisted reproductive technology in canid species. Theriogenology 2000;53:175– 86. [3] De Los Reyes M, Langea JDE, Miranda P, Palominos J, Barros C. Effect of human chorionic gonadotrophin supplementation during different culture periods on in vitro maturation of canine oocytes. Theriogenology 2005;64:1–11. [4] Kim MK, Fibrianto YH, Oh HJ, Jang G, Kim HJ, Lee KS, Kang SK, Lee BC, Hwang WS. Effects of estradiol-17b and progesterone supplementation on in vitro nuclear maturation of canine oocytes. Theriogenology 2005;63:1342–53. [5] Telfer EE. In vitro development of pig preantral follicles. Reprod Suppl 2001;58:81–90. [6] Figueiredo JR. Essential role of follicle stimulating hormone in the maintenance of caprine preantral follicle viability in vitro. Zygote 2007;15:173– 82.

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