- Email: [email protected]
Ovine secondary follicles vitrified out the ovarian tissue grow and develop in vitro better than those vitrified into the ovarian fragments
Theriogenology 85 (2016) 1203–1210
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Ovine secondary follicles vitrified out the ovarian tissue grow and develop in vitro better than those vitrified into the ovarian fragments Franciele Osmarini Lunardi a, *, Francisco Leo Nascimento de Aguiar a, Ana Beatriz Graça Duarte a, Valdevane Rocha Araújo a, Laritza Ferreira de Lima a, Naiza Arcângela Ribeiro de Sá a, Hudson Henrique Vieira Correia a, Sheyla Farhayldes Souza Domingues b, Cláudio Cabral Campello a, Johan Smitz c, José Ricardo de Figueiredo a, Ana Paula Ribeiro Rodrigues a a
Laboratory of Manipulation of Oocytes and Ovarian Pre-antral Follicles (LAMOFOPA), Faculty of Veterinary Medicine, Ceará State University, Fortaleza, CE, Brazil b Laboratory of Wild Animal Biology and Medicine, Federal University of Pará, Belem, Brazil c Follicle Biology Laboratory, Center for Reproductive Medicine, UZ Brussel, Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 August 2015 Received in revised form 8 October 2015 Accepted 30 October 2015
Cryopreservation of preantral follicles is a promising technique to preserve female fertility. The aim of this study was to evaluate the effect of vitrification on the development of secondary follicles included in ovarian tissue or isolated after microdissection. An important end point included is the capacity of grown oocytes to resume meiosis. Sheep ovarian cortexes were cut into fragments and split into three different groups: (1) fresh (control): secondary follicles isolated without any previous vitrification; (2) folliclevitrification (follicle-vit): secondary follicles vitrified in isolated form; and (3) tissuevitrification (tissue-vit): secondary follicles vitrified within fragments of ovarian tissue (in situ former) and subsequently subjected to isolation. From the three groups, isolated secondary follicles were submitted to IVC for 18 days. After IVC, cumulus-oocyte complexes (COCs) were harvested from follicles. As an additional control group, in vivo grown, in vivo-grown COCs were collected from antral ovarian follicles. All, recovered COCs were matured and the chromatin configuration was evaluated. Data were analyzed by ANOVA, and the means were compared by Student-Newman-Keuls test, and by chisquare. Differences were considered to be significant when P < 0.05. Isolated preantral follicles from all treatments had normal morphology, antrum formation, and low follicle degeneration after IVC. The growth rate between control and follicle-vit did not differ (P > 0.05), and was higher (P < 0.05) than for tissue-vit. The percentage of follicles that decreased diameter during IVC was significantly higher in tissue-vit than the in folliclevit. Recovery rate of oocytes from normal follicles was higher in follicle-vit than in tissuevit. Furthermore, oocyte viability was lower in tissue-vit than other treatments, and follicle-vit did not differ from control and in vivo grown. The percentage of oocytes meiosis resuming was not different between treatments except for in vivo grown. After vitrification, only follicle-vit showed metaphase I oocyte. We conclude that secondary follicles vitrified after isolation displayed a better follicular growth rate, oocyte viability,
Keywords: Cryopreservation Follicular development Oocyte maturation Ovary Ovine/sheep
* Corresponding author. Tel.: þ55 85 31019861; fax: þ55 85 31019859. E-mail address: [email protected] (F.O. Lunardi). 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.10.043
1204
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
percentage of oocytes reaching the metaphase I stage, and fewer follicles with decreased diameter after IVC. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Cryopreservation of ovarian tissue is nowadays considered a feasible option for fertility preservation. However, some authors have indicated the risk of reintroduction of cancer cells after transplantation [1–3]. Thus, the IVC of isolated secondary follicles after cryopreservation is considered a reasonable alternative to avoid this and to obtain fertilizable oocytes [4]. Appropriate protocols for IVC of noncryopreserved preantral follicles had already been reported in murine, in which viable offspring were obtained [5]. Nevertheless, in nonhumane primates species [6,7] or livestock animals, such as caprine [8] and ovine [9], only a limited and variable number of embryos arising from preantral follicle oocytes cultured in vitro have been obtained until today. Although the higher results in animal models being plenty supportive, in humane species the results are not suitable, considering reports of low frequency of antrum formation of preantral follicles grown in vitro [10]. Notwithstanding, only in murine, embryos were obtained from previously cryopreserved ovarian tissue, with successful reports [11,12]. In latter studies, vitrification was used as the first choice method for ovary cryopreservation. Vitrification constitutes the ultra-rapid cooling of biological material to very low temperatures and the use of high concentrations of intracellular cryoprotectant agents. These particular conditions allow a solidification, or amorphous liquid state of vitrification solution, which inhibits nucleation and ice crystals growth [13,14]. The ovarian tissue is constituted by different cellular types that vary in size, distribution, and quantity and by many follicular development stages cells (granulosa and theca cells) that form the barrier to the oocytes. All these structures represent a huge hurdle to an optimal permeability for cryoprotectant agents and could result in difficulties to an adequate oocyte protection at liquid nitrogen temperature (196 C). Recently, our group reported that secondary sheep follicles could be vitrified effectively, enclosed or not in ovarian cortex. After thawing, these follicles (previously isolated or isolated after ovarian fragment vitrification) are capable to form an antrum when cultured in vitro for 6 days and to be similar to fresh noncultured follicles without vitrification [15]. However, because of the short IVC that was applied, it was not possible to ascertain if oocytes enclosed in secondary follicles were capable to survive a long-term culture, needed to reach the development stage of oocyte to resume meiosis. The present study aimed to vitrify secondary follicles enclosed in or isolated from ovarian tissue and to evaluate relevant end points: post-thaw follicular survival, growth, antrum formation and hormone production after 18 days of culture, and oocyte viability and maturation.
This experiment was approved and performed under the guidelines of Ethics Committee for Animal Use of State University of Ceará. Unless otherwise mentioned, all chemicals used in the present study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.1. Source of ovaries Ovaries (n ¼ 30) were collected at abattoirs from 15 adult mixed-breed ewes. Immediately postmortem, under aseptic conditions, the ovaries were washed in 70% alcohol for 10 seconds, followed by two washes in HEPES buffered minimum essential medium (MEM) supplemented with 100-mg/mL penicillin and 100-mg/mL streptomycin. Each pair of ovaries was transported to the laboratory within 4 hours into tubes containing 15 mL of MEM HEPES at 4 C. 2.2. Experimental design At the laboratory, ovaries were cleaned from surrounding fat and fibrous tissue, and their cortexes were recovered and fragmented into pieces (1–2 mm thick) and split into three different groups: (1) fresh (control): secondary follicles were isolated without any previous vitrification; (2) follicle-vitrification (follicle-vit): secondary follicles were vitrified in isolated form; and (3) tissuevitrification (tissue-vit): secondary follicles (formed in situ) were vitrified within fragments of ovarian tissue and isolated subsequently after warming. From all groups, isolated secondary follicles were in vitro cultured for 18 days. During IVC, the levels of hormones secretion (progesterone and estradiol), antrum formation, and growth rate were measured. After IVC, the cumulus-oocyte complexes (COCs) were recovered from cultured follicles and from in vivo grown and submitted to IVM. In addition, the viability and chromatin configuration of the oocytes were also evaluated. 2.3. Ovarian cortex fragmentation and follicle isolation The ovarian cortex was sectioned into pieces of 1 to 2 mm thickness under sterile conditions in a petri dish containing MEM HEPES supplemented with antibiotics (100-mg/mL penicillin and 100-mg/mL streptomycin). Fragments obtained were transferred to another petri dish containing fresh medium and then visualized under a stereomicroscope (Nikon SMZ 645; Tokyo, Japan; magnification: 100). Once localized, secondary follicles, with average diameter of 325.07 mm (range 150–350 mm), were mechanically isolated by microdissection using a 26-G needle attached to 1-mL syringe. Only those follicles with the oocyte-follicle complex visible, surrounded by several layers of granulosa cells with intact basement membrane and no antral cavities were selected for this study.
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
2.4. IVC of sheep secondary follicles Secondary follicles were transferred into drops of 100 mL of culture medium under mineral oil in petri dishes (60 15 mm) and cultured for 18 days (one follicle/drop) at 39 C and 5% CO2 in air. The culture medium used was composed of MEM alpha (a-MEM) supplemented with 10-mg/mL insulin, 5.5-mg/mL transferrin, 5-ng/mL selenium, 2-mM glutamine, 2-mM hypoxanthine, 3-mg/mL bovine serum albumin, 50-mg/mL ascorbic acid, 50-ng/mL leukemia inhibitory factor, and 50-ng/mL Kit ligand. Fresh media were prepared immediately before use and were incubated for 1 hour prior to use. Every 6 days, 100 mL of medium was replaced with fresh medium. This medium was chosen on the basis of our previous study [15]. 2.5. Morphologic analysis and assessment of in vitro follicular growth At Day 0, 6, 12, and after 18 days of IVC, the percentage of morphologically normal follicles and follicular diameters from control, follicle-vit, and tissue-vit were determined. Follicles were classified as morphologically normal if they presented an intact basement membrane, no extrusion of the COCs from the follicle, bright and homogeneous granulosa and theca cells. Follicle degeneration was recognized when the oocytes and surrounding cells were darkened, had misshaped oocytes or decreased follicle diameter. Antral cavity formation was defined as a visible translucent cavity within the granulosa cell layers. Follicular diameter was measured at the basement membrane (from the major and minor axes) of each follicle with the aid of an ocular micrometer inserted into a stereomicroscope (SMZ 645 Nikon; Tokyo, Japan; magnification: 100) on Days 0, 6, 12, and 18 of IVC. The average of these two measurements was used to determine the follicle diameter only in morphologically normal follicles. The daily mean increase in follicular diameter (daily follicular growth rate) was calculated as the diameter of morphologically normal follicles at Day 18 minus the diameter of the same follicle at Day 0, divided by the total number of days in culture. 2.6. Vitrification protocol used to ovarian isolated follicles and fragments Ovarian fragments (1–2 mm thick) and isolated secondary follicles were exposed to different concentrations (12.5%, 25%, 50%, and 100%) of vitrification solution composed by MEM HEPES supplemented with 10% fetal bovine serum (FBS), 2.60-M acetamide, 2.62-M dimethylsulfoxide, 1.31-M 1, 2-propanediol, and 0.0075-M polyethylene glycol. Samples were exposed to the first two concentrations (12.5% and 25%) of vitrification solution for 5 minutes at room temperature; then to the next two concentrations (50% and 100%) of vitrification solution for 15 minutes (for fragments) or 5 minutes (for isolated follicles) at 4 C. After, samples (either ovarian fragments or isolated follicles) were placed on the surface of a metal cube partially submerged in liquid nitrogen and transferred to cryovials (10 follicles or 10 fragments each) in accordance with protocol adapted from
1205
Lunardi et al. [15]. Samples were kept into liquid nitrogen (196 C) for 6 days. 2.7. Warming protocol of ovarian follicles and fragments Cryovials containing samples of ovarian tissue and isolated secondary follicles were removed from liquid nitrogen and exposed to room temperature for 1 minute and immersed in a water bath (37 C) for 30 seconds. Soon after, removal of cryoprotectants was performed by immersion of ovarian fragments or secondary follicles in washing solutions, three baths of 5 minutes each with the solution composed out of MEM HEPES plus 10% FBS and decreasing concentrations of sucrose (0.5, 0.25, and 0.0 M) in accordance with protocol adapted from Lunardi et al. [16]. 2.8. Steroid production To evaluate 17b-estradiol (pmol/L) and progesterone (nmol/L) concentrations during IVC, the total of 30 pooled samples of conditioned medium from 60 growing follicles (two follicles per medium, 10 samples per group) from control and each of the vitrification groups at Days 6, 12, and 18 was retrieved and stored at 80 C. The concentrations of 17b-estradiol (pmol/L) and progesterone (nmol/L) were measured by chemiluminescence using an immunoassay system (Vitros Eci/EciQ Immunodiagnostic System; Johnson & Johnson Company, Ortho-Clinical Diagnostics, Buckinghamshire, UK) according to manufacturer’s instructions. 2.9. IVM of COCs At the end of the culture period, the COCs obtained from in vitro cultured were carefully harvested from intact follicles using 26-G needles and from extrude follicles under a stereomicroscope. From in vivo grown control, in vivo-grown COCs were collected from antral ovarian follicles. Oocytes surrounded by at least one compact layer of cumulus cells were selected for IVM. The recovery rate was calculated by dividing the number of COCs by the number of viable follicles at Day 18 of culture multiplied by 100. The selected COCs were washed in medium composed of tissue culture medium 199 (TCM199) plus HEPES supplement with pyruvate (0.911 mM/L) and 10% FBS followed by IVM medium. The maturation medium was composed by tissue culture medium 199 plus sodium bicarbonate supplemented with 0.5mg/mL recombinant bovine follicle-stimulating hormone (Nanocore, Campinas, São Paulo, Brazil), 5-mg/mL luteinizing hormone, 1-mg/mL 17b-estradiol, 10-ng/mL recombinant epidermal growth factor, 0.911-mM/L pyruvate, 100-mM/L cysteamine, 50-ng/mL recombinant insulin-like growth factor I, and 1% bovine serum albumin. After washing, COCs were transferred to 50-mL drops of maturation medium under mineral oil and then incubated for 36 to 40 hours at 39 C with 5% CO2 according to Luz et al. [17]. 2.10. Assessment of oocyte viability and chromatin configuration After IVM, the COCs were denuded from surrounding expanded cumulus cells by manual pipetting in TCM199
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
HEPES containing 0.1% hyaluronidase and subjected to viability analysis. To this end, the oocytes were incubated in 100-mL drops of 2-mM ethidium homodimer-1 supplemented with 4-mM calcein-acetoxymethyl (calcein-AM), 0.5% glutaraldehyde, and 10-mM Hoechst 33342 at room temperature for 30 minutes. After this, the oocytes were washed in TCM199 HEPES and were visualized under fluorescence microscopy (Nikon Eclipse 80i; Tokyo, Japan; magnification: 40). Oocytes were considered viable if the cytoplasm was positively stained with calcein-AM (green) and not stained with ethidium homodimer-1 (red). The emitted fluorescent signals of calcein-AM and ethidium homodimer-1 were collected at 488 nm and 568 nm, respectively. In addition, oocytes were stained with Hoechst 33342 (Molecular Probes; Invitrogen, Karlsruhe, Germany) and then analyzed for chromatin configuration being emitted fluorescent signals at 483 nm. This dye was used to analyze the oocyte’s chromatin configuration through observation of the intact germinal vesicle (GV), meiotic resumption (including GV breakdown [GVBD], metaphase I [MI], anaphase I, or telophase I), or nuclear maturation (metaphase II [MII]). 2.11. Statistical analysis Data for discrete variables (morphologically normal follicles, antrum formation, recovery rate and chromatin configuration of oocytes) were analyzed as dispersion of frequency using chi-square test. Otherwise, when the observed frequency was equal or less than five units, Fisher’s exact test was applied. In both cases, results were expressed as percentages. Data for continuous variables (follicular diameter, growth rate after culture, and hormonal dosages) Kruskal–Wallis and unpaired t test were initially evaluated for homocedasticity and normal distribution of the residues, by Bartlett’s and Shapiro–Wilk tests, respectively. Confirmed both requirements underlying analysis of variance, the effects of treatment, time of culture, and treatment by time interaction were analyzed using PROC MIXED of SAS (2002), including repeated statement to account for autocorrelation between sequential measurements. The model was Yijk ¼ m þ Ri þ Fj þ Tk þ (RT)ik þ eijk, where Yijk is the observation of the jth follicle in the ith treatment at the kth time of culture, m is the overall mean, Ri is the ith treatment, Fj is the random effect of the jth follicle within the ith treatment, Tk is the kth time of culture, (RT)ik is the treatment by time interaction term, and eijk is the random residual effect. Comparisons among treatments or times were further analyzed by the Student-Newman-Keuls test. A probability of P < 0.05 indicated a significant difference, and results were expressed as mean standard deviation (oocyte diameter) or expressed as mean standard error of the mean (follicle diameter, growth rate). 3. Results 3.1. Assessment of morphologically normal follicles and antral cavity formation during culture A total of 154 secondary follicles were distributed randomly to groups control (n ¼ 50), follicle-vit (n ¼ 40),
and tissue-vit (n ¼ 64) and in vitro cultured for 18 days. The percentage of morphologically normal follicles was similar between follicle-vit (97.5%, n ¼ 39/40) and tissue-vit (93.75%, n ¼ 60/64) being both higher (P < 0.05) than in control group (78%, n ¼ 39/50) (Fig. 1). At the end of culture period, some follicles showed rupture of the basal membrane and, consequently, released their COCs. In control (20%, n ¼ 10/50), there was an increase (P < 0.05) in the percentage of extruded oocytes when compared to tissue-vit (1.56%, n ¼ 1/64). Interestingly, the follicle-vit (n ¼ 0/40) did not present follicle extrusion. The percentage of degenerated follicles was similar (P > 0.05) among the three groups (control, 2%; follicle-vit, 2.5%; and tissue-vit, 4.69%). Except for follicle-vit, the percentage of antrum formation increased significantly from Day 6 to 18 of culture. A significantly higher percentage of antrum formation was observed in the tissue-vit compared to the control (Fig. 2).
3.2. Follicle diameter and daily growth rate before and after vitrification and IVC of ovine preantral follicles Follicle diameter increased significantly from Day 0 to 18 of culture only in the control and follicle-vit groups. However, no differences were observed among the groups regardless the culture time (P > 0.05; Fig. 3). The Figure 4A illustrates the follicular daily growth rate in the three groups throughout the period of IVC. The control (7.05 1.07 mm/day, n ¼ 21) and follicle-vit (5.02 0.63 mm/day, n ¼ 38) were similar between themselves and higher (P < 0.05) than tissue-vit (2.42 0.32 mm/day, n ¼ 44). The Figure 4B shows the percentage of follicles with decreased diameter throughout the IVC. The percentage of follicles with decreased diameter in follicle-vit (2.56%, n ¼ 1/39) was lower (P < 0.05) than control (46.15%, n ¼ 18/39) and in tissue-vit (26.67%, n ¼ 16/60). 100
Aa Aa Aa
Aa Aa Aa
Aa Ab
Aa Aab Ab
Normal follicles (%)
1206
Bb
80
60
40
20
0
Day 0
Day 6
Control
Follicle-Vit
Day 12
Day 18
Tissue-Vit
Fig. 1. Percentage of isolated morphologically normal sheep preantral follicles without vitrification (control, n ¼ 50) and after vitrification of isolated follicles (follicle-vit, n ¼ 40) or ovarian tissue (tissue-vit, n ¼ 64) at Days 0, 6, 12, and 18 of IVC. ABDifferent uppercase letters indicate statistically significant differences among groups (P < 0.05) within the same day. abDifferent lowercase letters indicate statistically significant differences among days of culture (P < 0.05) within the same group.
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
B
E
100 μm
C
100 μm
D
Antrum formation (%)
A
1207
Aa Ab
80 60
ABa Aa
Aa Aa
100
Ba Bab
Bb
40 20 0
Day 6 100 μm
100 μm
Control
Day 12
Follicle-Vit
Day 18
Tissue-Vit
Fig. 2. Normal sheep secondary follicle before (Day 0) IVC (A) or cultured for 6 days (B), 12 days (C), or 18 days (D) in follicle-vit group. Note beginning of antrum formation at Day 6 of IVC. (E) Percentage of antrum formation of sheep preantral follicles without vitrification (control, n ¼ 39) and after vitrification of isolated follicles follicle-vit, n ¼ 39) or ovarian tissue (tissue-vit, n ¼ 60) at Days 6, 12, and 18 of IVC. ABDifferent uppercase letters indicate statistically significant differences among groups (P < 0.05) within the same day. abDifferent lowercase letters indicate statistically significant differences among days of culture (P < 0.05) within the same group.
3.3. Steroid production The follicles from control produced higher (P < 0.01) estradiol levels than vitrified groups. Besides, only in the control, estradiol production increased significantly from Day 6 to 18 of culture, only in follicle-vit, progesterone levels increase significantly from Day 6 to 18 (Table 1).
3.4. Oocyte viability and diameter, recovery rate of oocytes cultured in vitro, and meiotic stages of sheep oocytes from preantral follicles after long-term culture The recovery rate of oocytes from normal intact– cultured follicles was similar between vitrified and control.
500
Aa Aab
Follicular diameter (μm)
450 400 350
Aa
Abc Aa
Aa Aa
Aa
Aa
Ac Ab Aa
300 250
However, the follicle-vit had a higher (P < 0.05) percentage of recovered oocytes when compared to tissue-vit. Recovered oocytes from antral follicles that grown in vivo (in vivo grown) had larger (P < 0.05) diameter than oocytes cultured in vitro. Moreover, oocytes from control (fresh cultured secondary follicles) had higher (P < 0.05) diameter when compared to oocytes from follicle-vit and tissue-vit groups. The vitrification procedure of secondary follicles (isolated or enclosed on ovarian stroma) did not affect the oocyte size after IVM (Table 2). After IVM, all oocytes were incubated with fluorescent labels calcein-AM, ethidium homodimer-1, and Hoechst 33342 to evaluate viability and chromatin configuration, respectively. Tissue-vit group had higher (P < 0.05) oocyte degeneration rate (33.33%) when compared to other groups (in vivo grown, 6.66%; control, 8.69%, and follicle-vit, 7.69%). When evaluating chromatin configuration, the percentage of meiotic resumption (GVBD þ MI þ MII) in control, follicle-vit, and tissue-vit did not differ, but they were lower (P < 0.05) than in vivo grown. In vitrified groups, only follicle-vit had one oocyte in MI. As expected, in vivo grown oocytes showed a significantly higher percentage of MII oocyte (Table 2) than in control (Fig. 5).
200 150
4. Discussion
100 50 0 Day 0
Day 6
Control
Follicle-Vit
Day 12
Day 18
Tissue-Vit
Fig. 3. Follicular diameter (mm; mean standard error of the mean) of sheep preantral follicles without vitrification (control, n ¼ 21) and after vitrification of isolated follicles (follicle-vit, n ¼ 38) or ovarian tissue (tissuevit, n ¼ 44) at Days 0, 6, 12, and 18 of IVC. AThe uppercase letter indicates statistically significant differences among groups (P < 0.05) within the same day. abcDifferent lowercase letters indicate statistically significant differences among days of culture (P < 0.05) within the same group.
In the present study, the follicular development and the oocyte IVM were evaluated after two different forms of vitrification of sheep secondary follicles: vitrification in isolated form (without much stroma) or vitrification in in situ form (within ovarian tissue fragments). Therefore, secondary follicles were first isolated from ovarian fragments and then vitrified, i.e., the follicle-vitrification group (follicle-vit) or fragments of ovarian tissue that were first vitrified, followed by isolation of the secondary follicles, composing the tissue-vitrification group (tissue-vit).
1208
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
9,0
B A
Daily growth rate (μm/day)
8,0 7,0
A
6,0 5,0 4,0
B 3,0 2,0 1,0
Follicles with decreased diameter (%)
A
0,0
70 60
A
50
40
B
30 20 10
C
0
Control
Follicle-Vit
Tissue-Vit
Control
Follicle-Vit
Tissue-Vit
Fig. 4. (A) Daily growth rate (mm/day; mean standard error of the mean) of sheep preantral follicles without vitrification (control, n ¼ 21) and after vitrification of isolated follicles (follicle-vit, n ¼ 38) or ovarian tissue (tissue-vit, n ¼ 44) after 18 days of IVC and (B) Percentage of sheep preantral follicles with decreased diameter from control (n ¼ 18), follicle-vit (n ¼ 1), and tissue-vit (n ¼ 16) after 18 days of culture. ABCDifferent uppercase letters indicate statistically significant differences among groups (P < 0.05).
Our data showed that the follicles from follicle-vit had higher follicular growth rate, oocyte viability, and recovery rate. Similar results were reported by Hatami et al. [4] in mice, in which the vitrification of isolated preantral follicles was better than vitrification of the whole ovary. These authors observed better follicular survival, antrum formation, and oocyte maturation rates. These results may be due to different conditions under which follicles are submitted when vitrified, within or outside of the ovarian stroma. Follicles and oocytes vitrified included in ovarian tissue may not be fully penetrated by intracellular cryoprotectants, because of the large number of cells that surround them. Moreover, the differences among the various cell types present in the ovarian tissue can interfere with the permeability of cryoprotectants, leading follicles to an increased susceptibility to injuries, during cooling. The ovarian fragments were w2 mm thickness, whereas their secondary follicle has an average diameter of 312.57 mm, thereby, the spread of the cryoprotectant into the tissue, during the period of exposure to vitrification solutions, may not have occurred homogeneously, resulting in an irregular dehydration leaving a substrate for ice formation [14,18]. A previous study showed a high percentage of follicles with decreasing diameter in culture of 6 days when they
had previously undergone vitrification [15]. In the present study, we found that when the follicles were vitrified in isolated form (follicle-vit) only 2.56% had a diameter reduction, which was lower than the reduction of follicles vitrified in the tissue (tissue-vit) or fresh follicles (control). Reduction in follicular diameter may occur because of dehydration and osmotic variations suffered during vitrification [19]. This is an acute phenomenon and their effect must be temporary. Follicular diameter should be restored after a few hours of IVC. Studies indicate that if the diameter is not reestablished (as we observed in this study even after 18 days of IVC) it can be considered an indication of follicular degeneration [6,7,20]. In the present study, the follicle-vit (5.02 0.63 mm; 372.0 100.0 pmol/L) treatment had follicular growth rate similar to the control group. However, the estradiol production on the same treatment was lower than control (7.05 1.07 mm; 4729.0 1200.0 pmol/L). The variation in estradiol production may be due to spontaneous luteinization of these follicles cells and not characterize the proliferative or follicular phase of normal estrous cycle. Although, luteinized cells are not typical of follicles that will be recruited and will gain dominance in the normal ovarian folliculogenesis. Xing et al. [21] reported a lower estradiol production and a greater diameter in rats isolated
Table 1 17b-estradiol (pmol/L; mean SEM) and progesterone (nmol/L) concentration (mean SEM) in pooled media collected on the different days during longterm culture of preantral follicles in control (n ¼ 20), follicle-vit (n ¼ 20), and tissue-vit (n ¼ 20) groups. Groups
Control Follicle-vit Tissue-vit ab
Estradiol
Progesterone
Day 6
Day 12
Day 18
Day 6
Day 12
Day 18
1989.0 1270.0bA 306.0 31.0aB 301.0 38.0aB
2792.0 1800.0abA 263.0 24.0bC 353.0 61.0aB
4729.0 1200.0aA 372.0 100.0aB 375.0 180.0aB
d 9.0 6bB 16 6.0aB
10.0 3aB 12.0 3bA 13.0 2.0bA
7.0 1.2aC 25.0 7aA 15.0 1.0aB
Within a row, values without a common superscript differed among days (P < 0.01). Within a column, values without a common superscript differed among groups (P < 0.01). Abbreviation: SEM, standard error of the mean. ABC
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
1209
Table 2 Oocyte viability (%) and diameter (mm SD), recovery rate of oocytes cultured in vitro (%), and meiotic stages (%) of sheep oocytes from preantral follicles after long-term culture (18 days) in control, follicle-vit, and tissue-vit groups. Groups
No. of oocytes recovered/No. of normal follicles (%)
No. of viable oocytes/No. of oocytes (%)
In vivo grown Control Follicle-vit Tissue-vit
d 23/39 (58.97)AB 26/39 (66.67)A 24/60 (40.00)B
28/30 21/23 24/26 16/24
(93.3)A (91.3)A (92.3)A (66.7)B
Mean oocyte diameter (mm)
131.10 105.99 93.94 90.40
23.07A 9.18B 12.84C 14.28C
No. of oocytes with meiosis resumption/No. of viable oocytes (%)
No. of GVBD oocytes/No. of oocytes meiosis resumption (%)
No. of MI oocytes/No. of oocytes meiosis resumption (%)
No. of MII oocytes/No. of oocytes meiosis resumption (%)
26/28 (92.85)A 8/21 (38.09)B 11/24 (45.83)B 6/16 (37.5)B
9/26 (34.61)B 4/8 (50.00)AB 10/11 (90.90)A 6/6 (100)A
1/26 (3.84)B 3/8 (37.50)A 1/11 (9.09)AB 0/6 (0.0)
16/26 (61.53)A 1/8 (12.50)B 0/11 (0.0) 0/6 (0.0)
ABC
Different uppercase letters indicate statistically significant differences (P < 0.05) among groups (column). Abbreviations: GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II; SD, standard deviation.
secondary follicles after vitrification. Otherwise, during vitrification process, the cells can decrease cell metabolism and consequently decrease the estradiol production in an attempt to compensate the potential damage. Moreover, Silva et al. [22] and Cecconi et al. [23] previously reported that estradiol synthesis and follicular growth pattern are not strictly dependent on each other. Both treatments with vitrified follicles in our experiment had lower production of estradiol than the control, probably because the internal theca cells, and even part of granulosa cells have been possibly damaged by the toxic effect of cryoprotectants. The internal theca cells secrete androstenedione, an androgenic precursor that is transferred through the basal lamina to the granulosa cells culminating in the production of testosterone, which is then converted to estradiol by aromatase [24]. These cells damage during vitrification procedures compromises the intrinsic machinery of hormone production, and certainly, the oocyte growth and competence for development will also be damaged. Therefore, it becomes necessary deeper studies of methods that preserve the functionality of theca and granulosa cells after vitrification to ensure and to maintain adequate production of estradiol to support the long term culture and better maturation rate. In this study, the oocytes from follicles previously vitrified showed significantly smaller diameter, after IVM, than the oocytes from follicles in vitro cultured without prior vitrification. In a prior study, it has been shown that most oocytes vitrified in isolated secondary follicles cease their growth in the first 6 days of culture, and vitrified oocytes in secondary follicles enclosed in ovarian tissue, not only stop growing, in the same period of IVC, but also reduce in diameter [15].
In the tissue-vit group, the cooling of the oocyte is slow and gradual, as occurs in peripheral region to the interior of the follicle, where is situated the oocyte [4]. In follicle-vit group, the oocyte is free of barriers such as the ovarian stroma, so the cooling is faster. As a high cooling rate is associated with the absence of ice crystal formation [18], we suggest that this might be the reason why the oocytes from follicle-vit had a higher post-thaw viability than oocytes from tissue-vit group. Other studies [25,26] have also demonstrated that the final oocyte diameter from in vitro cultured follicles remains smaller than the diameter of oocytes from in vivo grown follicles. This may be an early sign of degeneration, possibly because of injuries in the connecting structures between oocyte and surrounding granulosa cells because it is known that the vitrification pronouncedly reduces the oocyte microvilli [15]. The loss of these microvilli may impair the oocyte-somatic cells communication and reduces their growth characteristics [21]. This study showed for the first time that sheep oocytes from vitrified secondary follicles (follicle-vit) cultured in vitro, for 18 days, are able to resume meiosis (GV breaks), and also reach the stage of MI, after IVM, although with lower rate compared to in vivo grown oocytes. Although many efforts and promising results have been reported by different teams regarding the in vitro development of preantral follicles in different species [10], we believe that the conditions of IVC of these follicles, which ensure the full oocyte growth need to be improved especially to oocytes previously submitted to cryopreservation. We also believe that the determination of an ideal system for the IVC of preantral follicles cryopreserved or not is absolutely necessary, whereas the transplantation of
Fig. 5. Viable oocyte in metaphase I after IVM from follicle-vit group after IVC for 18 days. Brightfield (A), Calcein-AM (B), and Hoechst 33342 (C).
1210
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
ovarian tissue offers risks of reintroduction of cancer cells to patients in remission. Considering that isolated follicles display a better follicular growth rate, oocyte viability, and fewer follicles with decreased diameter IVC, after vitrification, we conclude that with our current method of vitrification, the strategy of vitrification after isolation of secondary follicles provided a better substratum for growing the enclosed oocytes. Acknowledgments This work was supported by CNPq. Franciele Osmarini Lunardi is a recipient of a grant from CAPES Brazil. In addition, Ana Paula Ribeiro Rodrigues and José Ricardo de Figueiredo are recipients of a grant from CNPq Brazil. Johan Smitz is Especial Visitor Researcher from CAPES. References [1] Shaw JM, Bowles J, Koopman P, Wood EC, Trounson AO. Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to graft recipients. Hum Reprod 1996;11:1668–73. [2] Dolmans M-M, Marinescu C, Saussoy P, Van Langendonckt A, Amorim C, Donnez J. Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe. Blood 2010;116:2908–14. [3] Rosendahl M, Andersen MT, Ralfkiaer E, Kjeldsen L, Andersen MK, Andersen CY. Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia. Fertil Steril 2010;94: 2186–90. [4] Hatami S, Zavareh S, Salehnia M, Lashkarbolouki T, Ghorbanian MT, Karimi I. The impact of alpha lipoic acid on developmental competence of mouse vitrified pre-antral follicles in comparison to those isolated from vitrified ovaries. Iran J Reprod Med 2014;12:57– 64. [5] O’Brien MJ, Pendola JK, Eppig JJ. A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biol Reprod 2003; 68:1682–6. [6] Xu J, Bernuci MP, Lawson MS, Yeoman RR, Fisher TE, Zelinski MB, et al. Survival, growth, and maturation of secondary follicles from prepubertal, young, and older adult rhesus monkeys during encapsulated three-dimensional culture: effects of gonadotropins and insulin. Reproduction 2010;140:685–97. [7] Xu J, Lawson MS, Yeoman RR, Pau KY, Barrett SL, Zelinski MB, et al. Secondary follicle growth and oocyte maturation during encapsulated three-dimensional culture in rhesus monkeys: effects of gonadotrophins, oxygen and fetuin. Hum Reprod 2011;26:1061–72. [8] Saraiva MV, Rossetto R, Brito IR, Celestino JJ, Silva CM, Faustino LR, et al. Dynamic medium produces caprine embryo from preantral follicles grown in vitro. Reprod Sci 2010;17:1135–43.
[9] Arunakumari G, Shanmugasundaram N, Rao VH. Development of morulae from the oocytes of cultured sheep preantral follicles. Theriogenology 2010;74:884–94. [10] Telfer EE, Zelinski MB. Ovarian follicle culture: advances and challenges for human and nonhuman primates. Fertil Steril 2013;99: 1523–33. [11] Hasegawa A, Mochida N, Ogasawara T, Koyama K. Pup birth from mouse oocytes in preantral follicles derived from vitrified and warmed ovaries followed by in vitro growth, in vitro maturation, and in vitro fertilization. Fertil Steril 2006;86:1182–92. [12] Wang XQ, Catt S, Pangestu M, Temple-Smith P. Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births. Reproduction 2011;141:183–91. [13] Liebermann J, Nawroth F, Isachenko V, Isachenko E, Rahimi G, Tucker MJ. Potential importance of vitrification in reproductive medicine. Biol Reprod 2002;67:1671–80. [14] Zhou XM, Qiao WT, Zhang XL, Liu Z, Gao DY. Physical modeling of flow boiling in microchannels and its induced vitrification of biomaterials. Int J Heat Mass Transfs 2015;83:659–64. [15] Lunardi FO, Chaves RN, de Lima LF, Araújo VR, Brito IR, Souza CE, et al. Vitrified sheep isolated secondary follicles are able to grow and form antrum after a short period of in vitro culture. Cell Tissue Res 2015;362:241–51. [16] Lunardi FO, Araujo VR, Faustino LR, Carvalho AA, Braga Goncalves RF, Bass CS, et al. Morphologic, viability and ultrastructural analysis of vitrified sheep preantral follicles enclosed in ovarian tissue. Small Rumin Res 2012;107:121–30. [17] Luz VB, Araujo VR, Duarte AB, Celestino JJ, Silva TF, MagalhaesPadilha DM, et al. Eight-cell parthenotes originated from in vitro grown sheep preantral follicles. Reprod Sci 2012;19:1219–25. [18] Devismita D, Kumar A. Effect of cryoprotectant on optimal cooling rate during cryopreservation. Cryobiology 2015;70:53–9. [19] Paynter SJ, Cooper A, Fuller BJ, Shaw RW. Cryopreservation of bovine ovarian tissue: structural normality of follicles after thawing and culture in vitro. Cryobiology 1999;38:301–9. [20] Ting AY, Yeoman RR, Lawson MS, Zelinski MB. In vitro development of secondary follicles from cryopreserved rhesus macaque ovarian tissue after slow-rate freeze or vitrification. Hum Reprod 2011;26: 2461–72. [21] Xing W, Zhou C, Bian J, Montag M, Xu Y, Li Y, et al. Solid-surface vitrification is an appropriate and convenient method for cryopreservation of isolated rat follicles. Reprod Biol Endocrinol 2010;8:42. [22] Silva CM, Castro SV, Faustino LR, Rodrigues GQ, Brito IR, Rossetto R, et al. The effects of epidermal growth factor (EGF) on the in vitro development of isolated goat secondary follicles and the relative mRNA expression of EGF, EGF-R, FSH-R and P450 aromatase in cultured follicles. Res Vet Sci 2013;94:453–61. [23] Cecconi S, Barboni B, Coccia M, Mattioli M. In vitro development of sheep preantral follicles. Biol Reprod 1999;60:594–601. [24] Walters KA. Role of androgens in normal and pathological ovarian function. Reproduction 2015;149:R193–218. [25] Hu Y, Betzendahl I, Cortvrindt R, Smitz J, Eichenlaub-Ritter U. Effects of low O(2) and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture. Hum Reprod 2001; 16:737–48. [26] Segers I, Adriaenssens T, Ozturk E, Smitz J. Acquisition and loss of oocyte meiotic and developmental competence during in vitro antral follicle growth in mouse. Fertil Steril 2010;93:2695–700.
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Ovine secondary follicles vitrified out the ovarian tissue grow and develop in vitro better than those vitrified into the ovarian fragments Franciele Osmarini Lunardi a, *, Francisco Leo Nascimento de Aguiar a, Ana Beatriz Graça Duarte a, Valdevane Rocha Araújo a, Laritza Ferreira de Lima a, Naiza Arcângela Ribeiro de Sá a, Hudson Henrique Vieira Correia a, Sheyla Farhayldes Souza Domingues b, Cláudio Cabral Campello a, Johan Smitz c, José Ricardo de Figueiredo a, Ana Paula Ribeiro Rodrigues a a
Laboratory of Manipulation of Oocytes and Ovarian Pre-antral Follicles (LAMOFOPA), Faculty of Veterinary Medicine, Ceará State University, Fortaleza, CE, Brazil b Laboratory of Wild Animal Biology and Medicine, Federal University of Pará, Belem, Brazil c Follicle Biology Laboratory, Center for Reproductive Medicine, UZ Brussel, Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 August 2015 Received in revised form 8 October 2015 Accepted 30 October 2015
Cryopreservation of preantral follicles is a promising technique to preserve female fertility. The aim of this study was to evaluate the effect of vitrification on the development of secondary follicles included in ovarian tissue or isolated after microdissection. An important end point included is the capacity of grown oocytes to resume meiosis. Sheep ovarian cortexes were cut into fragments and split into three different groups: (1) fresh (control): secondary follicles isolated without any previous vitrification; (2) folliclevitrification (follicle-vit): secondary follicles vitrified in isolated form; and (3) tissuevitrification (tissue-vit): secondary follicles vitrified within fragments of ovarian tissue (in situ former) and subsequently subjected to isolation. From the three groups, isolated secondary follicles were submitted to IVC for 18 days. After IVC, cumulus-oocyte complexes (COCs) were harvested from follicles. As an additional control group, in vivo grown, in vivo-grown COCs were collected from antral ovarian follicles. All, recovered COCs were matured and the chromatin configuration was evaluated. Data were analyzed by ANOVA, and the means were compared by Student-Newman-Keuls test, and by chisquare. Differences were considered to be significant when P < 0.05. Isolated preantral follicles from all treatments had normal morphology, antrum formation, and low follicle degeneration after IVC. The growth rate between control and follicle-vit did not differ (P > 0.05), and was higher (P < 0.05) than for tissue-vit. The percentage of follicles that decreased diameter during IVC was significantly higher in tissue-vit than the in folliclevit. Recovery rate of oocytes from normal follicles was higher in follicle-vit than in tissuevit. Furthermore, oocyte viability was lower in tissue-vit than other treatments, and follicle-vit did not differ from control and in vivo grown. The percentage of oocytes meiosis resuming was not different between treatments except for in vivo grown. After vitrification, only follicle-vit showed metaphase I oocyte. We conclude that secondary follicles vitrified after isolation displayed a better follicular growth rate, oocyte viability,
Keywords: Cryopreservation Follicular development Oocyte maturation Ovary Ovine/sheep
* Corresponding author. Tel.: þ55 85 31019861; fax: þ55 85 31019859. E-mail address: [email protected] (F.O. Lunardi). 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.10.043
1204
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
percentage of oocytes reaching the metaphase I stage, and fewer follicles with decreased diameter after IVC. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Cryopreservation of ovarian tissue is nowadays considered a feasible option for fertility preservation. However, some authors have indicated the risk of reintroduction of cancer cells after transplantation [1–3]. Thus, the IVC of isolated secondary follicles after cryopreservation is considered a reasonable alternative to avoid this and to obtain fertilizable oocytes [4]. Appropriate protocols for IVC of noncryopreserved preantral follicles had already been reported in murine, in which viable offspring were obtained [5]. Nevertheless, in nonhumane primates species [6,7] or livestock animals, such as caprine [8] and ovine [9], only a limited and variable number of embryos arising from preantral follicle oocytes cultured in vitro have been obtained until today. Although the higher results in animal models being plenty supportive, in humane species the results are not suitable, considering reports of low frequency of antrum formation of preantral follicles grown in vitro [10]. Notwithstanding, only in murine, embryos were obtained from previously cryopreserved ovarian tissue, with successful reports [11,12]. In latter studies, vitrification was used as the first choice method for ovary cryopreservation. Vitrification constitutes the ultra-rapid cooling of biological material to very low temperatures and the use of high concentrations of intracellular cryoprotectant agents. These particular conditions allow a solidification, or amorphous liquid state of vitrification solution, which inhibits nucleation and ice crystals growth [13,14]. The ovarian tissue is constituted by different cellular types that vary in size, distribution, and quantity and by many follicular development stages cells (granulosa and theca cells) that form the barrier to the oocytes. All these structures represent a huge hurdle to an optimal permeability for cryoprotectant agents and could result in difficulties to an adequate oocyte protection at liquid nitrogen temperature (196 C). Recently, our group reported that secondary sheep follicles could be vitrified effectively, enclosed or not in ovarian cortex. After thawing, these follicles (previously isolated or isolated after ovarian fragment vitrification) are capable to form an antrum when cultured in vitro for 6 days and to be similar to fresh noncultured follicles without vitrification [15]. However, because of the short IVC that was applied, it was not possible to ascertain if oocytes enclosed in secondary follicles were capable to survive a long-term culture, needed to reach the development stage of oocyte to resume meiosis. The present study aimed to vitrify secondary follicles enclosed in or isolated from ovarian tissue and to evaluate relevant end points: post-thaw follicular survival, growth, antrum formation and hormone production after 18 days of culture, and oocyte viability and maturation.
This experiment was approved and performed under the guidelines of Ethics Committee for Animal Use of State University of Ceará. Unless otherwise mentioned, all chemicals used in the present study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.1. Source of ovaries Ovaries (n ¼ 30) were collected at abattoirs from 15 adult mixed-breed ewes. Immediately postmortem, under aseptic conditions, the ovaries were washed in 70% alcohol for 10 seconds, followed by two washes in HEPES buffered minimum essential medium (MEM) supplemented with 100-mg/mL penicillin and 100-mg/mL streptomycin. Each pair of ovaries was transported to the laboratory within 4 hours into tubes containing 15 mL of MEM HEPES at 4 C. 2.2. Experimental design At the laboratory, ovaries were cleaned from surrounding fat and fibrous tissue, and their cortexes were recovered and fragmented into pieces (1–2 mm thick) and split into three different groups: (1) fresh (control): secondary follicles were isolated without any previous vitrification; (2) follicle-vitrification (follicle-vit): secondary follicles were vitrified in isolated form; and (3) tissuevitrification (tissue-vit): secondary follicles (formed in situ) were vitrified within fragments of ovarian tissue and isolated subsequently after warming. From all groups, isolated secondary follicles were in vitro cultured for 18 days. During IVC, the levels of hormones secretion (progesterone and estradiol), antrum formation, and growth rate were measured. After IVC, the cumulus-oocyte complexes (COCs) were recovered from cultured follicles and from in vivo grown and submitted to IVM. In addition, the viability and chromatin configuration of the oocytes were also evaluated. 2.3. Ovarian cortex fragmentation and follicle isolation The ovarian cortex was sectioned into pieces of 1 to 2 mm thickness under sterile conditions in a petri dish containing MEM HEPES supplemented with antibiotics (100-mg/mL penicillin and 100-mg/mL streptomycin). Fragments obtained were transferred to another petri dish containing fresh medium and then visualized under a stereomicroscope (Nikon SMZ 645; Tokyo, Japan; magnification: 100). Once localized, secondary follicles, with average diameter of 325.07 mm (range 150–350 mm), were mechanically isolated by microdissection using a 26-G needle attached to 1-mL syringe. Only those follicles with the oocyte-follicle complex visible, surrounded by several layers of granulosa cells with intact basement membrane and no antral cavities were selected for this study.
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
2.4. IVC of sheep secondary follicles Secondary follicles were transferred into drops of 100 mL of culture medium under mineral oil in petri dishes (60 15 mm) and cultured for 18 days (one follicle/drop) at 39 C and 5% CO2 in air. The culture medium used was composed of MEM alpha (a-MEM) supplemented with 10-mg/mL insulin, 5.5-mg/mL transferrin, 5-ng/mL selenium, 2-mM glutamine, 2-mM hypoxanthine, 3-mg/mL bovine serum albumin, 50-mg/mL ascorbic acid, 50-ng/mL leukemia inhibitory factor, and 50-ng/mL Kit ligand. Fresh media were prepared immediately before use and were incubated for 1 hour prior to use. Every 6 days, 100 mL of medium was replaced with fresh medium. This medium was chosen on the basis of our previous study [15]. 2.5. Morphologic analysis and assessment of in vitro follicular growth At Day 0, 6, 12, and after 18 days of IVC, the percentage of morphologically normal follicles and follicular diameters from control, follicle-vit, and tissue-vit were determined. Follicles were classified as morphologically normal if they presented an intact basement membrane, no extrusion of the COCs from the follicle, bright and homogeneous granulosa and theca cells. Follicle degeneration was recognized when the oocytes and surrounding cells were darkened, had misshaped oocytes or decreased follicle diameter. Antral cavity formation was defined as a visible translucent cavity within the granulosa cell layers. Follicular diameter was measured at the basement membrane (from the major and minor axes) of each follicle with the aid of an ocular micrometer inserted into a stereomicroscope (SMZ 645 Nikon; Tokyo, Japan; magnification: 100) on Days 0, 6, 12, and 18 of IVC. The average of these two measurements was used to determine the follicle diameter only in morphologically normal follicles. The daily mean increase in follicular diameter (daily follicular growth rate) was calculated as the diameter of morphologically normal follicles at Day 18 minus the diameter of the same follicle at Day 0, divided by the total number of days in culture. 2.6. Vitrification protocol used to ovarian isolated follicles and fragments Ovarian fragments (1–2 mm thick) and isolated secondary follicles were exposed to different concentrations (12.5%, 25%, 50%, and 100%) of vitrification solution composed by MEM HEPES supplemented with 10% fetal bovine serum (FBS), 2.60-M acetamide, 2.62-M dimethylsulfoxide, 1.31-M 1, 2-propanediol, and 0.0075-M polyethylene glycol. Samples were exposed to the first two concentrations (12.5% and 25%) of vitrification solution for 5 minutes at room temperature; then to the next two concentrations (50% and 100%) of vitrification solution for 15 minutes (for fragments) or 5 minutes (for isolated follicles) at 4 C. After, samples (either ovarian fragments or isolated follicles) were placed on the surface of a metal cube partially submerged in liquid nitrogen and transferred to cryovials (10 follicles or 10 fragments each) in accordance with protocol adapted from
1205
Lunardi et al. [15]. Samples were kept into liquid nitrogen (196 C) for 6 days. 2.7. Warming protocol of ovarian follicles and fragments Cryovials containing samples of ovarian tissue and isolated secondary follicles were removed from liquid nitrogen and exposed to room temperature for 1 minute and immersed in a water bath (37 C) for 30 seconds. Soon after, removal of cryoprotectants was performed by immersion of ovarian fragments or secondary follicles in washing solutions, three baths of 5 minutes each with the solution composed out of MEM HEPES plus 10% FBS and decreasing concentrations of sucrose (0.5, 0.25, and 0.0 M) in accordance with protocol adapted from Lunardi et al. [16]. 2.8. Steroid production To evaluate 17b-estradiol (pmol/L) and progesterone (nmol/L) concentrations during IVC, the total of 30 pooled samples of conditioned medium from 60 growing follicles (two follicles per medium, 10 samples per group) from control and each of the vitrification groups at Days 6, 12, and 18 was retrieved and stored at 80 C. The concentrations of 17b-estradiol (pmol/L) and progesterone (nmol/L) were measured by chemiluminescence using an immunoassay system (Vitros Eci/EciQ Immunodiagnostic System; Johnson & Johnson Company, Ortho-Clinical Diagnostics, Buckinghamshire, UK) according to manufacturer’s instructions. 2.9. IVM of COCs At the end of the culture period, the COCs obtained from in vitro cultured were carefully harvested from intact follicles using 26-G needles and from extrude follicles under a stereomicroscope. From in vivo grown control, in vivo-grown COCs were collected from antral ovarian follicles. Oocytes surrounded by at least one compact layer of cumulus cells were selected for IVM. The recovery rate was calculated by dividing the number of COCs by the number of viable follicles at Day 18 of culture multiplied by 100. The selected COCs were washed in medium composed of tissue culture medium 199 (TCM199) plus HEPES supplement with pyruvate (0.911 mM/L) and 10% FBS followed by IVM medium. The maturation medium was composed by tissue culture medium 199 plus sodium bicarbonate supplemented with 0.5mg/mL recombinant bovine follicle-stimulating hormone (Nanocore, Campinas, São Paulo, Brazil), 5-mg/mL luteinizing hormone, 1-mg/mL 17b-estradiol, 10-ng/mL recombinant epidermal growth factor, 0.911-mM/L pyruvate, 100-mM/L cysteamine, 50-ng/mL recombinant insulin-like growth factor I, and 1% bovine serum albumin. After washing, COCs were transferred to 50-mL drops of maturation medium under mineral oil and then incubated for 36 to 40 hours at 39 C with 5% CO2 according to Luz et al. [17]. 2.10. Assessment of oocyte viability and chromatin configuration After IVM, the COCs were denuded from surrounding expanded cumulus cells by manual pipetting in TCM199
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
HEPES containing 0.1% hyaluronidase and subjected to viability analysis. To this end, the oocytes were incubated in 100-mL drops of 2-mM ethidium homodimer-1 supplemented with 4-mM calcein-acetoxymethyl (calcein-AM), 0.5% glutaraldehyde, and 10-mM Hoechst 33342 at room temperature for 30 minutes. After this, the oocytes were washed in TCM199 HEPES and were visualized under fluorescence microscopy (Nikon Eclipse 80i; Tokyo, Japan; magnification: 40). Oocytes were considered viable if the cytoplasm was positively stained with calcein-AM (green) and not stained with ethidium homodimer-1 (red). The emitted fluorescent signals of calcein-AM and ethidium homodimer-1 were collected at 488 nm and 568 nm, respectively. In addition, oocytes were stained with Hoechst 33342 (Molecular Probes; Invitrogen, Karlsruhe, Germany) and then analyzed for chromatin configuration being emitted fluorescent signals at 483 nm. This dye was used to analyze the oocyte’s chromatin configuration through observation of the intact germinal vesicle (GV), meiotic resumption (including GV breakdown [GVBD], metaphase I [MI], anaphase I, or telophase I), or nuclear maturation (metaphase II [MII]). 2.11. Statistical analysis Data for discrete variables (morphologically normal follicles, antrum formation, recovery rate and chromatin configuration of oocytes) were analyzed as dispersion of frequency using chi-square test. Otherwise, when the observed frequency was equal or less than five units, Fisher’s exact test was applied. In both cases, results were expressed as percentages. Data for continuous variables (follicular diameter, growth rate after culture, and hormonal dosages) Kruskal–Wallis and unpaired t test were initially evaluated for homocedasticity and normal distribution of the residues, by Bartlett’s and Shapiro–Wilk tests, respectively. Confirmed both requirements underlying analysis of variance, the effects of treatment, time of culture, and treatment by time interaction were analyzed using PROC MIXED of SAS (2002), including repeated statement to account for autocorrelation between sequential measurements. The model was Yijk ¼ m þ Ri þ Fj þ Tk þ (RT)ik þ eijk, where Yijk is the observation of the jth follicle in the ith treatment at the kth time of culture, m is the overall mean, Ri is the ith treatment, Fj is the random effect of the jth follicle within the ith treatment, Tk is the kth time of culture, (RT)ik is the treatment by time interaction term, and eijk is the random residual effect. Comparisons among treatments or times were further analyzed by the Student-Newman-Keuls test. A probability of P < 0.05 indicated a significant difference, and results were expressed as mean standard deviation (oocyte diameter) or expressed as mean standard error of the mean (follicle diameter, growth rate). 3. Results 3.1. Assessment of morphologically normal follicles and antral cavity formation during culture A total of 154 secondary follicles were distributed randomly to groups control (n ¼ 50), follicle-vit (n ¼ 40),
and tissue-vit (n ¼ 64) and in vitro cultured for 18 days. The percentage of morphologically normal follicles was similar between follicle-vit (97.5%, n ¼ 39/40) and tissue-vit (93.75%, n ¼ 60/64) being both higher (P < 0.05) than in control group (78%, n ¼ 39/50) (Fig. 1). At the end of culture period, some follicles showed rupture of the basal membrane and, consequently, released their COCs. In control (20%, n ¼ 10/50), there was an increase (P < 0.05) in the percentage of extruded oocytes when compared to tissue-vit (1.56%, n ¼ 1/64). Interestingly, the follicle-vit (n ¼ 0/40) did not present follicle extrusion. The percentage of degenerated follicles was similar (P > 0.05) among the three groups (control, 2%; follicle-vit, 2.5%; and tissue-vit, 4.69%). Except for follicle-vit, the percentage of antrum formation increased significantly from Day 6 to 18 of culture. A significantly higher percentage of antrum formation was observed in the tissue-vit compared to the control (Fig. 2).
3.2. Follicle diameter and daily growth rate before and after vitrification and IVC of ovine preantral follicles Follicle diameter increased significantly from Day 0 to 18 of culture only in the control and follicle-vit groups. However, no differences were observed among the groups regardless the culture time (P > 0.05; Fig. 3). The Figure 4A illustrates the follicular daily growth rate in the three groups throughout the period of IVC. The control (7.05 1.07 mm/day, n ¼ 21) and follicle-vit (5.02 0.63 mm/day, n ¼ 38) were similar between themselves and higher (P < 0.05) than tissue-vit (2.42 0.32 mm/day, n ¼ 44). The Figure 4B shows the percentage of follicles with decreased diameter throughout the IVC. The percentage of follicles with decreased diameter in follicle-vit (2.56%, n ¼ 1/39) was lower (P < 0.05) than control (46.15%, n ¼ 18/39) and in tissue-vit (26.67%, n ¼ 16/60). 100
Aa Aa Aa
Aa Aa Aa
Aa Ab
Aa Aab Ab
Normal follicles (%)
1206
Bb
80
60
40
20
0
Day 0
Day 6
Control
Follicle-Vit
Day 12
Day 18
Tissue-Vit
Fig. 1. Percentage of isolated morphologically normal sheep preantral follicles without vitrification (control, n ¼ 50) and after vitrification of isolated follicles (follicle-vit, n ¼ 40) or ovarian tissue (tissue-vit, n ¼ 64) at Days 0, 6, 12, and 18 of IVC. ABDifferent uppercase letters indicate statistically significant differences among groups (P < 0.05) within the same day. abDifferent lowercase letters indicate statistically significant differences among days of culture (P < 0.05) within the same group.
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
B
E
100 μm
C
100 μm
D
Antrum formation (%)
A
1207
Aa Ab
80 60
ABa Aa
Aa Aa
100
Ba Bab
Bb
40 20 0
Day 6 100 μm
100 μm
Control
Day 12
Follicle-Vit
Day 18
Tissue-Vit
Fig. 2. Normal sheep secondary follicle before (Day 0) IVC (A) or cultured for 6 days (B), 12 days (C), or 18 days (D) in follicle-vit group. Note beginning of antrum formation at Day 6 of IVC. (E) Percentage of antrum formation of sheep preantral follicles without vitrification (control, n ¼ 39) and after vitrification of isolated follicles follicle-vit, n ¼ 39) or ovarian tissue (tissue-vit, n ¼ 60) at Days 6, 12, and 18 of IVC. ABDifferent uppercase letters indicate statistically significant differences among groups (P < 0.05) within the same day. abDifferent lowercase letters indicate statistically significant differences among days of culture (P < 0.05) within the same group.
3.3. Steroid production The follicles from control produced higher (P < 0.01) estradiol levels than vitrified groups. Besides, only in the control, estradiol production increased significantly from Day 6 to 18 of culture, only in follicle-vit, progesterone levels increase significantly from Day 6 to 18 (Table 1).
3.4. Oocyte viability and diameter, recovery rate of oocytes cultured in vitro, and meiotic stages of sheep oocytes from preantral follicles after long-term culture The recovery rate of oocytes from normal intact– cultured follicles was similar between vitrified and control.
500
Aa Aab
Follicular diameter (μm)
450 400 350
Aa
Abc Aa
Aa Aa
Aa
Aa
Ac Ab Aa
300 250
However, the follicle-vit had a higher (P < 0.05) percentage of recovered oocytes when compared to tissue-vit. Recovered oocytes from antral follicles that grown in vivo (in vivo grown) had larger (P < 0.05) diameter than oocytes cultured in vitro. Moreover, oocytes from control (fresh cultured secondary follicles) had higher (P < 0.05) diameter when compared to oocytes from follicle-vit and tissue-vit groups. The vitrification procedure of secondary follicles (isolated or enclosed on ovarian stroma) did not affect the oocyte size after IVM (Table 2). After IVM, all oocytes were incubated with fluorescent labels calcein-AM, ethidium homodimer-1, and Hoechst 33342 to evaluate viability and chromatin configuration, respectively. Tissue-vit group had higher (P < 0.05) oocyte degeneration rate (33.33%) when compared to other groups (in vivo grown, 6.66%; control, 8.69%, and follicle-vit, 7.69%). When evaluating chromatin configuration, the percentage of meiotic resumption (GVBD þ MI þ MII) in control, follicle-vit, and tissue-vit did not differ, but they were lower (P < 0.05) than in vivo grown. In vitrified groups, only follicle-vit had one oocyte in MI. As expected, in vivo grown oocytes showed a significantly higher percentage of MII oocyte (Table 2) than in control (Fig. 5).
200 150
4. Discussion
100 50 0 Day 0
Day 6
Control
Follicle-Vit
Day 12
Day 18
Tissue-Vit
Fig. 3. Follicular diameter (mm; mean standard error of the mean) of sheep preantral follicles without vitrification (control, n ¼ 21) and after vitrification of isolated follicles (follicle-vit, n ¼ 38) or ovarian tissue (tissuevit, n ¼ 44) at Days 0, 6, 12, and 18 of IVC. AThe uppercase letter indicates statistically significant differences among groups (P < 0.05) within the same day. abcDifferent lowercase letters indicate statistically significant differences among days of culture (P < 0.05) within the same group.
In the present study, the follicular development and the oocyte IVM were evaluated after two different forms of vitrification of sheep secondary follicles: vitrification in isolated form (without much stroma) or vitrification in in situ form (within ovarian tissue fragments). Therefore, secondary follicles were first isolated from ovarian fragments and then vitrified, i.e., the follicle-vitrification group (follicle-vit) or fragments of ovarian tissue that were first vitrified, followed by isolation of the secondary follicles, composing the tissue-vitrification group (tissue-vit).
1208
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
9,0
B A
Daily growth rate (μm/day)
8,0 7,0
A
6,0 5,0 4,0
B 3,0 2,0 1,0
Follicles with decreased diameter (%)
A
0,0
70 60
A
50
40
B
30 20 10
C
0
Control
Follicle-Vit
Tissue-Vit
Control
Follicle-Vit
Tissue-Vit
Fig. 4. (A) Daily growth rate (mm/day; mean standard error of the mean) of sheep preantral follicles without vitrification (control, n ¼ 21) and after vitrification of isolated follicles (follicle-vit, n ¼ 38) or ovarian tissue (tissue-vit, n ¼ 44) after 18 days of IVC and (B) Percentage of sheep preantral follicles with decreased diameter from control (n ¼ 18), follicle-vit (n ¼ 1), and tissue-vit (n ¼ 16) after 18 days of culture. ABCDifferent uppercase letters indicate statistically significant differences among groups (P < 0.05).
Our data showed that the follicles from follicle-vit had higher follicular growth rate, oocyte viability, and recovery rate. Similar results were reported by Hatami et al. [4] in mice, in which the vitrification of isolated preantral follicles was better than vitrification of the whole ovary. These authors observed better follicular survival, antrum formation, and oocyte maturation rates. These results may be due to different conditions under which follicles are submitted when vitrified, within or outside of the ovarian stroma. Follicles and oocytes vitrified included in ovarian tissue may not be fully penetrated by intracellular cryoprotectants, because of the large number of cells that surround them. Moreover, the differences among the various cell types present in the ovarian tissue can interfere with the permeability of cryoprotectants, leading follicles to an increased susceptibility to injuries, during cooling. The ovarian fragments were w2 mm thickness, whereas their secondary follicle has an average diameter of 312.57 mm, thereby, the spread of the cryoprotectant into the tissue, during the period of exposure to vitrification solutions, may not have occurred homogeneously, resulting in an irregular dehydration leaving a substrate for ice formation [14,18]. A previous study showed a high percentage of follicles with decreasing diameter in culture of 6 days when they
had previously undergone vitrification [15]. In the present study, we found that when the follicles were vitrified in isolated form (follicle-vit) only 2.56% had a diameter reduction, which was lower than the reduction of follicles vitrified in the tissue (tissue-vit) or fresh follicles (control). Reduction in follicular diameter may occur because of dehydration and osmotic variations suffered during vitrification [19]. This is an acute phenomenon and their effect must be temporary. Follicular diameter should be restored after a few hours of IVC. Studies indicate that if the diameter is not reestablished (as we observed in this study even after 18 days of IVC) it can be considered an indication of follicular degeneration [6,7,20]. In the present study, the follicle-vit (5.02 0.63 mm; 372.0 100.0 pmol/L) treatment had follicular growth rate similar to the control group. However, the estradiol production on the same treatment was lower than control (7.05 1.07 mm; 4729.0 1200.0 pmol/L). The variation in estradiol production may be due to spontaneous luteinization of these follicles cells and not characterize the proliferative or follicular phase of normal estrous cycle. Although, luteinized cells are not typical of follicles that will be recruited and will gain dominance in the normal ovarian folliculogenesis. Xing et al. [21] reported a lower estradiol production and a greater diameter in rats isolated
Table 1 17b-estradiol (pmol/L; mean SEM) and progesterone (nmol/L) concentration (mean SEM) in pooled media collected on the different days during longterm culture of preantral follicles in control (n ¼ 20), follicle-vit (n ¼ 20), and tissue-vit (n ¼ 20) groups. Groups
Control Follicle-vit Tissue-vit ab
Estradiol
Progesterone
Day 6
Day 12
Day 18
Day 6
Day 12
Day 18
1989.0 1270.0bA 306.0 31.0aB 301.0 38.0aB
2792.0 1800.0abA 263.0 24.0bC 353.0 61.0aB
4729.0 1200.0aA 372.0 100.0aB 375.0 180.0aB
d 9.0 6bB 16 6.0aB
10.0 3aB 12.0 3bA 13.0 2.0bA
7.0 1.2aC 25.0 7aA 15.0 1.0aB
Within a row, values without a common superscript differed among days (P < 0.01). Within a column, values without a common superscript differed among groups (P < 0.01). Abbreviation: SEM, standard error of the mean. ABC
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
1209
Table 2 Oocyte viability (%) and diameter (mm SD), recovery rate of oocytes cultured in vitro (%), and meiotic stages (%) of sheep oocytes from preantral follicles after long-term culture (18 days) in control, follicle-vit, and tissue-vit groups. Groups
No. of oocytes recovered/No. of normal follicles (%)
No. of viable oocytes/No. of oocytes (%)
In vivo grown Control Follicle-vit Tissue-vit
d 23/39 (58.97)AB 26/39 (66.67)A 24/60 (40.00)B
28/30 21/23 24/26 16/24
(93.3)A (91.3)A (92.3)A (66.7)B
Mean oocyte diameter (mm)
131.10 105.99 93.94 90.40
23.07A 9.18B 12.84C 14.28C
No. of oocytes with meiosis resumption/No. of viable oocytes (%)
No. of GVBD oocytes/No. of oocytes meiosis resumption (%)
No. of MI oocytes/No. of oocytes meiosis resumption (%)
No. of MII oocytes/No. of oocytes meiosis resumption (%)
26/28 (92.85)A 8/21 (38.09)B 11/24 (45.83)B 6/16 (37.5)B
9/26 (34.61)B 4/8 (50.00)AB 10/11 (90.90)A 6/6 (100)A
1/26 (3.84)B 3/8 (37.50)A 1/11 (9.09)AB 0/6 (0.0)
16/26 (61.53)A 1/8 (12.50)B 0/11 (0.0) 0/6 (0.0)
ABC
Different uppercase letters indicate statistically significant differences (P < 0.05) among groups (column). Abbreviations: GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II; SD, standard deviation.
secondary follicles after vitrification. Otherwise, during vitrification process, the cells can decrease cell metabolism and consequently decrease the estradiol production in an attempt to compensate the potential damage. Moreover, Silva et al. [22] and Cecconi et al. [23] previously reported that estradiol synthesis and follicular growth pattern are not strictly dependent on each other. Both treatments with vitrified follicles in our experiment had lower production of estradiol than the control, probably because the internal theca cells, and even part of granulosa cells have been possibly damaged by the toxic effect of cryoprotectants. The internal theca cells secrete androstenedione, an androgenic precursor that is transferred through the basal lamina to the granulosa cells culminating in the production of testosterone, which is then converted to estradiol by aromatase [24]. These cells damage during vitrification procedures compromises the intrinsic machinery of hormone production, and certainly, the oocyte growth and competence for development will also be damaged. Therefore, it becomes necessary deeper studies of methods that preserve the functionality of theca and granulosa cells after vitrification to ensure and to maintain adequate production of estradiol to support the long term culture and better maturation rate. In this study, the oocytes from follicles previously vitrified showed significantly smaller diameter, after IVM, than the oocytes from follicles in vitro cultured without prior vitrification. In a prior study, it has been shown that most oocytes vitrified in isolated secondary follicles cease their growth in the first 6 days of culture, and vitrified oocytes in secondary follicles enclosed in ovarian tissue, not only stop growing, in the same period of IVC, but also reduce in diameter [15].
In the tissue-vit group, the cooling of the oocyte is slow and gradual, as occurs in peripheral region to the interior of the follicle, where is situated the oocyte [4]. In follicle-vit group, the oocyte is free of barriers such as the ovarian stroma, so the cooling is faster. As a high cooling rate is associated with the absence of ice crystal formation [18], we suggest that this might be the reason why the oocytes from follicle-vit had a higher post-thaw viability than oocytes from tissue-vit group. Other studies [25,26] have also demonstrated that the final oocyte diameter from in vitro cultured follicles remains smaller than the diameter of oocytes from in vivo grown follicles. This may be an early sign of degeneration, possibly because of injuries in the connecting structures between oocyte and surrounding granulosa cells because it is known that the vitrification pronouncedly reduces the oocyte microvilli [15]. The loss of these microvilli may impair the oocyte-somatic cells communication and reduces their growth characteristics [21]. This study showed for the first time that sheep oocytes from vitrified secondary follicles (follicle-vit) cultured in vitro, for 18 days, are able to resume meiosis (GV breaks), and also reach the stage of MI, after IVM, although with lower rate compared to in vivo grown oocytes. Although many efforts and promising results have been reported by different teams regarding the in vitro development of preantral follicles in different species [10], we believe that the conditions of IVC of these follicles, which ensure the full oocyte growth need to be improved especially to oocytes previously submitted to cryopreservation. We also believe that the determination of an ideal system for the IVC of preantral follicles cryopreserved or not is absolutely necessary, whereas the transplantation of
Fig. 5. Viable oocyte in metaphase I after IVM from follicle-vit group after IVC for 18 days. Brightfield (A), Calcein-AM (B), and Hoechst 33342 (C).
1210
F.O. Lunardi et al. / Theriogenology 85 (2016) 1203–1210
ovarian tissue offers risks of reintroduction of cancer cells to patients in remission. Considering that isolated follicles display a better follicular growth rate, oocyte viability, and fewer follicles with decreased diameter IVC, after vitrification, we conclude that with our current method of vitrification, the strategy of vitrification after isolation of secondary follicles provided a better substratum for growing the enclosed oocytes. Acknowledgments This work was supported by CNPq. Franciele Osmarini Lunardi is a recipient of a grant from CAPES Brazil. In addition, Ana Paula Ribeiro Rodrigues and José Ricardo de Figueiredo are recipients of a grant from CNPq Brazil. Johan Smitz is Especial Visitor Researcher from CAPES. References [1] Shaw JM, Bowles J, Koopman P, Wood EC, Trounson AO. Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to graft recipients. Hum Reprod 1996;11:1668–73. [2] Dolmans M-M, Marinescu C, Saussoy P, Van Langendonckt A, Amorim C, Donnez J. Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe. Blood 2010;116:2908–14. [3] Rosendahl M, Andersen MT, Ralfkiaer E, Kjeldsen L, Andersen MK, Andersen CY. Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia. Fertil Steril 2010;94: 2186–90. [4] Hatami S, Zavareh S, Salehnia M, Lashkarbolouki T, Ghorbanian MT, Karimi I. The impact of alpha lipoic acid on developmental competence of mouse vitrified pre-antral follicles in comparison to those isolated from vitrified ovaries. Iran J Reprod Med 2014;12:57– 64. [5] O’Brien MJ, Pendola JK, Eppig JJ. A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biol Reprod 2003; 68:1682–6. [6] Xu J, Bernuci MP, Lawson MS, Yeoman RR, Fisher TE, Zelinski MB, et al. Survival, growth, and maturation of secondary follicles from prepubertal, young, and older adult rhesus monkeys during encapsulated three-dimensional culture: effects of gonadotropins and insulin. Reproduction 2010;140:685–97. [7] Xu J, Lawson MS, Yeoman RR, Pau KY, Barrett SL, Zelinski MB, et al. Secondary follicle growth and oocyte maturation during encapsulated three-dimensional culture in rhesus monkeys: effects of gonadotrophins, oxygen and fetuin. Hum Reprod 2011;26:1061–72. [8] Saraiva MV, Rossetto R, Brito IR, Celestino JJ, Silva CM, Faustino LR, et al. Dynamic medium produces caprine embryo from preantral follicles grown in vitro. Reprod Sci 2010;17:1135–43.
[9] Arunakumari G, Shanmugasundaram N, Rao VH. Development of morulae from the oocytes of cultured sheep preantral follicles. Theriogenology 2010;74:884–94. [10] Telfer EE, Zelinski MB. Ovarian follicle culture: advances and challenges for human and nonhuman primates. Fertil Steril 2013;99: 1523–33. [11] Hasegawa A, Mochida N, Ogasawara T, Koyama K. Pup birth from mouse oocytes in preantral follicles derived from vitrified and warmed ovaries followed by in vitro growth, in vitro maturation, and in vitro fertilization. Fertil Steril 2006;86:1182–92. [12] Wang XQ, Catt S, Pangestu M, Temple-Smith P. Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births. Reproduction 2011;141:183–91. [13] Liebermann J, Nawroth F, Isachenko V, Isachenko E, Rahimi G, Tucker MJ. Potential importance of vitrification in reproductive medicine. Biol Reprod 2002;67:1671–80. [14] Zhou XM, Qiao WT, Zhang XL, Liu Z, Gao DY. Physical modeling of flow boiling in microchannels and its induced vitrification of biomaterials. Int J Heat Mass Transfs 2015;83:659–64. [15] Lunardi FO, Chaves RN, de Lima LF, Araújo VR, Brito IR, Souza CE, et al. Vitrified sheep isolated secondary follicles are able to grow and form antrum after a short period of in vitro culture. Cell Tissue Res 2015;362:241–51. [16] Lunardi FO, Araujo VR, Faustino LR, Carvalho AA, Braga Goncalves RF, Bass CS, et al. Morphologic, viability and ultrastructural analysis of vitrified sheep preantral follicles enclosed in ovarian tissue. Small Rumin Res 2012;107:121–30. [17] Luz VB, Araujo VR, Duarte AB, Celestino JJ, Silva TF, MagalhaesPadilha DM, et al. Eight-cell parthenotes originated from in vitro grown sheep preantral follicles. Reprod Sci 2012;19:1219–25. [18] Devismita D, Kumar A. Effect of cryoprotectant on optimal cooling rate during cryopreservation. Cryobiology 2015;70:53–9. [19] Paynter SJ, Cooper A, Fuller BJ, Shaw RW. Cryopreservation of bovine ovarian tissue: structural normality of follicles after thawing and culture in vitro. Cryobiology 1999;38:301–9. [20] Ting AY, Yeoman RR, Lawson MS, Zelinski MB. In vitro development of secondary follicles from cryopreserved rhesus macaque ovarian tissue after slow-rate freeze or vitrification. Hum Reprod 2011;26: 2461–72. [21] Xing W, Zhou C, Bian J, Montag M, Xu Y, Li Y, et al. Solid-surface vitrification is an appropriate and convenient method for cryopreservation of isolated rat follicles. Reprod Biol Endocrinol 2010;8:42. [22] Silva CM, Castro SV, Faustino LR, Rodrigues GQ, Brito IR, Rossetto R, et al. The effects of epidermal growth factor (EGF) on the in vitro development of isolated goat secondary follicles and the relative mRNA expression of EGF, EGF-R, FSH-R and P450 aromatase in cultured follicles. Res Vet Sci 2013;94:453–61. [23] Cecconi S, Barboni B, Coccia M, Mattioli M. In vitro development of sheep preantral follicles. Biol Reprod 1999;60:594–601. [24] Walters KA. Role of androgens in normal and pathological ovarian function. Reproduction 2015;149:R193–218. [25] Hu Y, Betzendahl I, Cortvrindt R, Smitz J, Eichenlaub-Ritter U. Effects of low O(2) and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture. Hum Reprod 2001; 16:737–48. [26] Segers I, Adriaenssens T, Ozturk E, Smitz J. Acquisition and loss of oocyte meiotic and developmental competence during in vitro antral follicle growth in mouse. Fertil Steril 2010;93:2695–700.