In general, follicles are cultured in attachment and
non-attachment systems. In an attachment culture system, the follicle architecture and the communications of
follicular cells are disrupted because of the attachment
of granulosa cells to the culture dish, as the mentioned
phenomenon might negatively affect the growth and development of the follicles, especially in large mammals
In recent years, alginate (ALG) hydrogel has been
largely used in biomedical and tissue engineering applications (
Moreover, it is shown that the molecular support provided by different cell types could affect follicle growth
and development as well as the physical mechanics of
the matrix (
Although in many studies the impacts of different
ALG concentrations and the rule of OCs on follicle
development have been investigated, there is no study
that evaluates these two parameters simultaneously to
find the best ALG concentration for the culture of both
preantral follicles and OCs. Hence, in the first step of
the present factorial study, the morphology, diameter,
survival, antrum formation and maturation of preantral
follicles encapsulated and cultured in different concentrations of ALG hydrogel, in the absence or presence of
co-encapsulated OCs (OCs and +OCs-respectively),
are evaluated. Then, in the second step, to understand
the effects of OCs on the quality of cultured follicles and
their oocytes, the preantral follicles were cultured in the
best concentration of ALG in the absence or presence
of OCs, and were investigated in terms of the amounts
of DNA fragmentation in the follicular cells. Since gap
junction proteins play a significant role in the folliculogenesis process via transferring nutrients, ions and
some nucleotides among follicular cells and oocyte, the
changes in their expression might somehow affect follicle development (
In the first step of this experimental study, preantral follicles were isolated from mice ovaries in five independent replicates, randomly allocated to encapsulate in 0.5, 0.75 and 1% ALG hydrogels in the absence or presence of OCs, and cultured for 13 days. The diameter and morphological appearance of developing follicles were analyzed on days 1, 6 and 13 of culture. Additionally, on day 13, the survival rate of the follicles was calculated and healthy follicles were evaluated with regard to their antrum formation rate. Then, antral follicles were induced by 2.25 IU/ml human chorionic gonadotropin (hCG, Choriomon, Switzerland) and 20-22 hours later, on day 14 of culture, the developmental stages of the obtained oocytes were assessed. After determining the best concentration of ALG based on the larger diameter, higher survival, antrum formation, and maturation rates, in the second step of the study this concentration was used for culturing preantral follicles in the absence or presence of OCs. On day 13 of culture, antral follicles were fixed for investigation of DNA fragmentation and assessment of Cx37 and Cx43 protein levels. Conditioned media from the follicle cultures were collected in three replicates for the measurement of estradiol, progesterone, and androstenedione secretions. Finally, after hCG induction, MII oocytes were collected and evaluated in terms of their cortical granule distribution, meiotic spindle organization, and chromosomal alignment.
Female NMRI mice (Pasteur Institute, Iran) were housed in the animal facility of Royan Institute under standard housing conditions, with controlled temperature (20-25°C) and lighting (12 hours light: 12 hours dark). They were handled pursuant to the ethical guidelines set by Royan Institute (ethical permission number: IR.ACECR.ROYAN.REC.1395.93).
Twenty three-four-week-old immature mice were sacrificed by cervical displacement, and their ovaries were isolated in an aseptic condition and placed in ice-cold base medium containing Dulbecco's Modified Eagle’s medium (DMEM, Gibco, UK), penicillin (Gibco, UK), streptomycin sulfate (Gibco, UK), sodium bicarbonate (NaHCO3, Sigma, USA) and 10% fetal bovine serum (FBS, Gibco, UK). Next, the ovaries were cleaned of the bursa and adipose tissue under a stereomicroscope (SZ61, Olympus, Japan). Oocytes and granulosa cells were removed from the ovaries by puncturing follicles with two 29G insulin syringes and then discarded. The remnants were chopped and incubated for 45 minutes at 37°C in 200 µl per ovary of collagenase solution containing 4 mg/ml collagenase IV (Gibco, UK) in serum-free base medium. During this time, the ovarian tissue pieces were pipetted up and down at least 20 times every 10-15 minutes to mechanically disrupt them. To stop the enzymatic activity an equivalent volume of base medium was added to the samples. The isolated cell solution was then filtered through a sterilized 40 µm filter mesh (Falcon, Mexico) and centrifuged at 1800 rpm for 5 minutes. The obtained cells were washed and the final pellet was re-suspended in a known volume of base medium. The cells were transferred to T25 culture flasks containing 4 ml of base medium supplemented with 1% insulin-transferrin-selenium (ITS, Gibco, UK), 1% L-glutamine (Sigma, USA), 1% non-essential amino acids (Gibco, UK) and 0.1% β-mercaptoethanol (Sigma, USA), and then incubated at 37°C in a water-saturated atmosphere of 95% air and 5% CO2 until they reach full confluency. Next, OCs were trypsinized and washed and the viable cells were counted with a trypan blue staining and a Neubauer chamber. Afterward, 1ml aliquots of the cells (5×10<sup>5</sup> cells/ml) were stored in 10% DMSO (Sigma, USA)/FBS at -80°C for later use.
A total of thirty 12-14-day-old mice were sacrificed by cervical displacement. Mouse ovaries were mechanically dissected under a stereomicroscope at 37°C, using two 29G needles attached to 1ml insulin syringes, and placed in alpha minimum essential medium (α-MEM, Gibco, UK) supplemented with penicillin, streptomycin, NaHCO3 , and 10% FBS. Only intact preantral follicles with 2-3 layers of granulosa cells and 100-130 µm in diameter were chosen and divided randomly into experimental groups.
To make a 1.0% (w/v) ALG solution, 10 mg/ml alginic
acid sodium salt (Sigma, USA), 25 mM 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES, Sigma, USA)
and 150 mM sodium chloride (NaCl, Sigma, USA) were
dissolved in deionized water, filtered through a sterilized
0.22 µm filter (Millipore, USA) (
In the first step of the study, groups of 109.83 ± 7.59 preantral follicles were individually encapsulated in 0.5, 0.75 and 1% ALG solutions and in the absence or presence of OCs, in five independent replicates. For cell encapsulation, about 5×103 OCs per follicle were mixed with hydrogel solutions and pipetted in 5-µl droplets on sterile ultra-low attachment culture dishes (Dow Corning, USA). The concentration of OCs in each droplet was determined based on the best results obtained in our pilot study. Afterwards, follicles were individually placed in the 5-µl droplets, cross-linking solution was gently pipetted on top of each droplet, and then incubated at 37°C for 2 minutes. After incubation, the beads were washed with α-MEM medium and then placed into 96-well plates (TPP, Switzerland). Each well contained one bead in 100 µl culture medium [α-MEM supplemented with 5% FBS, 1% ITS, 10 mIU/ml follicle stimulating hormone (FSH, Merck, Germany)]. Lastly, plates were incubated in a 5% CO2 incubator at 37°C for 13 days and 50 µl of the medium was replenished every 3-4 days.
Morphological features and the diameters of developing
follicles were assessed on days 1, 6, 13 of culture. The
diameters were determined as the mean of two perpendicular measurements of each follicle using ImageJ software (U.S. National Institutes of Health) (
In the second step of the study, some preantral follicles were encapsulated in the ultimate concentration of ALG hydrogel from the first step, either in the absence or presence of OCs, and were cultured similar to the first step. On day 13 of culture, survived antral follicles were fixed in 4% paraformaldehyde overnight at 4°C; then the follicles were rinsed twice in PBS, dehydrated in increasing concentrations of ethanol and embedded in paraffin. Next, 5 µm thick slices were prepared and mounted on adhesion slides for DNA and protein analyses. Three sections per group, taken from the middle of three random follicles, were selected for either DNA fragmentation or Cx37 and Cx43 protein expression detection. To prepare the sections, they were first deparaffinized at 60°C for 30-40 minutes, washed in xylene solution for 20 minutes, and rehydrated by rinsing in serially diluted ethanol and water bath.
Strand breaks of DNA in apoptotic cells were detected
by terminal deoxynucleotidyl transferase-mediated dUTP
nick end labeling (TUNEL) assay utilizing the In Situ Cell Death Detection Kit, TMR Red (Roche Diagnostics GmbH,
Mannheim, Germany). The procedure was done according
to the manufacturer's protocol and previous descriptions
Red fluorescence was visualized in TUNEL-positive cells. Sections from mouse ovarian tissue were incubated with DNase I recombinant [5 U/ml in 50 mM Tris- HCl, 1mg/ ml bovine serum albumin (BSA), pH=7.5] and were used as a positive control. Negative control sections were incubated with the label solution rather than TUNEL reaction mixture.
To quantify the DNA fragmented follicular cells, all cells found in the sections, either for TUNEL staining or DAPI counterstaining, were counted using ImageJ cell counter plugin; then, the percentage of TUNEL-positive cells was computed.
After deparaffinization and rehydration, antigen retrieval was performed by incubating the sections of the antral follicles in 0.01 M sodium citrate buffer (pH=6.0, Sigma, USA) for 1 hour in a 96°C oven. The sections were washed two times in PBS-T, and incubated in 10% goat and donkey serums (Sigma, USA) diluted in PBS for 1 hour at 37°C to block non-specific protein bindings in Cx37 and Cx43 immunostainings, respectively. After two PBS washes, the sections were incubated overnight at 4°C with primary antibodies against Cx37 [Primary rabbit polyclonal antibody (ab181701, Abcam, UK)] and Cx43 [primary mouse monoclonal antibody (C8092, Sigma, USA)]. Both primary antibodies were diluted 1:400 in related blocking solutions. Then, the sections were rinsed with PBS carefully and incubated with secondary antibodies [(goat anti-rabbit IgG (H+L) crossadsorbed (Alexa Fluor 488, A11034, Thermo Fisher Scientific, USA) and donkey anti-mouse IgG H&L (Alexa Fluor 488, ab150105, Abcam, UK)] for 1 hour at 37°C. Both secondary antibodies were diluted 1:1000 in related blocking solutions. Lastly, the sections were washed, counterstained with DAPI for 1 minute, and inspected under an inverted fluorescence microscope (Eclipse 50i, Nikon, Japan). Sections from rat heart tissue were used as a positive control. For the negative control, ovarian sections were processed without the primary antibodies.
On day 13 of culture, the level of estradiol (E2), progesterone (P4) and androstenedione (A4) hormones were measured in conditioned media collected from 30 cultured antral follicles per group, in three replicates. The hormonal measurement was conducted using mouse ELISA kits (Bioassay Technology Laboratory, China) and according to the kits’ instruction. Data were adjusted for every follicle by dividing each of the measured hormonal secretions by the number of the follicles. According to the kits’ datasheets, the sensitivity of the assay for E2, P4 and A4 were 1.51 ng/L, 0.28 ng/ml and 0.022 ng/ml, respectively.
Following the assessment of DNA fragmentation and
protein expressions, cultured antral follicles were induced
by hCG as was explained in detail in the determination
of oocytes meiotic maturation section, and then a total
number of 20 MII oocytes (10 oocytes per group) were
collected in three replicates. Also, 10
Statistical differences in follicle survival rates, antrum formation rates, oocyte maturation rates, oocyte abnormality rates and DNA fragmentation were analyzed using GENMOD procedure including function link logit in the model. GENMOD procedure produced odds ratio (OR) as the strength of difference between the groups. Data associated with follicle diameters were analyzed by MIXED procedure including RANDOM and REPEATED statements in the model to identify between and within covariances, respectively. Data pertaining to hormonal secretions were analyzed using GLM procedure. In addition, LSMEANS statement was included in the model to perform multiple comparisons. All analyses were conducted in SAS version 9.4 (SAS Institute Inc., NC, USA). Differences were considered significant at P<0.05.
Assessment of the morphologies and diameters of the follicles on days 1, 6 and 13 of culture indicated no significant
difference in follicles encapsulated in different concentrations of ALG, either in the absence or presence of OCs.
On the other hand, follicles which were co-cultured with
OCs had a more spherical shape and relatively larger diameters than the non-co-cultured ones. On day 13 of culture,
for instance, the difference in the diameters of 0.5%-OCs
and 0.5%+OCs groups was significant (347.18 ± 10.63 vs.
418.14 ± 21.89 µm, respectively, P<0.05,
Growth of preantral follicles encapsulated and cultured in 0.5, 0.75 and 1% alginate hydrogels in the absence or presence of ovarian cells (OCs and +OCs-respectively). A. Morphological changes and B. Diameter of the survived follicles on days 1, 6 and 13 of culture. Data are presented as the mean diameter ± standard error. Data points a and b differ significantly (P<0.05, scale bar: 100 µm).
Assessment of follicle survival rates on day 13 of culture
indicated that there was a linear trend towards a better survival
rate with lowering ALG concentration, in both the absence and
presence of the OCs. However, the difference between 0.5%-
OCs group and both 0.75%-OCs and 1%-OCs groups reached
statistical significance (71.87 vs. 52.52 and 40%, respectively,
Surprisingly, in the absence of OCs, the proportion of the
follicles developed to antral stage was higher in 1% group as
compared to the 0.75 % group (75 vs. 59.61%, respectively,
P<0.05); while in the presence of OCs there was no significant
difference between the groups. On the other hand, all -OCs
groups had a relatively lower antrum formation rate than the
+OCs ones. Nevertheless, the difference reached statistical
difference in 0.5 and 0.75% groups, only (69.59 vs. 88.57%
and 59.61 vs. 77.65% for-OCs and +OCs groups, respectively, P<0.05,
The evaluation of oocytes obtained from 0.5, 0.75 and 1%
ALG-cultured antral follicles showed that there was no significant difference between groups regarding the rates of GV and
GVBD/MII oocytes, either in the absence or presence of OCs.
However, 0.5% ± OCs groups had a higher rate of degenerated
oocytes than the 1% ± OCs ones (P<0.05). Also, it was clear
that the oocytes of 0.5%+OCs and 0.75%+OCs groups were
more likely to break down their GVs and develop to GVBD or
MII stages as compared to the -OCs groups (P<0.05,
Based on the first step results, 0.5% ALG hydrogel is
potentially best suited for preantral follicle culture, either
in the absence or presence of the OCs. In the second step,
the evaluation of DNA fragmentation in 0.5% ALG ± OCcultured antral follicles revealed that although a negligible percentage of the follicular cells was TUNEL-positive
in both groups, follicles that were co-cultured with OCs
demonstrated a relatively lower percentage of DNA fragmentation compared to the non-co-cultured ones (2.2 ±
0.7 vs. 3.9 ± 0.7%, respectively,
Immunofluorescence staining for Cx37 and Cx43 are displayed in Figure 2B and C as shown, the strong immuno-labeling of Cx37 and Cx43 were observed on granulosa cells of both -OCs and +OCs groups, while qualitatively, no remarkable difference was observed between the two groups.
Hormonal secretion data indicated that there was no
significant difference between the groups in the levels
of E2 and A4 hormones; however, the level of P4 in the
+OCs group was significantly higher than that in the -OCs
group (3.2 ± 0.4 vs. 1.8 ± 0.2 ng/ml, respectively, P<0.05,
Development of preantral follicles cultured in 0.5, 0.75 and 1% alginate hydrogels in the absence and presence of OCs for 14 days
|Groups||Survival rate||Antrum formation rate||Oocyte maturation|
|0.5%||69/96 (71.87)A||105/129 (81.39)||48/69 (69.59)a||93/105 (88.57)b||14/48 (29.16)a||8/93 (8.60)b||28/48 (58.33)a||69/93 (74.19)b||6/48 (12.5)A||16/93 (17.20)A|
|0.75%||52/99 (52.52)B||94/138 (68.11)||31/52 (59.61)Aa||73/94 (77.65)b||12/31 (38.70)a||5/73 (6.84)b||17/31 (54.83)a||63/73 (86.30)b||2/31 (6.45)||5/73 (6.84)|
|1%||40/100 (40)Ba||62/97 (63.91)b||30/40 (75)B||55/62 (88.70)||6/31 (19.35)||9/55 (16.36)||24/31 (77.41)||44/55 (80)||1/31 (3.22)B||2/55 (3.63)B|
OCs; Culture in the absence of ovarian cells, +OCs; Culture in the presence of ovarian cells, GV; Germinal vesicle, GVBD; Germinal vesicle breakdown, and MII; Metaphase II. Data are presented as n (%). A vs. B in each column and a vs. b in each row differ significantly (P<0.05).
Quality assessment of antral follicles encapsulated and cultured in 0.5% alginate hydrogel in the absence or presence of ovarian cells (OCs and +OCs-respectively), on day 13 of culture. A. TUNEL staining to detect DNA fragmentation in follicular cells. TUNEL-positive cells are stained in red and nuclei in blue (DAPI), B, and C. Immunofluorescence staining to label connexin 37 (Cx37) and connexin 43 (Cx43) proteins; both Cx37 and Cx43 proteins are stained in green and nuclei in blue with DAPI (scale bars: 100 µm).
Secretion of hormones by antral follicles encapsulated and cultured in 0.5% alginate hydrogel in the absence or presence of ovarian cells (OCs and +OCs-respectively). A. Estradiol (E2), B. Progesterone (P4), and C. Androstenedione (A4). Conditioned media were collected on day 13 of culture. Data are presented as mean ± standard error. Data points a and b are significantly different (P<0.05).
The normal and abnormal MII oocytes in terms of cortical granule distribution, meiotic spindle organization, and chromosomal alignment are shown in Figure 4. Our data revealed that all in vivo-developed oocytes had a normal cortical granule distribution; whereas 100 and 40% of the oocytes that were developed in 0.5%-OCs and 0.5%+OCs groups, respectively, were abnormal (lack of a cortical distribution of cortical granules) (P<0.05). Concerning meiotic spindle organization, under both in vivo and in vitro conditions, oocytes with abnormal spindle or chromosomal alignments were observed (disorganized spindle or misaligned chromosomes were considered as an indicator of spindle abnormality) (in vivo: 20%; 0.5%-OCs: 40%; 0.5%+OCs: 50%). Interestingly, the presence of OCs had no significant effect on the rate of abnormalities.
Immunofluorescence staining and abnormality assessment of corti- cal granules (CGs; red), meiotic spindle (Spdl; green) and chromosomes (Chrs; blue) in MII oocytes. Oocytes with cortical distributed cortical gran- ules, a well-organized spindle, and centrally aligned chromosomes were considered as normal oocytes (scale bar: 50 µm).
The aim of the present study was to simultaneously evaluate the effects of ALG concentration and OCs on the development and function of follicles.
To understand whether the proposed culture condition
is ideal for follicle development, the morphological characteristics, diameter, survival and antrum formation rates
of the cultured follicles and meiotic resumption of their
oocytes were assessed. Previous studies had shown that
the rigidity of the matrix used for encapsulation and culture of follicles changes all the above parameters (
In our study, in the absence of OCs, the survival rate
of the follicles cultured with 0.5% ALG was significantly
higher than that with 0.75 and 1% ALG. The results were
generally consistent with the study of Park et al. (
Interestingly, in the presence of OCs, we observed no
remarkable difference between 0.5, 0.75 and 1% ALGcultured follicles in terms of diameter, survival and antrum formation rates and oocyte maturation. Nonetheless,
similar to the cultures without OCs, the rate of oocyte degeneration was lower in the group with 1% ALG. However, the comparison of -OCs and +OCs groups showed
that the follicles in the +OCs groups had a more spherical
shape, a relatively larger diameter, higher survival rate,
better antrum formation, and higher GV to GVBD/MII
transition rates. The applied OCs in this study comprised
of a heterogeneous population of theca/interstitial cells,
endothelial cells of the blood vessels, immune cells such
as macrophages and smooth muscle cells, which produce
high levels of androgens, growth factors and cytokines
The cultured antral follicles in 0.5% ALG hydrogel (the best-suited hydrogel for follicle growth and development),
in the absence or presence of OCs, also were evaluated for
DNA fragmentation, Cx37 and Cx43 protein expressions,
hormonal secretions and the quality of their oocytes. Data
showed that only a small percentage of the follicular cells
were TUNEL-positive after 13 days of culture, either in
the absence or presence of OCs. Since the percentage of
the TUNEL-positive cells was less than 10%, according
to the classification explained in the previous studies (
On the other hand, there was strong immunolabeling of
both Cx37 and Cx43, which are two important gap junction proteins in the follicles, in both the absence and presence of OCs. Cx37 and Cx43 are responsible for transportation of nutrients and growth factors essential for the
growth and development of the follicles (
Furthermore, the evaluation of hormonal secretion by
0.5% ALG-cultured antral follicles showed that the follicles that were co-cultured with OCs secreted more P4
than the non-co-cultured ones. Earlier studies have found
that macrophages enhance progesterone production in the
granulosa cells of follicles (
Finally, to assess the cytoplasmic and nuclear maturations of 0.5% ALG-developed oocytes, the distribution
pattern of cortical granules, the formation of the meiotic
spindle and the alignment of chromosomes were evaluated and compared with the in vivo-developed ones. As
reported in previous studies, in a normal mature oocyte,
cortical granules represent a uniform cortical distribution
and the meiotic spindle is also well-assembled. Moreover,
a normal mature oocyte contains the correct number and
set of chromosomes (
In the present study, we showed that both rigidity and concentration of ALG hydrogel influenced the survival rate of the follicles. Indeed, there was a linear trend toward a better survival rate with the lower ALG concentration, in either absence or presence of OCs. Nonetheless, the concentration of ALG did not significantly affect the diameter, antrum formation and maturation rate of the follicles. However, it could be concluded that among 0.5, 0.75 and 1% ALG, the hydrogel with lower concentration is the most suitable for the mouse preantral follicle culture. Moreover, it seems that OCs positively influence follicle diameter, survival, antrum formation and maturation rate, and hormonal secretions. Hence, OCs could be successfully applied to the follicle culture systems in order to improve their culture conditions.