Characteristics of Freshly Isolated and Cryopreserved Human Ovarian Granulosa and Cumulus Cells

Utilization of granulosa and cumulus cell (GCC) complex could be prospective for co-culture of gametes and embryos as a part of assisted reproductive technologies (ART), that makes important their cryopreservation. The study was performed in GCCs of women aged 25–39, who underwent the treatment of infertility by IVF. Cryopreservation of GCC suspension was carried out using 1.5 M 1,2-propane diol solution. The samples were cooled with rate of 0.3 deg/min from 25 down to –6°C, thereafter the ice crystal initiation was done, from –6 down to –35°C the cooling rate was 1 deg/min, after that the samples were plunged into liquid nitrogen and stored then at –196°C. Following thawing the GCCs retained their main features: ability for adhesion and proliferation, and hormone production. During culture of GCCs its development was delayed for 1–2 days if compared with freshly isolated cells. In 7 days after explanting the cultures of frozen-thawed GCCs appeared nearly the same as primary cultures of corresponding terms of in vitro culture. The post thaw level of hormone production by GCCs was changed: decreased by 89.9% in case of estradiol, and increased by 81.4% for progesterone if compared with the indices of freshly isolated suspensions. The obtained results testify to a possibility to isolate the GCCs and then to store in low-temperature banks for prospective use in ART.

It is known that granulosa and cumulus cells are the mediators of hormone effect on meiotic transformations of chromosomes inside oocytes.For instance, it found previously that granulosa cells participated in conducting regulatory signals of prolactin and somatotropins to the cells of cumulus, having the mixed reception of the signals from these hormones [13,17].
Recently, the complex of infertility treatment in the frames of IVF actively involves the cryopreserved reproductive cells, and therefore an adequate culturing after thawing is nessessary [3,6,7].There would be expedient to preserve GCCs for their further application during culturing of frozen-thawed embryos.However, there are only a few published reports concerning cryopreservation of GCCs complex [8,22].
Above mentioned testifies to the fact that use of complex of GCCs after freezing can be prospective during co-culturing of gametes and embryos in assisted reproductive technologies.
The GCCs isolated from human ovary preovulatory follicles are quite new objects for cryobiological studies.Previous investigations were conducted in cellular complexes of ovarian stroma, granulosa and thecas, which were procured from the follicles of various development stages [15,18].Nowadays, due to the progress in assisted reproductive technologies, IVF in particular, the possibility arised to obtain some GCCs in the volume sufficient for experimental studies, cryopreservation and application in the programs of artificial fertilization.However, the tasks of preserving morphological integrity of these cells, their possible restoration after freeze-thawing, synthesis and secretion of sexual hormones have remained actual.
Cumulus cells are a special subpopulation of granulosa cells, differentiation of which is directed and supported by oocyte [17,19].Liu [16] demonstrated that cumulus, derived from oocytes at the germinal vesicle stage and metaphase I, differed by its morphofunctional characteristics from the one of mature oocytes, and showed a high level of apoptosis.It has been shown that morphofunctional state of GCCs could correlate with the quality of oocyte and its ability to fertilization, and early apoptosis of GCCs might accompany low quality of embryos in vitro [13].
On that basis, the research aim was to estimate morphofunctional properties of human granulosa cells and ovarian cumulus prior to and after cryopreservation under protection of 1,2-PD.

Materials and methods
The experiments were performed only in the cumulus obtained after denudation of oocytes being at the meiosis metaphase II.
The cells of granulosa and cumulus were obtained from the ovaries of women aged 25-39, treated for infertility by IVF with their informed written consent.Superovulation was induced by means of traditional long-term mean lutein protocol using agonists gonadotropin-releasing hormone Decapeptyl (Ferring, Germany) in a dose of 3.75 mg and recombinant follicle-stimulating hormone Gonal-F (Serono, Italy).As the trigger for complete maturation of follicles we used Ovitrelle (Serono) in a dose of 250 mg.Transvaginal puncture of follicles was performed under the control of ultrasound scanning system Phillips HD-11 (Germany).
After isolation of oocytes the follicular fluid with the rest of cells was transferred to cone-like plastic tubes and was kept at room temperature for one hour.After sedimentation of cells the supernatant was removed and three-fold washing of GCCs to remove erythrocytes and other debris cells was carried-out by re-suspending the sediment in 1 ml culturing medium (Fertilization medium, COOK, Australia).The sizes of GCCs tissue fragments were (3.2 ± 0.6) mm 2 .
Primary suspension of GCCs was obtained by means of desaggregation of GCCs tissue fragments in hyaluronidase solution (Toscani, Spain).
To assess morphofunctional state of freshly isolated and cryopreserved suspension the GCCs were transferred into 0.8 ml of culturing medium and cultured under mineral oil in 4-well plate (Nunc, Germany) without changing the medium during 14 days in CO 2 incubator (Sanyo, Japan) at 6% CO 2 , 37°C and 95% humidity.Each 24 hrs the morphological state of GCCs was estimated using microscope Olympus IX-71 (Japan).
Functional parameters of cells, viz. the estradiol and progesterone secretion to culture medium, were assessed with immune enzyme analysis using Estradiol Elisa Kit and Progesterone Elisa Kit (DRG, USA).
To determine the presence of lipid drops, which could be an indirect index of possible steroidogenesis, the GCCs were stained with Nile red (NR).Fluorescence of the cells was performed to the 7 th culturing day by means of confocal laser scanning microscope LSM 510 META (Carl Zeiss, Germany).The fluorescence was excited by He-Ne laser with 543 nm wavelength, emission was recorded in red band with maximum at 631 nm.
The numerical data were processed using the computer software Microsoft Excel and Origin 6.1.The data are presented as mean ± error of the mean.Significance of the differences was estimated using Student-Fisher criterion.

Results and discussion
After isolation and enzymatic disaggregation of GCCs fragments the primary suspension of the obtained cells represented the mixture of isolated cells of various size with predominantly round shape and cell aggregates with various dimensions.Only single erythrocytes were found in this heterogeneous suspension (Fig. 1A).It should be noted that together with the cells with distinctly contoured membrane, nucleus and light, somewhere granular cytoplasm the aggregates comprised dark coloured dead cells with impaired membrane and cell detritus.
Explantation of such primary suspension of GCCs in described conditions resulted in sedimentation of cell elements and aggregates to the bottom of cultural plate and adherence to it.During first day of culturing the majority of elements of primary suspension adhered to the bottom of cultural plate (82.0 ± 0.7)%.Herewith cell aggregates looked like round formations consisted of dark granulated cells.The part of cells preserved a rounded shape the rest demonstrated initial signs of flattening on a substrate (Fig. 1B).
Після розморожування суспензії КГК її вигляд та склад відрізнялися від первинної суспензії, Further culturing of primary culture of GCCs was accompanied with the flattening of several cells, adhered to substrate as well as migration of cells from aggregates and their flattening around an aggregate (Fig. 1C).
Following culturing resulted in the development of GCCs culture.To the 7 th culturing day the granulation of cells inside the aggregates enhanced.Centre of aggregates was as a rule multilayered and formed of cells with non-distinct contoured membrane.Periphery of aggregates was occupied mainly by flattened epithelioid cells, which migrated on a surface of substrate and formed a growth zone (Fig. 1D).
In some cases, especially when an aggregate comprised a small number of cells, these flattened nearly completely on a bottom surface of cultural plate, forming the monolayer areas of granulated cells.
After the 7 th day of GCCs culturing, their culture demonstrated gradual ageing, which signs were compactization of cells in terms of cytospheres formation and their detachment from the bottom of cultural plate.
Staying of the cells under culture conditions led to sedimentation and adherence of cell elements of GCCs to a substrate (Fig. 2B).
During 2-3 days after start of explantation the majority of cells and aggregates of the suspension adhered to the substrate and single cells appeared in the periphery of aggregates which migrated and flattened thereafter on a cultural plastics surface (Fig. 2C).These processes proceeded more slowly than those in primary cultures of GCCs, this enabled to suppose the presence of delayed in vitro development phase in cryopreserved GCCs, which was nessessary for reparation of non-lethal impairments.
Note: * -the differences are statistically significant if compared with primary culture of corresponding culture terms, p ≤ 0.05.
changes in primary culture at the same term, and which was mainly manifested in detachment of cell elements.Thus, during first 7 days of culture the freshly isolated and cryopreserved GCCs demonstrated the ability to adhere, flatten and migrate with the appearance of certain phenotype (cells of roundish shape with large nucleus and granular cytoplasm).Delay in culture development of the frozen-thawed cells from 1 st through 4 th experimental days could testify to the appearance of non-lethal impairments of cells resulted from the cryopreservation, in particular of their membranes, e. g. to the change in phase state of cell membrane lipids, disordered activity of membranebound enzymes etc.During further staying of cells in the culture conditions these impairments disappeared, which was confirmed with observation of the GCCs culture to the 7 th culture day.
The functional state specific for GCCs could be properly estimated only considering hormonopoiesis.Analysis of hormone producing function of freshly isolated and cryopreserved GCCs to the 1 st , 3 rd , 7 th culture days has shown that the secretion level of progesterone by frozen-thawed GCCs was significantly higher than in freshly isolated cells and estradiol one was significantly reduced (Table ).It could be seen, that ratio between the values of estradiol and progesterone production by GCCs was kept, but the absolute indices of hormone production by the cells altered.For instance, the secretion rate of estradiol following cryopreservation reduced by 89.9% in respect of these indices prior to cryopreservation.At the same time, the secretion level of progesterone in thawed GCCs increased by 81.4% vs. indices observed in the primary culture.
It is known that cytoplasm of GCCs normally contains a significant amount of lipid granules which depot cholesterol, which is a substrate to synthesize steroid hormones.Their presence could be visualised using fluorescent dye Nile red.
It is seen, that to the 7 th culture day the fluorescence intensity in individual cells could be different, depending likely on number and location of lipid drops.
After cryopreservation about (70 ± 7.1)% GCCs had bright fluorescence (Fig. 3B).The fluorescence intensity of cryopreserved GCCs was at the level of freshly isolated samples.The cells were of round shape with mani-  It is known that even optimal cryopreservation conditions could lead to appearance of non-lethal injuries in the survived cells, which considerably reduce the biological properties of cell cultures [21].However, these damages could be eliminated under certain culture conditions, allowing the cells to recover their morphofunctional features.
To date there are not enough reported data about the effect of cryopreservation factors on morphofunctional characteristics of isolated GCCs and these are of odd character, however, the cryopreservation of ovarian tissue fragments is described widely.
For instance, the fact of increasing proliferative properties and viability of fragments of human ovarian tissue fragments after cryopreservation has been found [4].In other study, goat ovarian fragments were slowly frozen in the solutions containing different intracellular cryoprotectants, namely ethylene glycol, propane diol and dimethyl sulfoxide [23].Ultrastructural analysis revealed morphologically normal preantral follicles in the control and cultured fragments prior to and after cryopreservation and short-term culturing [2].
Reduced tightness of intercellular contacts in GCCs was demonstrated after cryopreservation of some GCCs there [1].At the same time there are data as for the effect of cryoprotective solutions on DNA damage [15].Of interest is the fact that after equilibration of GCCs with the solutions of ethylene glycol (40%) and glycerol (28%) the number of DNA damages was lower than in freshly isolated cells.Moreover, after equilibration with cryoprotectants and
after further cryopreservation the authors have noted a high cell viability.
In the present study we assessed morphofunctional properties of GCCs, which were not the part of ovarian fragments, but represented the suspension of isolated cells.It has been established that during culture of freshly isolated cells their adhesion and migration were found to the 2 nd day.At the same time after cryopreservation under 1,2-PD protection the morphofunctional activity of cells was observed during the 3 rd and 4 th days.To the 7 th day of GCCs culture post thaw, numerous cells migrated to monolayer.Following culturing of cryopreserved GCCs till the 14 th day was accompanied by detachment of most cells and aggregates from the plate bottom which was the sign of culture ageing.
The findings testify to the fact that GCCs could be obtained and preserved under low temperature bank conditions with the aim of their further application for in vitro co-culture of gametes and embryos as a part of assisted reproductive technologies.

Conclusions
Low temperature preservation of GCCs under protection of 1.5 M 1,2-PD solution using slow freezing enabled the preservation of morphological integrity and functional activity of cells, the features of that were the ability of their adhesion, flattening, migration and proliferation under culturing conditions, as well as producing of steroid hormones.
fested granular cytoplasm.The presence of lipid inclusions in GCCs after cryopreservation could indirectly testify to a possible presence of cholesterol depot being an essential component to produce steroid hormones.