Mammalian ovarian tissues contain a large number of resting pool of primordial follicles, which
represent the reproductive potential of females.
However, the number of follicles is limited and
also gradually reduced during the female reproductive life (
Almost 70 years ago techniques were used by
O’Brien et al. (
The key advantage of this type of culture of ovarian fragments or whole ovary is that it preserves normal interactions between follicles at different sizes and stages and various extra follicular cell types within the ovarian tissue. However, the main disadvantage of this technique is that in large tissue fragments culture, degeneration and necrosis of tissues occur due to inadequate oxygenation. However additional investigations are required to improve this culture system.
The objective of the present study was to establish a mouse ovarian organ culture system where it could be used to evaluate a variety of factors which participate in folliculogenesis. During this organ culture, morphological and hormonal assessments of cultured ovaries were done.
In this experimental study, one week (n=15) and two weeks (n=5) old female mice obtained from National Medical Research Institute (NMRI) were housed and bred in the Central Animal House of Tarbiat Modares University. All animals were housed under a 12-hour light/12-hour dark regime at 22-24˚C.
The mice were sacrificed by cervical dislocation and their ovaries were dissected free of fat
and mesentery. For each one-week-old mouse, one
ovary was selected randomly and fixed immediately in Bouin’s solution (non-cultured control) and
the other was considered for
Each ovary (n=15) was cultured in 24-well plates with tissue culture well inserts (non-tissue culture treated, PICM 012 50, 0.4-µm pore size; Millipore Corp, Billerica, MA) in 0.4 ml α- MEM (Gibco, UK) supplemented with 5% fetal bovine serum (FBS), 100 mIU/ml recombinant follicle stimulating hormone (rFSH or Gonal-f, Serono, Switzerland), 1% insulin, transferrin and selenium (ITS, Gibco, UK), 100 IU/ml penicillin and 50 μg/ ml streptomycin. Approximately 400 μl of culture medium was added to the compartment below the membrane insert, such that ovaries on the membrane were covered with a thin film of medium. The ovaries were incubated at 37˚C and 5% CO2 for 7 days. Every other day, 150 μl media was replaced with fresh culture medium. The collected media were stored separately at -80˚C until undertaking the hormonal assay.
To assess the integrity of follicles after culturing, the follicular morphology was examined by histological staining. The non-cultured (control) ovaries from 7- and 14-day-old mice and cultured ovaries (for 7 days) from one-week-old mice were fixed in Bouin’s solution and were embedded in paraffin wax and serially sectioned at 5-μmthickness (n=5 for each group); every five sections of each ovary were mounted on glass slides, and stained with hematoxylin and eosin. All sections were examined using light microscopy at a magnification of ×400. For this study, stages of follicles have been classified and counted according to the method described previously
The survival rates of the isolated preantral follicles from cultured ovaries (n=30 in each group)
were determined using trypan blue staining. The
preantral follicles with 120 μm in diameter from
the ovaries were mechanically isolated using insulin-gauge needles under stereomicroscope. Only
follicles containing several layers of granulosa
cells with a centrally located, healthy, visible oocyte and a thin layer of theca cells were selected.
These isolated follicles were stained using trypan
blue (0.4%) (Sigma, St. Louis, MO). The follicles
were recorded as degenerated or survived: degenerated follicles stained blue and surviving ones not
Photographs of each ovary in all groups of study were prepared under Olympus (Tokyo, Japan) CK40 inverted microscope with an attached Olympus DP11 digital camera. All photographs were imported into ImageJ 1.33U software (National Institutes of Health, USA). Area was calculated in units of pixels and then converted to millimeter based on the pixel number to millimeter conversion ratio determined by measurements using the calibrated millimeter.
17-β estradiol (E2), progesterone (P4) and dehydroepiandrosterone (DHEA) were measured in collected media derived from ovarian culture during days 2, 4 and 6. The levels of 17-β estradiol (Monobind, USA, sensitivity=6.5 pg/mL) and progesterone (DiaPlus, USA, sensitivity=0.1 ng/ml) were measured by an enzyme immunoassay modified for the cell culture media. DHEA was measured with a commercial immunoenzymatic assay using antibodies directed against the α-subunit (Monobind, USA, sensitivity=20 pg/ml). These experiments were at minimum done in triplicates.
Statistical analysis was done with SPSS 16.0 software. The ovarian area and production of hormones were compared by one-way analysis of variance (ANOVA) and Tukey’s test. Student’s ttest was used to compare the proportion of follicular stages. P<0.05 was considered to be statistically significant.
The phase contrast morphology of ovaries is shown in fig 1. The appearance of growing follicles became apparent during the culture period and could be observed as swellings on the surface of the cultured ovary. The morphology of ovaries derived from 7-dayold mice before culture (non-cultured control) using the hematoxylin and eosin staining are shown in fig 2A, after one week culture in figs 2B and 2C and the ovaries of two-week-old mice in fig 2D. This figure showed that the structural organization of cultured ovaries was well preserved and appeared normal and it was similar to two-week-old mouse ovaries. Furthermore the necrosis area with atretic follicles (with a retracted oocyte and granulosa cells, pyknotic nucleus) was consistently seen in the central part of the cultured ovary
Photomicrographs of 7-day-old mouse ovary viewed under the invert microscope in non-cultured fresh samples (A) and during one week of culture (B-D).
Hematoxylin and eosin staining of fresh and cultured mouse ovarian organ. Non-cultured fresh one week old mouse ovary which contains mainly primordial follicles with a few primary and secondary follicles (A), one week old mouse ovary after 7 days of culture (B). Higher magnification of one week old mouse ovary after 7 days, more secondary follicles were observed (C). Non-cultured fresh 14-day-old mouse ovary (D).
The normality rates of follicles at various developmental stages within the ovaries of cultured
and non-cultured controls are presented in fig 3.
Ovaries derived from 7-day-old mice at the beginning of the culture contained mostly primordial
follicles (91.8 ± 0.2%), with a small proportion
of primary (5.8 ± 2.5%) and secondary follicles
(4.6 ± 0.4%). After 7 days of
Normality rates of follicles after 7 days of
The morphologies of preantral follicles after trypan blue staining are shown in fig 4. The survival rates of preantral follicles derived from cultured ovaries using negative trypan blue staining was 99.2% and this rate was 100% in the control group of 14-day-old mice ovaries. There was no significant difference between them (p>0.05).
The trypan blue staining of preantral follicles. A. The survived follicle was not stained and B. degenerated follicle was stained intensively.
The area of cultured ovaries increased significantly from 0.212 ± 0.05 mm2
on day 0 to 1.47 ±
on day 7 of culture (p<0.003,
Area of mouse ovary during 7 days of culture. *; There were significant differences between lengths of culture
The levels of E2, P4 and DHEA during different lengths of culture are compared in fig 6. The levels of E2 in culture media on day 2, 4 and 6 were 3381 ± 43, 6552 ± 214 and 12938 ± 684 pg/ml and the concentration of P4 on the same days were 55.7 ± 0.5, 111 ± 3 and 157 ± 14 ng/ml respectively. DHEA levels increased from day 2 to day 4 to day 6 of culture (6.8 ± 0.2, 14.5 ± 0.2 and 29 ± 0.5 ng/ml respectively).
The level of estradiol (A), progesterone (B) and dehydroepiandrosterone (C) in collected culture media of mouse ovary. *; There were significant differences between lengths of culture.
The establishment of a successful culture system for primordial follicle growth and development is essential to studies of
Data obtained from this histological study
showed that neonatal mouse ovary has a high percentage of primordial follicles. However, the proportion of these follicles decreased after 7 days of
Our observation confirmed that the ovarian culture conditions used in this study had a significant effect on follicular activation and growth. The culture of mouse whole ovary in α-MEM medium supplemented with 10% FBS and 1% ITS not only increased the number of large follicles but also the ovarian area, implicating that an increase in ovarian area during culture may represent follicular growth and development.
In comparison with a previous work on the culture of isolated follicles (
Ovarian steroid production is an indicator of
ovarian development and function. During the primordial follicle transition to the preantral follicle,
granulosa and theca cells synthesize estradiol and
androstenedione progressively, therefore measuring E2, P4 and DHEA could be an appropriate tool
to evaluate the functionality of these cells within
the ovary. The measurable steroidogenic function
of these cells may be due to the remarkable increase in proliferation and differentiation of follicle cells and aromatase activities in granulosa cells
during culture (
Results of this study show that