JBRA Assist. Reprod 2006;10(3):27-30
ARTIGO ORIGINAL
doi: 10.5935/1518-0557.2006.10.3.05
1*ARU, Maternity Hospital, Foresterhill, Aberdeen, AB252ZL, Scotland, UK. Tel: 0044-1224552879; fax: 1224-551072; e-mail: ogy414@abdn.ac.uk
2Centro de Reproducao Humana Nilo Frantz , Porto Alegre, Brasil
3Gestare Centro de Reproducao Humana, Novo Hamburgo, Brasil
ABSTRACT
The preservation of fertility in young women facing premature ovarian failure, particularly those with cancer, has augmented the interest in ovarian tissue cryopreservation.To evaluate the effects of one slow freezing and thawing protocol on the stromal components of the ovarian cortex, we performed specific histology staining techniques to collagen fibers and cabohydrates. Fresh and frozen-thawed sections of one bovine ovary were stained using Mallory´s phosphomolidic acid hematoxilyn for collagen fibers, Schiff´s periodic acid for carbohydrates and hematoxilin and eosin for control.The organization of collagen fibers, follicular basal membrane and chromatin condensation aspect of stromal cells nuclei were analysed after staining. In the frozen-thawed tissue, collagen fibers presented a disrupted thread-like pattern, leaving large spaces between them throughout the ovarian cortex, follicular basal membrane was dettached from the surrounding stroma in 33.4% of follicles classified as “shrunken” and the nuclei of the stromal cell population were mostly pycnotic with condensed chromatin and irregular shape. Our results show that cryopreservation causes damages to stromal elements of the ovarian cortex which may impair follicular survival, growth and ovulation after transplantation.
Key-words: cryopreservation, ovarian stroma, histological staining
RESUMOINTRODUCTION
The recent surge of interest in ovarian tissue banking reflects the importance and the need of fertility conservation for many women facing premature ovarian failure, particularly those with cancer. Survival rates for cancers that occur in young girls and women of reproductive age have improved dramatically in the last years. However, the treatment is associated with long-term effects, as severe depletion of the follicular store and premature menopause (Wallace et al., 1989; Meirow, 1999). Therefore, preservation of fertility in these women has become a relevant concern.
Several potential options are available to preserve the fertility of these patients (Meirow, 1999). More recently, greater attention has been focused on the preservation of ovarian fragments or whole ovaries for future transplantation (Picton et al., 2000). Ovarian tissue cryopreservation is still an experimental procedure, which may be a feasible option to preserve fertility in prepubertal girls and women, which cannot wait to undergo ovarian stimulation. Several studies have reported the damage caused to the follicular population after cryopreservation of ovarian grafts (Cortvrindt et al., 1996; Nisolle et al., 2000; Demirci et al., 2002; Lucci et al., 2004; Gandolfi et al, 2006) and a second key ovarian component, the cortical stroma, has received little attention. However, follicular survival, growth and ovulation after transplantation will depend, among other factors, on the efficient re-establishment of angiogenesis by the stromal elements of the ovarian cortex (Dissen et al., 1994).
The ovarian cortex is the peripheral zona containing follicles in various stages of development embedded in a loose connective tissue. A thick connective tissue layer the tunica albuginea lies immediately beneath the surface epithelium. It is formed by wellorganized dense collagen fibers, which become disrupted by the growth of ovarian follicles and corpus leteum (Dellmann & Eurell, 1998). Fibroblasts, which synthesize and export the collagen fibers into the extra cellular matrix and the fibers themselves, play a key role in ovulation.
The ovarian stroma is also well endowed with angiogenic factors synthesized by the stromal cells under gonadotrophic control (Dissen et al., 1994). It has been reported that more primordial follicles die of ischemia than of freezing injury after cryopreservation (Falconi et al., 2004), possibly due to an impaired or deficient angiogenesis caused by degeneration of stromal elements.
Basal laminas are important sheets of specialized extracellular matrix containing high quantities of glycoproteins, which form part of the reticular fibers that underlie and surround groups of cells, such as epithelia or endothelia, enabling these cells to orientate their basal/apical polarity and creating a microenvironment for them. Ovarian follicles are surrounded by a basal membrane from the early stages of primary follicle to the pre-ovulatory antral stage. Following ovulation the follicular basal lamina is degraded, but it may also participate on corpus luteum formation (Irving-Rodgers et al., 2004).
The objective of the present study was to evaluate by means of specific histological staining methods the alterations induced by cryopreservation in stromal components of the ovarian cortex, particularly the collagen fibers, the follicular basal membrane and stromal cells.
MATERIALS AND METHODS
Preparation of ovarian tissue
One bovine ovary ( 2-4 years old female) showing different stages of follicular development and a corpus luteum was obtained from an abattoir 30 minutes after slaughtering and transported in saline solution at 40.C to the laboratory whithin 3 hours.Initially, the whole ovary was split into two halves and most of the underlying medulla of each half-ovary was removed leaving a 3mm-thick cortex. Each half was processed into thin sheets of 2mm, which were then cut into small sections of 10mm. Fresh fragments were fixed immediately after trimming in Bouin´s fixative and were used as controls. The remaining pieces of tissue were frozen.
Freezing
The freezing protocol was similar to that described by Lucci and colleagues (2004) for cryopreservation of bovine ovarian tissue. The cryoprotectant solution consisted of 1.5 M propanediol (PROH) (Sigma Chemical, St. Louis MO, USA) prepared in Medium 199 (supplemented with HEPES and Hank’s salts; Cultilab, Campinas, SP, Brazil) containing 0.1M sucrose (S-188, Sigma Chemical, St. Louis MO, USA) and 0.4% bovine serum albumine (BSA, Sigma Chemical, St. Louis MO, USA).Ovarian cortex pieces were placed into 1.2ml cryogenic vials (Corning) containing 1ml of the cryoprotectant solution. Each vial received one piece of tissue. Vials were equilibrated at 20o.C for 20 minutes and then cooled from 20o.C to -7o.C at 1o.C/minute and held at this temperature for 10 minutes. At this point vials were manually seeded and then cooled at 0.3o. C/minute to -30o.C. The vials were plunged into liquid nitrogen at -196o.C and stored for three weeks before thawing.
Thawing
Vials were warmed in air at room temperature (approximately 25o.C) for 30 seconds and then immersed in water at 38o.C until the ice melted. Tissues were removed from the vials and washed three times (5 minutes each), twice in M 199+ containing decreasing concentrations of the cryoprotectant (0.75M and 0.375M) and sucrose (0.1M) and once in pure M199+.
Histological procedures
After thawing fragments were fixed in Bouin´s fixative for histology. Three different frozen/thawed tissue fragments and one fresh control were sectioned at 6 µm thickness.
Histological stainings
Mallory´s phosphomolybdic acid hematoxylin
Collagen fibers were stained by Mallory´s phosphomolybdic acid hematoxylin (Mallory, 1942). Briefly, sections were deparaffinized in an alcohol gradient and exposed to Zenker´s fluid for 20 minutes. After removal of the mercury precipitate by placing in alcoholic iodine solution for 5 minutes slides were cleared of iodine in 5% sodium thiosulfate (Merk, Germany) solution for further 5 minutes. After washing in tap water, slides were stained in phosphomolybdic acid hematoxylin for 3 hours at room temperature. Washing in tap water terminated the reaction. Slides were differentiated in alcohol 95% and dehydrated with 2 changes in alcohol 95%, and alcohol 100% and finally cleared with 2-3 changes of xylene before mounted in balsam of Canada (Reagen, São Paulo, Brasil). Analysis was made under an Olympus light microscope.
Periodic Acid Schiff Reaction
Follicular basal membrane was stained by the Periodic Acid- Schiff (PAS) reaction (MacManus, 1948) for carbohydrates. In brief, slides were exposed to the periodic acid solution (Merk, Germany) for 5 minutes and washed in distilled water. Slides were placed in Schiff´s leuco-fuchsin for 15 minutes, washed in tap water for 10 minutes and counterstained in Mayer´s hematoxylin for 5 minutes. Primordial, primary and pre-antral follicles were classified as “normal” or “shrunken” when they have a round or collapsed shape, respectively. The alterations on stromal components were described without any quantitative analysis as our aim was to detect elements of the cortical stroma using specific staining techniques. No description of the follicles themselves and the oocytes within them was made as that was part of another study.
RESULTS
The Mallory´s staining revealed bluish-gray collagen fibers with a thread-like organization leaving large spaces between them on the cortical surface of the frozen-thawed ovarian tissue. Overall, the cortical layer beneath the surface and the tunica albuginea looked vacuolated and disorganized compared with fresh controls. Collagen fibers presented a similar pattern in different sections of the three cryopreserved fragments analyzed.
Follicular basal membrane stained bright pink in all follicles from primordial to growing and antral ones. Only primordial, primary and secondary follicles were analyzed. When the follicular structure was not round, what we called “shrunken follicles”, PAS staining revealed a detachment of the basal membrane together with the follicular cells, from the surrounding stroma. This finding was clearly observed in 33.4% (34/101) shrunken follicles analyzed in frozen-thawed tissues. Similar follicular abnormality was observed in 6.6% (7/105) of the follicles in the fresh samples.
The nuclear morphology of stromal cells in fresh and cryopreserved tissues was analyzed after hematoxylineosin staining. Frozen-thawed ovarian samples presented elongated and pycnotic nuclei beneath the cortical surface. Control tissues presented elliptical-shaped nuclei, typical of fibroblasts with dispersed chromatin along the ovarian cortex .
DISCUSSION
The present study describes histological alterations on the ovarian cortex after cryopreservation. Our objective was to analyse stromal components of the ovarian cortex using specific staining techniques to evaluate the effects of one cryopreservation method, namely, the slow freezing/ rapid thawing in 1,2 propanediol. Mallory´s stain demonstrated that the slow freezing and thawing protocol used causes profound alterations on collagen fibers, which become thin, thread-like and apart from each other. The nuclei of fibroblasts among the collagen fibers showed a more pycnotic staining pattern than controls, indicative of cell damage, possibly cell death. Follicle basal membranes were detached from the surrounding stroma, together with the granulosa cells in follicles that were considered damaged in the frozen/thawed samples.
It would be interesting to know whether a culture period after thawing and before transplantation would have a positive effect specifically on collagen fibers and stromal cells allowing them to recover and to return to their original shape and organization. However, a recent work envolving ovarian tissue culture after cryopreservation (Kim et al., 2004) reported that the apoptotic rate of stromal cells was higher in frozen/thawed ovarian tissue compared with the fresh group after different incubation periods, indicating that cryopreservation may have caused irreversible injury to the ovarian cortex leading to cell death.
A previous study described the histological findings on human ovarian cortex after cryopreservation in 1,2 propanediol under various conditions (Gook et al., 1999). The authors assessed ovarian sections by light and electron microscopy and performed a detailed description of the observed follicular and oocyte abnormalities using one standard staining method with methylene blue. No mention was made on the collagen fibers organization although the pictures presented in the article show clear differences in their structure very similar to our data after freezing and thawing .
Collagen fibers form part of the loose connective tissue and the tunica albuginea of the ovarian cortex and play a fundamental role during follicle growth and ovulation (Dellman & Eurell, 1998). The extracellular matrix of connective tissue participates in a multitude of cell functions, including morphogenesis, survival, migration, proliferation, communication, metabolism and response to external stimuli. More important, recent reports based on histological and immunohistochemical findings suggest that components for new human primordial follicles, follicular cells and germ cells, differentiate sequentially and de novo from mesenchimal progenitor cells residing in the stroma of the ovarian tunica albuginea (Bukovsky et al, 2004; 2005). If these findings appear really true, the preservation of ovarian stromal components during cryopreservation is of fundamental importance to compensate for the follicular loss of a significant portion of the follicular pool after freezing and thawing and may ensure a longer reproductive lifespan than currently observed after transplantation of frozen/thawed ovarian fragments.
Damaged follicles and oocytes are the more common abnormality described in histological studies on the effects of cryopreservation in ovarian tissues. The present study focused on the follicular basal membrane and demonstrated by specific glycoprotein staining that part of the observed follicular abnormalities observed involves the basal membrane and may represent an irreversible damage to the follicular structure. Detachment of the follicular basal membrane from the surrounding stroma was also observed in 6.6% of the follicles in fresh samples. This observation suggests that this abnormality may not only be caused by the freezing/thawing procedure. However, this type of follicular damage was far more frequent in the cryopreserved samples indicating possibly an increased fragility of the tissue after cryopreservation. Considering the role that the basal membrane may play on the endothelial-mesenquimal transition of granulosa cells into luteal cells (Irving-Rodgers et al., 2004) the maintenance of its correct position and attachment to both granulosa and stromal cells is fundamental for subsequent ovarian function.
Antioxidant treatments have been proposed to improve follicular survival in fresh (Bukovsky et al, 2004) and frozen (Kim et al., 2004) ovarian grafts. Ascorbic acid, a cofactor for several hydroxylase enzymes and involved in collagen synthesis was efficient in decreasing apoptosis detected by TUNEL staining on stromal cells of fresh and frozen/thawed ovarian specimens incubated for 24 hours (Kim et al., 2004). We did not use a specific cell death staining technique, but the pycnotic nuclei observed among the altered collagen fibers may well represent dead fibroblasts observed immediatly after thawing. Actually, the picture presented by Kim et al., (2004) in their study on apoptosis in frozen/thawed ovarian tissue shows a very similar pattern of cortical stroma to the one we observed in the present work after specific staining for the collagen elements. Considering that collagen fibers and angiogenic factors responsable for the neovascularization of the ovarian grafts are synthesized by fibroblasts, the preservation of their integrity is of paramount importance while developing new cryothechnologies for the human ovary.
In conclusion, our results show that stromal components are profoundly damaged by the slow freezing/thawing procedure here employed, which may impair follicular survival and ovarian function after transplantation. Our findings extend the knowledge on the effects of cryopreservation on ovarian tissue.
AKNOWLEDGEMENTS
The authors wish to thank Prof Dr Matilde Achaval for allowing access to the Histology Laboratories of the Department of Morphological Sciences of the University of Rio Grande do Sul and for her expert advise. We are also thankful to Mrs. Eudira Luz da Silva for her technical support.
MacManus J.F.-Periodic acid Schiff’s reaction for carbohydrates. Stain Technology, 23: 99,1948.
Mallory F.B. In: Pathological Techniques, W.B. Saunders Co, Philadelphia, p47,1942.