Function of Ovarian Tissue Grafts After Hypothermic Storage : Importance of Incubation Medium Composition

There were comparatively analyzed the morphology, endocrine function and folliculogenesis of mature and neonatal rat ovarian tissue grafts after hypothermic storage (HS) in the media with different composition. For research performance the following methods were used: heterotopic transplantation (study of ovarian tissue structure and function after HS); immunoassay (determining the concentration of sex hormones in plasma); morphological method (evaluation of tissue graft structure integrity after HS). The composition of incubation medium was experimentally proven to be a key factor in follicular development and endocrine function of ovarian tissue grafts of different stages of histogenesis after HS. It was found that the use of phosphate-buffered saline for HS significantly reduced the follicular pool and decreased the steroidogenic function of grafts in both mature and neonatal ovarian tissue. It was shown that when using the mannitol-containing solution for HS, the number of follicles of different maturity degree and corpus luteum and the endocrine function after ovarian tissue transplantation were comparable with those after fresh tissue transplantation.

Эксперименты проводили в соответствии с Законом Украины «О защите животных от жес-Currently, the ovarian tissue transplantation is among the most promising methods for restoring reproductive function in women after chemo-and radiotherapy sessions [1,11,23].Herewith, by the time of ovarian tissue fragment revascularization, there was observed the death of 60-80% of follicles of the whole pool, among which 20-30% were damaged during extraction, transportation and storage [5,10].Hypothermic storage (HS) is known to be one of the main methods for organ and tissue preservation during transportation [6,8,24,26].For ovarian tissue HS one mostly applies such electrolyte media as normal saline solution, medium 199 [15], phosphate-buff ered saline, Braun-Collins solution [4].However, there are no reported data on the ovarian tissue integrity after HS in the mannitol-containing incubation media (Celsior, Custodiol) [26].
Of note is the fact that the mannitol use for brain [2], heart [16] and skeletal muscle [18] perfusion entails the reduction of necrotic zone in ischemized organs.N. Sagsoz [22] et al. in ovariantorsion model demonstrated the administration of 20% mannitol solution into the inferior vena cava to decrease the severity of ischemic tissue injuries in vivo.Our fi ndings testify to a protective eff ect of mannitol in HS of neonatal ovarian tissue [13].It is known that in mature ovarian tissue, the structure of oocyte cytoskeleton and the organization of microtubule system are diff erent from those in neonatal tissue, which may result in diff erent changes in morphological structure of tissue during HS [17,27].
Proceeding from the mentioned above this research was aimed to comparatively analyze the morphological structure and endocrine function of mature and neonatal ovarian tissue grafts after hypothermic storage in phosphate-buff ered saline and mannitol-containing solution.The neonatal ovarian tissue was isolated from 10-day-old rats under MBS-10 microscope (PA Rubin, USSR), then it was transferred into a Petri dish with saline, containing penicillin and streptomycin (100 U per 100 ml).Ovarian tissue of mature animal was fragmented (0.5-1 mm 3 ) with microsurgery scissors and placed into a sterile nutrient medium.The samples were subjected to HS for 24 hrs at 4°C in incubation media of diff erent composition: phosphate-buff ered saline (PBS) -130 mM NaCl, 20 mM KCl, 20 mM phosphate buff er at pH = 7.4; mannitol-containing solution (MCS) -250 mM mannitol (Dniprofarm, Ukraine), 10 mM NaCl, 20 mM KCl, 20 mM phosphate buff er at pH 7.4.

Materials and methods
To assess the growth and endocrine function of ovarian tissue after HS, the method of heterotopic transplantation under the left renal capsule was used.The ovarian tissue transplantation (0.15 mg/g of animal body weight) in all the groups was carried out to adult rats simultaneously with bilateral ovariectomy under sterile conditions and anesthesia.The transplantation procedure lasted 20 min.
Experimental animals were divided into the following groups: the group 1 -the transplantation of fresh neonatal ovarian tissue (subgroup 1A) and mature one (subgroup 1B), group 2 -transplantation of ovarian tissue after HS in PBS (neonatal and mature ones -subgroup 2A and 2B, respectively), group 3 -transplantation of ovarian tissue after HS in MCS (neonatal and mature ones -subgroup 3A and 3B, respectively).The control group comprised non-ovariectomized rats (C1) and those with bilateral ovariectomy (C2).To day 30 after transplantation the animals were sacrifi ced.
To determine the concentration of sex hormones, the blood was taken by intracardiac puncture, then centrifuged for 20 min at 3000g for plasma procurement.The estradiol and progesterone levels in blood plasma of recipient animals were determined with chemiluminescent enzyme immunoassay using the standard test kits 'ST AIA-PACK hsE2 G' and 'ST AIA-PACK PRO' (Tosoh Corporation, Japan) according to the instructions and enzyme immunoassay analyzer (Tosoh AIA 1200, Japan).
For morphological studies of grafts, the kidney was removed from a recipient animal, fi xed for 48 hrs in 10% formalin solution, then dehydrated and embedded in paraffi n blocks [14,20].Serial sections of the entire graft of 10 μm thickness were made from each kidney.Every third section was stained with hematoxylin and eosin.analysis, only the follicles with a visible nucleus were counted to exclude re-counting of the same follicle.
The follicles were identifi ed according to the A. Gougeon's classifi cation [7], where primordial follicle was the oocyte, surrounded by one layer of fl at granulosa cells; primary one was the oocyte, surrounded by a single layer of cuboidal granulosa cells; preantral one represented the oocyte, surrounded by more than two layers of granulosa cells, located on basal membrane, surrounded by single theca cells; antral one was the oocyte, increased in volume, surrounded by several layers of granulosa cells with formation of the cavity, containing follicular fl uid.
The ovarian tissue graft development was assessed by the change in its volume (length × width × thickness × 0.523) and the number of follicles in 1 mm 3 [25].The AxioVisionRelease 4.7.2 software (Carl Zeiss) was used for morphometric analysis.
The results were statistically processed using the Excel (Microsoft, USA) and Statistica v.6.0 (StatSoft, USA) software.Depending on the data distribution, either Student's t-test or Mann-Whitney U-test [3] were used to compare the samples.The results were presented as M ± m (p < 0.05).

Results and discussion
To day 30 of observation, the structure of grafts from fresh neonatal ovarian tissue (subgroup 1A) and ovarian tissue after HS in MCS (subgroup 3A) was established to have a similar character (Fig. 1A, C).
Histological sections revealed the follicles, inherent to both early (primordial and primary) and later (preantral and antral) stages of folliculogenesis, as well as the corpus luteum.When analyzing the morphological structure of the grafts of subgroups 1A and 3A there were revealed the follicular cysts, the inner wall of which was lined with several layers of elongated fi broblast-like cells with content (transparent or pale-stained with eosin) [21].When using PBS as HS medium (Fig. 1B), the tissue grafts (subgroup 2A) showed both sclerotic sites and well vascularized ovarian stroma, where the growing follicles were located.
The analysis of morphological structure of mature ovarian tissue grafts in animals of all the studied groups demonstrated the presence of follicles of diff erent maturity degrees and corpus luteum.

Результаты и обсуждение
In addition, the fi brosis of about 5-10% of tissue was revealed in the subgroup 2B graft, that was probably due to its lower survival rate during HS in PBS (Fig. 2B).
The follicle development in ovarian tissue grafts after HS in the media with diff erent composition was assessed by the follicle number per 1 mm 3 .In the grafts of subgroups 3A and 3B, the number of follicles per 1 mm 3 was 30.4 ± 5.2 and 36.5 ± 5.8, respectively, which was comparable to that in subgroups 1A and 1B, i. e. 38.6 ± 5.2 and 49.1 ± 7,4 respectively.At the same time, the studied index in subgroups 2A and 2B was signifi cantly lower vs. the control subgroups 1A and 1B (Fig. 3).
The presented fi ndings make it clear that the incubation medium composition is a key factor, determining follicular development and endocrine function of ovarian tissue grafts.The use of PBS as an incubation medium at HS signifi cantly reduced the follicular development and steroidogenic function of the grafts from both mature and neonatal ovarian tissue, but when using MCS for HS, these indices were comparable to the control ones (subgroups 1A and 1B).Consequently, under HS the destructive damages of the PBS-incubated ovarian tissue are of common character, having no relation to diff erent morphological structure of follicular unit and the follicle number per mm 3 of the initial material [9].A decrease in the injury rate of ovarian tissue when using MCS for HS may be explained by a protective eff ect of mannitol during ischemia, which is due to its osmotic properties.R.P. Paczynski et al. [19] experimentally proved the fact, that a multiple injection of mannitol in a postischemic period entailed the reduction of water amount in brain parenchyma and a decrease in interstitial pressure.R.J. Andrews et al. [2] demonstrated the mannitol administration to result in strengthened cerebral blood circulation and a decreased blood pressure in ischemized tissue, but no recovery of synaptic transmission of nerve impulses occurred.G.J. Magovern et al. [16] found кая передача нервных импульсов не восстанавливается.G.J. Magovern и соавт.[19] установили, что реперфузия сердца гиперосмолярными растворами маннитола улучшает диастолическую функцию желудочка по сравнению со стандартными кристаллоидными растворами.H.I. Karibe и соавт.выявили протективный эффект маннитола в условиях гипотермии и показали, что в результате их сочетаного применения не только уменьшается зона некроза головного мозга при ишемии, но и снижается нейрофункциональный дефицит [17].
Consequently, the study of the eff ect of various incubation media on morphological structure of neonatal and mature ovarian tissue fragments during HS, as well as on their functioning in a recipient's body after heterotopic transplantation in experimental bilateral ovariectomy, is relevant for reproductive medicine and cryobiology.

Conclusions
The MCS use for ovarian tissue HS has significant advantages as compared to PBS.It was experimentally proven that in the ovarian tissue grafts after HS in MCS, a follicular development comparable to that in fresh tissue grafts was observed.In ovariectomized recipient animals, transplanted with the ovarian tissue after a 24-hour HS, the endocrine function was restored.
проблеми кріобіології і кріомедицини problems of cryobiology and cryomedicine том/volume 29, №/issue 1, 2019 The research object was the ovarian tissue of 10-day and 3-month-old Wistar rats, housed in the animal facility of the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine (Kharkiv).The experiments were carried out in accordance with the Law of Ukraine 'On Protection of Animals Against Cruelty' (№3447-IV of February 21, 2006), agreed to the statements of European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientifi c Purposes (Strasburg, 1986) in compliance with the requirements of the Bioethics Committee of the Institute for Problems of Cryobiology and проблеми кріобіології і кріомедицини problems of cryobiology and cryomedicine том/volume 29, №/issue 1, 2019 Cryomedicine of the National Academy of Sciences of Ukraine.