Viability Of Ovarian Follicles Research Articles

Effects of erythropoietin on ischaemia-reperfusion when administered before and after ovarian tissue transplantation in mice

Research questionIs the optimal timing for administering erythropoietin to minimize ischaemic injury in ovarian tissue transplantation before ovary removal for cryopreservation and subsequent transplantation or after transplantation? DesignThirty Swiss mice (nu/nu) were divided into three groups: treatment control group (n = 10); erythropoietin before harvesting group (EPO-BH) (n = 10) and erythropoietin after transplantation group (EPO-AT) (n = 10). Animals underwent bilateral ovariohysterectomy and their hemiovaries were cryopreserved by slow freezing. At the same time, previously cryopreserved hemiovaries were transplanted subcutaneously in the dorsal region. Erythropoietin (250 IU/kg) and sterile 0.9% saline solution were administered every 12/12 h over 5 consecutive days in the EPO-AT and EPO-BH groups, respectively. ResultsAdministration of erythropoietin in the EPO-AT group improved the viability of ovarian follicles, reducing degeneration and increasing the number of morphologically normal growing follicles at 14 days after transplantation compared with the EPO-BH group (P = 0.002). This group also showed higher percentages of proliferative follicles at 7 days after transplantation (P ≤ 0.03), increased blood vessel count (P ≤ 0.03) and greater tissue area occupied by blood vessels at days 7 and 14 after transplantation (P ≤ 0.03), compared with hormone administration before cryopreservation (EPO-BH group) and the treatment control group. Additionally, treatment with erythropoietin before or after transplantation reduced fibrotic areas at 7 days after transplantation (P = 0.004). ConclusionErythropoietin treatment after transplantation reduced ischaemic damage in transplanted ovarian tissue, increased angiogenesis, maintenance of ovarian follicle proliferation and reduced fibrosis areas in the grafted tissue.

Is Ovarian Tissue Transport at Supra-zero Temperatures Compared to Body Temperature Optimal for Follicle Survival?

Transportation of ovarian cortex prior to freezing is used clinically; however, basic investigations of ovarian storage are limited and the question remains what temperature is optimal for transport over long distances and time periods. The aim of this study was to evaluate the rate of follicular loss over various time periods under two different temperatures and assess whether ovarian follicle viability is affected following cryopreservation and thawing subsequent to the transportation of ovarian tissue. Pig ovaries were transported at 4°C (n=10) or at 38°C (n=10) prior to cryopreservation. At 0, 4, 12 and 24 h tissues were fixed for histological examination and a LIVE/DEAD Assay. At the same time-points ovarian tissues were cryopreserved and analysed after thawing. Histological evaluation and LIVE/DEAD Assay of freshly transported ovarian tissue showed significantly better follicle survival at 4°C during transportation duration. In cryopreserved ovarian tissues the LIVE/DEAD Assay showed a significant difference in the number of intact and dead follicles at 24 h in favor of 4°C (p<0.05). Ovarian tissue transportation should be kept at a minimum to prevent potential damage.

53 Cryopreservation of Sheep Ovarian Tissue Using Different Cryoprotectants

Biological diversity is the key to maintaining life. The global trend in industrialization of agriculture bears in itself a set of risks. One of them is a reduction of a national genetic resource or gene pools of animals. Today, new methods of auxiliary reproductive technology permit using them for preserving not only the reproductive potential of the person, but also the biodiversity of wild and endangered species of farm animals. The art of cryobiology involves the addition of one or more cryoprotectants and change of time and temperature of exposition with tissue, and cooling speed. Therefore, the purpose of this work was to identify an optimum method of cryopreserving ovarian tissue of sheep using various cryoprotectants. Ovarian tissue from 8 indigenous Chuyi breed was transported to the laboratory at 36°C, divided into smaller pieces (5 × 3 × 1.0 mm) in the holding medium (Dulbecco’s PBS + 10% FCS), and randomly distributed into 8 groups. The pieces were equilibrated sequentially in freezing medium containing 0.25, 0.75 (5 min each), and 0.5 M sucrose with 1.5 M cryoprotectants (10 min) and placed in a cryo straw. The ovarian tissue was frozen in vapors of liquid nitrogen at a distance of 4 or 6 cm from the surface within 20 min with use of various cryoprotectants: 1.5 M dimethyl sulfoxide (DMSO), 1.5 M propylene glycol (PROH), 1.5 M ethylene glycol (EG), and 1.5 M glycerol (GL). After that, straws were plunged in liquid nitrogen. For thawing, straws were warmed in air at room temperature for 30 s and then immersed in a water bath at 37°C for 40 s. Then, ovarian pieces were transferred into the solution 0.75 M sucrose + 10% FCS + Dulbecco’s PBS (10 min, 37°C), then 0.3 M sucrose + Dulbecco’s PBS + 10% FCS (15 min, twice), and the holding medium (15 min). After thawing, the effects of cooling methods with different cryoprotectants on ovarian tissue morphology were evaluated by light microscopy after hematoxylin and eosin staining of tissue sections. The number of viable and damaged preantral follicles was counted. Data obtained in the experiments were analysed using Student’s t-test. Data are given as mean values ± standard errors. The level of significance was set at P &lt; 0.05. In groups that were frozen at 6 cm using 1.5 M DMSO, 54.6 ± 4.8% of follicles were morphologically normal. In groups that were frozen at 4 cm using 1.5 M GL and 1.5 M DMSO, 69 ± 3.0% and 58.4 ± 3.7% of follicles, respectively, were morphologically normal. Using 1.5 M PROH and 1.5 M EG did not yield good results. Differences were statistically significant compared with fresh control (99 ± 2.4%). The analysis of comparative histology showed that use of 1.5 M DMSO for freezing at distance of 6 cm or 1.5 M GL at 4 cm were more effective in terms of viability of ovarian follicles compared with other cryoprotectants.

42 THE EFFECT OF VARIOUS CRYOPROTECTIVE AGENTS AND SLOW COOLING RATE ON VIABILITY OF SHEEP OVARIAN TISSUE

Priority objects of protection in agrobiocenoses are grades of cultural plants and local breeds of the cultivated animals. The most general criteria for preservation of local breeds are viability, adaptability, a state of health, reproductive abilities, and unique genetic polymorphism at the molecular and morphological levels. Therefore, the aim of this study was to compare the effectiveness of different cryoprotectors on morphology of ovine ovarian tissue cryopreserved by a passive cooling method. Ovarian tissue from 10 indigenous Chuyi breed was transported to the laboratory within 30 min at 32 to 34°C, divided into smaller pieces (2.0 × 1.2 × 1.0 mm), and randomly distributed into 4 groups: (1) control (fresh tissue), (2) pieces after freezing/thawing with 1.5 M ethylene glycol (EG); (3) pieces after freezing/thawing with 1.5 M propanediol (PROH); (4) pieces after freezing/thawing with 1.5 M dimethyl sulfoxide (DMSO). The ovarian pieces were placed in a cryovial and equilibrated sequentially in freezing medium containing 0.25, 0.75, and 1.5 M cryoprotectors with 0.5 M sucrose (5 min each), precooled at 4°C, and stored in a −80°C freezer for 24 h. Then, the cryovials containing the ovarian pieces were placed in liquid nitrogen and stored (for 2 months) until thawing. The ovarian pieces were cultured in vitro for 7 days in TCM-HEPES+ 10% native ovine serum (NOS) (Seisenbayeva et al. 2015 Reprod. Fertil. Dev. 28, 193–194). After 7 days of culture, we evaluated the effects of passive cooling methods with different cryoprotectors on ovarian tissue morphology by light microscopy after hematoxylin and eosin staining of tissue sections. The number of viable and damaged follicles was counted. All morphologically normal primordial, primary, and secondary follicles had healthy and intact oocytes, each containing a round nucleus and clearly visible nucleolus surrounded by well-organised granulosa cells without a pyknotic nucleus. Integrity rate of tissue after treatment was evaluated by Student’s test. In groups 1, 2, 3, and 4, the mean densities of follicles per 1 mm3 was 17.0 ± 4.6a, 16.2 ± 7.2b, 13.0 ± 5.1c, and 11.9 ± 4.8d, respectively (ab, ac, ad P &gt; 0.05). For these groups, respectively, 69.2 ± 8.2%a , 61.7 ± 8.6%b , 52.4 ± 8.8%c and 48.5 ± 8.8%d preantral follicles were morphologically normal (ab, ac, ad P &gt; 0.05). We did not find significant differences between groups. The analysis of comparative histology shows that 1.5 M EG is more effective on viability of ovarian follicles than 1.5 M DMSO or 1.5 M PROH.

Influence of various cryoprotectors on survival of sheep’s ovarian tissuewith freezing in vapors of liquid nitrogen

Biological diversity is the key to maintaining life. Therefore preservation of biodiversity is important for sustaining a healthy Earth, but it also is immensely valuable tothe health and lifestyle of human society. Today new methods of auxiliary reproductivetechnology allow using them not only for preservation of reproductive potential of theperson, and also for preservation a biodiversity of wild and endangered species of farmanimals. Captive breeding programs, genetic resource banks and artificial reproductivetechniques have been suggested as important tools for conservation. Today, biotechnological methods, such as artificial insemination and transplantation of embryos are welldeveloped and are used in programs of animal husbandry and preservation. Receivingoocytes and embryos requires stimulation of ovaries on what some time depending ona species of animals usually is required. These methods are not acceptable in case ofemergency, and the in vivo received oocytes in smaller quantity do not guarantee themaximum storage of genetic material. Therefore an alternative method of preservation ofgenetic material is the cryopreservation of immature oocytes in the primordial follicleswhich are located in ovary cortex. The procedure of ovarian tissue cryopreservation permits conservation of hundreds of immature oocytes kept within the protective environment of the original ovarian tissue. An important advantage of this technique that thehormonal stimulation is not required in this case. Additionally, because the primordialfollicles are small and have a simple structure, they are much more tolerant to manipulation and to the freeze-thaw procedure compared with the large growing follicles. A keyelement of a good cryopreservation to cell survival is the physiochemical relationshipof heat and water transport between the intra- and extracellular environment. The art ofcryobiology involves the addition of one or more cryoprotectants which generally reduceboth the eutectic and freezing points. Therefore the purpose of this work is identificationof an optimum method of a cryopreservation of sheep‘s ovarian tissue using of variouscryoprotectors. In the present study, the cryopreservation of indigenous Chui population sheep’s ovarian tissue was conducted in vapors of liquid nitrogen at distance of 5cm from surface within 20 minutes with use of various cryoprotectors: 1,5 M dimethylsulfoxide (DMSO), 1,5 M propylene glycol (PG), 1,5 M ethylene glycol (EG) and 1,5 Mglycerol (HL). The analysis of comparative histology studying shows that use 1,5 M PGand 1,5 M DMSO has more effective effect on viability of ovarian follicles, than use 1,5M EG and 1,5 M HL.Key words: dimethyl sulfoxide, ethylene glycol, freezing, follicles, glycerol, ovariantissue, propylene glycol.

Effect of Previous Chemotherapy on the Quality of Cryopreserved Human Ovarian Tissue In Vitro.

BackgroundCryopreservation of ovarian tissue has been widely accepted as an option for fertility preservation among cancer patients. Some patients are exposed to chemotherapy prior to ovarian tissue cryopreservation. Consequently, assessment of the developmental capacity of human ovarian tissue after chemotherapy is of primary importance.MaterialsIn order to study the impact of previous chemotherapy on in vitro development and viability of ovarian follicles, quality control samples from 34 female cancer patients at median age of 15 years (range 1‒35), cryopreserved for fertility preservation before (n = 14) or after (n = 20) initiation of chemotherapy, were thawed and cultured for 7 days. The morphology and developmental stages of ovarian follicles were studied by light microscopy before and after culture. Possible associations between follicular densities, age and exposure to alkylating agents, expressed as cyclophosphamide equivalent dose (CED) were tested.ResultsExposure to chemotherapy significantly impaired the survival and development of ovarian follicles in culture. After seven days, significantly higher densities of intermediary, primary and secondary follicles and lower densities of atretic follicles was detected in the samples collected before chemotherapy. Increasing dose of alkylating agents was identified by multivariate linear regression analysis as an independent predictor of a higher density of atretic follicles, whereas increasing age of the patient predicted a better outcome with less follicle atresia and a higher density of maturing follicles.ConclusionThis study provides quantitative in vitro evidence of the impact of chemotherapy on developmental capacity of cryopreserved human ovarian tissue. The results indicate that fertility preservation should be carried out, if possible, before initiation of alkylating agents in order to guarantee better in vitro survival of ovarian follicles. In addition, ovarian samples from younger girls show lower viability and fewer developing follicles in culture.

123 Development of cryopreservation protocol for zebrafish ovarian fragments using controlled slow cooling

Cryopreservation of gametes is an important method of conservation of germplasm and has wide range of applications in aquaculture and fisheries management. Cryopreservation is the most efficient method for long term storage of genetic materials. Though fish sperm cryopreservation has been a success, developing successful protocols for eggs and embryo cryopreservation still remain elusive. Several studies have been undertaken on cryopreservation of isolated zebrafish ovarian follicles at different stages yet the protocols used lead to compromised viability. Cryopreservation of follicles within ovarian tissue has attracted considerable attention in recent years. It has become a valid alternative to cryopreservation of human oocytes. The present study was undertaken to determine the effect of cryopreservation on zebrafish ovarian tissue fragments containing the stage I and stage II follicles. The effect of cryopreservation medium, the effect of cryoprotectants, cooling rate and ovarian follicle viability assessment after controlled slow cooling were investigated. Ovarian follicle membrane integrity was assessed by trypan blue (TB) and fluorescein acetate-propidium iodide (FDA-PI) staining. ATP levels were measured to assess the metabolic status following cryopreservation. In these experiments ovarian tissue fragments were exposed to cryoprotectant solutions for 30 min at 22 °C and then were loaded into 0.5 ml plastic straws before placing in a programmable cooler (Planer KRYO 550). Ovarian tissue fragments incubated in cryoprotectant-free L-15 medium or KCl buffer were used as controls. The following cooling protocol was used: cooling at 0.3 °C/min, 0.4 °C/min, 1 °C/min, 2 °C/min, 4 °C/min and 7 °C/min from 20 °C to seeding temperature (−7.5 °C for 2 M methanol). Manual seeding and hold for 5 min, freezing from seeding temperature to −40 °C at 2 °C/min, from −40 °C to −80 °C at 10 °C/min and hold for 10 min, samples were then plunged in liquid nitrogen at −196 °C and held for at least 10 min. Samples were thawed using a water bath at 28 °C. Removal of cryoprotectant was conducted in four steps. The results from the present study indicated that the tissue fragments can be incubated in 2 M methanol + 20% FBS in L-15 medium for 30 min at room temperature and then frozen to −196 °C at post-seeding cooling rate 4 °C/min and the removal of the cryoprotectant in four steps. After cryopreservation the survival rate of the follicles was assessed using TB staining, FDA + PI staining and ATP assay. The survival rate after the cryopreservation procedure were 55.4 ± 2.3% for stage I and 68.2 ± 1.9% for stage II using TB staining and 48.2 ± 2.9% for stage I and 54.2 ± 2.6% for stage II follicles using FDA + PI staining. The results obtained from ATP assay showed compromised survival of the ovarian follicles. The present study also indicated that the use of non-permeating cryoprotectants is not beneficial for the zebrafish ovarian tissue cryopreservation since it decreased the survival rate of the follicles when compared to the use of permeating cryoprotectants. The results obtained after the in-vitro culture of the cryopreserved fragments showed that there was no growth of follicles obtained after in-vitro culture treatment and the survival rate of the cryopreserved follicles were significantly lower to those of the non-cryopreserved follicles. The results obtained in this study provide useful information for future cryopreservation protocol development. Source of funding: Funding support for the programme of research was provided by the strategic research funds of the iBEST, University of Bedfordshire Conflict of interest: None declared. tzhang@bournemouth.ac.uk

18. Vitrification of zebrafish (Danio rerio) ovarian follicles

Overfishing and environmental factors such as pollution have contributed to the significant decline of fish populations worldwide. Successful cryopreservation of fish gametes and embryos would have important applications in conservation and aquaculture. Although cryopreservation of fish semen has already been achieved successfully, cryopreservation of fish embryos and oocytes remains elusive. Despite the widespread use of vitrification for oocyte cryopreservation in humans and domestic mammals, vitrification of fish oocytes has not been studied to date. This study aimed at developing a cryopreservation protocol for stage III zebrafish ovarian follicles within fragment using vitrification. Vitrification ability of methanol, ethanol, Me 2 SO, propylene glycol and ethylene glycol were tested using a range of concentrations made up in 90% Leibovitz L-15 medium. Solutions were tested for vitrification ability using 0.25 ml plastic straw and a fibreplug™ (CryoLogic Ltd.) loaded by a pipette. The transparent glassy appearance during cooling and warming was used to identify vitrified solution. Based on results obtained from this stage, the vitrification ability of 24 combinations using the mentioned cryoprotectants was studied. Three vitrification solutions (VS) (V6B: 1.5 M methanol + 6.0 M ethylene glycol + 0.5 M sucrose; V1B: 1.5 M methanol + 4.5 M propylene glycol; and V2: 1.5 M methanol + 5.5 M Me 2 SO) were selected for subsequent experiments based on their vitrifying ability. For VS toxicity tests, ovarian follicles were equilibrated in L-15 medium containing 1.5 M methanol for 30 min at room temperature. After equilibration follicles were exposed to VS for 3 min in a stepwise manner. Afterwards the cryoprotectants were gradually removed in three steps and viability was assessed by trypan blue staining. For vitrification procedures, immediately after exposure to VS ovarian follicles were loaded in either plastic straw or fibreplug™ and plunged into liquid nitrogen, being stored for at least 20 min. The warming was performed at 28 °C and cryoprotectants were gradually removed in three steps. Ovarian follicle viability was assessed by three means: trypan blue staining, measurement of ATP level in the follicles and assessment of mitochondrial membrane potential and distribution by using JC-1 molecular probe. Results obtained from toxicity tests showed follicles viability of 74% for V1B; 30% for V2 and 78% for V6B. After vitrification, ovarian follicles presented a viability of 60% when fibreplug and V1B were used, V2 showed viability of 42% (using fibreplug), and using plastic straws V6B solution showed only 2% of viability. [ATP] soon after warming dropped down to 22% (V2) and 7% (V1B) and two hr later the concentration in V2 was 15% and for V1B a very low concentration of 3.5%. The viable follicles in V1B and V2 seemed to be opaque and morphologically intact, when assessed immediately after warming. These results are different to the results described so far in the literature, where zebrafish follicles were observed to become swollen and translucent even during the warming process after being exposed to controlled slow cooling protocols. However, 30 min after warming most of the follicles started to become semi-translucent and lightly swollen. A complete loss of internal structure pattern and also loss of fluorescence indicated follicles with no mitochondrial membrane potential after vitrification.

Development of in vitro culture method for early stage zebrafish (Danio rerio) ovarian follicles for use in cryopreservation studies

There have been no reported methods for in vitro growth of early stage ovarian follicles for fish and their cryopreservation is still under investigation. If cryopreservation of early stage ovarian follicles can be achieved, in vitro procedures for ovarian follicle culture, development, ovulation and fertilisation after cryopreservation would be needed. The aim of the present study was to develop an in vitro culture method for early stage zebrafish ovarian follicles for use after their cryopreservation. Procedures for in vitro culture of stage I (primary growth) and stage II (cortical alveolus) ovarian follicles were developed. The effects of concentration of L-15 medium, pH and the concentration of human chorionic gonadotropin (hCG) and activin A were studied. The results demonstrated that early stage zebrafish ovarian follicles can be cultured in vitro for 24 h, stage I and II ovarian follicles can grow to the sizes of early stage II and stage III ovarian follicles after hCG treatment. The method developed here is effective for assessing early stage zebrafish ovarian follicles growth competence in vitro. The results from the present study indicated that in vitro culture is the most reliable method for assessing ovarian follicle viability when compared with vital dye staining methods.

Use of an adenosine triphosphate assay, and simultaneous staining with fluorescein diacetate and propidium iodide, to evaluate the effects of cryoprotectants on hard coral ( Echinopora spp.) oocytes

The objective was to examine the effects of cryoprotectants on oocytes of hard corals ( Echinopora spp.) to obtain basic knowledge for cryopreservation procedures. Oocytes were exposed to various concentrations of cryoprotectants (0.25 to 5.0 M) for 20 min at room temperature (25 °C). Two tests were used to assess ovarian follicle viability: fluorescein diacetate (FDA) + propidium iodide (PI) staining, and adenosine triphosphate (ATP) assay. Both FDA + PI staining and ATP assay indicated that cryoprotectant toxicity to oocytes increased in the order methanol, dimethyl sulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG). The no observed effect concentrations for Echinopora spp. oocytes were 1.0, 0.5, 0.25, and 0.25 M for methanol, DMSO, PG, and EG, respectively, when assessed with FDA + PI. The ATP assay was more sensitive than FDA + PI staining (P < 0.05). Oocyte viability after 1.0 M methanol, DMSO, EG, or PG treatment for 20 min at room temperature assessed with FDA + PI tests and ATP assay were 88.9 ± 3.1% and 72.2 ± 4.4%, 66.2 ± 5.0% and 23.2 ± 4.9%, 58.9 ± 5.4% and 1.1 ± 0.7%, and 49.1 ± 5.1% and 0.9 ± 0.5%, respectively. We inferred that the ATP assay was a valuable measure of cellular injury after cryoprotectant incubation. The results of this study provided a basis for development of protocols to cryopreserve coral oocytes.

Development of cryopreservation protocols for early stage zebrafish ( Danio rerio) ovarian follicles using controlled slow cooling

Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, cryopreservation of early stage zebrafish ovarian follicles was studied for the first time using controlled slow freezing. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. Stages I and II ovarian follicles were frozen in 4 M methanol and 3 M DMSO in either L-15 medium or KCl buffer. Ovarian follicle viability was assessed using trypan blue, FDA + PI staining and ADP/ATP assay. The results showed that KCl buffer was more beneficial than L-15 medium, methanol was more effective than DMSO, optimum cooling rates were 2–4 °C/min, stepwise removal of cryoprotectant improved ovarian follicle viability significantly and stage I ovarian follicles were more sensitive to freezing. The results also showed that FDA + PI staining and ADP/ATP assay were more sensitive than TB staining. The highest follicle viabilities after post-thaw incubation for 2 h obtained with FDA + PI staining were 50.7 ± 4.0% although ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell damage. Studies are currently being carried out on in vitro maturation of these cryopreserved ovarian follicles.

Studies on chilling sensitivity of early stage zebrafish ( Danio rerio) ovarian follicles

Cryopreservation of fish gametes is of great importance in aquaculture, conservation and human genomic research. The creation of gamete cryobanks allows the storage of genetic material of targeted species for almost unlimited time periods. Cryopreservation has been successfully applied to fish sperm of many species, but there has been no success with fish embryos and oocytes. One of the obstacles to fish oocyte cryopreservation is their high chilling sensitivity and especially at subzero temperatures. Although studies on late stage oocyte cryopreservation has been carried out, there have been no reported studies on cryopreservation of early stage ovarian follicles. The aim of this study is to investigate the chilling sensitivity of early stage zebrafish ovarian follicles before developing protocols for their cryopreservation. Experiments were conducted with stage I (primary growth), stage II (cortical alveolus) and stage III (vetillogenesis) ovarian follicles, which were chilled in KCl buffer and L-15 medium for up to 144 h at −1 °C in a low temperature bath. Ovarian follicles were also exposed to 2 M methanol or 2 M DMSO in L-15 medium for up to 168 h at −1 and −5 °C, respectively. Control follicles were kept at 28 °C. Ovarian follicle viability was assessed using trypan blue staining. The results showed that stage I and II ovarian follicles are less sensitive to chilling than stage III follicles. These results were also confirmed following in vitro maturation of the chilled ovarian follicles. The results also showed that L-15 medium is more beneficial than KCl buffer for ovarian follicles at all stages. The presence of both methanol and DMSO reduced chilling sensitivity of ovarian follicles at all stages with methanol being the most effective. The study indicated that stage I and II follicles are less sensitive to chilling than stage III follicles, and that early stage zebrafish ovarian follicles may be better candidates for cryopreservation.

Ultrastructural changes in ovarian follicles of monkeys administered hexachlorobenzene

Abstract Objective To test hexachlorobenzene (hcb), an environmental pollutant, for its potential toxicity to the ovary. Design Nonhuman primates were orally administered the pollutant at doses between 0.01 and 10 mg of hcb/kg of body weight to test viability of ovarian follicles. At the end of dosing period, the monkeys received a compound that contained follicle-stimulating and luteinizing hormones to stimulate development of follicles that would be examined by electron microscopy. Animals Twenty, 6- to 13-year-old cynomolgus monkeys were randomly assigned to 5 groups. Procedure The hcb, in concentrations of 0.01, 0.1, 1.0, and 10.0 mg/kg, was orally administered with glucose in gelatin capsules for 13 weeks. Monkeys receiving capsules containing glucose only served as controls. After the 13th week, monkeys were given a compound that contained follicle-stimulating and luteinizing hormones daily during days 2 to 7 from the start of menses. On day 8 of the menstrual cycle, 5,000 IU of human chorionic gonadotropin was administered, and 35 to 38 hours later, 1 ovary from each monkey was obtained during laparotomy. Approximately 1-mm cubes of tissue from each ovary were harvested, fixed by immersion in buffered 2% glutaraldehyde, and processed for transmission electron microscopy. Results Ultrastructure of ovarian follicles was altered in the monkeys administered hcb. Lesions were observed in the follicles from monkeys given the lowest concentrations of hcb, and comprised condensed mitochondria in the developing ova and follicular cells that contained nuclei with deep indentations and abnormal accumulation of cytoplasmic lipid droplets. Alterations, such as herniation of the ooplasm, degeneration of the follicular cells, and appearance of abnormal spaces between follicular cells were observed in the follicles from monkeys of the 0.1 to 1.0 mg of hcb/kg dosage groups. The most relevant alterations were seen in the mitochondrion, an organelle that appeared to be most sensitive to the compound. Mitochondria were condensed, with abnormal intracristal spaces in the lower-dosage groups, and were markedly degenerated in the 10 mg/kg group. The effects of hcb were dose-related. Conclusion The hcb is a reproductive system toxicant, and its damaging effects may be a result of augmentation of lipid peroxidation, especially in the primary follicle, which abnormally affects cellular membranes and thus, impairs their permeability.