Apoptosis In Ovarian Tissue Research Articles

Assessment of Apoptosis-Related Genes in Autografted/ Vitrified Ovarian Tissue in Mice

Background: One of the new emerging techniques to preserve reproductive potential of cancer patients is cryopreservation of ovarian fragments prior to medical treatment and their re-transplantation after healing. In order to investigate and compare apoptosis in ovarian tissue, the current study has done while using different vitrification solutions. Methods: The ovaries of Balb/c mice were removed from the body. R ovaries autotransplanted and L ovaries vitrified and thawed in different solutions. Apoptosis-related genes (Bax, Bcl-2, Bid, P53) assessed by RT-PCR. Results: The results have shown that Bax and P53 expression showed significant changes in DCV3, but in DCV2 only p53 expression decreased considerably in vitrified-thawed group. Conclusion: We concluded that cryoprotectant solution is important for preserving tissue in vitrification process and in our study DCV2 showed better results than DCV3.

Evaluation of the therapeutic effects of arbutin on cisplatin‐induced ovarian toxicity in rats through endoplasmic reticulum stress and Nrf2 pathway

Arbutin (ARB) is a glycosylated hydroquinone with potent antioxidant effects. Although cisplatin (CP) is widely used in chemotherapy, its toxicity in healthy tissues, including ovotoxicity, is an insurmountable problem. This study aimed to evaluate the therapeutic effect of ARB against CP-related ovototoxicity by including nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in rats for the first time. Rats treated one dose of CP (5 mg/kg) on the first day, followed by ARB (5 and 10 mg/kg) for three days. Serum reproductive hormone levels were determined using ELISA kits. Oxidative stress (OS), inflammation, endoplasmic reticulum stress (ERS) and apoptosis markers in ovarian tissue were also determined colorimetrically. In addition, how CP affects Nrf2 pathway and the effect of ARB on this situation were also addressed. ARB treatment reduced the levels of markers of OS, inflammation, ERS and apoptosis in ovarian tissue of CP-stimulated animals. ARB regenerated the depleted antioxidant system by triggering Nrf2 pathway in the ovarian tissues of animals stimulated by CP. Histological findings also supported the therapeutic efficacy of ARB. The results indicate that ARB may have therapeutic effects against CP-induced reproductive toxicity with its Nrf2 activator potential. ARB should be tested in more extensive studies as a new generation chemopreventive candidate molecule.

The role of oxidative stress in chemotherapy-induced gonadotoxicity in a rat model, and the protective effects of Nigella Sativa oil on oxidative stress, the anti-Müllerian hormone level, and apoptosis.

This study aimed to determine the role of oxidative stress (OS) in carboplatin-induced gonadotoxicity and whether Nigella Sativa oil (NSO), an herbal antioxidant, has a protective effect on ovarian apoptosis, OS, and the anti-Müllerian hormone (AMH) level in a rat model. The study included 24 adult female rats that were divided into 4 treatment groups. Group A saline + saline (sham group); group B: NSO + saline; group C: saline + carboplatin; group D: NSO + carboplatin. Saline, NSO, and carboplatin were administered intraperitoneally 24 and/or 48 h before sacrification as 4 mL/kg, 4 mL/kg, and 80 mg/kg, respectively. Apoptosis, OS parameters, and AMH were measured. Oxidant levels and apoptosis were higher, whereas AMH and the antioxidants were lower in group C than in group A. Apoptosis, OS parameters, and AMH levels were negatively affected by chemotherapy (CTx) in group C whilst improvement in those parameters was observed in group D following NSO pretreatment. The levels of apoptosis and malondialdehyde (MDA), an OS parameter, in group D were lower than in group C as they declined from 34.3% to 8.65% (p = 0.002) and from 199.4 nmol/g tissue to 136.4 nmol/g tissue (p = 0.002), respectively. However, the slight increase in AMH level from 2.7 ng/mL to 3.5 ng/mL due to the NSO effect was not significant between groups C and D. The present findings show that carboplatin has adverse effects on AMH, ovarian tissue apoptosis, and OS parameters. NSO pretreatment might protect ovarian tissue and decrease CTx-induced ovarian injury by decreasing OS and apoptosis, but the protective effect of NSO on AMH is limited.

The ameliorative effect of CangFu Daotan Decoction on polycystic ovary syndrome of rodent model is associated with m6A methylation and Wnt/β-catenin pathway

Objective: This study investigates the effects of CangFu Daotan Decoction (CDD) on m6A methylation and Wnt/β-catenin pathway in rats with polycystic ovary syndrome (PCOS).Methods: The PCOS rat model was established by letrozole gavage. The rats were fed high-fat chow, and their body weight and blood glucose were recorded. The expressions of follicle-stimulating hormone(FSH), luteinizing hormone(LH), and testosterone(T) were quantified by ELISA. Chemical components in CDD were analyzed using UPLC-Q/TOF-MS. Based on network pharmacology methods, related targets of CDD on PCOS were screened. An enrichment analysis according to Tokyo Encyclopedia of Genes and Genomes (KEGG) was conducted to predict the potential signaling pathway of CDD in PCOS. The expressions of Wnt-1, β-Catenin, GSK-3β, C-MYC, Beclin1, LC3II, Bax, and PCNA were detected by western blotting. The expressions of Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, and Ythdf2 were monitored by RT-PCR. The expressions of Mettl3, Fto, and Ythdf1 were detected by western blotting.Results: Letrozole and a high-fat diet induced ovarian dysfunction in rats, which was attenuated by CDD. CDD decreased blood glucose, LH, and T concentrations and increased FSH expression in PCOS. After removing duplicates, a total of 71 compounds were identified by UHPLC-Q/TOF-MS, among which terpenoids and flavonoids account for the main proportion. The clustering analysis showed that the active site of CDD might be in the Wnt/β-catenin pathway. CDD decreased the expressions of Wnt-1, β-Catenin, GSK-3β, C-MYC, Beclin1, LC3II, and Bax and increased PCNA expression in the ovarian tissue of PCOS rats. CDD decreased the m6A gene expressions of Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, and Ythdf2 in peripheral blood and ovarian tissue of PCOS rats. CDD reduced the m6A proteins expressions of Mettl3, Fto, and Ythdf1 in the ovarian tissue of PCOS rats.Conclusion: CDD can regulate m6A modification and inhibit the Wnt/β-catenin signaling pathway in PCOS rats, thereby reducing body weight, lowering blood glucose levels, improving sex hormone disorders, and decreasing autophagy and apoptosis in ovarian tissue to promote the recovery of ovarian morphology.

Evaluating effect of acrylamide and ascorbic acid on oxidative stress and apoptosis in ovarian tissue of wistar rat

Acrylamide is one of the undesirable compounds created in food, which leads to oxidative stress. Under normal conditions of the body, there is a balance between the production and elimination of free radicals. Imbalance in this process causes oxidative stress. Ascorbic acid has antioxidant properties and can be used to prevent oxidative stress damage. In this study, we considered the effect of acrylamide as a substance that causes oxidative stress and ascorbic acid as an antioxidant. In this experiment, 28 rats were separated into 4 groups (n = 7). Mice were fed orally with acrylamide (10 mg/kg), ascorbic acid (200 mg/kg), both acrylamide and ascorbic acid, and water as AR, AA, AR&AA, and control groups, respectively. Blood and ovarian tissue samples were collected after the final feeding and anesthesia for hematological tests. Blood cells, anti-oxidation status and relative expression of BAX (Bcl-2 Associated X-protein), BCL-2 (B-cell lymphoma-2), Folliculogenesis Specific BHLH Transcription Factor (FIGLA), Follicle Stimulating Hormone Receptor (FSHR), and Klotho (KL) genes were assessed and compared between the study groups. Despite no change in the levels of other factors, white blood cells were significantly different between all groups. The total oxidant and antioxidant index had significant changes in the AR group compared to controls. Glutathione Peroxidase showed the least concentration in the AR group than others although this change was not significant. Gene expression of BAX, BCL-2, FIGLA, FSHR, and KL genes was not significant between the study groups (P > 0.05). The most ratio of BAX to BCL-2 belonged to the AR group compared to other groups. In conclusion, AR probably induces ovarian dysfunction by increasing the proportions of apoptosis-related genes, and the usage of antioxidants like AA can minimize its hazardous effects. However, more research is needed to uncover effective ways to limit AR exposure.

Stem cell–secreted factor therapy regenerates the ovarian niche and rescues follicles

Ovarian senescence is a normal age-associated phenomenon, but increasingly younger women are affected by diminished ovarian reserves or premature ovarian insufficiency. There is an urgent need for developing therapies to improve ovarian function in these patients. In this context, previous studies suggest that stem cell-secreted factors could have regenerative properties in the ovaries. This study aimed to test the ability of various human plasma sources, enriched in stem cell-secreted factors, and the mechanisms behind their regenerative properties, to repair ovarian damage and to promote follicular development. In the first phase, the effects of human plasma enriched in bone marrow stem cell soluble factors by granulocyte colony-stimulating factor mobilization, umbilical cord blood plasma, and their activated forms on ovarian niche, follicle development, and breeding performance were assessed in mouse models of chemotherapy-induced ovarian damage (n=7 per group). In addition, the proteomic profile of each plasma was analyzed to find putative proteins and mechanism involved in their regenerative properties in ovarian tissue. In the second phase, the most effective plasma treatment was validated in human ovarian cortex xenografted in immunodeficient mice (n=4 per group). Infusion of human plasma enriched bone marrow stem cell soluble factors by granulocyte colony-stimulating factor mobilization or of umbilical cord blood plasma-induced varying degrees of microvessel formation and cell proliferation and reduced apoptosis in ovarian tissue to rescue follicular development and fertility in mouse models of ovarian damage. Plasma activation enhanced these effects. Activated granulocyte colony-stimulating factor plasma was the most potent inducing ovarian rescue in both mice and human ovaries, and proteomic analysis indicated that its effects may be mediated by soluble factors related to cell cycle and apoptosis, gene expression, signal transduction, cell communication, response to stress, and DNA repair of double-strand breaks, the most common form of age-induced damage in oocytes. Our findings suggested that stem cell-secreted factors present in both granulocyte colony-stimulating factor-mobilized and umbilical cord blood plasma could be an effective treatment for increasing the reproductive outcomes in women with impaired ovarian function owing to several causes. The activated granulocyte colony-stimulating factor plasma, which is already enriched in both stem cell-secreted factors and platelet-enclosed growth factors, seems to be the most promising treatment because of its most potent restorative effects on the ovary together with the autologous source.

Ability of human umbilical cord mesenchymal stem cells to repair chemotherapy-induced premature ovarian failure.

Premature ovarian insufficiency (POI) and premature ovarian failure (POF) have become one of the major problems threatening women of childbearing age. Studies have shown that stem cells transplanted from bone marrow, umbilical cord, peripheral blood and amniotic fluid can migrate and proliferate to the ovary, promote ovarian function repair, increase the number of follicles and granulosa cells at all levels of ovary, improve endocrine function, and can differentiate into oocytes in specific ovarian environment to restore fertility to some extent. To study the ability of human umbilical cord mesenchymal stem cells (hUCMSCs) to repair ovarian injury after chemotherapy. A total of 110 female BALB/c mice (aged 7-8 wk old) with body masses of 16.0-20.0 g were selected. The mice were fed until 12 wk of age, and cyclophosphamide was administered by intraperitoneal injection for 14 consecutive days to induce premature ovarian failure in mice. Seventy-five mice with estrous cycle disorder were screened and randomly divided into 3 groups according to their body weight: model group, positive control group and hUCMSC group, and each group had 25 mice. Another 25 mice were used as negative controls. The mice in the hUCMSC group were injected with hUCMSCs in the tail vein, and the mice in the positive control group were given an oestradiol valerate solution and a medroxyprogesterone acetate solution in the tail vein. On the 1st, 15th, 30th, 45th, and 60th days after intravenous administration, vaginal smears were made to monitor the estrous cycles of the mice. The ovaries were weighed, and pathological sections were made to observe the morphology of the follicles; blood samples were collected to monitor the concentration of sex hormones (oestradiol and follicle-stimulating hormone). The estrous cycles of the model group mice were disrupted throughout the experiment. Mice in the hUCMSC group and the positive control group resumed normal estrous cycles. The ovarian weight of the model group mice continued to decline. The ovarian weight of the hUCMSC group mice and the positive control group mice decreased first and then gradually increased, and the ovarian weight of the hUCMSC group mice was heavier than that of the positive control group mice. The difference was statistically significant (P < 0.05). Compared with the negative control group, the model group experienced a decrease in oestradiol and an increase in follicle-stimulating hormone, and the difference was statistically significant (P < 0.05). Compared with the model group, the hUCMSC and positive control groups experienced a slight increase in oestradiol and a decrease in follicle-stimulating hormone; the difference was statistically significant (P < 0.05). The pathological examination revealed that the mouse ovaries from the model group were atrophied, the volume was reduced, the cortical and medullary structures were disordered, the number of follicles at all stages was significantly reduced, the number of atretic follicles increased, the number of primordial follicles and corpus luteum significantly decreased, and the corpus luteum had an irregular shape. Compared with those of the model group, the lesions of the hUCMSC and positive control groups significantly improved. hUCMSCs can repair ovarian tissue damaged by chemotherapy to a certain extent, can improve the degree of apoptosis in ovarian tissue, and can improve the endocrine function of mouse ovaries.

The production of nitric oxide in the coeliac ganglion modulates the effect of cholinergic neurotransmission on the rat ovary during the preovulatory period

The aim of the present work was to investigate whether the nitric oxide produced by the nitric oxide/nitric oxide synthase (NO/NOS) system present in the coeliac ganglion modulates the effects of cholinergic innervation on oxidative status, steroidogenesis and apoptotic mechanisms that take place in the rat ovary during the first proestrous. An ex vivo Coeliac Ganglion- Superior Ovarian Nerve- Ovary (CG-SON-O) system was used. Cholinergic stimulation of the CG was achieved by 10−6 M Acetylcholine (Ach). Furthermore, 400 μM Aminoguanidine (AG) – an inhibitor of inducible-NOS was added in the CG compartment in absence and presence of Ach. It was found that Ach in the CG compartment promotes apoptosis in ovarian tissue, probably due to the oxidative stress generated. AG in the CG compartment decreases the release of NO and progesterone, and increases the release of estradiol from the ovary. The CG co-treatment with Ach and AG counteracts the effects of the ganglionic cholinergic agonist on ovarian oxidative stress, increases hormone production and decreases Fas mRNA expression. These results suggest that NO is an endogenous modulator of cholinergic neurotransmission in CG, with implication in ovarian steroidogenesis and the apoptotic mechanisms that take place in the ovary during the preovulatory period in rats.

PROTECTIVE EFFECTS OF CISTANCHES HERBA AQUEOUS EXTRACT ON CISPLATIN-INDUCED PREMATURE OVARIAN FAILURE IN MICE

Background: Chemotherapeutic treatment of premenopausal women has been linked to premature ovarian failure (POF). Cistanches Herba (CH) is a commonly used male impotence and female infertility treatment in China; however, whether CH protects ovaries from chemotherapeutic drug-induced POF remains unclear. In this study, we investigated the protective effects of CH in a mouse model of chemotherapeutic drug-induced POF.&#x0D; Materials and Methods: We administered low- and high-concentration CH to cisplatin-induced POF mice for 2 weeks and determined body and ovarian weights, as well as serum follicle-stimulating hormone (FSH) and estradiol concentrations, to evaluate ovarian function. In addition, we evaluated the protective mechanisms of CH by detecting the levels of apoptosis-related proteins and evaluating markers of mitochondrial function.&#x0D; Results: In POF mice, we observed reduced body and ovarian weights; elevated serum FSH and attenuated estradiol concentrations; apoptosis of ovarian granulosa with concomitant changes in apoptosis-related proteins (including caspase-3, poly adenosine diphosphate-ribose polymerase, Bcl-2, and Bax); and mitochondrial dysfunction, such as a reduction in mitochondrial numbers, destruction of ultrastructural morphology, decrease in ATPase activity, and decreases in mitochondrial membrane potential and mitofusin-2 (a mitochondria dynamin-like GTPase). Significantly, CH reversed, to an extent, functional and morphologic injuries and ovarian tissue apoptosis by up-regulating the level of Mfn2 and the ratio of Bcl-2/Bax. Furthermore, CH reduced cisplatin-induced mitochondrial dysfunction in ovarian tissues.&#x0D; Conclusion: The present findings showed that CH inhibited cisplatin-induced POF through interactions between Mfn2 and Bcl-2/Bax proteins and, possibly, by up-regulation of Mfn2 expression. Ultimately, CH protects ovarian tissues from cisplatin-induced apoptosis.

Effect of Kineret® on ovarian ischemia reperfusion injury in a rat model.

To investigate the effect of Kineret® on ischemia reperfusion (IR) injury in rat ovaries. Rats were divided into four groups: ovarian IR (IRG); 50 mg/kg Kineret® + ovarian IR (KIR-50); 100 mg/kg Kineret® + ovarian IR (KIR-100); and sham operation (SOC). KIR-50 (n = 10) and KIR-100 (n = 10) groups received an intraperitoneal injection of Kineret® at doses of 50 and 100 mg/kg, respectively. IRG and SOC (n = 10) rat groups were given distilled water as solvent using the same method. The results were compared between the groups. In rats in which IR occurred, oxidant parameters, such as malondialdehyde (MDA) and myeloperoxidase (MPO), were increased, the level of proinflammatory interleukin 1 beta (IL-1β) was elevated and total glutathione (tGSH) as an antioxidant was decreased in the ovarian tissues. Administration of Kineret® at a dose of 100 mg/kg inhibited the increase of MDA, MOP and IL-1β and a decrease in tGSH caused by IR more significantly than administration of Kineret® at a dose of 50 mg/kg. In addition, 100 mg/kg Kineret® significantly decreased severe hemorrhage, degeneration and inflammatory signs in the follicular cells, caused by IR. Kineret® at 100 mg/kg markedly ameliorated increased apoptosis in ovarian tissue with IR more significantly than 50 mg/kg kineret. Our findings indicate that Kineret® might be useful in clinical practice for the treatment of damage that may occur as a result of ovarian torsion.

Follicular Histomorphometry and Evaluation of Ovarian Apoptosis in Queens of Different Age Groups

Background: In humans and bitchs the age is another factor that may affect the size of ovarian structures, verifying alterations in the quality of the pool and size of follicular structures, which can compromise the use of these structures for in vitro maturation. There are no reports correlating the morphometric characteristics of the follicles and ovarian apoptosis at different ages in cats. The aim of this study was to evaluate the histomorphometric parameters of follicular growth and the relationship with the occurrence of apoptosis in ovarian tissue of young, adult and senile queens.Materials, Methods &amp; Results: Eighteen domestic queens, multiparous, of different breeds and age groups were used in this study and divided into three groups according to their ages: five months to one year - young; (7.8 ± 1.0 months); one to six years - adults (2.8 ± 0.5 years); and more than six years - senile (8.0 ± 0.9 years). Vaginal cytology was performed in order to characterize the estrous phase associated with plasma concentrations of progesterone. The morphology and percentage of the vaginal epithelium cells were evaluated and queens were classified into estrous and non-estrus and plasma concentrations of progesterone were determined. Ovarian samples were collected after ovariohysterectomy to routine histological processin and all follicles were counted and categorized into two groups, non-atresic and atresic. The mean follicular and oocyte diameters were calculated between the measurement of the largest diameter and perpendicular diameter. The relationship between follicle and oocyte were determined using the measurements of diameter, area and perimeter. The apoptotic cells were detected and cells were considered positive when TUNEL reaction was detected. The morphometric index of 1039 follicles were evaluated. Primordial follicles in young animals showed larger diameter, follicular area and perimeter than the structures of adult queens, as well as the unilaminar primary follicles of the same group were larger compared with senile animals (P &lt; 0.05). Comparing adult and younger queens, the first showed a significant decrease of oocyte diameter in primary and unilaminar primary follicles, as well as oocyte area and perimeter (P &lt; 0.05). The values for follicular diameter, oocyte area and perimeter for multilaminar primary, secondary and pre-ovulatory structures did not present statistical differences between the groups (P &gt; 0.05). For the pre-ovulatory follicles there was no positive correlation between the oocyte growths regarding the follicles (P &gt; 0.05). Only in senile animals positive markers for apoptosis were identified in nuclei of primordial follicles. No significant differences concerning the number of follicles and Tunel positive cells were observed between groups (P &gt; 0.05).Discussion: Considering the importance of this study for greater knowledge in the basic aspects for reproductive biotechnologies, we verified that secondary follicles showed the largest diameters and younger animals the largest values for diameter, area and perimeter, suggesting that this age group could be ideal for the use and manipulation of oocytes. The process of follicular atresia is characterized by the occurrence of apoptosis, or programmed cell death when the organism begins to efficiently eliminate dysfunctional cells. The study of follicular apoptosis in small animals, especially in cats, is very important for the development of reproduction biotechnologies. Phenomenon of apoptosis showed no relationship with age in queens, occurring in a physiological, continuous and proportionate manner considering the number of nondominant follicles involved in each estrous cycle.

The effect of calorie restriction on the presence of apoptotic ovarian cells in normal wild type mice and low-plasma-IGF-1 Laron dwarf mice

BackgroundIt is known that caloric restriction extends lifespan and can minimize age-related dysfunction of the reproductive system. We became interested in how caloric restriction influences apoptosis, which is a crucial process that maintains ovarian cell homeostasis.MethodsWe examined ovarian cells in: 2.5-year-old wild type mice on caloric restriction (CR) or fed ad libitum (AL) and Laron dwarf mice (GHR-KO) at the same ages on CR or fed AL. Apoptosis was assessed by histochemical analysis on paraffin sections of ovarian tissue.ResultsMorphological and histochemical analysis revealed that CR improved reproductive potential in 2.5-year-old WT littermates and GHR-KO female mice, as indicated by the increased number of ovarian follicles. The level of apoptosis in ovarian tissue was higher in WT mice on a CR diet compared with WT mice on the AL diet. In GHR-KO mice, the level of apoptosis in ovaries was similar for mice on CR and on AL diets and bigger than in WT mice on CR.ConclusionsMorphological and histochemical analysis revealed a younger biological age of the ovaries in 2-year-old WT littermates and GHR-KO female mice on CR compared with animals fed AL.

Histological evaluation and in situ localization of apoptosis in fresh and cryopreserved ovarian tissue

Objective: Although apoptosis has significant impact on ovarian homeostasis and reproductive function, little is known about 1) the dynamics of this process; 2) the feasibility of using combined morphology and terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) for apoptosis detection and 3) the impact of cryopreservation on this process. We conducted this investigation to study these issues, using a porcine animal model.Design: Eight non-pregnant, adult, female farm pigs were utilized for this study. The bilateral oophorectomy was performed at the Cleveland Clinic Foundation Biological Resources Unit in accordance with their standard operating procedures.Materials/Methods: The ovarian tissues were divided into two parts; one part was immediately fixed while the other was cryopreserved using standard protocol for ovarian cortical strips. The cryopreserved specimens were subsequently thawed and then fixed in Bouin solution. All the specimens were sectioned, fixed and stained with H&E. Ovarian follicles were histologically categorized as healthy and atretic and evaluated for the presence of apoptosis. In situ examination of apoptosis was performed using TUNEL assay.Results: 1) Apoptosis was found in the atretic, but not in the healthy follicles; 2) the nuclei of the granulosa cells, but not those of theca or stromal cells, were TUNEL positive; 3) some cells with histological features of necrosis and apoptosis were TUNEL negative, and 4) the distribution of apoptosis was not different between cryopreserved tissue and freshly fixed tissue.Conclusions: 1) The presence of apoptosis in the atretic follicles suggests its involvement in follicular atresia; 2) necrosis may represent alternative pathway for granulosa cell death, and 3) combined histology and TUNEL assay is useful method in detecting apoptosis, and finally 4) cryopreservation does not affect the pattern of apoptosis in ovarian tissue.Supported by: None. Objective: Although apoptosis has significant impact on ovarian homeostasis and reproductive function, little is known about 1) the dynamics of this process; 2) the feasibility of using combined morphology and terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) for apoptosis detection and 3) the impact of cryopreservation on this process. We conducted this investigation to study these issues, using a porcine animal model. Design: Eight non-pregnant, adult, female farm pigs were utilized for this study. The bilateral oophorectomy was performed at the Cleveland Clinic Foundation Biological Resources Unit in accordance with their standard operating procedures. Materials/Methods: The ovarian tissues were divided into two parts; one part was immediately fixed while the other was cryopreserved using standard protocol for ovarian cortical strips. The cryopreserved specimens were subsequently thawed and then fixed in Bouin solution. All the specimens were sectioned, fixed and stained with H&E. Ovarian follicles were histologically categorized as healthy and atretic and evaluated for the presence of apoptosis. In situ examination of apoptosis was performed using TUNEL assay. Results: 1) Apoptosis was found in the atretic, but not in the healthy follicles; 2) the nuclei of the granulosa cells, but not those of theca or stromal cells, were TUNEL positive; 3) some cells with histological features of necrosis and apoptosis were TUNEL negative, and 4) the distribution of apoptosis was not different between cryopreserved tissue and freshly fixed tissue. Conclusions: 1) The presence of apoptosis in the atretic follicles suggests its involvement in follicular atresia; 2) necrosis may represent alternative pathway for granulosa cell death, and 3) combined histology and TUNEL assay is useful method in detecting apoptosis, and finally 4) cryopreservation does not affect the pattern of apoptosis in ovarian tissue. Supported by: None.

Cell death and its suppression in human ovarian tissue culture.

In women with premature ovarian failure, fertility may be preserved by ovarian tissue culture in vitro. However, techniques for tissue culture and follicle maturation have remained suboptimal. Our aim was to characterize ovarian tissue degeneration in cultures and to establish a model for cell death research in cultured ovarian tissue. Precise knowledge on the process resulting in cell death in cultured ovarian tissue will ultimately facilitate work aimed at improving long-term culture conditions. Ovarian tissue apoptosis was studied in a serum-free culture model in which nuclear DNA fragmentation was shown to occur within 24 h of the start of the culture. Activation of caspase-3 was detected in some stromal cells and a few oocytes. Since not all of the tissue exhibited signs of apoptosis and since DNA fragmentation increased over time, the tissue probably gradually dies by apoptosis. The antioxidant N-acetyl-L-cysteine (NAC; 25, 50 and 100 mmol/l) was found to inhibit this apoptosis. Thus, apoptosis appears to play a critical role in the degeneration of human ovarian cortical tissue cultures, and this cell death can be suppressed by NAC. The present tissue culture model can be used for identifying components capable of inhibiting cell death in vitro.