Apoptosis In Antral Follicles Research Articles

Local application of low level laser therapy in mice ameliorates ovarian damage induced by cyclophosphamide

The aim of the present study is to assess whether low level laser therapy (LLLT) can protect ovaries from chemotherapy-induced gonadotoxicity using a mice model of premature ovarian failure induced by cyclophosphamide (CTX). LLLT (64 J/cm2) increased the number of antral follicles whilst decreasing the number of atretic follicles compared to CTX alone. LLLT increased the number of primordial follicles compared with those in the CTX group but they did not differ from those in the control group. LLLT treatment increased the number of AMH-positive follicles compared to CTX alone. LLLT application increased ovarian weight, serum progesterone concentration and P450scc protein levels compared to CTX alone. LLLT reduced the apoptosis in antral follicles and the BAX/BCL-2 ratio compared to CTX alone. Vascular morphology, analysed by CD31 and α-SMA immunostaining, was restored in LLLT-treated ovaries compared to CTX alone. In conclusion, application of LLLT prior to CTX might serve as a promising and novel protocol to preserve female fertility in cancer survivors.

Stresses on Female Mice Impair Oocyte Developmental Potential: Effects of Stress Severity and Duration on Oocytes at the Growing Follicle Stage.

Although previous studies found that 1-time acute stress applied during follicle maturation impaired oocyte competence, it is unknown whether repeated chronic stress, which is known to cause animal behavioral adaptation, would damage oocytes when applied during follicle growth. In this study, female mice were exposed to repeated restraint stress (RRS) or unpredictable stress (UPS) for different days before equine chorionic gonadotropin injection to initiate oocyte prematuration development and to observe effects of different stressors on oocytes in the growing follicles. The results showed that although oocyte pre- and postimplantation development was unaffected when mice were exposed to RRS or UPS once a day for 4 days, development was impaired when mice were exposed to RRS for 8 or more days or to UPS twice a day for 4 days (4 × 2). The 4 × 2 UPS caused more oxidative stress in oocytes and severer apoptosis in antral follicles than did the 4-day RRS. The RRS mice were stressed consistently from days 1 to 23 of restraint, and the stress that a mouse had 4 × 2 UPS was severer than that from 4-day RRS. The results suggest that (1) the degree that a stress damages oocytes is the product of duration × severity of the stress; (2) RRS impaired oocyte developmental potential through cumulative effects on growing follicles; and (3) preantral follicles were not as sensitive to stress as antral follicles were.

Polyinosinic-Polycytidylic Acid Perturbs Ovarian Functions Through Toll-Like Receptor 3-Mediated Tumor Necrosis Factor A Production in Female Mice.

Viral infections may perturb ovarian functions and female fertility. Mechanisms underlying viral perturbation of ovarian functions are incompletely understood. This study found that intraperitoneal injection of polyinosinic-polycytidylic acid [poly (I:C)] in female mice inhibits estradiol synthesis and induces ovarian granulosa cell apoptosis. Poly (I:C) is a synthetic viral double-stranded RNA analog, which induces innate antiviral responses mimicking a viral infection through activation of pattern recognition receptors, including toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated gene 5. Injection of poly (I:C) significantly induced granulosa cell apoptosis in antral follicles and reduced antral follicle numbers. These effects were significantly diminished in Tlr3 knockout or tumor necrosis factor-alpha (Tnfa) knockout mice. We demonstrated that poly (I:C) induced TNFA production at a relatively high level in wild-type mice compared with that in Tlr3 knockout mice. Notably, TNFA neutralizing antibody significantly reduced poly (I:C)-induced ovarian dysfunction. In vitro assays confirmed that TNFA inhibits estradiol synthesis and induces granulosa cell apoptosis. Results provide novel insights into the mechanisms by which a mimicked viral infection perturbs ovarian functions in mice.

64 Low-level laser therapy in thyroid autoimmune disease treatment

The aim of the present study is to assess whether low level laser therapy (LLLT) can protect ovaries from chemotherapy-induced gonadotoxicity using a mice model of premature ovarian failure induced by cyclophosphamide (CTX). LLLT (64 J/cm2) increased the number of antral follicles whilst decreasing the number of atretic follicles compared to CTX alone. LLLT increased the number of primordial follicles compared with those in the CTX group but they did not differ from those in the control group. LLLT treatment increased the number of AMH-positive follicles compared to CTX alone. LLLT application increased ovarian weight, serum progesterone concentration and P450scc protein levels compared to CTX alone. LLLT reduced the apoptosis in antral follicles and the BAX/BCL-2 ratio compared to CTX alone. Vascular morphology, analysed by CD31 and α-SMA immunostaining, was restored in LLLT-treated ovaries compared to CTX alone. In conclusion, application of LLLT prior to CTX might serve as a promising and novel protocol to preserve female fertility in cancer survivors.

Roles of reactive oxygen species and antioxidants in ovarian toxicity.

Proper functioning of the ovary is critical to maintain fertility and overall health, and ovarian function depends on the maintenance and normal development of ovarian follicles. This review presents evidence about the potential impact of oxidative stress on the well-being of primordial, growing and preovulatory follicles, as well as oocytes and early embryos, examining cell types and molecular targets. Limited data from genetically modified mouse models suggest that several antioxidant enzymes that protect cells from reactive oxygen species (ROS) may play important roles in follicular development and/or survival. Exposures to agents known to cause oxidative stress, such as gamma irradiation, chemotherapeutic drugs, or polycyclic aromatic hydrocarbons, induce rapid primordial follicle loss; however, the mechanistic role of ROS has received limited attention. In contrast, ROS may play an important role in the initiation of apoptosis in antral follicles. Depletion of glutathione leads to atresia of antral follicles in vivo and apoptosis of granulosa cells in cultured antral follicles. Chemicals, such as cyclophosphamide, dimethylbenzanthracene, and methoxychlor, increase proapoptotic signals, preceded by increased ROS and signs of oxidative stress, and cotreatment with antioxidants is protective. In oocytes, glutathione levels change rapidly during progression of meiosis and early embryonic development, and high oocyte glutathione at the time of fertilization is required for male pronucleus formation and for embryonic development to the blastocyst stage. Because current evidence suggests that oxidative stress can have significant negative impacts on female fertility and gamete health, dietary or pharmacological intervention may prove to be effective strategies to protect female fertility.

Induction of Apoptosis by 9,10-Dimethyl-1,2-Benzanthracene in Cultured Preovulatory Rat Follicles Is Preceded by a Rise in Reactive Oxygen Species and Is Prevented by Glutathione1

The polycyclic aromatic hydrocarbon (PAH) 9,10-dimethyl-1,2-benzanthracene (DMBA) destroys primordial, primary, and secondary ovarian follicles in rodents, but its effects on antral follicles have received limited attention. PAHs are metabolized to reactive species, some of which can undergo redox cycling to generate reactive oxygen species (ROS). We previously showed that ROS initiate apoptosis in preovulatory follicles cultured without gonadotropin support and that glutathione (GSH) depletion induces apoptosis in the presence of gonadotropins. In the present study, we tested the hypothesis that DMBA induces apoptosis in preovulatory follicles, which is mediated by ROS and prevented by GSH. Preovulatory follicles were isolated from ovaries of 25-day-old rats 48 h after the injection of 10 IU of eCG and were cultured with DMBA in the presence of FSH for 2 to 48 h. DMBA induced granulosa cell (GC) and theca cell (TC) apoptosis at 48 h, as judged by TUNEL and activated caspase-3 immunostaining. DMBA treatment also increased the numbers of GCs and TCs that immunostained for the proapoptotic protein BAX. Follicular ROS levels were significantly increased in DMBA-treated follicles at 12, 24, and 48 h. GSH supplementation protected against and GSH depletion enhanced the induction of apoptosis in GCs and TCs by DMBA. These findings suggest that GSH is a critical protective mechanism against DMBA-induced apoptosis in antral follicles and that ROS generation may mediate DMBA-induced GC apoptosis.

Apoptosis in the Antral Follicles of Swamp Buffalo and Cattle Ovary: TUNEL and Caspase‐3 Histochemistry

The present study was carried out to investigate the pattern of apoptosis in the healthy antral and atretic follicles of Philippine swamp buffaloes (BU) in comparison with Holstein-Friesian (HF) cows. Paraffin sections of healthy follicles and various stages of atretic follicles were stained using the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labelling (TUNEL) method to detect DNA fragmentation and cleaved caspase-3 antibody to detect cells committed to undergo apoptosis. Five equidistant areas of a follicle were counted for the presence of TUNEL- and caspase-3-positive cells. Healthy follicles of BU and HF contained no TUNEL-positive cells in the granulosa and theca layer but showed some caspase-3 positivity. The granulosa layer of advanced atretic follicles showed a significantly higher frequency of caspase-3 positivity than the healthy and early atretic follicles in both breeds. The frequency of caspase-3-positive cells of BU was significantly higher than HF in the granulosa layer of healthy, early atretic and advanced atretic follicles. In the theca interna layer, BU and HF showed a significantly lower and higher frequency of TUNEL-positive cells in the late atretic follicles compared with advanced atretic follicle, respectively. However, the frequency of caspase-3-positive cells of both BU and HF in the late atretic follicles was significantly higher than the advanced atretic follicles in the theca interna layer. These results indicate that caspase-3 and DNA fragmentation is involved in the buffalo ovarian apoptotic process.

Relationship Between Low-Molecular-Weight Insulin-Like Growth Factor-Binding Proteins, Caspase-3 Activity, and Oocyte Quality1

Bovine follicular atresia is associated with the apoptosis of granulosa cells and the subsequent loss of oocyte competence through the reduction of cellular contact (e.g., gap junctions). Several components of the insulin-like growth factor (IGF) system are thought to affect follicular atresia. Whereas the IGF-binding proteins (IGFBPs) are present in varying quantities throughout follicular development, IGFBP-5 appears to be present only during atresia, in parallel with its regulation in other tissue remodeling systems. However, to our knowledge, no connection has yet been made between atresia, low-molecular-weight IGFBP content, and oocyte quality in the bovine ovary. Caspases are actively involved in ovarian follicular atresia, and apoptosis in antral follicles is caspase-3-dependent. Hence, the aim of the present study was to investigate the use of these factors in the assessment of oocyte quality and developmental potential. Oocytes were aspirated, morphologically classified, and individually matured in vitro. The follicular fluid and granulosa cells of these follicles were analyzed for IGFBP profile and caspase-3 activity, respectively. A significant correlation was found between the presence of low-molecular-weight IGFBPs in bovine follicular fluid and caspase-3 activity of granulosa cells isolated from individual follicles. The highest percentage of development to the blastocyst stage was observed in oocytes from slightly atretic follicles. This group of oocytes contained an equal proportion of oocytes at grades 1-3. These data demonstrate that low-molecular-weight IGFBP profile is a more reliable method than the traditional morphological assessment of oocytes and can be used as an effective marker of developmentally competent oocytes. Importantly, these results have implications for the use of noninvasive follicular fluid markers in the selection of competent oocytes to improve outcomes of in vitro fertilization.

Vasoactive intestinal peptide-mediated suppression of apoptosis in the ovary: potential mechanisms of action and evidence of a conserved antiatretogenic role through evolution.

Vasoactive intestinal peptide (VIP)-containing nerve fibers are present in ovarian follicles at all stages of development, and VIP, acting primarily via the cAMP pathway, has been reported to modulate many aspects of granulosa cell function. Herein we examined the effects of VIP and its potential mechanisms of action on apoptosis in antral follicles isolated from ovaries of gonadotropin-primed immature rats and incubated in vitro under serum-free conditions. Additionally, the effects of VIP on apoptosis in isolated avian granulosa cells incubated in vitro were used as a comparative model system to determine whether the ability of VIP to modulate apoptosis in the ovary has been conserved through evolution. Genomic DNA extracted from incubated rat antral follicles exhibited extensive levels of internucleosomal DNA cleavage characteristic of cell death via apoptosis. Treatment of follicles with VIP (1-1000 nM) caused a dose-dependent reduction in the extent of apoptotic DNA breakdown, with a maximal effect achieved with 100 nM VIP. Provision of the adenylyl cyclase activator, forskolin (10 microM), mimicked the inhibitory effect of VIP on apoptosis and concomitantly increased intrafollicular cAMP accumulation, suggesting a role for the cAMP pathway in mediating the immediate actions of VIP on follicular cell survival. Moreover, treatment of rat antral follicles with insulin-like growth factor-binding protein 3 (3 micrograms/ml) partially antagonized the ability of VIP (100 nM) to suppress apoptosis, suggesting involvement of endogenous insulin-like growth factor I in mediating the downstream actions of VIP in incubated rat antral follicles. To further confirm that VIP and activation of the cAMP pathway prevented atresia, individual rat antral follicles incubated for 24 h in the absence or presence of VIP (100 nM) or forskolin (10 microM) were fixed, embedded, and sectioned for morphological analysis. Follicles fixed immediately after isolation from equine CG-primed rat ovaries were classified as morphologically healthy, consistent with the absence of biochemical evidence for apoptosis (e.g. oligonucleosomes) in this pool of follicles. Follicles incubated for 24 h in the absence of tropic support displayed extensive granulosa cell pyknosis and disorganization characteristic of follicles at a moderate stage of atresia. Inclusion of VIP or forskolin maintained the morphological health status of incubated follicles at that resembling healthy follicles fixed immediately after isolation from ovaries of equine CG-primed rats. Lastly, extensive levels of internucleosomal DNA cleavage were also detected in avian granulosa cells incubated for 6 h under serum-free conditions.(ABSTRACT TRUNCATED AT 400 WORDS)