Oocyte Degeneration Research Articles

Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro

Hydrogen peroxide (H2O2) is known to induce cell cycle arrest and apoptosis in various somatic cell types cultured in vitro. We hypothesize that this reactive oxygen species (ROS) could modulate cell cycle and induce morphological features characteristics of apoptosis in oocytes cultured in vitro. To test this hypothesis, immature and mature oocytes were cultured in medium containing various doses of H2O2 with or without caspase-3 inhibitor for various times. The treatment of H2O2 induced germinal vesicle break down (GVBD) in all immature oocytes followed by initiation of shrinkage. Some of immature oocytes (but not mature oocytes) also showed membrane blebbing. On the other hand, H2O2 treatment inhibited first polar body emission in mature oocytes just prior to initiation of shrinkage. The cytoplasmic granulation and fragmentation into apoptotic bodies were observed in mature oocytes during later stages of H2O2 treatment. The shrinkage was induced by H2O2 in a dose- and time-dependent manner in both immature and mature oocytes. Although, H2O2-induced degeneration was observed in both immature and mature oocytes after 2.0 hrs of treatment, immature oocytes were more susceptible to undergo quick shrinkage, membrane blebbing and degeneration. Co-addition of caspase-3 inhibitor prevented shrinkage and degeneration of both immature and mature oocytes except membrane blebbing that was observed at higher doses of H2O2 after 1.0 hr of culture. Treatment of H2O2 induced bax protein expression (3 times), DNA fragmentation and caspase-3 activity (2.5 times) in oocytes undergoing morphological apoptotic changes. These findings clearly suggest that H2O2 induced GVBD in immature oocytes, inhibited first polar body extrusion in mature oocytes prior to initiation of morphological changes characteristic of apoptosis such as shrinkage, membrane blebbing and cytoplasmic fragmentation prior to degeneration.

Expression of glutaredoxin (thioltransferase) in the rat ovary during the oestrous cycle and postnatal development

Glutaredoxins (Grxs) are low-molecular-weight proteins which participate in redox events in association with glutathione (GSH) and are involved in a variety of cellular processes. It is known that oxidative stress plays important physiological roles within the ovary. In the present study, we have prepared specific antibodies against rat Grx and have used them to localize the protein in the ovaries of rats during postnatal development and during the oestrous cycle, by immunohistochemical methods. We have also performed a quantitative analysis of Grx by ELISA and Western blotting in homogenates of whole ovaries of cycling and pseudopregnant rats. We have found a prominent presence of Grx in the oocytes and in corpora lutea (CL) during developmental and oestrous cycle changes. Grx was absent from the oocytes in the first days of postnatal life when marked oocyte degeneration takes place, but its presence was very conspicuous in the cytoplasm of oocytes in healthy and attretic follicles in rats from 10 days of age onward, independently of the day of oestrous cycle. Follicular cells were negative. Grx immunostaining in the CL was strong in infiltrating macrophages and in a population of steroidogenic cells that survived the apoptotic burst in regressing CL and in CL remnants, but was faint or absent in young CL of the current cycle and in CL during pseudopregnancy. Grx content and oxidoreductase activity in whole ovaries increased significantly during the phase transition from proestrous to oestrous along the cycle. These results support a role of Grx in the maintenance of functional oocytes and in luteal cells surviving the regression process, probably as a consequence of the demonstrated deglutathionylating function of this protein in an antioxidant and antiapoptotic context.

Gonadal development and diandric protogyny in two populations of Dascyllus reticulatus from Madang, Papua New Guinea

Two Dascyllus reticulatus populations from Madang, Papua New Guinea exhibited diandric protogyny. In both populations, gonads began as undifferentiated, and then developed oocytes in the primary growth stage and an ovarian lumen. From this ovarian state or from more developed ovaries containing oocytes beyond the primary‐growth stage, some gonads developed into testes. The first sign of testicular development was degeneration of oocytes, degeneration of oocytes in the primary growth stage in ovarian gonads and degeneration of oocytes of all growth stages present including the primary growth stage in ovaries, which was then followed by development of spermatogenic tissue. In both populations, most of the fish that had gonads with degenerating oocytes were smaller than the smallest mature females, indicating that development towards testes was mostly initiated in immature gonads containing only pre‐vitellogenic oocytes. On some occasions, however, females as large as other mature females also had gonads with degenerating oocytes, suggesting that development towards testes may have occurred in mature ovaries as well. This latter notion is further strengthened by the discovery of a fish having a gonad that contained both degenerating vitellogenic oocytes and developing spermatogenic tissue. Taken together, these results suggest that D. reticulatus can exhibit diandric protogyny, because testes in D. reticulatus developed from juvenile gonads as well as from mature ovaries.

Inhibitory effects of the androgenic gland on ovarian development in the mud crab Scylla paramamosain

Isolation and characterization of androgenic hormone in decapod crustaceans depend on an effective bioassay of its action. In the present study, the effect of androgenic gland on ovarian development in the mud crab Scylla paramamosain was investigated with a view to develop a bioassay for androgenic hormone. Ovarian regression with degeneration of oocytes occurred in some female crabs implanted with androgenic gland in vivo. In vitro incubation of ovarian tissues at secondary vitellogenesis in extract of androgenic gland resulted in a significant decrease in amino acid uptake by the tissues. We propose that this inhibitory effect could be established as an effective bioassay for the isolation of androgenic hormone in the mud crab.

225 IN VITRO MATURATION OF LION OOCYTES

The potential for rescuing immature oocytes from the ovaries of females of rare species of felids that die or undergo ovariohysterectomy has been reported in only two studies (Johnston et al. 1991 Biol. Reprod. 45, 898-906; Jewgenow et al. 1997 J. Reprod. Fertil. Suppl. 51, 33-39), in which oocytes were maturated in complex media (Eagle's MEM, M199, respectively). The domestic cat is used as a research model for endangered species but there may be some differences that perturb the adaptation of in vitro maturation (IVM), fertilization, or culture methods of lion oocytes compared to cat oocytes. Therefore, in the present study we evaluated the in vitro response of lion oocytes to our system for in vitro maturation of domestic cat oocytes in a simple medium (Merlo et al. 2005 Theriogenology 63, 2032-2039). A 14-year-old lioness, referred to our clinic because of pyometra, underwent ovariohysterectomy. After premedication with acepromazine 0.1 mg/kg i.m. (Prequillan; ATI, Bologne, Italy) and ketamine 5 mg/kg i.m. (Ketavet 100; Intervet, Boxmeer, The Netherlands), anesthesia was induced with ketamine 0.05 mg/kg i.v. and diazepam 0.02 mg/kg i.v. (Diazepam; Intervet) and maintained with isoflurane (Forane; Abbott, Rome, Italy) after intubation. Ovaries were removed and stored in saline solution at room temperature until collection of oocytes (within 1 h). A total of 53 small follicles (2-4 mm), five corpora lutea and a 15-mm follicle were present on the ovaries. Oocytes were collected by aspirating visible follicles of each ovary with a 21-ga needle connected to a vacuum pump (K-MAR-5100; Cook Australia, Brisbane, Australia) at -75 mmHg; then CL were removed and the ovaries were minced with a scalpel blade in a 60-mm petri dish containing HEPES-SOF (H-SOF) for recovery of additional oocytes. A total of 45 oocytes were recovered, of which 19 were degenerate (42.2%); of the remaining 26, 12 were fully surrounded by cumulus cells, 9 had only corona radiata, and 5 were denuded. Degenerate oocytes were discarded and all other oocytes were washed twice in H-SOF and matured in a 35 mm petri dish containing SOFaaBSA 5 mg/mL plus 0.1 IU of porcine FSH-LH (Pluset; Laboratorios Calier, Barcelona, Spain), 25 �L/mL insulin-transferrin-selenium (ITS) (Sigma, Madrid, Spain), 1.2 mm l-cysteine (Sigma), and 25 ng/mL epidermal growth factor (EGF) (Sigma) for 24 h in a humidified atmosphere of 5% CO2 in air at 38.5�C. After maturation, cumulus cells were removed by pipetting oocytes into a 0.25% trypsin solution for 2 min. Then, denuded oocytes were washed once in H-SOF plus 10% FCS to inactive trypsin and twice in H-SOF before being stained with Hoechst 33342 (10 �g in 10 mL PBS) for 30 min at room temperature. After washing in PBS, oocytes were observed using fluorescence microscopy to determine maturation rate. Oocytes in telophase I or metaphase II were considered mature. Of 26 oocytes, 19 (73.1%) were mature and 7 (26.9%) were at the GV stage. These results demonstrated that lion oocytes can undergo successful IVM at a frequency that is similar to that of cat oocytes cultured in the same system (76.9%, P > 0.05). Furthermore, the maturation rate obtained in a simple medium was higher or similar to those previously reported (mean: 22.9% and 69.7% respectively, by Johnston et al. 1991 and Jewgenow et al. 1997).

Evidence that estrogen regulates the sex change of honeycomb grouper (Epinephelus merra), a protogynous hermaphrodite fish

Circulating estradiol-17beta (E2) levels decrease precipitously during female to male (protogynous) sex change in fish. Whether this drop in E2 levels is a cause or consequence of sex change is still largely unknown. The present study treated adult female honeycomb groupers (Epinephelus merra) with aromatase inhibitor (AI, Fadrozole), either alone or in combination with E2, to investigate the role of estrogen in protogynous sex change. Control fish had ovaries undergoing active vitellogenesis; the gonads of AI-treated fish had already developed into testes, which produced sperm capable of fertilization. In contrast, co-treatment of fish with E2 completely blocked AI-induced sex reversal. AI treatment significantly reduced circulating levels of E2, whereas the addition of E2 to AI prevented the loss. The plasma androgen (testosterone and 11-ketotestosterone) levels were increased in the AI-treated fish, while the levels in the E2-supplemented fish were low compared to controls. Present results show that E2 plays an important role in maintaining female sex of hermaphrodite fishes, and that the inhibition of E2 synthesis causes oocyte degeneration leading to testicular differentiation in the ovary.

311 ULTRASTRUCTURAL ANALYSIS OF OVINE PREANTRAL FOLLICLES CULTURED IN THE PRESENCE OF INDOLE ACETIC ACID, EGF, AND FSH

Previous studies from our team demonstrated that ovine primordial follicles are successfully activated in vitro after culturing in medium supplemented with 40 ng/mL indole acetic acid (IAA); besides that, the addition of IAA and epidermal growth factor (EGF) or EGF and follicle stimulating hormone (FSH) to the culture media were the most effective treatments to sustain the health and viability of activated ovine primordial follicles during in vitro culture. In this work, follicular quality was assessed only by histological studies; there is a great need to evaluate ultrastructural changes occurring in primordial follicles during activation in vitro. The aim of this study was to investigate the ultrastructural characteristics of preantral follicles after culturing of cortical slices in media containing IAA, EGF, and FSH, alone and in combination. Ovaries (n = 8) from adult merino ewes were collected at local slaughterhouses. Small pieces of ovarian fragments were removed for transmission electron microscopy (TEM). These pieces of ovarian cortex were cultured in culture dishes containing 1 mL aliquots of culture medium at 39�C with 5% CO2 in air. The media used were: (T1) Minimum essential medium (MEM) supplemented with ITS (insulin 6.25 �g/mL, transferrin 6.25 �g/mL and selenium 6.25 ng/mL), 0.23 mM pyruvate, 2 mM glutamine, 2 mM hypoxantine, 1.25 mg/mL bovine serum albumin (BSA), and antibiotics (100 ��g/mL penicillin and 100 ��g/mL streptomycim) (MEM+, control medium); (T2) MEM+IAA (40 ng/mL); (T3) MEM+IAA + epidermal growth factor (EGF) (100 ng/mL; Sigma, St. Louis, MO, USA); or (T4) MEM+EGF+FSH (100 ng/mL). After 6 days of culture of cortex tissue fragments in media, ultrastructural analysis was performed on preantral follicles (n = 3 in each treatment) included in a small cortical fragments. Preantral follicles were classified according to the stage of development as primordial follicles or as developing follicles. Preantral follicles cultured in supplemented media for 6 days were ultrastructurally normal. Their oocytes had an intact nucleus and cytoplasm that contained heterogeneous-sized lipid droplets, and numerous round or elongated mitochondria with intact parallel cristae were observed. Occasionally these organelles were associated with smooth endoplasmic reticulum (SER). Rough endoplasmic reticulum (RER) was rarely found. The cytoplasm of granulosa cells contained a large number of mitochondria and abundant RER. In contrast, follicles cultured in MEM+ (control) had a large number of vacuoles in the oocyte cytoplasm and excessive clustering of the chromatin material in the nucleus, suggesting an initial process of oocyte degeneration. In conclusion, the presence of IAA, EGF, FSH and their combinations helped to maintain ultrastructural integrity of ovine preantral follicles in cortical slices cultured in vitro.

Bypassing the effect of zona pellucida changes on embryo formation following cryopreservation of bovine oocytes

Objective: To evaluate the effects of zona pellucida changes following the cryopreservation of bovine oocytes using two different techniques. Design: Prospective randomised study. Setting: University research facility and IVF research laboratory, NURTURE, University of Nottingham, UK. Intervention: 508 bovine oocytes were collected from abattoir-derived ovaries and cryopreserved by slow freezing using standard straws and a new vitrification method using our self-constructed cryoloops. After thawing, the oocytes were inseminated by ICSI and standard IVF. Main outcome measures: Oocyte degeneration and oocyte cleavage. Results: Cleavage rates after IVF in fresh oocytes was higher (49.5%) than the cryopreservation groups (slow freezing: 15.4% and vitrification: 25.8%), whereas after ICSI, the rates were similar (fresh: 17.3%, slow freezing: 9.4%, vitrification: 16.0%). Conclusions: Zona pellucida changes after oocyte cryopreservation significantly reduce the embryo formation rates after IVF. ICSI bypasses this complication and in selected species it could be an appropriate method of insemination.

Induction of polycystic ovary by testosterone in immature female rats: Modulation of apoptosis and attenuation of glucose/insulin ratio.

Polycystic ovarian syndrome is seen in 5% of fertile aged women. However, there is no satisfactory PCOS model in experimental animals. To induce polycystic ovary phenotype in immature female rats, Wistar rats 21 days of age were injected daily with testosterone propionate 1 mg/100 g body weight dissolved in propylene glycol or propylene glycol for up to 35 days. Seven days of injection with testosterone (T) resulted in the appearance of large cystic follicles and a dramatic accumulation of multi-layer preantral follicles. At 42 days of age puberty in control animals was evident by the appearance of corpora lutea. In contrast in T treated animals no corpora lutea formation was seen even at the age of 56 days. Progesterone in the control animals was elevated at the age of 42 days in contrast with the T treated animals in which progesterone remained low (20% of control). While during 14 days of T injection most of the follicles did not have progressive apoptosis, at 21-35 days of injection (42-56 days of age) the vast majority of follicles became apoptotic. Progressive degeneration of oocytes was evident in T treated animals reaching 70-85% of total oocytes at 21-35 days of T injection compared to 30-40% in control animals. Western blot analysis of ovarian homogenates revealed gradual decrease in Bcl-2 content, evident at 28 and 35 days of T injection compared to control animals. Interestingly, the fasting glucose/insulin ratio was dramatically reduced in T treated animals following 14 days of testosterone treatment compared to controls. Our data suggest that T injection to immature female rats can induce polycystic ovaries, block ovulation and attenuate progesterone production. Moreover, normal/low glucose and high insulin blood levels in the testosterone treated rats raises the possibility that elevated androgens can lead to insulin resistance in this experimental PCOS model.

Gonadal development in the coral reef damselfish Dascyllus flavicaudus from Moorea, French Polynesia

The present study investigated the sexual pattern of the yellow-tailed dascyllus, Dascyllus flavicaudus (Randall and Allen), through histological examination of the process of gonadal differentiation and maturation and through male-removal experiments in the laboratory. The study was conducted in Moorea, French Polynesia (17°32′S; 149°50′W), from 31 December 1994 to 11 March 1995. For gonadal histology, two populations were sampled, one consisting of large aggregations (n=54) and the other consisting of small- to medium-sized groups around isolated corals (n=55). An additional small sample (n=21) was also collected from a population that consisted of small groups around isolated corals. After an initially undifferentiated state, gonads of D. flavicaudus developed an ovarian lumen, followed by oocytes in the primary growth stage. From this ovarian state or from more developed ovaries with cortical-alveolus stage oocytes, some gonads developed into testes through degeneration of oocytes and development of spermatogenic tissue. In all three populations, spermatogenic tissue developed only in gonads that contained pre-vitellogenic oocytes (termed as mixed stage 1–2 gonads). The two main populations did not differ in their expression of sex change despite differences in their social organization. In both populations, size of individuals with mixed stage 1–2 gonads overlapped mainly with the size range of immature females, which suggests that functional female-to-male sex change was rare. The hypothesis that D. flavicaudus is primarily gonochoristic is further supported by removal experiments in laboratory groups, in which removal of a dominant male(s) failed to induce sex change in any of the high-ranking females. Yet, in all three field populations, some fish with mixed stage 1–2 gonads were found within the size ranges of mature females, which indicates possible occurrences of functional sex change. These conflicting results indicate that it may be premature to draw any definitive conclusions about the sexual pattern of D. flavicaudus.

DDT in zebra mussels from Lake Maggiore (N. Italy): level of contamination and endocrine disruptions

The DDT contamination of Lake Maggiore (Northern Italy) has been monitored since a serious pollution event occurred in 1996. To assess the environmental risk associated with this contamination, bioaccumulation data coupled with histopathological markers were evaluated on zebra mussel populations from two different contaminated sites from April 2001 to April 2002. Biomonitoring results showed high DDT pollution in 2001 because of a flood which transported DDTs still contained in the sediments of a polluted river to the lake. DDT concentrations reached values of 4–5 μg/g lipids, higher than those recorded in other industrialized countries but comparable to levels measured in developing ones. In the ovaries of the most highly polluted mussels, histological analyses showed a delay in oocyte maturation and a high incidence of pathological pictures mainly referable to oocyte degeneration and haemocytic infiltration. Moreover, despite the presence of mature sperms, in 2001 first male gamete release occurred about 2 months later than in females. These results indicated a neuroendocrine interference of DDT on Dreissena polymorpha reproduction and also showed that these invertebrates can be successfully used to evaluate ecological implications due to exposure to endocrine disruptors in freshwater environments.

Effects of sex steroids on gonadal development and gender determination in the sea scallop, Placopecten magellanicus

Previous work has demonstrated the presence of various sex steroids in molluscs and suggested their possible functions in the control of reproduction. However, their potential roles in early sexual development have remained unexplored. The results of the present study showed that injections of estradiol, testosterone, progesterone, and dehydroepiandrosterone (DHEA) all accelerated gonadal differentiation and shifted sex ratios toward more males in the sea scallop. Morphological changes were also observed in the females of steroid-injected groups. Estradiol appeared to stimulate oocyte growth while testosterone appeared to induce degeneration of oocytes. No parallel changes were noted in males of steroid-injected groups. Hermaphroditic animals were also found in the progesterone- and DHEA-injected groups. Together, these results support the hypothesis that sex steroids are involved in invertebrate reproduction and may be exploited for gender manipulation in bivalve aquaculture.

What are determinants of the oocyte degeneration rate after intracytoplasimic sperm injection (ICSI)?

Background and Significance: Interest has evolved in determining the factors associated with oocyte degeneration after ICSI. Limited reports indicated that oocyte degeneration was associated with the ICSI technique. Objective: This purpose of this study was to determine whether oocyte degeneration was technician dependent in an experienced laboratory. Baseline characteristics and physician-adjustable ovarian stimulation variables were also evaluated. Design: Prospective study. Materials and Methods: Six thousand seven hundred eighty-nine intracytoplasmic injection procedures were analyzed to determine whether the oocyte degeneration rate was technician dependent. 230 independent, down-regulated cycles were examined to assess which predictors may be associated with the oocyte degeneration rate. Multivariate analyses were performed using the generalized linear model routines in Stata, Ver 7.0 (Stata Corporation, College Station, TX) to perform logistic regression allowing for clustering by cycle. The models were determined by backward elimination in a guided fashion, starting with a model including all the predictors (ICSI technician, stripping technician, baseline characteristics (age, day 3 FSH/ E2, AFC), physician-adjustable variables (start dose, total dose, number of mature oocytes retrieved, E2 on day of hCG, predicted E2/large follicle, number of 13–17mm (medium) and ≥18mm (large) follicles on the day of hCG, size of the lead follicle), then eliminating those that were not statistically significant one at a time guided by the previous literature and clinical experience. Tests were declared statistically significant for a two-sided p-value < 0.05. Results: Neither the ICSI technician nor the stripping technician was a determinant of the oocyte degeneration rate. However, the day 3 FSH, number of mature oocytes retrieved, and E2/large follicle were significant independent predictor of oocyte degeneration rate. The day 3 FSH was positively associated with the degeneration rate, and the number of mature oocytes retrieved and the predicted E2/large follicle were negatively associated with the degeneration rate. Physician-adjustable variables were not associated with the degeneration rate. Tabled 1numdegCoef.Std. Err.zP>|z|[95% Conf. Interval]Age.0065215.02394280.270.785−.0404056.0534486AFC.0019983.01526470.130.896−.0279199.0319165Day 3 E2.0036408.00231791.570.116−.0009023.0081838#MII Oocytes−.0335009.0142233−2.360.019−.0613781−.0056237Day 3 FSH.0437193.02188112.000.046.0008332.0866053Start Dose.0255645.14112290.180.856−.2510313.3021603Total Dose−.0081025.0112635−0.720.472−.0301787.0139736Pred E2−.0023453.0008457−2.770.006−.0040029−.0006878_cons−2.003593.8960944−2.240.025−3.759906−.2472804Pred E2 = E2 day of hCG/(#medium follicles * 0.75 + #large follicles) (Standard errors scaled using square root of Pearson X2-based dispersion). Open table in a new tab Pred E2 = E2 day of hCG/(#medium follicles * 0.75 + #large follicles) (Standard errors scaled using square root of Pearson X2-based dispersion). Conclusion: The ICSI or stripping technician did not impact the oocyte degeneration rate. The data suggests that oocyte degeneration is a function of oocyte aging and physicians cannot alter the inherent occyte degeneration rate within a down-regulated cycle.

What are determinants of the oocyte degeneration rate after intracytoplasimic sperm injection (ICSI)?

Background and Significance: Interest has evolved in determining the factors associated with oocyte degeneration after ICSI. Limited reports indicated that oocyte degeneration was associated with the ICSI technique.

Germline stem cells and follicular renewal in the postnatal mammalian ovary

A basic doctrine of reproductive biology is that most mammalian females lose the capacity for germ-cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. Here we show that juvenile and adult mouse ovaries possess mitotically active germ cells that, based on rates of oocyte degeneration (atresia) and clearance, are needed to continuously replenish the follicle pool. Consistent with this, treatment of prepubertal female mice with the mitotic germ-cell toxicant busulphan eliminates the primordial follicle reserve by early adulthood without inducing atresia. Furthermore, we demonstrate cells expressing the meiotic entry marker synaptonemal complex protein 3 in juvenile and adult mouse ovaries. Wild-type ovaries grafted into transgenic female mice with ubiquitous expression of green fluorescent protein (GFP) become infiltrated with GFP-positive germ cells that form follicles. Collectively, these data establish the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary.

Roles of gonadotropins and meiosis-activating sterols in meiotic resumption of cultured follicle-enclosed mouse oocytes

This study describes a model for short-term culture of intact mouse follicles under serum-free conditions. Follicles were either obtained from immature mice receiving no ovarian stimulation (i.e. no eCG-primed protocol, group I) or from mice undergoing ovarian stimulation (i.e. eCG-primed protocol, group II). Follicles were grouped according to size (100–170, 180–200, 210–250, 260–350 and 360–400 μm, respectively) and cultured for 24 h (group I) or for only 6 h (group II). Induced meiotic resumption of follicle-enclosed oocytes were evaluated following stimulation with gonadotropins (i.e. FSH and hCG), AY9944-A-7, an inhibitor of Δ14-reductase, and RS-21745, an inhibitor of lanosterol 14α-demethylase; both enzymes affect synthesis of the meiosis activating sterols (MAS) that induce oocyte maturation. The frequency of oocyte degeneration was also recorded. In group I, FSH (10–200 IU l −1) and AY9944-A-7 (5, 25 and 50 μM) separately induced resumption of meiosis in oocytes derived from follicles with a diameter of 180–400 μm. hCG (1.0 and 10 IU ml −1) exhibited a similar but weaker effect on oocytes present in follicles with a diameter of 260–400 μm. Irrespective of follicular diameter oocytes obtained from follicles in group II responded to hCG and FSH by resuming meiosis. FSH (50 IU l −1) alone or hCG (10 IU ml −1) alone both increased the GVBD percentage of oocytes enclosed in follicles with a diameter 260–400 μm, but the response to hCG was not significant compared to control. FSH (50 IU l −1) combination with hCG (10 IU ml −1) showed an additive effect raising the rate of GVBD after 6 h culture. Addition of 50 or 100 μM RS-21745 was able to attenuate gonadotropins-induced resumption of meiosis to below background levels. In conclusion, the ability of FSH to induce meiotic resumption of follicle-enclosed mouse oocytes is correlated to follicle size, being most pronounced in larger follicles. hCG caused a similar but less pronounced effect. The ability of RS-21745 to inhibit and the ability of AY9944-A-7 to enhance oocyte maturation of follicle-enclosed oocytes support the concept of FSH employing MAS as a downstream signal transduction molecule for initiation of oocyte maturation in mice.

Exposure to L-ascorbic acid or alpha-tocopherol facilitates the development of porcine denuded oocytes from metaphase I to metaphase II and prevents cumulus cells from fragmentation.

It is known that alpha-tocopherol (vitamin E) and L-ascorbic acid (vitamin C) can modulate many biochemical processes intracellularly or extracellularly as antioxidants. The objective of the present study was to investigate the effects of alpha-tocopherol and L-ascorbic acid on porcine oocyte meiotic maturation, viability and the functions of cumulus cells. In two independent experiments, porcine oocytes with or free from cumulus cells were exposed to different levels of alpha-tocopherol (0, 10, 100 and 200 microM) or L-ascorbic acid (0, 50, 250 and 750 microM). Cumulus expansion, cumulus cell DNA fragmentation, meiotic maturation and degeneration of oocytes were assessed 48 h after in vitro culture. The results showed that: (1) neither alpha-tocopherol nor L-ascorbic acid influenced cumulus expansion but both prevented cumulus cell DNA fragmentation. (2) Alpha-tocopherol lowered the percentage of denuded oocytes (DOs) arrested at germinal vesicle stage (GV). Among the oocytes undergoing germinal vesicle breakdown (GVBD) proportion, fewer DOs treated by alpha-tocopherol were at metaphase I (MI) and more at metaphase II (MII). L-ascorbic acid caused lower percentage of DOs arrested at GV stage and higher percentage of DOs undergoing GVBD, especially at MII. The influences of alpha-tocopherol and L-ascorbic acid were not obvious in cumulus-enclosed oocytes (CEOs). (3) Both vitamins compromised the viability of CEOs and DOs. These results indicate that exposure to alpha-tocopherol or L-ascorbic acid promotes the development of porcine DOs from MI to MII and prevents cumulus cell DNA fragmentation at certain levels, especially 10 microM alpha-tocopherol or 250 microM L-ascorbic acid.

Aromatase inhibitor induces complete sex change in the protogynous honeycomb grouper (Epinephelus merra).

The protogynous hermaphrodite fish change sex from female to male at the certain stages of life cycle. The endocrine mechanisms involved in gonadal restructuring throughout protogynous sex change are not clearly understood. In the present study, we implanted maturing female honeycomb groupers with nonsteroidal aromatase inhibitor (AI), Fadrozole (0, 1, and 10 mg/fish) and examined changes in gonadal structures and serum levels of sex steroid hormones 2(1/2) months after implantation. The ovaries of control females had oocytes undergoing active vitellogenesis, whereas AI caused females to develop into functional males. These males had testes, which were indistinguishable in structure from those of normal males, but bigger in size, and completed all stages of spermatogenesis including accumulation of large amount of sperm in the seminiferous tubules. AI significantly reduced the serum levels of estradiol-17beta (E2) and increased levels of testosterone (T), 11-ketotestosterone (11-KT), and 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP). Further, AI suppressed in vitro production of E2, and stimulated the production of T and 11-KT in the ovarian fragments of mature female. In the honeycomb grouper, suppression of both in vitro and in vivo production of E2 and degeneration of oocytes by AI suggests that AI induces complete sex change through inhibition of estrogen biosynthesis, and perhaps, subsequent induction of androgen function.

Administration of increasing amounts of gonadotrophin compromises preimplantation development of parthenogenetic mouse embryos

The aim of the present study was to examine the effect of ovarian stimulation with increasing amounts of pregnant mare's serum gonadotrophin (PMSG) on preimplantation development of diploid parthenogenetic embryos in vitro. Administration of 5, 10 and 20 IU PMSG significantly increased the number of oocytes obtained per mouse in a dose-dependent manner. The amount of PMSG administered did not alter the proportion of degenerate oocytes. However, there was a significant decrease in the proportion of 8-cell/compacted embryos after 53 h of culture with the administration of increasing amounts of PMSG. Proportion of embryos reaching at the blastocyst stage after 79 h of culture was reduced significantly in both the 10 and 20 IU PMSG groups. Reduced blastocyst development after 96 h of culture, however, was significant only in the 20 IU PMSG group. Total blastocyst, trophectoderm and inner cell mass numbers were also reduced significantly with the administration of 20 IU PMSG. It is concluded on the basis of these observations that preimplantation development of diploid parthenogenetic oocytes, which depends virtually entirely on maternal molecules accumulated during oogenesis along with gene products derived from the maternal genome, is compromised with the administration of increasing amounts of PMSG.

Melatonin as a new drug for improving oocyte quality.

Background: Although recent technical advances have benefited infertile couples, inadequate embryo development as a result of poor quality oocytes still contributes to infertility. The purpose of the present study was to evaluate melatonin as a drug for improving oocyte quality in such cases. Methods: Twenty-seven women from whom fewer than three fertilized embryos were grown and who failed to fall pregnant in previous treatment cycles were enrolled in the current prospective clinical study. Subjects took 1mg or 3mg tablets of melatonin orally at 22:00h from the fifth day of the previous menstrual cycle to the day they were injected with human chorionic gonadotropin. The numbers of mature follicles, retrieved oocytes, degenerate oocytes, and fertilized embryos were compared to their previous data without melatonin (the control cycle). Results: Intrafollicular melatonin concentrations were significantly increased, and intrafollicular lipid peroxide concentrations showed a tendency towards lower levels in the 3mg melatonin treatment cycles compared with the control cycles. The number of degenerate oocytes was significantly reduced, and the number of fertilized embryos showed a tendency towards an increase in the 3mg cycle compared to the control cycle. Three women succeeded in falling pregnant. Conclusion: Melatonin is likely to become the drug of choice for improving oocyte quality in women who cannot fall pregnant because of poor quality oocytes. (Reprod Med Biol 2003; 2: 139-144).