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Constant progress in the diagnosis and treatment of cancer disease has increased the number and prognosis of cancer survivors. However, the toxic effects of chemotherapy and radiotherapy on ovarian function have resulted in premature ovarian failure. Patients are, therefore, still expecting methods to be developed to preserve their fertility successfully. Several potential options are available to preserve fertility in patients who face premature ovarian failure, including immature or mature oocyte and embryo cryopreservation. However, for children or prepubertal women needing immediate chemotherapy, cryopreservation of ovarian tissue is the only alternative. The ultimate aim of this strategy is to implant ovarian tissue into the pelvic cavity (orthotopic site) or in a heterotopic site once oncological treatment is completed and the patient is disease free. Transplantation of ovarian tissue with sufficiently large numbers of follicles could potentially restore endocrine function and allow multiple cycles for conception. However, the success of ovarian tissue transplantation still has multiple challenges, such as the low number of follicles in the graft that may affect their longevity as well as the survival of the tissue during ex vivo processing and subsequent transplantation. Therefore, this review aims to summarize the achievements of ovary grafting and the potential techniques that have been developed to improve ovarian graft survival.
Recently, the existence of a mechanism for neo-oogenesis in the ovaries of adult mammals has generated much controversy within reproductive biology. This mechanism, which proposes that the ovary has cells capable of renewing the follicular reserve, has been described for various species of mammals. The first evidence was found in prosimians and humans. However, these findings were not considered relevant because the predominant dogma for reproductive biology at the time was that of Zuckerman. This dogma states that female mammals are born with finite numbers of oocytes that decline throughout postnatal life. Currently, the concept of neo-oogenesis has gained momentum due to the discovery of cells with mitotic activity in adult ovaries of various mammalian species (mice, humans, rhesus monkeys, domestic animals such as pigs, and wild animals such as bats). Despite these reports, the concept of neo-oogenesis has not been widely accepted by the scientific community, generating much criticism and speculation about its accuracy because it has been impossible to reproduce some evidence. This controversy has led to the creation of two positions: one in favour of neo-oogenesis and the other against it. Various animal models have been used in support of both camps, including both classic laboratory animals and domestic and wild animals. The aim of this review is to critically present the current literature on the subject and to evaluate the arguments pro and contra neo-oogenesis in mammals.
It is well documented that both epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) are critical for porcine oocyte maturation, however, little information is known about their mechanism of action in vitro. To gain insight into the mechanisms of action of the optimum doses of EGF and GDNF on porcine oocyte maturation, porcine cumulus–oocyte complexes (COCs) were matured in defined porcine oocyte medium supplemented with EGF, GDNF or a combination of both at varying concentrations (0–100 ng/ml) for 44 h. Nuclear and cytoplasmic maturation were determined in terms of nuclear stage after DNA staining with Hoechst and cortical granule distribution after lectin labeling, respectively. Mature oocytes were subsequently collected for gene expression analysis or subjected to in vitro fertilization and cultured for 7 days. The results showed that EGF and/or GDNF, when administered in a certain dose (50 ng/μl) to the maturation medium, not only effectively improved the synchronization of nuclear and cytoplasmic maturation processes within the oocyte, but enhanced expression of their corresponding receptors in mature oocytes (P < 0.05). Moreover, supplementation with an optimal combination of EGF + GDNF resulted in elevation of TFAM transcripts as well as a decrease of caspase-3 transcripts compared with the other studied groups (P < 0.05). Collectively, our results indicate that treatment of porcine oocytes with specific-dose combinations of EGF and GDNF stimulates oocyte quality and competence by transcriptional modulation of genes involved in oocyte survival and competence.
The worldwide consumption of red wine, nuts and grapes has resulted in increased human exposure to resveratrol, which could affect reproductive function. However, the effect of resveratrol on in vitro culture of early-stage ovarian follicles has never been investigated. The aims of the present study were to evaluate the effect of resveratrol on sheep secondary follicle morphology, growth, DNA fragmentation, intracellular levels of glutathione (GSH) and active mitochondria. Secondary follicles were isolated from the ovaries and cultured for 18 days in supplemented α-MEM+ (control medium) or in control medium containing resveratrol (2, 10 or 30 µM). The parameters analyzed were morphology, antrum formation, follicle diameter, DNA fragmentation, GSH levels and mitochondrial activity. After 18 days, all resveratrol groups significantly decreased the percentages of morphologically normal follicles compared with the control group (α-MEM+). Antrum formation was higher in both α-MEM+ and 2 µM resveratrol groups than in the 10 µM resveratrol group. In addition, 30 µM resveratrol increased the percentage of oocytes with DNA damage compared with the control. Oocytes from follicles treated with 10 or 30 µM resveratrol significantly decreased intracellular GSH levels compared with the 2 µM resveratrol group. Moreover, follicles in α-MEM+ (control) showed more active mitochondria than those in 10 or 30 µM resveratrol. In conclusion, ovine isolated secondary follicles are able to grow to the antral stage after in vitro culture in medium containing 2 µM resveratrol, maintaining the same rates of DNA damage, GSH levels and mitochondrial function as the control medium. However, the addition of 30 µM resveratrol increased DNA fragmentation and oxidative stress through decreasing mitochondrial activity.
Breeding and larval performance of novel hybrids from reciprocal crosses of Asian catfish Pangasianodon hypophthalmus (Sauvage, 1878) and African catfish Clarias gariepinus (Burchell, 1822) were investigated in this study. Spawning was by hormonal injection of brood fish, artificial fertilization, and incubation in triplicate aquarium tanks (0.5 × 0.5 × 0.5 m3) with continuous aeration. Reciprocal crosses (♀C. gariepinus × ♂P. hypophthalmus and ♀P. hypophthalmus × ♂C. gariepinus) had lower hatchability (≤50%) than their pure siblings (≥75%). Fish from all crosses survived until the juvenile stage but survival at 35 days post hatching (dph) was higher for pure C. gariepinus sib. ♀C. gariepinus × ♂P. hypophthalmus was observed to be less resistant to degradation of water quality than the other crosses, however it had higher body weight compared with the other crosses that showed similar performance. Morphological comparison of surviving juvenile at 35 dph, showed that all ♀P. hypophthalmus × ♂C. gariepinus and 13% of the ♀C. gariepinus × ♂P. hypophthalmus exhibited the very same morphology as that of their maternal parent species, while the other portion of the ♀C. gariepinus × ♂P. hypophthalmus cross exhibited morphological traits that were intermediate between those of both parent species. This study been the first successful attempt to hybridize both species and therefore, laid the groundwork for further studies on the aquaculture potentials of the novel hybrids.
We examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P < 0.05). The blastocyst formation rate was higher in the group treated with AZD5438 for 4 h than in the groups treated with AZD5438 for 2 or 6 h (42.8% vs. 38.6% and 37.2%, respectively; P > 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.
DNA methylation is an important form of epigenetic regulation in mammalian development. Methyl-CpG-binding domain protein 1 (MBD1) and methyl-CpG-binding domain protein 2 (MeCP2) are two members of the MBD subfamily of proteins that bind methylated CpG to maintain the silencing effect of DNA methylation. Given their important roles in linking DNA methylation with gene silencing, this study characterized the coordinated mRNA expression and protein localization of MBD1 and MeCP2 in embryos and placentas and aimed to analysis the effects of MBD1 and MeCP2 on transgenic cloned goats. Our result showed that MBD1 expression of transgenic cloned embryo increased significantly at the 2–4-cell and 8–16-cell stages (P < 0.05), then decreased at the morula and blastocyst stages (P < 0.05); MeCP2 expression in transgenic cloned embryo was significant decreased at the 2–4-cell stage and increased at the 8–16-cell stage (P < 0.05). Placenta morphology analysis showed that the cotyledon number of deceased transgenic cloned group (DTCG) was significantly lower than that the normal goats (NG) and in the live transgenic cloned goats (LTCG) (P < 0.05). MBD1 and MeCP2 were clearly detectable in the placental trophoblastic binucleate cells by immunohistochemical staining. Moreover, MBD1 and MeCP2 expression in DTCG was significant higher than in the NG and the LTCG (P < 0.05). In summary, aberrant expression of methylation CpG binding proteins MBD1 and MeCP2 was detected in embryonic and placental development, which reflected abnormal transcription regulation and DNA methylation involved in MBD1 and MeCP2. These findings have implications in understanding the low efficiency of transgenic cloning.
Following ovulation, oocytes undergo a time-dependent deterioration in quality referred to as post-ovulatory ageing. Although various factors influence the post-ovulatory ageing of oocytes, oxidative stress is a key factor involved in deterioration of oocyte quality. Artemisia asiatica Nakai ex Pamp. has been widely used in East Asia as a food ingredient and traditional medicine for the treatment of inflammation, cancer, and microbial infections. Recent studies have shown that A. asiatica exhibits antioxidative effects. In this study, we investigated whether A. asiatica has the potential to attenuate deterioration in oocyte quality during post-ovulatory ageing. Freshly ovulated mouse oocytes were cultured with 0, 50, 100 or 200 μg/ml ethanol extracts of A. asiatica Nakai ex Pamp. After culture for up to 24 h, various ageing-induced oocyte abnormalities, including morphological changes, reactive oxygen species (ROS) accumulation, apoptosis, chromosome and spindle defects, and mitochondrial aggregation were determined. Treatment of oocytes with A. asiatica extracts reduced ageing-induced morphological changes. Moreover, A. asiatica extracts decreased ROS generation and the onset of apoptosis by preventing elevation of the Bax/Bcl-2 expression ratio during post-ovulatory ageing. Furthermore, A. asiatica extracts attenuated the ageing-induced abnormalities including spindle defects, chromosome misalignment and mitochondrial aggregation. Our results demonstrate that A. asiatica can relieve deterioration in oocyte quality and delay the onset of apoptosis during post-ovulatory ageing.
Allicin (AL) regulates the cellular redox, proliferation, viability, and cell cycle of different cells against extracellular-derived stress. This study investigated the effects of allicin treatment on porcine oocyte maturation and developmental competence. Porcine oocytes were cultured in medium supplemented with 0 (control), 0.01, 0.1, 1, 10 or 100 μM AL, respectively, during in vitro maturation (IVM). The rate of polar body emission was higher in the 0.1 AL-treated group (74.5% ± 2.3%) than in the control (68.0% ± 2.6%) (P < 0.1). After parthenogenetic activation, the rates of cleavage and blastocyst formation were significantly higher in the 0.1 AL-treated group than in the control (P < 0.05). The reactive oxygen species level at metaphase II did not significantly differ among all groups. In matured oocytes, the expression of both BAK and CASP3, and BIRC5 was significantly lower and higher, respectively, in the 0.1 AL-treated group than in the control. Similarly, the expression of BMP15 and CCNB1, and the activity of phospho-p44/42 mitogen-activated protein kinase (MAPK), significantly increased. These results indicate that supplementation of oocyte maturation medium with allicin during IVM improves the maturation of oocytes and the subsequent developmental competence of porcine oocytes.
The yolk syncytial layer (YSL) of Teleostei is a dynamic multifunctional temporary system. This paper describes the YSL structure of Misgurnus fossilis (Cobitidae) during its early developmental stages, studied using histological methods. YSL formation is prolonged. From the late blastula stage, the basal surface of the YSL is uneven and has protuberances, but becomes smoother during development. There are syncytial ‘islands’ with 1–2 yolk syncytial nuclei in the yolk mass. During epiboly, gastrulation and early segmentation, loach YSL is of different thickness in different regions along the dorso-ventral and antero-posterior axes of an embryo. The YSL is thickened in the dorsal region of gastrulae compared with the ventral region. Although the development of M. fossilis is similar to the development of zebrafish, there are important differences in YSL formation and organization that await further study and analysis. The study of YSL organization contributes to our knowledge of teleost developmental diversity and to the biology of temporary structures.
Japanese fancy mouse, mini mouse or pet mouse are common names used to refer to strains of mice that present with different colour varieties and coat types. Although many genetic studies that involve spotting phenotype based on the coat have been performed in these mice, there are no reports of quantitative data in the literature regarding testis structure and spermatogenic efficiency. Hence, in this study we researched testis function and spermatogenesis in the adult Japanese fancy mouse. The following values of 68 ± 6 mg and 0.94 ± 0.1% were obtained as mean testis weight and gonadosomatic index, respectively. In comparison with other investigated mice strains, the fancy mouse Leydig cell individual size was much smaller, resulting in higher numbers of these cells per gram of testis. As found for laboratory mice strains, as a result of the development of the acrosomic system, 12 stages of the seminiferous epithelium cycle have been described in this study. The combined frequencies of pre-meiotic and post-meiotic stages were respectively 24% and 64% and very similar to the laboratory mice. The more differentiated germ cell types marked at 1 h or 9 days after tritiated thymidine administration were preleptotene/leptotene and pachytene spermatocytes at the same stage (VIII). The mean duration of one spermatogenic cycle was 8.8 ± 0.01 days and the total length of spermatogenesis lasted 37.8 ± 0.01 days (4.5 cycles). A high number of germ cell apoptosis was evident during meiosis, resulting in lower Sertoli cell and spermatogenic efficiencies, when compared with laboratory mice strains.
The aim of this study was to analyse the morphology and allometry of larvae belonging to five potamodromous species. Five breeding species belonging to the order Characiformes [Salminus brasiliensis (Cuvier, 1816), Leporinus steindachneri, Eigenmann, 1907, Prochilodus lineatus (Valenciennes, 1837), Prochilodus vimboides (Kner,1859) and Brycon insignis, Steindachner, 1877] were used to obtain larvae samples during the pre-flexing, post-flexing, and juvenile developmental stages. When we observed the degree-hour (DH) amplitude time values, we found three developmental groups based on allometry and morphometrics within the period between the pre-flexing and post-flexing phases. Group 1 consists of the species S. brasiliensis and B. insignis, Group 2 consists of P. lineatus and P. vimboides, and Group 3 consists of L. steindachneri. Group 1 requires less development time and has more slender larvae. Group 2 has a moderate development time and larvae with a more rounded shape. Group 3 presents a greater development time and an intermediate larval morphology. It was possible to classify the larvae through cross-validated discriminant analyses based on seven morphometric variables with 90% accuracy in B. insignis, 83% in L. steindachneri, 91% in P. lineatus, 80% in P. vimboides, and 96% in S. brasiliensis. These results indicate larval characteristics that can be used for the taxonomic identification of the icthyoplankton.
Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.
The objectives of the present studies were to investigate the developmental capacity of dromedary camel oocytes selected by brilliant cresyl blue (BCB) staining and to investigate the expression of select transcripts in germinal vesicle (GV) stage oocytes. These transcripts included BMP15 and GDF9 as important transcripts for folliculogenesis and oocyte development, Zar1 and Mater as maternal transcripts required for embryonic development, Cyclin B1 and CDK1 as cell cycle regulators and Oct4 and STAT3 as transcription factors. Dromedary camel oocytes were retrieved from ovaries collected at a local slaughterhouse. After exposure to BCB staining, cumulus–oocyte complexes (COCs) from BCB+, BCB− and control (selected based on morphological criteria) groups were subjected to in vitro maturation, in vitro fertilization and in vitro culture. For gene expression studies, after BCB staining cumulus cells were stripped off and the completely denuded GV stage oocytes were used for RT-PCR analysis of selected transcripts. BCB+ oocytes showed higher maturation, and fertilization rates compared with BCB− and control groups. Indices of early embryonic development, namely, cleavage at 48 hours post insemination (hpi), and development to morula at day 5 and day 7 blastocyst rates were also significantly higher in the BCB+ group. RT-PCR revealed a higher expression of BMP15, GDF9, Zar1, Mater, Cyclin B1, CDK1, OCT4 and STAT3 in good quality oocytes that stained positively for BCB (BCB+). Collectively, results provide novel information about the use of BCB screening for selecting good quality oocytes to improve in vitro embryo production in the dromedary camel.
This study aimed to examine the gonadal morphology of diploid and triploid fish through stereological analysis. Triploid individuals were obtained after temperature shock (40°C for 2 min) at 2 min post-fertilization and reared until 175 days post-fertilization (dpf). Intact eggs were used to obtain the diploids. Gonads were collected for histological analysis at 83, 114, 144 and 175 dpf. Diploid females and males presented normal oogenesis and spermatogenesis through all the experimental period. Conversely, stereological analysis revealed that triploid females were sterile and oogonia were the prevalent cell type in the ovaries. Triploid males presented increased amounts of spermatocyte cysts and a large area of lumen when compared with diploids and in addition the amount of spermatozoa was lower than that observed for diploids. However, some triploid males presented spermatogenesis similar to diploids. Therefore, we concluded that triploidization is an interesting alternative to produce sterile individuals in A. altiparanae.