Supraphysiological doses of nandrolone decanoate disrupts spermatogenesis but did not interfere on embryo rate.

The aim of this study was to investigate the effects of 10 mg/kg/week of nandrolone decanoate (DECA - Deca Durabolin®) on body composition, hormonal levels, spermatic parameters, redox status, and morphometric parameters of testicle and epididymis; furthermore, the fertility capacity of Wistar rats was measured thought in vitro fertilization (IVF). The animals (n = 16) were divided into two groups: control group (CTRL, n = 8), which received only vehicle composed by peanut oil and 10% of the benzoic alcohol and nandrolone decanoate group (DECA, n = 8), which received intramuscular injections of DECA for 8 weeks, both groups were treated for 8 weeks. The results demonstrate significative decrease in visceral fat, testosterone levels, and thiol content on epididymis, reduction on normal sperm parameters, and deleterious effect on testicles and epididymis tissue morphology showing reduction of germ height and luminal diameter on the DECA group. Thus, it can be concluded that high doses of nandrolone decanoate impairs male reproductive parameters.

ABO blood group and link to COVID-19: A comprehensive review of the reported associations and their possible underlying mechanisms.

ABO blood group is long known to be an influencing factor for the susceptibility to infectious diseases, and many studies have been describing associations between ABO blood types and COVID-19 infection and severity, with conflicting findings. This narrative review aims to summarize the literature regarding associations between the ABO blood group and COVID-19. Blood type O is mostly associated with lower rates of SARS-CoV-2 infection, while blood type A is frequently described as a risk factor. Although results regarding the risk of severe outcomes are more variable, blood type A is the most associated with COVID-19 severity and mortality, while many studies describe O blood type as a protective factor for the disease progression. Furthermore, genetic associations with both the risk of infection and disease severity have been reported for the ABO locus. Some underlying mechanisms have been hypothesized to explain the reported associations, with incipient experimental data. Three major hypotheses emerge: SARS-CoV-2 could carry ABO(H)-like structures in its envelope glycoproteins and would be asymmetrically transmitted due to a protective effect of the ABO antibodies, ABH antigens could facilitate SARS-CoV-2 interaction with the host' cells, and the association of non-O blood types with higher risks of thromboembolic events could confer COVID-19 patients with blood type O a lower risk of severe outcomes. The hypothesized mechanisms would affect distinct aspects of the COVID-19 natural history, with distinct potential implications to the disease transmission and its management.

Acute effect of high-intensity interval training exercise on redox status in the ovaries of rats fed a high-fat diet.

This study demonstrates the effect of a single high-intensity interval training (HIIT) session on the redox status of rat ovaries with excess adiposity. Forty Wistar female rats (mean (±s.e.m.) weight 94.40 ± 13.40 g) were divided into two groups and fed either a standard diet (SD) or a high-fat diet (HFD) for 62 days. At the end of this period, the rats were subjected to a single HIIT session and were killed 24 h after exercise. Both groups subjected to exercise (SDex and HFDex) generated a significantly higher antioxidant environment by presenting a higher thiol content, which represents a lower oxidation rate of GSH than their respective controls (SD and HFD). The percentage of morphologically normal primary follicles decreased, whereas that of antral follicles increased, in the SDex group. In addition, the HFD group had a higher percentage of degenerated antral follicles than the SD and SDex groups. Cells immunoreactive for α-smooth muscle actin were seen in the cortical stroma and thecal layer enclosing late secondary and tertiary follicles in all groups. Moreover, heme oxygenase and cytochrome P450 family 19 subfamily A member 1 (Cyp19A1) labelling was seen in all antral follicles. Progesterone concentrations were significantly higher in the HFDex than SDex group. In conclusion, this study indicates that a single session of HIIT may result in an improvement in ovary redox status because of metabolic muscle activity by inducing physiological adaptation after exercise in a paracrine manner.

5-Fluorouracil disrupts ovarian preantral follicles in young C57BL6J mice.

PURPOSE: 5-Fluorouracil (5-FU), an anti-cancer drug, has been used for hepatoblastoma (HB) chemotherapy in children, who may have impaired  ovarian follicle pool reserve with lasting effects to reproduction. Therefore, this study aimed to investigate 5-FU effects on survival, growth, and morphology of ovarian preantral follicles from C57BL6J young mice. METHODS: Experiments were carried-out both in vivo and in vitro. Mice were treated with 5-FU injection (450 mg/kg i.p) or saline and sacrificed 3 days after to obtain ovaries for histology and molecular biology. Ovaries for in vitro studies were obtained from unchallenged mice and cultured under basic culture medium (BCM) or BCM plus 5-FU (9.2, 46.1, 92.2 mM). Preantral follicles were classified according to developmental stages, and as normal or degenerated. To assess cell viability, caspase-3 immunostaining was performed. Transcriptional levels for apoptosis (Bax, Bcl2, p53, Bax/Bcl2) and Wnt pathway genes (Wnt2 and Wnt4) were also analyzed. Ultrastructural analyses were carried-out on non-cultured ovaries. In addition, ß-catenin immunofluorescence was assessed in mouse ovaries. RESULTS: The percentage of all-types normal follicles was significantly lower after 5-FU challenge. A total loss of secondary normal follicles was found in the 5-FU group. The highest 5-FU concentrations reduced the percentage of cultured normal primordial follicles. Large vacuoles were seen in granulosa cells and ooplasm of preantral follicles by electron microscopy. A significantly higher gene expression for Bax and Bax/Bcl2 ratio was seen after 5-FU treatment. A marked reduction in ß-catenin immunolabeling was seen in 5-FU-challenged preantral follicles. In the in vitro experiments, apoptotic and Wnt gene transcriptions were significantly altered. CONCLUSION: Altogether, our findings suggest that 5-FU can deleteriously affect the ovarian follicle reserve by reducing preantral follicles survival.

Ultra-diluted Folliculinum 6 cH impairs ovine oocyte viability and maturation after in vitro culture.

This study investigated the effect of Folliculinum 6 cH on the oocyte meiosis resumption and viability rates, progesterone production and mitochondrial activity after in vitro maturation of cumulus-oocyte complexes (COCs) in sheep. Sheep ovaries were collected at a local slaughterhouse and COCs were recovered by slicing technique. The selected COCs were maturated in TCM199 (Control treatment), or control medium supplemented with 0.05% ethanol (v/v) (the vehicle of the homeopathic preparation - Ethanol treatment) or with Folliculinum 6 cH. After 24 h of in vitro maturation (IVM), oocytes were mechanically denuded and incubated with Hoechst 33342 and MitoTracker (0.5 µM) Orange CMTMRos for analysis of viability and chromatin configuration, and mitochondrial activity, respectively. The results showed that Folliculinum 6 cH addition increased oocyte degeneration and reduced meiotic resumption compared to the control (P < 0.05). Interestingly, the percentages meiotic resumption and oocyte maturation were lower in the Folliculinum 6 cH treatment compared to its vehicle (Ethanol treatment) (P < 0.05). On the other hand, when the treatments were compared, higher mitochondrial activity was observed in the Ethanol treatment (P < 0.05). In conclusion, contrary to its vehicle, the addition of Folliculinum 6 cH to the IVM medium promoted oocyte degeneration and affected negatively the mitochondrial distribution, impairing meiosis resumption.

Natural antioxidants in the vitrification solution improve the ovine ovarian tissue preservation.

The present study evaluated the effect of the addition of antioxidants anethole (AN) and robinin (RO) in the vitrification solution, and the in vitro incubation (IVI) medium of ovine ovarian tissue. Ovarian fragments were vitrified without antioxidant (VWA) or with different concentrations of AN (30, 300 and 2000 µg/mL) or RO (0.125, 0.25 and 0.50 mg/mL), followed by IVI (24 h). Histological analyses showed that the percentage of morphologically normal preantral follicles (MNPF) in AN 2000 did not differ from RO 0.125 or fresh ovarian tissue (CTR). Subsequently, ovarian fragments were vitrified in the presence of AN 2000 and RO 0.125 followed by IVI without or with (AN 2000+ and RO 0.125+) the same antioxidants. The follicular activation in all treatments was significantly increased as compared to the CTR. The stroma cell density (SCD) in all the vitrified fragments was significantly lower than the CTR. However, in the AN 2000 and RO 0.125 this parameter was significantly higher when compared to the VWA. The reactive oxygen species (ROS) in the ovarian cortex of the AN 2000 or AN 2000+ were significantly reduced in comparison with the CTR while the intracellular ROS levels of AN 2000 and CTR were similar. The total antioxidant capacity (TAC) in RO 0.125 was significantly higher than that of VWA, AN 2000 and AN 2000+. According to the results, the use of antioxidants (AN or RO) only in the vitrification solution of ovine ovarian tissue is recommended, due to their better preservation of the SCD. Moreover, AN 2000 best maintains the follicular morphology, while RO 0.125 has a high TAC.

Incorporation of Nitroprusside on Silica Nanoparticles-A Strategy for Safer Use of This NO Donor in Therapy.

Silica-based nanoparticles have been developed as powerful platforms for drug delivery and might also prevent undesired side effects of drugs. Here, a fast method to synthesize positively charged mesoporous silica nanoparticles (ζ = 20 ± 0.5 mV, surface area = 678 m2 g-1, and 2.3 nm of porous size) was reported. This nanomaterial was employed to anchor sodium nitroprusside (SNP), a vasodilator drug with undesired cyanide release. A remarkable incorporation of 323.9 ± 7.55 µmol of SNP per gram of nanoparticle was achieved, and a series of studies of NO release were conducted, showing efficient release of NO along with major cyanide retention (ca. 64% bound to nanoparticle). Biological assays with mammalian cells showed only a slight drop in cell viability (13%) at the highest concentration (1000 µM), while SNP exhibited an LC50 of 228 µM. Moreover, pharmacological studies demonstrated similar efficacy for vasodilation and sGC-PKG-VASP pathway activation when compared to SNP alone. Altogether, this new SNP silica nanoparticle has great potential as an alternative for wider and safer use of SNP in medicine with lower cyanide toxicity.

Fraction of Auxemma oncocalyx and Oncocalyxone A Affects the In Vitro Survival and Development of Caprine Preantral Follicles Enclosed in Ovarian Cortical Tissue.

BACKGROUND: Auxemma oncocalyx and its main component oncocalyxone A (onco A) have a high level of antioxidant and antitumor activity, but there are no studies on the action of both of these drugs regarding folliculogenesis. MATERIAL AND METHODS: Caprine ovarian tissue fragments were fixed (non-cultured control) or cultured for 1 or 7 days in α-MEM+ alone (cultured control) or supplemented with dimethyl sulfoxide (DMSO; 20% v/v), bone morphogenetic protein 15 (BMP-15; 100 ng/ml), doxorubicin (DXR; 0.3 g/ml), or different concentrations of A. oncocalyx (1.2, 12, or 34 g/ml) or onco A (1, 10, or 30 g/ml). We analyzed for follicular morphology and growth, apoptosis (terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) assay), and cell proliferation (silver staining of argyrophilic nucleolus organizer regions (AgNOR) and test for proliferating cell nuclear antigen (PCNA)). RESULTS: A. oncocalyx and onco A (in a concentration-dependent manner) and DXR decreased (P < 0.05) the number of morphologically normal follicles, with no effect (P > 0.05) on follicular growth. A. oncocalyx reduced (P < 0.05) the percentage of normal follicles compared to onco A, whereas DXR, A. oncocalyx 1.2 g/ml, and onco A 1 g/ml increased (P < 0.05) the percentage of TUNEL-positive follicles. DXR decreased (P < 0.05) the number of nucleolus organizer regions. CONCLUSION: A. oncocalyx and onco A affected the in vitro caprine folliculogenesis in a concentration-dependent manner. Onco A (1 g/ml) has a less harmful effect than DXR on goat preantral follicle survival.

Ovine secondary follicles vitrified out the ovarian tissue grow and develop in vitro better than those vitrified into the ovarian fragments.

Cryopreservation of preantral follicles is a promising technique to preserve female fertility. The aim of this study was to evaluate the effect of vitrification on the development of secondary follicles included in ovarian tissue or isolated after microdissection. An important end point included is the capacity of grown oocytes to resume meiosis. Sheep ovarian cortexes were cut into fragments and split into three different groups: (1) fresh (control): secondary follicles isolated without any previous vitrification; (2) follicle-vitrification (follicle-vit): secondary follicles vitrified in isolated form; and (3) tissue-vitrification (tissue-vit): secondary follicles vitrified within fragments of ovarian tissue (in situ former) and subsequently subjected to isolation. From the three groups, isolated secondary follicles were submitted to IVC for 18 days. After IVC, cumulus-oocyte complexes (COCs) were harvested from follicles. As an additional control group, in vivo grown, in vivo-grown COCs were collected from antral ovarian follicles. All, recovered COCs were matured and the chromatin configuration was evaluated. Data were analyzed by ANOVA, and the means were compared by Student-Newman-Keuls test, and by chi-square. Differences were considered to be significant when P < 0.05. Isolated preantral follicles from all treatments had normal morphology, antrum formation, and low follicle degeneration after IVC. The growth rate between control and follicle-vit did not differ (P > 0.05), and was higher (P < 0.05) than for tissue-vit. The percentage of follicles that decreased diameter during IVC was significantly higher in tissue-vit than the in follicle-vit. Recovery rate of oocytes from normal follicles was higher in follicle-vit than in tissue-vit. Furthermore, oocyte viability was lower in tissue-vit than other treatments, and follicle-vit did not differ from control and in vivo grown. The percentage of oocytes meiosis resuming was not different between treatments except for in vivo grown. After vitrification, only follicle-vit showed metaphase I oocyte. We conclude that secondary follicles vitrified after isolation displayed a better follicular growth rate, oocyte viability, percentage of oocytes reaching the metaphase I stage, and fewer follicles with decreased diameter after IVC.

Connexin 37 and 43 gene and protein expression and developmental competence of isolated ovine secondary follicles cultured in vitro after vitrification of ovarian tissue.

Cryoinjuries caused by vitrification of tissues and organs lead to the loss of membrane proteins that mediate intercellular communications, such as connexins 37 (Cx37) and 43 (Cx43). Thus, the present study aimed to evaluate ovine Cx37 and Cx43 gene and protein expressions and developmental competence by in vitro-cultured secondary follicles retrieved from vitrified ovarian tissue. Ovarian fragments for the same ovary pair were distributed into six treatments: (1) fresh ovarian tissue (FOT); (2) vitrified ovarian tissue (VOT); (3) isolated follicles from fresh ovarian tissue (FIF); (4) isolated follicles from vitrified ovarian tissue; (5) isolated follicles from fresh ovarian tissue followed by in vitro culture (CFIF); (6) isolated follicles from vitrified ovarian tissue followed by in vitro culture (CVIF). In all treatments, Cx37 and Cx43 gene and protein expression patterns were evaluated by reverse transcription polymerase chain reaction and immunocytochemistry. In addition, secondary follicles were analyzed according to follicular integrity and growth, apoptosis, and cell proliferation. In vitro-cultured secondary follicles (CFIF and CVIF) were evaluated based on morphology (extruded follicles), antrum formation, and viability. The percentage of intact follicles was higher, whereas antrum formation, oocyte extrusion rate, and follicle viability were lower in CVIF than in CFIF treatment (P < 0.05). Terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphates nick end-labeling assay demonstrated that apoptosis was absent in FIF, whereas follicles from all other treatments showed positive labeling. Cell proliferation index was higher in isolated follicles from vitrified ovarian tissue and CVIF treatments than in follicles from FIF. Expression of Cx43 messenger RNA was lower in CVIF treatment when compared with follicles from all other treatments (P < 0.05). Follicle Cx37 messenger RNA levels did not show alterations in any treatment (P > 0.05). Cx37 and Cx43 immunolabeling was localized mainly on granulosa cells and oocytes, respectively. In conclusion, isolation of ovine secondary follicles could be done successfully after vitrification of ovarian tissue, and the basement membrane integrity remained intact after in vitro culture. Although the gene and protein expression of Cx37 did not change after vitrification of ovarian tissue, Cx43 turned out to be altered in secondary follicles after vitrification and in vitro culture.

The base medium affects ultrastructure and survival of bovine preantral follicles cultured in vitro.

The aim of this study was to determine the effectiveness of minimum essential medium alpha modification (α-MEM), tissue culture medium 199 (TCM-199), and McCoy's medium (McCoy's) on IVC of preantral follicles included in the bovine ovarian cortex (in situ). Bovine ovarian fragments were cultured in α-MEM, TCM-199, or McCoy supplemented ((+)) with glutamine, insulin, transferrin, selenium, ascorbic acid, BSA, penicillin, streptomycin, and HEPES buffer in 24-well plates, at 37 °C and 5% CO2 for 1 or 7 days. The morphology of follicles, normal, primordial and development (primary and secondary), as well as viability and morphometric variables of follicles and oocytes were assessed. The morphology and morphometry of preantral follicles were analyzed by ANOVA followed by the Tukey and Dunnett tests, and viability variables were determined by the chi-square test. The results showed that TCM-199(+) reduced significantly (P < 0.05) the percentage of morphologically normal and viable follicles after 7 days of culture compared to the control. Similar results were observed in McCoy(+), in which the percentage of viable follicles after 7 days of culture was significantly lower (P < 0.05) than in the control. However, it was similar (P > 0.05) between α-MEM(+) and TCM-199(+). Moreover, follicular diameters in McCoy(+) and TCM-199(+) were significantly smaller (P < 0.05) than in control and α-MEM(+) after 7 days of culture. In addition, the ultrastructure of preantral follicles was similar between the control and α-MEM(+) after 7 days of culture. In conclusion, α-MEM(+) showed to be the most effective medium to preserve morphology, morphometry and ultrastructure of bovine preantral follicles, ensuring their viability and growth after in situ culture.

Vitrified sheep isolated secondary follicles are able to grow and form antrum after a short period of in vitro culture.

The risk of reintroducing malignant cells after ovarian graft into patients following post-cancer treatment is an obstacle for clinical applications (autotransplantation). In this context, in vitro follicle culture would be an alternative to transplantation in order to minimize such risks. Therefore, the aim of this study was to compare the development of secondary follicles after vitrification in isolated form (without stroma) with vitrification in in situ form (within fragments of ovarian tissue). Follicles were first isolated from ovarian fragments from mixed-breed ewes and then vitrified; these comprised the Follicle-Vitrification group (Follicle-Vit), or fragments of ovarian tissue were first vitrified, followed by isolation of the follicles, resulting in the Tissue-Vitrification group (Tissue-Vit). Control and vitrified groups were submitted to in vitro culture (6 days) and follicular morphology, viability, antrum formation, follicle and oocyte diameter, growth rate, ultrastructural characteristics and cell proliferation were evaluated. The percentages of morphologically normal follicles and antrum formation were similar among groups. Follicular viability and oocyte diameter were similar between Follicle-Vit and Tissue-Vit. The follicular diameter and growth rate of Follicle-Vit were similar to the Control, while those of Tissue-Vit were significantly lower compared to the Control. Both vitrified groups had an augmented rate of granulosa cellular proliferation compared to Control. Secondary follicles can be successfully vitrified before or after isolation from the ovarian tissue without impairing their ability to survive and grow during in vitro culture.

In vitro culture of bovine preantral follicles: a review.

Preantral follicles are the majority of the ovarian follicle population and their use as a source of homogeneous oocytes for bovine reproductive biotechnologies could result in a substantial advance in this field. However, while in other species embryos and offspring have been produced, in bovine species the results have been limited to the follicular activation of small (primordial) preantral follicles and formation of early antral follicles from large (secondary) preantral follicles after in vitro culture. Therefore, this review will highlight the basic aspects of bovine folliculogenesis by focusing on preantral follicles, the methods of harvesting preantral follicles, the main results from in vitro follicular culture during the last 20 years, and the potential candidate substances (basic supplements, growth factors, and hormones) for improving the efficiency of in vitro follicle growth.

Dynamized follicle-stimulating hormone affects the development of ovine preantral follicles cultured in vitro.

OBJECTIVE: To evaluate the effect of dynamized follicle-stimulating hormone (FSH) on the survival, activation and growth of ovine preantral follicles (PFs) in vitro. METHODS: Ovarian fragments were cultured for 1 or 7 days in alpha minimum essential medium (α-MEM(+)) control in the absence or presence of alcohol (Al control) or FSH (6cH, 12cH and 30cH) added at intervals of 24 or 48 h. The ovarian fragments were processed, coded and analyzed by a blinded observer by classical histology (CH), fluorescence microscopy (FM) and transmission electron microscopy (TEM). RESULTS: After 7 days of culture, the group which to which FSH 6cH was added at 24 h intervals showed better rates of follicle survival and activation compared to α-MEM(+) control or Al control (p < 0.05). This group also showed higher follicle and oocyte growth than α-MEM(+) control (p < 0.05). FM and TEM techniques confirmed that FSH 6cH promoted viability and ultrastructural integrity of follicles after 7 days of culture. CONCLUSIONS: FSH 6cH (24 h) treatment maintained the viability, and promoted the activation and in vitro growth of ovine PFs.

Insulin-like growth factor II (IGF-II) and follicle stimulating hormone (FSH) combinations can improve the in vitro development of grown oocytes enclosed in caprine preantral follicles.

OBJECTIVE: Evaluate the possible role of IGF-II alone or in association with FSH on in vitro development of isolated caprine preantral follicles. METHODS: Preantral follicles (≥150 µm) were isolated from goat ovaries and cultured for 18 days in basic αMEM medium (control) or supplemented with IGF-II alone at 20 or 50 ng/ml, named IGF20 and IGF50, respectively, or in combination with recombinant FSH (FSH, IGF20F or IGF50F). During in vitro culture, the follicles were analyzed by using morphology criteria, antrum formation and growth rate as parameters. After 18 days of follicular culture, oocytes equal to or larger than 110 µm were used for in vitro maturation (IVM). Oocyte viability and meiosis resumption were assessed by fluorescence microscopy after labeling with calcein-AM, ethidium homodimer and Hoechst 33342. RESULTS: The IGF20 treatment was the only treatment capable of maintaining the percentage of morphologically normal follicles from D0 until D6 and from D12 to D18 (p>0.05), while in all other treatments the percentage of morphologically normal follicles decreased progressively during 18 days of in vitro culture (p<0.05). At D18, all treatments with IGF-II or FSH resulted in a significantly higher percentage of normal follicles when compared to αMEM alone. The IGF50F treatment provided a significantly higher early antrum formation rate when compared to αMEM and FSH alone. The addition of IGF-II alone (20 or 50 ng/ml) or in combination with FSH prevented oocyte degeneration after IVM. Moreover, the FSH treatment demonstrated a lower percentage of oocyte degeneration when compared to control (4.35% vs. 26.3%, respectively; p<0.05). Regarding meiosis resumption, the IGF20F treatment was the only treatment that significantly differed from αMEM alone. All treatments except the control (αMEM alone) presented oocytes at metaphase II. CONCLUSION: IGF-II associated with FSH stimulated in vitro follicular development, oocyte viability and meiotic resumption of caprine oocytes after IVM.

Goat ovarian follicles express different levels of mRNA for inhibin-ßA subunit and activin-A stimulates secondary follicle growth in vitro / Folículos ovarianos caprinos expressam diferentes níveis de RNAm para subunidade ßA da inibina e ativina-A promove o crescimento de folículos secundários in vitro

This study investigated the levels of messenger ribonucleic acids (mRNA) for inhibin-ßA subunit in goat primordial, primary and secondary follicles, as well as in cumulus-oocyte complexes (COCs) and mural granulosa / theca cells of antral follicles. The effects of activin-A (100ng mL-1) and/or follicle stimulating hormone (FSH, 50ng mL-1) on growth and expression of mRNA for activin-A and FSH receptor (FSH-R) in secondary follicles cultured for six days were evaluated. The data showed that the expression of inhibin-ßA is lower in secondary follicles than in primary follicles and is higher in large antral follicles than in small antral follicles. After culture, activin-A and/or FSH promoted growth of secondary follicles, while FSH increased the levels of mRNA for inhibin-ßA, and activin-A increased the levels of FSH-R mRNA. In conclusion, mRNA for inhibin-ßA is expressed at different levels in pre-antral and antral follicles and activin-A acts as a stimulator of the FSH-R expression in goat follicles. On its turn, the expression of inhibin-ßA is stimulated by FSH, which together with activin-A promotes secondary follicle growth in-vitro.

Este estudo investigou os níveis de ácidos ribonucleicos (RNAm) para a subunidade ßA da inibina em folículos primordiais, primários e secundários caprinos, bem como em complexos cumulus-oócitos (CCOs) e células da granulosa mural/teca de folículos antrais. Além disso, avaliaram-se os efeitos da ativina-A (100ng mL-1) e/ou hormônio folículo estimulante (FSH, 50ng mL-1) sobre o crescimento e a expressão do RNAm para inibina-ßA e receptores de FSH (FSH-R) em folículos secundários cultivados por seis dias. Os dados mostraram que a expressão de inibina-ßA é menor em folículos secundários do que em folículos primários e é maior em grandes folículos antrais que nos pequenos folículos antrais. Após o cultivo, ativina-A e/ou FSH promoveram o crescimento de folículos secundários. Enquanto o FSH aumentou os níveis de RNAm para inibina-ßA, a ativina-A aumentou os níveis de RNAm para FSH-R. Em conclusão, a inibina-ßA é expressa em diferentes níveis em folículos pré-antrais e antrais e a ativina-A atua como um estimulador da expressão de FSH-R em folículos caprinos. Por sua vez, a expressão de inibina-ßA é estimulada pelo FSH, que, juntamente com ativina, promove o crescimento de folículos secundários in vitro.

Restoring fertility after ovarian tissue cryopreservation: a half century of research.

Tissue transplantation and in vitro ovarian follicle culture have been investigated as alternative techniques to restore fertility in young women who are facing fertility-threatening diseases or treatments following ovarian tissue cryopreservation. Although transplants of fresh or frozen ovarian tissue have successfully yielded healthy live births in different species including humans, the risks of reintroducing cancer cells back into the patient, post treatment, have limited its clinical purpose. The in vitro ovarian follicle culture minimizes these risks and provides a way to harvest more mature oocytes, however its clinical translation has yet to be determined. Not only is it possible for tissue cryopreservation to safeguard fertility in cancer patients, this technique also allows the maintenance of germplasm banks for animals of high commercial value or for those animals that are at risk of extinction. Given the importance of managing female genetic material, this paper reviews the progress of the methods used to preserve and restore female fertility in different species to demonstrate the results obtained in the past 50 years of research, the current achievements and the future directions on this field.

Importance of vascular endothelial growth factor (VEGF) in ovarian physiology of mammals.

Ovarian folliculogenesis in mammals is a complex process. Several compounds have been tested during in vitro culture of follicular cells for a better understanding of the mechanisms and factors related to ovarian folliculogenesis in mammals. From these compounds, vascular endothelial growth factor (VEGF) can be highlighted, as it is strongly associated with angiogenesis and, in recent years, its presence in ovarian cells has been investigated extensively. Previous studies have shown that the presence of VEGF protein, as well as mRNA expression of its receptor 2 (VEGFR-2) increases during follicular development. Therefore, it is likely that the interaction between VEGF and VEGFR-2 is crucial to promote follicular development. However, few studies on the influence of this factor on follicular development have been reported. This review addresses aspects related to the structural characterization and mechanism of action of VEGF and its receptors, and their biological importance in the ovary of mammals.

Steady-state level of epidermal growth factor (EGF) mRNA and effect of EGF on in vitro culture of caprine preantral follicles.

Our aim was to verify the steady-state level of epidermal growth factor (EGF) mRNA in goat follicles at various developmental stages and to investigate the influence of EGF on the survival, antrum formation and growth of secondary follicles cultured for 6 days. Primordial, primary and secondary goat follicles and small and large antral follicles were obtained to quantify EGF mRNA by real-time reverse transcription with the polymerase chain reaction. The influence of EGF and the presence or absence of follicle-stimulating hormone (FSH) on the development of secondary follicles and on mRNA expression for EGF and FSH receptor (FSH-R) was determined after 6 days of culture. Survival, antrum formation and follicular diameter were evaluated every other day of culture. EGF mRNA levels in secondary follicles were significantly higher than those in primordial follicles, whereas in small and large antral follicles, EGF mRNA levels in cumulus-oocyte complexes (COCs) were significantly higher than in granulosa/theca cells. During culture, EGF in the presence or absence of FSH increased the follicular daily growth rate of secondary follicles when compared with that in enriched alpha minimal essential medium. FSH, EGF or both reduced EGF mRNA levels, whereas EGF reduced FSH-R mRNA levels after follicle culture for 6 days. Thus, EGF mRNA levels are higher in secondary follicles than in earlier stages, with both FSH and EGF promoting the growth of goat secondary follicles. EGF and/or FSH reduce EGF mRNA levels, whereas EGF decreases FSH-R mRNA levels, in cultured secondary follicles.

Follicular interactions affect the in vitro development of isolated goat preantral follicles.

The aim of this study was to evaluate the influence of the number of follicles per drop (one or three) and antral follicles on in vitro development of isolated goat preantral follicles. Preantral follicles were isolated through microdissection and distributed individually (control) or in groups of three follicles (treatment) in microdroplets of α-MEM with or without 1000 ng/ml follicle stimulating hormone (FSH) for Experiments 1 and 2, respectively. Experiment 3 was divided into four treatments according to the presence of one or three preantral follicles, associated or not with antral follicles. After culture, oocytes were retrieved from morphologically normal follicles and submitted to in vitro maturation (IVM) and live/dead fluorescent labelling. Results of Experiment 1 (basic medium without FSH) showed that culture of preantral follicles in groups enhances viability, growth and antrum formation after 12 days. However, in the presence of FSH (Experiment 2), only the recovery rate of fully grown oocytes for IVM was significantly affected by grouping of follicles. In Experiment 3, in general, co-culture of preantral follicles with an early antral follicle had a detrimental effect on viability, antrum formation and production of oocytes for IVM. In conclusion, the performance of in vitro culture of goat preantral follicles is affected by the number of follicles per drop, the presence of an antral follicle and FSH.