Melatonin reduces oxidative stress and improves follicular morphology in feline (Felis catus) vitrified ovarian tissue.

Ovarian tissue vitrification is associated with multiple events that promote accumulation of ROS (reactive oxygen species) which culminate in follicular apoptosis. Thus, this study was aimed at evaluating the role of melatonin in vitrification and culture of feline (Felis catus) ovarian tissue. In phase 1, domestic cat ovaries were fragmented into equal circular pieces of 1.5 mm diameter by 1 mm thickness and divided into four groups (fresh control and 3 treatments). The treatments were exposed to vitrification solutions supplemented with melatonin at 0 M, 10-9 M, and 10-7 M, then vitrified-warmed, histologically evaluated and assayed for ROS. Consequently, phase 2 experiment was designed wherein ovarian fragments were divided into two groups. One group was exposed to vitrification solution without melatonin and the other with 10-7 M melatonin supplementation, then vitrified-warmed and cultured for ten days with fresh ovarian fragments as control prior to assessment for histology, immunohistochemistry (Ki-67, MCM-7 and caspase-3) and ROS. Concentration of ROS was lower (p = 0.0009) in 10-7 M supplemented group in addition to higher proportion of grade 1 follicles. After culture, proportions of intact and activated follicles were higher (p < 0.05) in melatonin supplemented group evidenced by higher expression of Ki-67 and MCM-7. Follicular apoptosis was lower in melatonin supplemented group. In conclusion, melatonin at 10-7 M concentration preserved follicular morphological integrity while reducing ROS concentration in vitrified-warmed feline ovarian tissue. It has also promoted the follicular viability and activation with reduced apoptosis during in vitro culture of vitrified-warmed feline ovarian tissue.

Alpha lipoic acid controls degeneration and ensures follicular development in ovine ovarian tissue cultured in vitro.

This study aims to evaluate the effects of adding alpha lipoic acid (ALA) to the in vitro ovarian tissue culture medium, either fresh or after vitrification/warming. For this purpose, 10 ovaries from five adult sheep were used. Each pair of ovaries gave rise to 16 fragments and were randomly distributed into two groups: fresh (n = 8) and vitrified (n = 8). Two fresh fragments were fixed immediately and considered the control, while another six were cultured in vitro for 14 days in the absence; presence of a constant (100 µM/0-14 day) or dynamic (50 µM/day 0-7 and 100 µM/day 8-14) concentration of ALA. As for the vitrified fragments, two were fixed and the other six were cultured in vitro under the same conditions described for the fresh group. All the fragments were subjected to morphological evaluation, follicular development and stromal density (classical histology), DNA fragmentation (TUNEL), senescence (Sudan Black), fibrosis (Masson's Trichome), and endoplasmic reticulum stress (immunofluorescence). Measurements of the antioxidant capacity against the free radicals 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and estradiol (E2) levels in the culture medium was performed. The results showed that in the absence of ALA, in vitro culture of vitrified ovarian fragments showed a significant reduction (P < 0.05) in follicular morphology and increased the presence of senescence and tissue fibrosis (P < 0.05). Dynamic ALA maintained E2 levels unchanged (P > 0.05) until the end of vitrified ovarian tissue culture and controlled the levels of ABTS and DPPH radicals in fresh or vitrified cultures. Therefore, it is concluded that ALA should be added to the vitrified ovarian tissue in vitro culture medium to reduce the damage that leads to loss of ovarian function. To ensure steroidogenesis during in vitro culture, ALA should be added dynamically (different concentrations throughout culture).

VEGFA165 can rescue excess steroid secretion, inflammatory markers, and follicle arrest in the ovarian cortex of High A4 cows†.

A population of cows with excess androstenedione (A4; High A4) in follicular fluid, with follicular arrest, granulosa cell dysfunction, and a 17% reduction in calving rate was previously identified. We hypothesized that excess A4 in the ovarian microenvironment caused the follicular arrest in High A4 cows and that vascular endothelial growth factor A would rescue the High A4 phenotype. In trial 1, prior to culture, High A4 ovarian cortex (n = 9) had greater numbers of early stage follicles (primordial) and fewer later-stage follicles compared to controls (n = 11). Culture for 7 days did not relieve this follicular arrest; instead, High A4 ovarian cortex had increased indicators of inflammation, anti-Mullerian hormone, and A4 secretion compared to controls. In trial 2, we tested if vascular endothelial growth factor A isoforms could rescue the High A4 phenotype. High A4 (n = 5) and control (n = 5) ovarian cortex was cultured with (1) PBS, (2) VEGFA165 (50 ng/mL), (3) VEGFA165B (50 ng/mL), or (4) VEGFA165 + VEGFA165B (50 ng/mL each) for 7 days. Follicular progression increased with VEGFA165 in High A4 cows with greater early primary, primary, and secondary follicles than controls. Similar to trial 1, High A4 ovarian cortex secreted greater concentrations of A4 and other steroids and had greater indicators of inflammation compared to controls. However, VEGFA165 rescued steroidogenesis, oxidative stress, and fibrosis. The VEGFA165 and VEGFA165b both reduced IL-13, INFα, and INFß secretion in High A4 cows to control levels. Thus, VEGFA165 may be a potential therapeutic to restore the ovarian steroidogenic microenvironment and may promote folliculogenesis.

Rutin promotes activation and reduces apoptosis of primordial follicles by regulating Akt phosphorylation after in vitro culture of ovine ovarian tissue.

The aims of this study were to analyze the effects of different concentrations of rutin on primordial follicle survival and development after in vitro culture of sheep ovarian tissue, and to verify the possible involvement of the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway in the rutin actions. Ovarian fragments were fixed for histological analysis (fresh control) or cultured in α-minimum essential medium alone (α-MEM+: control medium) or in α-MEM+supplemented with different concentrations of rutin (0.1; 1 or 10 µg/mL) for 7 days. Inhibition of the PI3K activity was performed in fragments cultured with 50 µM LY294002. Thereafter, immunohistochemistry was performed to evaluate the expression of cleaved caspase-3 (apoptosis) and Akt phosphorylation (p-Akt). The results showed that 1 µg/mL rutin has a greater percentage of normal follicles (P < 0.05) than those of α-MEM+ and other rutin treatments. In addition, 1 µg/mL rutin maintained the follicular apoptosis similar (P > 0.05) to that of the fresh control and lower than α-MEM+ and 10 µg/mL rutin. All rutin concentrations increased (P < 0.05) follicular activation compared to fresh control and α-MEM+. Furthermore, follicular and oocyte diameters increased (P < 0.05) only after culture with 1 µg/mL rutin. After PI3K inhibition, there was a reduction (P < 0.05) of rutin follicular effects. In conclusion, rutin at 1 µg/mL reduces apoptosis, promotes activation and growth of sheep primordial follicles through the modulation of the PI3K/Akt signaling pathway after in vitro culture of ovine ovarian tissue.

Effects of a gel culture system made of polysaccharides (xanthan gum and locust bean gum) on in vitro bovine oocyte development and gene expression of the granulosa cells.

Xanthan gum (XG) and locust bean gum (LBG) are nontoxic polysaccharides that produce culture substrates. The present study examined the effect of XG-LBG gel on in vitro bovine oocyte growth and gene expression in granulosa cells. Oocytes and granulosa cell complexes (OGCs) were cultured in vitro on plastic culture plate (Plate) or XG-LBG gel for 16 days. OGCs formed a dome-like cavity surrounding the oocytes on plate but formed a spherical follicle structure on XG-LBG gel. The total granulosa cell numbers of the OGCs and their survival rate was greater for OGCs cultured on XG-LBG gel than for those cultured on plate. Oocytes grown on XG-LBG gels had higher lipid and mitochondrial content, as well as a larger diameter, than their plate counterparts. When oocytes grown in vitro were subjected to in vitro maturation and fertilization, the normal fertilization rate was significantly higher for oocytes developed on XG-LBG gel than that of oocytes cultured on the plate counterpart. RNAseq of the granulosa cells revealed that genes associated with focal adhesion, phosphatidylinositol 3'-kinase-Akt and Hippo signaling, and regulation of actin cytoskeleton were upregulated in granulosa cells of OGCs cultured on XG-LBG gel compared with those cultured on plate.

Gallic acid promotes the in vitro development of sheep secondary isolated follicles involving the phosphatidylinositol 3-kinase pathway.

This study was conducted to evaluate the effect of addition of gallic acid as the single antioxidant to the base medium for in vitro culture of sheep secondary follicles and if the phosphatidylinositol 3-kinase (PI3K) pathway is involved in the action of gallic acid. Secondary follicles were isolated and cultured for 12 days in α-MEM supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium, and ascorbic acid (control medium: α-MEM+) or in α-MEM supplemented with BSA, insulin, glutamine, hypoxanthine and different concentrations of gallic acid (25, 50 or 100 µM), thus replacing transferrin, selenium and ascorbic acid in the medium. Follicle morphology, glutathione (GSH), and mitochondrial activity, and meiotic resumption were evaluated. Furthermore, inhibition of PI3K pathway was performed by pretreatment with LY294002. After 12 days of culture, the follicle survival in a medium containing 100 µM gallic acid was similar (P > 0.05) to α-MEM+ and greater (P < 0.05) compared with other gallic acid concentrations. Antrum formation, follicle diameter, GSH, and mitochondrial activity, and meiotic resumption, however, were greater (P < 0.05) when 100 µM gallic acid was included in the α-MEM+ culture medium compared with the control medium. Furthermore, LY294002 inhibited (P < 0.05) follicle survival, development, and meiotic resumption stimulated by 100 µM gallic acid. In conclusion, concentration of 100 µM of gallic acid can be a substitute for transferrin, selenium, and ascorbic acid in the base medium during in vitro culture of sheep secondary follicles, inducing follicle development likely through the PI3K pathway.

The effect of DDE and dieldrin and their parental substances (DDT and aldrin) on PGE2 and PGF2α release from bovine endometrial explants collected during 120 to 180 days of the gestational period.

Dieldrin and DDE are environmental metabolites of the organochlorine pesticides aldrin and DDT, respectively. During pregnancy, these chemicals can quickly infiltrate through the placental barrier, accumulate in amniotic fluid and fetus, and act as endocrine disruptors (EDs). The aim of this study was to investigate the effect of DDE and dieldrin and their parental substances at concentrations of 1 and 10 ng/ml on secretion of PGE2 and PGF2α from bovine endometrial explants (120-150 and 151-180 days of pregnancy) after 24 hr of incubation with EDs. The mRNA expression of COX2, PGES and PGFS and the concentrations of PGE2 and PGF2α were measured. EDs did not affect (p>0.05) COX2 gene expression, but DDT and DDE decreased (p⟨0.05) PGES expression and PGE2 secretion in the explants from 120-150 days of pregnancy. Depending on the dose, DDT and DDE increased (p⟨0.05) PGFS expression and PGF2α secretion from the explants from 120-150 days and decreased PGF2α secretion (p⟨0.05) from the explants from 151-180 days of pregnancy. Aldrin and dieldrin decreased (p⟨0.05) PGFS expression and PGF2α secretion from all explants. In summary, EDs disrupt the secretion of PGE2 and PGF2α by influencing the gene expression of PGES and PGFS.

Effects of insulin on IGF-1 receptors in equine lamellar tissue in vitro.

Although it is understood that equine endocrinopathic laminitis can be triggered by high concentrations of insulin, it is unclear whether this represents a direct action on lamellar tissue via insulin receptors (InsR), an interaction with IGF-1 receptors (IGF-1R), or some other, indirect action. This uncertainty is because of the reported scarcity of InsR in lamellar tissue and the low affinity of insulin for equine IGF-1R. In the present study, the effects of insulin and IGF-1 (as a positive control) were examined using lamellar explants isolated from the hooves of healthy horses and incubated in cell culture medium for between 2 min and 48 h. In this system, a low physiological concentration of IGF-1 (10 nM; 1.31 ng/mL) caused a marked increase in the appearance of phosphorylated IGF-1R after 5 min (P < 0.05), and this effect was blocked by a human anti-IGF-1R monoclonal antibody (mAb). However, a high concentration of insulin (10 nM; 1,430 µIU/mL) appeared to cause dephosphorylation of the IGF-1R after 5 min (P < 0.01), 15 min, and 30 min (P < 0.001). Using 3H-thymidine as a marker, it was also demonstrated that insulin and IGF-1-stimulated cell proliferation in lamellar explants over the same concentration range as each other (1-100 nM), implying that each peptide acts via its own receptor (P < 0.001). Conversely, the effect of both peptides could be blocked using a selective anti-IGF-1R mAb (P < 0.001), implying that insulin acts via IGF1-R (either directly or indirectly). Notwithstanding this conundrum, the results demonstrate that insulin acts directly on lamellar tissue and suggest that a therapeutic anti-IGF-1R mAb could be useful in treating or preventing endocrinopathic laminitis.

Supportive techniques to investigate in vitro culture and cryopreservation efficiencies of equine ovarian tissue: A review.

During the reproductive lifespan of a female, only a limited quantity of oocytes are naturally ovulated; therefore, the mammalian ovary possesses a substantial population of preantral follicles available to be handled and explored in vitro. Hence, the manipulation of preantral follicles enclosed in ovarian tissue aims to recover a considerable population of oocytes of high-value animals for potential application in profitable assisted reproductive technologies (ARTs). For this purpose, the technique of preantral follicle in vitro culture (IVC) has been the most common research tool, achieving extraordinary results with offspring production in the mouse model. Although promising outcomes have been generated in livestock animals after IVC of preantral follicles, the quantity and quality of embryo production with those oocytes are still poor. In recent years, the mare has become an additional model for IVC studies due to remarkable similarities with women and livestock animals regarding in vivo and in vitro ovarian folliculogenesis. For a successful IVC system, several factors should be carefully considered to provide an optimum culture environment able to support the viability and growth of preantral follicles enclosed in ovarian tissue. The cryopreservation of the ovarian tissue is another important in vitro manipulation technique that has been used to preserve the reproductive potential in humans and, in the future, may be used in highly valuable domestic animals or endangered species. Several improvements in cryopreservation protocols are necessary to support the utilization of ovarian tissue of different species in follow-up ARTs (e.g., ovarian fragment transplantation). This review aims to provide an update on the most current advances regarding supportive in vitro techniques used in equids to evaluate and manipulate preantral follicles and ovarian tissue, as well as methodological approaches used during IVC and cryopreservation techniques.

Persian onager (Equus hemionus onager) endometrial explant cryopreservation and in vitro culture.

Assisted reproduction of endangered equids, such as Persian onagers (Equus hemionus onager), is vital for species conservation. Little is known about Persian onager reproductive functions, including functions of the uterine endometrium. Recently, successful cryopreservation of the domestic mare endometrium was reported, but there is no information on cryo-sensitivity or in vitro culture of endometrial tissues of any non-domestic equid. In the present study, endometrial explants from Persian onagers were cryopreserved and cultured in vitro for 5 days. There was no difference between endometrial explants when 10% and 20% dimethyl sulfoxide (DMSO) was used for cryopreservation. Cell viability and structural integrity were comparable to fresh tissue. Abundance of estrogen receptor-α (ESR1) and progesterone receptor (PGR) mRNA transcript in endometrial explants was less in most treatment groups compared to the fresh tissue control. There was variation in E-cadherin mRNA abundance in endometrial explants among treatment groups with some treatment groups having a lesser abundance compared to the control group. The abundance of Ki67 mRNA transcript of endometrial explants was not different among treatment groups compared to the control group. Results indicate that DMSO is a suitable cryoprotectant for the Persian onager endometrium, and in vitro culture in a liquid-gas interface can maintain Persian onager endometrial explants for as long as 5 days. Findings allow for a greater understanding of reproductive mechanisms in vitro for this endangered species and other domestic equids including donkeys.

Luteinizing hormone and ovarian steroids affect in vitro prostaglandin production in the equine myometrium and endometrium.

Prostaglandins (PGs) play crucial roles in the regulation of the oestrus cycle and establishment of pregnancy in animals. Luteinizing hormone (LH) and ovarian steroids are involved in regulating endometrial PG production in many species. Their effects on PG production and associated pathways in the mare myometrium and endometrium are the subjects of our interest. This study aimed to evaluate the specific effects of LH and ovarian steroids on equine myometrial and endometrial tissues on (i) PGE2 and PGF2α secretion and (ii) transcription of genes encoding specific enzymes responsible for PG synthesis, such as prostaglandin-endoperoxide synthase (PTGS2), PGE2 synthases (PGES), PGF2α synthases (PGFS), and PGI2 synthases (PGIS), using equine myometrial and endometrial explants. Equine myometrial and endometrial tissues were collected at the mid-luteal (n = 6) and follicular (n = 6) phases of the oestrus cycle and were exposed to: (1) vehicle (control), (2) arachidonic acid (AA, 50 ng/mL, positive control), (3) LH (10 ng/mL), (4) progesterone (P4, 10-7M) and (5) 17-ß oestradiol (E2, 10-9M) for 24 h. After exposure, PGF2α and PGE2 concentrations were determined using direct enzyme immunoassays. Alterations in PG synthase mRNA expression were determined using RT-qPCR. After 24 h, LH and P4 increased PGE2 and PGF2α secretion by myometrial tissues at the mid-luteal phase (P < 0.05), whereas PG secretion was augmented by LH and E2 during the follicular phase (P < 0.01). In contrast, LH and E2 increased PGE2 and PGF2α secretion by endometrial tissues during the mid-luteal phase (P < 0.05), while E2 enhanced PGE2 secretion during the follicular phase of the oestrus cycle (P < 0.01). These results indicate that LH and ovarian steroids modulate PG production in equine myometrial and endometrial tissues and affect PG synthase expression at the mRNA level. We conclude that the equine myometrium is an alternative source of PG production and participates in the regulation of uterus function during the oestrus cycle.

Evaluation of the recombinant proteins RlpB and VacJ as a vaccine for protection against Glaesserella parasuis in pigs.

BACKGROUND: Glaesserella parasuis, the causative agent of Glӓsser's disease, is widespread in swine globally resulting in significant economic losses to the swine industry. Prevention of Glӓsser's disease in pigs has been plagued with an inability to design broadly protective vaccines, as many bacterin based platforms generate serovar or strain specific immunity. Subunit vaccines are of interest to provide protective immunity to multiple strains of G. parasuis. Selected proteins for subunit vaccination should be widespread, highly conserved, and surface exposed. RESULTS: Two candidate proteins for subunit vaccination (RlpB and VacJ) against G. parasuis were identified using random mutagenesis and an in vitro organ culture system. Pigs were vaccinated with recombinant RlpB and VacJ, outer membrane proteins with important contributions to cellular function and viability. Though high antibody titers to the recombinant proteins and increased interferon-γ producing cells were found in subunit vaccinated animals, the pigs were not protected from developing systemic disease. CONCLUSIONS: It appears there may be insufficient RlpB and VacJ exposed on the bacterial surface for antibody to bind, preventing high RlpB and VacJ specific antibody titers from protecting animals from G. parasuis. Additionally, this work confirms the importance of utilizing the natural host species when assessing the efficacy of vaccine candidates.

Effects of melatonin on the in vitro growth of early antral follicles and maturation of ovine oocytes.

This study aimed to evaluate the effect of melatonin on the in vitro culture and maturation of isolated sheep early antral follicles. Isolated early antral follicles were cultured for 12 d in α-minimum essential medium (MEM+) alone (control) or α-MEM+ added with fixed different concentrations (100, 500, or 1,000 pg/mL) or a sequential concentration of melatonin (MelSeq; day 6 = 100; day 12 = 500 pg/mL). The percentage of morphologically normal follicles was higher (P < 0.05) in 500 pg/mL melatonin than the other treatments at 6 d. Mel 500 also showed a higher rate of fully grown oocytes (P < 0.05) than other treatments. After in vitro culture, reactive oxygen species (ROS) levels in oocytes were similar between Mel 500 and MelSeq, with both being lower (P < 0.05) than other treatments. Oocytes cultured in both Mel 500 and Mel 1000 showed glutathione peroxidase levels similar (P > 0.05) to the control group and higher (P < 0.05) than other treatments. Mitochondrial activity was similar (P > 0.05) among control, Mel 500, and Mel 1000 treatments. Mel 500 treatment presented a higher percentage of germinal vesicle breakdown oocytes than the control group and similar percentages to the other treatments. Follicles cultured in melatonin followed by oocyte maturation with the addition of 500 pg/mL melatonin in maturation medium showed increased (P < 0.05) levels of mitochondrial activity compared to α-MEM+ alone. In conclusion, the concentration of 500 pg/mL of melatonin promotes development and decreases ROS levels of ovine oocytes from in vitro grown early antral follicles. Moreover, melatonin increases mitochondrial activity and promotes the acquisition of meiotic competence of these oocytes.

Activation of goat primordial follicles in vitro: Influence of alginate and ovarian tissue.

The present study aimed to evaluate the effect of three culture systems on caprine primordial follicle activation in vitro: follicles cultured either in the isolated form within alginate (Isolated follicles + Alginate treatment), or enclosed in ovarian tissue (in situ), with or without alginate (Fragment + Alginate, and Fragment alone treatments, respectively). After culture, the Isolated follicles + Alginate treatment presented a percentage of morphologically normal follicles (MNF) similar to both the non-cultured control and the Fragment Alone treatments. Nevertheless, Fragment + Alginate treatment showed a significant reduction in the number of MNF when compared to the other treatments. Regarding follicle development, our results showed that regardless of the alginate, the presence of ovarian tissue limited primordial follicle activation during in vitro culture. Remarkably, the Isolated primordial follicle + Alginate treatment was the only one that significantly promoted follicle activation and increased both follicle and oocyte diameters during IVFC, pointing out a higher cell proliferation. In conclusion, the presence of ovarian tissue with or without alginate limited follicle development (activation) after culture. Nevertheless, when primordial follicles were isolated and encapsulated in alginate they presented suitable survival rates, higher rates of follicle activation and continued to grow throughout the culture period.

Evaluation of allogeneic freeze-dried platelet lysate in cartilage exposed to interleukin 1-ß in vitro.

BACKGROUND: Platelet-rich plasma (PRP) as well as other platelet-derived products have been used as a potential disease-modifying treatment for musculoskeletal diseases, such as osteoarthritis (OA). The restorative properties of such products rely mainly on the high concentrations of growth factors, demonstrating encouraging results experimentally and clinically. Yet, the autologous blood-derived nature of the PRP product lead to limitations that precludes it's widespread use. The main limitations for PRP use are; product variability, the need for minimum laboratory settings in most cases, and the need for storage at low temperatures to preserve its properties. Based on these limitations, the objective of this study was to investigate an allogeneic off-the-shelf platelet lysate (PL) in cartilage exposed to interleukin 1ß (IL-1ß). For this purpose, blood and cartilage were harvested from eight skeletally mature and healthy horses. Blood was processed into PL aliquots and divided into three groups (Frozen, Freeze-dried and Filtered freeze-dried), used in autologous and allogeneic conditions and in three different concentrations (1.5, 3 and 6-fold). Different PL preparations were then applied in cartilage culture with interleukin-1 beta and cultured for 10 days. Cartilage and media samples were collected and analyzed for total GAG and 35SO4-labeled GAG content. RESULTS: No significant differences between the controls and PL groups in cartilage and media were demonstrated. The effects of PL on cartilage matrix were concentration dependent and intermediate concentrations (3-fold) in PL showed increased 35SO4-labelled GAG in cartilage. CONCLUSION: In conclusion, the allogeneic freeze-dried PL presented equivalent effects compared to frozen autologous PL. Intermediate platelet concentration on average demonstrated improved results, demonstrating less GAG loss compared to other concentrations.

Effects of melatonin on morphology and development of primordial follicles during in vitro culture of bovine ovarian tissue.

This study aims to investigate the effect of melatonin on activation, growth and morphology of bovine primordial follicles, as well as on stromal cells density in ovarian tissues after in vitro culture. Ovarian fragments were cultured in α-MEM+ alone or supplemented with melatonin (250, 500, 1,000 or 2,000 pM) for a period of six days. Non-cultured and cultured tissues were processed for histological analysis; according to developmental stages, follicles were classified as primordial or growing follicles. These follicles were further classified as morphologically normal or degenerated. Ovarian stromal cell density was also evaluated. The percentages of primordial and developing follicles, as well as those classified of normal follicles, were compared by Fisher's exact test, and the differences were considered significant when p < .05. The results showed that the presence of 1,000 and 2,000 pM melatonin in culture medium promoted a reduction in the percentage of primordial follicles and an increase in the percentage of development follicles, when compared to follicles cultured in control medium. On the other hand, the presence of 250 or 500 pM melatonin did not show a significant effect on the percentage of primordial and developing follicles. Besides that, the presence of 500, 1,000 and 2,000 pM melatonin maintained the percentage of normal follicles similar to those seen uncultured control. Moreover, tissues cultured in presence of 1,000 pM melatonin showed a higher percentage of normal follicles when compared to follicles cultured in the presence of 250 pM melatonin. It was observed a similar profile of stromal density in both uncultured tissues and those cultured in vitro in the presence of melatonin. In conclusion, melatonin (1,000 and 2,000 pM) promotes bovine primordial follicles activation and maintains the stromal cell density during in vitro culture of ovarian cortical tissue.

Effect of oxygen tension on in vitro viability and development of dog follicles enclosed within the ovarian cortex.

Oxygen concentration has been shown to influence in vitro viability and growth of ovarian follicles. The present study examined the effect of oxygen tension on in vitro development of dog follicles enclosed within the ovarian cortex. Ovaries were obtained from domestic dogs (age, 8 months to 2 years), and cortical fragments were recovered. The cortices were then incubated on 1.5% (w/v) agarose gel blocks within a 4-well culture plate containing Eagle Minimum Essential Medium (MEM). Ovarian follicles within the tissues were processed for histology and assessed for follicle density, viability and diameter immediately after collection (Control) or after 2 or 5 days of in vitro incubation. Apoptotic cells were assessed using TUNEL assay. Comparisons of follicular viability and diameter were performed using analysis of variance followed by Tukey's test (p < 0.05). Comparisons of follicle density and apoptosis among treatments were conducted using Non-parametric Kruskal-Wallis test followed by Friedman's test (p < 0.05). No difference (p > 0.05) in follicle density was observed among groups at Day 2 of in vitro culture. However, the density of follicles within cortices cultured in 20% oxygen for 5 days significantly reduced compared to the Control and those incubated in 5% concentration. The viability of cultured follicles in all treatments decreased (p < 0.05) compared to the Control after 2 days incubation, and this value further reduced (p < 0.05) in 20% oxygen group at Day 5. There were no differences in the percentages of apoptotic follicles between the two treatment groups (p > 0.05). Nevertheless, after 5 days of culture, the percentage of TUNEL-positive follicles increased significantly (p < 0.05) in cortices incubated in 20% oxygen environment. In conclusion, our findings demonstrated that 5% oxygen level was superior to 20% concentration in sustaining in vitro viability of dog follicles enclosed within the ovarian cortex.

Growth differentiation factor-9 improves development, mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles.

This study analysed the effect of growth differentiation factor-9 (GDF-9) on the in vitro culture of isolated ovine secondary follicles. The follicles were cultured in α-MEM supplemented with BSA, insulin, glutamine, hypoxanthine, transferrin, selenium, ascorbic acid and FSH (α-MEM+ -control medium) or α-MEM+ supplemented with 1, 10, 50 or 100 ng/ml GDF-9. Next, the oocytes were destined to in vitro maturation (IVM). After 12 days of culture, there were no differences regarding the percentage of normal follicles, antrum formation and follicle diameter between the treatments (p > 0.05). The rates of fully grown oocytes (≥110 µm) were higher (p < 0.05) in 100 ng/ml GDF-9 than other treatments, except for 10 ng/ml of GDF-9 (p > 0.05). Treatment containing 100 ng/ml GDF-9 showed higher (p < 0.05) mitochondrial activity than the control group. Moreover, 100 ng/ml GDF-9 showed more oocytes in MI than α-MEM+ , 1 or 50 ng/ml GDF-9 (p < 0.05). In conclusion, 100 ng/ml GDF-9 increased the growth, mitochondrial function and meiotic resumption of oocytes from in vitro grown sheep secondary follicles.

Leptin induced in vitro development of ovarian follicles in sheep is related to the expression of P450 aromatase and steroidogenesis.

The objective of this study was to test the hypothesis that Leptin induced in vitro growth in preantral follicles in sheep involves modulation of P450 aromatase expression and steroidogenesis. Accordingly, the expression of P450 aromatase gene was studied in the cumulus cells and oocytes isolated from different stages of preantral follicles (PFs') grown in vivo, cultured in TCM 199B, TCM 199B + Leptin (10 ng/ml) (TCM199BL) or a standard PF culture medium supplemented with Leptin (10 ng/ml) (SML). Ovarian follicles grown in vivo or in SML expressed P450 aromatase both in cumulus cells and oocytes at all the development stages. In the oocytes from PFs' grown in vitro, P450 expression was consistently lower than in those from in vivo grown follicles at all except the preantral stage. The patterns of expression of aromatase gene in the cumulus cells from in vivo grown and the PFs' cultured in TCM 199BL were similar. Significantly higher levels of progesterone production were supported by SML at all the development stages than the other two media. Oestradiol concentration in the spent TCM 199B and SML showed a significant increase as the development progressed from preantral to large antral stage. However, such increase was not sustained beyond early antral stage in the PFs' cultured in TCM199BL. It is concluded that Leptin modulates the expression P450 aromatase while supporting the in vitro development of the ovarian follicles in sheep.

Effect of sphingosine-1-phosphate on cryopreserved sheep testicular explants cultured in vitro.

Complete spermatogenesis has been achieved in vitro in mouse testicular explants with resulting sperm used to produce pups after Intra Cytoplasm Sperm Injection and Embryo Transfer. In the present study, we evaluated the influence of sphingosine-1-phosphate (S1P) on spermatogenesis of frozen-thawed lamb testis explants in vitro. Thawed testicular pieces were cultured for 12 d on agarose blocks in serum-free growth medium containing 0, 2, 5 or 10 µM S1P. At the end of D6 and D12, some pieces were fixed and processed for histology. Other pieces were processed for RNA isolation and quantitation of proliferation (PCNA, Ki67) and differentiation (PLZF) markers and genes involved in S1P signaling (S1PR1, SGPL1, SGPP1, AKT1 and NFKBIA) by qPCR. Histology revealed an increase (P < 0.05) in seminiferous cord (SC) diameter under all culture conditions, except 5 and 10 µM S1P by D6. In the presence of 5 µM S1P, percentage of gonocytes decreased (P < 0.05) by D6 (control, 24.9% vs. S1P, 10.3%) with a concomitant increase (P < 0.05) in spermatogonia formation (control, 74.4% vs. S1P, 88.1%). S1P induced PCNA or Ki67 expression by D6, whereas PLZF was up-regulated (P < 0.05) by D6 in 2 µM S1P and D12 in 5 & 10 µM S1P. Expression of SGPL1 and SGPP1 increased 4-12-fold in tissues cultured in 10 µM S1P by D12 compared to D12 control. AKT1 and NFKBIA mRNA expression was low (P < 0.05) in 5 and or 10 µM S1P treatments on D6. These results demonstrate that S1P promotes germ cell proliferation during first week of culture and may exert an anti-apoptotic influence on the seminiferous cord in sheep testicular explants in vitro.