Ham F12 Research Articles

Novel combination with maca improves sperm parameters in vitro of asthenozoospermic men.

Infertility is an inability to conceive after a reasonable period of time (12 months) without the use of contraception or due to a person's incapacity to procreate, whether independently or with a spouse. Problems with the production and maturation of sperm are the most common causes of male infertility additionally; the motility is the major functional character that determines the fertilizing ability of spermatozoa. Therefore the goal of this study is to get better certain sperm function parameters in vitro of asthenozoospermic patient. The World Health Organization (WHO) and many studies considered the infertility as a disease and so many couples complaining from unsuccessful assisted reproductive technologies (ART) procedures to overcome their problem. The goal of this study is to improve certain sperm function feature in vitro of asthenozoospermic semen patients by using combination of motility inducing namely; Maca, L-carnitine and Pentoxifylline that enhance the medium to improve certain sperm characters that might be utilized for ART centers. Semen aliquots were collected from ninety patients with asthenozoospermia who participated in present study, the volume of semen samples with normal ejaculate when was ranged between 1.4-6ml and can be measured by using a measure pipette or conical graduated tube; Inclusion criteria was Asthenozoospermia, oligozoospermia and teratozoospermia men, Infertile idiopathic men also, fertile normozoospermic men. While Exclusion criteria was Azoospermic men, Alcoholic, Patients under treatment with antibiotics and men with Varicocele.The samples split into two equal groups at random. Using Ham's F12 medium, one portion served as the control group, and the other was the treatment group, which was mixing by combining the following ingredients, Maca powder extracts (Lepidium meyenii) (M) 1 mg/ml, 0.5 mg/ml of L-Carnitine (LC), and 10 mg/ml of Pentoxifylline (PTX). The data were analyzed using Statistical Package for Social Sciences (SPSS) version 23.0. The descriptive statistics including frequency, range, mean and standard error, Data from treated and control groups were expressed as mean ± SEM and to compare value between experimental and control groups using Students t-test. Layering approach is used to investigate sperm parameters before and after in vitro activation. The information showed a very large (p< 0.001) increase in active sperm motility grade A, percentage of progressive motility with significant increase in morphologically normal sperm (MNS) with decreased in DNA fragmentation index after activation by layering technique with novel combination medium compared to Hams F12 medium and before activation. The present work stated that novel combination medium (LC, maca and PXT) have potential effects to improve sperm characters in male infertility factors and suggested to be used for sperm preparation and activation in ART programs.

Cadherin switching in oral squamous cell carcinoma: A clinicopathological study.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide as it represents the sixth most common cancer. Numerous molecular mechanisms have been explained to regulate OSCC progression, including epithelial-mesenchymal transition (EMT). Cadherin switching is the pivotal process that controls EMT in which E-cadherin reduces while N-cadherin elevates. This work aimed to clarify the role of cadherin switching in OSCC. Thirty paraffin-embedded tissue blocks of OSCC including six cases with lymph node metastasis were subjected to immunohistochemical staining using antibodies against E&N-cadherins. Cell cultures were performed using OSCC cell lines (SCC-15/SCC-25) from the human tongue. F-12K medium (Kaighn's Modification of Ham's F12 Medium) was added as EMT inducing media. E&N-cadherin mRNA gene expression levels were detected by real time-polymerase chain reaction (RT-PCR). Cadherin switching through N-cadherin elevation and E-cadherin reduction was evaluated at the histopathologic level in primary and metastatic OSCC as well as at the genetic level within OSCC cell culture. Cadherin switching showed a significant correlation between E&N-cadherins at different histopathological grades of OSCC and in metastatic OSCC. Moreover, the level of mRNA gene expression of E&N-cadherins in human 15 SCC and 25 SCC cell lines with EMT-inducing media exhibited a significant correlation. Cadherin switching is a crucial event in the EMT process. It may be used as a significant tool in the study of OSCC progression. Cadherin switching plays a significant role in the invasion and metastasis of OSCC.

Effect of Pre-Incubation of Cryopreserved Sperm with either Kisspeptin or Glutathione to Mitigate Freeze-Thaw Damage.

Sperm cryopreservation reduces sperm quality. Kisspeptin (KP) has beneficial effects on sperm functions. This study compares the effect of KP and Glutathione (GSH) on mitigating the detrimental effects of the freeze-thaw cycle on sperm. An experimental study was conducted in Birjand (Iran) during 2018-2020. Thirty normal swim-up semen samples were treated with Ham's F10 medium (negative control), 1 mM GSH (positive control), or KP (10 µM) for 30 min before freezing. The motility, acrosome reaction, capacitation, and DNA quality of the frozen-thawed sperms were assessed according to the WHO guidelines. Statistical analysis was performed using paired t test, one-way analysis of variance, and least significant difference. Pre-incubation with KP significantly increased the percentage of sperm motility (34.00±6.7, P=0.003) compared to the control (20.44±7.4) and GSH-treated (31.25±12.2) aliquots. The frequency of non-capacitated spermatozoa was significantly higher in the KP-treated group (98.73%) than in the control (96.46%) and GSH-treated (96.49%) aliquots (P<0.001). The percentage of acrosome-intact spermatozoa in the KP-treated group (77.44%) was significantly higher than the control (74.3%) and GSH-treated (74.54%) groups (P<0.001). The sperm frequency with normal histone in the KP-treated group (51.86%) and with normal protamine (65.39%) was significantly higher than the controls (P=0.001 and P=0.002, respectively). The percentage of TUNEL-positive sperm was significantly lower in the KP-treated group (9.09±2.71) than both GSH-treated (11.22±2.73) and control (11.31±2.2) groups (both P=0.002). Pre-incubation with KP protects sperm motility and DNA integrity from the detrimental effect of the freeze-thaw cycle. KP is suitable as a pre-treatment to control sperm quality during freezing-thawing.

Adaptation process of engineered cell line FCHO/IL-24 stably secreted rhIL-24 in serum-free suspension culture

Interleukin-24 (IL-24) displays tumor cell-specific proliferation inhibition in vitro and in vivo. Recombinant human IL-24 (rhIL-24) has significantly higher activity, yet significantly lower expression level in mammalian cells than in bacteria. To further realize therapeutic potential of IL-24, we enhanced rhIL-24 expression in mammalian cell systems by adapting engineered Flp-InTMCHO/IL-24 (FCHO/IL-24) cells (adherent cultured in Ham's F12 medium with 10% serum) to serum-free suspension culture. First, MTT assay showed that among four different media (F12, DMEM/F12, 1640 and DMEM), DMEM/F12 medium was the most suitable media for lower-serum adherent culture. Then, cells were adherently cultured in DMEM/F12 with serum concentration reduced from 10% to 0.5% in a gradient manner. Compared to cells in 10% serum, cells in 0.5% serum displayed significantly lower relative cell viability by 40%, increased G0/G1 phase arrest (8.5 ± 2.4%, p < 0.05), decreased supernatant rhIL-24 concentration by 73%, and altered metabolite profiles, such as glucose, lactate and ammonia concentration. Next, the cells were directly adapted to 0.5% serum suspension culture in 125 mL shake flask at 119 rpm with the optimal cell seeding density of 5 × 105 cells/mL (3.3 times higher than that of adherent culture), under which the concentration of rhIL-24 in culture medium was stable at 3.5 ng/mL. Finally, cells adapted to 0.5% serum proliferated better in serum-free medium Eden™-B300S with higher rhIL-24 expression level compared to CDM4CHO. The successful adaptation of engineered cells FCHO/IL-24 laid foundation for adapting cells from adherent culture to suspension serum-free culture to mass produce rhIL-24 protein for therapeutic purposes.

Regulation of cellular communication network factor 1 by Ras homolog family member A in bovine steroidogenic luteal cells.

Development of the corpus luteum (CL) requires the growth of a new capillary network from preexisting vasculature, a process known as angiogenesis. Successful building of this capillary network occurs through a sequence of cellular events-differentiation, proliferation, migration, and adhesion-which are regulated by a suite of angiogenic proteins that includes cellular communication network factor 1 (CCN1). We previously reported that the expression of CCN1 was highest in luteal tissue obtained from the early-cycle, 4-d-old bovine CL (i.e., corpus hemorrhagicum) compared to the mid- and late-cycle CL. In the present study, we treated steroidogenic bovine luteal cells from early-cycle CL with luteinizing hormone (LH), but it had no effect on CCN1 expression. Direct stimulation of the canonical LH pathway with forskolin and dibutyryl-cyclic adenosine monophosphate (cAMP), however, inhibited CCN1 mRNA expression. In endothelial cells, stimulation of Ras homolog family member A (RhoA) induces CCN1 expression, whereas RhoA inactivation inhibits it. Yet, it is unknown if regulation of CCN1 in steroidogenic luteal cells works likewise. We hypothesized that a similar mechanism of CCN1 regulation exists in bovine luteal cells and that thrombin, a known RhoA activator, may be a physiologic trigger for this mechanism in the early-cycle CL. To test this hypothesis, ovaries were collected from lactating dairy cows on days 3 or 4 of the estrous cycle, and corpora lutea were dissected and dissociated. Steroidogenic luteal cells were suspended in defined Ham's F12 medium, supplemented with insulin/transferrin/selenium and gentamicin, and seeded into 6-well plates. After 24 h, spent medium was replaced with fresh Ham's F12, and the cells were cultured for 24 to 48 h. Cells were treated for 2 h with defined medium, 10% fetal bovine serum (FBS), thrombin (1, 5, 10 U/mL), or Rho Activator II (0.25, 1, 2 μg/mL). Cells were then lysed for RNA extraction, followed by cDNA generation, and quantitative polymerase chain reaction (qPCR). Thrombin (1, 5, 10 U/mL; n = 3) and Rho Activator II (0.25, 1, 2 μg/mL; n = 6) increased (P < 0.05) CCN1 mRNA expression. In summary, CCN1 in bovine steroidogenic luteal cells was induced by thrombin and appeared to be regulated in a Rho-dependent manner. Future work will elucidate the signaling partners downstream of Rho which leads to CCN1 gene expression.

Phase transitions and mechanisms of cryoprotection of serum-/xeno-free media based on dextran and dimethyl sulfoxide

The development of serum-/xeno-free media may help avoid the drawbacks of using serum and its components, such as probable contamination, instability of composition, or difficulty in sterilization. The objectives of this research were to investigate the use of combinations of a permeating cryoprotective agent (Me2SO) and non-permeating polymers (polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, hydroxyethyl starch, dextran) for cryopreservation of interstitial cells (ICs) of rat testis, and to propose the mechanism of cryoprotection of such compositions. In the course of this study, the best combination was 100 mg/ml dextran (M.m. 40 kDa) (Dex40) with 0.7 M Me2SO in Ham's F12. The ICs were additionally cooled and warmed to different end temperatures (−30, −50, −50 and −196 °C) to determine which temperature intervals contributed most to the IC loss. Then, the cryoprotective action of this serum-/xeno-free medium was investigated in comparison with serum or albumin-containing media by differential scanning calorimetry (DSC) and thermomechanical analysis (TMA). The results showed that the medium based on Dex40 did not decrease the amount of ice formed. However, it could undergo other phase separation and phase transformation to form glassy states. Potential cell-damaging physical processes such as eutectic crystallization/melting, recrystallization of NaCl and/or Me2SO derivatives, found in serum-containing media and taking place in specific temperature intervals, were not observed in the Dex40 based media. This was in good correlation with indicators of cell survival. Additionally, the application of Dex40 allowed using Me2SO in lower concentrations (0.7 M) than required for serum-containing media (1.4 M), which may decrease the toxicity of serum-/xeno-free media.

Ultra-Rapid Freezing Preserves Morphofunctional Integrity and Fertilizing Ability of Epididymal Cat Spermatozoa.

Vitrification and ultra-rapid freezing, which are more commonly used for oocytes and embryos, have recently been applied to spermatozoa in an attempt to make semen cryopreservation in field conditions easier compared to conventional freezing. It is well-known that in case of unexpected death of rare and wild animals, preserving epididymal spermatozoa from isolated testicles represents a great chance of salvaging male germplasm for future use in assisted reproductive technologies. The aim of this study was to evaluate the morphofunctional integrity of cat epididymal spermatozoa ultra-rapid frozen in pellets or straws with two different extenders [E1 (Tris buffer with 20% egg yolk and 0.25 M sucrose) or E2 (Ham's F10 with 1% bovine serum albumin and 0.4 M sucrose)] and to test whether spermatozoa preserved by the best combination were able to fertilize oocytes and produce embryos in vitro by intracytoplasmic sperm injection (ICSI) of in vitro matured cat oocytes. The results showed that E1 and E2 in straw or pellet were comparable (at warming, about 30% normal morphology, 45% intact membranes, and 20% intact acrosomes), except for post-warming motility that was better maintained along time by E1 pellet (21.7 ± 7.4% at warming and 3.6 ± 2.9% after 6 h). Such spermatozoa could fertilize conspecific oocytes and support embryonic development (cleavage 35.5%) as well as frozen control spermatozoa (cleavage 54.29%, p = 0.22). In conclusion, cat epididymal spermatozoa better maintained their morphofunctional features after ultra-rapid freezing with E1 and could successfully produce embryos in vitro after ICSI. This underscores their usefulness as cryobanked material for fertility and biodiversity preservation purposes.

Evaluation of cytotoxic effects of fungal origin nanosilver particles on oral cancer cell lines: An in vitro study.

Oral cancer is often associated with poor prognosis and it is found that conventional treatment options cause severe side effects, adjacent tissue disfigurement, and loss of function. Recently, silver nanoparticles (AgNPs) paved their path for cancer treatment. This study aimed to investigate cytotoxic effects of fungal procured AgNPs on oral squamous cell carcinoma (SCC-9) cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The silver nanoparticles were biosynthesized using the fungi Fusarium semitectum. Cell lines were cultured in a 1:1 ratio of Dulbecco's Modified Eagle's medium and Ham's F12 medium and subcultured in a T-75 cm2 flask. Cell count was adjusted to 1 × 105 cells/ml; 50,000 cells/well were seeded into a 96 well plate and incubated at 37°C, for 24 h in 5% CO2 humidified conditions. AgNPs (1.75-50 μl/ml) were added to the plates and further incubated at 37°C for another 24 h. Medium containing cells without AgNPs were used as a control group. Later, 20 μl of MTT was added to each well and incubated for 6 h at above-mentioned conditions. About 0.1 mL of Dulbecco's Modified Eagle's Medium solution was added to each well to solubilize formazan. The absorbance was measured using a Tecan reader at 540 nm. The experiment was repeated thrice independently. The percentage (%) inhibition of growth and the AgNP's concentration that prevents the cell growth by 50% (IC50) were determined. Significant dose-dependent inhibition of the growth of SCC-9 cell lines was seen and IC50 was found at 12 μl/ml concentration of AgNPs. Biosynthesized AgNPs of fungal origin exhibit effective anticancer properties against the SCC-9 cell line.

Effects of branched-chain amino acids on glucose uptake and lactose synthesis rates in bovine mammary epithelial cells and lactating mammary tissue slices

Even though supplementations of essential AA (EAA) are often related to increased lactose yields in dairy cows, underlying mechanisms connecting EAA availability to the mammary glands and lactose synthesis are poorly understood. The objective of this study was to examine the effects of branched-chain AA (BCAA) including Leu, Ile, and Val on (1) glucose transporter (GLUT1) abundance and glucose uptake, (2) the abundance of proteins regulating lactose synthesis pathway, and (3) fractional synthesis rates of lactose (FSR) using bovine mammary epithelial cells (BMEC) and mammary tissues slices (MTS). The BMEC (n = 4) were allocated randomly to regular Dulbecco's Modified Eagle Medium with Ham's F12 (DMEM/F12) medium (+EAA) or +EAA deficient (by 90%) in all EAA (-EAA), all BCAA (-BCAA), only Leu (-Leu), only Ile (-Ile) or only Val (-Val). Western immunoblotting analyses, depletion of glucose in media, and a proteomic analysis were performed to determine the abundance of GLUT1 in the cell membrane, net glucose uptake, and the abundance of enzymes involved in lactose synthesis pathway in BMEC, respectively. The MTS (n = 6) were allocated randomly to DMEM/F12 medium having all EAA and 13C-glucose at concentrations similar to plasma concentrations of cows (+EAAp), and +EAAp deprived of all BCAA (-BCAAp) or only Leu (-Leup) for 3 h. The 13C enrichments of free glucose pool in MTS (EGlu-free) and the enrichments of glucose incorporated into lactose in MTS and media [ELactose-bound (T&M)] were determined and used in calculating FSR. In BMEC, -BCAA increased the fraction of total GLUT1 translocated to the cell membrane and the fraction that was potentially glycosylated compared with +EAA. Among individual BCAA, only -Leu was associated with a 63% increase in GLUT1 translocated to the cell membrane and a 40% increase in glucose uptake of BMEC. The -BCAA tended to be related to a 75% increase in the abundance of hexokinase in BMEC. Deprivation of Leu tended to increase glucose uptake of MTS but did not affect EGlu-free, ELactose-bound (T&M), or FSR relative to +EAAp. On the other hand, -BCAAp did not affect glucose uptake of MTS but was related to lower ELactose-bound (T&M), or FSR relative to +EAAp. Considering together, decreasing Leu supply to mammary tissues enhances GLUT1 and thus glucose uptake, which, however, does not affect lactose synthesis rates. Moreover, the deficiency of other BCAA, Ile, and Val alone or together with the deficiency of Leu seemed to decrease lactose synthesis rates without affecting glucose uptake. The data also emphasize the importance of addressing the effect of the supply of other nutrients to the mammary glands than the precursor supply in describing the synthesis of a milk component.

Impact of biological and artificial seminal fluids on sperm parameters and DNA status in asthenozoospermic ejaculates

The chemical composition and physiological properties of seminal fluid (SF) affect sperm quality. The objective was to investigate the effects of in vitro exposure of artificial seminal fluid (ASF) and biological seminal fluid (SF) on sperm quality. Asthenozoospermic ejaculates (n=20) were divided into two aliquots. The first aliquot was centrifuged for obtaining asthenozoospermic SF. The second aliquot was processed with density gradient centrifugation (DGC), and the pellet was diluted separately with following media: (a) ASF; (b) Ham's F10; (c) normozoospermic SF; and (d) asthenozoospermic SF. Sperm parameters and DNA status were assessed after DGC, as well as 2 and 24hr after incubation. The data showed that sperm progressive motility, viability and DNA integrity were significantly higher in ASF than Ham's F10 medium immediately after DGC. At 2 and 24hr, the progressive motility was significantly decreased in biological SF compared with ASF and Ham's F10. DNA fragmentation index (DFI) was significantly lower in normozoospermic SF than asthenozoospermic SF and Ham's F10 at time 2hr. In conclusion, normal SF showed the protective role on sperm DNA structure. Moreover, ASF preserved sperm motility better than biological SF during 24hr, despite being similar to normal SF regarding DNA integrity preservation in short time.

The effect of dilution rate and successive semen collections on sperm quality and sexual motivation of sexually mature South African Merino rams.

This study investigated whether dilution rate and successive semen collections influenced sperm viability, morphology, motility and male sexual motivation in sexually mature South African Merino rams (SAMR). Semen was collected from 11 rams up to either sperm or behavioural exhaustion. Semen was then immediately serial diluted to make 0, 2, 4, 8 and 16× dilutions with Hams F10 diluent. Following dilution, sperm motility was evaluated using computer-assisted sperm analysis (SCA®), while sperm morphology and viability were assessed using nigrosin-eosin staining and SYBR14/PI, respectively. Male sexual motivation was recorded by reaction time to first mount, while male dexterity was calculated as the ratio of mounts to ejaculations. An increase of dilution rate did not affect sperm motility (P > 0.05) but resulted in a significant decrease in the percentage of live normal sperm (P < 0.05). Furthermore, while sperm concentration and number decreased with semen collection number (P < 0.05), no effect was detected on sperm viability, morphology and motility (P > 0.05), except for average curvilinear velocity which showed a biphasic trend (P < 0.05). Mating success and reaction time were negatively affected by successive semen collections (P < 0.05), while male dexterity was unaffected (P > 0.05). Nevertheless, relatively high numbers of motile sperm (>2 billion) were collected up to the 4th successive semen collection, with a short initial reaction period (<25 s) and good success rate (>65%). SAMR thus withstood frequent semen collections without affecting their sperm reserves or sexual motivation. Further studies are required to investigate optimal conditions for semen collection and artificial insemination in this breed.

Anti-cancer Effects of Pomegranate Seed Oil on Esophageal Cancer Cell Line (KYSE-30)

Background: Esophageal cancer is the eighth most common cancer and the sixth most frequent cancer-related death. Despite improvements in treatment approaches, it has remained one of the most challenging cancers for treatment. Thus, it is necessary to introduce novel and efficient methods for treatment. In this regard, the anti-cancer and antioxidant properties of pomegranate have been more concerned. Objectives: The present study aimed to assess the potential cytotoxicity effects of pomegranate on the esophageal cancer cell line. Methods: We cultured esophageal cancer cell line (KYSE-30) and fibroblast cell line (HF2FF) as normal cell line in RPMI-1640/Ham's F12 and RPMI-1640 medium, respectively, with different concentrations of pomegranate seed oil (2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/mL). Then, we evaluated the cytotoxic effects of pomegranate seed oil via morphologic observation and MTT assay after 24, 48, and 72 hours. The wound-healing assay was used for the evaluation of the mobility and migration potential of the treated cells. Results: The results of the MTT assay showed the cytotoxicity and growth inhibitory effects of pomegranate seed oil on esophageal cancer cells. The viability of tumor cells was significantly reduced compared with the untreated control cells and normal cells (P &lt; 0.05), which were dose and time-dependent. The results of the wound-healing assay indicated that pomegranate seed oil could reduce the migration ability of KYSE-30 cells. Conclusions: According to the results, pomegranate seed oil seems to have cytotoxicity effects on esophageal cancer cells. Thus, it might be considered a new, cheap, and safe treatment option for esophageal cancer.

Studying the effect of adding growth factors to the autologous melanocyte keratinocyte suspension in segmental vitiligo

Addition of different growth factors to the medium used in autologous melanocyte-keratinocyte transplantation procedure (MKTP) was reported in the literature. The aim of the current study was comparison of response to MKTP in segmental vitiligo (SV) with and without adding growth factors to the suspension medium. Eighteen cases with SV were randomly divided into two groups. In group A: Ham F12 medium was used for suspension and in group B: 5 ng/mL recombinant basic fibroblast growth factor (bFGF) and 25 mg/500 mL 3'5' cyclic adenosine monophosphate (cAMP) were added to the medium. All cases received NB-UVB twice weekly for 24 weeks. The area of vitiligo lesions was measured before and after therapy by point-counting technique and complications were recorded. Excellent response (90%-100% repigmentation) occurred in 5/9 cases (56%) in group A and 7/9 cases (78%) in group B (with growth factors). A significant decrease in the area of treated lesions before and after therapy was found in both groups A and B (P = .0012 and .0004, respectively), however, a higher percentage of reduction in area of vitiligo was seen in group B cases (70% in group A vs 90% in group B; P value: .028). Marginal halo was seen in five cases in group A and six in group B. In conclusion addition of bFGF and cAMP to MKTP medium improved the results of the procedure. It could be considered if economically feasible.

Protective Effect of Gallic Acid on Testicular Tissue, Sperm Parameters, and DNA Fragmentation against Toxicity Induced by Cyclophosphamide in Adult NMRI Mice.

This study aimed to investigate the protective effect of Gallic acid (GA) on the Cyclophosphamide (CP) toxicity induced in the reproductive system. After a pilot study for dose responses of Gallic acid ,Forty adult male NMRI mice were divided into 5 groups (n=8): control, sham (NaCl Serum: 0.2mL per day), CP (15 mg kg-1 per week; IP), GA (12.5 mg kg-1 per day ; IP) and GA (12.5 mg kg-1 per day ; IP) +CP(15 mg kg-1 per week; IP). After treatment, the left testis was detached and used for Histological examination and right testis used for Malondialdehyde (MDA) measures. Left caudal epididymis was placed in the Ham's F10 medium and released spermatozoa were used in order to analyze sperm parameters. Sperm DNA fragmentation was assessed by Sperm Chromatin Dispersion (SCD) method. In the CP group, there was a significant increase in the sperm DNA fragmentation (% 57.89 ± 23.91) compared with control group (% 24.52 ± 10.27). That was significantly improved by GA (12.5 mg kg-1 per day ; IP) in GA+CP group (% 28.4 ± 8.85) compared to CP group (p< .001).A significant increase was reported about MDA levels in CP group (6.26 ± 2.59) in compared with the control group (4.30 ± 2.05), But GA (3.24 ± 1.33) decreased it in GA+ CP group(p< .01). The histopathological investigation revealed marked testicular atrophy in CP group, whereas GA diminished these deviations (P< .05). Gallic acid can modify the reproductive toxicity of cyclophosphamide in NMRI mice and increase the antioxidant capacity of testis tissue.

Effect of Human Testicular Cells Conditioned Medium on In Vitro Maturation and Morphology of Mouse Oocytes.

BackgroundTesticular cell conditioned medium (TCCM) has been shown to induce female germ cell development in vitro from embryonic stem cells (ESCs). Testicular cells (TCs) secrete a variety of growth factors such as growth differentiation factor-9 (GDF-9), bone morphogenetic protein 4 (BMP-4), stem cell factor (SCF), leukemia inhibitory factor (LIF), and other, that could improve oocyte maturation. Here we have investigated the effect of human TCCM (hTCCM) on in vitro maturation (IVM) and morphology of mouse oocytes.Materials and MethodsIn this experimental study, 360 germinal vesicle (GV) oocytes were obtained from NMRI mice, aged 4-6 weeks that had received 5 IU pregnant mare's serum gonadotropin (PMSG) 48 hours before. GV oocytes were subjected to IVM. 120 GV oocytes were cultured in each medium; hTCCM as the test group, DMEM + 20%FBS as the control group and Ham’s F10 + HFF medium as the sham group. The rates of the IVM and perivi- telline space (PVS) changes were recorded at 8, 16 and 24 hours after culture. The metaphase II (MII) oocytes were subjected for in vitro fertilization (IVF) and the fertilization rate was evaluated after 1, 2, and 3 days.ResultsThere was a significant difference between the maturation rates in hTCCM (31.67% MII) and the control [0% MII, P<0.05, (7.5% MI, 52.5% deg. and 40%GV)] groups but there was not a significant difference between the maturation rates in hTCCM and the sham group (53.33% MII, P>0.05). IVF success rate for MII oocytes obtained from IVM in the hTCCM group was 28.94% (n=11). Our data showed that hTCCM is an effective medium for GV oocyte growth and maturation compared to the control medium.ConclusionOur findings show that TCCM supports oocyte IVM in mice and affect oocyte morphology.

Effects of temperature and storage time on the motility, viability, DNA integrity and apoptosis of processed human spermatozoa.

The aim of this study was to evaluate motility, viability, DNA integrity and apoptosis of spermatozoa when washed semen samples were kept for up to 12days at 4-6°C and 25°C. In this experimental study, 26 normozoospermic semen samples were washed twice in Modified Ham's F10 and resuspended in IVF fertilisation medium. Half of the specimens were stored at 4-6°C, and the other half was kept at 25°C for 12days. The proportions of viable, motile, spermatozoa with double-stranded DNA and apoptotic spermatozoa were examined during storage time. Apoptosis was measured using annexin V-PI staining followed by flow cytometry. Results showed that sperm motility and viability decreased during 12days of sample storage (p<.001). There was no significant difference between the two temperatures in terms of motility and viability for up to 2days (p<.05). The percentage of spermatozoa with double-stranded DNA remained unchanged during the 12days of storage at both temperatures (p>.05). Although there was no difference between the two temperatures in terms of motility, viability and apoptosis during the first two days of storage, storage of spermatozoa at 4-6°C is better than storage for a longer period than storage at 25°C. Sperm DNA resisted against denaturation during storage.

32 Bovine embryo cryopreservation in a chemically defined medium

Embryo cryopreservation media often contain an animal-derived component, such as bovine serum albumin (BSA) or fetal calf serum. However, the industry is faced with the issue of composition variability between batches and, most importantly, the risk of pathogen transmission. The aim of this study was to compare the effectiveness of two embryo cryopreservation ethylene glycol-based media: IMV's freezing medium with BSA (IMV Technologies) and a chemically defined medium containing STEMALPHA.CRYO3, called CRYO3 (Ref 5617, Stem Alpha). CRYO3 is produced according to EU good manufacturing practice, ensuring the composition and quality of the product. Bovine morulae were collected invivo and split into two groups to be slow frozen in both media. All results were analysed with chi-squared tests with Yates's correction. For the invitro approach of this study, frozen morulae (n=86) were thawed, cultured in medium (1:1:1 mixture of RPMI, Dulbecco's modified Eagle's medium, and Ham's F10) at 38.5°C with 5% CO2, and monitored every 24h for 72h to evaluate survival and expansion rates. Survival rate was higher in the CRYO3 group than in the BSA group (74 and 50%, respectively; P&amp;lt;0.05). Expansion and hatching rates followed the same trend: 38 and 14%, respectively, at 48h (P&amp;lt;0.05) and 40 and 18% at 72h (P&amp;lt;0.05). For the invivo approach of this study, frozen morulae (n=123) were thawed and transferred on the field to evaluate pregnancy and calving rates. Field results showed no significant difference between CRYO3 and the BSA-based medium for pregnancy rates (73 and 63%; P=0.3006) nor for the delivery rates (64 and 55%; P=0.3614).The trial was conducted with different AI teams and in 10 commercial herds, thus adding robustness to the data. This study has shown that CRYO3 can replace BSA in an ethylene glycol cryopreservation medium for invivo-produced bovine embryos. Therefore, this product eliminates some sanitary risk in embryo commerce, which is a concern in an international context.

231 Novel protocol for the invitro maintenance and expansion of adult epidermal LGR5+ stem cells

Currently, the standard for treatment of full-thickness skin wounds is skin grafts or bioengineered skin substitutes; however, this method is limited by the amount of intact donor skin and lack of follicles and architecture. Thus, a protocol is needed for the expansion and differentiation of adult epidermal and hair follicle stem cells for use in scaffold mediated tissue engineering. Recently, we developed a transgenic porcine model in which H2B-GFP is under the control of the LGR5 promoter. LGR5 is an established marker of stem cells, meaning this model can be used to track the development and behaviour of these cells. The focus of this project was to create a novel culture method for the maintenance and expansion of LGR5+ epidermal adult stem cells utilising the green fluorescent protein (GFP) tag. Single cell epidermal stem cells were isolated from porcine skin using dispase II (10mgmL−1; Sigma) and trypsin (0.05%; Corning). Porcine fetal fibroblasts (PFF) or mouse embryonic fibroblasts (MEF) were grown to 95% confluence in a 6-well plate. Feeder layer cells were mitotically inactivated by incubation with mitomycin C (Sigma Aldrich, 10μgmL−1). Three different media were tested: basal medium [Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, Corning; Ham's F12, ThermoFisher; fetal bovine serum, Gemini Bio-Products], basal media + 5-azacytidine (Sigma Aldrich) and CHIR99021 (Tocris), or basal media + keratinocyte growth supplements (transferrin, hydrocortisone, T3, adenine, insulin, cholera toxin; Sigma Aldrich, epidermal growth factor; R&amp;amp;D Systems). Epidermal cells were plated in each medium for both PFF and MEF feeder layers. Experiments were performed in technical duplicates and replicated 3 times. On Day 9, total numbers of colonies in each well were counted and number of GFP-positive cells were quantified using ImageJ (National Institutes of Health). Results in Table 1 show that overall, the MEF feeder layer was able to support a higher rate of growth (P&amp;lt;0.05) and maintain the LGR5+ lineage at a higher proportion under all of the experimental conditions (P&amp;lt;0.05). In the growth-supplemented media, MEFs had fewer colonies than PFFs, but MEF colonies were, on average, 2.5 times larger (P&amp;lt;0.05). Conditions containing 5-aza and CHIR were the only conditions to maintain the LGR5+ lineage on the feeder layer. Statistically significant differences (P&amp;lt;0.05) were determined using two-way ANOVA, followed by Tukey's HSD test. Next, LGR5+ cells will be plated in media containing additional growth factors to stimulate expansion, while using CHIR and 5-aza to maintain the LGR5+ lineage. This protocol could be used in scaffolds to create three-dimensional growth of skin invitro and lead to better grafts for burn victims. Table 1.Growth of LGR5+ cells in different media including 5-azacytidine (5-aza), CHIR 99021 (CHIR), and keratinocyte growth supplements Group1 Basal medium (BM) BM + 5-aza+ CHIR BM + growth supplements No. of colonies/well MEF 127.7±40.8AB 189.3±16.9A 87.3±14.6B PFF 65.0±14.1A 83.3±17.0AB 148±33.7B Average no. of GFP+ cells per frame MEF 0.5±0.8B 65.7±18.4A 1.8±1.7B PFF 0.9±1.0B 22.6±4.5A 0.3±0.6B A,BValues within rows with different superscripts differ (P ≤ 0.05). 1MEF=mouse embryonic fibroblasts; PFF=porcine fetal fibroblasts.

Epigenetic regulation of osteoblastogenesis by blackcurrant fruit extracts in vitro and in vivo

Osteoporosis is a major public health concern and is characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures. Osteoporosis and osteopenia affect over 45 million Americans annually with an estimated cost of &gt; $20 billion USD. Over 50% of women and ~13% of men over the age of 50 will suffer an osteoporosis‐related fracture in their lifetime resulting in loss of independence, health and productivity, thus the research and discovery of compounds that can enhance bone formation would be clinically significant. Blackcurrants (Ribes nigrum) are native to Central and Eastern Europe and Northern Asia and are used in traditional medicine for the treatment of cardiovascular disease, bone and muscle disorders, diabetes, viral infections and inflammation. The berries contain four major anthocyanins: delphinidin‐3‐O‐glucoside, delphinidin‐3‐O‐rutinoside, cyanidin‐3‐O‐glucoside, cyanidin‐3‐O‐rutinoside. We have previously shown that blackcurrant extracts (BCE) increase myoblastogenesis, and in this work, we investigate its effects in cultured hFOB human osteoblasts and osterix/Sp7:mCherry transgenic medaka. hFOB human osteoblasts were obtained from ATCC and cultured in 1:1 mixture of Ham's F12 Medium Dulbecco's Modified Eagle's medium, with 2.5 mM L‐glutamine (without phenol red). Double transgenic medaka (dT; osterix/Sp7:mCherry were maintained at UIC under protocol #17‐166. Larvae at 9–12 dpf were staged and then treated for 5 days with the extracts and compounds (1–10 mg/ml). Osteoblast formation and distribution were analyzed by fluorescence microscopy, bone mineralization was visualized by calcein. The results of this investigation demonstrate that BCE and delphinidin‐3‐glucose increased osteoblast proliferation and reduced apoptosis in cultured human osteoblasts (hFOB) by reducing Bax and p53 expression and altering HDAC1, HDAC3, SIRT 3 and PGC1amRNA expression. In osterix/Sp7:mCherry medaka, BCE treatment (10 mg/ml) increased osteoblast proliferation by increasing osterix/Sp7 expression. These data suggest that BCE and D3G increase osteoblastogenesis through epigenetic regulation of HDACs and mitochondrial biogenesis.Support or Funding InformationNCCIH at the National Institutes of HealthThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acai fruit extracts and anthocyanins alter osteoblast proliferation and the expression of HDAC1 and osterix/Sp7 in vitro and in vivo

Musculoskeletal injuries and disorders are the leading cause of disability in the United States and affect more than one out of every two persons in the United States age 18 and over, and nearly three out of four persons aged 65 and over. Bone health is critical to the overall health and quality of life for Americans, and greater efforts are needed to address the burdens associated with osteoporosis and related bone diseases. Evidence from a variety of studies strongly points to a positive link between fruit and vegetable consumption and indices of bone and joint health, although the mechanisms are not well understood. In this work we investigate the effects of Acai fruit extract (AFE) and anthocyanidins on osteoblastogenesis in vitro and in vivo. hFOB human osteoblasts were obtained from ATCC and cultured in 1:1 mixture of Ham's F12 Medium Dulbecco's Modified Eagle's medium, with 2.5 mM L‐glutamine (without phenol red). An extract of acai fruits (AFE) was prepared in methanol and purified cyanidin‐3‐glucoside were purchased. Double transgenic medaka (dT; osterix/Sp7:mCherry were maintained at UIC under protocol #17‐166. Larvae at 9–12 dpf were staged and then treated for 5 days with the extracts and compounds (1–10 mg/ml). Osteoblast formation and distribution were analyzed by fluorescence microscopy, bone mineralization was visualized by calcein. The results show that AE and cyanidin‐3‐glucoside (C3G) increased osteoblast proliferation and reduced apoptosis in hFOB 1.19 osteoblasts induced by serum and glucose starvation. Gene expression data showed that C3G increased HDAC1 expression, increased BCL‐2 and reduced BAX mRNA and caspase 3/7 in apoptotic hFOB cells. AFE increased the proliferation of osteoblasts in dT osx/Sp7:mCherry medaka by enhancing the expression of osx/Sp7. Osterix (also known as transcription factor Sp7) is an osteoblast‐specific transcription factor which activates a repertoire of genes during differentiation of preosteoblasts into mature osteoblasts and osteocytes. AFE and C3G enhanced proliferation of hFOB cells, reduced hFOB apoptosis induced by serum and glucose starvation by via epigenetic regulation of HDAC1, and by increasing BCl‐2 and reducing the expression of BAX and caspase 3/7. The AFE enhanced osteoblast proliferation in osx/Sp7:mCherry medaka, and increased osx/sp7 expression.Support or Funding InformationNCCIH at NIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.