scholarly journals In vitro growth and steroidogenesis of dog follicles are influenced by the physical and hormonal microenvironment

Reproduction ◽  
2011 ◽  
Vol 142 (1) ◽  
pp. 113-122 ◽  
Author(s):  
N Songsasen ◽  
T K Woodruff ◽  
D E Wildt
Keyword(s):  
Structural Integrity ◽  
Cell Function ◽  
Separate Experiment ◽  
In Vitro Growth ◽  
Follicle Growth ◽  
Steroid Production ◽  
Follicle Diameter ◽  
Equine Chorionic Gonadotropin ◽  
Over Time

The present study examined the influences of the physical and hormonal microenvironment on in vitro growth and steroidogenesis of dog follicles. Follicles were enzymatically isolated and individually encapsulated in 0.5% (w/v; n=17) or 1.5% (n=10) alginate and cultured with 0.5 IU/ml equine chorionic gonadotropin for 192 h. In a separate experiment, follicles were encapsulated in 0.5% alginate and cultured with 0 (n=22), 1 (n=23), 10 (n=20) or 100 (n=21) μg/ml FSH for 240 h. Follicle diameter and steroid production were assessed every 48 h in both studies. Follicles encapsulated in the 0.5% alginate grew faster (P<0.05) than those cultured in the 1.5% concentration. Oestradiol (E2) and progesterone (P4) increased consistently (P<0.05) over time, and follicles in the 1.5% alginate produced more (P<0.05) P4 than those in the 0.5% solution. Follicles cultured in the highest FSH concentration (100 μg/ml) increased 100% in size after 240 h compared with 50 to 70% in lower dosages. E2 concentration remained unchanged over time (P>0.05) across FSH dosages. However, P4 increased (P<0.05) as culture progressed and with increasing FSH concentration. Results demonstrate that dog follicles cultured in alginate retain structural integrity, grow in size and are hormonally active. Lower alginate and increasing FSH concentrations promote in vitro follicle growth. However, the absence of an E2 rise in follicles cultured in FSH alone suggests the need for LH supplementation to support theca cell differentiation and granulosa cell function.

Follicular size and stage and gonadotropin concentration affect alginate-encapsulated in vitro growth and survival of pre- and early antral dog follicles

2017 ◽  
Vol 29 (2) ◽  
pp. 262 ◽  
Author(s):  
Jennifer Nagashima ◽  
David E. Wildt ◽  
Alexander J. Travis ◽  
Nucharin Songsasen
Keyword(s):  
Developmental Stage ◽  
Cell Activity ◽  
Domestic Dog ◽  
In Vitro Growth ◽  
Follicle Growth ◽  
Growth And Survival ◽  
Alginate Encapsulation ◽  
Follicular Size ◽  
Over Time

Understanding stage-specific requirements of mammalian folliculogenesis is limited in the domestic dog. The present study examined the effects of two potential regulators of dog follicle growth and survival in vitro, namely the original stage of the follicle (i.e. preantral (≤230 µm diameter) vs early antral (diameter from >230 to ≤330 µm) and FSH and/or LH concentrations. After isolation and alginate encapsulation, follicles were cultured in 0, 1, 10 or 100 µg mL–1 FSH and 0, 1 or 10 ng mL–1 LH for 20 days. Regardless of stage, FSH promoted growth, but LH did the same only in the absence of FSH. Production of 17β-oestradiol and progesterone was detectable, indicating theca cell activity. The greatest growth occurred in preantral (mean (± s.d.) 61.4 ± 25.9%) versus antral (42.6 ± 20.3%) follicles, but neither developmental stage nor gonadotropin affected survival. Antrum detection was minimal due, in part, to antral collapse, and oocytes exhibited an increasingly pale appearance and chromatin degeneration over time. The results demonstrate that pre- and early antral stage dog follicles encapsulated in alginate grow significantly in vitro. However, because FSH and LH alone or in combination fail to promote antrum development, the next step is identifying factors that enhance antral expansion.

224. Platelet derived growth factors and receptors in the rat ovary contribute towards preantral follicle growth

2005 ◽  
Vol 17 (9) ◽  
pp. 87
Author(s):  
L. S. Sleer ◽  
C. C. Taylor
Keyword(s):  
Growth Factors ◽  
Preantral Follicle ◽  
Follicle Growth ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Follicle Diameter ◽  
The Family ◽  
Rat Ovary ◽  
In Cells

In this study, the family of platelet derived growth factors (PDGF) and receptors were identified and characterized in the rat ovary and a role in contributing towards growth of preantral follicles was revealed. Real-time polymerase chain reaction revealed the presence of mRNA for all platelet derived growth factors (PDGF-A, PDGF-B, PDGF-C and PDGF-D) and receptors (PDGF-Rα and PDGF-Rβ). In situ hybridization identified oocytes of primordial/primary follicles and cells of the theca layer as a source of PDGF-B, PDGF-C and PDGF-D mRNA. Protein expression was explored through immunohistochemistry. In rats aged days 0 and 4, PDGF-Rα, PDGF-A and PDGF-C immunoreactivity was observed within oocyte clusters, and PDGF-Rβ and PDGF-B immunoreactivity in cells surrounding oocyte clusters. In primordial follicles, PDGF-Rβ and PDGF-C was observed in the oocyte, and PDGF-Rα and A in the either the oocyte or pregranulosa cells. In primary follicles, PDGF-A, PDGF-C, PDGF-Rα and PDGF-Rβ are expressed in the oocyte. PDGF-Rβ is also expressed in cells surrounding primordial and primary follicles, possibly the precursors to theca cells. In secondary and antral follicles, all four PDGF isoforms and both receptors are expressed in either theca or vascular cells of the theca layer, and PDGF-Rα and A are also expressed in some granulosa cells in rats aged day 20 and older. A role in preantral follicle growth was identified by in vitro culture of preantral follicles. Preantral follicles cultured in serum free medium increased in diameter by 11.0 ± 1.57% over 5 days. Addition of PDGF-AA, PDGF-AB or PDGF-BB to the medium resulted in increases in follicle diameter after 5 days of 18.32 ± 2.18%, 17.72 ± 2.3% and 17.6 ± 1.81%, respectively, representing a significant increase over control diameters. In summary, this study has identified and characterized the presence and localization of all members of the family of platelet derived growth factors and receptors in the rat ovary and revealed a role for these growth factors in positively influencing early follicle growth.

428. Normal follicle growth and maturation in a three-dimensional in vitro culture system: follicular environment manipulation and assessment of oocyte outcomes

2008 ◽  
Vol 20 (9) ◽  
pp. 108
Author(s):  
K. R. Dunning ◽  
L. K. Akison ◽  
D. L. Russell ◽  
R. J. Norman ◽  
R. L. Robker
Keyword(s):  
Culture System ◽  
Cumulus Cell ◽  
Three Dimensional ◽  
Oocyte Quality ◽  
Metabolic Effects ◽  
Follicle Growth ◽  
Cumulus Expansion ◽  
Follicle Diameter

In vivo, the oocyte matures in a niche environment surrounded by somatic cells, and later in ovarian follicular development, by follicular fluid. Maternal diet influences the environment in which an oocyte matures but the mechanisms by which an altered metabolic profile, such as hyperinsulinemia, affects oocyte quality are not known. We investigated the use of a three dimensional follicle culture system allowing direct manipulation of the follicular environment thus circumventing systemic hormonal and metabolic effects. Secondary follicles (113.4 ± 1.02µm, n = 54) were isolated from mice at d12, encapsulated individually in 2µl of alginate matrix, and cultured in aMEM/5%FCS/10 mIU/mL LH/100 mIU FSH at 37°C/5%CO2, with media sampling and replacement every second day. Following 12 days of culture there was a significant 3-fold increase in follicle diameter (320 ± 10.1µm, n = 51). Histological analysis showed normal follicular morphology and antrum formation. Analysis of oestradiol (15.0ng/mL), androstenedione (7.8ng/mL) and progesterone (23.7ng/mL) in the media at d12 confirmed normal steroidogenesis and differentiation. Treatment of follicles with an ovulatory stimulus (1.5IU/mL hCG/5ng/mL Egf), resulted in cumulus expansion and hyaluronan localising to the cumulus oocyte complex (COC) and follicular basement membrane. These analyses were consistent with follicle growth and induction of ovulation in vivo. Further, COCs isolated from follicles and matured in vitro (IVM) in the presence of Egf and FSH, underwent cumulus expansion (CEI 2.8 ± 0.2) and were capable of fertilisation and blastocyst development. LH did not induce IVM COC expansion (CEI 1.36 ± 0.2), reflecting the normal in vivo differentiation process. However, culturing follicles in high insulin (5ug/mL) led to a significant increase in the degree of IVM cumulus expansion in response to LH (CEI 2.1 ± 0.3) indicating inappropriate cumulus cell differentiation, which may lead to poorer oocyte quality. These results demonstrate that this technique recapitulates normal in vivo folliculogenesis and is useful for manipulation of the follicular environment and assessment of oocyte outcomes.

Hypoxia limits mouse follicle growth in vitro

2016 ◽  
Vol 28 (10) ◽  
pp. 1570 ◽  
Author(s):  
J. M. Connolly ◽  
M. T. Kane ◽  
L. R. Quinlan ◽  
P. Dockery ◽  
A. C. Hynes
Keyword(s):  
Ascorbic Acid ◽  
Ovarian Follicle ◽  
Maximum Diameter ◽  
Mouse Serum ◽  
Follicle Growth ◽  
Follicle Culture ◽  
Follicle Diameter ◽  
Serum Type

Ovarian follicle culture is useful for elucidation of factors involved in the regulation of follicular function. We examined the effects of gas phase oxygen concentration, an oil overlay, serum type and medium supplementation with FSH, insulin–transferrin–selenium (ITS) and l-ascorbic acid on cultured preantral mouse follicle growth in a spherical, non-attached follicle culture system. Follicle growth in 5% oxygen was significantly (P < 0.01) inferior to growth in 20% oxygen in terms of follicle diameter. This was likely due to hypoxia, as evidenced by significantly (P < 0.05) increased follicle secretion of vascular endothelial growth factor (VEGF), a marker of cell hypoxia. Follicular growth was not (P > 0.05) affected by an oil overlay, ITS supplementation or serum type. Culture in medium with 5% mouse serum, 1 IU mL–1 FSH, 25 μg mL–1 l-ascorbic acid and 20% oxygen without an oil overlay supported the growth of follicles to a maximum diameter of 380 μm in 6 days. Compared with mature preovulatory mouse follicles in vivo that often have diameters >500 μm within the same time frame, in vitro-grown follicles clearly exhibit limited growth. Thus, adequate oxygenation is an essential factor in the process of optimising follicle growth.

121 ALGINATE-FIBRIN GEL MATRIX PROMOTES IN VITRO GROWTH OF DOG SECONDARY FOLLICLES

2012 ◽  
Vol 24 (1) ◽  
pp. 173 ◽  
Author(s):  
N. Songsasen ◽  
C. Guzy ◽  
D. E. Wildt
Keyword(s):  
Dynamic Mechanical Properties ◽  
Dimensional Structure ◽  
Follicle Development ◽  
Follicle Growth ◽  
Fibrin Gel ◽  
Chi Square ◽  
Paracrine Factors ◽  
Encapsulated Cells ◽  
Follicle Diameter

A previous study from our laboratory has demonstrated that preantral follicles from the dog that are cultured in alginate are able to grow and produce steroid hormones (Songsasen et al. 2011 Reproduction 142, 113–122). Here we investigated the influence of using a combination of alginate and a degradable biomaterial, fibrin, on dog follicle development in vitro. We hypothesised that the alginate and fibrin gel matrix would be superior to alginate alone because the former has dynamic mechanical properties that permit more expansive follicle development than the inert alginate-only system. Secondary follicles (128–220 μm in diameter) were collected from the ovaries of 4 prepubertal dogs (<6 months of age) and encapsulated in 0.5% alginate (n = 26) or 0.5% alginate + 12.5 mg mL–1 of fibrin (n = 22). Follicles were cultured for 12 days at 38.5°C in 100 μL of α-minimal essential medium + 2 mM of glutamine + 5.5 μg mL–1 of insulin + 5.5 μg mL–1 of transferrin + 6.7 ng mL–1 of selenium + 10 μg mL–1 of FSH and 1 ng mL–1 of LH + 3 mg mL–1 of polyvinyl alcohol. Follicle diameter was monitored and half of the medium exchanged every 48 h. Follicle survival was assessed based on ability to increase in size, as well as on oocyte and granulosa cell morphology. Comparisons of follicle growth rate for each culture day between the 2 treatments were conducted using Student's t-test and among culture days within the same group using ANOVA followed by a Holm-Sidak multiple comparison. Follicle survival was compared using a chi-square test. In both groups, follicles maintained the 3-dimensional structure and increased (P < 0.05) in size as culture period progressed. However, follicles encapsulated in alginate + fibrin grew larger (P < 0.05) than those in alginate alone. Specifically, follicles in alginate + fibrin were doubled in size by 12 days compared with a 60% increase for alginate alone. There were no differences (P > 0.05) in follicle survival between the 2 groups (27.0 and 38.1% for alginate and alginate-fibrin, respectively). Results demonstrate that a dynamic alginate-fibrin matrix enhances in vitro follicle growth. We suspect that the mechanism involved is related to facilitating expansion capacity. Specifically, it is likely that nondegradable alginate offers physical, but eventually restrictive, support to encapsulated cells. By contrast, in the gel combination, the fibrin degrades due to cell-secreted proteases that, in turn, permit more robust follicle expansion. Low follicle survival (<40%) in both treatments emphasises the need for more studies to identify influential endocrine/paracrine factors that enhance follicle growth and production of competent oocytes. Funded by NIH-KO1RR020564.

scholarly journals Size-specific follicle selection improves mouse oocyte reproductive outcomes

Reproduction ◽  
2015 ◽  
Vol 150 (3) ◽  
pp. 183-192 ◽  
Author(s):  
Shuo Xiao ◽  
Francesca E Duncan ◽  
Lu Bai ◽  
Catherine T Nguyen ◽  
Lonnie D Shea ◽  
...  
Keyword(s):  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Reproductive Outcomes ◽  
Follicle Growth ◽  
Follicle Diameter ◽  
Size Groups ◽  
Cell Embryo ◽  
Embryo Stage ◽  
Follicle Selection

Encapsulated in vitro follicle growth (eIVFG) has great potential to provide an additional fertility preservation option for young women and girls with cancer or other reproductive health threatening diseases. Currently, follicles are cultured for a defined period of time and analyzed as a cohort. However, follicle growth is not synchronous, and culturing follicles for insufficient or excessive times can result in compromised gamete quality. Our objective is to determine whether the selection of follicles based on size, rather than absolute culture time, better predict follicle maturity and oocyte quality. Multilayer secondary mouse follicles were isolated and encapsulated in 0.25% alginate. Follicles were cultured individually either for defined time periods or up to specific follicle diameter ranges, at which point several reproductive endpoints were analyzed. The metaphase II (MII) percentage after oocyte maturation on day 6 was the highest (85%) when follicles were cultured for specific days. However, if follicles were cultured to a terminal diameter of 300–350 μm irrespective of absolute time in culture, 93% of the oocytes reached MII. More than 90% of MII oocytes matured from follicles with diameters of 300–350 μm showed normal spindle morphology and chromosome alignment, 85% of oocytes showed two pronuclei after IVF, 81% developed into the two-cell embryo stage and 38% developed to the blastocyst stage, all significantly higher than the percentages in the other follicle size groups. Our study demonstrates that size-specific follicle selection can be used as a non-invasive marker to identify high-quality oocytes and improve reproductive outcomes during eIVFG.

scholarly journals IN VITRO NUTRITION OF ALSTROEMERIA

HortScience ◽  
1990 ◽  
Vol 25 (9) ◽  
pp. 1100e-1100
Author(s):  
Mark A. Smith ◽  
Mark P. Bridgen
Keyword(s):  
Calcium Chloride ◽  
Linear Response ◽  
Growth And Development ◽  
In Vitro Growth ◽  
Fresh Weight ◽  
Gelling Agents ◽  
Best Response ◽  
Low Levels ◽  
Over Time

In vitro growth and development of Alstroemeria `Cornell Pink' were evaluated on media containing different amounts of CaCl2, MgS O4, FeSO4, NO3, or NH4. Six levels of calcium chloride were originally examined (from 0 to 75 mM); the low levels proved to be most beneficial. Subsequent experiments used CaCl2 levels from 0 to 3.0 mM. Again, the low levels were most productive. Two experiments, with different gelling agents, were designed for MgSO4. The levels ranged from 0 to 15 mM. The 15 mM level produced explants with the greatest fresh weight. Three experiments were used to study the effect of FeSO4. The range was the same in all of the experiments (0 to 1 mM), but the increments and the gelling agents differed. In all three experiments, the 1 mM level proved to be toxic. The group with treatments from 0.01 to 0.5 mM had the best response over time. Both experiments with nitrogen found no response to different NO3:NH4 ratios. A positive linear response to rate was found within the range studied (20 to 80 mM).

scholarly journals Modulation of Endocannabinoid Tone in Osteoblastic Differentiation of MC3T3-E1 Cells and in Mouse Bone Tissue over Time

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1199
Author(s):  
Magdalena Kostrzewa ◽  
Ali Mokhtar Mahmoud ◽  
Roberta Verde ◽  
Federica Scotto di Carlo ◽  
Fernando Gianfrancesco ◽  
...  
Keyword(s):  
Osteoblast Differentiation ◽  
Cannabinoid Receptors ◽  
Bone Growth ◽  
Cell Function ◽  
Bone Cell ◽  
Mature Adult ◽  
Age Related ◽  
Over Time

Bone is a highly complex and metabolically active tissue undergoing a continuous remodeling process, which endures throughout life. A complex cell-signaling system that plays role in regulating different physiological processes, including bone remodeling, is the endocannabinoid system (ECS). Bone mass expresses CB1 and CB2 cannabinoid receptors and enzymatic machinery responsible for the metabolism of their endogenous ligands, endocannabinoids (AEA and 2-AG). Exogenous AEA is reported to increase the early phase of human osteoblast differentiation in vitro. However, regarding this cell context little is known about how endocannabinoids and endocannabinoid-related N-acylethanolamines like PEA and OEA are modulated, in vitro, during cell differentiation and, in vivo, over time up to adulthood. Here we characterized the endocannabinoid tone during the different phases of the osteoblast differentiation process in MC3T3-E1 cells, and we measured endocannabinoid levels in mouse femurs at life cycle stages characterized by highly active bone growth (i.e., of juvenile, young adult, and mature adult bone). Endocannabinoid tone was significantly altered during osteoblast differentiation, with substantial OEA increment, decline in 2-AG and AEA, and consistent modulation of their metabolic enzymes in maturing and mineralized MC3T3-E1 cells. Similarly, in femurs, we found substantial, age-related, decline in 2-AG, OEA, and PEA. These findings can expand existing knowledge underlying physiological bone cell function and contribute to therapeutic strategies for preventing bone-related metabolic changes accruing through lifespan.

Structural integrity after cold storage of human epidermal model in hypothermosol: A correlative ultrastructural and fluorescent assay

1994 ◽  
Vol 52 ◽  
pp. 270-271
Author(s):  
Henry H. Eichelberger ◽  
John G. Baust ◽  
Robert G. Van Buskirk
Keyword(s):  
Cold Storage ◽  
Structural Integrity ◽  
Culture Media ◽  
Cell Structure ◽  
Natural State ◽  
Sodium Cacodylate ◽  
Ruthenium Tetroxide ◽  
Cacodylate Buffer ◽  
Before And After

For research in cell differentiation and in vitro toxicology it is essential to provide a natural state of cell structure as a benchmark for interpreting results. Hypothermosol (Cryomedical Sciences, Rockville, MD) has proven useful in insuring the viability of synthetic human epidermis during cold-storage and in maintaining the epidermis’ ability to continue to differentiate following warming.Human epidermal equivalent, EpiDerm (MatTek Corporation, Ashland, MA) consisting of fully differentiated stratified human epidermal cells were grown on a microporous membrane. EpiDerm samples were fixed before and after cold-storage (4°C) for 5 days in Hypothermosol or skin culture media (MatTek Corporation) and allowed to recover for 7 days at 37°C. EpiDerm samples were fixed 1 hour in 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.2). A secondary fixation with 0.2% ruthenium tetroxide (Polysciences, Inc., Warrington, PA) in sodium cacodylate was carried out for 3 hours at 4°C. Other samples were similarly fixed, but with 1% Osmium tetroxide in place of ruthenium tetroxide. Samples were dehydrated through a graded acetone series, infiltrated with Spurrs resin (Polysciences Inc.) and polymerized at 70°C.

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