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.

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.

scholarly journals 326FOLLICULAR SIZE, BUT NOT STAGE OF REPRODUCTION OR SEASON, INFLUENCES MEIOTIC MATURATION OF DOMESTIC DOG OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 282 ◽  
Author(s):  
N. Songsasen ◽  
R. Spindler ◽  
D.E. Wildt
Keyword(s):  
Follicular Development ◽  
Domestic Dog ◽  
Experimental Conditions ◽  
Nuclear Maturation ◽  
Follicular Size ◽  
Time Of Year ◽  
Equine Chorionic Gonadotropin ◽  
The Impact ◽  
Meiotic Competence

The current in vitro maturation system (IVM) for dog oocytes is inefficient. On the average, only 15% of ovarian oocytes complete nuclear maturation in vitro. For unknown reasons, the ability of oocytes to develop to the metaphase II stage (MII) varies markedly among bitches (Songsasen et al., 2002, Mol. Reprod. Dev. 62, 407–415). The objective of this study was to identify the cause(s) underlying these significant variations in nuclear maturation. Initially, we retrospectively analyzed data obtained during the past 3 years;; 1661 oocytes were obtained from 74 bitches where stage of reproduction for the donor was known based on ovarian morphology. Oocytes were cultured in TCM 199+0.1% polyvinyl alcohol at 38.5°C in 5% CO2 in humidified air under various experimental conditions. Analysis of variance (ANOVA) was performed to compare differences in meiotic competence of oocytes obtained at various reproductive stages and during different seasons. Stage of reproduction did not influence meiotic abilities of oocytes. Percentages of oocytes obtained during proestrus/estrus (n=468 oocytes), diestrus/metestrus (n=333), anestrus (n=331) or prepuberty (6–8 months of age, n=479) and developing to MII were 17.9±2.9%, (mean±SEM), 24.0±6.0%, 20.8±4.7%, and 17.8±5.2%, respectively (P&gt;0.05). A similar analysis across seasons (spring, summer, fall, winter) also indicated no influence of time of year on nuclear maturation (P&gt;0.05). Because there is a known strong link between follicular growth and meiotic competence of goat oocytes (De Smedt et al., 1994 J. Exp. Zool. 269, 128–139), we also examined the impact of follicular size on nuclear maturation. The cortex of ovaries from 15 bitches was horizontally dissected (5mm thickness) so follicles could be observed and divided into three classes: (1) &lt;0.5mm diameter (n=60); (2) ≥0.5 to &lt;1mm (n=110); and (3) 1–2mm (n=72). Follicles were separated according to these size classes;; oocytes were recovered and cultured in TCM 199+0.25mM pyruvate, 2mM glutamine, 25mM β-mercaptoethanol, 10ng/mL epidermal growth factor (Basal TCM) supplemented with 0.5IU/mL equine chorionic gonadotropin for 1h. Oocytes then were cultured in Basal TCM for 48h before staining with 1% orcein to assess nuclear status. Follicular size influenced meiotic competence of the oocytes (ANOVA, P&lt;0.05). Mean percentages of MII oocytes were 14.2±7.2, 15.6±4.5, and 30.9±8.2, for oocytes recovered from &lt;0.5-mm, ≥0.5 to &lt;1-mm and 1–2-mm diameter follicles, respectively. This study revealed that stage of reproduction and season have no impact on in vitro nuclear maturation of the dog oocyte. However, the findings demonstrate that dog oocytes acquire meiotic competency during follicular development. Because the source of most dog oocytes for IVM are small follicles, results suggest that oocytes may be incapable of completing nuclear maturation under in vitro conditions that are designed for fully-grown oocytes.

scholarly journals In vitro development of mechanically and enzymatically isolated cat ovarian follicles

2021 ◽  
Vol 2 (1) ◽  
pp. 35-46
Author(s):  
Jennifer B Nagashima ◽  
Andrea M Hill ◽  
Nucharin Songsasen
Keyword(s):  
Fertility Preservation ◽  
Mammalian Species ◽  
Ovarian Follicles ◽  
Theca Cell ◽  
Follicle Development ◽  
Follicle Growth ◽  
Growth And Survival ◽  
Isolation Methods ◽  
Mechanical Isolation

Graphical Abstract Isolation of ovarian follicles is a key step in culture systems for large mammalian species to promote the continued growth of follicles beyond the preantral stage in fertility preservation efforts. Still, mechanical isolation methods are user-skill dependent and time-consuming, whereas enzymatic strategies carry increased risk of damaging theca cell layers and the basement membranes. Here, we sought to determine an optimal method to rescue domestic cat (Felis catus) early antral and antral stage follicles from ovarian tissue and to evaluate the influence of isolation strategy on follicle development, survival, and gene expression during 14 days of in vitro culture in alginate hydrogel. Mechanical isolation was compared with 90 min digestion in 0.7 and 1.4 Wünsch units/mL Liberase blendzyme (0.7L and 1.4L, respectively). Mechanical isolation resulted in improved follicle growth and survival, and better antral cavity and theca cell maintenance in vitro, compared with 1.4L (P < 0.05) but displayed higher levels of apoptosis after incubation compared with enzymatically isolated follicles. However, differences in follicle growth and survival were not apparent until 7+ days in vitro. Expressions of CYP19A1, GDF9, LHR, or VEGFA were similar among isolation-strategies. Cultured follicles from all isolation methods displayed reduced STAR expression compared with freshly isolated follicles obtained mechanically or via 0.7L, suggesting that prolonged culture resulted in loss of theca cell presence and/or function. In sum, early antral and antral stage follicle development in vitro is significantly influenced by isolation strategy but not necessarily observable in the absence of extended culture. These results indicate that additional care must be taken in follicle isolation optimizations for genome rescue and fertility preservation efforts. Lay summary The ovary contains hundreds of eggs with only a select few developing from an immature stage through to ovulation over the course of an animal's lifetime. Rescue of eggs from this pool, and the ability to grow them in culture to a mature stage, would be incredibly valuable for fertility preservation efforts in both humans and endangered species. Currently, the isolation of ovarian follicles (eggs with their surrounding helper cells) is a key step in culture systems for large mammalian species, to promote continued growth. Yet, isolation methods may affect the follicle’s future developmental capacity. We evaluated two isolation strategies, mechanical micro-dissection (needle/scalpel blade) and enzymatic digestion (using Liberase blendzyme) on ovaries of domestic cats obtained via routine spay procedures. Mechanically isolated follicles displayed improved growth, survival, and indications of developmental competence in 14-day culture, compared with high concentration (1.4 Wünsch units/mL) enzyme-isolated follicles. However, mechanical isolation was not different from low (0.7 Wünsch units/mL) enzyme for these metrics, or for expression of key genes indicative of follicular cell functions. Further, differences in follicle growth/survival were not apparent until 7+ days in culture. Thus, ovarian follicle isolation strategies influence developmental potential in culture, and extended culture will be required to identify optimal methods for fertility preservation efforts.

scholarly journals Hypoxia-mediated carbohydrate metabolism and transport promote early-stage murine follicle growth and survival

2014 ◽  
Vol 306 (8) ◽  
pp. E893-E903 ◽  
Author(s):  
Yogeshwar Makanji ◽  
David Tagler ◽  
Jennifer Pahnke ◽  
Lonnie D. Shea ◽  
Teresa K. Woodruff
Keyword(s):  
Target Genes ◽  
Early Stage ◽  
Fold Increase ◽  
Underlying Mechanism ◽  
Culture Conditions ◽  
Follicle Growth ◽  
Growth And Survival ◽  
Lactate Levels

Oxygen tension is critical for follicle growth and metabolism, especially for early-stage follicles, where vascularity is limited. Its role and underlying mechanism in the in vitro activation and maturation of immature to ovulatory follicles is largely unknown. In this study, early secondary (110 μm) murine follicles were isolated and encapsulated in alginate hydrogels to replicate the in vivo environment of the growing/maturing follicle. Encapsulated follicles were cultured for 8 days at either 2.5 or 20% O2. Survival (2.6-fold) and growth (1.2-fold) were significantly higher for follicles cultured at 2.5% compared with 20% O2. Using a mouse hypoxia-signaling pathway qRT-PCR array and GeneGo Metacore analysis, we found that direct target genes of the hypoxia-activated HIF1-complex were significantly upregulated in follicles cultured for 8 days at 2.5% compared with 20% O2, including the carbohydrate transport and metabolism genes Slc2a3, Vegfa, Slc2a1, Edn1, Pgk1, Ldha, and Hmox1. Other upregulated genes included carbohydrate transporters ( Slc2a1, Slc2a3, and Slc16a3) and enzymes essential for glycolysis ( Pgk1, Hmox1, Hk2, Gpi1, Pfkl, Pfkp, Aldoa, Gapdh, Pgam1, Eno1, Pkm2, and Ldha). For follicles cultured at 2.5% O2, a 7.2-fold upregulation of Vegfa correlated to an 18-fold increase in VEGFA levels, and a 3.2-fold upregulation of Ldha correlated to a 4.8-fold increase in lactate levels. Both VEGFA and lactate levels were significantly higher in follicles cultured at 2.5% compared with 20% O2. Therefore, enhanced hypoxia-mediated glycolysis is essential for growth and survival of early secondary follicles and provides vital insights into improving in vitro culture conditions.

Mechanisms Regulating In Vitro Follicle Growth and Differentiation Are Partially Conserved Between the Mouse and Domestic Dog.

2009 ◽  
Vol 81 (Suppl_1) ◽  
pp. 623-623
Author(s):  
Nucharin Songsasen ◽  
Teresa K. Woodruff ◽  
David E. Wildt
Keyword(s):  
Domestic Dog ◽  
Follicle Growth

253 LACK OF ABILITY TO REGULATE KINASE ACTIVITIES MAY BE RESPONSIBLE FOR MEIOTIC INCOMPETENCE IN THE DOG OOCYTE

2008 ◽  
Vol 20 (1) ◽  
pp. 206 ◽  
Author(s):  
N. Songsasen ◽  
P. Stein ◽  
R. M. Schultz ◽  
A. J. Travis ◽  
D. E. Wildt ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Domestic Dog ◽  
Kinase Assay ◽  
Pcr Analysis ◽  
Activity Levels ◽  
Expression Levels ◽  
Follicular Size

The size of donor follicles influences meiotic maturation of oocytes, including those of the domestic dog (Songsasen and Wildt 2005 Mol. Reprod. Dev. 72, 113–119). Maturation promoting factor (MPF, CDK1) and mitogen activating protein kinase (MAPK) play a pivotal role in regulating meiosis in several species (Abrieu et al. 2001 J. Cell Sci. 114, 257–267). Accordingly, we determined (1) MPF and MAPK activities, and (2) mRNA expression of cell cycle genes, including CDK1, CCNB1, and CDC25 in oocytes obtained from small (<1 mm diameter), medium (1 to 2 mm) and large (>2 mm) follicles. In Study 1, ovarian oocytes were classified into three groups (based on these sizes) and then cultured (38.5�C in 5% CO2) for 0, 24, or 48 h in TCM-199 (+25 µm β-mercaptoethanol, 10 ng mL–1 epidermal growth factor, 0.25 mm pyruvate, 2.0 mm glutamine, and 0.1% polyvinyl alcohol). Oocytes were denuded, assessed for nuclear status, and stored individually at –80�C until MPF and MAPK activities were assayed using a double kinase assay. Kinase activities of in vitro-matured (IVM) oocytes were expressed as a ratio of MPF and MAPK to that in metaphase I (MI) oocytes flushed from the oviducts of estrous bitches. In Study 2, oocytes (n = 20/follicular size class) were immediately frozen at –80�C; RNA was extracted, reverse transcribed, and subjected to quantitative real-time PCR analysis. Expression levels of each transcript were normalized to levels of endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Statistical analyses were performed using ANOVA followed by the Holm-Sidak test for multiple comparisons. Both MPF and MAPK activities varied among follicular size classes. Kinase activities increased in oocytes from large follicles upon meiotic resumption, with germinal vesicle (GV) oocytes expressing the lowest levels compared to their metaphase II (MII) counterpart (GV: 25.9 � 5.2% v. MII: 211.5 � 19.5% and 36.4 � 9.1% v. 150.7 � 22.9% for MPF and MAPK, respectively; P < 0.01). For medium follicles, MPF increased (P < 0.01) as oocytes progressed from GV (23.3 � 6.3%) to MII (243.4 � 55.5%). However, MAPK levels remained constant until the MI stage, and then increased (P < 0.01) in MII oocytes. For small follicles, MPF increased (P < 0.05) at the MI stage and then remained constant until meiosis was completed, whereas MAPK activities remained constant after GVBD. Kinase activity levels were not different (P < 0.05) between MI and MII oocytes harvested from the three follicular classes. Interestingly, follicular size had no effect (P > 0.05) on expression levels of cell cycle transcripts. These findings suggest that the compromised developmental competence of dog oocytes from small follicles likely is related to the oocytes' inability to regulate MAPK activity during meiotic resumption.

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 Multiple follicle culture supports primary follicle growth through paracrine-acting signals

Reproduction ◽  
2013 ◽  
Vol 145 (1) ◽  
pp. 19-32 ◽  
Author(s):  
J E Hornick ◽  
F E Duncan ◽  
L D Shea ◽  
T K Woodruff
Keyword(s):  
Feedback Mechanism ◽  
Follicle Growth ◽  
Primary Follicle ◽  
Growth And Survival ◽  
Follicle Culture ◽  
Secreted Factors ◽  
Secondary Stage ◽  
Survival And Growth

In vitro follicle growth in alginate hydrogels is a unique and versatile method for studying ovarian and follicle biology that may also have implications for fertility preservation. Current culture systems support the development of isolated mouse follicles from the secondary stage onward. However, it has been a challenge to grow smaller follicles in vitro due to the dissociation of the oocyte from companion somatic cells. Recent work has demonstrated that coculturing primary follicles with mouse embryonic fibroblasts or ovarian stromal cells supports follicle survival and growth. In this study, we demonstrate that follicles themselves can exert a beneficial coculture effect. When primary follicles were cultured in groups of five or ten (multiple follicle culture), there was increased growth and survival. The multiple follicle culture approach maintained follicle integrity and resulted in the formation of antral stage follicles containing meiotically competent gametes. The growth and survival of primary follicles were highly number dependent, with the most significant enhancement observed when the largest number of follicles was grown together. Our data suggest that the follicle unit is necessary to produce the secreted factors responsible for the supportive effects of multiple follicle culture, as neither denuded oocytes, oocyte-secreted factors, nor granulosa cells alone were sufficient to support early follicle growth in vitro. Therefore, there may be signaling from both the oocyte and the follicle that enhances growth but requires both components in a feedback mechanism. This work is consistent with current in vivo models for follicle growth and thus advances the movement to recapitulate the ovarian environment in vitro.

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