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Dog reproductive cycle is unique among other mammals in that females experience long and variable periods of ovarian inactivity. Neuroendocrine controls of the reproductive cycle have been thoroughly studied in the dog. However, there is little information regarding endocrine, paracrine and autocrine controls of dog ovarian folliculogenesis. Advancements in the understanding of mechanisms regulating dog ovarian follicle development will be helpful in the establishment of an approach to control cyclicity in this species. Furthermore, such information will likely be useful for the establishment of an in vitro follicle culture system to preserve fertility of genetically valuable disease models or endangered canids. This review highlights current knowledge on dog folliculogenesis with emphasis on endocrine, paracrine and autocrine controls of follicular development. [ABSTRACT FROM AUTHOR]

The aims of the present study were to determine the effects of insulin, in vitro , on: (1) the viability and growth of domestic cat ovarian follicles; (2) mRNA expression of genes regulating steroidogenesis (cytochrome P450 family 17 subfamily, A polypeptide 1 (Cyp17a1), cytochrome P450 family 19 subfamily, A polypeptide 1 (Cyp19a1) and steroidogenic acute regulatory protein (Star)) and water transport (aquaporins (AQPs) Aqp1 , Aqp3 , Aqp7 , Aqp9); and (3) steroid production (17β-oestradiol (E2), progesterone (P4), androstenedione (A4)). Cat secondary follicles were isolated from ovarian cortices and cultured in 0 (Control), 1 or 10 µg mL−1 insulin for 14 days (Day 0 = culture onset). Follicle and oocyte viability (based on neutral red staining), diameter and antrum formation were assessed every 72 h and at the end of incubation (Day 14). Expression of steroidogenic and water transport genes was evaluated on Days 0, 6 and 12, and E2, P4 and A4 concentrations in the culture medium were determined on Day 12. By Day 14, 1 and 10 µg mL−1 insulin had significantly promoted (P < 0.05) both antrum formation in a mean (± s.e.m.) 26.9 ± 9.0% and 78.0 ± 10.0% of follicles respectively, and follicle growth (diameter 151.4 ± 4.5 and 169.9 ± 10.5 µm respectively) compared with Control (antrum formation in 3.3 ± 3.3% of follicles and follicle diameter 129.1 ± 6.6 µm). High insulin (10 µg mL−1) treatment increased follicle viability compared with Control (86.0 ± 9.8% vs 38.1 ± 10.9% respectively; P < 0.05). However, insulin had no beneficial effect (P > 0.05) on oocyte diameter. Cyp17a1 expression on Days 6 and 12 was higher (P < 0.05) in follicles cultured in the low (1 µg mL−1) compared with high (10 µg mL−1) insulin treatment, with no significant difference between low or high insulin vs Control groups. Star expression was higher (P < 0.01) in the low insulin compared with Control group on Day 6, but Star was undetectable in the high insulin group by Day 12. Compared with high insulin, low insulin increased (P < 0. 05) Aqp1 expression on Day 6, but there were no significant differences between these two groups on Day 12. In contrast, high insulin decreased (P < 0.05) Aqp9 transcript levels compared with Control. Only P4 production was affected by insulin, with P4 concentrations in the medium being higher (P < 0. 05) in the low compared with high insulin and Control groups. In summary, the findings indicate that insulin promotes cat ovarian follicle growth and survival in vitro , including enhanced antrum formation, with the likely mechanism involving temporal expression of Cyp17a1 , Star and Aqp9 genes. Insulin is a key regulator of the growth and differentiation of secondary follicles. The aim of the present study was to determine the effects of insulin on in vitro viability and growth of domestic cat ovarian follicles. The findings indicate that insulin promotes cat ovarian follicle survival and growth and affects the temporal expression of the cytochrome P450 family 17 subfamily, A polypeptide 1 (Cyp17a1), steroidogenic acute regulatory protein (Star), and aquaporin 9 (Aqp9) genes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Disruption of the communication between the oocyte and granulosa cells is one of the major causes of poor development of in vitro grown ovarian follicles and oocytes. The present study investigated the effect of two cAMP modulators, cilostamide and forskolin, on in vitro growth of isolated dog secondary follicles and enclosed oocytes, communication between the gamete and surrounding granulosa cells, expression of GJA1 and GDF9 , as well as cAMP level. Secondary follicles were incubated with cilostamide or forskolin alone or a combination of 20 μM cilostamide +1 μM forskolin, and the diameter of the incubated follicles and enclosed oocytes assessed every 72 h. Gap junction activity, GJA1 and GDF9 expression and cAMP level were assessed on Days 6 and 12 and transzonal projection (TZP) density was evaluated on Day 12. Neither cilostamide nor forskolin alone enhanced in vitro growth of dog follicles and the enclosed oocytes (P > 0.05). However, these two cAMP modulators dose dependently sustained gap junction activity and stimulated cAMP production compared with the non-supplemented control. Cilostamide at the high dosage (20 μM) also upregulated GJA1 expression. The combination of cilostamide and forskolin supported oocyte growth during the first 9 days and upregulated GJA1 and GDF9 expression at Day 12 of in vitro culture. This combination treatment also sustained gap junction activity, cAMP production, and increased TZP function (calcein intensity: TZP density ratio). The findings indicated that a combination of cilostamide and forskolin supported growth and survival of oocytes enclosed within cultured follicles by sustaining cAMP production and gap junction activity. • Supplementation of cilostamide and/or forskolin enhances cAMP production from dog secondary follicles. • Cilostamide dose-dependently upregulated genes associated with gap junction communication. • The combination of cilostamide and forskolin supports oocyte growth as well as improves TZP functionality. [ABSTRACT FROM AUTHOR]

Abstract Understanding regulators of folliculogenesis remains limited in the domestic dog (Canis familiaris), which challenges our ability to develop in vitro follicle culture systems for canid genome rescue efforts. Here, we investigated the influence of activin on dog follicle development and survival, oocyte quality, and FSH receptor expression in culture. Preantral (150 - ≤230 μm diameter), early antral (231 - ≤330 μm), and antral (>330–550 μm) stage follicles were encapsulated in a fibrin-alginate hydrogel with 0, 100, or 200 ng/ml rhActivin plus 0, 0.1, 1, or 10 μg/ml FSH for 12 or 21 d of in vitro culture. All follicle groups increased in diameter (P < 0.05) with activin acting synergistically with FSH to improve (P < 0.05) growth and antral cavity expansion (to >630 μm) in early antral and antral cohorts. This complementary effect was not linked to changes in FSHR mRNA expression (P > 0.05). Although not influencing (P > 0.05) follicle survival or transzonal projection (TZP) density in shorter term 12 d culture, activin in the presence of 1 ng/ml FSH maintained TZP density from the 12–21 d interval. Activin also increased oocyte diameter and improved nuclear integrity compared to un-supplemented controls. These results indicate that activin acts synergistically with FSH to promote growth and antral cavity expansion of the dog follicle in vitro , information useful to formulating an effective culture microenvironment for this species. Highlights • Dog ovarian early antral and antral, but not preantral stage, follicles respond to activin supplementation in vitro. • FSH and activin's synergistic promotion of growth and antral cavitation is not mediated via changes in FSHR expression. • Activin supplementation does not influence domestic dog follicle survival during culture. • Activin facilitates preservation of dog oocyte nuclear integrity and trans-zonal projection density in vitro. [ABSTRACT FROM AUTHOR]

In the present study we examined the effects of stem cell factor (SCF; 50 vs 100 ng mL-1) alone or in combination with epidermal growth factor (EGF; 100 ng mL-1) on: (1) the in vitro viability and growth of cat follicles within ovarian cortices; (2) phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) phosphorylation; and (3) c-kit and FSH receptor (FSHr) mRNA expression. At 100 ng mL-1, SCF increased (P⩽0.05) the percentage and size of secondary follicles after 14 days of in vitro culture and sustained AKT phosphorylation after 3 days incubation. EGF suppressed this beneficial effect and reduced (P⩽0.05) the percentage of structurally normal follicles and FSHr expression when combined with 100 ng mL-1 SCF. Expression of c-kit mRNA was higher (P⩽0.05) in the presence of 100 ng mL-1 SCF compared with fresh follicles and cohorts cultured under other conditions. A c-kit inhibitor suppressed follicle growth and reduced AKT phosphorylation. Collectively, the results demonstrate that SCF promotes cat follicle development by upregulating c-kit mRNA expression and AKT phosphorylation. EGF suppresses the stimulating effect of SCF, leading to downregulation of FSHr expression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]