L-Carnitine Supports the In Vitro Growth of Buffalo Oocytes.

This study aimed to determine the effect of L-carnitine on the growth and subsequent nuclear maturation of buffalo small growing oocytes (92−108 µm in diameter) in vitro. Oocyte-granulosa cell complexes (OGCs) were dissected from early antral follicles of slaughtered buffaloes and cultured in in vitro growth (IVG) medium with the supplementation of different concentrations (0, 1.25, 1.875 or 2.5 mM) of L-carnitine for 6 days. The results revealed that L-carnitine increased the diameter of buffalo oocytes in vitro. The degeneration rate was significantly (p < 0.05) lower in 2.5 mM of L-carnitine-treated oocytes (10%) than others (55%, 45% and 32.5% in 0, 1.25 and 1.875 mM of L-carnitine-supplemented groups, respectively). The OGCs showed antrum-like structures significantly (p < 0.05) higher in the 2.5 mM of L-carnitine group (74.0%) than the 0- and 1.25-mM groups (34.6% and 38.1%, respectively). Furthermore, in vitro grown oocytes were placed in in vitro maturation (IVM) medium for 24 h to examine meiotic competence of in vitro grown oocytes with L-carnitine. The L-carnitine (1.875 and 2.5 mM) treated oocytes showed a higher rate of nuclear maturation up to the metaphase II (MII) stage and a lower rate of degeneration. In conclusion, L-carnitine enhances the growth, prevents degeneration, promotes the formation of antrum-like structures and supports nuclear maturation of buffalo oocytes in vitro.

Effects of estradiol on in vitro maturation of buffalo and goat oocytes.

PURPOSE: The effects of estradiol on oocyte development seem to be varied among species. The present study investigated the effects of 17ß-estradiol on in vitro maturation of buffalo and goat oocytes. METHODS: Cumulus oocyte complexes (COCs) were aspirated from large antral follicles of slaughtered buffalo and goat ovaries. COCs were cultured in TCM-199 medium supplemented with 0, 0.5, 1, and 1.5 µg/mL of 17ß-estradiol for in vitro maturation. Then, oocytes were used for the examination of state of nuclear maturation and cumulus expansion. RESULTS: In both species, oocytes treated with 17ß-estradiol showed higher cumulus expansion rate than control (0 µg/mL treated). In buffalo, the percentage of oocytes matured to the metaphase II (MII) stage increased in the concentration-dependent manner of 17ß-estradiol. Similarly, estradiol positively influenced nuclear maturation of goat oocytes in vitro. CONCLUSIONS: Estradiol has promoting effects on normalprogress of in vitro oocyte meiosis in buffalos and goats.

Interaction between growing oocytes and granulosa cells in vitro.

BACKGROUND: Oocyte growth is accompanied by follicular development in mammalian ovaries. Since the discovery of two oocyte-derived factors, growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15), knowledge of the bidirectional communication between oocytes and granulosa cells for ovarian function and fertility has been accumulated. In addition, the growth culture system of oocytes has been improved, further promoting the studies on the communication between oocytes and granulosa cells in vitro. METHODS: We provide an overview of the role of granulosa cells in oocyte growth and the role of oocytes in follicular development along with our recent findings in culture experiments of bovine growing oocytes. MAIN FINDINGS: Granulosa cells supply nutrients and metabolites through gap junctions to oocytes and secrete paracrine signals to regulate oocytes. Oocytes regulate granulosa cell proliferation and differentiation and induce antrum formation via GDF9 and BMP15. CONCLUSION: Oocytes actively participate in various aspects of follicular development, including antrum formation via the oocyte-derived factors GDF9 and BMP15, whose synthesis is probably regulated by granulosa cells. In vitro studies will reveal the precise communication loop between oocytes and granulosa cells that facilitates the coordinated development of oocytes and granulosa cells in the follicles.

Effects of stem cell factor on in vitro growth of buffalo oocytes.

Stem cell factor (SCF) plays important roles in primordial follicle activation, oocyte growth and survival, granulosa cell proliferation, theca cell recruitment, and ovarian steroidogenesis. The aim of this study was to investigate the effect of SCF on in vitro growth of buffalo oocytes. Oocyte-granulosa cell complexes (OGCs) were dissected from early antral follicles of slaughtered buffalo ovaries and cultured for 6 days in media supplemented with 0, 50 or 100 ng/mL SCF. In vitro grown oocytes were further cultured for in vitro maturation for 24 h. The results showed that SCF significantly (P < 0.05) increased oocyte diameter in vitro. The percentages of surviving oocytes were 60, 81 and 92 in 0, 50 and 100 ng/mL SCF supplemented group, respectively. SCF promoted formation of antrum-like structures in culture. The results also showed that SCF enhanced the maturation of in vitro grown buffalo oocytes. Here, 14% in vitro grown oocytes reached metaphase II (MII) stage in 50 ng/mL SCF supplemented group, whereas the percentage was increased to 26% in 100 ng/mL SCF treated group. These results show that SCF supports the growth, viability and nuclear maturation of buffalo oocytes in vitro.

GDF9 and BMP15 induce development of antrum-like structures by bovine granulosa cells without oocytes.

The role of oocytes in follicular antrum formation is not well understood. We examined the effect of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the formation of antrum-like structures by cultured bovine oocyte-granulosa cell complexes (OGCs). OGCs containing growing oocytes (105‒115 µm in diameter) were collected from early antral follicles (1.2‒1.8 mm) and used to prepare oocytectomized complexes (OXCs) and granulosa cell complexes (GCs). The mRNAs of GDF9 and BMP15 were expressed in the oocytes, but not in the granulosa cells. The complexes were cultured for five days with or without GDF9 and BMP15 either alone or in combination. The OGCs maintained their complex integrity and developed antrum-like structure, whereas OXCs and GCs neither maintained their integrity nor developed any antrum-like structure without growth factors. GDF9 or BMP15 alone increased the integrity of these complexes and induced antrum-like structures in OXCs and GCs. Moreover, the combination of GDF9 and BMP15 was more potent for both phenomena in all types of complexes. In OXCs and GCs cultured without GDF9 and BMP15 or with BMP15 alone, outgrowing granulosa cells differentiated into fibroblast-like cells. The combination of GDF9 and BMP15 suppressed the appearance of fibroblast-like cells in OXCs and GCs during incubation. Instead, the granulosa cells appeared rhomboid and pebble-like in shape, similar to those in OGCs cultured without supplementation of GDF9 and BMP15. These results suggest that oocytes maintain complex integrity by preventing granulosa cell differentiation and participate in follicular antrum formation via GDF9 and BMP15.

Inhibition of PDE3A sustains meiotic arrest and gap junction of bovine growing oocytes in in vitro growth culture.

Bovine growing oocytes with a diameter of 105-115 µm from early antral follicles (1.2-1.8 mm) are able to resume meiosis, but lack the competence to mature to metaphase II. To confer full maturation competence onto the oocytes, culture systems which can support their growth and prevent their meiotic resumption during culture are needed. In this study, we cultured growing oocytes for 5 days to examine the effects of different phosphodiesterase (PDE) inhibitors on meiotic arrest and acquisition of full maturation competence of growing oocytes, and their gap junctional communication with cumulus cells. Growing oocyte-cumulus complexes (OCCs) were cultured with 3-isobutyl-1-methylxanthine (IBMX; broad-spectrum PDE inhibitor), rolipram (PDE4D inhibitor), cilostamide and milrinone (PDE3A inhibitors). The mean diameters of oocytes increased similarly in all groups. IBMX, cilostamide and milrinone induced antrum formation by OCCs and maintained meiotic arrest of oocytes during culture, whereas rolipram neither promoted antrum formation nor maintained oocyte meiotic arrest. Gap junctional communication between oocytes and cumulus cells was maintained by IBMX and cilostamide, but not by rolipram as judged by the transfer of injected lucifer yellow dye from oocytes to cumulus cells. In subsequent in vitro maturation, oocytes grown with IBMX, cilostamide and milrinone showed full maturation competence. These results suggest that PDE3A inhibition maintains the meiotic arrest of bovine growing oocytes and sustains their gap junctional communication with cumulus cells for 5 days, thereby contributing to their acquisition of full maturation competence.