In current systems for production of bovine embryos in vitro, the frequency of
development to the blastocyst stage and subsequently production of live offspring is
lower than that observed in vivo. Oocytes are aspirated from a wide range of follicle
sizes (2-8 mm in diameter) for in vitro maturation and fertilisation. During
development oocytes increase in size with enlargement of follicles, this may reflect
different cytoplasmic maturation levels of the oocytes. Oocytes from larger follicles
have a higher developmental competence. In other word, oocytes from small antral
follicles which are more abundant on the ovaries, have an intrinsic deficiency in some
vital factors, which are important for embryonic development. In vivo, follicle enclosed
oocytes are arrested at germinal vesicle (GV) stage. When aspirated from the follicle
they are released from the inhibitory function of the follicle wall and develop to
metaphase of the second (Mil) meiotic division. It was hypothesised that if oocytes
could be cultured in vitro under conditions which maintain GV arrest, prior to in vitro
maturation, then the oocyte may have the opportunity to synthesise or modify the
factors which are required for subsequent embryonic development.
To this end a system was established for culture of intact large antral follicles of 3-8mm
in diameter for different periods of time up to 7 days. The majority of oocytes (96.8%)
recovered following 24 hours of follicle culture remained at the GV stage. After
recovery, 80% of these oocytes resumed meiosis and developed to the Mil stage.
However, with increasing periods of follicle culture, both the number of oocytes
remaining arrested at GV stage and also the number able to resume meiotic maturation
were significantly reduced. On the basis of these results, follicle culture for a period of
24 hours was used for the following experiments. In a comparative study, follicle
culture derived oocytes resulted in a significantly higher rate of blastocyst production
than directly aspirated oocytes used as the control group. However, there was no difference in embryo quality based on total cell number. It was concluded that oocytes
gain a higher developmental competence during follicle culture.
Histological and ultrastructural aspects of both the follicles and oocytes were analysed
following different periods of follicle culture. With increasing periods of follicle
culture, the number of pycnotic cells in the granulosa and cumulus compartments
increased and some oocytes resumed meiosis within the follicle. The cumulus cells
became expanded and abnormal chromatin condensation was observed in the GV. The
ultrastructure of oocytes was analysed using transmission electron microscopy.
Immature oocytes recovered following 24 hours of follicle culture and exhibited
characteristics typical of immature oocytes. However, oocytes recovered following
longer periods of follicle culture, showed some abnormal ultrastructure. It was
concluded that follicle culture up to 24 hours has no detrimental effect on oocyte
quality and structure.
The patterns of total and de novo proteins of oocytes were studied. During oocyte
maturation, some new protein bands were observed as evidenced by both 1D and 2DSDS PAGE followed by silver staining. There were differences in both the protein
profile and concentrations of some individual proteins between immature oocytes from
aspirated and follicle cultured groups. Any differences in de novo protein synthesis
during follicle culture could not be demonstrated by J5S methionine labelling due to
technical difficulties. Following maturation, oocytes from both groups showed identical
protein profiles. It was concluded that follicle culture has no detrimental effect on
protein synthesis in the oocyte.
Insulin is a known regulator of ovarian function and is also a follicular survival factor.
The effects of addition of insulin (5pg/ml) during bovine antral follicle culture on
oocyte quality and development, protein profile and Insulin-like Growth Factor Binding
Proteins (IGFBPs) expression were examined. Insulin adversely affected the cleavage
rate, however, there were no differences in the proportion of cleaved embryos which
subsequently developed to blastocyst stage. The ultrastructure and total protein profile
of oocytes from the insulin positive group indicated that these oocytes gain a
cytoplasmic structure similar to Mil oocytes. IGFBPs profiles in follicular fluid and
culture media in small (<4mm) and large (>4mm) follicles were demonstrated. Two
strong bands of 25 (IGFBP-4) and 34 (IGFBP-2) kDa disappeared from both the
follicular fluid and culture media in large follicles. Since the concentrations of these
proteins have been reported to increase during follicular atresia, it was concluded that
insulin prevents follicular atresia in larger follicles and promotes cytoplasmic features
of oocyte maturation. However, insulin does not improve oocyte quality or pre
implantation embryo development in vitro.
Apoptosis is known to be the mechanism by which large follicles become atretic. Many
hormones and growth factors have been reported as follicular survival factors in vivo.
The effects of various factors on prevention of apoptosis in the granulosa cells from
large antral follicle were assessed both individually and in combinations by adding to
the culture media during 2 days of follicle culture. Growth hormone (GH) and
luteinising hormone (LH), did not prevent apoptosis. In contrast, IGF-I and FSH
supported follicle survival and addition of a combination of oestradiol, FSH, LH, IGFI, EGF and GH completely prevented apoptosis. When the above combination was
added to the culture media in the presence or absence of 10% FCS there were no
differences between the two groups, whilst follicles cultured in the absence of both
showed a severe pattern of apoptotic cell death.
In conclusion, culture of bovine large antral follicles in vitro for up to 24 hours
maintain GV arrest and has no subsequent detrimental effect on oocyte quality. In fact
during this culture period it appears that the oocytes acquire a greater developmental
competence perhaps by de novo synthesis of proteins, mRNAs or other factors which
are necessary for subsequent embryonic development or by post translational or post
transcriptional modifications. The addition of factors which support follicular survival
to the culture media did not improve the rate of subsequent embryo production
following follicle culture. Therefore to improve oocyte quality other known or
unknown factor (s) may be required to supplement the culture media. Alternatively
improvements may be achieved through changes in the composition of culture media or
the follicle culture system.