Final follicular maturation in the cow and its effects on the developmental potential of the oocyte

Abstract

The use of assisted reproduction techniques can generate up to 27 (superovulation, SO) or 50 (in vitro embryo production, IVP) calves per cow per year instead of only one calf per cow per year after normal mating. That is due to the possibility to use more than one oocyte per estrous cycle; namely on average 15 (SO) and 60 (IVP). However, using these techniques, the efficiency per used oocyte decreases dramatically. Instead of the efficient use of oocytes during natural mating, only 30% efficiency is reached with SO. Furthermore, IVP generates only 8 calves per 100 used immature oocytes. This loss in efficiency might be due to deviations that occur during the final maturation of the oocytes. In this thesis concepts of final follicular maturation in unstimulated normally cyclic cows are applied to maturation during SO and IVP in order to gain better understanding of deviations in follicle and oocyte development using these techniques. Also preovulatory follicular development after SO is used as a model to study maturation in vivo. Chapter 1 introduces the subject. Deviations in preovulatory follicular development as induced using SO or as a result of oocyte collection and maturation during IVP are discussed against the background of the course of follicular maturation in unstimulated normally cyclic cows. In cows treated for SO the period of preovulatory follicular development is reduced compared to this period in unstimulated normally cyclic cows. Also, after SO an asynchrony of development is found within the population of stimulated follicles as well as between the follicle and its oocyte. For IVP, oocytes are usually collected from 3-6 mm follicles. These follicles lack part of the processes of follicular growth and selection. Furthermore, during in vitro maturation (IVM) of the collected immature oocytes, deviations in cytoplasmic maturation are frequently found. In chapter 2, the effect of prolongation of the period of preovulatory follicular development after SO on the heterogeneity of the population of preovulatory follicles and their oocytes with respect to the potential to mature, to ovulate, to be fertilized and to develop into embryos was investigated. In eCG-stimulated heifers, the spontaneous occurrence of the LH surge was suppressed with a norgestomet ear implant and at a later time a LH surge was induced using GnRH. The protocol resulted in a LH surge at the desired time in 100% of the cases. Prolongation of the period of preovulatory follicular development from 42.4 to 53.8 h increased ovulation rates with 25%. It was suggested that the heterogeneity of the follicular population, as is present in normally stimulated heifers at the time of the spontaneous LH surge, was reduced. The increased ovulation rates did not coincide with an increased number of embryos at day 7 after fertilization. The treatment with norgestomet did not adversely affect Chapter 8?Chapter 8 122 final maturation and fertilization. However, the treatment might have disturbed early embryonic development by altering the secretory activity of the cells of the epithelium of the oviduct. The superovulation protocol with LH surge induction (chapter 2) was used in the studies described in chapter 3 and 4 to obtain oocytes at a fixed stage of development in vivo. In chapter 3, it was investigated to what extend IVM contributes to limiting yields of viable embryos in currently used IVP programs. The use of in vivo matured oocytes, collected from eCG-stimulated heifers at 22-24 h after the LH surge, instead of in vitro matured oocytes collected from 2-8 mm sized follicles, for IVF and IVC improved blastocyst formation and hatching with 100%. Additionally, also the progress of embryonic development was different for the two groups of oocytes; both blastocyst formation and hatching of blastocysts progressed slower for in vivo matured oocytes than for in vitro matured oocytes. The decreased embryonic development after IVM (chapter 3) might be due to the maturation conditions per se or to a difference in startcompetence of the oocytes collected from 2-8 mm sized follicles when compared to the oocytes from preovulatory follicles generated in eCG-treated cows. In chapter 4 the significance of the conditions during maturation for efficient IVP was tested using oocytes for in vivo maturation and IVM which presumably had an equivalent startcompetence. Therefore, heifers were stimulated with eCG using the same protocol as described in chapter 2. From part of the heifers, oocytes from preovulatory follicles were collected at the presumptive time of the LH surge and subsequently subjected to IVM. From the other heifers, oocytes from preovulatory follicles were collected 22-24 h after the occurrence of an induced LH surge. Both groups of oocytes were subjected to IVF and IVC. Blastocyst formation and hatching rates were significantly lower after in vitro maturation than after in vivo maturation of the oocytes. It was concluded that the conditions during IVM are an important factor responsible for limited yield after IVP. In this study, the progress of embryonic development was similar for both groups of oocytes and was conform to that as observed for the in vivo matured oocytes in chapter 3. Since in that study in vitro matured oocytes from 2-8 mm follicles developed at a faster rate than in vivo matured oocytes from preovulatory follicles it was suggested that oocytes undergo certain changes during follicular development from 2-8 mm to preovulatory stage at onset of final maturation which may be involved in programming embryonic development. Both in the studies described in chapter 3 and 4, a large variation in blastocyst formation between individual heifers was found, which is probably due to differences in response to superovulatory treatment. When in the group of heifers donating the in vitro matured oocytes (chapter 4) heifers with exceptional follicular function on the basis of estradiol-17ß and progesterone concentrations in the follicular fluid (FF) were excluded, rates of blastocyst formation increased indicating that the steroid producing capacity of the follicular wall influences oocyte quality. In chapter 5 and 6 preovulatory follicular development after eCG-treatment for SO was used as a model to study the possible role of the insulin-like growth factor (IGF)?Summary 123 system during final maturation in vivo. Earlier results from in vitro studies suggested a stimulatory role of IGF-I on growth and differentiation of granulosa cells and on final maturation of the oocyte. IGF binding proteins (IGFBPs) are suggested to decrease bioavailability of IGF-I but also individual functions of these IGFBPs on follicle and oocyte maturation can not be excluded. In chapter 5, levels of IGF-I and IGFBPs in the FF of eCG-stimulated follicles with ‘normal’ and ‘deviant’ follicular function on the basis of steroid hormones in the FF were compared at several times during final maturation. Final maturation in eCG-stimulated preovulatory follicles with ‘normal’ function was characterized by rather constant levels of IGF-I and IGFBP3 in the FF. Low molecular weight IGFBPs (LMW IGFBPs, i.e. IGFBP2, -4 and -5) were absent in FF at the time the oocyte undergoes germinal vesicle breakdown (GVBD) and reaches metaphase I (MI). Just prior to ovulation, in 15% of the FF of the follicles with ‘normal’ function, LMW IGFBPs were found. While the levels of IGF-I and IGFBP3 in the fluid of ‘deviant’ follicles were not different when compared to eCG-stimulated follicles with ‘normal’ function, the presence of LMW IGFBPs was different in these follicles; at the onset of final maturation, 35% of the ‘deviant’ follicles contained LMW IGFBPs. All these follicles, except one, were classified ‘deviant’ due to too low estradiol-17ß concentrations, probably a sign of atresia. During final maturation, LMW IGFBPs were occasionally found (GVBD, MI) or absent (just prior to ovulation) in ‘deviant’ follicles. It was concluded that only a permissive role of IGF-I in final maturation can be expected. It was also concluded that, as in unstimulated, normally cyclic cows, low estradiol-17ß concentration in the FF of large follicles (> 8mm) coincided with the presence of LMW IGFBPs. The appearance of LMW IGFBPs in the FF of follicles with ‘normal’ follicular function just prior to ovulation indicated a different role of the IGF system during ovulation and corpus luteum formation. Because different IGFBP patterns were found in the FFs of eCG-stimulated follicles with ‘deviant’ follicular function and of concurrently developing follicles with ‘normal’ follicular function, eCG stimulated follicles were used to study the regulation of the presence of IGFBPs during preovulatory follicular development (Chapter 6). Using reverse transcriptase-polymerase chain reaction (RT-PCR), mRNA levels for LMW IGFBPs were analyzed (semi-quantitatively) in eCG-stimulated preovulatory follicles at the onset of maturation with ‘normal’ or ‘deviant’ function, on the basis of estradiol-17ß concentrations in their FF. No or small differences in gene expression were found. Therefore, it was concluded that the earlier found difference in the presence of LMW IGFBPs in FF could not be explained by a difference in gene expression for these IGFBPs in the wall of the follicle. Thus, regulation of the presence of these proteins has to take place at the level of translation or protein degradation. Finally, in chapter 8, the results of the performed experiments are discussed against the background of the current knowledge and existing hypotheses on final follicular maturation. Possible directions of future research in order to improve the efficiency of assisted reproduction are discussed.