Reviews of Reproduction (2000) 5, 122–130

Oocyte maturation and ovum quality in pigs

M. G. Hunter

Division of Animal Physiology, School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics LE12 5RD, UK

Oocyte quality in pigs is defined as the potential of that oocyte to develop into a viable off- spring. There is increasing evidence that the programming of the oocyte must be completed before leaving the ovarian follicle, including both nuclear and cytoplasmic maturation. Pig oocytes matured in vitro under basic conditions are deficient in some, as yet unidentified, cyto- plasmic factors and thus developmentally incompetent. This developmental incompetence can be overcome to some extent by follicular supplementation (with follicular fluid or granulosa cells) of the culture medium, emphasizing the importance of somatic signals during oocyte maturation. Furthermore, evidence is accumulating that the status of the follicle has a critical impact on the competence of the oocyte in vitro and in vivo and this has been demonstrated by co-culture with follicles at different maturational stages or from breeds with enhanced embryo survival. It now also appears that manipulation of maternal nutrition before mating or oocyte collection can enhance embryo survival in vivo and oocyte developmental competence in vitro, presumably by altering follicular secretions and hence the environment in which the oocyte is nurtured. Identification of both the key follicular factors influencing pig oocyte quality and reliable markers of oocyte quality will undoubtedly yield major improvements in embryo survival in vivo and embryo production in vitro.

The capacity for fertilization and development is acquired by Oocyte and follicle growth the oocyte after a long period of growth and development. This process involves both the synthesis of cytoplasmic components The embryonic gonad in the pig embryo is first observable at and a rearrangement and reduction in the chromosomes. These 24–26 days after mating, although the primordial germ cells are two processes are linked and their timing is critical to ensure noted as early as 18 days after mating in the region of the that the oocyte reaches nuclear and cytoplasmic maturation germinal ridge (Black and Erickson, 1968). Mitotic divisions of simultaneously. Embryonic development is readily compro- germ cells are seen from day 13 of embryonic life until about mised by faulty oocyte maturation. Meiosis begins in early fetal 7 days after birth. The number of germ cells increases markedly life but is arrested and remains at the diplotene stage until the from 5000 at 20 days after mating to a peak of 1100 000 at oocyte either degenerates during atresia or resumes meiosis 50 days after mating. Thereafter, mitotic activity ceases and just before ovulation. Pig oocytes in preovulatory follicles com- some germ cells are lost by necrosis. Meiosis begins as early as plete the first meiotic division about 36–40 h after the gonado- day 40 and, by about 35 days after birth, all oogonia are in the trophin surge, before ovulation, and then enter a second period prophase of the first meiotic division (the germinal vesicle of arrest which usually persists until the egg is activated by stage; Fig. 1). These eggs are surrounded by a flat layer of sperm fusion at fertilization. Macromolecules stored in the granulosa cells and this population of primordial follicles is oocyte are responsible for the progression through the first cell approximately 500 000 at birth (Black and Erickson, 1968) and divisions until the embryonic genome takes over, although decreases to 420 000 at puberty (Gosden and Telfer, 1987). Even relatively little is known about the accumulation of the stored before birth, groups of primordial follicles start to develop into molecular programme. Increasing evidence indicates that the primary follicles and the first antral follicle has been observed programming of the oocyte must be complete before leaving at 70 days after birth (Mauleon, 1978; Oxender et al., 1979; the ovarian follicle and also that the stage of differentiation of Fig. 1). Morbeck et al. (1992) estimated using histology that the the follicle has a critical impact on the quality of the pig oocyte, time span to develop from a primordial into an antral follicle that is, its potential to develop into a viable offspring. Both the was 84 days, and that a further 19 days were required to reach naturally occurring follicular heterogeneity and the manipu- preovulatory status. This estimate was confirmed in our lab- lation of the maturational environment surrounding the pig oratory using ovarian autografted pigs, in which, as is the case oocyte in vivo and in vitro have provided new information in ewes (Gosden et al., 1994), all but the primordial and early on the hormone and follicular factors that regulate the final primary follicles in the ovarian graft regressed (R. Webb and maturation of the oocyte. This review will summarize current M. G. Hunter, unpublished). These studies showed that ani- knowledge on the mechanisms controlling pig oocyte matu- mals subsequently returned to oestrus approximately 3 months ration and highlight recent advances in elucidating the somatic after grafting, thus supporting the proposed time scale to de- signals that influence oocyte quality. velop from primordial to preovulatory status (Fig. 2). During © 2000 Journals of Reproduction and Fertility 1359-6004/2000 Downloaded from Bioscientifica.com at 09/30/2021 07:23:49PM via free access Oocyte quality in pigs 123

Germ cell Meiosis Mitotic divisions All oocytes First antral Graafian mitotic divisions begins completed in GV stage follicle follicles Puberty Conception 1st ovulation Day 13 Day 40 Day 0 Day 7 Days 20Ð35 Day 70 5Ð6 p.c. p.c. Birth months

Fig. 1. Timing of ovarian development in pigs from conception to puberty and first ovulation. Meiosis (40 days after mating) and mitotic divisions (7 days after birth) refer to the oocyte. (Based on Mauleon, 1978; Oxender et al., 1979.) p.c.: post coitum. this period of development, numerous morphological and secreted in a temporal fashion. Potential candidates are the functional changes occur in the oocyte itself, many of which various growth factors, including GDF-9 and other members involve interactions between the oocyte and its surrounding of the transforming growth factor β family, plasminogen acti- follicular cells. vator and members of the interleukin family. The growth of oocytes in mammals occurs in two distinct and characteristic phases. In the first, the growth of the follicle Meiotic maturation and oocyte is coincident and correlated in a positive and linear manner and, in the second, the size of the oocyte remains con- Pig follicles < 0.7 mm in diameter contain oocytes incapable of stant despite a continuation of follicular growth (Fig. 2). The completing meiotic resumption and, although some oocytes oocyte does not acquire the competence to complete the first from follicles between 0.8 and 1.6 mm are capable of maturing meiotic division until it has reached almost the maximum adult in vitro, not all from follicles > 1.7 mm in diameter progress to size (Moor and Warnes, 1978). During the growth phase, pig the metaphase II stage after 48 h of culture. Even the larger oocytes increase in diameter from approximately 30 to 120 µm follicles (> 3 mm) contain some oocytes that cannot reach the and growth is almost complete (115 µm) in 1.8 mm follicles normal configuration in culture (Motlik et al., 1984; Fig. 2). (Motlik and Fulka, 1986, Fig. 2). Evidence indicates that somatic Meiotically competent oocytes become arrested in growth cell–oocyte interactions via gap junctions are essential for the phase 2 (G2) and meiotic competence is defined as the ability to provision of substrates for energy metabolism and, hence, overstep the G2 checkpoint and complete meiosis. Denuded in- oocyte growth. The somatic cell compartment, in addition to competent oocytes can become partially nuclear competent supplying nucleosides, amino acids and phospholipids, main- during culture without significant growth (Fulka et al., 1994). tains ionic balance and mRNA stability in the maturing oocyte. The ability to complete the metaphase I to metaphase II tran- The rate of oocyte growth is related directly to the number of sition after incubation in culture coincides with the cessation of granulosa cells coupled to it (Herlands and Schultz, 1984) as nucleolar transcriptional activity during growth (McGaughey the granulosa cells effectively increase the surface area of the et al., 1979). If only fully grown oocytes are competent to be- oocyte. The surface to volume ratio of the oocyte is increased come embryos after fertilization, this could indicate that a limit- and hence the rate of entry of small molecules crucial for ing factor is accumulated to a critical concentration at the end growth and development is also increased. In addition to these of the growth phase. In addition to providing metabolic sup- interactions mediated by gap junctions, oocyte development is port, the preovulatory follicle generates signals required for the regulated by paracrine signals from the granulosa cells and, completion of maturation, in response to the LH surge. The LH while denuded oocytes cannot grow, some development is surge results in the elimination of one or more inhibitory sub- possible when co-cultured with soluble factors from granulosa stances such as oocyte maturation inhibitor (OMI) and the cells (Cecconi et al., 1996). Exogenous factors that may promote elimination of these inhibitory substances leads to the acti- oocyte growth and survival in a variety of species include c-kit, vation of cyclins, phosphatases and kinases, which are required c-kit ligand and stem cell factor (Packer et al., 1994; Tanikawa for nuclear maturation to be achieved. et al., 1998). Furthermore, a gene has been identified in mice In contrast to mouse oocytes, an active transcription and (GDF-9) which is expressed exclusively in the oocyte and translation is required for chromatin condensation and germi- which appears to be required for the early stages of folliculo- nal vesicle breakdown in pig oocytes. When resumption of genesis (Dong et al., 1996). Nevertheless, the precise signal for meiosis occurs, it is accompanied and regulated by a sub- the initiation of oocyte and follicular growth in pigs remains stantial increase in the cytosolic kinase activity of the oocyte. unknown, but clearly requires further investigation. Oocyte– A pivotal component of this activity is cyclin B-p34cdc2, also granulosa cell communication is bi-directional, and pig oocytes called maturation promoting factor (MPF) (for review, see secrete a cumulus expansion-enabling factor (Singh et al., 1993; Dekel, 1996). MPF is a serine–threonine protein kinase involved Nagyova et al., 1997) in addition to a soluble factor that regu- in cell cycle regulation and MPF activity in pig oocytes is lates cumulus cell steroidogenesis (Coskun et al., 1995), sup- assessed by measurement of histone H1 kinase activity. This presses luteinization and promotes proliferation of granulosa activation triggers a series of reactions leading ultimately to cells in culture (Brankin et al., 1999). Similar data have also nuclear membrane breakdown, chromosome condensation and been produced in mice (Vanderhyden and Tonary, 1995) and spindle formation, events that are vital to support successful cows (Armstrong et al., 1999), and it appears that the factor is fertilization and early embryo development. Christmann et al.

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84 days 14 days 19 days

Primordial Antrum > 3 mm Preovulatory follicle formation follicle

Oocytes meiotically Some oocytes Most oocytes meiotically competent incompetent competent 120 µm 120 µm

Oocyte diameter 30 µm 100 µm 115 µm 120 µm LH surge

Follicle diameter/ Preantral 0.7 mm 1.8 mm ≥ 5 mm 8Ð10 mm 8Ð10 mm development Preovulatory Ovulatory

Fig. 2. Follicle and oocyte growth and the acquisition of meiotic competence in pigs. The days reported for the various developmental stages are based on development in vivo (Morbeck et al., 1992).

(1994) showed that inadequacies in these cell cycle molecules been shown to be activated before or concurrently with MPF in limit meiotic competence and, specifically, that concentrations pig oocytes (Naito and Toyoda, 1991). MAPK values increase of p34cdc2 are limiting in small oocytes in preantral follicles and steadily up to the metaphase II stage and MAPK activity re- that, in growing oocytes, the inadequacy lies in MPF kinase acti- mains high even when MPF activity shows a transient drop vation rather than in the absolute concentrations of p34cdc2 during anaphase and telophase. Unlike MPF activity, MAP (Hirao et al., 1995). MPF activity remains high in the metaphase kinase activity remains high at the extrusion of the polar body. II stage, when the second meiotic arrest occurs. After fertil- The activities of MPF and MAPK in relation to oocyte nuclear ization or parthenogenetic activation, the activity decreases and maturation are illustrated (Fig. 3). meiosis II is completed (Kikuchi et al., 1995). This decrease in Although current knowledge indicates that MAPK and MPF MPF activity occurs concomitantly with the exit from meta- are involved in the regulation of the maturation process, their phase I and II and indicates that the inactivation of MPF is precise physiological relevance for oocyte maturation has not necessary for this progression. Funahashi et al. (1996) showed been fully established. Further information will aid in the that the MPF activity of pig oocytes matured in vitro was influ- understanding of the processes involved in cytoplasmic matu- enced by the culture medium, and a correlation between MPF ration and should help improve culture conditions. The pig activity in metaphase II oocytes and their ability to develop oocyte is the ideal system in which to study these events pronuclei was reported by Naito et al. (1992). Thus, it may be since pig oocytes require almost twice the time of other large that the determination of MPF activity could be used as an domestic animals, such as cattle, to transform the germinal indicator of cytoplasmic maturation of pig oocytes and, hence, vesicle into condensing chromatin (the germinal vesicle stage of oocyte ‘quality’. is 20–24 h and progression occurs to the metaphase II stage Although it is clear that MPF plays an important role in in 40–46 h in vitro (Motlik and Fulka, 1976)). This delay in vitro driving the meiotic resumption, other kinases are activated be- reflects a comparable situation in vivo and the extended duration fore or concurrently with MPF during reinitiation of meiosis. of the germinal vesicle stage compared with that in other species One group of these kinases are the mitogen-activated protein facilitates investigation. kinases (MAPK) that phosphorylate cytoskeletal proteins and nuclear lamin, both of which play a pivotal role in meiotic cell Oocyte maturation in vitro divisions (Wehrend and Meinecke, 1998). Activation of two MAPK isoforms (ERK1 and ERK2) with different molecular Although pig oocytes matured in vitro can be penetrated by weights has been described during pig oocyte maturation spermatozoa under appropriate conditions, low rates of pro- (Inoue et al., 1995) and this activation requires active protein nuclear formation and a high incidence of polyspermy occur synthesis (Meinecke et al., 1997). In addition, members of the and, therefore, the developmental potential, as determined by family of the mitogen-activated protein (MAP) kinases have the quality of the oocyte, after fertilization is disappointingly

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Table 1. Summary of factors shown to improve nuclear or cytoplasmic maturation of pig oocytes in vitro as assessed by maturation to meta- MII phase II, fertilization, male pronuclear formation, cleavage or blastocyst development

Factor References MI MAPK Follicular factors Follicular fluid Niwa, 1993; Yoshida et al., 1992; GV A/T Funahashi and Day, 1997a

Enzyme activity MPF Follicle shells/ granulosa cells Mattioli et al.,1988a; Ding and Foxcroft, 1992; Liu et al., 1997

Time in culture Follicle conditioned media Mattioli et al., 1988b; Ding and Foxcroft, 1994a

Steroids* Fig. 3. Activities of maturation promoting factor (MPF) and mitogen Progesterone, oestradiol Thibault et al., 1987; activated protein kinase (MAPK) during nuclear maturation of Mattioli et al., 1998a pig oocytes in vitro (adapted from Wehrend and Meinecke, 1998). Androgen Petr et al., 1996; Coskun et al., 1997 GV, germinal vesicle; MI, metaphase I; A/T, anaphase and telo- phase; MII, metaphase II. Gonadotrophins LH/FSH Liu et al., 1997; Illera et al., 1998 low. This finding implies that conditions in vitro are vastly in- Growth factors ferior to those in vivo for reasons that are not fully compre- IGF-I Xia et al., 1994; Illera et al., 1998 hended. While the importance of hormones in the ovary is well known, the influence of the follicle on the oocyte is poorly EGF Ding and Foxcroft, 1994b; understood. In early studies, oocytes were matured in vitro in Illera et al., 1998 medium containing only bovine serum albumin or serum and Others normal male pronuclear formation did not occur. At that time it was concluded that, for normal fertilization to occur, the oocyte TIMP-1 Funahashi and Day, 1997b had to experience germinal vesicle breakdown in vivo (Motlik Glucocorticoids Yang et al., 1999 and Fulka, 1986) and that pig oocytes matured in vitro were de- Cysteine/cysteamine Yoshida 1993; Grupen et al., 1995 ficient in some cytoplasmic factors. Now that successful culture techniques for the early development of pig embryos from the Hyaluronic acid Miyoshi et al., 1999 one-cell to the blastocyst stage have been developed, efforts to produce embryos in vitro have focused on the development of a *In some instances, steroids were reported to inhibit pig oocyte maturation. EGF: epidermal growth factor; IGF-I: insulin-like growth factor I; TIMP-1: tissue system for efficient in vitro maturation (IVM) and in vitro fertil- inhibitor of metalloproteinase 1 ization (IVF) of good quality oocytes. The cellular relationship between the oocyte and somatic fol- licular cells is fundamental to oocyte maturation (Moor et al., 1990) and follicular cells play a critical role in the regulation of (Mattioli et al., 1988a). A major advantage of this persistent in- oocyte meiotic arrest and the resumption of meiosis, in ad- teraction between cumulus cells and oocyte is the acquisition of dition to providing nutrients. Pig oocytes require follicle cell the factors in the ooplasm required to decondense penetrated stimulation for at least the first 32 h of maturation (Moor et al., spermatozoa and form male pronuclei. Inability to perform this 1990) and the role of the cumulus cells is to maintain functional function has often been the major abnormality of pig oocytes intercellular coupling between the oocyte and the follicular matured in vitro. However, it has become apparent that the in- compartment (Mattioli et al., 1988a). Studies on the maturation cidence of male pronuclear formation is highly correlated with of pig oocytes in vitro by co-culture with either follicular cells the glutathione content of the oocyte (Yoshida et al., 1993) and or follicular fluid (Niwa, 1993; Sirard et al., 1993) indicate that that the addition of cysteine during IVM increases glutathione follicular cells secrete factors that play a crucial role in sup- content (Grupen et al., 1995). Therefore, the previous problem porting oocyte cytoplasmic maturation. Even when cumulus of failure in male pronuclear formation appears to have been cells are present, the addition of follicle shells to cultures of due, at least in part, to oxidative stress. cumulus-enclosed oocytes confers normal condensation com- Factors shown to influence pig oocyte nuclear and/or cyto- petence (Mattioli et al., 1988b; Moor et al., 1990). The precise plasmic maturation in vitro are summarized (Table 1). This mechanism by which follicular and granulosa cell factors influ- summary is based on improvements observed after the ad- ence the process of oocyte maturation remains to be clarified, dition of the above components to IVM systems and it is con- although it is likely to be attributable, at least in part, to the ceivable that they reflect an indirect effect on follicle–cumulus positive influence of somatic cells on cumulus–oocyte coupling cells rather than a direct effect on the oocyte itself. Although

Downloaded from Bioscientifica.com at 09/30/2021 07:23:49PM via free access 126 M. G. Hunter transcripts and proteins have been detected in other mam- maintained (Eppig and O’Brien, 1996). Culture systems for malian oocytes for receptors such as oestrogen (Wu et al., 1992), pig follicles are at a less advanced stage of development activin (Manova et al., 1995), c-kit (Manova et al., 1993) and (Greenwald and Moor, 1989) partly owing to their larger TNF-α (Naz et al., 1997), they have yet to be demonstrated in physical size, although culture of preantral follicles for 16 days pig oocytes. The presence of the oestrogen receptor is particu- produced some oocytes that were meiotically competent larly interesting as oestradiol is the major steroid produced by and penetrated by spermatozoa in vitro (Hirao et al., 1994). the preovulatory follicle. Mattioli et al. (1998b) showed that the Oocyte–granulosa cell complexes isolated from sheep ovaries lack of oestradiol during the initial phase of pig oocyte matu- have been grown to antral size in vitro and contain viable ration in vitro was associated with subsequent developmental oocytes (Newton et al., 1999) and, in our laboratory culture of failure. Conversely, the prolific Meishan pig, which is proposed pig preantral follicles for 30 days, have produced antrum to produce oocytes of improved quality (see below), has a formation and viable oocytes (G. Shuttleworth, unpublished). higher concentration of oestradiol in the follicular fluid that Similar results have been reported using pig oocyte–cumulus– surrounds the oocyte. Indeed, recent data from our laboratory granulosa cell complexes recovered from early antral follicles indicate that the presence of the pure oestrogen antagonist (Shen et al., 1997). Advances in ‘culture technology’ are being ICI 182,780 in the maturation medium significantly reduced made and further progress will be facilitated by improved the number of oocytes reaching metaphase II (R. Kerr and understanding of the mechanisms regulating growth of the M. G. Hunter, unpublished). Furthermore, the oestrogen recep- follicle wall. tor, which is a ligand-activated transcription factor of which there are two forms (α and β) can also be activated by non- Oocyte maturation in vivo steroidal factors. Growth factors are particularly important in controlling oestrogen receptor function and both insulin-like Implications of follicle maturation and heterogeneity growth factor I (IGF-I) and epidermal growth factor (EGF) activate transactivation function through post-translational Only a very small fraction of competent oocytes retained processing of the receptor (Ignar-Trowbridge et al., 1996). As a within the follicle resume meiosis each cycle in vivo. In pigs, result of this interaction between IGF and EGF, it is possible several (14–20) growing follicles survive through to ovulation that oocyte maturation and quality are affected by growth fac- in each cycle, although it has been demonstrated that the fol- tors (see Table 1) through an effect on oestrogen receptor licular population is morphologically and biochemically het- function. The ability to manipulate growth factor availability in erogeneous (Foxcroft and Hunter, 1985) and even follicles of follicular fluid in vivo via nutrition (see below) provides the similar size can show marked differences in follicular fluid opportunity to study further the direct role of growth factors steroid concentrations, granulosa cell number and LH recep- on oocyte maturation in the preovulatory follicle. tors. This asynchrony persists from the time of follicle recruit- In addition to steroids and growth factors, tissue inhibitors ment through to the time of ovulation and beyond and, in of metalloproteinases (TIMPs) have been indicated as con- follicles recovered between the LH surge and ovulation, dif- ferring developmental competence on pig oocytes during the ferences in the luteinization response and oocyte maturation maturation phase (Funahashi et al., 1997b). TIMP-1 is a major are already apparent (Hunter and Wiesak, 1990). Furthermore, secretory product of pig preovulatory granulosa (Smith et al., the maturational status of the follicle used for co-culture or to 1994) and theca (Shores, 1999) cells and, after the preovulatory provide cells or fluid had a critical influence on the cytoplasmic LH surge, mRNA encoding TIMP-1 is increased. The presence maturation of pig oocytes (Ding and Foxcroft, 1992, 1994a), of TIMP-1 from 20–44 h of culture for IVM of pig oocytes en- with larger, more mature follicles producing better quality hanced the competence to develop to the blastocyst stage with- oocytes as determined by sperm penetration and male pro- out affecting factors associated with fertilization. It is currently nuclear formation. This finding reinforces the critical influence unknown how TIMP-1 promotes the ability of pig oocytes to of the follicle on oocyte maturation and has implications for develop to the blastocyst stage, and clearly this warrants oocyte quality in vivo. Extensive studies (Pope et al., 1990; Pope, further study. These findings emphasize the range of somatic 1992) provided convincing evidence that the preovulatory de- signals that influence oocyte maturation and also that the velopment of pig follicles and oocytes has consequences for actions they exert during oocyte maturation may not become embryo development and survival. The maturational state of evident until blastocyst formation. The modification of con- oocytes in the periovulatory period is associated with their de- ditions during IVM as described above has improved the velopment as early zygotes and, furthermore, later ovulating incidence of blastocyst formation after IVM, IVF and in vitro oocytes develop into the smallest embryos in utero and are more culture significantly, and now > 30% of pig oocytes matured vulnerable to the changing uterine environment. Therefore, it in vitro develop to the blastocyst stage (Funahashi and Day, appears that, in pigs, folliculogenesis directs embryogenesis, at 1977). least in part, by influencing oocyte quality (Pope, 1992). A potentially useful alternative approach is the culture of immature follicles to a stage at which mature oocytes can be Oocyte quality in Meishan pigs harvested. This approach would produce large numbers of oocytes and, in addition to improving IVF and embryo transfer, Chinese Meishan pigs are characterized by their prolificacy, would greatly facilitate transgenesis and gene targeting. This producing an average of three or four more piglets per litter in vitro approach has already been used succesfully to pro- than European breeds (Haley and Lee, 1993). Substantial dif- duce live pups after culture of mouse primordial follicles to ferences in the characteristics of follicle maturation between maturity, during which the three-dimensonal architecture was Meishan and Large White hybrid pigs have been observed.

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These differences include a smaller preovulatory follicle di- ameter, but an increased follicular fluid oestradiol concen- tration and aromatase activity in both granulosa and theca Age of animal tissues in Meishan animals (Hunter et al., 1993). Meishan fol- licles also produce more cAMP in response to LH and luteinize more rapidly in response to hCG administration (Downey and Breed/genotype Driancourt, 1994) or the endogenous LH surge (Hunter et al., 1996). Furthermore, more oocytes mature to the metaphase II stage within the estimated 7 h before ovulation in Meishan than in Large White hybrid animals (Faillace and Hunter, 1994). Nutrition In female pigs managed under normal husbandry con- ditions, fertilization rates are high and most prenatal death (approximately 30%) occurs during the embryonic period be- Environment fore day 30 of pregnancy. The precise timing of this loss has been much studied and, although reports do not all agree, the general consensus is that most embryo death occurs between days 12 and 18 of pregnancy. Several studies indicate that Fig. 4. Summary of physiological situations influencing oocyte the prolificacy of Meishan pigs is due primarily to enhanced quality in vivo. Short arrows represent the intrafollicular routes of early embryonic survival (for review, see Haley and Lee, 1993), communication to the oocyte via granulosa–theca cell secretions, although the precise mechanisms by which this is achieved re- follicular fluid composition and cumulus cell coupling and action. main unclear. However, it is likely that the different pattern of follicle maturation and hence oocyte quality in Meishan pigs is an important component. The LH surge occurs later relative to has a much greater influence on subsequent embryo survival the onset of behavioural oestrus in Meishan than in Large than diet after mating. Ashworth et al. (1999) showed that White hybrid animals (Hunter et al., 1993) and the admin- gilts that consumed 2.8 × maintenance rations during the istration of hCG at the onset of oestrus decreases embryo sur- oestrous cycle preceding mating had had a higher percentage vival in Meishan animals (Hunter and Picton, 1995). of embryos that survived, and that blastocysts recovered from These observations led to the hypothesis that the Meishan these gilts were larger with enhanced metabolic and secretory follicle may provide a ‘better’ environment for oocyte matu- activity in vitro. These gilts also exhibited less within-litter ration than Large White hybrid follicles, which may contribute variation in development, which may provide a means of re- to the prolificacy of Meishan pigs by producing oocytes of ducing within-litter variability in birth weight and vitality. improved ‘quality’. A preliminary study indicated that more Although the specific mechanisms responsible have not yet Meishan than Large White hybrid oocytes progressed to been determined, it is likely that the quality of the oocytes metaphase II when cultured with a follicle shell of the and hence their developmental capacity was influenced by same breed. A further experiment was carried out to address nutritionally induced changes in follicle development. Support this issue: oocytes recovered from the abattoir were matured for this concept has been provided by the experiments of Zak in the presence of conditioned media generated by the incu- et al. (1997) in which sows fed to appetite during week 4 of bation of everted preovulatory follicles recovered from either lactation had more large oestrogenic follicles and higher em- Meishan or Large White hybrid animals, followed by fertil- bryo survival than sows receiving only 50% rations during the ization in vitro. A total of 1343 oocytes was cultured and results same period. Significantly, more oocytes from the re-fed group showed that both sperm penetration rates and male pronuclear than from the restricted-diet group progressed to metaphase II formation rates were significantly higher in oocytes matured in vitro. A large number of abattoir-derived oocytes were in Meishan-conditioned media than those matured in Large matured in follicular fluid from re-fed or restricted sows to White hybrid-conditioned media (Xu et al., 1998). This finding test further the hypothesis that dietary modification of the pre- supports the hypothesis that differences in the maturational ovulatory follicular environment affects oocyte quality. The status of Meishan compared with Large White hybrid follicles fluid from the re-fed animals was better able to support oocyte results in an improved ability of the follicles to support maturation than fluid from the restricted animals, again sup- oocyte maturation and produce oocytes of improved quality porting the hypothesis that nutrition affects follicle secretions (Fig. 4). The challenge remaining is to identify the factors that, in turn, influence oocyte quality. The precise nature of responsible, for example by two-dimensional electrophoresis these altered secretions remains to be elucidated, although the of proteins secreted into the conditioned media by follicles of insulin–IGF system is a likely candidate. Therefore, nutrition- each breed, as used by Driancourt et al. (1996) to investigate ally mediated effects on oocyte maturation and quality appear sheep follicle secretions. to be central to embryo survival and the programming of fetal growth trajectory (Fig. 4). Nutritional influences on oocyte quality Other influences on oocyte quality Evidence is accumulating that indicates that maternal nu- trition before conception has a major impact on pregnancy out- Environmental stressors such as temperature and season come in cattle (McEvoy et al., 1997) and pigs (Ashworth et al., affect embryo viability, and seasonal variations in fertility 1999) and, furthermore, that nutritional status before mating occur in the UK. Although there are several mechanisms that

Downloaded from Bioscientifica.com at 09/30/2021 07:23:49PM via free access 128 M. G. Hunter could account for this reduction, alterations in follicle growth Ashworth CJ, Antipatis C and Beattie L (1999) Effect of pre- and post-mating and hence oocyte ‘quality’ have been proposed (Wetteman and nutritional status on hepatic function, progesterone concentration uterine protein secretion and embryo survival in Meishan pigs Reproduction, Bazer, 1985; Fig. 4). The timing of insemination relative to ovu- Fertility and Development 11 67–73 lation is also important and the ageing of oocytes results in re- Black JL and Erickson BH (1968) Oogenesis and ovarian development in the duced fertilization and, in cases in which fertilization does prenatal pig Anatomical Record 161 45–56 occur, in reduced embryo viability (Hunter, 1967). Bolamba D, Dubuc A, Dufour JJ and Sirard MA (1996) Effects of gonado- Exogenous hormone administration is used frequently to tropin treatment on ovarian follicle growth, oocyte quality and in vitro fertilization of oocytes in prepubertal gilts Theriogenology 46 717–726 promote follicle growth, although it may affect oocyte quality. Brankin V, Webb R and Hunter MG (1999) Factor(s) secreted by the porcine Pinkert et al. (1989) showed that zygotes recovered from super- oocyte modulate granulosa cell growth and steroidogenesis Journal of ovulated prepubertal gilts had a reduced capacity for de- Reproduction and Fertility Abstract Series 23 66 velopment in vitro compared with zygotes from pubertal gilts. Cecconi S, Rossi G, De Felici M and Colonna R (1996) Mammalian oocyte growth in vitro is stimulated by soluble factor(s) produced by preantral In another study, gonadotrophin priming of prepubertal granulosa cells and by Sertoli cells Molecular Reproduction and Development gilts increased the number of large follicles, but the effect 44 540–546 on the proportion of good quality oocytes varied depending on Christmann L, Jung T and Moor RM (1994) MPF components and meiotic the precise hormonal stimulation used (Bolamba et al., 1996). competence in growing pig oocytes Molecular Reproduction and Development Gilts bred at their first oestrus are less prolific than gilts 38 85–90 Coskun S, Uzumcu M, Lin Y, Friedman CI and Alak BM (1995) Regulation of that experience two or more cycles before mating. Although granulosa cell steroidogenesis by the porcine oocyte and preliminary char- this can be attributed, at least in part, to a lower ovulation rate acterization of oocyte-produced factor(s) Biology of Reproduction 53 670–675 in pubertal gilts, embryo transfer experiments have shown that Coskun S, Friedman CI and Alk BM (1997) Differential effect of testosterone embryos recovered from donors mated at their first oestrus sur- on porcine oocyte maturation Biology of Reproduction Supplement 56 330 Dekel N (1996) Protein phosphorylation/dephosphorylation in the meiotic vived less well than those mated at their second oestrus cell cycle of mammalian oocytes Reviews of Reproduction 1 82–88 (Archibong et al., 1992). This phenomenon is probably caused Ding J and Foxcroft GR (1992) Follicular heterogeneity and oocyte matu- by differences in oocyte ‘quality’ and Koenig and Stormshak ration in vitro in pigs Biology of Reproduction 47 648–655 (1993) reported that more cytogenetically immature oocytes Ding J and Foxcroft GR (1994a) Conditioned media produced by follicular were ovulated at the first compared with at the third oestrus. shells of different maturity affect maturation of pig oocytes Biology of Reproduction 50 1377–1384 Ding J and Foxcroft GR (1994b) Epidermal growth factor stimulates oocyte maturation in pigs Molecular Reproduction and Development 39 30–40 Conclusions Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N and Matzuk MM The developmental competence of pig oocytes matured and (1996) Growth differentiation factor 9 is required during early ovarian folliculogenesis Nature 383 531–535 fertilized in vitro has been enhanced through modification of Downey BD and Driancourt MA (1994) Morphological and functional the culture conditions of oocytes during IVM and this has been characteristics of preovulatory follicles in large white and Meishan gilts achieved primarily by mimicking active communication be- Journal of Animal Science 72 2099–2106 tween the oocyte and follicular cells. The differential effect of Driancourt MA, Gormon T, Phan Thanh L and Boomarov O (1996) Analysis co-culture with follicles at different maturational stages is an by two-dimensional electrophoresis of proteins secreted by sheep ovarian follicles: effects of the FecB gene, follicle size and atresia Journal of important finding and indicates that the heterogeneous intra- Reproduction and Fertility 107 69–77 follicular environment to which oocytes are exposed in a poly- Eppig JJ and O’Brien MJ (1996) Development in vitro of mouse oocytes from ovular species such as the pig results in oocytes of different primordial follicles Biology of Reproduction 54 197–207 quality and developmental potential. Evidence indicates that Faillace LS and Hunter MG (1994) Follicle development and oocyte matu- ration during the immediate preovulatory period in Meishan and white the status of the follicle, as determined for example by matu- hybrid gilts Journal of Reproduction and Fertility 101 571–576 rational stage, breed or maternal nutrition, has a critical influ- Foxcroft GR and Hunter MG (1985) Basic physiology of maturation in the ence on oocyte quality in vivo presumably due to alterations in pig Journal of Reproduction and Fertility Supplement 33 1–19 the somatic signals. The identification of the critical follicular Fulka J, Jr, Bradshaw J and Moor RM (1994) Meiotic cycle checkpoints in factors from the large number implicated will enable controlled, mammalian oocytes Zygote 2 351–354 *Funahashi H and Day BN (1997) Advances in in vitro production of pig predictable maturation to be achieved in vivo and in vitro. embryos Journal of Reproduction and Fertility Supplement 52 271–283 Further research is needed to determine ‘markers’ of oocyte Funahashi H, Stumpf TT, Kim NH and Day B (1996) Low salt maturation quality so that oocytes with adequate developmental com- medium enhances the histone H1 kinase activity of porcine oocytes at the petence can be identified before fertilization. end of in vitro maturation Journal of Reproduction and Development 42 109–115 Funahashi H, Cantley C and Day BN (1997a) Synchronisation of meiosis in porcine oocytes by exposure to dibutyryl cyclic AMP improves develop- Collaboration with numerous colleagues and students is gratefully mental competence following in vitro fertilization Biology of Reproduction acknowledged. 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