MOLECULAR REPRODUCTION AND DEVELOPMENT 29:385-391(1991)
Review Article Oviduct Function in Pigs, With Particular Reference to the Pathological Condition of Polyspermy R.H.F. HUNTER University of Edinburgh, Edinburgh, Scotland
ABSTRACT Because the exceptionally high incidence of polyspermic fertilisation has been emphasised as a major defect in systems of in vitro fertilisation in pigs, the aetiology of the condition has been analysed in a series of experiments in vivo in the search for a common underlying cause and possible means of mitigation. Whereas the defense mechanism against polyspermy in pig oocytes is classically viewed as a zona reaction, more recent evidence suggests a secondary block at the vitelline surface. Both blocks may be compromised in situations leading to polyspermy, although deleterious influences seem to be expressed principally in an inadequate zona block, as judged by the presence of perivitelline sperma-
tozoa. Postovulatory aging of mammalian oocytes prior to sperm penetration leads to polyspermy, as can be demonstrated in pig eggs. The primary lesion may concern the cortical reaction, owing to a delayed and incomplete exocytosis of the vesicular contents. Eggs ovulated after gonadotrophin treatment during the luteal phase of the cycle show a high incidence of polyspermic penetration (60.6%),as do those shed at estrus in animals treated with progesterone systemically (40%)or by local microinjections in the oviduct wall (32.3%).Whereas progesterone may be modifying interactions of the gametes and responses of the egg organelles in all four above experimental situations, enhanced numbers of spermatozoa ascending a more patent isthmus appear to be the principal cause of polyspermy. Support for this hypothesis comes from isthmus resection with re-anastomosis of remaining portions of the oviduct (32.4% polyspermy), and surgical insemination directly into the oviducts (33.8%polyspermy). Recent experiments draw attention to a role for oviduct epithelial glycoproteins in modulating the extent of polyspermy in vitro. The role of such oviduct macromolecules in gamete interactions in vivo is considered.
Key Words: Fertilisation, Spermatozoa, Insemination, Progesterone, Glycoprotein
INTRODUCTION The stimulus for preparing this previously unpublished lecture as a manuscript has been the recent paper by Nagai and Moor (1990), with the exciting suggestion that oviduct secretory products (macromolecules) derived from co-culture of epithelial cells with gametes may act to influence the incidence of 0 1991 WILEY-LISS, INC.
polyspermy in pig eggs fertilised in vitro. This proposition and the underlying concern with polyspermy in pig eggs seem worth setting in an historical perspective, albeit a somewhat personal one. Physiological processes in the oviduct that influence mammalian fertilisation have been reviewed in recent years in a number of key publications (Gwatkin, 1977; Bedford, 1982; Fraser, 1984; Fraser and Ahuja, 1988; Hunter, 1988; Yanagimachi, 1988). By contrast, the etiology of polyspermic fertilisation has received very little attention, even though polyspermy may arise in vivo under diverse experimental conditions. This latter statement will be illustrated in the following review by specific reference to observations in the domestic pig. This is an invaluable animal in studies of fertilisation, since (1)relatively large numbers of eggs are ovulated from the two ovaries, and (2) the time of ovulation can be regulated with precision. Polyspermy refers to penetration of the vitellus-the cytoplasm of the egg-by more than one spermatozoon. Unlike the situation in fish and birds, polyspermy is known to be a pathological condition in placental mammals, usually causing very early death of the zygote (Beatty, 1957; Bomsel-Helmreich, 1965). It has been recognised, since at least 1954, that the block to polyspermy-the egg’s defense mechanism-is located in the zona pellucida of most mammals so far studied, and not primarily at the surface of the vitelline membrane as occurs in the rabbit egg (Braden et al., 1954). In fact, a block at the level of the zona pellucida in pig eggs could have been judged from photographs of histologically sectioned eggs published at the turn of the century (Assheton, 1898); these show very many sperm heads embedded in the substance of the zona pellucida. More recently, it has been appreciated that there may also be a secondary block to polyspermy functioning at the surface of the vitelline membrane in pig eggs (Hunter and Nichol, 1988; Hunter, 1990a). Two further sentences ofjustification for focussing on polyspermic fertilisation may be appropriate at this stage. First, polyspermy remains a relatively frequent Received March 4, 1991; accepted March 13, 1991. Address reprint requests to Dr. R.H.F. Hunter, 32 Gilmour Road, Edinburgh EH16 5NT, Scotland. This paper was an invited lecture presented a t McGill University, Montreal, March 1990.
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R.H.F. HUNTER
occurrence in pig eggs, and may be a significant cause of embryonic loss which commonly attains an incidence of 3040% in this species (Perry, 1954; Hanly, 1961; Polge, 1978). Second, in all the recent studies of in vitro fertilisation, polyspermic penetration is by far and away the largest abnormality if fertilisation occurs at all (reviewed by Hunter, 1990a).
HISTORICAL PERSPECTIVE The first publications specifically concerned with polyspermic fertilisation of pig eggs appeared during the 1950s, and all three reports associated the polyspermic condition with delayed (i.e., late) matings and postovulatory aging of the egg before sperm penetration. The incidence of polyspermy in these reports extended from 10 to 29% (Pitkjanen, 1955; Hancock, 1959; Thibault, 1959). However, none of these investigators knew when ovulation had occurred, so the incidence of polyspermy could not be related to the age of egg at initial penetration. But when the author commenced his own research studies in 1962, two techniques were available that offered much greater precision in studies of mammalian fertilisation, and could certainly be applied to the examination of vitelline events in the pig. These were (1)the technique of phase-contrast microscopy of whole-mount preparations (Austin and Smiles, 1948; Chang, 1952), little exploited on the eggs of farm animals, enabling nuclear structures to be visualized in fixed and stained preparations without resorting to the relatively laborious procedures of serial histology; and (2) the ability to control precisely the time of ovulation with an injection of a luteinizing hormone (LH) or human chorionic gonadotropin (hCG) preparation given to animals in late proestrus when a responsive population of mature Graafian follicles would be available. Such an injection anticipates and mimicks the endogenous surge of gonadotropic hormones to programme resumption of meiosis and collapse of the follicles.
POSTOVULATORY AGING OF EGGS By combining the two techniques mentioned above, while working in the laboratory of Professor E.J.C. Polge at the Animal Research Station in Cambridge, the postovulatory age of pig eggs at the time of initial sperm penetration was regulated. An increasing incidence of polyspermic fertilisation occurred with increasing age of the eggs, reaching a highest mean figure of 15.4% (Table 1). However, the animals had been artificially inseminated with relatively low numbers of spermatozoa (1-2 x lo8 celldm1 in approximately 100 ml of fresh semen), and the polyspermic condition observed was principally one of dispermy or trispermy (Hunter, 1967a). The nature of the postovulatory degeneration that enhanced an egg’s susceptibility to polyspermic penetration was not clarified, but was nonetheless suspected to involve vitelline organelles and the plasmalemma rather than primarily the zona pellucida, although a diminishing effective-
ness of the cortical response would be expressed in the latter (Szollosi, 1975).
LUTEAL-PHASE OVULATIONS A second experiment concerned eggs resulting from ovulations induced during the luteal phase of the estrous cycle with precisely imposed gonadotrophin treatments. Many reports had appeared during the 1940s and 1950s, especially from the Wisconsin laboratory of L.E. Casida, documenting the infertility associated with luteal phase ovulations in the major farm species of cow, sheep, and pig and during pseudopregnancy in rabbits (Casida et al., 1940,1943;Murphree et al., 1944, 1947; Boyarsky et al., 1947; Tanabe et al., 1949;Rowson, 1951;Spalding et al., 1955).One possible interpretation of such infertility was as an extreme form of delayed insemination-the postovulatory aging egg syndrome-which would effectively represent the situation in the early luteal phase. The regimen used for predictably inducing ovulation during the luteal phase of the porcine estrous cycle was a subcutaneous injection of 1,500 IU PMSG on day 5 of the cycle, followed by an intramuscular injection of 500 IU hCG on day 9; ovulation occurred on day 11. Such animals did not exhibit behavioural estrus at the time of induced ovulation, so mating was not possible. Instead, they were inseminated artificially 4-18 hr before ovulation with 120 ml of fresh undiluted semen. In the 160 penetrated eggs recovered from 14 animals, the incidence of polyspermic penetration was 60.6%, and the degree of the polyspermic condition ranged from 2 to 7 accessory male pronuclei as well as from 2 to >50 swollen and unswollen sperm heads (Hunter, 1967b). Such extensive polyspermy appeared to offer a plausible explanation for the widely reported infertility during the luteal phase of the estrous cycle.
EGG TRANSPLANTATION STUDIES Arising from the previous study came a proposition that bears on a putative action of oviduct luminal fluids, that is of secretions from oviduct epithelial cells (Nagai and Moor, 1990). In fact, it represented the author’s own initial interest in a physiological role for such fluids. As a result of many discussions, the notion arose that the exceptionally high incidence of polyspermic fertilisation may have been due primarily to an influence of luteal-phase oviduct fluids on the mechanism of the block to polyspermy, the composition of oviduct luminal fluid being dominated by the high titres of circulating progesterone. In brief, the hypothesis was that luteal-phase oviduct fluid had rendered the zona reaction less effective. Support for this hypothesis was obtained from two sets of egg transplantation experiments, each involving 12 animals (R.H.F. Hunter, C. Polge, and L.E. Rowson, unpublished observations). Eggs released from mature follicles during estrus were transplanted into the oviducts of animals artificially inseminated on day 11 of the luteal phase. This manipulation induced extensive polyspermy, with
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387
TABLE 1. Demonstration of the Influence of Postovulatory Aging of Eggs Before Sperm Penetration on the Incidence of Normal Fertilisation and That of Polyspermic Fertilisation* Estimated age of egg at sperm penetration (hr)
Eggs examined and stained
0 4 8 12 16 20
Eggs fertilised normally
(N)
(W
Eggs polyspermic
141 152 166 155 149 155
90.8 92.1 94.6 70.3 48.3 50.9
0.0 1.3 1.2 6.8 15.4 11.1
(%I
Adapted from Hunter (1967a).
transplanted eggs exhibiting 3 or 4 large male pronuclei, together with a variable number of sperm heads in the vitellus and spermatozoa also frequently being observed in the perivitelline space (Fig. la,b). The reciprocal experiment of transplanting eggs derived from ovulations during the luteal phase of the oestrous cycle into the oviducts of mated oestrous animals resulted in morphologically normal fertilisation (Hunter, 1965). These observations led to the conclusion that, in pigs at least, eggs shed during the luteal phase were not intrinsically defective and that the oviduct luminal environment under the influence of high progesterone titres was, by some unspecified action, downgrading the effectiveness of the block to polyspermy.
SYSTEMIC ADMINISTRATION OF PROGESTERONE
PROGESTERONE TITRES AND SPERM NUMBERS During 1967 and 1968, the author was restricted to working on small laboratory rodents in Massachusetts but he had nonetheless devoted some thought as to the means whereby circulating progesterone might be influencing the physiology of pig oviducts. At least as reasonable an explanation for the high incidence of polyspermy as an effect mediated by oviduct fluid on the zona pellucida would have been that of a direct influence of progesterone on the myosalpinx and on the mucosa of the oviduct wall. Whether of endogenous or exogenous origin, high titres of progesterone could have been acting to diminish the extent of oedema in the mucosa and also to relax the smooth muscle coats of the duct so that the lumen would be much increased in patency. This, in turn, could permit large numbers of spermatozoa to pass to the site of fertilisation at the ampullary-isthmic junction, with a concomitant increase in the risk of polyspermic fertilisation due to closely synchronous penetration of the zona pellucida by two or more spermatozoa. Critical factors influencing the incidence of polyspermy would include the number of competent spermatozoa achieving the surface of the zona pellucida and the actual rate of establishment of the block to polyspermy after activation of the cortical response. This possibility of an excess of competent spermatozoa at the site of fertilisation acting to promote pathological fertilisation was tested in a number of ways.
At this stage of the work (1965), the author became a postdoctoral student in North America, whereas Professor B.N. Day moved from Columbia, Missouri, to Cambridge, England, specifically to learn the procedures of phase-contrast microscopy and egg transplantation for studies in farm animals. The notion that progesterone in some manner acted to influence the normality of fertilisation had remained an important theme in Cambridge, not least since orally active progestagens were being used to synchronise estrus in farm animals, frequently with poor or very poor resultant fertility (Dziuk and Polge, 1962; Polge and Dziuk, ISTHMUS RESECTION AND POLYSPERMY 1965; Dziuk, 1965). The next logical experiment was A surgical approach was to remove most of the therefore performed by Day and Polge (1968), that of injecting inseminated animals systemically with a isthmus by resection and then anastomose the remainsolution of progesterone in oil (100 mg progesterone) ing portions of the oviduct so that the regulatory role of 24-36 hr before ovulation and then examining eggs the isthmic portion of the duct on sperm transport to recovered from the oviducts and/or uterus by phase- the site of fertilisation was largely overcome. This contrast microscopy. Two significant findings were experiment was performed during 1969 with a skilled reported: a 3640% incidence of polyspermy and an French surgeon at the Station de Physiologie Animale, accelerated passage of eggs through the oviducts into Jouy-en-Josas, and the incidence of polyspermy in eggs shed into the surgically shortened oviduct of 18 pigs the uterus.
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R.H.F. HUNTER
Fig. 1. a,b. Whole-mount preparations of pig eggs seen under phase-contrast with many spermatozoa visible in both the perivitelline space and zona pellucida. After fixing and staining, these eggs were found also to exhibit polyspermic penetration of the vitellus, with unswollen sperm heads and pronuclei in the same preparation.
c,d. Multiple sperm penetration of the vitellus demonstrable in spontaneously ovulated pig eggs viewed as stained preparations under the phase-contrast microscope. Coalescence of nuclear chromatin has led to the formation of aggregates between adjoining sperm head material.
OVIDUCT FUNCTION IN PIGS was 32.4%compared with 0.9% in 114 control eggs from the contralateral oviduct (Hunter and Lkglise, 1971). These findings therefore strengthened the argument that passage of elevated numbers of competent spermatozoa to the site of fertilisation increases the risk of polyspermic fertilisation.
MICROINJECTIONS OF PROGESTERONE SOLUTION A further experiment involving a progesterone treatment was to inject locally microdroplets of a solution of the steroid in oil (total of 1 mg progesterone in 0.1 ml oil) under the serosal layer of the oviduct wall in the vicinity of the utero-tuba1junction and distal portion of the isthmus some 8-12 hr before ovulation. The contralateral oviduct acted as a control, receiving a similar distribution of microinjections but only of the oil vehicle. The results based on 344 eggs from 30 animals revealed an incidence of polyspermy of 32.3% in the progesterone-treated oviduct compared with 9.7% in the control oviduct (Hunter, 1972). Local microinjections of the progesterone solution at a greater interval before ovulation might perhaps have increased the incidence of polyspermic fertilisation, since this would have permitted a longer interval in which to achieve relaxation of the myosalpinx and reduction of oedema in the mucosa, thereby increasing the patency of the duct. Whilst polyspermy was considered to have originated in this experiment principally from increased numbers of spermatozoa reaching the site of fertilisation, a direct effect of progesterone on the membranes of the gametes was not excluded. In any event, synchrony of development among the two or three male pronuclei suggested simultaneous or almost simultaneous penetration of the vitellus by several spermatozoa.
INSEMINATION INTO OVIDUCTS
389
noted for hamster eggs in vitro (Barros et al., 1972). On the basis of further analysis of the process of capacitation of boar spermatozoa in vivo highlighting the synergism between uterus and oviducts and the time relationships for sperm surface modifications in each of these compartments (Hunter and Hall, 1974a,b), suitably timed surgical insemination of sperm suspensions directly into the isthmus enabled a condition of massive polyspermy to be demonstrated (Hunter, 1976). Indeed, the extent of sperm penetration into the vitellus was frequently so overwhelming (>80 spermatozoa) that aggregates of sperm head chromatin were usually conspicuous (Fig. lC,D).
SUSCEPTIBILITY OF OOCYTES TO POLYSPERMY A conclusion that can be drawn from all these in vivo experiments, and that still appears tenable, is that the number of capacitated spermatozoa available to confront the newly ovulated eggs is the principal factor regulating the incidence and extent of polyspermic fertilisation. Under conditions of enhanced but no2 overwhelming numbers of competent spermatozoa in the oviducts, it would appear that approximately 30% of ovulated pig eggs are especially susceptible to polyspermy (Table 21, in part due to a slow instigation of a full zona reaction or to some related abnormality focussing on the properties of the zona pellucida or vitelline membrane. Indeed, as a working hypothesis, the suggestion has been offered that these 30-35% of spontaneously ovulated pig eggs represent those that may succumb to early embryonic death. Susceptibility to polyspermic penetration could thus be a means of revealing or identifying, albeit retrospectively, this particular population of eggs (Hunter, 1979, 1990a). Finally, with reference to the above studies on abnormal fertilisation in porcine oocytes, the wheel has now turned almost full circle in at least one sense, for the incidence and degree of polyspermy can be used to monitor the extent of the population of capacitated boar spermatozoa in the immediate vicinity of recently ovulated eggs (Hunter and Nichol, 1988). The experiments also raise the question of precisely how the oviducts normally regulate with such precision the number of competent spermatozoa reaching the surface of the eggs after a spontaneous mating so that the polyspermic condition is avoided. Such systems of regulation in the isthmic portion of the duct have been discussed in some detail elsewhere (Suarez, 1987; Smith and Yanagimachi, 1990; Hunter, 1990b)and will not be repeated in this brief review. By contrast, the observations by Nagai and Moor (1990) on pig eggs fertilised in vitro raise substantial questions that must bear on the preliminaries to fertilisation in vivo.
A final logical experiment was to note the influence of introducing suspensions of spermatozoa directly into the lumen of the oviducts at different times before ovulation. This had been performed by Polge et al. (1970)as a means of avoiding a deleterious influence of the uterus on frozen-thawed boar spermatozoa. Although polyspermic penetration was observed in their study, the experiment had not been specifically designed to examine the influence of sperm numbers in the oviducts on the incidence of polyspermy. However, this was done in a subsequent study in which surgical introduction of a 0.05-ml suspension of freshly ejaculated spermatozoa (1.7-2.4 X lo8 cells/ml) through the utero-tuba1 junction into the distal isthmus 12 hr before ovulation resulted in 33.8% polyspermy in 88 eggs compared with 5.2% polyspermy in 77 eggs recovered from the contralateral side after insemination into OVIDUCT GLYCOPROTEINS VIS A VIS the tip of the uterine horn (Hunter, 1973). Polyspermy POLYSPERMY was represented mainly by dispermy and trispermy, As mentioned at the outset, Nagai and Moor (1990) and there was no suggestion of a continued penetrability of the zona pellucida during a period of hours, as suggested that oviduct secretory products (macromole-
390
R.H.F. HUNTER TABLE 2. Incidence of Polyspermic Fertilisation Exhibited in Mature Pigs in Various Experimental Situations After Mating or Insemination at Estrus*
Treatment Delayed mating Delayed mating Delayed insemination Tubal surgery Progesterone microinjections Tubal insemination
examined Eggs N 53 41 149
Eggs polyspermic N %
Reference
34
6 12 23 11
11.0a 29.2 15.4 32.4
Thibault (1959) Hancock (1959) Hunter (1967a) Hunter and Lkglise (1971)
198 77
64 26
32.3 33.8
Hunter (1972) Hunter (1973)
*Adapted from Hunter (1979). aA further 21% of eggs were considered digynic, giving a total of 32%.
cules) may act to reduce the incidence of polyspermy, as judged by their experiments on pig eggs fertilised in vitro in the presence of oviduct cells. If this beneficial influence of oviduct epithelial cells can be demonstrated to be the case in a consistent and predictable manner, (1)the mechanism of action of the secretory products, and (2) the identification of the molecules specifically involved are worth serious pursuit. It is by no means certain that the beneficial effect is biochemical as distinct from largely a physical one but, assuming that it is, how might the macromolecules exert such an effect? Based on the finding of Brown and Cheng (1986) that glycoproteins elaborated by the porcine oviduct bind t o the zona pellucida of newly ovulated pig eggs, then such glycoproteins could conceivably act to reduce the rate of simultaneous nenetration of the zona pellucida or indeed of the envefoping cumulus matrix by two Or more spermatozoa, thereby reducing the chances of polyspermic fertilisation. An alternative is that a glYcoProtein On the zona pellucida, followed by its entry into the perivitelline space (Verhage and Fazleabas, 19901,facilitates either a more svnchronous exocvtosis of the cortical granule contents or expedites andaugments physiological responses of the zona substance to cortical granule material (Barros and Yanagimachi, 1971; Gwatkin et al., 1973), leading more efficiently to establishment of a functional block to polyspermy. Irrespective of which of these or indeed other possibilities, such as an influence on the sperm plasmalemma to stabilise and reduce the numbers becoming capacitated simultaneously, ultimately turns out to be correct, interpretation of the egg transplantation studies described earlier in this review may also need to take into account these putative influences of oviduct fluid glycoproteins rather than focussing simply on the number of competent sperm cells available at the site of fertilisation. Perhaps the most challenging problem in the context of the above review is to elaborate the precise molecular mechanisms in the physiological situation that enable the heads of boar spermatozoa to enter the zona pellucida of activated eggs, but then t o become unable to pass through the innermost portion of the zona to
compromise the genetic composition of the zygote. A secondary requirement would be to clarify temporal aspects of establishment of the block to polyspermy and its effectiveness under different glycoprotein treatment regimens, a study that would clearly be best achieved in vitro using the work of Nagai and Moor (1990) as a starting point. This final remark is not intended to infer that the time course of blocking mechanisms in vitro would necessarily correspond precisely to those functioning in vivo for, in the latter situation, eggs would be rotated by cilia in the presence of a dynamic secretory epithelium under the influence of gonadal endocrine activity. The response to components of oviduct luminal fluid might therefore be anticipated as somewhat different and possibly more effective.
ACKNOWLEDGMENTS Most of the studies by the present author referred to in the above review were supported by grants from the Agricultural Research Council (UK), for which grateful acknowledgment is made. I also wish to thank Mrs. Frances Anderson for kindly typing the manuscript. REFERENCES Assheton R (1898): The development of the pig during the first ten days. QJ Microsc Sci 41:329-359. Austin CR, Smiles J (1948):Phase-contrast microscopy in the study of fertilisation and early development of the rat egg. J R Microsc SOC 68:13-19. Barros C, Vliegenthart AM, Franklin LE (1972): Polyspermic fertilisation of hamster eggs in vitro. J Reprod Fertil 28:117-120. Barros C, Yanagimachi R (1971): Induction of the zona reaction in golden hamster eggs by cortical granule material. Nature 233:268269. Beatty RA (1957): “Parthenogenesis and Polyploidy in Mammalian Development.” Cambridge: Cambridge University Press. Bedford JM (1982): Fertilisation. In CR Austin, RV Short (eds): “Reproduction in Mammals.” Cambridge: Cambridge University Press, Vol 1,pp 128-163. Bomsel-Helmreich 0 (1965): Heteroploidy and embryonic death. In G.E.W. Wolstenholme, M O’Connor (eds): “Preimplantation Stages of Pregnancy.” Ciba Foundation Symposium. London: Churchill, pp 246-267. Boyarsky LH, Baylies H, Casida LE, Meyer RK (1947): Influence df progesterone upon the fertility of gonadotrophin treated female rabbits. Endocrinology 41:312-321.
OVIDUCT FUNCTION IN PIGS Braden AWH, Austin CR, David HA (1954): The reaction of the zona pellucida to sperm penetration. Aust J Biol Sci 7:391-409. Brown CR, Cheng WTK (1986):Changes in composition of the porcine zona pellucida during development of the oocyte to the 2- to 4-cell embryo. J Embryol Exp Morphol 92:183-191. Casida LE, Nalbandov A, McShan WH, Meyer RK, Wisnicky W (1940):Potential fertility of artificially matured and ovulated ova in cattle. Proc Am SOCAnim Prod 33:302-304. ‘Casida LE, Meyer RK, McShan WH, Wisnicky W (1943): Effects of pituitary gonadotrophins on the ovaries and the induction of superfecundity in cattle. Am J Vet Res 4:76-94. Chang MC (1952): Fertilizability of rabbit ova and the effects of temperature in vitro on their subsequent fertilisation and activation in vivo. J Exp Zool 121:351-381. Day BN, Polge C (1968): Effects of progesterone on fertilisation and egg transport in the pig. J Reprod Fertil 17:227-230. Dziuk PJ (1965): Response of sheep and swine to treatments for the control of ovulation. In “Proceedings of Conference on Oestrous Cycle Control in Domestic Animals.” University of Nebraska, USDA Miscellaneous Publ. No. 1005, pp 50-57. Dziuk PJ, Polge C (1962): Fertility in swine after induced ovulation. J Reprod Fertil 4:207-208. Fraser LR (1984): Mechanisms controlling mammalian fertilisation. Oxford Rev Reprod Biol6:174-225. Fraser LR, Ahuja KK (1988):Metabolic and surface events in fertilisation. Gamete Res 20:491-519. Gwatkin RBL (1977): “Fertilisation Mechanisms in Man and Mammals.’’ New York: Plenum Press. Gwatkin RBL, Williams DT, Hartmann J F , Kniazuk M (1973): The zona reaction of hamster and mouse eggs: Production in vitro by a trypsin-like protease from cortical granules. J Reprod Fertil32:259265. Rancock JL (1959): Polyspermy of pig ova. Anim Prod 1:103-106. Hanly S (1961): Prenatal mortality in farm animals. J Reprod Fertil 2:182-194. Hunter RHF (1965): Studies on ovulation and fertility in the pig. PhD dissertation and abstract of dissertation, University of Cambridge. Hunter RHF (1967a): The effects of delayed insemination on fertilisation and early cleavage in the pig. J Reprod Fertil 13:133-147. Hunter RHF (1967133: Polyspermic fertilisation in pigs during the luteal phase of the estrous cycle. J Exp Zool 165:451-460. Hunter RHF (1972): Local action of progesterone leading to polyspermic fertilisation in pigs. J Reprod Fertil 31:433444. Hunter RHF (1973): Polyspermic fertilisation in pigs after tubal deposition of excessive numbers of spermatozoa. J Exp Zool 183:5764. Hunter RHF (1976): Sperm-egg interactions in the pig: Monospermy, extensive polyspermy, and the formation of chromatin aggregates. J Anat 122:43-59. Hunter RHF (1979): Ovarian follicular responsiveness and oocyte quality after gonadotrophic stimulation of mature pigs. Ann Biol Anim Biochim Biophys 19:1511-1520. Hunter RHF (1988):“The Fallopian Tubes: Their Role in Fertility and Infertility.” Berlin: Springer-Verlag. Hunter RHF (1990a): Fertilisation of pig eggs in vivo and in vitro. J Reprod Fertil 4O(suppl):211-226. Hunter RHF (1990b): Physiology of the Fallopian tubes, with special reference to gametes, embryos and microenvironments. In JLH Evers, M J Heineman (eds): “From Ovulation to Implantation.” Amsterdam: Excerpta Medica, International Congress Series No. 917, pp 101-119.
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Hunter RHF, Hall J P (1974a): Capacitation of boar spermatozoa: Synergism between uterine and tubal environments. J Exp Zool 1881203-214. Hunter RHF, Hall J P (197413): Capacitation of boar spermatozoa: The influence of post-coital separation of the uterus and Fallopian tubes. Anat Rec 180:597-604. Hunter RHF, Leglise PC (1971): Polyspermic fertilisation following tubal surgery in pigs, with particular reference to the role of the isthmus. J Reprod Fertil 24:233-246. Hunter RHF, Nichol R (1988): Capacitation potential of the Fallopian tube: A study involving surgical insemination and the subsequent incidence of polyspermy. Gamete Res 21:255-266. Murphree RL, Warwick EJ, Casida LE, McShan WH (1944):Potential fertility of ova from ewes treated with gonadotrophins. J Anim Sci 3:12-2 1. Murphree RL, Warwick EJ, Casida LE, McShan WH (1947):Influence of reproductive stage upon the fertility of gonadotrophin-treated female rabbits. Endocrinology 41:308-311. Nagai T, Moor RM (1990): Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilised in vitro. Mol Reprod Dev 26:377382. Perry JS (1954): Fecundity and embryonic mortality in pigs. J Embryol Exp Morphol 2:30%322. Pitkjanen IG (1955): Ovulation, fertilisation, and early embryonic development in the pig. Izv Akad Nauk SSR Ser Biol 3:120-131. Polge C (1978): Fertilisation in the pig and the horse. J Reprod Fertil 54:461470. Polge C, Dziuk P (1965): Recovery of immature eggs penetrated by spermatozoa following induced ovulation in the pig. J Reprod Fertil 9:357-358. Polge C, Salamon S, Wilmut I (1970): Fertilising capacity of frozen boar semen following surgical insemination. Vet Rec 87:424428. Rowson, LE (1951): Methods of inducing multiple ovulation in cattle. J Endocrinol 7:260-270. Smith TT, Yanagimachi R (1990): The viability of hamster spermatozoa stored in the isthmus of the oviduct: The importance of sperm-epithelium contact for sperm survival. Biol Reprod 42:450457. Spalding JF, Berry RO, Moffit JG (1955): The maturation process of the ovum of swine during normal and induced ovulations. J Anim Sci 14:609-620. Suarez SS (1987):Sperm transport and motility in the mouse oviduct: Observations in situ. Biol Reprod 36:203-210. Szollosi D (1975): Mammalian eggs aging in the Fallopian tubes. In R J Blandau (ed): “Aging Gametes.” Basel: S Karger, pp 9 g 1 2 1 . Tanabe TY, Warnick AC, Casida LE, Grummer RH (1949):The effects of gonadotrophins administered to sows and gilts during different stages of the estrual cycle. J Anim Sci 8:550-557. Thibault C (1959):Analyse de la fecondation de I’oeuf de la truie apres accouDlement ou insemination artificielle. Ann Zootechnol Ser D (suppi):165-177. Verhage HG, Fazleabas AT (1990): Steroid-dependent oviduct secretions in the primate. In H Croxatto (ed):“Biology of the Mammalian Oviduct.” Proceedings of the International Symposium, Santiago (abst 9). Yanagimachi R (1988): Mammalian fertilisation. In E Knobil, J Neil1 (eds):“The Physiology of Reproduction.” New York: Raven Press, pp 135-185.
Review Article Oviduct Function in Pigs, With Particular Reference to the Pathological Condition of Polyspermy R.H.F. HUNTER University of Edinburgh, Edinburgh, Scotland
ABSTRACT Because the exceptionally high incidence of polyspermic fertilisation has been emphasised as a major defect in systems of in vitro fertilisation in pigs, the aetiology of the condition has been analysed in a series of experiments in vivo in the search for a common underlying cause and possible means of mitigation. Whereas the defense mechanism against polyspermy in pig oocytes is classically viewed as a zona reaction, more recent evidence suggests a secondary block at the vitelline surface. Both blocks may be compromised in situations leading to polyspermy, although deleterious influences seem to be expressed principally in an inadequate zona block, as judged by the presence of perivitelline sperma-
tozoa. Postovulatory aging of mammalian oocytes prior to sperm penetration leads to polyspermy, as can be demonstrated in pig eggs. The primary lesion may concern the cortical reaction, owing to a delayed and incomplete exocytosis of the vesicular contents. Eggs ovulated after gonadotrophin treatment during the luteal phase of the cycle show a high incidence of polyspermic penetration (60.6%),as do those shed at estrus in animals treated with progesterone systemically (40%)or by local microinjections in the oviduct wall (32.3%).Whereas progesterone may be modifying interactions of the gametes and responses of the egg organelles in all four above experimental situations, enhanced numbers of spermatozoa ascending a more patent isthmus appear to be the principal cause of polyspermy. Support for this hypothesis comes from isthmus resection with re-anastomosis of remaining portions of the oviduct (32.4% polyspermy), and surgical insemination directly into the oviducts (33.8%polyspermy). Recent experiments draw attention to a role for oviduct epithelial glycoproteins in modulating the extent of polyspermy in vitro. The role of such oviduct macromolecules in gamete interactions in vivo is considered.
Key Words: Fertilisation, Spermatozoa, Insemination, Progesterone, Glycoprotein
INTRODUCTION The stimulus for preparing this previously unpublished lecture as a manuscript has been the recent paper by Nagai and Moor (1990), with the exciting suggestion that oviduct secretory products (macromolecules) derived from co-culture of epithelial cells with gametes may act to influence the incidence of 0 1991 WILEY-LISS, INC.
polyspermy in pig eggs fertilised in vitro. This proposition and the underlying concern with polyspermy in pig eggs seem worth setting in an historical perspective, albeit a somewhat personal one. Physiological processes in the oviduct that influence mammalian fertilisation have been reviewed in recent years in a number of key publications (Gwatkin, 1977; Bedford, 1982; Fraser, 1984; Fraser and Ahuja, 1988; Hunter, 1988; Yanagimachi, 1988). By contrast, the etiology of polyspermic fertilisation has received very little attention, even though polyspermy may arise in vivo under diverse experimental conditions. This latter statement will be illustrated in the following review by specific reference to observations in the domestic pig. This is an invaluable animal in studies of fertilisation, since (1)relatively large numbers of eggs are ovulated from the two ovaries, and (2) the time of ovulation can be regulated with precision. Polyspermy refers to penetration of the vitellus-the cytoplasm of the egg-by more than one spermatozoon. Unlike the situation in fish and birds, polyspermy is known to be a pathological condition in placental mammals, usually causing very early death of the zygote (Beatty, 1957; Bomsel-Helmreich, 1965). It has been recognised, since at least 1954, that the block to polyspermy-the egg’s defense mechanism-is located in the zona pellucida of most mammals so far studied, and not primarily at the surface of the vitelline membrane as occurs in the rabbit egg (Braden et al., 1954). In fact, a block at the level of the zona pellucida in pig eggs could have been judged from photographs of histologically sectioned eggs published at the turn of the century (Assheton, 1898); these show very many sperm heads embedded in the substance of the zona pellucida. More recently, it has been appreciated that there may also be a secondary block to polyspermy functioning at the surface of the vitelline membrane in pig eggs (Hunter and Nichol, 1988; Hunter, 1990a). Two further sentences ofjustification for focussing on polyspermic fertilisation may be appropriate at this stage. First, polyspermy remains a relatively frequent Received March 4, 1991; accepted March 13, 1991. Address reprint requests to Dr. R.H.F. Hunter, 32 Gilmour Road, Edinburgh EH16 5NT, Scotland. This paper was an invited lecture presented a t McGill University, Montreal, March 1990.
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occurrence in pig eggs, and may be a significant cause of embryonic loss which commonly attains an incidence of 3040% in this species (Perry, 1954; Hanly, 1961; Polge, 1978). Second, in all the recent studies of in vitro fertilisation, polyspermic penetration is by far and away the largest abnormality if fertilisation occurs at all (reviewed by Hunter, 1990a).
HISTORICAL PERSPECTIVE The first publications specifically concerned with polyspermic fertilisation of pig eggs appeared during the 1950s, and all three reports associated the polyspermic condition with delayed (i.e., late) matings and postovulatory aging of the egg before sperm penetration. The incidence of polyspermy in these reports extended from 10 to 29% (Pitkjanen, 1955; Hancock, 1959; Thibault, 1959). However, none of these investigators knew when ovulation had occurred, so the incidence of polyspermy could not be related to the age of egg at initial penetration. But when the author commenced his own research studies in 1962, two techniques were available that offered much greater precision in studies of mammalian fertilisation, and could certainly be applied to the examination of vitelline events in the pig. These were (1)the technique of phase-contrast microscopy of whole-mount preparations (Austin and Smiles, 1948; Chang, 1952), little exploited on the eggs of farm animals, enabling nuclear structures to be visualized in fixed and stained preparations without resorting to the relatively laborious procedures of serial histology; and (2) the ability to control precisely the time of ovulation with an injection of a luteinizing hormone (LH) or human chorionic gonadotropin (hCG) preparation given to animals in late proestrus when a responsive population of mature Graafian follicles would be available. Such an injection anticipates and mimicks the endogenous surge of gonadotropic hormones to programme resumption of meiosis and collapse of the follicles.
POSTOVULATORY AGING OF EGGS By combining the two techniques mentioned above, while working in the laboratory of Professor E.J.C. Polge at the Animal Research Station in Cambridge, the postovulatory age of pig eggs at the time of initial sperm penetration was regulated. An increasing incidence of polyspermic fertilisation occurred with increasing age of the eggs, reaching a highest mean figure of 15.4% (Table 1). However, the animals had been artificially inseminated with relatively low numbers of spermatozoa (1-2 x lo8 celldm1 in approximately 100 ml of fresh semen), and the polyspermic condition observed was principally one of dispermy or trispermy (Hunter, 1967a). The nature of the postovulatory degeneration that enhanced an egg’s susceptibility to polyspermic penetration was not clarified, but was nonetheless suspected to involve vitelline organelles and the plasmalemma rather than primarily the zona pellucida, although a diminishing effective-
ness of the cortical response would be expressed in the latter (Szollosi, 1975).
LUTEAL-PHASE OVULATIONS A second experiment concerned eggs resulting from ovulations induced during the luteal phase of the estrous cycle with precisely imposed gonadotrophin treatments. Many reports had appeared during the 1940s and 1950s, especially from the Wisconsin laboratory of L.E. Casida, documenting the infertility associated with luteal phase ovulations in the major farm species of cow, sheep, and pig and during pseudopregnancy in rabbits (Casida et al., 1940,1943;Murphree et al., 1944, 1947; Boyarsky et al., 1947; Tanabe et al., 1949;Rowson, 1951;Spalding et al., 1955).One possible interpretation of such infertility was as an extreme form of delayed insemination-the postovulatory aging egg syndrome-which would effectively represent the situation in the early luteal phase. The regimen used for predictably inducing ovulation during the luteal phase of the porcine estrous cycle was a subcutaneous injection of 1,500 IU PMSG on day 5 of the cycle, followed by an intramuscular injection of 500 IU hCG on day 9; ovulation occurred on day 11. Such animals did not exhibit behavioural estrus at the time of induced ovulation, so mating was not possible. Instead, they were inseminated artificially 4-18 hr before ovulation with 120 ml of fresh undiluted semen. In the 160 penetrated eggs recovered from 14 animals, the incidence of polyspermic penetration was 60.6%, and the degree of the polyspermic condition ranged from 2 to 7 accessory male pronuclei as well as from 2 to >50 swollen and unswollen sperm heads (Hunter, 1967b). Such extensive polyspermy appeared to offer a plausible explanation for the widely reported infertility during the luteal phase of the estrous cycle.
EGG TRANSPLANTATION STUDIES Arising from the previous study came a proposition that bears on a putative action of oviduct luminal fluids, that is of secretions from oviduct epithelial cells (Nagai and Moor, 1990). In fact, it represented the author’s own initial interest in a physiological role for such fluids. As a result of many discussions, the notion arose that the exceptionally high incidence of polyspermic fertilisation may have been due primarily to an influence of luteal-phase oviduct fluids on the mechanism of the block to polyspermy, the composition of oviduct luminal fluid being dominated by the high titres of circulating progesterone. In brief, the hypothesis was that luteal-phase oviduct fluid had rendered the zona reaction less effective. Support for this hypothesis was obtained from two sets of egg transplantation experiments, each involving 12 animals (R.H.F. Hunter, C. Polge, and L.E. Rowson, unpublished observations). Eggs released from mature follicles during estrus were transplanted into the oviducts of animals artificially inseminated on day 11 of the luteal phase. This manipulation induced extensive polyspermy, with
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387
TABLE 1. Demonstration of the Influence of Postovulatory Aging of Eggs Before Sperm Penetration on the Incidence of Normal Fertilisation and That of Polyspermic Fertilisation* Estimated age of egg at sperm penetration (hr)
Eggs examined and stained
0 4 8 12 16 20
Eggs fertilised normally
(N)
(W
Eggs polyspermic
141 152 166 155 149 155
90.8 92.1 94.6 70.3 48.3 50.9
0.0 1.3 1.2 6.8 15.4 11.1
(%I
Adapted from Hunter (1967a).
transplanted eggs exhibiting 3 or 4 large male pronuclei, together with a variable number of sperm heads in the vitellus and spermatozoa also frequently being observed in the perivitelline space (Fig. la,b). The reciprocal experiment of transplanting eggs derived from ovulations during the luteal phase of the oestrous cycle into the oviducts of mated oestrous animals resulted in morphologically normal fertilisation (Hunter, 1965). These observations led to the conclusion that, in pigs at least, eggs shed during the luteal phase were not intrinsically defective and that the oviduct luminal environment under the influence of high progesterone titres was, by some unspecified action, downgrading the effectiveness of the block to polyspermy.
SYSTEMIC ADMINISTRATION OF PROGESTERONE
PROGESTERONE TITRES AND SPERM NUMBERS During 1967 and 1968, the author was restricted to working on small laboratory rodents in Massachusetts but he had nonetheless devoted some thought as to the means whereby circulating progesterone might be influencing the physiology of pig oviducts. At least as reasonable an explanation for the high incidence of polyspermy as an effect mediated by oviduct fluid on the zona pellucida would have been that of a direct influence of progesterone on the myosalpinx and on the mucosa of the oviduct wall. Whether of endogenous or exogenous origin, high titres of progesterone could have been acting to diminish the extent of oedema in the mucosa and also to relax the smooth muscle coats of the duct so that the lumen would be much increased in patency. This, in turn, could permit large numbers of spermatozoa to pass to the site of fertilisation at the ampullary-isthmic junction, with a concomitant increase in the risk of polyspermic fertilisation due to closely synchronous penetration of the zona pellucida by two or more spermatozoa. Critical factors influencing the incidence of polyspermy would include the number of competent spermatozoa achieving the surface of the zona pellucida and the actual rate of establishment of the block to polyspermy after activation of the cortical response. This possibility of an excess of competent spermatozoa at the site of fertilisation acting to promote pathological fertilisation was tested in a number of ways.
At this stage of the work (1965), the author became a postdoctoral student in North America, whereas Professor B.N. Day moved from Columbia, Missouri, to Cambridge, England, specifically to learn the procedures of phase-contrast microscopy and egg transplantation for studies in farm animals. The notion that progesterone in some manner acted to influence the normality of fertilisation had remained an important theme in Cambridge, not least since orally active progestagens were being used to synchronise estrus in farm animals, frequently with poor or very poor resultant fertility (Dziuk and Polge, 1962; Polge and Dziuk, ISTHMUS RESECTION AND POLYSPERMY 1965; Dziuk, 1965). The next logical experiment was A surgical approach was to remove most of the therefore performed by Day and Polge (1968), that of injecting inseminated animals systemically with a isthmus by resection and then anastomose the remainsolution of progesterone in oil (100 mg progesterone) ing portions of the oviduct so that the regulatory role of 24-36 hr before ovulation and then examining eggs the isthmic portion of the duct on sperm transport to recovered from the oviducts and/or uterus by phase- the site of fertilisation was largely overcome. This contrast microscopy. Two significant findings were experiment was performed during 1969 with a skilled reported: a 3640% incidence of polyspermy and an French surgeon at the Station de Physiologie Animale, accelerated passage of eggs through the oviducts into Jouy-en-Josas, and the incidence of polyspermy in eggs shed into the surgically shortened oviduct of 18 pigs the uterus.
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Fig. 1. a,b. Whole-mount preparations of pig eggs seen under phase-contrast with many spermatozoa visible in both the perivitelline space and zona pellucida. After fixing and staining, these eggs were found also to exhibit polyspermic penetration of the vitellus, with unswollen sperm heads and pronuclei in the same preparation.
c,d. Multiple sperm penetration of the vitellus demonstrable in spontaneously ovulated pig eggs viewed as stained preparations under the phase-contrast microscope. Coalescence of nuclear chromatin has led to the formation of aggregates between adjoining sperm head material.
OVIDUCT FUNCTION IN PIGS was 32.4%compared with 0.9% in 114 control eggs from the contralateral oviduct (Hunter and Lkglise, 1971). These findings therefore strengthened the argument that passage of elevated numbers of competent spermatozoa to the site of fertilisation increases the risk of polyspermic fertilisation.
MICROINJECTIONS OF PROGESTERONE SOLUTION A further experiment involving a progesterone treatment was to inject locally microdroplets of a solution of the steroid in oil (total of 1 mg progesterone in 0.1 ml oil) under the serosal layer of the oviduct wall in the vicinity of the utero-tuba1junction and distal portion of the isthmus some 8-12 hr before ovulation. The contralateral oviduct acted as a control, receiving a similar distribution of microinjections but only of the oil vehicle. The results based on 344 eggs from 30 animals revealed an incidence of polyspermy of 32.3% in the progesterone-treated oviduct compared with 9.7% in the control oviduct (Hunter, 1972). Local microinjections of the progesterone solution at a greater interval before ovulation might perhaps have increased the incidence of polyspermic fertilisation, since this would have permitted a longer interval in which to achieve relaxation of the myosalpinx and reduction of oedema in the mucosa, thereby increasing the patency of the duct. Whilst polyspermy was considered to have originated in this experiment principally from increased numbers of spermatozoa reaching the site of fertilisation, a direct effect of progesterone on the membranes of the gametes was not excluded. In any event, synchrony of development among the two or three male pronuclei suggested simultaneous or almost simultaneous penetration of the vitellus by several spermatozoa.
INSEMINATION INTO OVIDUCTS
389
noted for hamster eggs in vitro (Barros et al., 1972). On the basis of further analysis of the process of capacitation of boar spermatozoa in vivo highlighting the synergism between uterus and oviducts and the time relationships for sperm surface modifications in each of these compartments (Hunter and Hall, 1974a,b), suitably timed surgical insemination of sperm suspensions directly into the isthmus enabled a condition of massive polyspermy to be demonstrated (Hunter, 1976). Indeed, the extent of sperm penetration into the vitellus was frequently so overwhelming (>80 spermatozoa) that aggregates of sperm head chromatin were usually conspicuous (Fig. lC,D).
SUSCEPTIBILITY OF OOCYTES TO POLYSPERMY A conclusion that can be drawn from all these in vivo experiments, and that still appears tenable, is that the number of capacitated spermatozoa available to confront the newly ovulated eggs is the principal factor regulating the incidence and extent of polyspermic fertilisation. Under conditions of enhanced but no2 overwhelming numbers of competent spermatozoa in the oviducts, it would appear that approximately 30% of ovulated pig eggs are especially susceptible to polyspermy (Table 21, in part due to a slow instigation of a full zona reaction or to some related abnormality focussing on the properties of the zona pellucida or vitelline membrane. Indeed, as a working hypothesis, the suggestion has been offered that these 30-35% of spontaneously ovulated pig eggs represent those that may succumb to early embryonic death. Susceptibility to polyspermic penetration could thus be a means of revealing or identifying, albeit retrospectively, this particular population of eggs (Hunter, 1979, 1990a). Finally, with reference to the above studies on abnormal fertilisation in porcine oocytes, the wheel has now turned almost full circle in at least one sense, for the incidence and degree of polyspermy can be used to monitor the extent of the population of capacitated boar spermatozoa in the immediate vicinity of recently ovulated eggs (Hunter and Nichol, 1988). The experiments also raise the question of precisely how the oviducts normally regulate with such precision the number of competent spermatozoa reaching the surface of the eggs after a spontaneous mating so that the polyspermic condition is avoided. Such systems of regulation in the isthmic portion of the duct have been discussed in some detail elsewhere (Suarez, 1987; Smith and Yanagimachi, 1990; Hunter, 1990b)and will not be repeated in this brief review. By contrast, the observations by Nagai and Moor (1990) on pig eggs fertilised in vitro raise substantial questions that must bear on the preliminaries to fertilisation in vivo.
A final logical experiment was to note the influence of introducing suspensions of spermatozoa directly into the lumen of the oviducts at different times before ovulation. This had been performed by Polge et al. (1970)as a means of avoiding a deleterious influence of the uterus on frozen-thawed boar spermatozoa. Although polyspermic penetration was observed in their study, the experiment had not been specifically designed to examine the influence of sperm numbers in the oviducts on the incidence of polyspermy. However, this was done in a subsequent study in which surgical introduction of a 0.05-ml suspension of freshly ejaculated spermatozoa (1.7-2.4 X lo8 cells/ml) through the utero-tuba1 junction into the distal isthmus 12 hr before ovulation resulted in 33.8% polyspermy in 88 eggs compared with 5.2% polyspermy in 77 eggs recovered from the contralateral side after insemination into OVIDUCT GLYCOPROTEINS VIS A VIS the tip of the uterine horn (Hunter, 1973). Polyspermy POLYSPERMY was represented mainly by dispermy and trispermy, As mentioned at the outset, Nagai and Moor (1990) and there was no suggestion of a continued penetrability of the zona pellucida during a period of hours, as suggested that oviduct secretory products (macromole-
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R.H.F. HUNTER TABLE 2. Incidence of Polyspermic Fertilisation Exhibited in Mature Pigs in Various Experimental Situations After Mating or Insemination at Estrus*
Treatment Delayed mating Delayed mating Delayed insemination Tubal surgery Progesterone microinjections Tubal insemination
examined Eggs N 53 41 149
Eggs polyspermic N %
Reference
34
6 12 23 11
11.0a 29.2 15.4 32.4
Thibault (1959) Hancock (1959) Hunter (1967a) Hunter and Lkglise (1971)
198 77
64 26
32.3 33.8
Hunter (1972) Hunter (1973)
*Adapted from Hunter (1979). aA further 21% of eggs were considered digynic, giving a total of 32%.
cules) may act to reduce the incidence of polyspermy, as judged by their experiments on pig eggs fertilised in vitro in the presence of oviduct cells. If this beneficial influence of oviduct epithelial cells can be demonstrated to be the case in a consistent and predictable manner, (1)the mechanism of action of the secretory products, and (2) the identification of the molecules specifically involved are worth serious pursuit. It is by no means certain that the beneficial effect is biochemical as distinct from largely a physical one but, assuming that it is, how might the macromolecules exert such an effect? Based on the finding of Brown and Cheng (1986) that glycoproteins elaborated by the porcine oviduct bind t o the zona pellucida of newly ovulated pig eggs, then such glycoproteins could conceivably act to reduce the rate of simultaneous nenetration of the zona pellucida or indeed of the envefoping cumulus matrix by two Or more spermatozoa, thereby reducing the chances of polyspermic fertilisation. An alternative is that a glYcoProtein On the zona pellucida, followed by its entry into the perivitelline space (Verhage and Fazleabas, 19901,facilitates either a more svnchronous exocvtosis of the cortical granule contents or expedites andaugments physiological responses of the zona substance to cortical granule material (Barros and Yanagimachi, 1971; Gwatkin et al., 1973), leading more efficiently to establishment of a functional block to polyspermy. Irrespective of which of these or indeed other possibilities, such as an influence on the sperm plasmalemma to stabilise and reduce the numbers becoming capacitated simultaneously, ultimately turns out to be correct, interpretation of the egg transplantation studies described earlier in this review may also need to take into account these putative influences of oviduct fluid glycoproteins rather than focussing simply on the number of competent sperm cells available at the site of fertilisation. Perhaps the most challenging problem in the context of the above review is to elaborate the precise molecular mechanisms in the physiological situation that enable the heads of boar spermatozoa to enter the zona pellucida of activated eggs, but then t o become unable to pass through the innermost portion of the zona to
compromise the genetic composition of the zygote. A secondary requirement would be to clarify temporal aspects of establishment of the block to polyspermy and its effectiveness under different glycoprotein treatment regimens, a study that would clearly be best achieved in vitro using the work of Nagai and Moor (1990) as a starting point. This final remark is not intended to infer that the time course of blocking mechanisms in vitro would necessarily correspond precisely to those functioning in vivo for, in the latter situation, eggs would be rotated by cilia in the presence of a dynamic secretory epithelium under the influence of gonadal endocrine activity. The response to components of oviduct luminal fluid might therefore be anticipated as somewhat different and possibly more effective.
ACKNOWLEDGMENTS Most of the studies by the present author referred to in the above review were supported by grants from the Agricultural Research Council (UK), for which grateful acknowledgment is made. I also wish to thank Mrs. Frances Anderson for kindly typing the manuscript. REFERENCES Assheton R (1898): The development of the pig during the first ten days. QJ Microsc Sci 41:329-359. Austin CR, Smiles J (1948):Phase-contrast microscopy in the study of fertilisation and early development of the rat egg. J R Microsc SOC 68:13-19. Barros C, Vliegenthart AM, Franklin LE (1972): Polyspermic fertilisation of hamster eggs in vitro. J Reprod Fertil 28:117-120. Barros C, Yanagimachi R (1971): Induction of the zona reaction in golden hamster eggs by cortical granule material. Nature 233:268269. Beatty RA (1957): “Parthenogenesis and Polyploidy in Mammalian Development.” Cambridge: Cambridge University Press. Bedford JM (1982): Fertilisation. In CR Austin, RV Short (eds): “Reproduction in Mammals.” Cambridge: Cambridge University Press, Vol 1,pp 128-163. Bomsel-Helmreich 0 (1965): Heteroploidy and embryonic death. In G.E.W. Wolstenholme, M O’Connor (eds): “Preimplantation Stages of Pregnancy.” Ciba Foundation Symposium. London: Churchill, pp 246-267. Boyarsky LH, Baylies H, Casida LE, Meyer RK (1947): Influence df progesterone upon the fertility of gonadotrophin treated female rabbits. Endocrinology 41:312-321.
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Hunter RHF, Hall J P (1974a): Capacitation of boar spermatozoa: Synergism between uterine and tubal environments. J Exp Zool 1881203-214. Hunter RHF, Hall J P (197413): Capacitation of boar spermatozoa: The influence of post-coital separation of the uterus and Fallopian tubes. Anat Rec 180:597-604. Hunter RHF, Leglise PC (1971): Polyspermic fertilisation following tubal surgery in pigs, with particular reference to the role of the isthmus. J Reprod Fertil 24:233-246. Hunter RHF, Nichol R (1988): Capacitation potential of the Fallopian tube: A study involving surgical insemination and the subsequent incidence of polyspermy. Gamete Res 21:255-266. Murphree RL, Warwick EJ, Casida LE, McShan WH (1944):Potential fertility of ova from ewes treated with gonadotrophins. J Anim Sci 3:12-2 1. Murphree RL, Warwick EJ, Casida LE, McShan WH (1947):Influence of reproductive stage upon the fertility of gonadotrophin-treated female rabbits. Endocrinology 41:308-311. Nagai T, Moor RM (1990): Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilised in vitro. Mol Reprod Dev 26:377382. Perry JS (1954): Fecundity and embryonic mortality in pigs. J Embryol Exp Morphol 2:30%322. Pitkjanen IG (1955): Ovulation, fertilisation, and early embryonic development in the pig. Izv Akad Nauk SSR Ser Biol 3:120-131. Polge C (1978): Fertilisation in the pig and the horse. J Reprod Fertil 54:461470. Polge C, Dziuk P (1965): Recovery of immature eggs penetrated by spermatozoa following induced ovulation in the pig. J Reprod Fertil 9:357-358. Polge C, Salamon S, Wilmut I (1970): Fertilising capacity of frozen boar semen following surgical insemination. Vet Rec 87:424428. Rowson, LE (1951): Methods of inducing multiple ovulation in cattle. J Endocrinol 7:260-270. Smith TT, Yanagimachi R (1990): The viability of hamster spermatozoa stored in the isthmus of the oviduct: The importance of sperm-epithelium contact for sperm survival. Biol Reprod 42:450457. Spalding JF, Berry RO, Moffit JG (1955): The maturation process of the ovum of swine during normal and induced ovulations. J Anim Sci 14:609-620. Suarez SS (1987):Sperm transport and motility in the mouse oviduct: Observations in situ. Biol Reprod 36:203-210. Szollosi D (1975): Mammalian eggs aging in the Fallopian tubes. In R J Blandau (ed): “Aging Gametes.” Basel: S Karger, pp 9 g 1 2 1 . Tanabe TY, Warnick AC, Casida LE, Grummer RH (1949):The effects of gonadotrophins administered to sows and gilts during different stages of the estrual cycle. J Anim Sci 8:550-557. Thibault C (1959):Analyse de la fecondation de I’oeuf de la truie apres accouDlement ou insemination artificielle. Ann Zootechnol Ser D (suppi):165-177. Verhage HG, Fazleabas AT (1990): Steroid-dependent oviduct secretions in the primate. In H Croxatto (ed):“Biology of the Mammalian Oviduct.” Proceedings of the International Symposium, Santiago (abst 9). Yanagimachi R (1988): Mammalian fertilisation. In E Knobil, J Neil1 (eds):“The Physiology of Reproduction.” New York: Raven Press, pp 135-185.
