FERTILIZATION of SHEEP OVA FOLLOWING MECHANICAL PENETRATION of the ZONA PELLUCIDA* by N

FERTILIZATION OF SHEEP OVA FOLLOWING MECHANICAL PENETRATION OF THE ZONA PELLUCIDA* By N. W. MooREt

Many spermatozoa may become attached to the zona pellucida of the ovine ovum, but rarely does more than one penetrate the zona. Thus, polyspermy is uncommon, apart from when "aged" eggs (i.e. eggs more than 36 hr old after ovula tion) are exposed to sperm (Killeen 1969). In most species polyspermy is fatal (Beatty 1957) and in the ewe polyspermic ova do not appear to develop beyond the formation of pronuclei (I. D. Killeen and N. W. Moore, unpublished data). The block to penetra tion of the zona by more than one sperm-the zona reaction-must occur rapidly, but little is known of the mechanisms involved. In this experiment micromanipula tion techniques were used to study the response of the zona to mechanical penetration and to examine the fertilizing capacity of spermatozoa placed directly within the perivitelline space.

Experimental Recently ovulated ova were collected from the fallopian tubes of donor ewes 24-36 hr after they were first mated to vasectomized rams. The donors were treated with an equine anterior pituitary extract to induce multiple ovulation (Moore and Shelton 1964). The ova were treated as shown in Table 1 and then they were transferred to recipient ewes. Ova were either not treated and then either transferred to recipients inseminated with freshly ejaculated sperm (group A), or the zonae were penetrated with glass needles (see Fig. 1) and then transferred to inseminated recipients (group B) or non·inseminated recipients (group C), or freshly ejaculated sperm (group D) or sperm recovered from the uteri of naturally mated ewes (group E) were injected directly into the perivitelline space and the ova were then transferred to non·inseminated recipients. Freshly ejaculated sperm were collected by artificial vagina and diluted 20-40 times in Dulbecco phosphate buffert enriched with 10% heterologous sheep serum (D+I0%S). "Uterine sperm" were recovered from oestrous ewes 6-8 hr after they had been mated to fertile rams. The body and uterine horns were flushed with 10 ml D + 10 %S and the flushings were then centrifuged for 10 min (300-400 g) and the sedimented sperm suspensions were used for insemination. A micropipette of approximately 5 pm internal diameter was used for the injection of sperm into ova and an attempt was made to place at least one progressively motile sperm in the perivitelline space of each ovum. In a further group of ova (group F) the zonae were penetrated with a micropipette and a similar small volume of D+I0%S, as used for sperm insemination, was injected into the perivitelline space. The ova were then transferred to inseminated recipients. * Manuscript received 25 October 1971. t Department of Animal Husbandry, University of Sydney; present address: University of Sydney Farms, Private Bag, Camden, N.S.W. 2570. t Commonwealth Serum Laboratories, Melbourne; composition (g/litre): N aCI 8·0; KCIO'2; Na2HP04 1·15; KH2P04 0·2; CaCI20·1; MgCI20·1.

Awt. J. bioi. Sci., 1972,25, 433-6 434 SHORT COMMUNICATIONS

Penetration of the zona and insemination of ova were carried out using Leitz micromani pulators and microscope ( X 100-200) with the ova held in cavity slides in D +lO%S. Immediately after treatment, or in the case of untreated ova 1 hr after collection, the ova were transferred to the fallopian tubes of recipients which had been first mated to vasectomized rams within 12 hr of their respective donors. Only those recipients which had ovulated from only one ovary were used and ova were transferred to the contralateral fallopian tube. 5-8 ova were transferred to each tube. In groups A, B, and F 0·01 ml freshly ejaculated semen, diluted 1 : 1 with D+IO%S, was injected into the tip of the lumen of the uterine horn corresponding to the fallopian tube to which ova were transferred. Either at 18-24 or 48 hr after transfer portion of the uterine horn and fallopian tube to which ova had been transferred were flushed with normal saline and ova present in the flushings were examined as fresh specimens and then again after staining with 2% orcein.

Results and Discussion A large proportion of ova transferred to inseminated recipients following no treatment (group A), after penetration of the zona (group B), or after injection of D+lO%S into the perivitelline space (group F) were apparently normally fertilized (Table 1). The majority of ova recovered 48 hr after transfer were of eight cells, those recovered 18-24 hr after transfer were mostly of two cells (Fig. 2).

TABLE 1 NUMBER OF OVA FERTILIZED FOLLOWING TREATMENT AND SUBSEQUENT TRANSFER TO RECIPIENT EWES

No. of ova Treatment Treatment of Percentage Group ,-- ..-A...--___----, of ovum recipient fertilized Treated Recovered Fertilized

A Nil Inseminated 80 47 35 74 B Zona Inseminated 81 59 41 69 penetrated C Zona Not inseminated 42 29 0 0 penetrated D Inseminated Not inseminated 39 26 0 0 with fresh sperm E Inseminated Not inseminated 43 28 0 0 with "uterine sperm" F Inseminated Inseminated 32 21 13 62 with sperm diluent

The majority of fertilized ova which were recovered after penetration of the zona had numerous sperm attached to their zonae, but none showed any evidence of polyspermy. Thus, mechanical penetration of the zona did not evoke tIle zona reaction, nor did penetration interfere with the subsequent initiation of the zona reaction. Therefore, it would seem that the simple mechanical stimulation provided by sperm during penetration of the zona is not the effective stimulus evoking the zona reaction. In sheep there is strong evidence for the need for capacitation of sperm and the process is probably completed in less than It hr (Mattner 1963; Killeen 1969). SHORT COMMUNICATIONS 435

Fig. I.-Recently ovulated ovum held in glass crook and zona pellucida penetrated by glass needle. x180. Fig. 2.-·0va recovered from inseminated recipient ewe 18 hr after penetration of zonae pellucida by glass needles. One ovum cleaved, three with two nuclei but not yet cleaved, one (lower centre) with two pronuclei. Orcein stained. x 260. 436 SHORT COMMUNICATIONS

Therefore, it would seem reasonable to assume that the "uterine sperm" were capaci tated. However, neither they, nor freshly ejaculated sperm, placed within the perivitelline space, appeared to be able to fertilize ova (Table 1). It is unlikely that the flushing and centrifugation procedures involved in collecting uterine sperm had any gross effect upon their subsequent fertilizing ability. Uterine sperm injected into either the fallopian tubes or uteri have been shown to readily fertilize sheep ova (Killeen 1969; Killeen and Moore 1971). In one experiment Killeen and Moore (1971) recorded 61 % of ova fertilized after uterine insemination of uterine sperm and 53% of the fertilized ova developed into lambs. Neither the sperm-injection procedures nor the presence of the sperm diluent in the perivitelline space had any gross effect upon the subsequent fertilizability of ova. 62% of ova recovered from inseminated recipients following the injection of D+lO%S into the perivitelline space showed apparently normal cleavage (Table 1). The results suggest that the major purpose of capacitation in the sheep is not simply one of developing within sperm the ability to penetrate the zona. It may be that passage through the zona is necessary to develop full fertilizing capacity in ram sperm. In this context there may be differences between species. In the mouse Lin (1969) recovered two-cell ova from recipient female mice following the injection of motile sperm into the perivitelline space. Unfortunately, no details were provided on either treatment of sperm before injection or on the proportion of ova fertilized.

Acknowledgment The study was supported by grants from the Australian Research Grants Com mittee and the Rural Credits Development Fund. Grateful acknowledgment is made to Miss Patricia Glenn for technical assistance.

References BEATTY, R. A. (1957).-"Parthenogenesis and Polyploidy in Mammalian Development." (Cam bridge Univ. Press.) KILLEEN, 1. D. (1969).-Studies on fertilization and development of the ovine ovum. Ph.D. Thesis, University of Sydney. KILLEEN, 1. D., and MOORE, N. W. (1971).-J. Reprod. Pert. 24, 63. LIN, T. P. (1969).-Proc. Soc. Study Reprod., 2nd Ann. Mtg Univ. Calif. p. 19. MATTNER, P. E. (1963).-Nature, Lond., 199, 772. MOORE, N. W., and SHELTON, J. N. (1964).-J. Reprod. Fert. 7, 79.