Sperm Penetration of Rat Eggs in Vitro After Dissolution of Zona Pellucida by Chymotrypsin

NATURE. VOL. 220. NOVEMBER 9. 1968 589

(crosses 3 and 4 in Table 1) 13 non-Sb to 120 Sb individuals tivity but are independently controlled by a single pair survived. The streptomycin sensitive strains, especially of alleles exhibiting incomplete dominance. The FU 8train 11, may not have been completely homozygous resistant mice did not therefore exhibit a greater ability for the strf! gene, or certain minor gene modifiers might to break down uracil. 5-Fluoro-2' deoxyuridine 5' -mono have been in operation. The gene(s) for streptomycin phosphate, a metabolite of FU, inhibits the enzyme sensitivity appears to be recessive and to be located on the thymidylate synthetase in bacterial, mammalian and third chromosome . neoplastic cells, preventing growth". l!'U is incorporated The chromosome on which the flU factor is located was into RNA in many tissues, but the consequences have not !!imilarly determined. Reciproca l crosses were made alwa ys been clear'. The selection of Drosophila strains, between Pm: Sb/Xa (FU sensitive) and FU resistant specific in their reaction to FU, should provide a system individuals. The Fl survival rate was very low although for investigation of the mechanisms through which this all expected phenotypes were observed. The l!\ Pm : Sb chemical exerts its effects in animals. males were first crossed to flus females, which had been This work was supported by grants from the US selected from the same original stock as the flur strain, National Institutes of Health. and then backcrossed to flur females. The results are E. J. D. was supported by a genetics training grant shown in Table 2. Very few progeny were obtained in and E. G. by a rese.arch career development award, those crosses involving sensitive females, the survivors both from the National Institutes of Health, US Public being either Pm : Sb+ or Pm+ : Sb+. The back crosses to Health Service. flur females gave a much better survival rate. Practically E. J. DUKE· no individuals with the third chromosome marked by the E. GLASSMAN Sb gene survived, indicating that sensitivity to FU is associated with the third chromosome. The flur /flus Department of Biochemistry, heterozygote has a very low level of survival on this con and the Genetics Curriculum, centration of FU. This is evident in the low survival rate University of North Carolina. observed in the initial F 1 generation and in the number Received AprIl 11 ; revised September 5, 1968. of progeny obtained in crosses 1 and 2 (Table 2). In addi • Present addr~'8: Department of Zoology. University College, Belfleld, tion, the Sb survivors in crosses 3 and 4 (Table 2) could be StlUorgan Road, Dublin 4, Ireland. r s flu /flu h eterozygotes. The results are consistent with , Keller, E. C .• and Glassman. E ., Nature, 208, 202 (1965). the hypothesis that resistance to FU is semi·dominant • Davis. J., GUbert, W., and Gorlnl, L., Proe. US Nat . .dead. Sci., 61, 88S and is caused by a gene(s) carried on the third chromosome. 1196.). • Bridges, C. B., and Brehme, K. S., Carnegie Imt. Wash. Publ., 652 (1944). • Birgard, R ., and Heidelberger, C., Biochemistry, 6, 3339 (1966). I Dagg, C. P .• .dmer. Zool., S, 223 IIIJ63). Table 2. CHROMOSOMAL LOCATION 011' flu GENE • Dagg, C. P., Coleman, D. L .• and Fraser, G. M., Genetics, 49, 979 (1964.). Cross A 'i''i' fln. Sperm Penetration of Rat Eggs in vitro x 'i'\?- after Dissolution of Zona Pellucida Cross 2 uterus could penetrate the vitelline surface, activate the eggs and lead to the formation of pronuclei. All sensitive stocks should be continually t ested and This communication reports the procedures and results all resistant stocks maintained on the appropriate con of this experiment. centrations of drug media. Eggs were obtained from either adult or 27-30 day old Streptomycin sensitivity is recessive in Drosophila immature Wistar rats. Adult rats kept in 14 h of light but dominant in E. coli2• It is now important to ascertain and 10 h of darkness and showing regular oestrous cycles whether sensitivity in the two is related biochemically. were killed at 0500-0700 h on the day after a proestrous Work is progressing on the effects of streptomycin on in vaginal smear had been taken. Immature rats were vitro mechanisms of protein synthesili involving strepto· injected subcutaneously with 30 10 of PMS 44-48 h mycin sensitive and resistant stocks. before an intraperiton&al injection of 20 10 of BCG and The mechanisms through which FU exercises its toxic killed 14-16 h later. Their oviducts were placed in a effect have been investigated (see ref. 4 for review of small watchglass and covered with warm mineral oil that literature). Intraperitoneal injection of FU into certain had been bubbled with 5 per cent CO, in air and stored strains of ten-day pregnant mice results in the develop at 37° C. The eggs in cumulus oophorus were dissected ment of malformed foetuses". Genetic crosses indicated out from the oviduct and the oviduct was discarded. An that resistance is recessive but that more than one gene approximately equal volume (about 0·1-1·0 !J.l.) of various is involved. In a subsequent study' the rates of uracil- proteolytic enzymes known to be effective for dissolving 2_HC degradation to HCO. in these and many other the zona",7 was mixed immediately with the cumulus clot strains of mice were m easured. The differences in degrada under the mineral oil and incubated at 37° C under 5 per

tion rate were not related to fluorouracil resistance or sensi- cent CO 2 in air for 30 min. At the end of the incuba.tion,

min resulted in twenty penetrated eggs (29 per cent, two dispermic and one trispermic) out of sixty-nine zona-free eggs. After many trials, it was found that 0·02 per cent chymotrypsin (ex-chymotrypsin type 2. Sigma) was the best for the manipulation of eggs and was thus used throughout this experiment. In the immature rats thirty-four out of 235 (14·1) pCI' cent) '1ona-free eggs were pene trated by epididymal sperm, while thirty out of 100 eggs wero penetrated by uterine sperm. Although there is a significant difference in favour of uterine sperm, seven teen of thirty-two (53 per cent) eggs were penetrated by epididyrmtl sperm, and eight, een of thirty-eight (47 per eent) eggs were penetrated by uterine sperm in those spon taneously ovulatcd by adult rats. Thus Fig. 1. A, Zona-free rat egg with all enlarged sperm head (arrow) and the seconc\ polar body (c) almost abstricted, pretreated with chymotrypSin, 6 h after inselll;nation with whether the discrepancy is a consequence of epididymal sperm, stained with Lacmoid, x 250. B. Zona-free egg with fertilizing sperm the origin of spcrm eggs, or of a slight tail (arrow), male pronucleus (a), female pronucleus (I», and the .econd polar body (c). Pretreated with chymotrypsin, 6 h after insemination with uterine sperm. unstltined x 250. difference in the manipulation of thc eggs is still not clear, but none of 250 eggs the mixture was highly viscous and contained dispersed with zona in the control group was penetratcd. cells and zona-free eggs. A small drop of sperm sus In the earIy trials, when proteolytic E;nzyrncs werE- not pension containing 1,500-6,000 active sperm/fl.!. was added, thc motility of the sperm declined rapidly within added by means of a glass pipette under the mineral oil 1- 2 h in the presence of the egg clot. Introduction of and the suspension incubated again for 4- 8 h. The sperm chymotrypsin or trypsin into the medium markedly sllspensions were prepared by adding a drop of dense improved the motility of sperm in the first few hours, sperm, obtained either from the epididymis or from the and the motility usually lasted even to the end of the uterus of a female mated 6- 8 h previously, to 0·4--2 ml. incubation. The physiological mechanism of such im of TC 199 (Difco) supplemented with 100 IU/m!. of peni provement is still obscure. cillin G potassium and 50 fl.g/m!. of streptomycin sulphate. Rat sperm is known to need capacitation in the female After illcubation tho eggs were washed twice in Hanks tract for a few hours beforo the eggs can be penetrated·,,, solution containing 1 mg/m!. of polyvinylpyrrolidone. and uterine sperm can penetrate eggs a few hours sooner They wero mounted on a glass slide, pressed gently with than epididymal Bpermo. As the work rep:->rted here has a cover slip and examined under a phase contrast micro shown, it seems that either capacitation of sperm can be scope. A penl-trated egg was judged by the presence of achicved in a short time in the presence of chymotrypsin an enlarged sperm head or of male and female pronuclei or that the capacitation of sperm is necessary only for in the vitellus with a sperm tail inside the vitellus (Fig. 1). the penetration of zona pellucida. In most cases the distal part of the tail remained outside To test the possibility that chymotrypsin is capable of the vitellus and often showed slow wave motion. capacitating sperm or of facilitating sperm penetration, Table I shows that pretreatment of eggs with hyal epididymal sperm, instead of the eggs, were preincubated uronidase neither dissolved the zona pellucida nor in a medium containing 0·005--0·02 per cent chymotrypsin facilitated sperm penetration. Only three out of fifty for 0'5 to 6 h. The medium used for this preincubation five (5 per cent) zona-free eggs were penetrated when was Ham's FlO (Difco) supplemented with 0·5 per cent pronase was used for preincubation of oggs. Pretreatment bovine crystallinc albumin, which waB found to be one with hyaluronidase followed by chymotrypsin resulted of the best synthetic media for maintaining motility and in sperm penetration in three of thirty-one eggs. Pre survival of rat sperm at 37° C under 5 per cent CO. in lllcubation of eggs with 0·02 per cent trypsin (Trypsin air. The zona pellucida of eggs was again dissolved and 1 : 300, Nutritional Biochemical Corporation) for 10--20 a small proportion (5-16 per CEnt) of 348 zona-frGe eggs was penetrated. Insemination with sperm preincubated Table 1. In vitro SPERM PENETRATION OF RAT EGGS AFTER DISSOLUTION OF in a lower concentration of chymotrypsin (0'0001 per ZONA cent), however , did not cause the dissolution of zona, and No. of No. of No. (per cent) none of the seventy-four eggs was penetrated. Thus the Treatmcnts females eggs penetrated action of chymotrypsin is chi&fly to remove the mE-ch Control, addition of various animal fluids 76 844 0 anical barrier of the zona for sperm pcnetmtion. Control. no enzymes: Almost all the eggs, penetrated or not, had between ten Eggs from inunature rats with cpi. and 200 sperm heads attached to their surface (Fig. 1), dldymal sperm 7 137 0 Eggs from adult rats with uterine but among 158 penetrated eggs only ten were poly sperm 10 90 0 spermic. This indicates a strong vitelline block during Treatmen t of eggs with various enzymes: sperm pcnetration even in the absence of zona pellucida. 0·02-0'1 per cent hyaluronidase 7 70 0(0) 0·02 per cent pronase 5 55 3 (5) The sperm head in the vitellus showed various stages Hyaluronidase followed by 0·02 per of transformation into a male pronucleus. Of twenty cent chymotrypsin 31 3 (10) 0·02 per cent trypsin for 10--20 min 5 69 20 (29) nine penetrated eggs examined 4 to 5 h after insemina Treatment of eggs with 0·02 per cent tion, seven had sperm hcad showing the first change after chymotrypsin for 15-30 min: penetration, that is, there was decreased opacity of sperm Eggs from immature rats with epididymal sperm 13 235 34 (14,5) head and the detachment of pcrforatorium. The second with uterine sperm 6 100 30 (30) meiosis in these eggs was at telophase, showing the Eggs from adul t rats with epididymal sperm 4 32 17 (53) aetivation of these eggs. The other twenty-two eggs had with uterine sperrn 4 38 18 (47) either an enlarged sperm h ead (eleven eggs) or a m ale Treatment of epididymal sperm in pronucleus (eleven eggs). Enlarged sperm heads wero 0'005- 0'02 per cent ch~'motrypsill for 30 min 7 118 19(16) usually difficult to see in the fresh preparation, but could for 6(}-90 min 4 88 6 (7) be seen distinctly after staining with 0·25 per cent Lacmoid for 12(}-150 min 4 61 4 (7) for 240-360 min 4 81 4 (5) in 45 per cent acetic acid. The male pronucleus at 1!he

© 1968 Nature Publishing Group NATURE. VOL. 220. NOVEMBER 9, 1968 591 early stage was Hot sphtrical but elongated, retaining potent in the male rat (Table 1). This ester is effective the original curvature. In all these twenty-two eggs the when administered either by injection or orally, and five second polar body was almost completely abstricted but consecutive daily doses of 100 mgjkg by stomach tube still connected to the vitellus. Either a densely packed to the male rat seem to be tho minimal amount required mass of female chromatin or It small female pronucleus for a clear-cut action. The predominant effect is a "func could bc found near the second polar body. tional" type of sterilizing action' involving post-meiotic Seventy penetrated eggs were examined between 5·5 cells (spermatids and spermatozoa), so that intact motile and 8 h after insemination; only seven of them had a sperm remain available, but are rendered incompetent. sperm head at the stage of chaJlging, and male and female pronuclei wer6 seen in sixty-three eggs. Usually Tai,\e l. TOT';'L WEEKLY OFFSPRISQ FROM MAL" RATS AND )lICE TREATEI> the male pronucleus at the late stage was round and WITH TRIM~:THYLl'HOSl'II';'TE contained a single large nucleolus, while the female pro nucleus was smaller and also containcd only one nucleolus. Weeks from firs t dose 1o 1 2 3 4 5 6 7 8 9 10 11 12 According to Austin , it takes 3 h from sperm pene 5 rats 5 x 250 mg/kg tration to the first appearance of primary nucleoli in the p.o. 24 0 (l 0 0 20 14 15 20 16 10 23 enlarged sperm head and about 5 h from sperm penetration 5 x 100 mg/kg p.o. 3 0 0 5 30 32 30 19 5 41 27 8 mice 5 x 1 g/kg p.o. 0 0 5 32 46 64 49 t o the development of a spherical male pronucleus con 5 x 1 g/kg i.p. 0 8 22 34 15 25 taining a single nucleolus. If the same time interval for pronuclear development is assumed in this case, sperm penetration would be estimated to have occurred between This antifertility effect resembles that of lnethyl 1 and 3 h after insemination in most cases. Furthermore, methanesulphonate (MMS)6,7, but there are certain differ when superovulated eggs were examined between 0·5 and ences. The sulphonic ester is more effective when given 1·5 h after insemination with epididymal sperm, all ninety intraperitoneally, whereas the phosphate ester seems to four eggs were surrounded by numerous vigorously moving be equally active when given by mouth or by injection. sperm, but none was penetrated. It seems that 1-3 h There are also indications that the phosphate ester has an were required before the sperm could penetrate vitellus inhibitory action on the early stages of spermatogenesis, in this experiment. Although sperm penetration into the which is not the case with MMS. At room temperature vitellus, activation of eggs, and the formation of pronuclei TMP is quite stable in solution whereas MMS hydrolyscs were achieved in our experimcntal conditions, such zona spontaneously. Another unexpected finding is that tri free eggs were unable to develop further and showed isopropylphosphate has no action in the mouse, although signs of degeneration after a second incubation for 18 h. isopropyl methane-sulphonate exerts a powerful anti Nevertheless, this experiment has shown that passing spermatogenic action against the proliferative stages of this B through the zona pellucida is not a prerequisite for the process in both the mouse and rat6- • fusion of gametes and the formation of pronuclei. Trimethylphosphate has been found to be almost devoid This work was supported by grants from the US Public of anticholinesterase activity'; in a variety of rodents it Health Service and tho Ford Foundation. is not particularly toxic, and doses which are lethal when given orally vary according to different investigators. In Y. TOYODA the rat, 1·8 gjkg of trimethylphosphate introduced intra M. C. CHANG venously was reported to be tolerable, and 2·4 gjkg 10 Worcester Foundation for Experimental Biology, produced transient anaesthesia and death within 24 h • Shrewsbury, Massachusetts. The lethal dose administered orally is given as 1·65 ml.jkgll, Received August 13; revised September 16, 1968. a nd in our experiments, using redistilled commercial matorial, 5 x 500 mg/kg given per 08 was just tolerable to t Dauzier, L., Thibault, C., and Winterburger, S., eR Acad. Sci., 238, 844 (1954). male rats, although 1 gjkg by the same route was lethal • Chang, 101. C., Natw'e, 184,466 (1959). after four doses . , Yanaglmachi, R., and Chang, M. C., Natw'e, 200,281 (1963). Using 32P_TMP we have found that after oral or intra 4 Barros, C., and Austin, C. Ro, J. Exp. Zool., 166, 317 (1967). peritoneal administration, only one radioactive metabolite, , Austin, C. R., Austral. J. Sci. Res., 4, 581 (1951). dimothylphosphate (DMP), is found in mouse urine and • Chang, M. C., and Hunt, D. M., Exp. Cell Res., 11, 497 (1956). , Mintz, B., 8cience, 148, 1232 (1965). in the bladder after 3 h. The rat also rapidly metabolizes • Austin, C. R., Austral. J. BioI. Sci., 7, 179 (1954). oral TMP to DMP, bllt the conversion occurs much more • Noyes, R. W., WeBtern J. SUTg. Obstet. Gynec., 61, 342 (1953). slowly when TMP is given intraperitoneally so that both \0 Austin, C. R ., J . Roy. Microse. 80c., 71, 295 (1951). compounds are found in the urine up to 16 h after treat ment. There is no evidence of further degradation to inorganic phosphate or even to monomethylphosphate in either species. In a preliminary in vitro study, rat kidney did not metabolize TMP, but both liver and intestinal tissue carried out the conversion. Uniformly labellcd Antifertility Action and Metabolism of 14C-TMP enabled the identification of S-methyl eysteine as a further urinary m etabolite which is produced Trimethylphosphate in Rodents slowly by the rat, but appears within a fcw hours in mouse RECENT studies with hexamethylphosphoramide (HMPA) urIne. and its analogues have shown that its antifertility activi Trimethylphosphate is used as a methylating agent in ties in insects1 and rodents' require the presence of all the organic laboratoryl2. Our study, however, demon six methyl groups. Thus although pentamethylphosphor strates a selective biological effect against post-meiotic amide is a metabolite of HMPA in both the housefly' and male germ cells. Further studies will decide whether a the rodent' it does not possess significant antifertility background of dominant lethal mutational cffects mili activity in either species. tates against any further useful development. This observation led us to investigate the antifertility The methyl group is associated with a variety of effect of simplc phosphate esters, although comparative responses in its relationship to a n antifertility action studies of the relationship between structure and activity against spcrmatogenic cells. III HMPA the effect is an in HMPA and its intermediates in which the dimethyl antispermatogenic action predominantly affecting sperma amino groups had been replaced by methoxyl groups were tids and spermatocytes2. Through MMS the same group not encouraging. Trimethylphosphate (TMP) did, how mcdiates a functional sterilizing action on spermatids ever, show antifertility activity in the male mouse at and sperm, thus inducing infertility with little delay. a fairly high concentration, but was considerably more Finally, in TMP the methyl group is involvcd in inducing