MOLECULAR REPRODUCTION AND DEVELOPMENT 27:332-336 (1990)
Zona Drilling Enhances Fertilization by Mouse Caput Epididymal Sperm WASSIM C. WAZZAN, RALPH B.L. GWATKIN, AND ANTHONY J. THOMAS, JR. Department of Urology and Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio ABSTRACT Spermatozoa from the caput epididymis are known to be much less capable of fertilization when compared to sperm from more distal segments of the epididymis. The purpose of this study was to determine if two micromanipulative techniques, zona drilling (ZD) and a modification of partial zona dissection (PZD), could be used to enhance fertilization with caput epididymal sperm. A mouse in vitro fertilization model was used. Inseminating oocytes with 500-1,000 sperm/ oocyte from the cauda epididymis as a control resulted in fertilization of 98 of 300 (32.6%) oocytes. Of those fertilized, 47 developed to the blastocyst stage (47.9%). Caput sperm fertilized 13 of 116 ( 1 1.2%) nonmanipulated oocytes. Only 1 of 13 developed into a blastocyst, while with oocyte ZD, caput sperm fertilized 24 of 144 ( 1 6.7%) oocytes, 50% of those fertilized developing to blastocyst (P = 0.0129). When modified PZD was performed on oocytes, only one of 23 was fertilized, with no blastocyst development. These results indicate that acid Tyrode ZD enhances both fertilization and early embryonal development when caput epididymal sperm are used for insemination. These mouse studies suggest that ZD or other micromanipulation techniques may prove clinically useful in men with proximal epididymal obstruction where only caput sperm are available.
cases to improve their fertilizing ability. In humans, some pregnancies were established after "high" vasoepididymostomies (Schoysman and Bedford, 1986; Silber, 1989) and in cases of sperm aspiration from the caput epididymis for in vitro fertilization (Silber et al., 1989). McLaughlin and Shur (1987) showed that mouse caput epididymal sperm can acquire the ability to bind to the zona pellucida after washing them free of caput epididymal fluid. However, these investigators did not evaluate the fertilizing ability associated with the acquisition of zona binding capacity. A possible means of enhancing the efficiency of in vitro fertilization by caput epididymal sperm is to assist fertilization mechanically. In this study, we assessed the value of two micromanipulative procedures, zona drilling (ZD) (Gordon and Talansky 1986) and a modified partial zona dissection (PZD) (Malter and Cohen 19891, for enhancing in vitro fertilization of mouse oocytes with caput epididymal sperm.
MATERIALS AND METHODS Sperm Processing
The epididymides of C3D2F1/J adult male mice (Jackson Laboratories, Bar Harbor, ME) were dissected. The caput and cauda segments were separately transferred to 2 ml of IVF medium (Hogan et al., 1986) supplemented with 30 mg/ml bovine serum albumin (BSA fraction V, Sigma Chemical Co., St. Louis, MO). A sperm preparation technique essentially similar to Key Words: Caput epididymis, Micromanipulathat described by McLaughlin and Shur (1987) was tion, In vitro fertilization used. Each epididymal segment was punctured more than 20 times with a 25-gauge needle and reciprocated to release the sperm. Suspensions were filtered INTRODUCTION Investigation of several species has shown that caput through Nitex mesh to remove the debris and the epididymal spermatozoa are immature and have a very sperm resuspended in 10 ml of prewarmed IVF melow fertilizing potential (see review by Bedford, 1988). dium. Caput sperm suspensions were washed three Recent studies have shown that maturation can be achieved to varying degrees in vivo and in vitro. Thus, short-term ligations of the epididymis (Bedford, 1967, 1988; Horan and Bedford, 1972), sperm treatment with androgen dependant epididymal proteins (OrgebinCrist and Fournier Delpech, 1982; Cuasnicu et al., Received March 28, 1990; accepted June 18, 1990. 1984; Gonzalez Echeverria et al., 19841,and sperm cocul- Address reprint requests to Dr. Anthony J. Thomas, Jr., Department of Urology, The Cleveland Clinic Foundation, 9500 Euclid Avenue. ture with epididymal epithelial cells (Moore and Hart- Cleveland, OH 44106. man, 1986; Moore et al., 1986) were shown to induce Dr. Ralph B.L. Gwatkin, is now at ReproGene, 25460 Bryden Road, the maturation process of spermatozoa and in some Beachwood, OH 44122.
0 1990 WILEY-LISS, INC.
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Fig. 1. Zona drilling. A mouse oocyte held on the tip of a holding pipet (left).A micropipet loaded with acid Tyrode’s has just dissolved a hole through the zona (right). x 450. (Note tip of injection needle a t the surface of the zona pellucida and not in the perivitelline space.)
times by centrifugation at 800g for 10 minutes at room temperature. The supernatant was discarded each time and the pellet resuspended in 10 ml of fresh medium. The final pellets were resuspended in 1 ml of IVF medium usually a t concentrations of 1-5 x lo5 sperm/ml. Cauda sperm suspensions were resuspended in the same supernatant after each centrifugation then finally diluted to yield the same caput sperm concentrations. Sperm counts were determined on a hemocytometer. All suspensions were incubated at 37°C under 5% CO,, 5% 02,and 90% N2 for 1-1.5 hour to allow for capacitation.
Systems Corp., Greenvale, NY). Holding pipets were made of 1 mm glass capillary tubes hand pulled and fire polished using a Narishige microforge to yield and outside tip diameter of 80-100 km and a n inside tip diameter of 10-15 km. The injection and dissection needles were made of thin-walled glass capillaries containing a filament and pulled on a Narishige vertical puller to yield a n outside tip diameter of 3 km. Micromanipulation procedures were performed on a prewarmed microscope stage in a Petri dish with a rectangular 200 kl M2 drop under silicone oil. The two procedures were performed a s follows:
Oocytes Collection C3B6F1/J female mice (5-6 weeks: Jackson) were induced to superovulate by intraperitoneal injection of 5 i.u. PMSG (Sigma Chemical Co., St. Louis, MO) followed 48-50 hours later by 5 i.u. hCG (Sigma Chemical Co., St. Louis, MO). Animals were sacrificed a t 1213 hours after hCG by ether overdose. Oviducts were excised and cumulus released in M16 medium (Whittingham, 1971) with 4 mg/ml BSA and 0.1% hyaluronidase. Oocytes were then washed three times in M16. The mature oocytes were distributed between three media drops under silicon oil (Dow Corning 200 Fluid, Dow Corning Corp. Midland, MI). One drop contained M16 for in vitro fertilization (nonmanipulation group), another contained M2 medium (Hogan et al., 1986) for ZD, and the third M2 with 0.05 M sucrose (Sigma Chemical Co., St. Louis, MO) for PZD.
Zona Drilling (Fig. 1.) The technique we used was essentially similar to that described by Gordon and Talansky (1986). With the oocyte grasped by the holding pipet, a n injection needle loaded with acid Tyrode’s solution (pH 2.3) was pressed gently and tangentially against the zona pellucida releasing the acid stream. The zona was seen to flatten followed by a tiny bleb and rupture exposing the vitellus at the point of acid application. After drilling, each oocyte was moved to the far end of the drop to separate it from the nondrilled oocytes.
Micromanipulation The equipment consisted of a Nikon inverted microscope carrying Narishige micromanipulators (Medical
Modified Partial Zona Dissection (Fig. 2.) The technique used was a modification of that described by Malter and Cohen (1989). The dissecting needles were prepared as were the needles for zona drilling, but their tips were not broken and no acid was used. The dissection procedure used was as follows. With the oocytes held steady, a microneedle was inserted into the zona pellucida at a point away from the polar body. The needle was then pushed through the
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Fig. 2. Partial zona dissection. A: Introducing the needle. B: Rubbing the oocyte against the holding pipet to enlarge the holes. External diameter of needle at the point of entry is 15-20 pm. x 450.
perivitelline space avoiding the vitellus until it punctured the zona on the opposing wall. A slit between the two openings was not made as in the technique of Malter and Cohen (1989). However, the dissection needle was thrust to a point where its outside diameter was 15-20 pm in order to enlarge opening in the zona a t the entry point of the needle to the same size. The oocyte was then released from the holding pipet and carried on the dissection needle to be rubbed in several directions against the surface of the holding pipet in a n attempt to further widen the two openings in the zona.
Insemination and Embryo Development Following micromanipulation, oocytes were washed three times in MI6 medium. Each of the three groups of oocytes, i.e., nonmanipulated, ZD, and PZD, were further subdivided between the two dishes, each to be inseminated with either caput or cauda sperm suspension at a ratio of 500-1,000 sperm/egg. Insemination dishes were incubated for 4 hours after which oocytes were transferred to culture dishes containing MI6 medium with 4 mg/ml bovine serum albumin. These were incubated and examined under light microscopy at 24 hours for two-cell cleavage as presumptive evidence for fertilization. Further examination was carried out a t 48, 72, and 96 hours to look for development into blastocysts. A total of nine experiments were performed. PZD was performed in the last four experiments only. In each experiment a small number of ZD oocytes were not inseminated to note the effect of ZD alone on the activity of oocytes.
Statistical Analysis Usually a chi-square test was used to analyze data. When chi-square was not appropriate because of a
small sample size, Fisher’s exact test was used instead. The data presented in Tables 1-3 was obtained by combining results of each of nine experiments. Comparisons of interest were performed for each individual experiment. These results were not different from those obtained by doing overall analyses.
RESULTS Fertilization and development rates of nonmanipulated oocytes are shown in Table 1. Ninety-eight of three hundred oocytes (32.6%) were fertilized when inseminated with cauda sperm as compared to 13 of 116 (11.2%) oocytes exposed to caput sperm. Normal development to the blastocyst stage occurred in 47.9% of the cauda sperm 2-cell embryos, while only 1 of 13 (7.7%) caput 2-cell embryos developed into a blastocyst. Eight 2-cell embryos showed fragmentation and four did not cleave any further. The results of fertilization and development after micromanipulation of oocytes inseminated with cauda sperm are shown in Table 2. Fifty-seven ZD oocytes out of eighty-seven (65.5%) were fertilized and 75.4% of these developed into blastocysts, showing a clear advantage over the nonmanipulated group (P= 0.002). After PZD, 19 of 42 (45.2%) eggs were fertilized and 11 of 19 (57.9%) developed to blastocyst. While there appeared to be some advantage in this group over the nonmanipulated oocytes, no statistical difference was found. The results of experiments with caput epididymal sperm are detailed in Table 3. Twenty-four ZD oocytes out of one-hundred forty-four (16.7%) fertilized. This is statistically not different from the 11.2% in our nonmanipulated group. The development rates, however, between the two groups were significantly different.
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epididymis has been associated with a n increased rate of embryonic loss thought to be due to delayed fertilization (Orgebin-Crist, 1967, 1968; Orgebin-Crist and Total Jahad, 1977) or abnormal activation (Brackett et al., number of Blastocysti oocytes 2-cell (%) 2-cell (%) 1978). In our experiments with nonmanipulated Cauda 300 98 (32.6) 47/98 (47.9) oocytes, a fertilization rate of 11.2% was observed with Caput 116 13 (11.2) 1/13 (7.7) caput epididymal sperm, but only 1 out of 13 two-cell embryos developed normally to the blastocyst stage. These findings are in accordance with those of the TABLE 2. Effect of Micromanipulation on Fertilization above mentioned studies. With Cauda Sperm When zona drilling was performed on oocytes to be inseminated with caput sperm, the fertilization rate Total did not significantly improve. However a remarkable number of Blastocvstsi Procedure oocytes 2-ce11 (%I 2-ce11?%) improvement was seen in the blastocyst development No manipulation 300 98 (32.6) 47/98 (47.9)".b rate (50%vs. 7.7%). A plausable explanation for this Zona drilling 87 57 (65.5) 43/57 (75.3)" phenomenon is the fact that ZD allows for earlier Partial z o n i sperm egg fusion and thus fertilization and pronuclear dissection 42 19 (45.2) 11/19 (57.9)' formation (Talansky and Gordon, 1988). Early fertili"P = 0.002 (significant). zation may be a n important factor in decreasing the bP= 0.526 (not significant). rate of embryonic loss. A further explanation of the advantage of ZD is that eliminating the zona barrier and exposing the oolemma could provide a selective TABLE 3. Effect of Micromanipulation on Fertilization advantage for fertilization to the very few caput sperWith Caput Sperm matozoa that may be functionally mature. In this Total study, zona drilling also showed a significant enhancenumber of Blastocyst/ ment of fertilization in the oocyte group inseminated 2-cell (%) Procedure oocytes 2-cell (%) with Cauda sperm. This confirms prior reports of the No manipulation 116 13 (11.2) 1/13 ( 7 . 7 ) " ~ ~efficacy of ZD in assisting in vitro fertilization (Gordon Zona drilling 144 24 (16.7) 12/24 (50)" and Talansky, 1986; Talansky and Gordon, 1988; Partial zona Conover and Gwatkin, 1988; Depypere et al., 1988). dissection 23 l(4.3) Oil ( O P From this study, our modification of PZD does not "P = 0.0129 (significant). appear to provide the same advantage as ZD. With the bP= 1.000 (not significant). oocytes inseminated with cauda sperm only slightly better fertilization rates were observed, but they were not statistically significant. With caput sperm, no benFifty percent (12/24) with ZD as opposed to 7.7% (1/13) eficial effect of PZD was observed. The difference in in the nonmanipulated group formed blastocysts outcome of ZD as opposed to PZD may be due to the (P=O.O129). Of the 12 two-cell embryos that did not nature of the opening created. Zona drilling by acid reach the blastocyst stage, 3 cleaved to the 4-cell stage, results in a much wider hole than PZD (personal ob4 failed to cleave beyond the 2-cell stage, and 5 showed servation, Ng et al., 1990). ZD also exposes the egg early fragmentation. In the PZD group only one out of plasma surface to the surrounding milieu at the site of 23 oocytes showed a 2-cell embryo with no further pro- the opening (Gordon and Talansky, 1986). PZD does gression. Of the 30 oocytes that were zona drilled, but not show this feature, since the holes tend to reseal and not inseminated, only one showed evidence of parthevitellus is shrunken by sucrose. nogenetic activation resulting in a 2-cell stage with fragmentation of both blastomeres. CONCLUSIONS TABLE 1. In Vitro Fertilization by Cauda and Caput Epididymal Sperm
DISCUSSION In this study, we evaluated the impact of zona drilling and a modification of partial zona dissection on the fertilization of mouse oocytes using caput and cauda epididymal sperm. Saling (1982) showed that mouse caput sperm bind very poorly to the zona pellucida and even to the oolemma in zona free oocytes. In the hamster, caput epididymal sperm nuclei injected into eggs were not able to develop to pronuclei within the time period studied (Uehara and Yanagimachi, 1976). In the rabbit, fertilization with immature sperm from the
Our results suggest that ZD using acid Tyrode's solution enhances in vitro fertilization by mouse caput epididymal spermatozoa which are immature in their majority. This enhancement occurs in the form of improved preimplantation embryonic development. The mechanism by which ZD effects this improvement is probably related to allowing earlier gamete fusion and fertilization. The relevance of these findings to clinical application is in cases of continued male infertility after proximal vasoepididymostomies or direct sperm aspiration from the caput epididymis. Acid ZD in humans, however, has been reported to be associated with
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an increased rate of oocyte damage (Ng et al., 1989), polyspermy (Gordon et al., 19881, and abnormal cleavage patterns (Talansky and Gordon, 1988). It is yet to be determined whether other mechanical micromanipulation techniques, e.g., microinsemination of sperm into the perivitelline space (see Ng et al., 19901, enhance human fertilization and development with immature epididymal sperm as ZD did with the mouse in our study.
REFERENCES Bedford JM (1967): Effects of duct ligation on the fertilizing ability of spermatozoa from different regions ofthe rabbit epididymis. J E~~ Zoo1 166:217-222. Bedford J M (1988): The bearing of epididymal function in strategies for in vitro fertilization and gamete intrafallopian transfer, Ann NY Acad Sci 541:284-291. Brackett BG, Hall JL, Oh Y-K (1978): In vitro fertilizing ability of testicular, epididymal, and ejaculated rabbit spermatozoa. Fertil Steril 29571-582. Conover Jc, Gwatkin RBL (1988): Fertilization of zOna drilled oocytes treated with a monoclonal antibody to the zona glycoprotein ZP3. J EXPZoo1 2471113-118. cuasnicu ps, ~~~~~l~~Echeverria F, piazza AD, pineiro L, ~ l J A (1984):Epididymal proteins mimic the androgenic effect on zona pellucida recognition by immature hamster spermatozoa. J Reprod Fertil 71:427-431. De Pepypere HT’ McLaughlin KJ’ RF’ Warnes GM’ Matthews CD (1988): Comparison of zona cutting and zona drilling as techniques for assisted fertilization in the mouse J Reprod Fertil 84:205-211. Gonzalez Echeverria F, Cuasnicu PS, Piazza A, Pineiro L, Blaquier JA (1984): Addition of androgen free epididymal protein extract increases the ability of immature hamster spermatozoa to fertilize in vivo and in vitro. J Reprod Fertil 71:433-437. Gordon JW, Talansky BE (1986): Assisted fertilization by zona drilling: A mouse model for correction OfoligOsPermia. J ExP ZOO1 239: 347-354. Gordon JW, Grunfeld L, Garrisi GJ, Talansky BE, Richards C, Laufer LN (1988): Fertilization of human oocytes by sperm from infertile males after zona pellucida drilling. Fertil Steril 50:68-73. Hogan B, Costantini F, Lacy E (1986): “Manipulating the Mouse Embryo: A Laboratory Manual.” New York: Cold Spring Harbor Laboratory. Horan AH, Bedford JM (1972): Development of the fertilizing ability of spermatozoa in the epididymis of the Syrian hamster. J Reprod Fertil 30:417-432.
Malter HE, Cohen J (1989): Partial zona dissection of the human oocyte : A non traumatic method using micromanipulation to assist zona pellucida penetration. Fertil Steril 51:139-148. McLaughlin JD, Shur BD (1987):Binding of caput epididymal mouse sperm to the zona pellucida. Dev Biol 124:557-561. Moore HDM, Hartman TD (1986): In vitro development of fertilizing ability of hamster epididymal spermatozoa after co-culture with epithelium from the proximal cauda epididymis. J Reprod Fertil 78:347-352. Moore HDM, Hartman TD, Smith CA (1986):In vitro culture of hamster epididymal epithelium and induction of sperm motility. J Reprod Fertil 78:327-336. Ng SC, Bongso A, Chang SI, Sathananthan H, Ratnam SS (1989): Transfer of human sperm into the perivitelline space of human oocytes after zona drilling or zona puncture. Fertil Steril 52:73-78. Ng SC, Bongso A, %thananthan H, Ratnam SS (1990):Micromanipulation: Its relevance to human in vitro fertilization. Fertil Steril 53:203-219’ Orgebin-Crist MC (1967): Maturation of spermatozoa in the rabbit epididymis. Fertilizing ability and embryonic mortality in does inseminated with epididymal spermatozoa. Ann Biol Anim Biochem Biophys 7:372-389. Orgebin-Crist MC (1968): Maturation of spermatozoa in the rabbit epididymis: Delayed fertilization in doses inseminated with epididymal spermatozoa‘ Reprod Fertil 16:29-40’ Orgebin-Crist MC, Jahad N (1977): Delayed cleavage of rabbit ova after fertilization by Young ePididYmal spermatozoa. Bid Reprod ~ ~ ~ i ~ ~ 16:358-362. Orgebin-Crist MC, Fournier-Delpech S (1982): Sperm-egg interaction. Evidence for maturational changes during epididymal transit. J Androl 3:429-433. Saling PM (1982): Development of the ability to bind to zonae pellucidae. Reversible immobilization of mouse spermatozoa by lanthanum. Biol Reprod 26:429-436, Schoysman RG, Bedford JM (1986):The role of human epididymis in sperm maturation and sperm storage as reflected in the consequences of epididymovasostomy~Fertil Steril 46:293-299, Silber sJ (1989): Role of epididymis in sperm maturation, Urology 33:47-51. Silber SJ, Ord T, Balmaceda J, patrizio p, Asch RH (1989): congenital absence ofthe vas deferens: Studies on the fertilizing ability of human epididymal sperm [abstract]. Read at the 45th annual meeting of the American Fertility Society, San Francisco, CA. Talansky BE, Gordon JW (1988): Cleavage characteristics of mouse embryos inseminated and cultured after zona pellucida drilling. Gamete Res 21:277-287. Uehara T, Yanagimachi R (1977): Behavior of nuclei of testicular, caput and cauda epididymal spermatozoa injected into hamster eggs. Biol Reprod 16:315-321. Whittingham DG (1971): Culture of mouse ova. J Reprod Fertil [Suppll 14:7-21.
Zona Drilling Enhances Fertilization by Mouse Caput Epididymal Sperm WASSIM C. WAZZAN, RALPH B.L. GWATKIN, AND ANTHONY J. THOMAS, JR. Department of Urology and Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio ABSTRACT Spermatozoa from the caput epididymis are known to be much less capable of fertilization when compared to sperm from more distal segments of the epididymis. The purpose of this study was to determine if two micromanipulative techniques, zona drilling (ZD) and a modification of partial zona dissection (PZD), could be used to enhance fertilization with caput epididymal sperm. A mouse in vitro fertilization model was used. Inseminating oocytes with 500-1,000 sperm/ oocyte from the cauda epididymis as a control resulted in fertilization of 98 of 300 (32.6%) oocytes. Of those fertilized, 47 developed to the blastocyst stage (47.9%). Caput sperm fertilized 13 of 116 ( 1 1.2%) nonmanipulated oocytes. Only 1 of 13 developed into a blastocyst, while with oocyte ZD, caput sperm fertilized 24 of 144 ( 1 6.7%) oocytes, 50% of those fertilized developing to blastocyst (P = 0.0129). When modified PZD was performed on oocytes, only one of 23 was fertilized, with no blastocyst development. These results indicate that acid Tyrode ZD enhances both fertilization and early embryonal development when caput epididymal sperm are used for insemination. These mouse studies suggest that ZD or other micromanipulation techniques may prove clinically useful in men with proximal epididymal obstruction where only caput sperm are available.
cases to improve their fertilizing ability. In humans, some pregnancies were established after "high" vasoepididymostomies (Schoysman and Bedford, 1986; Silber, 1989) and in cases of sperm aspiration from the caput epididymis for in vitro fertilization (Silber et al., 1989). McLaughlin and Shur (1987) showed that mouse caput epididymal sperm can acquire the ability to bind to the zona pellucida after washing them free of caput epididymal fluid. However, these investigators did not evaluate the fertilizing ability associated with the acquisition of zona binding capacity. A possible means of enhancing the efficiency of in vitro fertilization by caput epididymal sperm is to assist fertilization mechanically. In this study, we assessed the value of two micromanipulative procedures, zona drilling (ZD) (Gordon and Talansky 1986) and a modified partial zona dissection (PZD) (Malter and Cohen 19891, for enhancing in vitro fertilization of mouse oocytes with caput epididymal sperm.
MATERIALS AND METHODS Sperm Processing
The epididymides of C3D2F1/J adult male mice (Jackson Laboratories, Bar Harbor, ME) were dissected. The caput and cauda segments were separately transferred to 2 ml of IVF medium (Hogan et al., 1986) supplemented with 30 mg/ml bovine serum albumin (BSA fraction V, Sigma Chemical Co., St. Louis, MO). A sperm preparation technique essentially similar to Key Words: Caput epididymis, Micromanipulathat described by McLaughlin and Shur (1987) was tion, In vitro fertilization used. Each epididymal segment was punctured more than 20 times with a 25-gauge needle and reciprocated to release the sperm. Suspensions were filtered INTRODUCTION Investigation of several species has shown that caput through Nitex mesh to remove the debris and the epididymal spermatozoa are immature and have a very sperm resuspended in 10 ml of prewarmed IVF melow fertilizing potential (see review by Bedford, 1988). dium. Caput sperm suspensions were washed three Recent studies have shown that maturation can be achieved to varying degrees in vivo and in vitro. Thus, short-term ligations of the epididymis (Bedford, 1967, 1988; Horan and Bedford, 1972), sperm treatment with androgen dependant epididymal proteins (OrgebinCrist and Fournier Delpech, 1982; Cuasnicu et al., Received March 28, 1990; accepted June 18, 1990. 1984; Gonzalez Echeverria et al., 19841,and sperm cocul- Address reprint requests to Dr. Anthony J. Thomas, Jr., Department of Urology, The Cleveland Clinic Foundation, 9500 Euclid Avenue. ture with epididymal epithelial cells (Moore and Hart- Cleveland, OH 44106. man, 1986; Moore et al., 1986) were shown to induce Dr. Ralph B.L. Gwatkin, is now at ReproGene, 25460 Bryden Road, the maturation process of spermatozoa and in some Beachwood, OH 44122.
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Fig. 1. Zona drilling. A mouse oocyte held on the tip of a holding pipet (left).A micropipet loaded with acid Tyrode’s has just dissolved a hole through the zona (right). x 450. (Note tip of injection needle a t the surface of the zona pellucida and not in the perivitelline space.)
times by centrifugation at 800g for 10 minutes at room temperature. The supernatant was discarded each time and the pellet resuspended in 10 ml of fresh medium. The final pellets were resuspended in 1 ml of IVF medium usually a t concentrations of 1-5 x lo5 sperm/ml. Cauda sperm suspensions were resuspended in the same supernatant after each centrifugation then finally diluted to yield the same caput sperm concentrations. Sperm counts were determined on a hemocytometer. All suspensions were incubated at 37°C under 5% CO,, 5% 02,and 90% N2 for 1-1.5 hour to allow for capacitation.
Systems Corp., Greenvale, NY). Holding pipets were made of 1 mm glass capillary tubes hand pulled and fire polished using a Narishige microforge to yield and outside tip diameter of 80-100 km and a n inside tip diameter of 10-15 km. The injection and dissection needles were made of thin-walled glass capillaries containing a filament and pulled on a Narishige vertical puller to yield a n outside tip diameter of 3 km. Micromanipulation procedures were performed on a prewarmed microscope stage in a Petri dish with a rectangular 200 kl M2 drop under silicone oil. The two procedures were performed a s follows:
Oocytes Collection C3B6F1/J female mice (5-6 weeks: Jackson) were induced to superovulate by intraperitoneal injection of 5 i.u. PMSG (Sigma Chemical Co., St. Louis, MO) followed 48-50 hours later by 5 i.u. hCG (Sigma Chemical Co., St. Louis, MO). Animals were sacrificed a t 1213 hours after hCG by ether overdose. Oviducts were excised and cumulus released in M16 medium (Whittingham, 1971) with 4 mg/ml BSA and 0.1% hyaluronidase. Oocytes were then washed three times in M16. The mature oocytes were distributed between three media drops under silicon oil (Dow Corning 200 Fluid, Dow Corning Corp. Midland, MI). One drop contained M16 for in vitro fertilization (nonmanipulation group), another contained M2 medium (Hogan et al., 1986) for ZD, and the third M2 with 0.05 M sucrose (Sigma Chemical Co., St. Louis, MO) for PZD.
Zona Drilling (Fig. 1.) The technique we used was essentially similar to that described by Gordon and Talansky (1986). With the oocyte grasped by the holding pipet, a n injection needle loaded with acid Tyrode’s solution (pH 2.3) was pressed gently and tangentially against the zona pellucida releasing the acid stream. The zona was seen to flatten followed by a tiny bleb and rupture exposing the vitellus at the point of acid application. After drilling, each oocyte was moved to the far end of the drop to separate it from the nondrilled oocytes.
Micromanipulation The equipment consisted of a Nikon inverted microscope carrying Narishige micromanipulators (Medical
Modified Partial Zona Dissection (Fig. 2.) The technique used was a modification of that described by Malter and Cohen (1989). The dissecting needles were prepared as were the needles for zona drilling, but their tips were not broken and no acid was used. The dissection procedure used was as follows. With the oocytes held steady, a microneedle was inserted into the zona pellucida at a point away from the polar body. The needle was then pushed through the
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Fig. 2. Partial zona dissection. A: Introducing the needle. B: Rubbing the oocyte against the holding pipet to enlarge the holes. External diameter of needle at the point of entry is 15-20 pm. x 450.
perivitelline space avoiding the vitellus until it punctured the zona on the opposing wall. A slit between the two openings was not made as in the technique of Malter and Cohen (1989). However, the dissection needle was thrust to a point where its outside diameter was 15-20 pm in order to enlarge opening in the zona a t the entry point of the needle to the same size. The oocyte was then released from the holding pipet and carried on the dissection needle to be rubbed in several directions against the surface of the holding pipet in a n attempt to further widen the two openings in the zona.
Insemination and Embryo Development Following micromanipulation, oocytes were washed three times in MI6 medium. Each of the three groups of oocytes, i.e., nonmanipulated, ZD, and PZD, were further subdivided between the two dishes, each to be inseminated with either caput or cauda sperm suspension at a ratio of 500-1,000 sperm/egg. Insemination dishes were incubated for 4 hours after which oocytes were transferred to culture dishes containing MI6 medium with 4 mg/ml bovine serum albumin. These were incubated and examined under light microscopy at 24 hours for two-cell cleavage as presumptive evidence for fertilization. Further examination was carried out a t 48, 72, and 96 hours to look for development into blastocysts. A total of nine experiments were performed. PZD was performed in the last four experiments only. In each experiment a small number of ZD oocytes were not inseminated to note the effect of ZD alone on the activity of oocytes.
Statistical Analysis Usually a chi-square test was used to analyze data. When chi-square was not appropriate because of a
small sample size, Fisher’s exact test was used instead. The data presented in Tables 1-3 was obtained by combining results of each of nine experiments. Comparisons of interest were performed for each individual experiment. These results were not different from those obtained by doing overall analyses.
RESULTS Fertilization and development rates of nonmanipulated oocytes are shown in Table 1. Ninety-eight of three hundred oocytes (32.6%) were fertilized when inseminated with cauda sperm as compared to 13 of 116 (11.2%) oocytes exposed to caput sperm. Normal development to the blastocyst stage occurred in 47.9% of the cauda sperm 2-cell embryos, while only 1 of 13 (7.7%) caput 2-cell embryos developed into a blastocyst. Eight 2-cell embryos showed fragmentation and four did not cleave any further. The results of fertilization and development after micromanipulation of oocytes inseminated with cauda sperm are shown in Table 2. Fifty-seven ZD oocytes out of eighty-seven (65.5%) were fertilized and 75.4% of these developed into blastocysts, showing a clear advantage over the nonmanipulated group (P= 0.002). After PZD, 19 of 42 (45.2%) eggs were fertilized and 11 of 19 (57.9%) developed to blastocyst. While there appeared to be some advantage in this group over the nonmanipulated oocytes, no statistical difference was found. The results of experiments with caput epididymal sperm are detailed in Table 3. Twenty-four ZD oocytes out of one-hundred forty-four (16.7%) fertilized. This is statistically not different from the 11.2% in our nonmanipulated group. The development rates, however, between the two groups were significantly different.
ZONA DRILLING AND CAPUT EPIDIDYMAL SPERM
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epididymis has been associated with a n increased rate of embryonic loss thought to be due to delayed fertilization (Orgebin-Crist, 1967, 1968; Orgebin-Crist and Total Jahad, 1977) or abnormal activation (Brackett et al., number of Blastocysti oocytes 2-cell (%) 2-cell (%) 1978). In our experiments with nonmanipulated Cauda 300 98 (32.6) 47/98 (47.9) oocytes, a fertilization rate of 11.2% was observed with Caput 116 13 (11.2) 1/13 (7.7) caput epididymal sperm, but only 1 out of 13 two-cell embryos developed normally to the blastocyst stage. These findings are in accordance with those of the TABLE 2. Effect of Micromanipulation on Fertilization above mentioned studies. With Cauda Sperm When zona drilling was performed on oocytes to be inseminated with caput sperm, the fertilization rate Total did not significantly improve. However a remarkable number of Blastocvstsi Procedure oocytes 2-ce11 (%I 2-ce11?%) improvement was seen in the blastocyst development No manipulation 300 98 (32.6) 47/98 (47.9)".b rate (50%vs. 7.7%). A plausable explanation for this Zona drilling 87 57 (65.5) 43/57 (75.3)" phenomenon is the fact that ZD allows for earlier Partial z o n i sperm egg fusion and thus fertilization and pronuclear dissection 42 19 (45.2) 11/19 (57.9)' formation (Talansky and Gordon, 1988). Early fertili"P = 0.002 (significant). zation may be a n important factor in decreasing the bP= 0.526 (not significant). rate of embryonic loss. A further explanation of the advantage of ZD is that eliminating the zona barrier and exposing the oolemma could provide a selective TABLE 3. Effect of Micromanipulation on Fertilization advantage for fertilization to the very few caput sperWith Caput Sperm matozoa that may be functionally mature. In this Total study, zona drilling also showed a significant enhancenumber of Blastocyst/ ment of fertilization in the oocyte group inseminated 2-cell (%) Procedure oocytes 2-cell (%) with Cauda sperm. This confirms prior reports of the No manipulation 116 13 (11.2) 1/13 ( 7 . 7 ) " ~ ~efficacy of ZD in assisting in vitro fertilization (Gordon Zona drilling 144 24 (16.7) 12/24 (50)" and Talansky, 1986; Talansky and Gordon, 1988; Partial zona Conover and Gwatkin, 1988; Depypere et al., 1988). dissection 23 l(4.3) Oil ( O P From this study, our modification of PZD does not "P = 0.0129 (significant). appear to provide the same advantage as ZD. With the bP= 1.000 (not significant). oocytes inseminated with cauda sperm only slightly better fertilization rates were observed, but they were not statistically significant. With caput sperm, no benFifty percent (12/24) with ZD as opposed to 7.7% (1/13) eficial effect of PZD was observed. The difference in in the nonmanipulated group formed blastocysts outcome of ZD as opposed to PZD may be due to the (P=O.O129). Of the 12 two-cell embryos that did not nature of the opening created. Zona drilling by acid reach the blastocyst stage, 3 cleaved to the 4-cell stage, results in a much wider hole than PZD (personal ob4 failed to cleave beyond the 2-cell stage, and 5 showed servation, Ng et al., 1990). ZD also exposes the egg early fragmentation. In the PZD group only one out of plasma surface to the surrounding milieu at the site of 23 oocytes showed a 2-cell embryo with no further pro- the opening (Gordon and Talansky, 1986). PZD does gression. Of the 30 oocytes that were zona drilled, but not show this feature, since the holes tend to reseal and not inseminated, only one showed evidence of parthevitellus is shrunken by sucrose. nogenetic activation resulting in a 2-cell stage with fragmentation of both blastomeres. CONCLUSIONS TABLE 1. In Vitro Fertilization by Cauda and Caput Epididymal Sperm
DISCUSSION In this study, we evaluated the impact of zona drilling and a modification of partial zona dissection on the fertilization of mouse oocytes using caput and cauda epididymal sperm. Saling (1982) showed that mouse caput sperm bind very poorly to the zona pellucida and even to the oolemma in zona free oocytes. In the hamster, caput epididymal sperm nuclei injected into eggs were not able to develop to pronuclei within the time period studied (Uehara and Yanagimachi, 1976). In the rabbit, fertilization with immature sperm from the
Our results suggest that ZD using acid Tyrode's solution enhances in vitro fertilization by mouse caput epididymal spermatozoa which are immature in their majority. This enhancement occurs in the form of improved preimplantation embryonic development. The mechanism by which ZD effects this improvement is probably related to allowing earlier gamete fusion and fertilization. The relevance of these findings to clinical application is in cases of continued male infertility after proximal vasoepididymostomies or direct sperm aspiration from the caput epididymis. Acid ZD in humans, however, has been reported to be associated with
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an increased rate of oocyte damage (Ng et al., 1989), polyspermy (Gordon et al., 19881, and abnormal cleavage patterns (Talansky and Gordon, 1988). It is yet to be determined whether other mechanical micromanipulation techniques, e.g., microinsemination of sperm into the perivitelline space (see Ng et al., 19901, enhance human fertilization and development with immature epididymal sperm as ZD did with the mouse in our study.
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Malter HE, Cohen J (1989): Partial zona dissection of the human oocyte : A non traumatic method using micromanipulation to assist zona pellucida penetration. Fertil Steril 51:139-148. McLaughlin JD, Shur BD (1987):Binding of caput epididymal mouse sperm to the zona pellucida. Dev Biol 124:557-561. Moore HDM, Hartman TD (1986): In vitro development of fertilizing ability of hamster epididymal spermatozoa after co-culture with epithelium from the proximal cauda epididymis. J Reprod Fertil 78:347-352. Moore HDM, Hartman TD, Smith CA (1986):In vitro culture of hamster epididymal epithelium and induction of sperm motility. J Reprod Fertil 78:327-336. Ng SC, Bongso A, Chang SI, Sathananthan H, Ratnam SS (1989): Transfer of human sperm into the perivitelline space of human oocytes after zona drilling or zona puncture. Fertil Steril 52:73-78. Ng SC, Bongso A, %thananthan H, Ratnam SS (1990):Micromanipulation: Its relevance to human in vitro fertilization. Fertil Steril 53:203-219’ Orgebin-Crist MC (1967): Maturation of spermatozoa in the rabbit epididymis. Fertilizing ability and embryonic mortality in does inseminated with epididymal spermatozoa. Ann Biol Anim Biochem Biophys 7:372-389. Orgebin-Crist MC (1968): Maturation of spermatozoa in the rabbit epididymis: Delayed fertilization in doses inseminated with epididymal spermatozoa‘ Reprod Fertil 16:29-40’ Orgebin-Crist MC, Jahad N (1977): Delayed cleavage of rabbit ova after fertilization by Young ePididYmal spermatozoa. Bid Reprod ~ ~ ~ i ~ ~ 16:358-362. Orgebin-Crist MC, Fournier-Delpech S (1982): Sperm-egg interaction. Evidence for maturational changes during epididymal transit. J Androl 3:429-433. Saling PM (1982): Development of the ability to bind to zonae pellucidae. Reversible immobilization of mouse spermatozoa by lanthanum. Biol Reprod 26:429-436, Schoysman RG, Bedford JM (1986):The role of human epididymis in sperm maturation and sperm storage as reflected in the consequences of epididymovasostomy~Fertil Steril 46:293-299, Silber sJ (1989): Role of epididymis in sperm maturation, Urology 33:47-51. Silber SJ, Ord T, Balmaceda J, patrizio p, Asch RH (1989): congenital absence ofthe vas deferens: Studies on the fertilizing ability of human epididymal sperm [abstract]. Read at the 45th annual meeting of the American Fertility Society, San Francisco, CA. Talansky BE, Gordon JW (1988): Cleavage characteristics of mouse embryos inseminated and cultured after zona pellucida drilling. Gamete Res 21:277-287. Uehara T, Yanagimachi R (1977): Behavior of nuclei of testicular, caput and cauda epididymal spermatozoa injected into hamster eggs. Biol Reprod 16:315-321. Whittingham DG (1971): Culture of mouse ova. J Reprod Fertil [Suppll 14:7-21.
