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Sperm Capacitation in Humans Is Transientand Correlates With Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11039-11043, November 1995 Physiology Sperm capacitation in humans is transient and correlates with chemotactic responsiveness to follicular factors (chemotaxis/fertilization/mammalian reproduction/acrosome reaction/sperm selection) ANAT COHEN_DAYAG*, ILAN TUR-KASPAt, JEHOSHUA DORt, SHLOMO MASHIACHt, AND MICHAEL EISENBACH*t *Department of Membrane Research and Biophysics, Weizmann Institute of Science, 76100 Rehovot, Israel; and tDepartment of Obstetrics and Gynecology, Sheba Medical Center, Tel Aviv University Medical School, 52621 Tel Hashomer, Israel Communicated by Julius Adler, University of Wisconsin, Madison, WI, August 11, 1995 ABSTRACT In humans, only a small fraction (2-12%) of reviews, see refs. 8-13)-seemed a reasonable possibility (3, a sperm population can respond by chemoattraction to fol- 7). This study investigates this possibility and finds a linkage licular factors. This recent finding led to the hypothesis that between sperm capacitation and chemotaxis. chemotaxis provides a mechanism for selective recruitment of functionally mature spermatozoa (i.e., of capacitated sperma- tozoa, which possess the potential to undergo the acrosome MATERIALS AND METHODS reaction and fertilize the egg). This study aimed to examine Spermatozoa. Human ejaculates were collected by mastur- this possibility. Capacitated spermatozoa were identified by bation from normal healthy donors and washed, as described their ability to undergo the acrosome reaction upon stimula- (2), with Biggers, Whitten, and Whittingham medium (14) tion with phorbol 12-myristate 13-acetate. Under capacitating supplemented with Hepes (1 mM, pH 7.4) (this solution is conditions, only a small portion (2-14%) of the spermatozoa hereafter referred to as BWW). To promote capacitation, the were found to be capacitated. The spermatozoa were then spermatozoa were resuspended in BWW to a concentration of separated according to their chemotactic activity, which re- 108 cells per ml and incubated for 2 h (unless indicated sulted in a subpopulation enriched with chemotactically re- otherwise) at 35°C or room temperature (there was no statis- sponsive spermatozoa and a subpopulation depleted of such tically significant difference in the percentage of capacitated spermatozoa. The level of capacitated spermatozoa in the spermatozoa between the two temperatures). former was -13-fold higher than that in the latter. The Sperm Separation. Separation of spermatozoa according to capacitated state was temporary (50 min < life span < 240 their chemotactic activity was carried out by a separation min), and it was synchronous with the chemotactic activity. A apparatus as described earlier (7). Briefly, the apparatus continuous process of replacement of capacitated/chemotac- consisted of two chambers connected by a tube: one chamber tic spermatozoa within a sperm population was observed. contained spermatozoa and the other contained an attractant. Spermatozoa that had stopped being capacitated did not The active fraction of follicular fluid which remains after become capacitated again, which indicates that the capaci- precipitation by 90% acetone (2), diluted 1:1000, was used as tated state is acquired only once in a sperm's lifetime. A total an attractant. The apparatus was incubated for 1 h ("the sperm population depleted of capacitated spermatozoa stopped separation time") during which net movement of spermatozoa being chemotactic. When capacitated spermatozoa reappeared, to the attractant-containing chamber was observed. As shown chemotactic activity was restored. These observations suggest earlier (7), the population of spermatozoa that migrated to the that spermatozoa acquire their chemotactic responsiveness as attractant-containing chamber was enriched with chemotactic part of the capacitation process and lose this responsiveness spermatozoa; the population of spermatozoa that remained in when the capacitated state is terminated. We suggest that the role the original chamber and did not follow the attractant was ofsperm chemotaxis in sperm-egg interaction in vivo may indeed depleted of chemotactic spermatozoa. The sperm suspensions be selective recruitment of capacitated spermatozoa for fertiliz- from both chambers were collected and washed twice by ing the egg. centrifugation (120 x g, 15 min). The original population was similarly washed as a control. Human spermatozoa are attracted to follicular factors in vitro, Chemotaxis Assays. The responsiveness of spermatozoa to and the attraction is correlated with egg fertilizability (1). The the attractant was determined, as described (2), in a sealed attraction results from chemotaxis and is accompanied by chamber (15) in which the spermatozoa choose between a well speed enhancement (ref. 2; for reviews, see refs. 3 and 4). containing the attractant and a well containing BWW. The Unlike the case of species with external fertilization in which sperm concentration used in the chemotaxis assays was 1-5 x most, if not all, of the sperm population is chemotactically 108 cells per ml. responsive (for reviews, see refs. 5 and 6), in humans only a Determination ofthe Fraction of Capacitated Spermatozoa. small fraction of the sperm population (2-12%) is chemotac- Capacitated spermatozoa were identified according to their tically responsive at any given time (7). The identity of the ability to undergo the acrosome reaction. In practice, we responsive spermatozoa in humans changes with time by determined the number of capacitated spermatozoa from the turnover: chemotactic spermatozoa lose their activity while difference between the number of acrosome-reacted cells others acquire it (7). This raised the possibility that sperma- before and after stimulation with phorbol 12-myristate 13- tozoa are selectively chemotactic only at a certain physiological acetate (PMA; Sigma), which is known to induce the acrosome stage. The capacitated stage-i.e., the stage at which sperma- reaction only in capacitated spermatozoa (16, 17). For stim- tozoa possess the potential to undergo the acrosome reaction ulation, spermatozoa (108 cells per ml) were incubated with (a release of proteolytic enzymes enabling sperm penetration PMA (5 ,uM from a stock solution in dimethyl sulfoxide; the through the egg coat) and to fertilize the egg (for recent Abbreviations: BWW, Biggers, Whitten and Whittingham medium The publication costs of this article were defrayed in part by page charge supplemented with Hepes; PKC, protein kinase C; PMA, phorbol payment. This article must therefore be hereby marked "advertisement" in 12-myristate 13-acetate. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 11039 Downloaded by guest on October 1, 2021 11040 Physiology: Cohen-Dayag et al. Proc. Natl. Acad. Sci. USA 92 (1995) final concentration of dimethyl sulfoxide in the sperm samples by collecting the spermatozoa that migrated to an attractant- did not exceed 0.5%) for 30 min at 35°C. Acrosome-reacted containing chamber. As a control for random separation, the spermatozoa were identified by the acrosomal marker fluo- separation procedure was repeated with BWW in place of the rescein isothiocyanate-conjugated Pisum sativum agglutinin attractant. As an additional control, we studied a total sperm (Sigma), using a modification of previously reported staining population that had been incubated with (but not separated methods (18-20). Briefly, spermatozoa (45 Al of 108 cells per by) the attractant. The level of capacitated spermatozoa in the ml) were washed (twice at 120 x g for 10 min at room chemotactically enriched subpopulation was 13 ± 4 (mean + temperature) with BWW lacking glucose and bovine serum SD of the enriched to depleted ratio of five experiments) times albumin, cooled on ice for 10 min, and then permeabilized by higher than that in the remaining, chemotactically depleted 95% methanol for 30 s. The acrosomal marker, conjugated P. subpopulation (Fig. 1A). In contrast, the percentages of sativum agglutinin (5 ,A of 1 mg/ml), was added, and the capacitated spermatozoa were similar in the subpopulations mixture was incubated on ice for an additional 30 min, followed and in the total population when the separation had been by washing with the same medium and fixation with 2% carried out substituting BWW for the attractant (Fig. 1B). The (vol/vol) formaldehyde for 30 min on ice. An aliquot of the incubation with the attractant did not affect the percentage of sperm suspension was smeared on a microscope slide, air capacitated spermatozoa (Fig. 1C), indicating that the in- dried, and then covered with a mounting fluid (Mowiol, creased percentage of capacitated spermatozoa in the chemo- Hoechst) and a cover glass. The slides were inspected using a tactically enriched subpopulation in Fig. 1A ("Passed" bar) is Zeiss Axiovert 35 microscope equipped with a x 100 oil the result of chemotactic attraction and not the result of the objective. Two fluorescence patterns were observed: a uniform incubation with the attractant during the separation. These fluorescence over the whole acrosomal region and fluores- results indicate that sperm separation by chemotaxis also cence restricted to the equatorial acrosomal region, indicative results in separation according to capacitation. Similar to the of acrosome-intact and acrosome-reacted spermatozoa, re- level of chemotactic cells in a total sperm population (7), the spectively (19, 20). Cells were inspected by an independent level of capacitated
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