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Spermatozoa After Sperm Residence in the Female Reproductive Tract E

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Spermatozoa After Sperm Residence in the Female Reproductive Tract E Detection of altered acrosomal physiology of cryopreserved human spermatozoa after sperm residence in the female reproductive tract E. Z. Drobnis, P. R. Clisham, C. K. Brazil L. W. Wisner, C. Q. Zhong and J. W. Overstreet ^Division of Reproductive Biology and Medicine, Department of Obstetrics and Gynecology, School of Medicine, and department of Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616-8659, USA At least some of the spermatozoa that remain motile following cryopreservation have sustained sublethal damage that reduces their functional capacity in vivo. Although it is believed that acrosomal damage is partly responsible for impaired sperm function in vivo, direct evidence for this hypothesis is lacking because spermatozoa have not been collected from the female reproductive tract for evaluation. In the study reported here, cervical mucus was collected from women 24 h after artificial insemination by cervical cup. For both cryopreserved and nonfrozen inseminates, spermatozoa within the cervical mucus and spermatozoa that migrated out of mucus into culture medium (t = 1 h) were viable and had intact acrosomes. However, although nonfrozen spermatozoa did not initially respond to induction of the acrosome reaction with follicular fluid, a significant proportion of cryopre- served spermatozoa did respond. These results demonstrate that cryopreservation increases the acrosomal lability of spermatozoa residing in the female reproductive tract. An in vitro test was developed to detect this form of cryodamage. Sperm-free mucus was collected before insemination and spermatozoa from the inseminate were allowed to swim into this column of mucus in vitro. Spermatozoa recovered from this mucus sample were compared with spermatozoa from the paired sample collected from the cervix 24 h later. This in vitro test could detect acrosomal lability in cryopreserved semen samples, and this approach may prove valuable for studying sublethal cryodamage to the acrosome. Introduction impaired in some species (Mattner et al, 1969; Lightfoot and Salamon 1970; Lineweaver et al, 1970; Pursei et al, 1978; Extensive research on cryopreservation has been carried out Saacke, 1982); (3) longevity or retention of cryopreserved the reduced with spermatozoa from many species over the last forty years. spermatozoa in female reproductive tract is Nevertheless, conception rates following artificial insemination (Mattner et al, 1969; Pursel et al, 1978; Saacke, 1982; Parrish and with cryopreserved spermatozoa are lower than those obtained Foote, 1986); and (4) conception rates achieved with can those using nonfrozen spermatozoa, even when equal numbers of cryopreserved spermatozoa approach for nonfrozen motile spermatozoa are inseminated. The lower fertility of spermatozoa if insemination is more carefully timed with cryopreserved spermatozoa suggests that those that survive respect to ovulation (Smith et al, 1981; Parrish and Foote, 1986) freezing and remain motile after thawing have impaired func¬ or spermatozoa are inseminated closer to the oviduct (Lightfoot tional competence in vivo. It is possible that all of the spermatozoa and Salamon, 1970; Byrd et al, 1990). have reduced function, or that the size of the functional The sublethal cryodamage that results in abnormal sperm population is reduced. In general, it is agreed that at least part function has proved difficult to detect in vitro. The lack of of this impaired function is due to abnormal sperm transport in laboratory methodology for detecting sperm cryodamage has the female reproductive tract, including decreased longevity been an impediment to studies of sperm cryobiology. Without of spermatozoa following insemination (Saacke, 1982; Hawk, good experimental endpoints, it is difficult to determine the 1987). Several lines of evidence support this hypothesis (1) mechanisms underlying diminished sperm function, and it is longevity during incubation in vitro is reduced for cryopre¬ difficult to develop improved techniques of sperm cryopreser¬ served spermatozoa (Saacke and White, 1972; Keel and Black, vation. Endpoints such as embryonic development and rate of 1980; Critser et al, 1987b); (2) transport to the oviduct of cell division that are useful for studying cryobiology in other motile, cryopreserved spermatozoa has been shown to be cellular systems are not applicable to spermatozoa. Assessment of viability alone is useful for monitoring cryodamage in many ^Correspondence. cells, but this approach cannot detect the sublethal changes Received ó November 1992. that can disrupt the highly complex function of mammalian Downloaded from Bioscientifica.com at 09/25/2021 08:23:20PM via free access spermatozoa. At the present time, the only effective measure¬ Materials and Methods ment of sperm function in vivo is the conception rate following insemination (Amann, 1989). With the exception of dairy cattle, Preparation of cryopreserved and fresh semen for insemination this approach cannot be used for initial testing of cryopreser¬ vation methods. In vitro assays of sperm function are required The six research subjects who provided semen for this study for preliminary studies to identify methods for testing in clinical were donors in the therapeutic artificial insemination pro¬ trials. gramme. After approval was obtained from the University of Cryopreserved spermatozoa have been shown to differ California, Davis Institutional Review Board, informed consent from nonfrozen spermatozoa by several laboratory criteria was obtained to use these samples for this research. Each donor are human (references examples for spermatozoa), including was screened to exclude genetic and health problems as the ultrastructure (Woolley and Richardson, 1978), percent¬ recommended by the American Fertility Society (1990), and of motile age spermatozoa (Critser et al, 1987b), swimming they were further screened for good semen quality and sperm velocity (Pilikian et al, 1982), metabolism (Ackerman and cryosurvival. Cryopreserved semen from these donors was Behrman, 1975), percentage of intact acrosomes (Pilikian and proven to be fertile in the artificial insemination programme. Critser Guerin, 1986; et al, 1987a; Centola et al, 1990), acro¬ The cryopreservative diluent was modified from the TEST- somal enzymes (Mack and Zaneveld, 1987), acrosomal func¬ yolk medium of Bolanos et al (1983). All reagents used were tion (Cross and Hanks, 1991), longevity (Critser et al, obtained from Sigma Chemical Company (St Louis, MO). The of cervical mucus 1987b), penetration (Ulstein, 1973), binding buffer solution contained 188.7 mmol Tes I , 84.8 mmol tris to the zona pellucida (Coddington et al, 1991), and fusion base I-1, 11.1 mmol glucose 1~\ 15 iu penicillin G ml-1 with zona-free hamster oocytes (Critser et al, 1987b). How¬ (sodium salt), and 25 iu streptomycin sulfate 1 in HPLC- ever, the relationship of these measures to sperm function grade water. Complete diluent was prepared by combining 80% the in female reproductive tract is not well understood. In (v/v) buffer solution and 20% fresh egg yolk. The yolk granules human semen, distinct subpopulations of spermatozoa can be were sedimented by centrifuging the diluent at 1000 # for distinguished on the basis of physiological characteristics 30 min. Approximately the upper two-thirds of the supernatant Davis (e.g. and Katz, 1988; Robertson et al, 1988; Mortimer solution was collected, and the pH was adjusted to 7.40 with and Camenzind, 1989; Menkveld et al, 1991). Measurements 1.0 mol NaOH l-1. After filtering the diluent through a sterile, that mean provide values for the entire population of 0.45 pm pore-size filter, 10 ml aliquots of diluent were frozen in spermatozoa in semen may not detect differences in the sub- 15 ml centrifuge tubes (Coming Glass Works, NY) and stored at populations of spermatozoa that are biologically important. 20°C. The diluent was thawed and warmed above 20°C — Assays that do not distinguish between motile and immotile before use. spermatozoa may be particularly insensitive for detecting Semen was collected by masturbation and was allowed to changes in spermatozoa that are functionally significant. liquify at room temperature. After liquefaction, the volume In vitro tests for sperm cryodamage must be carefully chosen was measured and the concentration and percentage of motile to approximate conditions encountered by the fertilizing spermatozoa in the semen were determined. For semen to be spermatozoon in vivo. The standard assays of sperm lon¬ cryopreserved, the total number of motile spermatozoa in the an increased of gevity may detect sensitivity cryopreserved ejaculate was calculated. Sperm 'freezability' was defined as the spermatozoa to conditions in vitro (e.g. pH, toxic components ratio of the percentage of motile spermatozoa after thawing to of seminal plasma), but these conditions may have little the percentage of motile spermatozoa in the fresh ejaculate. For relevance to the environment of the female reproductive each donor, the mean sperm freezability for the three previous tract. ejaculates was used to estimate motility loss during cryopreser¬ In the we experiments reported here, have studied the vation, and the semen was diluted to produce a predicted after motility and acrosomal status of cryopreserved human sperma¬ thawing insemination dose of 30 10a motile spermatozoa in tozoa following a period of residence in the female reproductive 0.5 ml. If the motile sperm density was inadequate to dilute at Such
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