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Cryopreservation of bull spermatozoa alters the perinuclear theca
Theriogenology 66 (2006) 1969–1975 www.journals.elsevierhealth.com/periodicals/the
Cryopreservation of bull spermatozoa alters the perinuclear theca Carmen Omega Martı´nez a, Marı´a de Lourdes Jua´rez-Mosqueda a,*, Jorge Herna´ndez a, Javier Valencia b a
Departamento de Morfologı´a, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Auto´noma de Me´xico, CP 04510 Me´xico DF, Mexico b Departamento de Reproduccio´n, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Auto´noma de Me´xico, CP 04510 Me´xico DF, Mexico Received 9 May 2005; accepted 25 October 2005
Abstract The perinuclear theca (PT) is involved in several important sperm functions leading to fertilization. The objective of this study was to investigate the effect of cryopreservation of bull spermatozoa on the integrity of the PT and the relationship between PT integrity and semen characteristics. Semen from seven bulls was evaluated before and after cryopreservation, comparing the integrity of the plasma membrane (hypo-osmotic test), percentage of live and dead spermatozoa (triple stain), acrosome integrity (triple stain) and the integrity of the PT (negative stain by electron microscopy). Cryopreservation of bull semen caused substantial damage to the PT; the proportion of spermatozoa with a damaged PT was 15.2% versus 52.5% (P < 0.05) in fresh versus frozenthawed spermatozoa, respectively. Furthermore, on average, 67.4% (range, 64–72%) of fresh spermatozoa were live, compared to 53.1% (range, 49–58%) for frozen-thawed spermatozoa; there was an inverse correlation between the percentage of live spermatozoa and the percentage with damage to the PT. Although 59.1% of frozen-thawed spermatozoa had an intact acrosome, only 43.7% of them still remained alive. In frozen-thawed semen, there was a high correlation (r = 0.69) between live spermatozoa with an intact acrosome and spermatozoa that maintained an intact PT. In conclusion, freezing/thawing of bull spermatozoa altered the PT and maintaining PT integrity may be necessary to maintain acrosome integrity. # 2006 Elsevier Inc. All rights reserved. Keywords: Perinuclear theca; Sperm cryopreservation; Semen thawing; Bull spermatozoa; Sperm viability
1. Introduction Although cryopreservation has been applied as a routine technique for processing bull spermatozoa for artificial insemination (AI), from 40 to 50% of spermatozoa do not survive the freezing-thawing processes, even when the most successful techniques * Corresponding author. Tel.: +52 55 5 622 58 93; fax: +52 55 5 622 59 02. E-mail address: [email protected] (M.d.L. Jua´rez-Mosqueda). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.10.028
are used [1]. However, fundamental biological properties that determine survival following cryopreservation have been largely ignored [2]. Therefore, although fertility with frozen-thawed bull semen is generally acceptable, the efficiency of semen cryopreservation is still relatively low [3]. Freezing and thawing damages living cells; membranes are particularly vulnerable during the cryopreservation process. The sperm membranes that are affected by cryopreservation include the plasma membrane and the outer acrosomal membrane [4–6]. Some studies have suggested the importance of the cytoskeleton for the
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support of the plasma and acrosomal membranes, and it is known that cytoskeletal elements are temperaturesensitive. In fact, cooling other cell types results in premature depolymerization of microfilaments (F-actin) [7]. Moreover, a recent study with boar spermatozoa has shown that the microfilament network appears to be important for volume regulation [8]. The perinuclear theca (PT) is the major cytoskeletal element of the head of bovine spermatozoa [9]. The PT is a rigid capsule that covers the nucleus of mammalian spermatozoa and it is believed to have a membranebinding role [10,11]. This extranuclear structure has been subdivided into two structurally continuous regions: apically the subacrosomal layer and caudally the postacrosomal sheath. Continuity between the PT and the nuclear matrix has been reported [12,13]. In addition, the postacrosomal sheath is a putative site for actin in a number of mammalian spermatozoa [14,15]. Conversely, Jua´rez-Mosqueda and Mu´jica [16] described that F-actin was implicated in the stabilization of the substructure of the apical region of the PT postacrosomal sheath of the guinea pig spermatozoa, as the PT-substructure disappears when it is treated with cytochalasin D (which disrupts F-actin filaments). A similar PT substructure was reported in bull, rabbit, pig, and sheep spermatozoa [16– 18]. Therefore, it would be worthwhile to examine the morphology of the PT-substructure to detect cryopreservation-induced damage. Routine techniques currently used to assess the viability of semen for AI are inadequate of detecting spermatozoa with reduced fertilization ability, especially when this is due to conditions involving the cytoskeletal elements of the sperm head [19,20]. To our knowledge, there are no published data regarding alteration of the PT of cryopreserved mammalian spermatozoa. The objective of the present study was to compare, using electron microscopy, PT morphology in fresh and frozen-thawed bull spermatozoa. We also evaluated the relationship between the morphological findings and the results of traditional laboratory techniques used to evaluate sperm viability (i.e. motility, viability, plasma membrane integrity, and acrosome integrity). 2. Materials and methods Unless otherwise indicated, reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.1. Semen collection and initial evaluation Semen samples were obtained from seven bulls from a semen processing center in Queretaro, Mexico. One
ejaculate was obtained from each bull using an artificial vagina. Each sample was divided in two fractions; one was processed for freezing and the other was immediately evaluated for sperm concentration, percentage of motile spermatozoa, and motility rate. Semen concentration was determined using a spectrophotometer (Espectronic 20, Bausch-Lomb, Rochester, NY, USA) after 10 mL of fresh semen was diluted in 5.8 mL of 0.9% NaCl. A small drop of semen was placed on a glass slide prewarmed to 37 8C and covered with a coverslide [21]. Progressive motility of spermatozoa and motility rate were assessed subjectively at 200 magnification, with a phase-contrast microscope (Zeiss, Oberkochen, Germany) with a heated stage (37 8C). Progressive motility was expressed as the percentage of motile spermatozoa (in increments of 5%), and motility rate (forward progression) was scored using a scale of 0 (lowest) to 3 (highest). All ejaculates fulfilled the minimum standard of progressive motility (70%). For the estimation of sperm morphology, a drop of semen was fixed with an equal volume of 2% glutaraldehyde in phosphatebuffered saline (PBS) [22], and smears were prepared on glass slides and examined (1000) under a light microscope. A total of 100 spermatozoa were examined to determine the percentages of normal spermatozoa and spermatozoa with primary or secondary defects. Primary defects were those likely to appear during spermatogenesis (e.g. acrosome defects, head alteration in shape or contour, proximal droplets, strongly folded tails), whereas secondary defects were those that occur after sperm release and during semen processing (e.g. free normal heads, distal droplets, simple bent tails) [23]. 2.2. Semen freezing and thawing The traditional two-step procedure was used to process samples. The semen was extended with an ultrapasteurized skim milk-based extender, supplemented with 0.25 g/L gentamicin, 0.05 g/L tylosin, 0.15 g/L lincomycin, and 0.3 g/L spectinomycin. The ejaculates were diluted to 200 106 cells/mL at 36 8C and cooled to 8 8C (over 2 h) in a water bath. Then the tubes were placed in a beaker with water at 5 8C for 15 min. Upon reaching 5 8C, the samples were further diluted by adding extender (14% glycerol) (v:v) at 5 8C every 10 min, to achieve a final glycerol concentration of 7% and 45 106 spermatozoa/0.5 mL. Thereafter, the diluted semen was equilibrated at 5 8C for a further 2 h period. After equilibration the semen was packaged in 0.5 mL French straws (Minitu¨b, Tiefenbach, Germany).
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The straws were cooled from +5 to 15 8C at a rate of 8 8C/min, and from 15 to 120 8C at 20 8C/min, using a controlled programmable freezer (PS4-2, Union Carbide, Indianapolis, IN, USA). The frozen straws were stored in liquid nitrogen at 196 8C and stored for at least 2 weeks. For thawing, straws were placed in a water bath at 35 8C for 30 s and motility was immediately determined. 2.3. Semen quality parameters Aliquots of the fresh and frozen-thawed semen were assessed. Two frozen straws per bull were evaluated. A microscope (Leica, model DMLS, Wetzlar, Germany) equipped with a camera (JVC, Model KY-F70B; Victor Co., Yokohama, Japan) and Automontage software (Synchroscopy, Frederick, MD, USA) were used to examine the samples by light microscopy. All assessments of semen characteristics were conducted by the same experienced individual. 2.3.1. Assessment of membrane integrity Sperm membrane integrity was assessed using the hypo-osmotic swelling (HOS) test [21]. Two semen samples from each ejaculate were used for the HOS test. Semen (100 mL) was added to 1 mL of a hypo-osmotic solution (100 mOsm/kg H2O) prepared with 75 mM fructose and 25 mM sodium citrate in distilled water. After incubation for 60 min at 37 8C, sperm swelling was assessed by placing 15 mL of a well-mixed sample on a warm slide (37 8C), which was covered with a cover glass before being observed under light microscopy at 400 magnification. One hundred spermatozoa per slide were assessed and the percentage of cells with curled tails (swelling/intact plasma membrane) was calculated. 2.3.2. Assessment of sperm viability and acrosome integrity Assessment of live and dead spermatozoa and proportion of acrosome-reacted spermatozoa was according with the triple stain protocol described by Talbot and Chacon [24]. Briefly, a 100 mL aliquot of sperm suspension was placed into a 2-mL test tube, and an equal amount of 2% Trypan blue dissolved in PBS was added. The test tubes were then kept in a water bath at 37 8C for 15 min, before centrifugation (280 g for 3 min). The supernatant was removed and the sperm pellet was resuspended in 2 mL distilled water and centrifuged again. After three centrifugations, a clear or pale blue suspension was obtained. Then the samples were centrifuged again and the pelleted spermatozoa
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were fixed in 1 mL of 3% glutraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 30 min at 4 8C. Afterward, the spermatozoa were washed once in distilled water with centrifugation (280 g for 3 min). The pellets were resuspended in PBS; 10–20 mL of this suspension was spread evenly on a glass slide on a surface of approximately 3 cm2 and air-dried. Slides were then exposed to 8% Bismark brown solution in distilled water (pH 1.8) for 15 min at 37 8C. The glass slides were rinsed with water and stained with 0.8% rose Bengal solution in 0.1 M Tris–HCl buffer (pH 5.3) for 1 min at room temperature. The glass slides were rinsed with water and examined (1000) under a light microscope. A total of 100 spermatozoa per slide were evaluated to estimate sperm viability and acrosome integrity. Rose stain over the acrosomal region and blue stain over the postacrosomal region were observed in acrosome-intact dead spermatozoa; no stain over the acrosomal region and blue stain over the postacrosomal region were observed in acrosome-reacted dead spermatozoa. Rose stain over the acrosomal region and brown stain over the postacrosomal region was observed in acrosome-intact live spermatozoa; no stain over the acrosomal region and brown stain over the postacrosomal region were observed in acrosomereacted live spermatozoa. 2.3.3. Assessment of PT integrity Both fresh and frozen-thawed semen samples from each ejaculate were processed for examination of the PT morphology by negative staining electron microscopy [25]. To expose the PT surface, the plasma membrane and the acrosome were solubilized with the nonionic detergent Brij 36-T (Canamex, Nuevo Leo´n, Mexico). Spermatozoa were washed in saline solution (154 mM) and adjusted to a concentration of 35 106 cells/mL. Then, Brij 36-T (1.2% final concentration) was added, and the spermatozoa were incubated for 5 min at room temperature, fixed in Karnowsky fixative for 20 min at room temperature. A drop of the sperm suspension was placed on collodion-carbon coated grids. Samples were stained with an aqueous 0.02% phosphotungstic acid (Merck, Darmstadt, Germany) and examined on a Zeiss EM-9 transmission electron microscope (Oberkochen, Germany) at 50 KV. For estimation of PT morphology, 50 cells were assessed and classified. The characteristic used to assess the PT was the morphological appearance of the substructure above the postacrosomal sheath (Fig. 1). Samples were classified as: intact or normal when the substructure appeared as a continuous row of papillae (Fig. 1B); altered when the substructure had some damage, such as the lack of some papilla and/or
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Fig. 1. Electron micrographs showing PT morphology of pre-freeze and post-thaw bull spermatozoa. Spermatozoa were treated with the nonionic detergent Brij 36-T that removes the whole plasma membrane, the nuclear membrane, and the acrosome, while the PT remains around the nucleus: (A) whole-mount preparation showing a negatively stained head of a non-Brij-treated bull sperm. The plasma membrane and acrosome (Ac) have a smooth appearance while the PT-substructure is not visible because the plasma membrane remains covering it. The inset shows a high-magnification view. (B–D) are showing whole-mount spermatozoa heads with its PT exposed (Brij-treated); (B) over the PT, the substructure is apparent (arrow) between the subacrosmal layer (SL) and the postacrosomal layer (PL) of the PT; it consists of a row of papillae circling the sperm head. Lower inset shows a high-magnification view of the substructure. Note that the papillae, which characterize the anterior margin of the postacrosomal layer, remain intact. Upper inset shows a thin section of Brij-treated spermatozoa. A shell, the perinuclear theca (*), is surrounding the condensed sperm nucleus (N); (C) whole-mount postthaw sperm head showing a damaged substructure. Note some discontinuities in the substructure (arrow) and the lack of some papillae; and (D) whole-mount post-thaw sperm head with the substructure absent (arrow).
continuity disruptions (Fig. 1C); and absent substructure, when the cells did not have this substructure (Fig. 1D). For thin sections, spermatozoa were fixed in Karnowsky fixative for 1 h. After postfixation with 1% (w/v) OsO4 in PBS buffer (pH 7.4) for 1 h, the cells were dehydrated through a graded ethanol series, embedded in Spurr’s resin, thin-sectioned, and double-
stained with uranyl acetate and lead nitrate prior to transmission electron microscopy. 2.4. Statistical analysis The values from fresh and frozen-thawed samples were compared with a Paired-Sample Sign test [26]. Pearson’s correlations were evaluated with the SAS
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computer software package (SAS Institute, Cary, NC, USA). 3. Results Freezing/thawing reduced (P < 0.05) the percentage of motile, membrane intact, live, and acrosome-intact spermatozoa. Moreover, after freezing/thawing, the proportions of spermatozoa with disrupted or absent PT substructure increased 39.7 and 7.8%, respectively (P < 0.05; Table 1). There were no significant correlations among the proportion of spermatozoa with normal PT substructure and other sperm viability tests in fresh semen, but the proportion of HOST positive spermatozoa had a high correlation with the proportion of live/ acrosome reacted spermatozoa (r = 0.93; P < 0.05). In frozen-thawed semen, the proportion of spermatozoa with normal PT substructure were correlated with the proportion of live/intact acrosome spermatozoa (r = 0.69; P < 0.05). The proportion of spermatozoa with absent PT substructure was negatively correlated with the proportion of live spermatozoa and live/ acrosome-intact spermatozoa (r = 0.76 and 0.81, respectively; P < 0.05) and tended to be correlated with the proportion of live/acrosome-reacted spermatozoa (r = 0.69; P < 0.08). 4. Discussion Improved understanding of the causes of the cryoinjury should improve the efficiency of semen preservation [1]. In the past years, a series of functional assays has been developed to determine the structural morphology and functional integrity of the plasma Table 1 Mean (S.D.) end points for fresh and frozen/thawed bull semen
Motility (%) Motility rate (0–3) HOS positive (%) Live/intact acrosomec (%) Live/acrosome reactedc (%) Dead/intact acrosomec (%) Dead/acrosome reactedc (%) PT substructure normal (%) PT substructure altered (%) PT substructure absent (%)
Fresh semen
Frozen/thawed semen
70 0.85a 2.5 0.4 67.6 0.6a 63.1 1.0a 4.3 0.7a 27.5 0.9a 5 1.0a 84.8 1.6a 12.0 0.9a 3.1 1.1a
55 1.0 b 54.3 2.5 b 43.7 1.4 b 9.4 0.3 b 15.4 1.1 b 31.4 0.9 b 47.4 2.6 b 41.7 1.8 b 10.8 3.4 b
HOS, hypoosmotic swelling test; PT, perinuclear theca. a Within a row, means with different superscripts differ (P < 0.05). b Within a row, means with different superscripts differ (P < 0.05). c Determined with trypan blue, Bismark brown, and rose Bengal.
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membrane and sperm acrosomal membrane [19]. However, the relationship between these parameters and fertility has not been clearly established [27]. This study demonstrated that the PT of bull spermatozoa is damaged by the cryopreservation process, supporting the proposal that the decrease in the viability of cryopreserved semen can be attributed not only to a primary damage to the plasma membrane [3,28,29], but also to damage to other internal elements, e.g. cytoskeleton [7]. The PT is a unique cytoskeletal, extranuclear structural element in the sperm head that is believed to have a membrane-binding role [9,10]. The present study showed for the first time, that the PT substructure of bull spermatozoa could be used as a morphological marker to assess PT integrity. It has been shown that the substructure is a stabilizing element of the PT [6]. In our study, alterations of the PT-substructure could be detected in every semen sample, regardless of fresh or frozen-thawed status. This finding was compatible with the general belief that certain low-frequency sperm defects will be present in any semen sample [30,31]. However, the frequency of PT defects was increased by freezing and thawing; the nature of these defects ranged from small alterations to a complete lack of the PTsubstructure. It was noteworthy that F-actin has been implicated in the PT-substructure stabilization of guinea pig spermatozoa [15]. In bull spermatozoa, actin has been observed in the anterior two-thirds of the postacrosomal region [32]. We infer that freezing and thawing may result in changes in the microfilament cytoskeleton (of which the PT-substructure base zone is composed), resulting in partial or complete loss of the substructure. It has already been reported that disruption of microfilaments by cytochalasin D leads to substructure loss in guinea pig spermatozoa [16]. Of importance is the recognition that cooling of the cells results in premature depolymerization of actin filaments [33,34]. Moreover, it has been reported that some PT components can be extracted by simple freeze/thawing of hamster spermatozoa [35]. It has been reported that acrosome integrity rather than sperm motility has a significant effect on in vitro fertilization rates when cryopreserved spermatozoa were evaluated [19,36]. In the present study, although a high percentage of spermatozoa (59.1 1.3%) had an intact acrosome after thawing, only 43.7 1.4% of them were alive; the latter percentage was similar to the percentage of spermatozoa that had their PT intact (47.4 2.6). Longo et al. [9] have suggested that PTproteins may contribute to the nucleus-acrosome association by providing some sort of intermembranous cement. Furthermore, failure in PT differentiation has
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been associated with acrosome-deficient rounded-head spermatozoa [37]. Although the correlation between frozen-thawed spermatozoa that conserved a normal PT and live spermatozoa with an intact acrosome was not significant, this study does not exclude the idea that if a spermatozoa conserves its PT intact, it could also keep the acrosome intact. To assess this relationship, it would be necessary to increase the number of samples studied. Conversely, there was a correlation between the number of spermatozoa with absent PT substructure and the number of live spermatozoa with an acrosomal reaction; these data were consistent with the observations of guinea pig spermatozoa [16], where a loss of the substructure was detected in spermatozoa that undergo an acrosomal reaction. In the present study, triple staining was used to classify live and dead spermatozoa with and without an acrosome. Some researchers point out that classic supravital stains are not appropriate for predicting the viability of thawed spermatozoa, because the presence of glycerol interferes with the differential stain between live and dead cells [38]. The results of this study do not agree with that view since the use of triple staining allowed identification of spermatozoa with and without an acrosome, and to distinguish between those that were alive or dead. A great disadvantage of the technique is the time that is required for assessing the sample. Based on the correlation between live spermatozoa with an intact acrosome and normal PT, this technique could be useful for assessing sperm quality. Finally, we do not discard the idea that damage to the PT could affect other sperm physiological functions. In recent years several authors have reported the participation of the PT in other important functions, such as the maintenance and formation of the functional plasma membrane domains of the sperm head, the stability of the nucleus, and the activation of the egg [39–41]. Collectively, we inferred that damage to the PT integrity has a major role in the decreased viability of frozenthawed semen. In conclusion, we demonstrated for the first time that the PT of bovine spermatozoa was damaged during cryopreservation; as the percentage of spermatozoa with an absent PT increased, the number of live spermatozoa decreased. We inferred that damage to the PT could contribute to the reduced viability of frozenthawed semen. Acknowledgements This work was supported by a grant from UNAM (PAPIIT IN-206702). We thank Dr. E. Camors and Dr.
W. Remberg (Semen Processing Center RECA, Queretaro, Mexico) for providing the bull semen and Dra. Ma. Elena Rese´ndiz for technical assistance. Thanks are due to the staff of the Unidad de Microscopia Electro´nica de la Facultad de Medicina Veterinaria y Zootecnia (FMVZ), de la Universidad Nacional Auto´noma de Me´xico (UNAM). We also thank Dr. Luis Zarco for correcting the English version of the manuscript. References [1] Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their postthawing function. Reprod Fertil Dev 1995;7:871–91. [2] Woods ER, Benson JD, Agca Y, Critser JK. Fundamental cryobiology of reproductive cells and tissues. Cryobiology 2004;48:146–56. [3] Holt WV. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 2000;53:47–58. [4] Leeuw F, Ching-Chen H, Colenbrander B, Verkleji A. Cold induced ultrastructural changes in bull and boar sperm plasma membranes. Cryobiology 1990;27:171–83. [5] Petrunkina AM, Gro¨pper B, To¨pfer-Petersen E, Gu¨nzel-Apel AR. Volume regulatory function and sperm membrane dynamics as parameters for evaluating cryoprotective efficiency of a freezing extender. Theriogenology 2005;63:1390–406. [6] Mc Ganny L, Yand H, Walterson M. Manifestations of cell damage after freezing and thawing. Cryobiology 1998;25:178– 85. [7] Watson PF. The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci 2000;60–61:481–92. [8] Petrunkina AM, Hebel M, Waberski D, Weitze KF, To¨pferPetersen E. Requirement for an intact cytoskeleton for volume regulation in boar spermatozoa. Reproduction 2004;127:105–15. [9] Longo FJ, Krohne G, Franke WW. Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids. A novel class of cytoskeletal elements. J Cell Biol 1987;105:1105– 20. [10] Korley R, Pouresmaeili F, Oko R. Analysis of the protein composition of the mouse sperm perinuclear theca and characterization of its major protein constituent. Biol Reprod 1997; 57:1426–32. [11] Courtens JL, Courot M, Flechon J. The perinuclear substance of boar, bull, ram and rabbit spermatozoa. J Ulrastruct Res 1976;57:54–64. [12] Oko R, Clermont Y. Origin and distribution of perforatorial proteins during spermatogenesis of the rat: an immunocytochemical study. Anat Rec 1991;230:489–501. [13] Bellve´ AR, Chandrika R, Martinova YS, Barth AH. The perinuclear matrix as a structural element of the mouse sperm nucleus. Biol Reprod 1992;47:451–65. [14] Yagy A, Paranko J. Actin, a-actin, and spectrin with specific associations with the postacrosomal and acrosomal domains of bovine spermatozoa. Anat Rec 1995;241:77–87. [15] Mu´jica A, Navarro-Garcı´a F, Herna´ndez-Gonza´lez EO, Jua´rez-Mosqueda ML. Perinuclear theca during spermatozoa maturation leading to fertilization. Microsc Res Tech 2003; 61:76–87.
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Cryopreservation of bull spermatozoa alters the perinuclear theca Carmen Omega Martı´nez a, Marı´a de Lourdes Jua´rez-Mosqueda a,*, Jorge Herna´ndez a, Javier Valencia b a
Departamento de Morfologı´a, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Auto´noma de Me´xico, CP 04510 Me´xico DF, Mexico b Departamento de Reproduccio´n, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Auto´noma de Me´xico, CP 04510 Me´xico DF, Mexico Received 9 May 2005; accepted 25 October 2005
Abstract The perinuclear theca (PT) is involved in several important sperm functions leading to fertilization. The objective of this study was to investigate the effect of cryopreservation of bull spermatozoa on the integrity of the PT and the relationship between PT integrity and semen characteristics. Semen from seven bulls was evaluated before and after cryopreservation, comparing the integrity of the plasma membrane (hypo-osmotic test), percentage of live and dead spermatozoa (triple stain), acrosome integrity (triple stain) and the integrity of the PT (negative stain by electron microscopy). Cryopreservation of bull semen caused substantial damage to the PT; the proportion of spermatozoa with a damaged PT was 15.2% versus 52.5% (P < 0.05) in fresh versus frozenthawed spermatozoa, respectively. Furthermore, on average, 67.4% (range, 64–72%) of fresh spermatozoa were live, compared to 53.1% (range, 49–58%) for frozen-thawed spermatozoa; there was an inverse correlation between the percentage of live spermatozoa and the percentage with damage to the PT. Although 59.1% of frozen-thawed spermatozoa had an intact acrosome, only 43.7% of them still remained alive. In frozen-thawed semen, there was a high correlation (r = 0.69) between live spermatozoa with an intact acrosome and spermatozoa that maintained an intact PT. In conclusion, freezing/thawing of bull spermatozoa altered the PT and maintaining PT integrity may be necessary to maintain acrosome integrity. # 2006 Elsevier Inc. All rights reserved. Keywords: Perinuclear theca; Sperm cryopreservation; Semen thawing; Bull spermatozoa; Sperm viability
1. Introduction Although cryopreservation has been applied as a routine technique for processing bull spermatozoa for artificial insemination (AI), from 40 to 50% of spermatozoa do not survive the freezing-thawing processes, even when the most successful techniques * Corresponding author. Tel.: +52 55 5 622 58 93; fax: +52 55 5 622 59 02. E-mail address: [email protected] (M.d.L. Jua´rez-Mosqueda). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.10.028
are used [1]. However, fundamental biological properties that determine survival following cryopreservation have been largely ignored [2]. Therefore, although fertility with frozen-thawed bull semen is generally acceptable, the efficiency of semen cryopreservation is still relatively low [3]. Freezing and thawing damages living cells; membranes are particularly vulnerable during the cryopreservation process. The sperm membranes that are affected by cryopreservation include the plasma membrane and the outer acrosomal membrane [4–6]. Some studies have suggested the importance of the cytoskeleton for the
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support of the plasma and acrosomal membranes, and it is known that cytoskeletal elements are temperaturesensitive. In fact, cooling other cell types results in premature depolymerization of microfilaments (F-actin) [7]. Moreover, a recent study with boar spermatozoa has shown that the microfilament network appears to be important for volume regulation [8]. The perinuclear theca (PT) is the major cytoskeletal element of the head of bovine spermatozoa [9]. The PT is a rigid capsule that covers the nucleus of mammalian spermatozoa and it is believed to have a membranebinding role [10,11]. This extranuclear structure has been subdivided into two structurally continuous regions: apically the subacrosomal layer and caudally the postacrosomal sheath. Continuity between the PT and the nuclear matrix has been reported [12,13]. In addition, the postacrosomal sheath is a putative site for actin in a number of mammalian spermatozoa [14,15]. Conversely, Jua´rez-Mosqueda and Mu´jica [16] described that F-actin was implicated in the stabilization of the substructure of the apical region of the PT postacrosomal sheath of the guinea pig spermatozoa, as the PT-substructure disappears when it is treated with cytochalasin D (which disrupts F-actin filaments). A similar PT substructure was reported in bull, rabbit, pig, and sheep spermatozoa [16– 18]. Therefore, it would be worthwhile to examine the morphology of the PT-substructure to detect cryopreservation-induced damage. Routine techniques currently used to assess the viability of semen for AI are inadequate of detecting spermatozoa with reduced fertilization ability, especially when this is due to conditions involving the cytoskeletal elements of the sperm head [19,20]. To our knowledge, there are no published data regarding alteration of the PT of cryopreserved mammalian spermatozoa. The objective of the present study was to compare, using electron microscopy, PT morphology in fresh and frozen-thawed bull spermatozoa. We also evaluated the relationship between the morphological findings and the results of traditional laboratory techniques used to evaluate sperm viability (i.e. motility, viability, plasma membrane integrity, and acrosome integrity). 2. Materials and methods Unless otherwise indicated, reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.1. Semen collection and initial evaluation Semen samples were obtained from seven bulls from a semen processing center in Queretaro, Mexico. One
ejaculate was obtained from each bull using an artificial vagina. Each sample was divided in two fractions; one was processed for freezing and the other was immediately evaluated for sperm concentration, percentage of motile spermatozoa, and motility rate. Semen concentration was determined using a spectrophotometer (Espectronic 20, Bausch-Lomb, Rochester, NY, USA) after 10 mL of fresh semen was diluted in 5.8 mL of 0.9% NaCl. A small drop of semen was placed on a glass slide prewarmed to 37 8C and covered with a coverslide [21]. Progressive motility of spermatozoa and motility rate were assessed subjectively at 200 magnification, with a phase-contrast microscope (Zeiss, Oberkochen, Germany) with a heated stage (37 8C). Progressive motility was expressed as the percentage of motile spermatozoa (in increments of 5%), and motility rate (forward progression) was scored using a scale of 0 (lowest) to 3 (highest). All ejaculates fulfilled the minimum standard of progressive motility (70%). For the estimation of sperm morphology, a drop of semen was fixed with an equal volume of 2% glutaraldehyde in phosphatebuffered saline (PBS) [22], and smears were prepared on glass slides and examined (1000) under a light microscope. A total of 100 spermatozoa were examined to determine the percentages of normal spermatozoa and spermatozoa with primary or secondary defects. Primary defects were those likely to appear during spermatogenesis (e.g. acrosome defects, head alteration in shape or contour, proximal droplets, strongly folded tails), whereas secondary defects were those that occur after sperm release and during semen processing (e.g. free normal heads, distal droplets, simple bent tails) [23]. 2.2. Semen freezing and thawing The traditional two-step procedure was used to process samples. The semen was extended with an ultrapasteurized skim milk-based extender, supplemented with 0.25 g/L gentamicin, 0.05 g/L tylosin, 0.15 g/L lincomycin, and 0.3 g/L spectinomycin. The ejaculates were diluted to 200 106 cells/mL at 36 8C and cooled to 8 8C (over 2 h) in a water bath. Then the tubes were placed in a beaker with water at 5 8C for 15 min. Upon reaching 5 8C, the samples were further diluted by adding extender (14% glycerol) (v:v) at 5 8C every 10 min, to achieve a final glycerol concentration of 7% and 45 106 spermatozoa/0.5 mL. Thereafter, the diluted semen was equilibrated at 5 8C for a further 2 h period. After equilibration the semen was packaged in 0.5 mL French straws (Minitu¨b, Tiefenbach, Germany).
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The straws were cooled from +5 to 15 8C at a rate of 8 8C/min, and from 15 to 120 8C at 20 8C/min, using a controlled programmable freezer (PS4-2, Union Carbide, Indianapolis, IN, USA). The frozen straws were stored in liquid nitrogen at 196 8C and stored for at least 2 weeks. For thawing, straws were placed in a water bath at 35 8C for 30 s and motility was immediately determined. 2.3. Semen quality parameters Aliquots of the fresh and frozen-thawed semen were assessed. Two frozen straws per bull were evaluated. A microscope (Leica, model DMLS, Wetzlar, Germany) equipped with a camera (JVC, Model KY-F70B; Victor Co., Yokohama, Japan) and Automontage software (Synchroscopy, Frederick, MD, USA) were used to examine the samples by light microscopy. All assessments of semen characteristics were conducted by the same experienced individual. 2.3.1. Assessment of membrane integrity Sperm membrane integrity was assessed using the hypo-osmotic swelling (HOS) test [21]. Two semen samples from each ejaculate were used for the HOS test. Semen (100 mL) was added to 1 mL of a hypo-osmotic solution (100 mOsm/kg H2O) prepared with 75 mM fructose and 25 mM sodium citrate in distilled water. After incubation for 60 min at 37 8C, sperm swelling was assessed by placing 15 mL of a well-mixed sample on a warm slide (37 8C), which was covered with a cover glass before being observed under light microscopy at 400 magnification. One hundred spermatozoa per slide were assessed and the percentage of cells with curled tails (swelling/intact plasma membrane) was calculated. 2.3.2. Assessment of sperm viability and acrosome integrity Assessment of live and dead spermatozoa and proportion of acrosome-reacted spermatozoa was according with the triple stain protocol described by Talbot and Chacon [24]. Briefly, a 100 mL aliquot of sperm suspension was placed into a 2-mL test tube, and an equal amount of 2% Trypan blue dissolved in PBS was added. The test tubes were then kept in a water bath at 37 8C for 15 min, before centrifugation (280 g for 3 min). The supernatant was removed and the sperm pellet was resuspended in 2 mL distilled water and centrifuged again. After three centrifugations, a clear or pale blue suspension was obtained. Then the samples were centrifuged again and the pelleted spermatozoa
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were fixed in 1 mL of 3% glutraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 30 min at 4 8C. Afterward, the spermatozoa were washed once in distilled water with centrifugation (280 g for 3 min). The pellets were resuspended in PBS; 10–20 mL of this suspension was spread evenly on a glass slide on a surface of approximately 3 cm2 and air-dried. Slides were then exposed to 8% Bismark brown solution in distilled water (pH 1.8) for 15 min at 37 8C. The glass slides were rinsed with water and stained with 0.8% rose Bengal solution in 0.1 M Tris–HCl buffer (pH 5.3) for 1 min at room temperature. The glass slides were rinsed with water and examined (1000) under a light microscope. A total of 100 spermatozoa per slide were evaluated to estimate sperm viability and acrosome integrity. Rose stain over the acrosomal region and blue stain over the postacrosomal region were observed in acrosome-intact dead spermatozoa; no stain over the acrosomal region and blue stain over the postacrosomal region were observed in acrosome-reacted dead spermatozoa. Rose stain over the acrosomal region and brown stain over the postacrosomal region was observed in acrosome-intact live spermatozoa; no stain over the acrosomal region and brown stain over the postacrosomal region were observed in acrosomereacted live spermatozoa. 2.3.3. Assessment of PT integrity Both fresh and frozen-thawed semen samples from each ejaculate were processed for examination of the PT morphology by negative staining electron microscopy [25]. To expose the PT surface, the plasma membrane and the acrosome were solubilized with the nonionic detergent Brij 36-T (Canamex, Nuevo Leo´n, Mexico). Spermatozoa were washed in saline solution (154 mM) and adjusted to a concentration of 35 106 cells/mL. Then, Brij 36-T (1.2% final concentration) was added, and the spermatozoa were incubated for 5 min at room temperature, fixed in Karnowsky fixative for 20 min at room temperature. A drop of the sperm suspension was placed on collodion-carbon coated grids. Samples were stained with an aqueous 0.02% phosphotungstic acid (Merck, Darmstadt, Germany) and examined on a Zeiss EM-9 transmission electron microscope (Oberkochen, Germany) at 50 KV. For estimation of PT morphology, 50 cells were assessed and classified. The characteristic used to assess the PT was the morphological appearance of the substructure above the postacrosomal sheath (Fig. 1). Samples were classified as: intact or normal when the substructure appeared as a continuous row of papillae (Fig. 1B); altered when the substructure had some damage, such as the lack of some papilla and/or
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Fig. 1. Electron micrographs showing PT morphology of pre-freeze and post-thaw bull spermatozoa. Spermatozoa were treated with the nonionic detergent Brij 36-T that removes the whole plasma membrane, the nuclear membrane, and the acrosome, while the PT remains around the nucleus: (A) whole-mount preparation showing a negatively stained head of a non-Brij-treated bull sperm. The plasma membrane and acrosome (Ac) have a smooth appearance while the PT-substructure is not visible because the plasma membrane remains covering it. The inset shows a high-magnification view. (B–D) are showing whole-mount spermatozoa heads with its PT exposed (Brij-treated); (B) over the PT, the substructure is apparent (arrow) between the subacrosmal layer (SL) and the postacrosomal layer (PL) of the PT; it consists of a row of papillae circling the sperm head. Lower inset shows a high-magnification view of the substructure. Note that the papillae, which characterize the anterior margin of the postacrosomal layer, remain intact. Upper inset shows a thin section of Brij-treated spermatozoa. A shell, the perinuclear theca (*), is surrounding the condensed sperm nucleus (N); (C) whole-mount postthaw sperm head showing a damaged substructure. Note some discontinuities in the substructure (arrow) and the lack of some papillae; and (D) whole-mount post-thaw sperm head with the substructure absent (arrow).
continuity disruptions (Fig. 1C); and absent substructure, when the cells did not have this substructure (Fig. 1D). For thin sections, spermatozoa were fixed in Karnowsky fixative for 1 h. After postfixation with 1% (w/v) OsO4 in PBS buffer (pH 7.4) for 1 h, the cells were dehydrated through a graded ethanol series, embedded in Spurr’s resin, thin-sectioned, and double-
stained with uranyl acetate and lead nitrate prior to transmission electron microscopy. 2.4. Statistical analysis The values from fresh and frozen-thawed samples were compared with a Paired-Sample Sign test [26]. Pearson’s correlations were evaluated with the SAS
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computer software package (SAS Institute, Cary, NC, USA). 3. Results Freezing/thawing reduced (P < 0.05) the percentage of motile, membrane intact, live, and acrosome-intact spermatozoa. Moreover, after freezing/thawing, the proportions of spermatozoa with disrupted or absent PT substructure increased 39.7 and 7.8%, respectively (P < 0.05; Table 1). There were no significant correlations among the proportion of spermatozoa with normal PT substructure and other sperm viability tests in fresh semen, but the proportion of HOST positive spermatozoa had a high correlation with the proportion of live/ acrosome reacted spermatozoa (r = 0.93; P < 0.05). In frozen-thawed semen, the proportion of spermatozoa with normal PT substructure were correlated with the proportion of live/intact acrosome spermatozoa (r = 0.69; P < 0.05). The proportion of spermatozoa with absent PT substructure was negatively correlated with the proportion of live spermatozoa and live/ acrosome-intact spermatozoa (r = 0.76 and 0.81, respectively; P < 0.05) and tended to be correlated with the proportion of live/acrosome-reacted spermatozoa (r = 0.69; P < 0.08). 4. Discussion Improved understanding of the causes of the cryoinjury should improve the efficiency of semen preservation [1]. In the past years, a series of functional assays has been developed to determine the structural morphology and functional integrity of the plasma Table 1 Mean (S.D.) end points for fresh and frozen/thawed bull semen
Motility (%) Motility rate (0–3) HOS positive (%) Live/intact acrosomec (%) Live/acrosome reactedc (%) Dead/intact acrosomec (%) Dead/acrosome reactedc (%) PT substructure normal (%) PT substructure altered (%) PT substructure absent (%)
Fresh semen
Frozen/thawed semen
70 0.85a 2.5 0.4 67.6 0.6a 63.1 1.0a 4.3 0.7a 27.5 0.9a 5 1.0a 84.8 1.6a 12.0 0.9a 3.1 1.1a
55 1.0 b 54.3 2.5 b 43.7 1.4 b 9.4 0.3 b 15.4 1.1 b 31.4 0.9 b 47.4 2.6 b 41.7 1.8 b 10.8 3.4 b
HOS, hypoosmotic swelling test; PT, perinuclear theca. a Within a row, means with different superscripts differ (P < 0.05). b Within a row, means with different superscripts differ (P < 0.05). c Determined with trypan blue, Bismark brown, and rose Bengal.
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membrane and sperm acrosomal membrane [19]. However, the relationship between these parameters and fertility has not been clearly established [27]. This study demonstrated that the PT of bull spermatozoa is damaged by the cryopreservation process, supporting the proposal that the decrease in the viability of cryopreserved semen can be attributed not only to a primary damage to the plasma membrane [3,28,29], but also to damage to other internal elements, e.g. cytoskeleton [7]. The PT is a unique cytoskeletal, extranuclear structural element in the sperm head that is believed to have a membrane-binding role [9,10]. The present study showed for the first time, that the PT substructure of bull spermatozoa could be used as a morphological marker to assess PT integrity. It has been shown that the substructure is a stabilizing element of the PT [6]. In our study, alterations of the PT-substructure could be detected in every semen sample, regardless of fresh or frozen-thawed status. This finding was compatible with the general belief that certain low-frequency sperm defects will be present in any semen sample [30,31]. However, the frequency of PT defects was increased by freezing and thawing; the nature of these defects ranged from small alterations to a complete lack of the PTsubstructure. It was noteworthy that F-actin has been implicated in the PT-substructure stabilization of guinea pig spermatozoa [15]. In bull spermatozoa, actin has been observed in the anterior two-thirds of the postacrosomal region [32]. We infer that freezing and thawing may result in changes in the microfilament cytoskeleton (of which the PT-substructure base zone is composed), resulting in partial or complete loss of the substructure. It has already been reported that disruption of microfilaments by cytochalasin D leads to substructure loss in guinea pig spermatozoa [16]. Of importance is the recognition that cooling of the cells results in premature depolymerization of actin filaments [33,34]. Moreover, it has been reported that some PT components can be extracted by simple freeze/thawing of hamster spermatozoa [35]. It has been reported that acrosome integrity rather than sperm motility has a significant effect on in vitro fertilization rates when cryopreserved spermatozoa were evaluated [19,36]. In the present study, although a high percentage of spermatozoa (59.1 1.3%) had an intact acrosome after thawing, only 43.7 1.4% of them were alive; the latter percentage was similar to the percentage of spermatozoa that had their PT intact (47.4 2.6). Longo et al. [9] have suggested that PTproteins may contribute to the nucleus-acrosome association by providing some sort of intermembranous cement. Furthermore, failure in PT differentiation has
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been associated with acrosome-deficient rounded-head spermatozoa [37]. Although the correlation between frozen-thawed spermatozoa that conserved a normal PT and live spermatozoa with an intact acrosome was not significant, this study does not exclude the idea that if a spermatozoa conserves its PT intact, it could also keep the acrosome intact. To assess this relationship, it would be necessary to increase the number of samples studied. Conversely, there was a correlation between the number of spermatozoa with absent PT substructure and the number of live spermatozoa with an acrosomal reaction; these data were consistent with the observations of guinea pig spermatozoa [16], where a loss of the substructure was detected in spermatozoa that undergo an acrosomal reaction. In the present study, triple staining was used to classify live and dead spermatozoa with and without an acrosome. Some researchers point out that classic supravital stains are not appropriate for predicting the viability of thawed spermatozoa, because the presence of glycerol interferes with the differential stain between live and dead cells [38]. The results of this study do not agree with that view since the use of triple staining allowed identification of spermatozoa with and without an acrosome, and to distinguish between those that were alive or dead. A great disadvantage of the technique is the time that is required for assessing the sample. Based on the correlation between live spermatozoa with an intact acrosome and normal PT, this technique could be useful for assessing sperm quality. Finally, we do not discard the idea that damage to the PT could affect other sperm physiological functions. In recent years several authors have reported the participation of the PT in other important functions, such as the maintenance and formation of the functional plasma membrane domains of the sperm head, the stability of the nucleus, and the activation of the egg [39–41]. Collectively, we inferred that damage to the PT integrity has a major role in the decreased viability of frozenthawed semen. In conclusion, we demonstrated for the first time that the PT of bovine spermatozoa was damaged during cryopreservation; as the percentage of spermatozoa with an absent PT increased, the number of live spermatozoa decreased. We inferred that damage to the PT could contribute to the reduced viability of frozenthawed semen. Acknowledgements This work was supported by a grant from UNAM (PAPIIT IN-206702). We thank Dr. E. Camors and Dr.
W. Remberg (Semen Processing Center RECA, Queretaro, Mexico) for providing the bull semen and Dra. Ma. Elena Rese´ndiz for technical assistance. Thanks are due to the staff of the Unidad de Microscopia Electro´nica de la Facultad de Medicina Veterinaria y Zootecnia (FMVZ), de la Universidad Nacional Auto´noma de Me´xico (UNAM). We also thank Dr. Luis Zarco for correcting the English version of the manuscript. References [1] Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their postthawing function. Reprod Fertil Dev 1995;7:871–91. [2] Woods ER, Benson JD, Agca Y, Critser JK. Fundamental cryobiology of reproductive cells and tissues. Cryobiology 2004;48:146–56. [3] Holt WV. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 2000;53:47–58. [4] Leeuw F, Ching-Chen H, Colenbrander B, Verkleji A. Cold induced ultrastructural changes in bull and boar sperm plasma membranes. Cryobiology 1990;27:171–83. [5] Petrunkina AM, Gro¨pper B, To¨pfer-Petersen E, Gu¨nzel-Apel AR. Volume regulatory function and sperm membrane dynamics as parameters for evaluating cryoprotective efficiency of a freezing extender. Theriogenology 2005;63:1390–406. [6] Mc Ganny L, Yand H, Walterson M. Manifestations of cell damage after freezing and thawing. Cryobiology 1998;25:178– 85. [7] Watson PF. The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci 2000;60–61:481–92. [8] Petrunkina AM, Hebel M, Waberski D, Weitze KF, To¨pferPetersen E. Requirement for an intact cytoskeleton for volume regulation in boar spermatozoa. Reproduction 2004;127:105–15. [9] Longo FJ, Krohne G, Franke WW. Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids. A novel class of cytoskeletal elements. J Cell Biol 1987;105:1105– 20. [10] Korley R, Pouresmaeili F, Oko R. Analysis of the protein composition of the mouse sperm perinuclear theca and characterization of its major protein constituent. Biol Reprod 1997; 57:1426–32. [11] Courtens JL, Courot M, Flechon J. The perinuclear substance of boar, bull, ram and rabbit spermatozoa. J Ulrastruct Res 1976;57:54–64. [12] Oko R, Clermont Y. Origin and distribution of perforatorial proteins during spermatogenesis of the rat: an immunocytochemical study. Anat Rec 1991;230:489–501. [13] Bellve´ AR, Chandrika R, Martinova YS, Barth AH. The perinuclear matrix as a structural element of the mouse sperm nucleus. Biol Reprod 1992;47:451–65. [14] Yagy A, Paranko J. Actin, a-actin, and spectrin with specific associations with the postacrosomal and acrosomal domains of bovine spermatozoa. Anat Rec 1995;241:77–87. [15] Mu´jica A, Navarro-Garcı´a F, Herna´ndez-Gonza´lez EO, Jua´rez-Mosqueda ML. Perinuclear theca during spermatozoa maturation leading to fertilization. Microsc Res Tech 2003; 61:76–87.
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