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H. Rodriguez-Martinez, S. Einarsson and B. Larsson

Summary. Intraluminal pressure microtransducers were placed at the uterotubal junction, the proximal isthmus, the ampullary—isthmic junction and the mid-ampulla. Spontaneous motility occurred throughout the oestrous cycle in all segments. During oestrus there were regular, high amplitude peristaltic waves in all segments, superimposed on basal activity. On Day 1 of the cycle the pattern was mostly antiperistaltic, presumably related to sperm transport. During the periovulatory period the number of peristaltic and antiperistaltic waves became equal, perhaps in relation to the transport of gametes to the fertilization site. During Day 3 there was no peristaltic activity; the motility patterns of the isthmus and ampullary—isthmic junction were similar (regular phasic contractions of high frequency and amplitude) while the ampullary motility was low. On Day 4, when the eggs enter the uterine lumen, the ampullary—isthmic junction and particularly the isthmus showed strong contraction waves (mostly peristaltic) superimposed on the basal phasic activity. This suggests an active role of the smooth muscle of the lower oviducal segments in ovum descent. During the mid- and late-luteal phases, the isthmus remained motile, with an irregular base line, but lost the pattern of basal contractions that dominated the activity during the first 4 days of the cycle. The ampulla showed low levels of spontaneous motility throughout the rest of the cycle.

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H Funahashi, H Ekwall, K Kikuchi and H Rodriguez-Martinez

The aim of this study was to determine the ultrastructure of cross-sectioned zonae pellucidae of in vitro-matured and ovulated pig oocytes before or after sperm penetration in vitro and in vivo, respectively. The in vitro and in vivo (ovulated) oocytes and zygotes (fertilized in vitro and in vivo) were fixed with glutaraldehyde either directly or after pretreatment with ruthenium red and saponin, processed and then examined using transmission electron microscopy. The thickness of the zona pellucida, as measured on the section of the specimens with largest diameter fixed with glutaraldehyde, differed between the in vivo (9.19 +/- 0.47 microm) and in vitro (5.95 +/- 0.51 microm) oocytes. The in vivo oocytes had a rather thick external mesh-like structure, whereas it was much thinner in the in vitro oocytes. This mesh-like external rim was less apparent in both in vivo and in vitro zygotes. Obvious differences in the density of the lattice formed by the fixed zonae pellucidae were visible between the outer and inner (ad-oolemmal) zonae. The outer area always formed a concentrically arrayed fibrillar network, whereas the inner area showed a much more compact, trabecule-like mesh. However, both areas, but particularly the outer network, were much more compacted after the zona reaction. Clear differences in the degree of fibrillar aggregation of the inner zona area were also observed between in vitro and in vivo zygotes, being much higher in the latter. This fibrillar network was more clearly visible in the zygotes pretreated with ruthenium red and saponin; the in vitro zygotes had a fibrillar, radially oriented set of parallel fibrils, whereas it was much more aggregated and trabecule-like in the in vivo zygotes. These results demonstrate that the fine structure of the zona pellucida and the zona reaction at sperm penetration differ between pig oocytes fertilized in vivo and in vitro. Moreover, the ultrastructure of the outer and inner pig zonae pellucidae has a different network organization.

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H. Rodriguez-Martinez, J. L. Courtens, U. Kvist and L. Plöen

Summary. Protamine was specifically demonstrated in boar spermatozoa collected from the rete testis, caput, corpus and cauda epididymidis and the ejaculate by immunoelectron microscopy, using anti-boar or anti-ram protamine antisera and an indirect post-embedding immunogold technique. Spermatozoa from all collection sites stained after incubation although with different degrees of labelling. Controls were negative. Labelling increased from the rete testis towards the epididymal corpus, where it was most intense, decreasing sharply thereafter. The weakest binding of the assayed antibodies was obtained in the ejaculated spermatozoa but it could be reversed by in-vitro induction of chromatin decondensation with sodium dodecyl sulphate and the metal-chelating EDTA. The finding of a significant decrease in the immunolabelling detected from the corpus epididymidis onwards indicates a critical point for the interaction between DNA and the protamines in boar spermatozoa during the epididymal maturation.

Keywords: spermatozoa; epididymis; protamine; immunocytochemistry; electron microscopy; boar

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B Strom Holst, B Larsson, C Linde-Forsberg and H Rodriguez-Martinez

Sperm binding to the zona pellucida is a prerequisite for fertilization, and tests that evaluate this function have been described for several species. When carrying out such tests in the canine species, ovaries or oocytes have to be stored to obtain a sufficient number of oocytes at the time of testing. In the present study, the sperm binding capacities of salt-stored oocytes and oocytes from deep frozen ovaries were measured and compared with that of fresh oocytes. Two different procedures for washing the sperm-oocyte complexes (gentle and tough) were used before evaluating the number of bound spermatozoa. The total number of oocytes that bound spermatozoa was significantly lower for both salt-stored and deep frozen oocytes compared with fresh oocytes. Significantly fewer spermatozoa bound to stored oocytes than to fresh oocytes (P </= 0.05) irrespective of washing procedure and there was no significant difference between the two methods of storage. Scanning electron microscopy revealed that the zona pellucida of fresh oocytes was sponge-like in appearance with fenestrations surrounded by a meshwork of filaments. In stored oocytes, the meshwork tended to be wider, with larger fenestrations. The thickness of the zona pellucida was 0.3-1.0 microm in salt-stored oocytes and 6-8 microm in oocytes from frozen ovaries, whereas it was 3-6 microm in fresh oocytes. Changes in morphology that occur under the various storage conditions tested might have contributed to the reduced sperm binding capacity of the stored oocytes. Although stored canine oocytes do not bind as many spermatozoa as do fresh oocytes, the former can be used in a zona pellucida binding assay, and the deep freezing of the ovaries appears to be a better storage method than storing oocytes in salt solution.

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B Strom Holst, B Larsson, C Linde-Forsberg and H Rodriguez-Martinez

Sperm binding to the zona pellucida is a prerequisite for fertilization, and tests that evaluate this function have been described for several species. When carrying out such tests in the canine species, ovaries or oocytes have to be stored to obtain a sufficient number of oocytes at the time of testing. In the present study, the sperm binding capacities of salt-stored oocytes and oocytes from deep frozen ovaries were measured and compared with that of fresh oocytes. Two different procedures for washing the sperm-oocyte complexes (gentle and tough) were used before evaluating the number of bound spermatozoa. The total number of oocytes that bound spermatozoa was significantly lower for both salt-stored and deep frozen oocytes compared with fresh oocytes. Significantly fewer spermatozoa bound to stored oocytes than to fresh oocytes (P </= 0.05) irrespective of washing procedure and there was no significant difference between the two methods of storage. Scanning electron microscopy revealed that the zona pellucida of fresh oocytes was sponge-like in appearance with fenestrations surrounded by a meshwork of filaments. In stored oocytes, the meshwork tended to be wider, with larger fenestrations. The thickness of the zona pellucida was 0.3-1.0 microm in salt-stored oocytes and 6-8 microm in oocytes from frozen ovaries, whereas it was 3-6 microm in fresh oocytes. Changes in morphology that occur under the various storage conditions tested might have contributed to the reduced sperm binding capacity of the stored oocytes. Although stored canine oocytes do not bind as many spermatozoa as do fresh oocytes, the former can be used in a zona pellucida binding assay, and the deep freezing of the ovaries appears to be a better storage method than storing oocytes in salt solution.

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B Strom Holst, B Larsson, C Linde-Forsberg and H Rodriguez-Martinez

Zona pellucida binding assays provide information about the fertilizing ability of spermatozoa. A zona-binding assay for canine spermatozoa using intact, denuded homologous oocytes has not been evaluated previously. In the present study, an assay using canine oocytes derived from frozen-thawed ovaries was evaluated using three types of semen: fresh untreated; killed; and a 50:50 mixture of untreated and killed spermatozoa. The assays were performed on 3 x 20 oocytes for each sperm treatment, using semen from pooled ejaculates (0.5 x 10(6) spermatozoa in each 50 microliter droplet containing five oocytes). There was a significant difference (P < 0. 001) between all treatments. Thereafter, the same procedure was used to evaluate methods of chilling and freeze-thawing of canine semen. There was a trend (P = 0.067) for more sperm binding after 1 day of chilling compared with after 4 days of chilling. Semen samples frozen using an extender (with or without the addition of Equex STM paste) were evaluated. Equex had a significant (P = 0.034) positive effect on the capacity of the spermatozoa to bind to the zona pellucida. In conclusion, the addition of a zona pellucida binding assay to established in vitro tests should give a better estimate of the damage caused by the various procedures when developing new techniques for chilling and freeze-thawing. Furthermore, the present study showed that chilling for 4 days tended to reduce the zona-binding capacity of the spermatozoon, and that Equex STM paste had a beneficial effect on the capacity of the frozen-thawed spermatozoon to bind to the zona pellucida.

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P Tienthai, M Yokoo, N Kimura, P Heldin, E Sato and H Rodriguez-Martinez

Hyaluronan is related to essential reproductive processes in pigs. Hyaluronan produced by cumulus cells builds, via specific cell surface receptors, an extracellular matrix responsible for cumulus cell cloud expansion during final oocyte maturation, a preparatory event for ovulation and fertilization. In addition, hyaluronan that has been localized in the pig oviduct both in the intraluminal fluid and on the surface of the lining epithelium of the preovulatory sperm reservoir, has proven beneficial during in vitro fertilization and embryo culture, thus indicating that it has a role in vivo. This study monitored the immunolocalization, protein determination and gene expression of the major cell surface hyaluronan receptor CD44 in the epithelial lining of the pig oviduct during selected stages of standing oestrus, in relation to spontaneous ovulation. The CD44 immunostaining in the lining epithelium was localized to the surface membrane and the supranuclear domain of mainly the secretory cells, particularly in the sperm reservoir of both treatment (inseminated) and control (non-inseminated) specimens. Up to four hyaluronan-binding protein (HABP) bands (60, 90, 100 and 200 kDa) were detected in the tubal epithelium, and the 200 kDa band was determined as CD44 by immunoblotting. The expression of CD44 mRNA was higher before than after ovulation (P < 0.05), most conspicuously in the uterotubal junction (UTJ). In addition, CD44 expression in the preovulatory UTJ and the ampullary-isthmic junction (AIJ) of control animals was higher than in those that were inseminated (P < 0.05 and P < 0.01 for UTJ and AIJ, respectively). The results demonstrate for the first time that the specific hyaluronan receptor CD44 is expressed by the oviduct epithelial cells during spontaneous oestrus, and is particularly abundant in the sperm reservoir before ovulation. Presence of spermatozoa in this segment seemed to downregulate the receptor. The variation in the expression of CD44 in relation to spontaneous ovulation and the presence of spermatozoa indicate that the hyaluronan CD44-signalling pathway may play a role in oviduct function during sperm storage and fertilization in pigs.

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C Ortega Ferrusola, L González Fernández, J M Morrell, C Salazar Sandoval, B Macías García, H Rodríguez-Martinez, J A Tapia and F J Peña

Lipid peroxidation (LPO) of stallion spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezability. Innate levels of LPO were very low in fresh spermatozoa but increased after thawing, a change that was largely stallion-dependent. The level of LPO in fresh spermatozoa was not correlated with that of the thawed spermatozoa. Negative correlations existed between LPO and intact membranes post-thaw (r=−0.789, P<0.001), and also between LPO and spermatozoa with high mitochondrial membrane potential (Δψm) post-thaw (r=−0.689, P<0.001). LPO was also highly and significantly correlated with caspase activity. The correlation between caspase activity in ethidium positive cells and LPO was r=0.772, P<0.001. This LPO is unlikely to represent, per se, a sign of cryopreservation-induced injury, but it is apparently capable of triggering ‘apoptotic-like changes’ that could result in the sub-lethal cryodamage often seen among surviving spermatozoa.

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S Sancho, I Casas, H Ekwall, F Saravia, H Rodriguez-Martinez, J E Rodriguez-Gil, E Flores, E Pinart, M Briz, N Garcia-Gil, J Bassols, A Pruneda, E Bussalleu, M Yeste and S Bonet

This study evaluated the effects of cooling, freezing and thawing on the plasma membrane integrity, kinetics and expression of two sugar transporters glucose transporter-3 and -5 (GLUT-3 and GLUT-5) in spermatozoa from Iberian boars. Semen samples were collected twice weekly from eight young, fertile Iberian boars of the ‘Entrepelado’ and ‘Lampiño’ breeds. The samples were suspended in a commercial extender and refrigerated to 17 °C for transport to the laboratory (step A), where they were further extended with a lactose–egg yolk-based extender and chilled to 5 °C (step B) prior to freezing in the presence of glycerol (3%). Spermatozoa were assessed for plasma membrane integrity and sperm motility at each of the steps, including post-thaw (step C). Aliquots were also prepared for immunocytochemical localisation of the sugar transporters (fixed and thin smears for transmission and scanning electron microscopy levels respectively) and for SDS–PAGE electrophoresis and subsequent western blotting, using the same antibodies (rabbit anti-GLUT-3 and anti-GLUT-5 polyclonal antibodies). The results showed lower percentages of progressively motile spermatozoa at step C in both breeds, while the percentage of live spermatozoa was significantly lower only in the ‘Entrepelado’ breed. The results obtained from electron microscopy clearly showed that Iberian boar spermatozoa expressed the hexose transporters, GLUT-3 and GLUT-5. The pattern of expression, in terms of location and concentration, was characteristic in each case but, in the case of isoform GLUT-5, it remained constant during the different steps of freezing–thawing protocol. These results indicate that cryopreservation affects the status of sperm cells of Iberian boars by altering the distribution of some membrane receptors and decreasing the percentage values of parameters linked to sperm quality.