Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa.

Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen-thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP-7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid-piece and principal piece domains in ejaculated spermatozoa. AQP-9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP-7, but not that of AQP-9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP-9 but relocated AQP-7 towards the acrosome. AQP-7, but not AQP-9, appears as a relevant marker for non-empirical studies of sperm handling.

Cryo-scanning electron microscopy discloses differences in dehydration of frozen boar semen stored in large containers.

In general, freezing in flat plastic polyethylene terephthalate (PET) bags (FlatPacks) at 50 degrees C/min gives better post-thaw viability, in terms of sperm motility and membrane integrity, than does freezing in plastic maxi-straws, probably owing to differences in cryobiology. To test the hypothesis that this better survival post-thaw relates to the degree of sperm dehydration during freezing, the present study investigated the structure of boar semen in a frozen state using cryo-scanning electron microscopy (cryo-SEM) to compare two different packages (FlatPacks and maxi-straws) for single artificial insemination (AI) doses, and three different freezing rates. The semen was split-sample frozen in maxi-straws or FlatPacks (both holding 5 ml) using 3% glycerol as cryoprotectant. Three freezing rates were applied from -5 degrees C to -100 degrees C, namely 2 degrees C/min, 50 degrees C/min and 1200 degrees C/min, the lattermost by plunging the samples into liquid nitrogen (LN(2)). The samples were thereafter fractured into LN(2) and larger areas of extra-cellular, unbound frozen water ('ice lakes') were measured to determine the degree of dehydration of the spermatozoa. These areas decreased in size with an increase in cooling rate, the differences in size being more dramatic for maxi-straws than for FlatPacks. Size of ice lakes was also influenced by location within package in relation to cooling rate, the central values being always smaller in maxi-straws than in Flatpacks (p < 0.05 at 2 degrees C/min and 50 degrees C/min) but not at 1200 degrees C/min, which suggested the FlatPack allows for more homogenous freezing of boar semen.

Effects of cryopreservation on semen quality and the expression of sperm membrane hexose transporters in the spermatozoa of Iberian pigs.

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 degrees C for transport to the laboratory (step A), where they were further extended with a lactose-egg yolk-based extender and chilled to 5 degrees 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.

Morphological features of spermatozoa of swamp buffalo AI bulls in Thailand.

The appearance and incidence of sperm abnormalities was studied in 115 ejaculates, collected periodically over 1 year covering all seasons from five mature, healthy swamp buffalo (Bubalus bubalis) bulls reared under tropical conditions and serving as the current source of semen for artificial insemination (AI) in Thailand. Light microscopy of stained smears was used to investigate sperm head shape morphology, while unstained wet smears were used to examine other sperm abnormalities. The most commonly found morphological aberrations were pear-shaped spermatozoa, knobbed acrosomes, proximal cytoplasmic droplets, simple bent tails and coiled tails under the head, whose ultrastructure (scanning electron microscopy) corresponded to what has been found in other species of bovidae, including varieties of buffalo. The mean prevalence (as least squares mean +/- SEM) of sperm abnormalities was low (below 15%), corresponding to healthy spermiograms. The younger bulls (<10 years old, n = 3) had less abnormalities than the older ones (10.1 +/- 0.6% versus 14.1 +/- 0.8%, P < 0.001, n = 2), including abnormalities of sperm head shape (1.1 +/- 0.3% versus 3.6 +/- 0.3, P < 0.001), acrosome defects with knobbed acrosomes (1.1 +/- 0.2% versus 1.2 +/- 0.3%, P < 0.001), spermatozoa with proximal cytoplasmic droplets (2.7 +/- 0.1% versus 1.4 +/- 0.2%, P < 0.001), defective mid-pieces (0.2 +/- 0.1% versus 0.3 +/- 0.1%) and abnormal sperm tails (3.1 +/- 0.3% versus 5.7 +/- 0.4%, P < 0.001). The within-bull effect of the year solely affected the incidence of pear-shaped spermatozoa while the incidences of abnormal contour, variable size of sperm head shapes, abnormal mid-piece and simple bent tail among bulls were affected by ejaculate (week of collection). Interaction between age and ejaculate affected only the prevalence of spermatozoa with proximal cytoplasmic droplets. In conclusion, the types of defects encountered were similar to those found in other bovidae, with a very low prevalence over the year the AI sires were followed through.

Transmission electron microscopy studies of the zona reaction in pig oocytes fertilized in vivo and in vitro.

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.

Involvement of oviduct in sperm capacitation and oocyte development in pigs.

An overview is presented on the structure and function of the pig oviduct in relation to sperm capacitation and oocyte development in vivo. In pigs, a functional sperm reservoir is established in the uterotubal junction-isthmus when sperm deposition occurs before ovulation. Capacitation is assumed to occur in this location, and spermatozoa progress towards the ampullary-isthmic junction at about the time of ovulation as a consequence of capacitation and hyperactivation. Preliminary data from our laboratory on viable spermatozoa retrieved from the sperm reservoir and the ampullary-isthmic junction of mated sows at pre- and periovulation oestrus showed that the largest subpopulation (60-90%) was of uncapacitated spermatozoa (using merocyanine-540), whereas 6-37% of the gated cells were capacitated spermatozoa. Incubation in a capacitation-inducing medium (bicarbonate-containing modified Brackett-Oliphant medium; mBO) for < 30 min effected capacitation readily, more markedly in ampullary-isthmic junction samples than in samples from the uterotubal junction, thereby indicating that uncapacitated spermatozoa responded to the addition of the effector bicarbonate at concentrations similar to those recorded in the periovulatory ampullary-isthmic junction in vivo. Addition of preovulatory isthmic oviductal fluid and hyaluronan under a similar incubation regimen maintained tubal sperm viability without obvious induction of capacitation. This finding indicates that, before ovulation, the intraluminal fluid of the sperm reservoir might delay sperm capacitation, perhaps because of its hyaluronan content. Evidence is presented that the sperm population in the oviduct undergoes capacitation under particular conditions in the upper tubal compartments. The diverse response of spermatozoa to capacitation stimuli helps to ensure full rates of fertilization in vivo. Data are also provided on the importance of final zona pellucida maturation in the pig oviduct to warrant proper zona pellucida reaction after sperm penetration, which would address in part the abnormal occurrence of polyspermy in in vitro fertilization of pigs.

Zona reaction in porcine oocytes fertilized in vivo and in vitro as seen with scanning electron microscopy.

Morphological changes in zona pellucidae (ZP) isolated from in vitro-matured (IVM) and ovulated porcine oocytes were compared before or after fertilization in vitro and in vivo, respectively, by using scanning electron microscopy (SEM). The ZP of some ovulated or IVM oocytes and in vivo- or in vitro-fertilized (IVF) zygotes were equally split into two halves while immersed in an enzyme-inhibitor solution, using a surgical blade. After washing, intact and ZP halves were fixed in 1% glutaraldehyde solution in 0.1 M cacodylate buffer, processed, and examined using SEM. The outer surface of ZP in ovulated oocytes had a mesh-like structure. The outer morphology in IVM oocytes was more smooth although the mesh-like structure was still visible at high magnification. In in vivo zygotes and IVM-IVF zygotes, this lysed, mesh-like structure was more obvious. The inner surface of ZP had some small depressions (orifices). The mean number of orifices per 100 micrometer(2) of ZP surface was larger in IVM oocytes as compared to ovulated ones. The number of orifices per 100 micrometer(2) decreased in IVM-IVF zygotes as compared to IVM oocytes; whereas, in vivo zygotes did not differ from ovulated oocytes. The mean diameter of intact ZP as well as their mean thickness was greater in ovulated oocytes than IVM oocytes. The mean thickness of the ZP was larger in ovulated oocytes than IVM ones. The ZP thickness was larger in zygotes than in in vivo oocytes, whereas that of IVM-IVF zygotes did not differ from that of IVM oocytes. These results indicate that the morphology of ZP and the ZP reaction at sperm penetration appears to be much different between IVM oocytes and ovulated ones.

Localization of calcium and zinc in the sperm storage tubules of chicken, quail and turkey using X-ray microanalysis.

Sperm storage tubules from the utero-vaginal junction of chickens, quails and turkeys were analysed for calcium and zinc using X-ray microanalysis of ultra-rapidly frozen tissue in a scanning electron microscope. This technique enabled the tubular fluid surrounding the stored spermatozoa and the intracellular content of the cells of the sperm storage tubules to be analysed separately and, by using standards with known concentrations, their elemental concentrations were estimated. The mean (+/- SEM) concentration of calcium in the tubular fluid from chickens, quails and turkeys was 17 +/- 3, 19 +/- 3 and 17 +/- 4 mmol kg(-1) wet weight, respectively. The intracellular calcium concentration of the cells of the tubules did not differ significantly from these values and was also similar in the mucosal epithelial cells of the utero-vaginal junction. Zinc was localized in the cells of turkey sperm storage tubules and tubular fluid, but at low concentrations. No zinc could be detected in corresponding structures from chickens and quails. The concentration of calcium in the tubular fluid is within the range known to inhibit the motility of spermatozoa, supporting this function for calcium during storage. Zinc is known to depress turkey sperm metabolism and it may also be involved in inducing quiescence of spermatozoa during storage in this species.

Retrograded high-amylose corn starch reduces cholic acid excretion from the small bowel in ileostomy subjects.

OBJECTIVE: To study the short-term effect of resistant starch (RS) from retrograded high-amylose corn starch (HACS) on the excretion of bile acids and nutrients from the small bowel in humans. DESIGN: Seven healthy ileostomists were given a controlled, constant diet during three days. On days 2 and 3, 100 g/d of one of two test-products--drum-dried ordinary corn starch and autoclaved retrograded HACS, providing 5 and 39 g RS/d, respectively--was given, in random order. Ileostomy effluents were collected for 24 h per day and analysed for wet weight, dry weight, energy, bile acids and nutrients. SETTINGS: In-patient study at the metabolic ward, Department of Clinical Nutrition, Sahlgrenska University Hospital, Göteborg. RESULTS: Consumption of retrograded HACS caused (1) a 42% lower mean excretion of cholic acid (P = 0.024); (2) a 42% lower mean wet weight concentration of bile acids (P < 0.001); (3) a 70% increased excretion of dry weight (P = 0.001); and (4) a 41% increased excretion of energy (P= 0.036) compared with consumption of drum-dried ordinary corn starch. CONCLUSION: The reduced ileal excretion and concentration of cholic acid would be protective regarding colon cancer risk in addition to the increased fermentation substrate provided by RS and other energy-yielding components.

Embedding resin space, water contents and chromatin compaction in rabbit sperm nuclei: electron microscopic X-ray spectrophotometry of a brominated probe.

EPON labelled with bromide was used to embed ejaculated rabbit spermatozoa, with the hypothesis that it replaces cell water. X-Ray spectrophotometric microanalysis of sperm nuclei, of egg yolk (an internal standard containing roughly 50% water) and of surrounding embedding resin, revealed that a part of the bromide was bound to the biological components. These latter were saturated when bromide was added in higher concentrations, and the increase in measured bromide could be used to calculate absolute resin contents in sperm nuclei which gives a mean value of 22.62%. Most nuclei (60.80%) were well condensed and displayed a mean resin space close to 17% of the total nuclear volume. The less condensed nuclei had a mean resin space close to 28%. The use of an internal standard revealed that calculated values were underestimated by 4%.

Fine structure and elemental composition of fresh and frozen dog spermatozoa.

Dog spermatozoa from fresh ejaculates and after freezing-thawing were air-dried on to grids and subjected to X-ray microanalysis or examined by scanning and transmission electron microscopy. The spermatozoa subjected to freezing-thawing presented major changes in their morphology, which included the loss of acrosomal contents, seen both by the swelling and rarefaction of the acrosome and the loss of electron-dense acrosomal material. In many cells the acrosomal damage, including the equatorial region, was conspicuous, with vesiculization of the acrosomal membranes. The plasmalemma remained, in most cases, apparently intact. X-ray microanalysis of the sperm aliquots showed a significant decrease in the amount of most selected elements after freezing-thawing. The extent to which concentrations of phosphorus and sulfur decreased in the sperm head region suggested that major changes in the chromatin occurred during the freeze-thaw process.

Comparison of nuclear hydration in boar spermatozoa before and after freeze-thawing: contrast analysis of cells embedded in bromide-labeled medium.

Epon labeled with bromide was used to embed ejaculated and freeze-thawed spermatozoa, with the hypothesis that it replaces most of cell water. Image analysis of relative contrasts between sperm nuclei and the surrounding medium revealed that when used in low concentrations, bromide is mostly adsorbed to the nuclear structures. For higher concentrations, the chromatin is saturated, and the increase in contrast can be used to calculate relative differences in the hydration of nuclei. Boar sperm nuclei are more hydrated after freeze-thawing than before.

Cryopreservation of boar semen. III: Ultrastructure of boar spermatozoa frozen ultra-rapidly at various stages of conventional freezing and thawing.

Ejaculated boar spermatozoa subjected to a conventional freezing and thawing process, were ultra-rapidly fixed, freeze-substituted and examined by electron microscopy to monitor the presence of real or potential intracellular ice and the degree of cell protection attained with the different extenders used during the process. Numerous ice crystal marks representing the degree of hydration of the cells were located in the perinuclear space of those spermatozoa not in proper contact with the extender containing glycerol (i.e. prior to freezing). The spermatozoa which were in proper contact with the extenders presented a high degree of preservation of the acrosomes, plasma membranes as well as the nuclear envelopes. No ice marks were detected in acrosomes before thawing, indicating that the conventional assayed cryopreservation method provided a good protection against cryoinjury. The presence of acrosomal changes (internal vesiculization, hydration and swelling) in thawed samples however, raises serious questions about the thawing procedure employed.

Preliminary study of water and some element contents in boar spermatozoa, before, during and after freezing.

Boar semen was analysed by electron microscopy coupled to image analysis and X-ray energy dispersive spectroscopy, during the usual process for freezing and thawing in field conditions. Freeze-substitution and freeze-quenching permitted recording of real or potential intracellular ice before, during, and after freezing. Heads and flagella displayed two different osmotic properties before freezing. Heads were dehydrated progressively before and during freezing, while flagella were hydrated before freezing and were only dehydrated during freezing. All parts of the thawed cells were rehydrated. Ice crystal damage was mostly present in frozen mitochondria and axonemes and the acrosomes were strongly affected by thawing. The total amounts of Na, Cl, Ca, K, Mg, and Zn per cell were only elevated in frozen and thawed midpieces while the heads were permeable both to water and elements at that time.

Nucleus of the boar spermatozoon: structure and modifications in frozen, frozen-thawed, or sodium dodecyl sulfate-treated cells.

After cryosubstitution and Epon embedding, or after Nanoplast embedding and very thin sectioning, the chromatin of ejaculated or diluted boar spermatozoa appears to be formed of DNA fibers embedded in a quite homogeneous matrix. After sodium dodecyl sulfate (SDS) treatment, and to a lesser extent after freeze-thawing, the DNA fibers are present mostly between cords, probably proteinaceous in nature. The quantity of free sulfhydryl (SH) groups, as calculated from staining by DACM and flow fluorometry, is increased in thawed or SDS-treated cells. The quantity of NH2 groups, calculated from electron microscopy image analysis of alcoholic phosphotungstic acid-stained cells, is decreased in thawed nuclei. The DNA is more accessible to the fluorochrome ethidium bromide after freeze-thawing, and its sensitivity to HCl hydrolysis is modified, during the Feulgen-like staining procedure using acriflavine. The X-ray energy dispersive analysis of cryosections of nuclei indicates that the slight separation of DNA and nucleoproteins in freeze-thawed spermatozoa could result from a dramatic modification of the nuclear ionic environment during thawing.

Differentiation of the rat testis between 20 and 120 days of age.

The differentiation of the seminiferous epithelium was studied in 20-120-day-old Sprague-Dawley rats. Body weights and the weights of the testes, seminal vesicles, and kidneys were determined. The best way of defining the development of the testis was to denote the most differentiated germ cell present. Spermatogenesis is complete at 56 days of age, but the testis continues to grow up to 108 days of age. The development of certain parameters has been calculated, and it is very important to define the age of the animals in experimental studies and to include sufficient numbers of controls.

Differentiation of the rat seminiferous tubules between 13 and 19 days of age.

The differentiation of rat seminiferous tubules have been studied in 13 to 19 days old. Testicular weight and tubular cross-sectional area were more than doubled during this period. The percentage of tubules with more than 10 primary spermatocytes increased from 4% to 90%, and the lanthanum excluding ability of the inter-Sertoli cell junctions (the blood-testis barrier) showed a similar increase but two days later. ABP production in vitro increased more than twentyfold from day 13 to day 19 of age. It is concluded that the differentiation of Sertoli cell function and the appearance of primary spermatocytes are temporally correlated which supports the assumption that the function of the Sertoli cells is important for initiation and maintenance of spermatogenesis.

Specialized cell contacts and the blood-testis barrier in the seminiferous tubules of the domestic fowl (Gallus domesticus).

The ultrastructure of Sertoli-Sertoli and Sertoli-germ cell surface specializations in the domestic fowl was studied in material fixed by vascular perfusion through the thoracic aorta. Three main types of surface specializations were found between adjacent Sertoli cells. These are focal tight junctions, desmosome-like devices, and a specialization characterized by the presence of long and dilated subsurface cisternae of rough endoplasmic reticulum. Typical inter-Sertoli cell junctions similar to those of mammals were absent. Germ cells were attached to Sertoli cells mainly by desmosome-like devices of varying appearance. The junctions between Sertoli cells and elongating or elongated spermatids, "the mantle", consisted of only slight condensations of filamentous material in the Sertoli cell. The tight junctions between adjacent Sertoli cells were efficient in preventing lanthanum from passing towards the lumen beyond the level of the spermatogonia.