Cryo-scanning electron microscopy demonstrates that ice morphology is not associated with the post-thaw survival of domestic boar (Sus domesticus) spermatozoa: A comparison of directional and conventional freezing methods.

Directional freezing (in 2 or 10 ml hollow glass tubes) has been reported to improve post-thaw sperm survival parameters compared to conventional methods (in 0.5 ml straws). However, the biophysical properties that increase post-thaw survival are poorly understood. Therefore, the aim for the current study was to investigate the effect of ice morphology on the post-thaw survival of domestic boar spermatozoa directionally and conventionally cryopreserved in 0.5 ml straws. Ice morphology was quantitatively analyzed using a combination of cryo-scanning electron microscopy and Fiji Shape Descriptors. Multivariate analysis found a significant, non-linear effect (p < 0.05) of interface velocity on ice morphology, with an increase in both ice-lake size, as indicated by area and in aspect ratio, at an interface velocity of 0.2 mm/s. By contrast, post-thaw sperm survival (defined as spermatozoa with both intact plasma membranes and acrosomes) was biphasic, with peaks of survival at interface velocities of 0.2 mm/s (54.2 ± 1.9%), and 1.0 or 1.5 mm/s (56.5 ± 1.5%, 56.7 ± 1.7% respectively), and lowest survival at 0.5 (52.1 ± 1.6%) and 3.0 mm/s (51.4 ± 1.9%). Despite numerical differences in Shape Descriptors, there was no difference (p > 0.05) in the post-thaw survival between conventionally and directionally cryopreserved samples at optimal interface velocities of 1.0 or 1.5 mm/s. These findings suggest that: 1) ice morphology has little impact on post-thaw survival of boar spermatozoa, and 2) directional freezing in 0.5 ml straws (rather than 2 or 10 ml hollow glass tubes) may attenuate benefits of directional freezing.

Evaluation of boar and bull sperm capacitation and the acrosome reaction using flow cytometry.

Flow cytometry can be used to evaluate many sperm attributes and Dr. Duane Garner was influential in developing assays to understand sperm physiology and function. We review some of Dr. Garner's work and describe experiments that evaluate sperm capacitation using Dr. Garner's philosophy. In exploratory experiments, boar sperm were cryopreserved in lactose egg yolk (LEY) or Beltsville Freezing Extender 5 (BF5) and incubated in one capacitating medium. In another experiment, frozen-thawed bull sperm were incubated in TALP-Ca or CFDM1 capacitating media. In both experiments, sperm viability and capacitation were evaluated using multiple probes. Boar sperm frozen in LEY had greater survival rates (38%) than sperm frozen in BF5 (22%; P < 0.05) but did not capacitate as effectively as sperm in BF5 (P < 0.05). In Experiment 2, bull sperm survived to a greater extent when incubated in TALP-Ca than in CFDM1 (P < 0.05) and had greater capacitation for most parameters (P < 0.05). Of particular interest, 77% of sperm incubated in TALP-Ca had activated second messenger systems involved in capacitation, compared with < 5% of sperm incubated in CFDM1. The results indicate different freezing and capacitating media induce different responses to sperm capacitation and functions. If only sperm viability and acrosomal integrity were evaluated, these results would be interpreted very differently. Dr. Garner's philosophy of evaluating multiple sperm parameters was an impetus to determine unique treatment differences which help in understanding sperm capacitation, and design further experiments to determine how media content causes sperm physiology differences.

Motility and Fertility Evaluation of Thawed Frozen Stallion Semen After 24 Hours of Cooled Storage.

Breeding mares with cryopreserved semen requires specialized equipment for storage and thawing and more intensive mare management. The objectives of this study were (1) evaluate the longevity of frozen stallion semen once it had been thawed, extended, and maintained at 5°C for 48 hours in a passive cooling container, and (2) determine fertility potential of frozen semen that had been thawed, extended, and used to inseminate mares after 24 hours of cooled storage. Eight ejaculates were collected and aliquots were cooled in either INRA96 and CryoMax LE minus cryoprotectant at a concentration of 50 million total sperm/mL. The remainder of the ejaculate was frozen in CryoMax LE extender at a concentration of 200 million total sperm/mL. Semen was thawed using 1 of 3 thawing protocols, and diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE minus cryoprotectant and cooled to 5°C. Sperm motility was evaluated at 24 and 48 hours. Eight mares were inseminated over two estrous cycles using frozen semen that had been thawed, extended in INRA96, and cooled for 24 hours. There was no difference in progressive motility at 24 or 48 hours of cooled-storage post-thaw between the 3 thawing protocols. An overall per cycle pregnancy rate of 56% (9/16 cycles) was achieved using frozen-thawed semen that had been extended and cooled for 24 hours. In summary, frozen stallion sperm was thawed, extended, and cooled to 5°C for 24 hours and still maintained adequate (>30%) sperm motility and fertility.

Localisation of phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (WBP2 N-terminal like) on equine spermatozoa and flow cytometry quantification of PLCZ1 and association with cleavage in vitro.

Oocyte activation is initiated when a fertilising spermatozoon delivers sperm-borne oocyte-activating factor(s) into the oocyte cytoplasm. Candidates for oocyte activation include two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We localised PLCZ1 and WBP2NL/PAWP in stallion spermatozoa and investigated the PLCZ1 content and sperm parameters as well as cleavage after intracytoplasmic sperm injection (ICSI). PLCZ1 was identified as 71-kDa protein in the acrosomal and postacrosomal regions, midpiece and principal piece of the tail. Anti-WBP2NL antibody identified two WBP2NL bands (~28 and ~32kDa) in the postacrosomal region, midpiece and principal piece of the tail. PLCZ1 and WBP2NL expression was positively correlated (P=0.04) in sperm heads. Flow cytometry evaluation of PLCZ1 revealed large variations in fluorescence intensity and the percentage of positively labelled spermatozoa among stallions. PLCZ1 expression was significantly higher in viable than non-viable spermatozoa, and DNA fragmentation was negatively correlated with PLCZ1 expression and the percentage of positively labelled spermatozoa (P<0.05). The use of equine sperm populations considered to have high versus low PLCZ1 content resulted in significantly higher cleavage rates after ICSI of bovine and equine oocytes, supporting the importance of PLCZ1 for oocyte activation.

Effects of extender, cryoprotectants and thawing protocol on motility of frozen-thawed stallion sperm that were refrozen for intracytoplasmic sperm injection doses.

We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose-EDTA-egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk-egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially thawed to 5 °C, diluted and refrozen in SMEY containing 2, 4, 6 or 8% GLY or GMF. In Experiment 1, sperm motility was reduced after each cryopreservation cycle (P < 0.05). Extender type did not affect motility after refreezing (P > 0.05), but sperm initially thawed to 5 °C exhibited higher motility than sperm thawed to 37 °C (P < 0.05). In addition, sperm refrozen in SMEY containing MF or GMF exhibited higher motility than sperm refrozen in GLY alone (P < 0.05). In experiment 2, there was an interaction between CPA and CPA concentration (P < 0.05). Sperm refrozen with GMF had higher motility than refrozen sperm with GLY (P < 0.05), and while GLY concentration did not affect post-thaw motility (P > 0.05). Sperm refrozen with 6 or 8% GMF exhibited the highest motility (P < 0.05). In conclusion, sperm motility is best maintained when thawing and refreezing stallion sperm in low sperm concentration ICSI doses by initially thawing the sperm to 5 °C and diluting the sperm in a freezing extender with 8% GMF.

Cryopreservation of bison epididymal sperm: A strategy for improving post-thaw quality when collecting sperm in field conditions.

This study was conducted to optimize the cryopreservation of epididymal bison sperm harvested in the field. In the first experiment, epididymal bison sperm were treated with or without seminal plasma (n = 6) and cooled to 5 °C over 2 hours. In a separate experiment, glycerol was added at different times and sperm was held at 5 °C for different periods of time before cryopreservation (n = 11). In addition, epididymal sperm frozen with and without seminal plasma (n = 6) and after 4, 24, and 48 hours (n = 5) of equilibration at 5 °C, were evaluated for their in vitro fertilizing ability. Post-thaw motility of bison epididymal sperm was similar when cryopreserved with or without seminal plasma or when glycerol was added at either 0, 4, 24, or 48 hours before freezing (P > 0.05). However, sperm incubated at 5 °C for 24 hours before freezing exhibited higher percentages of motile sperm (44% vs. 35% for 4 hours or 48 hours, P < 0.05). Fertilization rates of bison oocytes were not different for any treatments. Chilling the whole epididymis for 24 or 48 hours resulted in complete loss of sperm viability. In conclusion, bison epididymal sperm can be chilled outside of the epididymis for at least 48 hours before cryopreservation without compromising post-thaw sperm motility providing flexibility for technicians performing field collections.

Cholesterol-loaded cyclodextrin in fresh goat sperm improves cryosurvival rates / Colesterol em sêmen caprino melhora a criopreservação

Este estudo teve como objetivo avaliar o efeito da adição do colesterol carreado com a ciclodextrina (CCC) sobre a melhoria da qualidade espermática após a descongelação. Trinta ejaculados foram diluídos, centrifugados e ressuspendidos com Tris para concentração de 120 x 106 células/mL. O sêmen foi tratado com 0, 0,75, 1,5, 3,0, 4,5, 6,0 ou 7,5 mg de CCC. Em seguida, as amostras foram resfriadas a 4°C durante 2 horas, diluídas com Tris-Gema de ovo e 2% de glicerol, envasadas e colocadas sobre o vapor do nitrogênio líquido (N2 ) por 20 min e depois mergulhadas no N2 . As amostras foram descongeladas a 37°C por 30s, e avaliadas quanto: o teste de termorresistência (TRT); motilidade progressiva utilizando CASA; teste hiposmótico e a capacidade de ligação dos espermatozoides à membrana perivitelina (MP). As variáveis foram analisadas por meio da ANOVA e os tratamentos comparados a 5% de probabilidade. A motilidade dos espermatozoides (50,4; 33,8 e 22,5%) foi maior nas amostras tratadas com 0,75 mg de CCC após 0, 60 e 120 min de incubação quando comparado com demais tratamentos (P<0,05). As amostras tratadas com 6,0 e 7,0 mg de CCC apresentaram maior dobramento de cauda que os demais tratamentos (P<0,05). A capacidade de ligação dos espermatozoides a MP foi maior no tratamento com 0,75 mg CCC comparado ao controle (166 vs 65; P <0,05). No entanto, quando o potencial de ligação dos espermatozoides foi determinado dividindo o número médio de espermatozoides ligados a MP sobre o percentual de espermatozoides móveis, o tratamento com CCC proporcionou maior eficiência (1,52) do que o controle (1,00; P < 0,05). A adição de 0,75 mg de CCC no sêmen fresco de caprino antes da criopreservação melhorou a motilidade espermática após a criopreservação até 2 horas.

Membrane modification strategies for cryopreservation.

Cell membranes can be modified using cyclodextrins loaded with lipids or unilamellar liposomes. Lipid choice can greatly influence the organization of the targeted membrane and result in a cell that is more capable of surviving cryopreservation due to altered membrane-phase transition properties or membrane reorganization that may alter the normal physiologic processes of the treated cell. The protocols described here explain the preparation of the cyclodextrins and liposomes, impact of the amount and type of lipids, and general principles for treating cells using either of these technologies.

Effects of milk proteins on sperm binding to the zona pellucida and intracellular Ca(2+) concentration in stallion sperm.

Objectives were to determine the effects of extracellular Ca(2+) and milk proteins on intracellular Ca(2+) concentrations in stallion sperm; and to determine the effects of single caseins on sperm binding to the zona pellucida (ZP). In Experiment I, sperm were incubated in media containing 2 or 4mM Ca(2+) and intracellular Ca(2+) concentration was determined after ionomycin treatment and long-term incubation (3h). Extracellular Ca(2+) concentrations (2 compared with 4mM) did not affect baseline intracellular Ca(2+) concentration of sperm. However, incubating sperm in a medium containing 4 compared with 2mM Ca(2+) resulted in greater (P<0.05) influx of Ca(2+) into sperm. In Experiment II, sperm incubated in media containing 1mg/mL of native phosphocaseinate (NP) or sodium caseinate (SC) showed similar baseline intracellular Ca(2+) and influx of Ca(2+) than control (TALP). In Experiment III, sperm-ZP binding assays were performed in TALP medium containing: no additions (TALP); 1mg/mL SC; 1 or 3mg/mL of α-casein; 1 or 3mg/mL of ß-casein; and 1 or 3mg/mL of κ-casein. The number of stallion sperm bound to bovine ZP was greatest (P<0.05) when SC was used. Co-incubation in media containing single caseins (α-, ß- or κ-casein) resulted in similar results to TALP; however, a dose effect (P<0.05) was observed for ß- and κ-caseins. In conclusion, extracellular Ca(2+) concentration and milk proteins did not affect baseline intracellular calcium in stallion sperm. It appears that ß- and κ-caseins may be responsible for enhancing sperm binding to ZP, but the mechanism remains unknown.

Overexpression of follistatin in the mouse epididymis disrupts fluid resorption and sperm transit in testicular excurrent ducts.

Activin is a well-established modulator of male and female reproduction that stimulates the synthesis and secretion of follicle-stimulating hormone. Nonpituitary effects of activin have also been reported, although the paracrine actions of this growth factor in several reproductive tissues are not well understood. To identify the paracrine functions of activin during mammary gland morphogenesis and tumor progression, we produced transgenic mice that overexpress follistatin (FST), an intrinsic inhibitor of activin, under control of the mouse mammary tumor virus (MMTV) promoter. Although the MMTV-Fst mice were constructed to assess the role of activin in females, expression of the transgene was also observed in the testes and epididymides of males. While all 17 transgenic founder males exhibited copulatory behavior and produced vaginal plugs in females, only one produced live offspring. In contrast, transgenic females were fertile, permitting expansion of transgenic mouse lines. Light and transmission electron microscopic examination of the transgenic testes and epididymides revealed impairment of fluid resorption and sperm transit in the efferent ducts and initial segment of the epididymis, as indicated by accumulation of fluid and sperm stasis. Consequently, a variety of degenerative lesions were observed in the seminiferous epithelium, such as vacuolation and early stages of mineralization and fibrosis. Sperm collected from the caudae epididymidis of MMTV-Fst males had detached heads and were immotile. Together, these data reveal that activin signaling is essential for normal testicular excurrent duct function and that its blockade impairs fertility. These results also suggest that selective inhibitors of activin signaling may provide a useful approach for the development of male contraceptives without compromising androgen synthesis and actions.

Effects of components of semen extenders on the binding of stallion spermatozoa to bovine or equine zonae pellucidae.

The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the sperm through Percoll did not increase sperm binding to the ZP but did remove any positive effect of EZM on sperm-ZP binding. In Experiment IV, exposure of either sperm or ZP to EZM before co-incubation did not increase sperm binding to ZP. In Experiment V, sperm diluted in TALP containing skim milk, EZM, or INRA96 bound more efficiently to the ZP than sperm diluted in TALP without milk proteins. In Experiment VI, sodium caseinate, native phosphocaseinate, and caseinoglycopeptide increased sperm binding to the ZP. In conclusion, diluents containing milk or milk proteins markedly enhanced the number of sperm bound to both equine and bovine ZP.

Treating ram sperm with cholesterol-loaded cyclodextrins improves cryosurvival.

Acceptable fertility using cryopreserved ram sperm is currently only achieved using laparoscopic intrauterine insemination. Improving the cryosurvival of ram sperm may permit greater fertility rates using more practical techniques. This study was conducted to determine if treating ram sperm with six different cyclodextrins pre-loaded with cholesterol (CLC), prior to cryopreservation increases sperm cryosurvival and if this technology can be used with neat semen. Subsequent experiments evaluated how adding CLC to sperm affected sperm cholesterol content, sperm osmotic tolerance limits, sperm post-thaw survival after incubation and the capacity of sperm to bind to zona pellucidae of cattle and sheep oocytes. Sperm treated with 2-hydroxypropyl-beta-cyclodextrin prior to cryopreservation exhibited greater percentages of motile sperm (62%) compared to the control (no CLC treatment) samples (43%, P<0.05), after thawing. In addition, samples treated with methyl-beta-cyclodextrin exhibited percentages of motile and viable sperm similar to samples treated with 2-hydroxypropyl-beta-cyclodextrin. Other CLC-treated samples were similar to the control. The CLC concentration that optimized sperm cryosurvival was 2mg CLC/120 x 10(6) sperm for both methyl-beta- and 2-hydroxypropyl-beta-cyclodextrin when added to neat semen prior to cryopreservation. Addition of 2mg CLC not only maintained greater percentages of motile sperm compared to the control samples, but maintained greater percentages of motile sperm during a 3h incubation after thawing. In addition, 2-hydroxypropyl-beta-cyclodextrin pre-loaded with cholesterol maintained greater percentages of viable sperm (33%), than control sperm (18%; P<0.05). Treating ram sperm with CLC increased the sperm cholesterol content>1.9-fold and although some cholesterol was lost from the sperm during cooling and cryopreservation, the cholesterol content remained greater in CLC-treated sperm after cooling and after thawing than in control sperm (P<0.05). In addition, CLC-treated sperm maintained greater percentages of motile sperm through a wide range of osmotic solutions (150 and 425 mOsm) while control sperm lost motility in solutions outside a more narrow range (270 to 370 mOsm). Greater numbers of CLC-treated sperm bound to zona pellucida than control sperm (P<0.05), although number of sperm binding cattle and sheep oocytes, was similar (P>0.05). In conclusion, treating ram sperm with CLC increases sperm cryosurvival rates and sperm longevity after thawing. It also increases the cholesterol content, osmotic tolerance, and zona-binding capabilities of sperm. Finally, CLCs can be added to neat semen, making this technology feasible for practical application using current cryopreservation techniques for ram semen.

The fertility of ram sperm held for 24h at 5 degrees C prior to cryopreservation.

Diluted ram sperm can be held for 24h at 5 degrees C prior to cryopreservation without impacting cryosurvival rates, however, the effects this storage has on subsequent fertility are unknown. These studies were conducted to evaluate the fertility of semen held for 24h (to mimic shipping semen to a cryopreservation center), prior to freezing. Semen from Suffolk rams (n=3 in experiment 1 and n=6 in experiment 2) with initial motility of greater than 70%, were diluted to 200 x 10(6)sperm/mL, in one step, with a Tris-egg yolk-glycerol diluent. In experiment 1, diluted samples were cooled to 5 degrees C over 2h, and then divided. Sperm in one fraction were loaded into 0.5mL straws, frozen (T0) and stored in liquid nitrogen until thawing. Sperm in the second fraction were held at 5 degrees C for 24h (T24) before being frozen. In experiment 2 ejaculates were collected and divided into two fractions. Sperm in one fraction were treated with cholesterol-loaded cyclodextrin (CLC) and sperm in the other served as control. Both fractions were diluted, cooled, and cryopreserved as described in experiment 1. Stage of the estrous cycle was synchronized in ewes (n=196) using controlled internal drug releasing devices (CIDR) for 12d and at CIDR removal each ewe was administered PMSG (500IU in experiment 1 and 350IU in experiment 2) immediately before insemination. Ewes were stratified by age and randomly assigned to one of the semen treatments; experiment 1: Fresh (F), T0, or T24; experiment 2: F, T24, or CLC, and inseminated laparoscopically 56h after CIDR removal. Differences in fertility were detected between experiments, but not for treatments within experiments. Differences in fertility were also observed due to ewe age, with the 3-year-old ewes having the greatest fertility (50.7%) and 6-year-old ewes having the least fertility (9.6%; P<0.05). Differences in the prolificacy rates due to semen treatment were also observed but differences due to ewe age were not detected. Therefore, sperm can be held at 5 degrees C for 24h prior to cryopreservation without altering sperm fertility.

Classification of hyperactivated spermatozoa using a robust Minimum Bounding Square Ratio algorithm.

A method for automatically identifying and classifying hyperactivated spermatozoa trajectories is described. This physiologically-based computerized algorithm captures the motion behavior of sperm during hyperactivation. A novel Minimum Bounding Square Ratio (MBSR) algorithm classifies spermatoza as hyperactivated, transitional or progressive. Classification boundaries were established on selected trajectory data from a single stallion and then tested on random trajectories of sperm from other stallions. MBSR classified sperm in a robust and effective manner.

Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol.

Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cyclodextrin (CLC), subjected to anisotonic solutions and spermatozoal motility analyzed. The spermatozoa were then returned to isotonic conditions and the percentages of motile spermatozoa again determined. CLC treatment increased the osmotic tolerance limit of stallion spermatozoa in anisotonic solutions and when returned to isotonic conditions. The second and third experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. In experiment two, stallion spermatozoa were determined to behave as linear osmometers. In experiment three, spermatozoa were treated with CLC, incubated with different cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and their volume excursions measured during cryoprotectant removal at 5 degrees and 22 degrees C. Stallion spermatozoa were less permeable to the cryoprotectants at 5 degrees C than 22 degrees C. Glycerol was the least permeable cryoprotectant in control cells. The addition of CLC's to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants. Therefore, adding cholesterol to spermatozoal membranes reduces the amount of osmotic stress endured by stallion spermatozoa during cryopreservation.

Effect of sex-sorting on the ability of fresh and cryopreserved bull sperm to undergo an acrosome reaction.

Previous studies indicate that sex-sorted sperm exhibit different physiology, including fertilizing capacity, from non-sorted sperm. However, differences between X- and Y-bearing sperm in their ability to undergo an acrosome reaction have never been investigated. This study determined the ability of non-sorted and sex-sorted sperm to undergo the acrosome reaction prior to and after cryopreservation. Sperm were treated with dilauroylphosphatidylcholine (PC12) to induce the acrosome reaction and the percentages of live-acrosome-reacted sperm and dead sperm were evaluated. The X- and Y-bearing sperm reacted similarly to the PC12 treatment, regardless of whether sperm were assessed prior to or after cryopreservation. Fresh control sperm exhibited lower percentages of live sperm (60%) than either X- or Y- sorted sperm (69-74%, P<0.05). Percentages of live control sperm were also lower after thawing (29-35%) than sex-sorted sperm (55-58%, P<0.05). Control and sex-sorted fresh sperm responded similarly to PC12 treatment. However, sex-sorted cryopreserved sperm exhibited higher percentages of live-acrosome-reacted sperm (23%) than control sperm (9%, P<0.05) after 40 min without PC12 treatment. In addition, cryopreserved control sperm treated with 79 microM PC12 exhibited higher percentages of live-acrosome-reacted sperm than sex-sorted sperm. In conclusion, X- and Y-bearing sperm responded similarly to PC12 treatment. In addition, fresh sexed and non-sorted sperm responded similarly to PC12 treatment. However, cryopreserved sex-sorted sperm underwent an acrosome reaction more rapidly in the absence of PC12 (over a 40 min period) than the non-sorted sperm. Therefore, sex-sorting induced changes in sperm membranes that accelerated the acrosome reaction process in sperm after cryopreservation.

Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival.

Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sperm were maintained after thawing when 1.5 mg CLC was added to sperm from stallions whose sperm do not survive freezing well, compared to control sperm from those same stallions (67% vs. 50%; P<0.05). Addition of CLCs increased the percentages of membrane intact sperm surviving cryopreservation compared to untreated sperm for all stallions (P<0.05). The amount of cholesterol that incorporated into the membranes of the sperm cells increased in a polynomial fashion (R2=0.9978) and incorporated into all sperm membranes. In addition, there was a significant loss of cholesterol from sperm membranes after cryopreservation; however, addition of CLCs to sperm prior to cryopreservation maintained higher cholesterol levels in the sperm after freezing and thawing than untreated sperm (P<0.05). Addition of CLCs also resulted in more sperm binding to the zona pellucida of bovine oocytes after cryopreservation than control sperm (48 vs. 15; P<0.05). In conclusion, CLCs improved the percentage of post-thaw viability in equine sperm as well as increased the number of sperm that bind to zona pellucida. Addition of CLCs to stallion sperm prior to cryopreservation is a simple procedure that increases the cryosurvival of cells.

Effect of adding cholesterol to bull sperm membranes on sperm capacitation, the acrosome reaction, and fertility.

When cholesterol is added to sperm membranes before cryopreservation, higher percentages of motile and viable cells are recovered after thawing. However, because one of the first steps in sperm capacitation is cholesterol efflux from the sperm plasma membrane, adding cholesterol to enhance cryosurvival may retard sperm capacitation. These studies evaluated the ability of sperm treated with cholesterol-loaded cyclodextrins (CLC) to capacitate, acrosome react, and fertilize oocytes. Control (non-CLC-treated) and CLC-treated sperm were treated with heparin, dilauroylphosphatidylcholine (PC12), or calcium ionophore A23187 (A23187) to capacitate and induce the acrosome reaction. Sperm capacitation, assessed by an increase in intracellular calcium level, and acrosome-reacted sperm were measured using flow cytometry. Fresh CLC-treated sperm cells underwent capacitation and/or the acrosome reaction at rates different from control samples, and the differences detected were dependent on the method used to induce sperm capacitation and the acrosome reaction. After cryopreservation, however, CLC-treated and control sperm underwent capacitation and the acrosome reaction at similar rates regardless of the method used to induce capacitation and the acrosome reaction. The primary concern for CLC-treated sperm, however, is whether this treatment would affect in vitro or in vivo fertility. Adding either control or CLC-treated cryopreserved sperm to bovine oocytes in vitro resulted in similar oocyte cleavage rates and blastocyst formation rates. In addition, when inseminated into heifers, pregnancy rates for control and CLC-treated sperm were also similar. Therefore, treating bull sperm with CLC permits greater numbers of sperm to survive cryopreservation while preserving the fertilizing potential of each individual sperm.

Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK.

Specialized membrane microdomains known as lipid rafts are thought to contribute to G-protein coupled receptor (GPCR) signaling by organizing receptors and their cognate signaling molecules into discrete membrane domains. To determine if the GnRHR, an unusual member of the GPCR superfamily, partitions into lipid rafts, homogenates of alpha T3-1 cells expressing endogenous GnRHR or Chinese hamster ovary cells expressing an epitope-tagged GnRHR were fractionated through a sucrose gradient. We found the GnRHR and c-raf kinase constitutively localized to low density fractions independent of hormone treatment. Partitioning of c-raf kinase into lipid rafts was also observed in whole mouse pituitary glands. Consistent with GnRH induced phosphorylation and activation of c-raf kinase, GnRH treatment led to a decrease in the apparent electrophoretic mobility of c-raf kinase that partitioned into lipid rafts compared with unstimulated cells. Cholesterol depletion of alpha T3-1 cells using methyl-beta-cyclodextrin disrupted GnRHR but not c-raf kinase association with rafts and shifted the receptor into higher density fractions. Cholesterol depletion also significantly attenuated GnRH but not phorbol ester-mediated activation of extracellular signal-related kinase (ERK) and c-fos gene induction. Raft localization and GnRHR signaling to ERK and c-Fos were rescued upon repletion of membrane cholesterol. Thus, the organization of the GnRHR into low density membrane microdomains appears critical in mediating GnRH induced intracellular signaling.