Sperm Membrane Cholesterol Research Articles

Assessment of quality in specific fractions of Large White Yorkshire boar semen

Boar semen is voluminous and ejaculated as jets or fractions of pre-sperm, sperm rich (SRF) and post-sperm rich fractions. Recent studies have reported more resilient characteristics of sperm in initial portions of SRF towards cold shock and cryopreservation. The present study was conducted to assess the quality of specific fractions of SRF, namely, first 10mL of SRF (F1) and rest of SRF (F2) in Large white Yorkshire (LWY) boar semen. Ejaculates were collected using gloved-hand technique and were subjected to quality assessments of volume, pH, sperm progressive motility, concentration, plasma membrane integrity, abnormality, acrosome integrity and sperm membrane cholesterol. Upon statistical analysis, significant differences were noticed in volume, pH, sperm concentration and sperm membrane cholesterol between fractions of the ejaculate.

Effect of cholesterol supplementation on cryosurvival of goat spermatozoa.

Aim:Sperm membrane cholesterol influences cryodamage during cryopreservation. The present study was carried out to evaluate the effect of varying cholesterol levels in Tris based extenders on the freezability of sexually healthy Malabari buck semen.Materials and Methods:A total of 48 ejaculates from two adults healthy sexually healthy Malabari bucks were utilized for the study. The collected and pooled ejaculates were divided into four groups with Group I serving as Control - I, Group II and III were treated with 1 mg and 2 mg of cholesterol-loaded-cyclodextrin (CLC)/120 × 106 spermatozoa, respectively, and Group IV, treated with 1 mg methyl-β-cyclodextrin (MβCD) served as Control - II. Manual freezing was carried out to cryopreserve the treated and control spermatozoa.Results:Treatment of semen samples with CLC resulted in improved maintenance of sperm motility at pre-freeze and post-thaw stages of cryopreservation without affecting hypo-osmotic swelling response. Treatment of semen with 1 mg of CLC/120 × 106 spermatozoa was observed to be better than treatment with 2 mg of CLC/120 × 106 spermatozoa. In general, MβCD treatment was found to result in significantly lower sperm characteristics than those of Control - I and CLC treatment at pre-feeze and post-thaw stages and when incubated up to 4 h.Conclusion:Cholesterol treatment of sexually healthy Malabari buck semen was found to hold promise for improving cryopreservability of spermatozoa.

71 EFFECT OF CHOLESTEROL-LOADED CYCLODEXTRINS ON PRE- AND POST-THAW FUNCTION OF FELID SPERM

Propagation of genetically diverse felid populations would benefit from more effective assisted reproduction strategies, including enhanced methods for sperm cryopreservation. In felids, sperm cryopreservation has been improved by substituting soy-lecithin for egg yolk in cryomedium (Vick et al. 2012 Theriogenology 78, 2120–2128). In other species, such as elephants (Kiso et al. 2012 Reprod., Fert. Dev. 24, 1134–1142) and cattle (Purdy et al. 2004 Cryobiology 48, 36–45), the addition of cholesterol-loaded cyclodextrins (CLC) to sperm before freezing has been shown to produce superior cryopreservation results. In this study, our objectives were to (1) assess cholesterol content of cat sperm membranes and capacitation status following incubation with CLC; (2) evaluate post-thaw sperm motility, acrosome status, and fertility in vitro following CLC treatment and freezing in a soy-based cryomedium; and (3) conduct a preliminary assessment of cholesterol content in nondomestic cat sperm. Freshly collected domestic cat sperm (n = 2 males, 3–4 ejaculates/male) were incubated with CLC (0, 1.5, or 3.0 mg mL–1), and cholesterol levels were measured using an Amplex Red Cholesterol Assay. Sperm aliquots from each CLC concentration were treated with calcium ionophore (2 μM, 30 min) during in vitro incubation and stained with fluorescein isothiocyanate/PNA to evaluate induced acrosomal loss. To assess post-thaw parameters, cat sperm treated with CLC were frozen in straws using soy-lecithin cryomedium, thawed, and cultured in vitro over time. To evaluate fertility, oocytes were collected laparoscopically from gonadotropin-treated domestic cats (n = 7 females, 147 oocytes total) and inseminated with low numbers of thawed-frozen sperm pretreated with 0 or 1.5 mg mL–1 CLC. Data were analysed using ANOVA and mean differences assessed with Fisher l.s.d. or chi-squared analysis. Sperm cholesterol levels were increased (P < 0.05) after exposure to both 1.5 and 3.0 mg mL–1 CLC. Prefreeze motility was decreased (P < 0.05) and capacitation was delayed at 3.0 mg mL–1 CLC relative to treatment with 0 or 1.5 mg mL–1 CLC. Both post-thaw motility and percentage of acrosome intact sperm were reduced (P < 0.05) with the highest CLC concentration, but results were similar (P > 0.05) for 0 and 1.5 mg mL–1 CLC. Fertilization percentages did not differ (P > 0.05) between treatment groups (0 CLC, 33.3%, 25/75; 1.5 mg mL–1 CLC, 26.4%, 19/72). Preliminary results from a single cheetah (Acinonyx jubatus) and single fishing cat (Prionailurus viverrinus) suggest that sperm membrane cholesterol may be lower compared to the domestic cat. Cholesterol content appeared to increase in both species after exposure to 1.5 mg mL–1 CLC. In summary, our findings suggest CLC treatment increased cholesterol content of felid sperm membranes. The higher CLC concentration was detrimental to sperm motility, capacitation, and post-thaw sperm traits. The lower CLC concentration did not improve post-thaw sperm function in domestic cats.Research supported by the Procter & Gamble Wildlife Conservation Scholarship Program.

121 ACROSOME REACTION AS EFFECT OF ADDITION OF CHOLESTEROL TO BOAR SPERM MEMBRANES

Altering the lipid composition of sperm plasma membranes not only affects the ability of sperm capacitation and acrosome reaction, it also affects the way sperm respond to cryopreservation. The objective was to determine if increasing sperm membrane cholesterol levels, by adding cholesterolloaded cyclodextrin (CLC) to boar sperm, alter the cryopreservation sperm to undergo acrosome reaction in vitro. The CLC was prepared as described by Purdy and Graham (2004) with some modification: 200 mg of cholesterol was dissolved in 1 mL of chloroform, and 1 g of methyl-β-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass Petri dish and the solvents removed using a hot plate for 24 h. The resulting crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL of BTS at 37°C. Ejaculates (n = 5) from 5 boars were collected, diluted 1:1 in Beltsville thawing solution, and kept for 2 h at 22°C. Afterward, the ejaculates were put at 15°C/ for 60 min. Later, the ejaculates were centrifuged at 15°C at 400g/10 min, the pellet was suspended to 120 million cells in cooled diluent (80 mL of lactose solution 11%, 20 mL of egg yolk) and divided in 2 treatments: control and 1.5 mg of CLC/mL. These treatments were incubated for 15 min at 15°C. The samples were cooled to 5°C/90 min period and diluted 1:1 with freeze diluent (72.5-mL lactose solution 11%, 6 mL of glycerol, 1.5 mL of Equex). The sperm were packaged into 0.5-mL straws and frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen. Straws were thawed in a water bath 37°C/30 s. A 90% Percoll solution was prepared by diluting 1 mL of 10× PBS with 9 mL of Percoll. A 35% Percoll solution was then prepared by diluting 90% Percoll (0.67 mL) with Medium 199 (1.33 mL). Frozen/thawed spermatozoa (2 mL) were then layered onto 2 mL of 35% Percoll solution in a 15-mL conical tube and centrifuged at 400g/5.5 min. The resulting pellet was suspended with Medium 199 to 100 million cells/mL, and the cells were stained with 5 μL of PI (1 mg mL-1 in water) and 10 μL of FITC-PNA (1 mg mL-1 in 10× PBS). The cells were incubated for 5 min at room temperature to allow PI and FITC-PNA to become incorporated. The acrosomal status of viable cells for each treatment was then determined by epifluorescence microscope at 400× magnification, and the percentage of acrosome reacted cells was calculated as the proportion of FITC-PNA stained and PI negative cells (acrosome reacted, live)/total live cells (PI negative, FITC-PNA positive and negative). Treatment differences for acrosome reaction were determined using ANOVA. The addition of CLC to boar sperm before cryopreservation resulted in higher acrosome reaction (28%) compared with control cells (22%; P < 0.05). Several studies evaluated the ability of bull and stallion sperm treated with CLC to capacity and acrosome react. Adding cholesterol might alter the plasma membrane structure, improving the acrosome reaction in CLC-treated boar spermatozoa. FAPEMIG, Piglandia, CNPq, FACEPE.

Estrogen Sulfotransferase Is Highly Expressed Along the Bovine Epididymis and Is Secreted Into the Intraluminal Environment

Estrogen is found in high concentrations in the excurrent duct, where it regulates the expression of genes involved in water reabsorption. Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes specific sulfonation with a high affinity for estrogens. Because sulfated estrogens do not bind to estrogen receptors, they are considered to be hormonally inactive. EST may thus determine where along the male tract estrogenic environment predominates. Sulfotransferase activity increases along the epididymis and may also play a role in sperm physiology during the epididymal transit. Using a bovine model, we investigated the distribution of EST along the excurrent duct and the possibility that sterols associated with spermatozoa can be substrates of this enzyme. Reverse transcription polymerase chain reactions showed that mRNA encoding EST was expressed in the testis and all along the epididymis. A highly specific antiserum was raised against the bovine recombinant EST and used in Western blots and immunohistologic studies. Western blots of tissue homogenates showed that EST was localized all along the excurrent duct with a higher signal in the caput and corpus epididymidis. EST was detectable in the intraluminal compartment only in the caput epididymidis, where it was associated with epididymosomes and spermatozoa. EST was undetectable in different fractions of fluids collected in the cauda segment. In immunohistologic studies, EST was restricted to the acrosomal region of the caput, but not the cauda epididymal spermatozoa, and detectable in the cytoplasm of the epithelium bordering the lumen all along the epididymis as well as in the rete testis and vas efferens. This enzyme was also associated with the nucleus in the caput and corpus as well as with the apical membrane of the corpus epididymal epithelium. When recombinant EST was incubated in vitro in the presence of caput and cauda spermatozoa, it was able to add sulfate to sperm membrane cholesterol. Our study shows that EST is present in both the intracellular and intraluminal compartments of the epididymis, suggesting that this enzyme plays different roles along the excurrent duct.

Relationship Between Standard Semen Parameters, Calcium, Cholesterol Contents, and Mitochondrial Activity in Ejaculated Spermatozoa From Fertile and Infertile Males

To correlate levels of cholesterol (CH), calcium (Ca2+), and mitochondrial activity (MA) with the standard semen parameters and to compare them between fertile and infertile men. We studied 151 semen samples from infertile (n = 60) or fertile (n = 91) males. Basic sperm parameters were analyzed. Ca2+ and CH concentrations on seminal plasma were determined by enzymoimmunoanalysis. Intracellular Ca2+ and CH concentrations in the sperm plasma membrane and mitochondrial activity by fluorometry. There was a significant positive correlation between sperm membrane CH and sperm morphology. Intracellular Ca2+ was lower in infertile patients compared to fertile. No differences were found regarding Ca2+ and CH concentrations in seminal plasma. MA is directly and strongly related with sperm motility. Intracellular concentrations of Ca2+ and the proportion of CH in the sperm membrane are two important markers of the sperm quality due to its direct relationship with sperm morphology and fertility potential.

The role of cholesterol efflux in regulating the fertilization potential of mammalian spermatozoa.

Following spermatogenesis and spermiogenesis, mammalian spermatozoa leaving the testis appear to be morphologically mature but clearly are immature from a functional standpoint; that is, they have acquired neither progressive motility nor the ability to fertilize a metaphase II‐arrested egg. Although progressive motility is acquired and signaling pathways mature during sperm transit through the epididymis, complete fertilization capacity in vivo is conferred only during residence in the female reproductive tract. Similar observations have been made using a variety of in vitro assays, suggesting that a series of events, some initiated by environmental cues, confer on sperm the ability to fertilize the egg. This acquired capacity to fertilize was first observed by Austin (1) and Chang (2), who demonstrated that freshly ejaculated sperm cannot fertilize eggs until they reside in the female reproductive tract for a finite period of time. All of the cellular events that allow the ejaculated sperm to fertilize an egg were subsumed into a single phenomenon that was termed “capacitation.” The ability to capacitate sperm in vitro has been of great importance to both scientists and clinicians, who have used it to study the basic biology of fertilization and to develop various assisted reproductive technologies for humans and other species. Work by many investigators has established that the process of fertilization, not surprisingly, represents a series of elegant intercellular communication and cellular activation events (3‐5). Sperm functions such as motility and capacitation in the female reproductive tract are likely modulated by environmental cues in the luminal fluid, as well as by interactions with oviductal epithelium or other female tissues (6). When sperm arrive in the oviduct and encounter the ovulated, metaphase II‐arrested egg enclosed in its cumulus cell matrix, a complex series of cell-cell and cell-ECM interactions ensues, initiating cellular signaling events that permit the fusion of the sperm and egg plasma membranes. Several of these cell-matrix and cell-cell interactions involve novel gamete surface proteins and matrices. Signal transduction events leading to gamete activation, in particular sperm acrosomal exocytosis and egg cortical granule secretion, share some features with signaling events described in somatic cells. Sperm membrane cholesterol efflux contributes to one such novel signaling mechanism that controls sperm capacitation, and the details of this effect are now beginning to be understood at the molecular level. Knowledge of how cholesterol efflux occurs in these cells, as well as how this efflux is integrated with transmembrane signaling to regulate sperm function, may reveal much about the fertilization process and may also provide insights into the role and dynamics of membrane cholesterol efflux in somatic cell function. Here, we offer a short overview of the role of cholesterol efflux in regulating sperm capacitation, with an aim toward identifying areas of future investigation that may ultimately pro

Current status of sperm cryopreservation: why isn't it better?

Cryopreservation extends the availability of sperm for fertilization; however, the fertilizing potential of the frozen-thawed sperm is compromised because of alterations in the structure and physiology of the sperm cell. These alterations, characteristics of sperm capacitation, are present in the motile population and decrease sperm life-span, ability to interact with female tract, and fertilizing ability. The etiology of such alterations may represent a combination of factors, such as inherited fragility of the sperm cell to withstand the cryopreservation process and the semen dilution. Although the former is difficult to address, approaches that make-up for the dilution of seminal fluid may be sought. The aim of this work is to review aspects of sperm cryopreservation paralleled by events of capacitation and evaluate the possible roles of sperm membrane cholesterol, reactive oxygen species, and seminal plasma as mediators of cryopreservation effects on sperm function.

Effects of methyl-beta-cyclodextrin on cryosurvival of boar spermatozoa.

Methyl-beta-cyclodextrin (MBCD), which leads to the stimulation of cholesterol efflux from the cell membrane, was examined for its ability to increase the cryosurvival of spermatozoa from Göttingen miniature pigs. The intactness of the acrosome and various motion parameters of spermatozoa after freezing and thawing were used to monitor cryosurvival. Spermatozoa were exposed to MBCD or a combination of MBCD and cholesterol-3-sulfate over a period of 3 hours while being cooled slowly from 25 degrees C to 5 degrees C, and were subsequently cryopreserved by the pellet method. After freezing and thawing, the acrosomal status was monitored by fluorescein isothiocyanate-labeled peanut agglutinin, and the motion parameters were assessed with a computer-assisted sperm motility analysis system. In post-thaw spermatozoa the values of the intact acrosome, motility, progressive motility, progressive velocity, straightness, and linearity of the cell path increased greatly with the concentration of MBCD (P < .05). In contrast, the lateral head displacement amplitude and the beat cross-frequency of post-thaw spermatozoa were not different among all treatments. On the contrary, the addition of cholesterol-3-sulfate to the diluent containing MBCD abolished the protective effect against freeze-thaw injury that MBCD provides to spermatozoa. These results indicate that cryosurvival of boar spermatozoa is enhanced by exposure to MBCD before freezing. This protective effect of MBCD may be due to active depletion of sperm membrane cholesterol.

Major proteins of bovine seminal plasma and high-density lipoprotein induce cholesterol efflux from epididymal sperm.

One of the hypotheses to explain the mechanism of capacitation involves the loss of sperm membrane cholesterol. Here, we studied whether or not the major proteins of bovine seminal plasma designated as BSP-A1, -A2, -A3, and -30-kDa (collectively called BSP proteins), which are implicated in sperm capacitation, induce cholesterol efflux. When epididymal sperm were labeled with [3H]cholesterol and incubated with bovine seminal plasma (0.05-2%) or BSP proteins (20-120 microg/ml) for 8 h, the sperm lost [3H]cholesterol (3.6-fold and 3-fold, respectively). The same results in the presence of BSP-A1/-A2 were obtained (3.5-fold) by direct determination of cholesterol on unlabeled epididymal sperm. Analysis of efflux particles by ultracentrifugation on a sucrose gradient revealed a single symmetrical peak of radioactivity at 1.14 g/ml. Immunoblotting of the fractions obtained from size-exclusion chromatography of the efflux particles showed that a portion of the BSP proteins were associated with [3H]cholesterol. Heparin (12 microg/ml) alone did not stimulate cholesterol efflux. In contrast, high-density lipoprotein (HDL, 100 microg/ml) alone stimulated cholesterol efflux up to 3.1-fold after 8 h. When labeled epididymal sperm were preincubated for 20 min with BSP-A1/-A2 (120 microg/ml), washed, and incubated with HDL (100 microg/ml) for 8 h, the total cholesterol efflux of the sperm suspension was 51.8 +/- 5.0% compared to 39.3 +/- 1.2% when HDL alone was used. These results indicate that BSP proteins and HDL play an important role in the sperm sterol efflux that occurs during capacitation. Furthermore, the heparin-induced sperm capacitation did not involve the efflux of sperm membrane cholesterol.

Type II Domains of BSP-A1/-A2 Proteins: Binding Properties, Lipid Efflux, and Sperm Capacitation Potential

Bovine seminal plasma contains a family of major proteins (collectively called1BSP proteins) that potentiate sperm capacitation by binding to capacitation factors such as heparin and by stimulating sperm membrane cholesterol efflux. Here, we investigated the structure-function relationship of type II domains of BSP proteins. We isolated from a tryptic digest of citraconylated BSP-A1/-A2 proteins the intact second type II domain (domain b or Db). Similar to native protein, Db bound to heparin-Sepharose, p-aminophenylphosphorylcholine-Agarose and liposomes containing phosphatidylcholine. When assessed for biological function, Db did not stimulate cholesterol efflux from human fibroblasts, a cell model for lipid efflux studies, and from bovine spermatozoa, or potentiate bovine sperm capacitation induced by heparin and high-density lipoproteins. Therefore, type II motifs of BSP proteins represent binding units for sperm membrane choline phospholipids and heparin but the second type II domain of BSP-A1/-A2 alone is not sufficient to stimulate lipid efflux nor is sufficient to potentiate bovine sperm capacitation. Thus, the presence of both type II domains in BSP proteins is essential for the expression of functional properties, namely lipid efflux and sperm capacitation.