Inhibition of Acrosomal Enzymes by Gossypol Is Related to Its Antifertility Action

ＩｎｈｉｂｉｔｉｏｎｏｆＡｃｒｏｓｏｍａｌＥｎｚｙｍｅｓｂｙＧｏｓｓｙｐｏｌＩｓＲｅｌａｔｅｄｔｏＩｔｓＡｎｔｉｆｅｒｔｉｌｉｔｙＡｃｔｉｏｎＹＵＡＮＹｕ－ｙｉｎｇ;（袁玉英）ＺＨＡＮＧＹａｎ－ｌｉｎ;（张燕林）ＳＨＩＱｉ－ｘｉａｎ（石其贤）（Ｚｈｅ...     

A mechanical model for elongation of the acrosomal process in Thyone sperm

We present a mechanical model for the elongation of the acrosomal process in Thyone sperm based upon osmotically driven hydrostatic forces.     

Evaluation of rabbit sperm acrosomal integrity and fertilizing ability by use of vital stains.

Three staining procedures to detect sperm acrosome integrity were compared via electron microscopy. Stains were applied to epididymal, freshly ejaculated, in vivo capacitated, and sonicated sperm cells in addition to spermatozoa displaying sequentially removed plasma and outer and inner acrosomal membranes. Sequential membrane removal procedures resulted in removal of plasma membranes from 73% of all sperm cells, removal of plasma and outer acrosomal membranes from 74% of all sperm cells, and removal of plasma and outer and inner acrosomal membranes from 87% of all sperm cells as determined by electron microscopy. Live/dead staining results were not statistically different from subjective microscopic motility evaluations (P less than 0.005) for epididymal, sonicated, freshly ejaculated, and in vivo capacitated sperm samples. All three stains assessed were similarly capable of detecting the acrosome status of freshly ejaculated and of sonicated spermatozoa compared to data obtained by electron microscopy (P = 0.010). However, only the Bryan-Akruk stain afforded data that were closely correlated with data obtained via electron microscopy for all sperm types assessed; the latter included in vivo capacitated spermatozoa and sperm cells rendered free of plasma membranes. Results confirmed an earlier report by successfully effecting sequential removal of rabbit acrosomal membranes and documented use of the Bryan-Akruk acrosomal stain for evaluation of sperm cell populations for fertilizing ability. These findings should prove useful in further investigations of mechanisms involved in achievement of fertilizing ability by rabbit spermatozoa.     

Activation of mammalian oocytes by a factor obtained from rabbit sperm

In this study a fraction was prepared from rabbit sperm that activated rabbit and mouse oocytes following injection into the cytoplasm. The sperm factor activated oocytes exhibited cortical granule exocytosis, pronuclear formation, and cleavage. The sperm factor was soluble in aqueous solution and was not active extracellularly. Unlike most artificial activation methods that are only effective with aged oocytes, the sperm factor activated recently ovulated oocytes. The factor appears to be a protein or associated with a protein but not an acrosomal protein. Fractions from both mouse and bull sperm did not activate rabbit or mouse oocytes. Their inactivity may be owing to the techniques used to recover the fractions or differences between species in sperm morphology and fertilization processes. These observations support the hypothesis that oocyte activation is induced by a factor within sperm that is released into the cytoplasm of the oocyte at the time of sperm-oocyte fusion.     

Release of acrosomal enzymes following in vitro capacitation of ejaculated rabbit spermatozoa

Significant release of the acrosomal enzymes arylsulfatase, β-N-acetylhexosaminidase and hyaluronidase was observed following the treatment of ejaculated rabbit spermatozoa for 12 hours in 20% rabbit serum for inducing in vitro capacitation, and these sperm were capable of in vivo fertilization; however, the treatment of sperm for 15 minutes in high ionic strength (380 mOsm/kg) or low ionic strength medium (305 mOsm/kg) for in vitro capacitation did not result in any significant release of the above enzymes nor were the sperm capable of in vivo fertilization. Serum-treated spermatozoa remained significantly motile following the 12 hour treatment, 51% underwent the acrosome reaction and were capable of fertilizing 66% of the ova in vivo. Identical serum treatment of lysosomes from rabbit liver resulted in a comparable release of the lysosomal enzymes. Serum treatment for in vitro capacitation resulted in vesiculation of the anterior margin of half the spermatozoa, but left their inner acrosomal membranes and equatorial segments intact. A biochemical relationship between the release of acrosomal enzymes and capacitation is suggested.     

Secretion patterns and effect of prostate-derived granules on the sperm acrosome reaction of rabbit buck

There is increasing evidence that the particulate fraction of seminal plasma plays an important role in reproduction of several mammalian species. However, the origin and role of these granules in the physiology of rabbit spermatozoa is partially unknown. The aim of this study was to investigate the implication of prostate gland in the production and secretion of granules into the rabbit semen and the role of prostate-derived granules in the sperm acrosome reaction. Light and electron microscopy of the prostate gland showed that the anterior and middle tracts of the prostate (namely the proprostate and prostate, respectively) are chiefly implicated in the secretion of granules of different size: smaller granules (SG; 0.5 μm) and large granules (LG; 4 μm). Two major patterns of secretion were identified, based on electron microscope views: storage granules (large granules) seem to empty inner smaller granules directly into the duct by exocytosis, or the storage vesicle itself is released in toto into the ducts (diacytosis). In vitro experiments using granules from vasectomized rabbits, to exclude testicular origin of granules, showed that granules reduce the acrosome reaction of Percoll-selected spermatozoa, independently of the size. Interestingly, spermatozoa incubated with heat-treated granules showed a higher sperm acrosome reaction rate, suggesting a potential role of granule-derived proteins in this process. Inhibition of the acrosome reaction is a crucial event in rabbit reproduction; ejaculated spermatozoa have to wait for a long time (8-16 h) for egg availability in the female tract after mating. Taking together, our results demonstrate that prostate granules secreted either by exocytosis or diacytosis can preserve spermatozoa fertilizing ability, by preventing sperm acrosome reaction. The type of granule-derived proteins or other macromolecules implicated in this process should be further investigated.     

Inhibition of bull and rabbit sperm enzymes by alpha-chlorohydrin.

High concentrations of alpha-chlorohydrin were found to inhibit hyaluronidase, beta-glucuronidase, and aryl sulphatases in bull and rabbit spermatozoa, but not acrosin and neuraminidase. Preincubation of the enzyme and alpha-chlorohydrin was essential to achieve the maximum inhibition which was irreversible.     

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN₂) and then preserved in the LN₂. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN₂. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.     

Morphological and Biochemical Alterations of Abalone Testicular Germ Cells and Spawned Sperm and their Fertilizing Ability

In this study, we aimed to detect morphological and biochemical changes in developing germ cells (Gc), testicular sperm (Tsp), and spawned sperm (Ssp) using capacitation-associated characteristics. Gradual changes in the profiles of two membrane proteins, namely NaCl- and detergent-extractable proteins, were observed as compared Gc with Tsp and Tsp with Ssp. These membrane modifications were accomplished mostly through the introduction of new protein sets, both peripheral and integral, into Tsp and Ssp membranes. Activation of serine proteases, particularly in Ssp detergent-extracted proteins with the molecular masses of 38-130 kDa was evident and marked a major difference between Ssp and Tsp. An increase in the level of tyrosine phosphorylation of the proteins ranging from 15 to 20 kDa was noted in Tsp and remained constant in Ssp. Specifically, these three capacitation-associated characteristics could be detected in Ssp, possessing full fertilizing capacity. The lack of an activated proteolytic activity in Tsp resulted in a delayed fertilization, but not affected fertilizing ability. We believe that these characteristics should be advantageous in predicting abalone sperm fertilizing capability, particularly in cases when isolated germ cells or purified Tsp are used in place of spawned sperm in abalone aquaculture.     

Effects of flaxseed dietary supplementation on sperm quality and on lipid composition of sperm subfractions and prostatic granules in rabbit

Lipids are the main structural/functional components of the sperm, and their composition may undergo a series of modifications in relation to either physiologic events (capacitation and acrosome reaction) and/or diet. The goals of the current study were (1) to investigate whether a flaxseed (FS) dietary supplementation could affect the lipid and fatty acid profile of sperm subfractions and of prostatic granules (PGs) and (2) to evaluate the effects of dietary FS on rabbit buck semen quality. Accordingly, 20 adult New Zealand White rabbits were fed ad libitum a control diet (CO) or a diet supplemented with 5% extruded FS. Integration of diet with FS, as a consequence of the linolenic acid (C18:3n-3; LNA; 56%), increased the dietary n-3/n-6 ratio and resulted in a substantial rearrangement of sperm fatty acid composition at the subcellular level, mainly of polyunsaturated fatty acid (PUFA)n-3 (8.3% vs. 14.3%, P < 0.05). The lipid and fatty acid profiles of sperm tail membrane were the most affected, undergoing the following significant changes: (1) a reduction by half of linoleic acid (C18:2n-6; LA) and docosapentaenoic acid (22:5n-6; DPA), and a reduction of cholesterol (−70%); (2) a concomitant increase of LNA (+65%), docosahexaenoic acid (22:6n-3; DHA; +83%), and of oleic acid (C18:1n-9, +61%). As a consequence, the sperm of FS-fed rabbits had a twice higher n-3/n-6 ratio and phospholipid/cholesterol ratio compared with the control sperm. These changes might have been on the basis of the higher responsiveness to hypo-osmotic solution and, hence, the higher sperm track speed observed for the FS group. Also, the membrane integrity and viability of the LNA-enriched sperm were both improved. On the other hand, the presence of lignans in FS might have accounted for the reduction of sperm cholesterol in the semen of FS-treated rabbits. The responsiveness of sperm to acrosome reaction was not affected by the dietary treatment probably due to supranutritional level of vitamin E and to the higher number of PGs, which are known to play a key role in sperm capacitation. In conclusion, our data showed for the first time that the integration of FS into the rabbit diet may improve sperm quality by modifying the sperm lipid composition and that the sperm subfractions and the PGs respond differently to the FS-induced lipid manipulation.     

Phospholipids of rabbit and bull sperm membranes: Structural order parameter and steady-state fluorescence anistropy of membranes and membrane leaflets

The plasma (PM), outer acrosomal (OAM), and inner acrosomal membranes (IAM) were isolated from rabbit and bull spermatozoa and the major phospholipids characterized. Choline-containing phospholipids, phosphatidylcholine (PC) and sphingomyelin (SM), constituted more than 60% of the total phospholipids (TPL) in all membranes of both species. Approximately more than 50% of PC in membrane preparations contained some form of ether linkage. Compared to OAM and IAM, cholesterol to phospholipid molar ratio was highest in PM of both species. Contrarily, protein to phospholipid ratio for PM was lowest compared to other membranes. The sphingomyelin to phosphatidylcholine ratio increased in the direction from PM to OAM to IAM. The hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to examine both the steady-state fluorescence anisotropy parameters and structural order parameter S_DPH. The data showed higher rigidity in rabbit spermatozoa compared to bull spermatozoa (S_DPH = 0.7582 and S_DPH = 0.7326). In both species OAM had higher rigidity compared to the other two membranes (S_DPH(OAM) = 0.7809, S_DPH(PM) = 0.7308, and S_DPH(IAM) = 0.7481 for bull; S_DPH(OAM) = 0.8091, S_DPH(PM)= 0.7857, and S_DPH(IAM) = 0.7663 for rabbit). The inner leaflets of bull and rabbit spermatozoal membranes had significantly higher rigidity than the outer leaflets (for inner leaflet: rabbit-S_DPH(PM) = 0.8391, S_DPH(OAM) = 0.8149, and S_DPH(IAM) = 0.7675; bull-S_DPH(PM) = 0.8000, S_DPH(OAM) = 0.7990, and S_DPH(IAM) = 0.7990, and for outer leaflet: rabbit-S_DPH(PM) = 0.7021, S_DPH(OAM) = 0.7145, and S_DPH(IAM) = 0.6867; bull-S_DPH(PM) = 0.6986, S_DPH(OAM) = 0.5980, and S_DPH(IAM) = 0.7388). © 1993 Wiley-Liss, Inc.     

Effects of gossypol on the motility of mammalian sperm

The effects of the male contraceptive gossypol on the motility of mammalian spermatozoa are reviewed. The role of sperm motility in the processes of fertilization and the effect of the drug on these processes determine its effectiveness as a contraceptive. The promising male contraceptive potential of gossypol is discussed in the context of the serious adverse effects of the agent.     

Acrogranin,an acrosomal cysteine-rich glycoprotein,is the precursor of the growth-modulating peptides,granulins, and epithelins,and is expressed in somatic as well as male germ cells

Spermatogenesis is a unique system of differentiation involving cellular remodeling and the biogenesis of sperm-specific organelles. To study the biogenesis of one such organelle, the acrosome, we have been examining the gene expression, biosynthesis, and targeting of specific acrosomal proteins during mammalian spermatogenesis. An acrosomal marker that we recently purified and began characterizing is acrogranin, a 67,000-molecular-weight glycoprotein originally isolated from guinea pig testes. This glycoprotein is detected in pachytene spermatocytes and is found later in the acrosomes of developing spermatids and sperm. Immunoblotting of several tissues and immunofluorescent localization in frozen sections of guinea pig testes suggested that acrogranin was a germ cell-specific glycoprotein that was expressed meiotically and post-meiotically. However, Northern blot analysis demonstrated that the mRNA for acrogranin was ubiquitously expressed in all guinea pig and mouse tissues examined. Furthermore, the primary structures of guinea pig and mouse acrogranins, deduced from the cDNA sequences, reveal that this glycoprotein is a cysteine-rich molecule with a motif that is tandemly repeated seven times, very similar to that of the human epithelin/granulin precursor. We conclude that guinea pig and mouse acrogranins are homologues of the precursor of the human and rat epithelin/granulin peptides previously demonstrated to have growth-modulating properties. © 1993 Wiley-Liss, Inc.     

Comparative histochemical observations on the contributions of the acrosomal vesicle and granule to the acrosomal cap in the spermatozoa of mammals

Summary By applying the periodic acid-Schiff technique to frozen sections of material fixed in weak Bouin's solution and extracted with hot pyridine, the contributions of the acrosomal vesicle and granule to the acrosomal cap have been observed in the maturing spermatozoa of the American opossum, guinea pig, rabbit, marmoset and chimpanzee. In the spermatids of the opossum, the acrosomal granule is not developed and the thin, homogeneous carbohydrate layer of the acrosomal cap is derived from the concentrated material of the acrosomal vacuole. In the guinea pig, both the acrosomal vesicle and granule make the greatest contributions to the carbohydrates of the acrosomal cap; these continue to maintain their identity in the head of mature sperm. In the rabbit, marmoset and chimpanzee, the carbohydrate material of acrosomal vesicle origin is less developed and constitutes a thin layer in the lateral portions of the acrosomal cap; the carbohydrate material in its anterior portions is mainly derived from the acrosomal granule which becomes flattened; there is also a slight thickening of the intermediate layer of the acrosomal cap in this region. The distinction between the carbohydrate contributions from the acrosomal vesicle and granule disappears in the fully formed acrosomal cap which shows a homogeneously PAS-positive intermediate layer. In the chimpanzee, the acrosomal vesicle makes a relatively very small contribution to the carbohydrate layer of the acrosomal cap which is mainly formed by spreading of material from the acrosomal granule.     

Inhibition by gossypol of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis

Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca²⁺-dependent protein kinase from pig testis. Gossypol inhibited Ca²⁺-dependent activity of the enzyme without affecting its basal activity. The IC₅₀ value (concentration causing 50% inhibition) was 31 μM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP (K_i = 31 μM) or lysine-rich histone (K_i = 30μM), and competitively with respect to phosphatidylserine (K_i = 2.1 μM). With Ca²⁺, irrespective of the presence or absence of 1,3-diolein, the compound lowered V_max and increased the apparent K_a for Ca²⁺. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrate in the testis for the enzyme located in nucleosome), with an IC₅₀ value of 88 μM. These results suggested that a phospholipid-sensitive Ca²⁺-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis.     

Epitope topology and removal of mouse acrosomal plasma membrane by P12-targeted immunoaggregation

P12 is a Kazal-type trypsin inhibitor that has been purified from mouse seminal vesicle secretion. We observed a slight impact of P12 on sperm capacitation, and demonstrated the removal of plasma membrane overlaying the acrosome region by immunoaggregation of P12 on mouse sperm. Further, we compared the immunoreactivity of P12 antibody to ten P12 variants, including six single-site mutated mutants (R19L, Y21V, D22G, R43G, K44S, and R45T), two multisite mutated mutants (R43G/K44S/R45T and L50H/R52G/K53A), and two deletion mutants (Nd10 and Cd8) in which 10 and 8 residues were deleted from the N- and C-terminals, respectively. We found that the N-terminal region, 43RKR45, and the C-terminal region, but not R19, Y21, and D22, are involved in the three epitopes that reside on one side and are three-dimensionally distant from R19, Y21, and D22 on the P12 molecule. Based on the epitope topology, we elucidated the structural basis by which P12 antibody immunoaggregated P12 on sperm head.     

Study of nuclear and acrosomal sperm morphometry in ram using a computer-assisted sperm morphometry analysis fluorescence (CASMA-F) method

The aim of this study was to develop a new method that allows morphometric assessment of the sperm nucleus and acrosome in the ram using fluorescence microscopy and free software. The study was divided into three experiments. In the first experiment, semen smears from 20 ejaculates were fixed and labeled with a propidium iodide–pisum sativum agglutinin (PI/PSA) combination. Digital images of the sperm nucleus, acrosome, and whole sperm head were captured and analyzed using the ImageJ program. The computer-assisted sperm morphometry analysis fluorescence (CASMA-F) method used allowed the differentiation, capture, and morphometric analysis of most sperm nuclei, acrosomes, and whole heads with high precision and the assessment of the acrosomal status. In the second experiment, sperm nuclear morphometry by CASMA-F was compared by staining with the PI/PSA combination and staining with Hoechst 33342 as in previous studies. Similar results were obtained using both methods. In the third experiment, CASMA-F with PI/PSA was compared with a more conventional CASMA method (semen smears stained with Hemacolor (HEM) and processed with the ISAS commercial software, HEM). Spermatozoa displayed a bigger size when processed with CASMA-F than with HEM method in all primary sperm head morphometric parameters, but results using both methods were correlated. It was concluded that the CASMA-F method allows the simultaneous assessment of sperm nucleus, acrosome, and head in the ram.     

Store-operated calcium channels trigger exocytosis of the sea urchin sperm acrosomal vesicle

The acrosome reaction (AR) of sperm is a prerequisite for fusion with the egg. In sea urchins, the complete AR (CAR) consists of exocytosis of the acrosomal vesicle (AV) and polymerization of acrosomal actin to form the approximately 1 micro m long acrosomal process. The fucose sulfate polymer (FSP) of egg jelly stimulates Ca(2+) entry through two distinct Ca(2+) channels and induces the CAR. Here we report that the second channel is blocked by SKF96365 (SKF), an inhibitor of store-operated channels. SKF also blocks the thapsigargin (TG), trifluoperazine (TFP), and calmidizolium (CMZ) stimulated Ca(2+) entry into sperm. These data indicate that the second Ca(2+) channel is a store-operated channel (SOC) that may be regulated by calmodulin. The TG, TFP, and CMZ-induced intracellular Ca(2+) elevations are similar to those induced by FSP, but the sperm acrosomal process does not polymerize. An antibody to bindin, the major protein of the AV, showed that in a significant percentage of these drug-treated sperm, the AV had undergone exocytosis. When NH(4)Cl was added to increase intracellular pH, the TG-treated sperm polymerized actin to form the acrosomal process. We conclude that the second Ca(2+) channel of sea urchin sperm is a SOC that triggers AV exocytosis.