Pig zona pellucida 2 (pZP2) protein does not participate in zona pellucida formation in transgenic mice

The zona pellucida, an extracellular matrix surrounding mammalian oocytes, is composed of three or four glycoproteins. It is well known that the zona pellucida plays several critical roles during fertilization, but there is little knowledge about its formation. The purpose of this study is to examine whether a pig zona pellucida glycoprotein 2 (pZP2) would assemble with mouse zona pellucida. A transgene construct was prepared by placing a minigene encoding pZP2 downstream from the promoter of mouse ZP2. The result showed that the transgenic protein was synthesized in growing oocytes but not incorporated into the zona pellucida. Furthermore, the pZP2 transgene did not rescue the phenotype in ZP2-knockout zona-deficient mice. These results indicate that pZP2 does not participate in mouse zona pellucida formation and the zona pellucida is constituted from its component proteins in a molecular species-specific manner between mice and pigs.     

Human ZP4 is not sufficient for taxon-specific sperm recognition of the zona pellucida in transgenic mice

The molecular basis of human fertilization remains enigmatic. Mouse models are often used to study sperm-egg recognition, but the mouse zona pellucida surrounding ovulated eggs contains three proteins (ZP1, ZP2, and ZP3) whereas the human zona contains four (ZP1, ZP2, ZP3, and ZP4). Human sperm are fastidious and recognize human but not mouse eggs. Transgenic mouse lines were established to ascertain whether human ZP4 is the sole determinant of human sperm binding. Human ZP4 expressed in transgenic mice had a molecular mass similar to the range of native protein isoforms and was incorporated into the extracellular zona matrix. Transgenic females were fertile with normal litter sizes. Mouse sperm readily recognized transgenic ovulated eggs, but human sperm did not. We conclude that human ZP4 is not sufficient to support human sperm binding to the zona pellucida in transgenic mice and that other zona proteins may be needed for human gamete recognition.     

Evaluation of bovine zona pellucida characteristics in polarized light as a prognostic marker for embryonic developmental potential

It has previously been demonstrated that zona pellucida imaging of human oocytes using polarized light microscopy is a clinically applicable method for the noninvasive assessment of oocyte quality. This study was designed to investigate whether zona pellucida characteristics of bovine oocytes and zygotes in polarized light may similarly serve as a useful marker for developmental competence in bovine reproductive biotechnologies. Zona birefringence intensity parameters of 2862 oocytes/zygotes were objectively evaluated with an automatic analysis system and correlated with oocyte/zygote quality. In detail, immature oocytes of good quality assessed with brilliant cresyl blue staining showed significantly lower zona birefringence than poor-quality counterparts (P<0.001). After in vitro maturation and classification according to maturational status, the birefringence intensity parameters were significantly different in those oocytes that reached metaphase II compared with arrested stages (P<0.001). Following either parthenogenetic activation or IVF with subsequent in vitro culture in a well-of-the-well system until day 9, superior development as determined by cleavage, blastocyst formation, and hatching ability was associated with lower zona birefringence intensity parameters. When early zygote-stage embryos were selected and assorted in groups based on zona birefringence (high/medium/low), the group of embryos derived from high-birefringence zygotes displayed a significantly compromised developmental potential compared with low-birefringence zygotes. These results clearly show that developmentally competent bovine oocytes/zygotes exhibit lower zona birefringence intensity parameters. Therefore, birefringence imaging of zona pellucida is a suitable technique to predict bovine preimplantation embryo development.     

Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes

Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.     

Molecular evolution of the carboxy terminal region of the zona pellucida 3 glycoprotein in murine rodents

In mammals, before fertilization can occur, sperm have to bind to, and penetrate, the zona pellucida (ZP). In the laboratory mouse, which has been used as a model system for fertilization studies, sperm-ZP binding has been found to be mediated by a region at the carboxy terminal, encoded by exon 7 of the Zp3 gene. This region shows considerable interspecific sequence diversity with some evidence of adaptive evolution in mammals, suggesting that it may contribute to species-specific sperm-ZP binding. However, in a previous study of sequence diversity of ZP3 of three species of Australian murine rodents, we found an identical protein sequence of the region encoded by exon 7. Here, we expand this earlier study to determine the sequence diversity of this region in 68 out of the 130 species of Australasian murine rodents. Maximum likelihood analyses, using representatives of both New Guinean and Australian taxa, provide evidence of positive selection at three codons adjacent to, or within, the putative combining-site for sperm of ZP3, but this was not evident when the analysis was restricted to the Australian taxa. The latter group showed low levels of both intra- and inter-generic sequence divergences in the region encoded by exon 7 of Zp3, with little evidence that this region contributes to species specificity of sperm-ZP binding. These findings suggest that the selective forces acting on the Zp3 exon 7 region during the evolution of the Australasian murine rodents have been variable, and that positive selection has only occurred in a few lineages.     

Impact of feline zona pellucida glycoprotein B-derived synthetic peptides on in vitro fertilization of cat oocytes

Although immunocontraception based on porcine zona pellucida (ZP) proteins is widely applied in many species, it is not suitable for cat contraception due to the lack of cross-reactivity. Since the first ZP gene expressed during oocyte growth in domestic cats is ZPB, we assumed that immunization with feline ZPB (fZPB)-derived synthetic peptides may cause irreversible infertility, which would be preferable in stray cats. Thus, the present study evaluated the immunogenicity and the contraceptive potential of synthetic fZPB peptides. Antigenic epitope sequences were detected via epitope mapping using specific rabbit anti-fZP antibodies. Six peptides representing the recognized epitopes were synthesized subsequently. Two out of six peptides (ZPB amino acid residue 130-149 = P3 and 175-193 = P6) cross-reacted with anti-fZP antiserum in dot blot analysis and ELISA. Coupled to BSA, both peptides were utilized to produce specific antibodies in rats. Despite several booster injections the antibody titers monitored by ELISA did not exceed 1:5000. Both rat antisera were tested for contraceptive potential in cat in vitro maturation/in vitro fertilization (IVF). Antiserum against peptide P3 significantly inhibited sperm binding and fertilization of cat oocytes in vitro (57.3% of sperm binding; 41.5% of fertilization), whereas the inhibition by anti-P6 was not significant. Pre-incubation of sperm cells with both peptides before IVF failed to affect either sperm binding or fertilization (22.3 +/- 3.7 sperm/egg vs 25.5 +/- 5.8 for P3 and 20.7 +/- 4.0 for P6, respectively). In conclusion, antibodies directed against one of the two identified antigenic determinants of fZPB inhibited sperm binding and IVF and therefore showed promising results as a contraceptive. However, the specific immune response and anti-fertile properties of this synthetic vaccine have to be examined in vivo to verify the suitability of its components.     

Hardening of the zona pellucida of unfertilized eggs can reduce polyspermic fertilization in the pig and cow

One of the proposed mechanisms of polyspermy block is an increased resistance of the zona pellucida (ZP) to proteolytic digestion (ZP hardening) as a consequence of cortical granule exocytosis that occurs soon after fertilization. However, evidence is available that the zonae pellucidae of freshly ovulated pig and cow oocytes harden considerably before fertilization. It was thought that such pre-fertilization ZP hardening could be involved in the control of polyspermy, and its lack in the oocytes matured in vitro could be one of the reasons for the extremely high incidence of polyspermy in pig in vitro fertilization (IVF). To test this hypothesis, two different types of cross-linking reagents were employed and their effects on ZP hardening and IVF efficiency were examined. The sulfhydryl-reactive cross-linkers produced a slight hardening of ZP (P<0.001) of treated oocytes compared with control oocytes, and totally inhibited sperm penetration into pig oocytes after IVF. In the cow, sperm penetration into eggs was reduced to 10%. It is proposed that formation of disulfide bonds in ZP or blocking of SH groups in the oocyte plasma membrane proteins prevents sperm penetration. An amine-reactive cross-linker was then assayed and produced strong ZP hardening, increasing the incidence of monospermy in both pig and cow oocytes after fertilization. When the cross-linker concentration was optimized, a 45% improvement for pig IVF efficiency was reached. It is proposed that the observed physiological ZP hardening is a mechanism to control polyspermy, differentially affecting various mammalian species and can be imitated by the use of amine-reactive cross-linkers during IVF.     

Induction of final maturation by sperm penetration in canine oocytes

In contrast to oocytes of most mammals, the canine oocyte is at the germinal vesicle stage at ovulation. Moreover, the bitch is receptive to mating while immature oocytes are present in the oviducts. The aims of this study were to examine the influence of fertilization in immature oocytes on the resumption of meiosis, and the modification of both male and female chromatin in fertilized oocytes. Canine cumulus-oocyte complexes collected from routine ovariectomies were cultured in medium 199 with 20% fetal calf serum for 24 h, incubated in the same medium with fresh semen for 24 h, washed, cultured for a further 24 h and fixed. Control oocytes were cultured in the same medium but without spermatozoa for 24, 48 or 72 h. After fixation, chromatin was stained with propidium iodide and examined using laser scanning confocal microscopy. The data indicate that sperm penetration can occur in immature canine oocytes and that it induces resumption of meiosis. After 72 h of culture, the percentage of oocytes at the germinal vesicle stage was significantly lower in fertilized oocytes (40% versus 60.3% for control oocytes; P < 0.05) and the percentage of oocytes beyond metaphase I was significantly greater in fertilized oocytes (28.3% metaphase I and II, and two pronuclei versus 10.2% metaphase I and II for control oocytes; P < 0.01). Observation and measurement of the area of chromatin in fertilized oocytes showed an overall parallel condensation-decondensation of both female and male chromatin from the germinal vesicle stage to the pronuclear stage.     

Variations in modifications of sugar residues in hamster zona pellucida after in vivo fertilization and in vitro egg activation

Lectins were used as probes in conjunction with quantitative analysis to investigate the distribution of different carbohydrate residues in hamster zona pellucida and their possible modification patterns after in vivo fertilization and in vitro egg activation. Several lectins including HPA, WGA, RCA-I, PNA, DSA, BSAIB(4), DBA, AAA and MAA were used to label the zona pellucida of both unfertilized and fertilized eggs. With the exception of PNA and BSAIB(4), the same lectins were also used to label the zona pellucida of oocytes activated in vitro. A multicomparison quantitative analysis of the density of labelling in the inner and outer regions of the zona pellucida before and after fertilization in vivo, as well as after in vitro egg activation, was performed. Of all the lectins studied, preferential localization of labelling by RCA-I and DSA to the inner zona pellucida of unfertilized eggs was observed. After in vivo fertilization, there was an increase in labelling in the inner region of the zona pellucida when thin sections of fertilized oocytes were incubated with HPA, BSAIB(4) and AAA. Although increased labelling by RCA-I was observed, a significant decrease in labelling intensity was obtained with WGA and the sequence Neu-WGA in both the inner and outer zona pellucida of fertilized oocytes. A significant increase in the density of labelling with WGA was also observed after digestion with neuraminidase. In parallel, when hamster oocytes activated in vitro were compared with those fertilized in vivo, a difference in lectin-gold labelling was observed in both the inner and outer region of the zona pellucida. Labelling with HPA, WGA, DSA and MAA increased significantly in both the inner and outer regions of the zona pellucida, whereas labelling by DBA significantly decreased in the inner portion of the zona pellucida. After neuraminidase treatment, a significant increase in labelling density was observed when thin sections of in vitro-activated oocytes were incubated with WGA. These results demonstrate: (i) the post-fertilization modifications of major saccharidic determinants that may play a role in the sperm-egg interaction process of fertilization in vivo; and (ii) that the modified properties of zonae pellucidae of fertilized and in vitro-activated eggs resulting from the action of hydrolytic enzymes, as well as glycoproteins released through exocytosis of cortical granules, are not identical.     

Immunogenicity and contraceptive potential of three infertility-relevant zona pellucida 2 epitopes in the marsupial brushtail possum (Trichosurus vulpecula)

In a previous study, three infertility-relevant epitopes of possum ZP2 (Pep12 (amino acids 111-125), Pep31 (amino acids 301-315), and Pep44 (amino acids 431-445)) were identified using sera from possums (Trichosurus vulpecula) immunized with recombinant possum zona pellucida 2 (ZP2) constructs, and a synthetic peptide library of possum ZP2 protein. In this study, the three peptides were conjugated to keyhole limpet hemocyanin and 300 mug of each conjugated peptide were administered subcutaneously to female possums (n = 20 per peptide) in complete Freund's adjuvant. Immunogen doses were repeated 3 and 6 weeks later using incomplete Freund's adjuvant. Control animals were immunized with either phosphate-buffered saline only (n = 10) or 300 mug keyhole limpet hemocyanin (n = 10), administered with the same adjuvants. Serum antibodies from animals immunized against these three epitopes bound to the corresponding possum ZP2 peptides, recombinant possum ZP2 protein constructs, and native zona. Possum fertility was assessed following superovulation and artificial insemination. Peptides Pep12 and Pep31 had no significant effects on fertility parameters (P > 0.05). However, animals immunized with Pep44 had lower egg fertilization rates (immunized 19.5% versus control 60.5%, P < 0.05) and produced significantly fewer embryos than control animals (immunized 0.5 embryos versus control 2.4 embryos, P < 0.05). The number of Pep44-immunized females that produced embryos was reduced by 64%. Identification and characterization of possum infertility-relevant epitopes on possum ZP2 protein will assist development of safe, humane, and possum-specific immunocontraceptive vaccines for controlling the introduced possums in New Zealand.     

Effect of in vitrofertilization medium on the acrosome reaction,cortical reaction,zona pellucida hardening and in vitro development in pigs

Physiological events at the time of fertilization of pig oocytes may differ in vitro depending on the in vitro fertilization (IVF) medium. This hypothesis was tested by in vitro maturation of pig oocytes for 44 h in NCSU-37 medium and thereafter fertilization with frozen-thawed ejaculated spermatozoa. Three different IVF media (TCM-199, Tyrode's albumin lactate pyruvate (TALP) and Tris-buffered medium (TBM)) were used. For the acrosome reaction test, spermatozoa were incubated for 0-150 min in the three IVF media, and the proportion of live acrosome-reacted and acrosome-intact cells was determined by fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA) and propidium iodide (PI) staining. The cortical granule density of oocytes was evaluated by confocal microscopy, 2.5 and 5.0 h after culture in each medium in the presence or absence of spermatozoa. Zona pellucida resistance to pronase digestion was also determined in the same groups. The percentages of penetration, monospermy, male pronucleus formation, cleavage and blastocyst formation, and the number of cells per blastocyst after culture were determined. The results indicate that the acrosome reaction occurred much faster in TBM than in TCM-199 or TALP medium. Continuous cortical granule synthesis was observed in the three media when oocytes were incubated in the absence of spermatozoa. The presence of spermatozoa triggered the cortical reaction in a large proportion of oocytes fertilized in TCM-199 and TALP media. On the basis of the duration of pronase digestion, the zona pellucida of oocytes incubated in TCM-199 was harder (407.7 +/- 35.5 s) than that of oocytes cultured in TALP (235.4 +/- 18.2 s) or TBM (189.1 +/- 16.8 s). No zona pellucida hardening was noted in oocytes after insemination in any of the media. The percentages of penetration and cleavage were higher in oocytes cultured in TCM-199 and TALP than in TBM. The percentage of monospermy was higher in TCM-199 and TBM than in TALP. No effect of the medium was shown on the percentage of blastocyst formation or on the number of cells per blastocyst. In conclusion, the results highlight how differently the fertilization events take place in each IVF medium and how far these IVF media still are from achieving biological properties of gametes close to those observed in the physiological setting.     

A musty flavour defect in calcium caseinate due to chemical tainting by 2,4,6-tribromophenol and 2,4,6-tribromoanisole

A musty flavour defect in calcium caseinate that was due to tainting by 2,4,6-tribromoanisole (TBA) is described. The concentration of TBA in tainted calcium caseinate was between 2 and 560 μg kg^?1; substantially above the sensory threshold (0.02 μg kg^?1 in a 10% calcium caseinate solution). The TBA was derived from 2,4,6-tribromophenol (TBP) that had contaminated materials that were used in the calcium caseinate supply chain. TBP is readily converted to TBA by environmental fungi. Potential TBP sources identified in this investigation were timber shipping container floors, and slipsheets constructed from recycled plastics that were used for transporting stacks of calcium caseinate. The TBP sources identified were consistent with trends in the increased industrial use of this chemical in wood products and plastics.     

Contraception in mice immunized with recombinant zona pellucida subunit 3 proteins correlates with Th2 responses and the levels of interleukin 4 expressed by CD4+ cells

The immune responses and contraceptive effect in mice were tested following immunization with purified recombinant zona pellucida (ZP) proteins produced using a vaccinia (v) virus T7 mammalian expression system. Female BALB/c and CBA mice were immunized with recombinant mouse (m) ZP3 (vmZP3) or pig (p) ZPC (vpZPC) using Freund's adjuvants and boosted three times. Fertility and mean litter size were significantly reduced in groups of BALB/c mice immunized with recombinant vmZP3 and vpZPC compared with controls treated with Freund's adjuvants alone. In CBA mice, fertility and mean litter size were significantly reduced in groups of animals immunized with vmZP3 but not with vpZPC compared with the controls. Most infertile animals treated with vmZP3 and a single infertile BALB/c mouse treated with vpZPC lacked mature follicles in the ovaries, whilst no abnormalities were detected in the remaining vpZPC treated, fertile vmZP3 treated and control mice. All mice (both fertile and infertile) immunized with vmZP3 and vpZPC produced IgG antibodies, but the levels of total IgG, IgG1 and IgG2a did not correlate with infertility. All BALB/c and CBA mice immunized with vmZP3 and vpZPC showed greater delayed type hypersensitivity responses in the footpads after challenge with their respective antigens than controls, but these did not differ between the fertile and infertile mice. There was, however, a significant correlation between infertility and the levels of the Type 2 T helper cell (Th2) cytokine interleukin 4 produced by CD4+ cells from vmZP3 immunized mice in response to stimulation with vmZP3 and this did not apply to the levels of the Type 1 T helper cell (Th1) cytokine interferon gamma or the general proliferation response. The results support the conclusion that induction of Th2 responses in individual mice determines whether infertility develops in response to immunization with zona pellucida proteins.     

Intra-cytoplasmic sperm injection in a marsupial

Here we report the first use of intra-cytoplasmic sperm injection (ICSI) in a marsupial, the tammar wallaby (Macropus eugenii), to achieve in vitro fertilization and cleavage. A single epididymal spermatozoon was injected into the cytoplasm of each mature oocyte collected from Graafian follicles or from the oviduct within hours of ovulation. The day after sperm injection, oocytes were assessed for the presence of pronuclei and polar body extrusion and in vitro development was monitored for up to 4 days. After ICSI, three of four (75%) follicular and four of eight (50%) tubal oocytes underwent cleavage. The cleavage pattern was similar to that previously reported for in vivo fertilized oocytes placed in culture, where development also halted at the 4- to 8-cell stage. One-third of injected oocytes completed the second cleavage division, but only a single embryo reached the 8-cell stage. The success of ICSI in the tammar wallaby provided an opportunity to examine the influence of the mucoid coat that is deposited around oocytes passing through the oviduct after fertilization. The presence of a mucoid coat in tubal oocytes did not prevent fertilization by ICSI and the oocytes cleaved in vitro to a similar stage as follicular oocytes lacking a mucoid coat. Cell-zona and cell-cell adhesion occurred in embryos from follicular oocytes, suggesting that the mucoid coat is not essential for these processes. However, blastomeres were more closely apposed in embryos from tubal oocytes and cell-cell adhesion was more pronounced, indicating that the mucoid coat may be involved in maintaining the integrity of the conceptus during cleavage.     

Effect of beta-mercaptoethanol during in vitro fertilization procedures on sperm penetration into porcine oocytes and the early development in vitro

This study was carried out to determine the effects of beta-mercaptoethanol (bME) during a transient co-culture of gametes for 10 min, and/or the following culture until 6-9 h after insemination, on sperm penetration of porcine in vitro maturation (IVM) oocytes and the early development in vitro. When fresh spermatozoa were cultured in various concentrations of bME for 2 h, bME neutralized the stimulatory effect of caffeine-benzoate on sperm capacitation and the spontaneous acrosome reaction at 50-250 micromol/l. When 50 micromol/l bME were added during a transient co-culture of gametes for 10 min, the sperm penetration rate was reduced 9 h after insemination (70.5-82.0% vs 90.5-94.0% in the absence of bME), but the incidence of monospermic penetration was not affected. When 50 micromol/l bME were supplemented during culture after a transient co-culture, the sperm penetration rate was not affected, but the incidence of monospermy oocytes was increased (43.9-45.8% vs 31.7-34.3% in the absence of bME). The presence of bME following a transient co-culture minimized a decrease of oocyte glutathione content at 6 h after insemination (7.9 pmol/oocyte before in vitro fertilization (IVF), 6.7 pmol/oocyte in the presence of bME vs 5.5 pmol/oocyte in the absence of bME). When the distribution of cortical granules was evaluated 1 h after activation with calcium ionophore, mean pixel intensity of fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) at the cortex region was lower in the oocytes activated and cultured in the presence of 50 micromol/l bME. Although the presence of 50 micromol/l bME during a transient co-culture for 10 min and the following culture did not increased blastocyst formation (29.6-37.7%), 50 micromol/l bME during the following culture significantly increased the mean cell numbers per blastocyst (73.3-76.4 vs 51.2 in the presence and absence of bME respectively). These results demonstrate that supplementation with bME during IVF procedures, except during a transient co-culture period of gametes in the presence of caffeine, has a beneficial effect in maintaining the function of gametes, the incidence of normal fertilization and, consequently, the quality of IVF embryos.     

Acrosomal integrity and capacitation are not influenced by sperm cryopreservation in the giant panda

Sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. This study examined the influence of freeze-thawing on sperm function, specifically capacitation. Sperm from nine giant pandas were assessed before and after rapid (- 40 and - 100 degrees C/min) cryopreservation by incubation in HEPES-buffered Ham's F10 medium with and without the capacitation accelerators, 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP). At 0, 3 and 6 h of exposure, aliquots were assessed for sperm motility traits and capacitation, defined as the proportion of sperm with intact acrosomes following exposure to solubilised zonae pellucidae (ursid or felid) or calcium ionophore subtracted from the proportion of sperm with intact acrosomes before exposure. Although mean+/-S.E.M. sperm motility post-thaw (56.1 +/- 3.9% at 0 h) was less (P < 0.05) than pre-freeze (71.7 +/- 6.0%), there was no difference (P > 0.05) in the proportion of acrosome-intact sperm (fresh, 93.0 +/- 1.7% versus cryopreserved-thawed, 81.7 +/- 4.7% at 0 h). Incidence of capacitation was greater (P < 0.05) in fresh sperm incubated with capacitation accelerators IBMX and dbcAMP (9 h: 50.9 +/- 1.1) compared with fresh sperm incubated without accelerators (9 h: 41.2 +/- 1.1%). Frozen-thawed sperm preincubated without accelerators underwent capacitation (49.6 +/- 1.1%) to a greater extent (P < 0.05) compared with these fresh counterparts. Thawed samples with (9 h: 45.9 +/- 1.4%) and without accelerators (9 h: 41.2 +/- 1.1%) did not differ (P > 0.05) during the 9-h incubation. We conclude that giant panda spermatozoa (1) undergo capacitation in vitro with or without chemical accelerators and (2) withstand a rapid cryopreservation protocol, including retaining normal acrosomal integrity and functional capacitation ability.     

Zinc in mammalian sperm: a review

The relationship of zinc to morphologic, physiologic, and metabolic functions in the male reproductive system are summarized, and gaps in current understanding are pointed out. Semen and its constituents generally contain high zinc, although concentrations vary among animals and species; the relationships between zinc and fertility of semen is unclear. During zinc deficiency, retarded development of testicular growth involved marked atrophy of tubular epithelium and reduced deoxyribonucleic acid, ribonucleic acid, and protein, as well as reduced zinc contents of testis, epididymis, and dorsolateral prostate. Functions of zinc in hormone interrelationships are little understood, but zinc deficiency decreases output of pituitary gonadotrophins and androgen production, and zinc turnover involves testosterone as well as pituitary hormones. Metabolic regulation of sperm appears to be mediated through zinc as a regulator of enzyme activity in the semen. Within spermatozoa, zinc is closely associated with sulfhydryl groups and disulfide linkages and is concentrated in the tail. Control of motility of sperm by zinc apparently involves control of energy utilization through adenosine triphosphate systems involved in contraction and through regulation of phospholipid energy reserves. The many roles for zinc in the male reproductive system are extremely complex and scarcely understood. The importance of zinc contents of commonly utilized feedstuffs in relation to reproductive capabilities of the mammalian sperm remain unclear, although zinc deficiency in relation to male reproduction may be much more widespread than is recognized commonly.     

Cytoskeleton localization in the sperm head prior to fertilization

Three major cytoskeletal proteins, actin, tubulin and spectrin, are present in the head of mammalian spermatozoa. Although cytoskeletal proteins are implicated in the regulation of capacitation and the acrosome reaction (AR), their exact role remains poorly understood. The aim of this study was to compare the distribution of the sperm head cytoskeleton before and after the AR in spermatozoa representing a range of acrosome size and shape. Spermatozoa from the human and three rodents (rat, hamster and grey squirrel) were fixed before and after the AR in appropriate medium in vitro. Indirect immunofluorescent localization of cytoskeletal proteins was undertaken with antibodies recognizing actin, spectrin and alpha-tubulin. Preparations were counterstained with propidium iodide and examined by epifluorescent and confocal microscopy. Our results clearly demonstrated changes in localization of cytoskeleton during the AR, mainly in the apical acrosome with further changes to the equatorial segment and post-acrosomal regions. The pattern of cytoskeletal proteins in the sperm head of all the species was similar in respect to various sub-compartments. These observations indicated that the sperm head cortical cytoskeleton exhibits significant changes during the AR and, therefore, support the image of cytoskeletal proteins as highly dynamic structures participating actively in processes prior to fertilization.