A comparative study of sperm production in two species of Australian arid zone rodents (Pseudomys australis, Notomys alexis) with marked differences in testis size

The plains rat, Pseudomys australis, and the spinifex hopping mouse, Notomys alexis, show marked differences in the size of their testes and in the number of spermatozoa within the epididymides. In the present study, the dynamics of sperm production and the duration of sperm transit along the male excurrent ducts were compared between these two species. The durations of the cycle of the seminiferous epithelium, spermatogenesis and sperm transit were determined by tracking cells using autoradiography after [(3)H]thymidine incorporation. Daily sperm production was determined from counts of testicular spermatids after homogenization and further estimates of sperm transit were obtained by dividing sperm reserves within the various regions of the extratesticular ducts by the daily sperm production of the attached testis. In the plains rat, the mean duration of the cycle of the seminiferous epithelium was 11.2 days, the duration of spermatogenesis was 45 days, daily sperm production was 2.6 x 10(7) spermatozoa per gram of testis and epididymal transit of spermatozoa took approximately 9 days (caput 0.8 days; corpus 1.5 days; cauda 6.5 days). In contrast, in the hopping mouse, the mean duration of the cycle of the seminiferous epithelium was 14 days, the duration of spermatogenesis was 56 days and daily sperm production per gram of testis was < 1.0 x 10(7). Epididymal transit of spermatozoa was completed in about 4 days (caput + corpus < 1 day; cauda 3 days); however, spermatozoa may be stored for an additional 1.5-2.0 days in the vas deferens. These results indicate that, in addition to small testes, the hopping mouse shows a low efficiency of sperm production, a relatively long duration of spermatogenesis and rapid passage of spermatozoa through the epididymis, all of which contribute to low epididymal sperm counts. These data are considered in relation to interspecific differences in sperm competition.     

Fate of lactadherin P47 during post-testicular maturation and capacitation of boar spermatozoa

Polyclonal avian antibody was used partially to characterize the pig sperm lactadherin P47. P47 is a mosaic protein, composed of two epidermal growth factor (EGF)-like domains and two C1/C2 domains. P47 is homologous to the bovine mammary gland protein MGP 53/57 and mouse milk fat globule protein. Expression of P47 along the male genital tract and its localization on spermatozoa during post-testicular maturation and capacitation were studied. P47 was detected in the testis and in all parts of the epididymis by immunohistochemistry and by western blots of tissue extracts. By indirect immunocytochemistry, P47 was localized at the apical ridge of the sperm head in testicular, epididymal and ejaculated spermatozoa. The fluorescence intensity progressed during sperm transit from caput to cauda epididymis, probably caused by the ongoing expression and subsequent accumulation of P47 on the sperm surface. During the time course of capacitation, P47 appears to be unmasked by the release of coating proteins and appears to migrate from the apical ridge onto the entire acrosomal region, showing an intensive fluorescence pattern after 3 h capacitation in vitro. The kinetics of signal changes during in vitro capacitation were different in epididymal and ejaculated spermatozoa, indicating accelerated capacitational plasma membrane destabilization in epididymal spermatozoa.     

Beta-defensin 22 is a major component of the mouse sperm glycocalyx

Surface components of sperm isolated from the cauda epididymides were stabilized by whole sperm fixation for immunization of rabbits. The resulting immunoglobulins (Igs) recognized a single protein of 130 kDa (non-reduced) or 54-57 kDa (reduced) on western blots of cauda sperm. Igs recognized the same 54-57 kDa protein band on whole tissue blots of the corpus and cauda epididymidis and vas deferens. No immunoreactive bands were detected on blots of the prostate, seminal vesicles, testes, caput epididymis, or any of various non-reproductive tissues. Removal of sperm from the vas deferens prior to blotting eliminated the detection of the sperm antigen. Antibodies raised to synthetic peptides, identical in amino acid sequence to two unique spans of DEFB22, recognized the same 130/54-57 kDa antigen on western blots of both caudal sperm and the purified antigen isolated with the anti-sperm Ig. From indirect immunofluorescence, both the anti-sperm and anti-peptide Igs appeared to localize to the entire sperm surface, a pattern confirmed at the ultrastructural level. Real-time PCR identified the corpus epididymides as the major site of expression of DEFB22, with negligible expression in the testes, caput epididymides, and vas deferens. Immunostaining of epididymal sections showed DEFB22 being released into the lumen at the distal caput/proximal corpus, with sperm becoming intensely coated with DEFB22 as they reached the distal corpus. Most uterine sperm recovered from mice 4 h following copulation exhibited DEFB22 coating the entire sperm surface. By contrast, some sperm recovered from the oviduct and cumulus extracellular matrix showed loss of DEFB22 from the sperm head.     

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.     

Identification and characterization of ERp29 in rat spermatozoa during epididymal transit

The mammalian epididymis is able to create sequential changes in the composition of luminal fluid throughout its length, wherein spermatozoa undergo morphological, biochemical, and physiological modifications. Subsequently, spermatozoa acquire the ability for fertilization upon reaching the epididymal cauda. In this study, protein variations in Sprague-Dawley rat spermatozoa along the caput and caudal regions of epididymis were investigated by high-resolution two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry. From total protein spots on the 2DE maps, 43 spots were shown to be significantly modified as sperm traverse the epididymis, and seven unambiguous proteins were identified from them. Finally, using indirect immunofluorescence, we demonstrated that localization of one of these seven proteins, the endoplasmic reticulum protein (ERp29) precursor, which was first reported in mammalian spermatozoa, was apparently up-regulated as the sperm underwent epididymal maturation and expressed mainly on caudal sperm. Western blot analysis also revealed that ERp29 precursor, from both whole spermatozoa and membrane proteins, increased significantly as the sperm underwent epididymal maturation. Furthermore, the results from immunofluorescence-stained epididymal frozen sections demonstrated that ERp29 was localized in cytoplasm of epididymal epithelia, and the fluorescence intensity was significantly higher in the caudal epididymis than in the caput. These clues indicated that the ERp29 precursor, perhaps related to secretory protein synthesis and absorbed by spermatozoa, may play a vital role in sperm maturation during the epididymal transit, particularly, in the sperm/organelle membrane.     

Postnatal development and testosterone dependence of a rat epididymal protein identified by neonatal tolerization

A rat epididymal protein of 27 kDa was identified using neonatal tolerization. This study reports the production and characterization of a polyclonal antiserum to this protein. ELISA was used to demonstrate that this antiserum reacts strongly with epididymal sperm proteins, but has little or no reactivity with testicular proteins. Western blot analysis revealed that this polyclonal antiserum recognized a 27 kDa protein extracted from the corpus epididymidis as well as from spermatozoa from the corpus and cauda epididymides, and immunostaining revealed the presence of the protein in the corpus to cauda epididymides. Stronger reactivity was observed in the supranuclear region and stereocilla of principal cells of the corpus epididymidis and in the luminal content of the corpus and cauda epididymides. The testicular section showed no reactivity. Treatment with the antiserum resulted in time- and dose-dependent agglutination of rat spermatozoa. By indirect immunofluorescence, the antiserum localized proteins in the mid-piece region of rat spermatozoa. Studies were carried out to determine the age at which the protein first became apparent during postnatal development. The protein was expressed from day 40 onwards, as demonstrated by western blot analysis. The androgen regulation of this protein was ascertained by castration and supplementation studies. Expression of this protein showed a decline starting at day 14 after castration and by day 21 the protein was absent; however, androgen replacement resulted in the reappearance of the protein. The results of these studies indicate that the protein identified is specific to the epididymis, and is regulated by development and androgens. The importance of epididymis-specific proteins that are regulated by androgens in sperm maturation is discussed, and the need to ascertain the sequence of the protein and clone the cognate gene is indicated.     

Replacement of nuclear protein by histone in pig sperm nuclei during in vitro fertilization

Sperm-specific nuclear protamines are dissociated before decondensation of sperm nuclei during fertilization in pigs. In the present study, replacement of nuclear protein by histone in boar spermatozoa during in vitro fertilization was evaluated by immunohistochemistry using anti-histone antibody. First, the specificity of the antibody used in this study was examined. Immunohistochemistry of the testes and epididymides indicated that somatic nuclei, but not elongated spermatids or maturing spermatozoa, were immunoreactive. Furthermore, immunoreaction was diminished after the antibody had been preincubated with unfractionated histone, indicating that the antibody was specific for the somatic nuclear histone. Immunohistochemistry of serial sections of oocytes, which were matured and co-cultured with boar spermatozoa for 2 to 6 h indicated that, at 2 to 3 h after insemination, penetrating sperm nuclei in the condensed state were not immunoreactive. At 4 to 5 h after insemination, some of the condensed sperm nuclei were immunoreactive in part or over the whole area of the nucleus, and all of the decondensing nuclei and male pronuclei were immunoreactive. At 6 h after insemination, the decondensing sperm nuclei and well-developed male pronuclei were immunoreactive. These results imply that, in pigs, remodelling of sperm nuclear protein from protamine to histone is initiated at the time of sperm penetration, before onset of decondensation and male pronuclear formation.     

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.     

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

The aim of this study was to determine the ultrastructure of cross-sectioned zonae pellucidae of in vitro-matured and ovulated pig oocytes before or after sperm penetration in vitro and in vivo, respectively. The in vitro and in vivo (ovulated) oocytes and zygotes (fertilized in vitro and in vivo) were fixed with glutaraldehyde either directly or after pretreatment with ruthenium red and saponin, processed and then examined using transmission electron microscopy. The thickness of the zona pellucida, as measured on the section of the specimens with largest diameter fixed with glutaraldehyde, differed between the in vivo (9.19 +/- 0.47 microm) and in vitro (5.95 +/- 0.51 microm) oocytes. The in vivo oocytes had a rather thick external mesh-like structure, whereas it was much thinner in the in vitro oocytes. This mesh-like external rim was less apparent in both in vivo and in vitro zygotes. Obvious differences in the density of the lattice formed by the fixed zonae pellucidae were visible between the outer and inner (ad-oolemmal) zonae. The outer area always formed a concentrically arrayed fibrillar network, whereas the inner area showed a much more compact, trabecule-like mesh. However, both areas, but particularly the outer network, were much more compacted after the zona reaction. Clear differences in the degree of fibrillar aggregation of the inner zona area were also observed between in vitro and in vivo zygotes, being much higher in the latter. This fibrillar network was more clearly visible in the zygotes pretreated with ruthenium red and saponin; the in vitro zygotes had a fibrillar, radially oriented set of parallel fibrils, whereas it was much more aggregated and trabecule-like in the in vivo zygotes. These results demonstrate that the fine structure of the zona pellucida and the zona reaction at sperm penetration differ between pig oocytes fertilized in vivo and in vitro. Moreover, the ultrastructure of the outer and inner pig zonae pellucidae has a different network organization.     

Localization of CD9 in pig oocytes and its effects on sperm-egg interaction

CD9 is a cell surface protein that participates in many cellular processes, such as cell adhesion. Fertilization involves sperm and oocyte interactions including sperm binding to oocytes and sperm-oocyte fusion. Thus CD9 may play an essential role during fertilization in mammals. The present study was conducted to examine whether CD9 is present in porcine gametes and whether it participates in the regulation of sperm-oocyte interactions. The presence of CD9 in ovarian tissues, oocytes and spermatozoa was examined by immunohistochemistry, immunofluorescence and immunoblotting. Sperm binding and penetration of oocytes treated with CD9 antibody were examined by in vitro fertilization. The results showed that CD9 was present on the plasma membrane of oocytes at different developmental stages. A 24 kDa protein was found in oocytes during in vitro maturation by immunoblotting and its quantity was significantly (P < 0.001) increased as oocytes underwent maturation and reached the highest level after the oocytes had been cultured for 44 h. No positive CD9 staining was found in the spermatozoa. Both sperm binding to ooplasma and sperm penetration into oocytes were significantly (P < 0.01) reduced in anti-CD9 antibody-treated oocytes (1.2 +/- 0.2 per oocyte and 16.6% respectively) as compared with oocytes in the controls (2.5 +/- 0.4 per oocyte and 70.3% respectively). These results indicated that CD9 is expressed in pig oocytes during early growth and meiotic maturation and that it participates in sperm-oocyte interactions during fertilization.     

Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine

To reduce the incidence of polyspermic penetration, the effects of transient exposure of washed fresh spermatozoa to caffeine in a brief co-culture in vitro fertilization (IVF) system were examined. A pretreatment effect of spermatozoa with adenosine was also examined. When 5 mmol caffeine/l was supplemented during periods of co-culture and additional culture periods until 8 h after insemination, a shortened co-incubation period of gametes (30 denuded oocytes in 100 microl modified Medium 199-suspended spermatozoa at 2.5 x10(5) sperm/ml) from 30 to 5 min increased the monospermy rate in total mature oocytes examined. The number of spermatozoa binding to the zona surface was significantly lower in oocytes co-cultured for 5 min (33.1 +/- 2.2) than 8 h (207.6 +/- 13.7). A limited exposure of gametes to 5 mmol caffeine/l only during a transient co-culture period for 5 or 30 min significantly reduced the mean number of sperm cells that penetrated into the oocyte. Transient exposure of spermatozoa to caffeine for only 5 min increased the percentage of capacitated cells but not acrosome-reacted cells, as compared with a whole exposure treatment. Furthermore, preincubation of spermatozoa with 10 micromol adenosine/l for 90 min increased both the incidence of capacitated cells and the monospermy rate and consequently decreased the number of sperm cells that penetrated into the oocyte. In conclusion, these results have demonstrated that a new transient co-incubation IVF system, in which denuded oocytes are co-cultured with spermatozoa in medium containing caffeine for 5 to 30 min and then continuing the culture in caffeine-free medium, will reduce the incidence of polyspermic penetration. Preincubation of fresh spermatozoa with adenosine before the transient co-incubation IVF can also improve the monospermy rate. Furthermore, asynchrony in the morphology of sperm nuclei in polyspermic oocytes was reduced by the pretreatment with adenosine and a brief exposure to caffeine.     

Presence, processing, and localization of mouse ADAM15 during sperm maturation and the role of its disintegrin domain during sperm-egg binding

Successful fertilization requires gametes to complete several stages, beginning with maturation and transport along the male and female reproductive tracts and ending with the interaction between the sperm and the egg. This last step involves sperm-egg adhesion and membrane fusion. ADAMs (disintegrin and metalloprotease domain proteins) are a family of membrane-anchored glycoproteins that are thought to play diverse roles in cell-cell adhesion through their interaction with integrins. This study analyzes the presence, location, processing, and possible role of ADAM15 in mouse sperm. The presence of ADAM15 in mouse spermatozoa was detected by Western blotting, which revealed that ADAM15 is post-translationally processed, during epididymal sperm maturation and the acrosome reaction. The 35 kDa antigen present in the acrosome-reacted sperm is the last proteolytic product of the 110/75 kDa ADAM15 found in non-capacitated sperm. This 35 kDa protein contains the disintegrin domain. By indirect immunofluorescence, ADAM15 was identified in the acrosomal region and along the flagellum of mouse spermatozoa. In acrosome-reacted sperm, ADAM15 was lost from the acrosomal region, but remained diffusely distributed throughout the head and flagellum. Furthermore, the ADAM15 disintegrin domain (RPPTDDCDLPEF) partially inhibited fusion and almost completely inhibited sperm-oolemma adhesion. In conclusion, our data indicate that ADAM15 is present in the testis and in spermatozoa from the caput, corpus, and cauda epididymis, as well as in non-capacitated and acrosome-reacted gametes. Results also indicate that ADAM15 is processed during epididymal maturation and acrosome reaction and that it may play a role during sperm-egg binding.     

Detection of osteopontin on Holstein bull spermatozoa, in cauda epididymal fluid and testis homogenates, and its potential role in bovine fertilization

Osteopontin (OPN) is a secreted extracellular matrix phosphoprotein identified in various tissues and fluids including those of the male and female reproductive tracts. OPN was previously identified as a 55 kDa high fertility marker in Holstein bull seminal plasma, produced by the ampulla and the vesicular gland. The objectives of this study were to characterize OPN on ejaculated and cauda epididymal sperm using immunofluorescence and western blot analysis, and to assess the role of sperm OPN in fertilization. Solubilized sperm membrane proteins from ejaculated and cauda epididymal sperm were separated by 1D SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to bovine milk OPN. A 35 kDa protein was detected by this antibody in both ejaculated and cauda epididymal sperm membranes. Analyses also recognized OPN at 55 and 25 kDa in cauda epididymal fluid and testicular parenchyma homogenates respectively. Immunofluorescent analysis of ejaculated and cauda epididymal sperm showed OPN localization in a well-defined band in the postacrosomal region of the sperm head and also on the midpiece. Results of in vitro fertilization experiments showed that sperm treated with an antibody to OPN fertilized fewer oocytes than sperm treated with control medium while increasing incidence of polyspermy, suggesting a role of sperm-associated OPN in fertilization and a block to polyspermy. These studies demonstrate that OPN exists at multiple molecular weight forms in the bull reproductive tract and its presence on ejaculated sperm may signal its importance in fertilization by interacting with integrins or other proteins on the oocyte plasma membrane.     

Early history of in vitro fertilization

Although in vitro fertilization (IVF) is used widely for a variety of purposes, it is often not appreciated how this technology was developed. A large number of experiments beginning in 1878 contributed to the first successful reports of IVF over 75 years later. The discovery of sperm capacitation in 1951 was central to the development of IVF technology, and it was rapidly followed by the first convincing reports of IVF in several species. The ability to fertilize oocytes in vitro has allowed major advances to be made into understanding the mechanisms involved in fertilization and early development, and IVF now supports reproductive biotechnology in animals and in humans. This article is a historical review of key experiments that helped to provide the basis for present day IVF procedures, placed into context with current practice.     

Differential modulation of bovine epididymal activity by oxytocin and noradrenaline

Passage of spermatozoa through the epididymis and emission of sperm during ejaculation are based on spontaneous and induced contractions of epididymal peritubular muscle layers. This study deals with the ejaculation-relevant factors noradrenaline (NA) and oxytocin (OT) and their contractile effects in the course of the bovine epididymal duct. Muscle tension recording revealed excitatory effects of NA in all duct regions. A peculiarity was found in a duct section between the mid-cauda and ductus deferens, where the responsiveness to NA was particularly faint in comparison with the adjacent regions. NA-induced contraction was primarily mediated by postjunctional alpha(2)-adrenoceptors (ADRA) in the caput and corpus regions, and by alpha(1)-ADRA in the cauda region. Contrary to NA, OT exerted regionally varying effects. The peptide induced contraction in intact and epithelium-denuded caput as well as in epithelium-denuded corpus segments but had a relaxant net effect in intact corpus and proximal cauda segments. Within the mid-cauda, OT evoked strong contraction, which progressively decreased distally. Receptor specificity of the epididymal OT effects was verified using the selective OT receptor (OTR) agonist [Thr(4),Gly(7)]OT and vasopressin. OTR immunoreactivity was detected in the epididymal peritubular muscle wall and epithelial principal cells. RT-PCR analysis confirmed the presence of OTR in all duct regions. In summary, different contractile responses to OT and NA occur in the course of the epididymal duct, possibly preventing excessive sperm transport through the corpus and serving orthograde emission of sperm during ejaculation.     

Evaluation of calcium-free Tyrode's sperm capacitation medium for use in bovine in vitro fertilization

Our objective was to determine if a bovine sperm capacitation technique, developed with zona-free hamster oocytes, could be used for the in vitro fertilization of in vitro matured bovine zona-intact oocytes. Bovine cumulus-enclosed primary oocytes from 2- to 5-mm follicles were matured in tissue culture Medium 199 containing Earle's salts and bicarbonate and supplemented with 10% fetal calf serum, FSH (10 micrograms/ml), and estradiol-17 beta (1.5 microgram/ml) for 24 h at 37 degrees C under paraffin oil. Ejaculated bovine sperm, washed thrice in bovine serum albumin-saline (pH 7.6) and capacitated for 4 h in Ca(++)-free Tyrode's medium (pH 7.6), were diluted to 2 x 10(6) sperm/ml in Medium 199 supplemented with 10% fetal calf serum. Oocytes were added (10/500 microliters droplet) to this medium containing the capacitated sperm, freeze-thawed killed sperm, or no sperm and incubated for 8 h before transfer to fresh medium and then incubated for 40 h. At the end of each incubation, a portion of the oocytes were stained and evaluated for development or fertilization. After 24 h of culture, 49% of the oocytes had matured (metaphase II). Fertilization rates were 55.6% after exposure of all oocytes to Ca(++)-free Tyrode's capacitated sperm and 82.5% if only metaphase II oocytes were selected. The parthenogenetic controls were negative (1.4% and 0%). Therefore, the Ca(++)-free Tyrode's sperm capacitation technique can be used for bovine in vitro fertilization studies.     

Insights into the molecular basis of sperm-egg recognition in mammals

The zona pellucida surrounding the egg and pre-implantation embryo is required for in vivo fertility and early development. Explanatory models of sperm-egg recognition need to take into account the ability of sperm to bind to ovulated eggs, but not to two-cell embryos. For the last two decades, investigators have sought to identify an individual protein or carbohydrate side chain as the 'sperm receptor'. However, recent genetic data in mice are more consistent with the three-dimensional structure of the zona pellucida, rather than a single protein (or carbohydrate), determining sperm binding. The mouse and human zonae pellucidae contain three glycoproteins (ZP1, ZP2, ZP3) and, following fertilization, ZP2 is proteolytically cleaved. The replacement of endogenous mouse proteins with human ZP2, ZP3 or both does not alter taxon specificity of sperm binding or prevent fertility. Surprisingly, human ZP2 is not cleaved following fertilization and intact ZP2 correlates with persistent sperm binding to two-cell embryos. Taken together, these data support a model in which the cleavage status of ZP2 modulates the three-dimensional structure of the zona pellucida and determines whether sperm bind (uncleaved) or do not (cleaved).     

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.     

Evaluation of a TEST-yolk sperm capacitation system for use in bovine in vitro fertilization.

Bovine sperm acquire the ability to penetrate zona-free hamster oocytes (capacitation) after incubation in TEST-yolk buffer. Our objective was to determine whether such sperm could penetrate zona-intact bovine oocytes in vitro. Bovine cumulus enclosed oocytes from 2- to 5-mm follicles were incubated in maturation medium for 24 h at 37 degrees C. Ejaculated bovine semen was diluted 1: 10 in TEST-yolk buffer, cooled to 4 degrees C, and stored for 8 h to induce capacitation. Sperm were then washed thrice in pH 7.6, .15 M NaCl containing .1% bovine serum albumin V (37 degrees C) and diluted to 2 x 10(6) sperm/ml in fertilization medium. Droplets of fertilization medium containing capacitated sperm, killed sperm, or no sperm were made under paraffin oil. Oocytes (matured 24 h) were added and cocultured with sperm for 8 h and then transferred to fresh fertilization medium for 40 h. After 24 h, 53% of the oocytes had matured (metaphase II). The fertilization rate of the metaphase II oocytes (203) with TEST-yolk capacitated sperm was 87%, whereas the parthenogenetic controls were 2 and 0%, respectively. Therefore, TEST-yolk buffer can be used to capacitate bull sperm for in vitro fertilization.