The preference of fertilized (IVF) and somatic cell nuclear transfer (SCNT) presumptive zygotes for different media when cultured in vitro to the blastocyst stage was evaluated in this study. The experiment comprised two zygote production methods (IVF and SCNT) × two culture media (mSOF and G1.5/G2.5) factorial design in which culture droplets that contained approximate 30 presumptive zygotes formed the experimental plots for the assessment of cleavage and blastocyst development. There were 15 to 20 replicates (culture droplets) per treatment combination. Sub-samples 30 to 41 of the blastocysts produced were assessed for cell number and cell apoptosis. A further 10 blastocysts per treatment combination were used for quantitative real-time polymerase chain reaction (RT-PCR) to evaluate the relative abundance of Hsp70 and Bax mRNA. Presumptive zygotes produced by IVF were developmentally more competent than SCNT zygotes in terms of cleavage rate (66.9 vs. 57.0%; P < 0.05) and blastocyst development rates (blastocysts of presumptive zygotes 29.7 vs. 24.8%; blastocysts of cleaved zygotes 44.4 vs. 36.6%; P < 0.05). Over both zygote production systems, however, the results were similar whether culture was in mSOF or in G1.5/G2.5 media for cleavage rate (63.2 vs. 62.4%; P > 0.05) and blastocyst development rate (blastocysts of presumptive zygotes 26.4 vs. 25.7%; P > 0.05; blastocysts of cleaved zygotes 41.8 vs. 41.2%; P > 0.05). There was, however, a significant interaction between the method of zygote production and culture medium for the apoptotic index of blastocysts. The interaction was such that IVF-produced zygotes cultured in mSOF had a lower apoptotic index compared with those cultured in G1.5/G2.5 (4.7 ± 1.2% vs. 9.8 ± 0.9%; P < 0.05) whereas SCNT zygotes had a higher apoptotic index when cultured in mSOF compared with those cultured in G1.5/G2.5 (11.9 ± 1.5% vs. 4.5 ± 1.2%; P < 0.05). Moreover, RT-PCR analysis showed that embryos from IVF-produced zygotes cultured in mSOF had a lower expression level of stress-related and apoptosis genes (Hsp70 and Bax) than those cells cultured in G1.5/G2.5 medium, while SCNT-derived embryos cultured in mSOF had a higher expression level of these genes than those embryos cultured in G1.5/G2.5 medium. The results of this study show that bovine IVF- and SCNT-produced presumptive zygotes have different nutrient requirements for in vitro culture to the blastocyst stage of development. IVF-derived zygotes have a preference for mSOF as the culture medium whereas the G1.5/G2.5 medium is more suitable for the culture of bovine SCNT-derived zygotes.

The technique of sperm-mediated gene transfer (SMGT) can be used to delivery exogenous DNA into the oocyte. However, it has low repeatability and produces inconsistent results. In order to optimize this technique, it is necessary to study the mechanism by which DNA enters the sperm cell and integrates in the sperm genome. Furthermore, studies must focus in the maintenance of sperm cell viability and function. The aim of this study was to evaluate different SMGT protocols of sperm electroporation or capacitation (CaI) aiming to maintain sperm viability in the production of bovine embryos in vitro. Frozen–thawed semen was divided in two experimental groups (electroporation or CaI) and one control group (non-treated cells). For the electroporation method, five different voltages (100, 500, 750, 1000 or 1500 V) with 25 μF capacitance were used. For CaI treatment, combinations of two CaI concentrations (250 nM or 500 nM), two incubation periods of sperm cells with CaI (1 or 5 min) and two incubation periods that mimicked time of sperm cell interaction with exogenous DNA molecules (1 or 2 h) were evaluated. According to our data, electroporation and CaI treatments do not prevent sperm penetration and oocyte fertilization and can be an alternative method to achieve satisfactory DNA delivery in SMGT protocols.

Glutathione (γ-glutamyl-cysteinyl-glycine; GSH) is a ubiquitous intracellular free thiol that improves development of the male pronucleus at fertilisation and has also been implicated in promoting the development of preimplantation embryos. The objective of this study was to evaluate the effects of adding GSH or cysteine to the in vitro maturation medium on intracellular GSH amounts after in vitro maturation and fertilisation of prepubertal goat oocytes. Oocytes were matured in TCM199 medium supplemented with 10% bovine fetal serum, 1 mg/ml 17β-estradiol, 10 μg/ml o-FSH, 10 μg/ml LH and 50 mg/ml gentamicin. In vitro maturation medium was completed with two independent treatments: GSH at different concentrations (0, 0.25, 0.50 and 1.00 mM) and L-cysteine at different concentrations (0, 150, 300, 600 and 900 μM). After 27 h of culture at 38.5 °C in 5% CO2 in air, the nuclear stage was evaluated. Simultaneously, another sample of oocytes was frozen and the intracellular GSH level was evaluated with spectrophotometric methodology. Oocytes were inseminated with fresh semen (2-3 × 106 sperm/ml) in TALP medium supplemented with 1 mg/ml hypotaurine. Oocytes were fixed at 20 h post-insemination to evaluate the in vitro fertilisation. Oocytes matured in 1.00 mM GSH-supplemented medium exhibited higher amounts of intracellular GSH (3.23 pmol per oocyte). The percentage of normal fertilisation (17-27%) was similar for the treatment groups. In conclusion, the addition of 1.00 mM GSH to the maturation medium could be a useful method for increasing the intracellular GSH levels of prepubertal goat oocytes. However, this increase was not associated with a higher normal fertilisation rate of prepubertal goat oocytes.

This study aimed to evaluate the relationship between antral follicular count (AFC) and ovarian volume (OV), preantral follicular population and survival, meiotic progression and ultrastructure of cumulus–oocyte complexes (COCs) after in vitro maturation. In experiment 1, the relationship between AFC and preantral follicle population and survival was evaluated by classical histology. In experiment 2, the relationship among AFC, OV, ability of oocytes to resume meiosis and ultrastructure of in vitro matured bovine COCs was studied. A positive correlation (P < 0.05) between AFC and the numbers of healthy primordial, degenerate and total follicles was observed, as well as with healthy secondary follicles and total follicles. The numbers of grades I and II oocytes in ovaries of high AFC class were higher compared with those with intermediate or lower AFC. After in vitro maturation, COCs from ovaries of high AFC had a higher percentage of oocytes in metaphase II compared with those of intermediate and low AFC (P < 0.0001). Ovaries of intermediate AFC had a higher percentage of oocytes in metaphase II compared with ovaries with low AFC (P < 0.0001). The proportion of oocytes in metaphase I, telophase I and anaphase I in COCs from ovaries of intermediate AFC (26.04%) was higher (P < 0.05) compared with that seen in COCs of ovaries with high (8.55%) and low (14.15%) AFC. No differences in the ultrastructure of oocytes were seen. In conclusion, after in vitro maturation, cow ovaries with high AFC have higher numbers of oocytes that reach in metaphase II (MII), but they also have higher numbers of degenerated primordial and primary follicles.

The aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

The cumulus–oocyte–complexes (COCs) recovery rates with respect to reproductive status per sei (Balaenoptera borealis) and Bryde's (B. edeni) whales were determined in Experiment 1. The number of COCs recovered ranged from 16.0 to 30.6 and from 6.7 to 26.8 per sei and Bryde's whales, respectively. The effects of COCs grades and protein supplementation in embryo culture medium on development of in vitro fertilized (IVF) embryos were evaluated in sei and Bryde's whales in Experiment 2. The COCs were classified into either Grade A (COCs with five or more layers of compact cumulus cells) or Grade B (COCs with less than five layers of compact or expanded cumulus cells) before being cultured for IVM. The cleavage (12.0 to 19.5%), 4-cell (8.0 to 12.0%) and 8-cell (4.0 to 8.0%) formation rates in sei whales did not vary significantly between embryos derived from either grade A or B oocytes and between embryos cultured in either fetal whale serum (FWS)- or bovine serum albumin (BSA)-supplemented medium. The cleavage (4.0 to 14.8%), 4-cell (0.0 to 7.5%) and 8-cell (0.0 to 2.6%) formation rates in Bryde's whales did not vary significantly between embryos derived from either grade A or B oocytes and between embryos cultured in either FWS- or BSA-supplemented medium. The grade B oocytes cultured in FWS-supplemented medium developed to morula stage (1.1%) in sei whales. In conclusion, the present study indicates that IVF in sei whales is possible to achieve cleaved embryos developing to morula stage. This is the first in vitro embryo production attempt in sei and Bryde's whales.

Spermatogenesis is a complex differentiation process which is characterised, among other features, by conspicuous stage-specific nuclear events such as the pairing of homologous chromosomes coupled with the formation of synaptonemal complexes, the replacement of histones with sperm-specific proteins during spermiogenesis and, as a result, chromatin condensation and its inactivation in sperm cells. The chromatin of spermatogenic cells undergoes dramatic conformational changes upon differentiation from spermatogonia to mature spermatozoa. During the haploid stage of spermatogenesis, histones are gradually replaced, firstly by transition proteins and later by sperm-specific proteins. As a result of the high degree of condensation and inactivation of spermatid and sperm chromatin, Sertoli cells are responsible for the nourishment of germ cells with ribosomal RNA and nutritive substances.

Interspecies intracytoplasmic sperm injection has been carried out to understand species-specific differences in oocyte environments and sperm components during fertilization. While sperm aster organization during cat fertilization requires a paternally derived centriole, mouse and hamster fertilization occur within the maternal centrosomal components. To address the questions of where sperm aster assembly occurs and whether complete fertilization is achieved in cat oocytes by interspecies sperm, we studied the fertilization processes of cat oocytes following the injection of cat, mouse, or hamster sperm. Male and female pronuclear formations were not different in the cat oocytes at 6 h following cat, mouse or hamster sperm injection. Microtubule asters were seen in all oocytes following intracytoplasmic injection of cat, mouse or hamster sperm. Immunocytochemical staining with a histone H3-m2K9 antibody revealed that mouse sperm chromatin is incorporated normally with cat egg chromatin, and that the cat eggs fertilized with mouse sperm enter metaphase and become normal 2-cell stage embryos. These results suggest that sperm aster formation is maternally dependent, and that fertilization processes and cleavage occur in a non-species specific manner in cat oocytes.

Stable ovarian function is a key factor in the performance of the reproductive system. In contrast, some ovarian function-related diseases, such as polycystic ovarian syndrome, premature ovarian failure (POF), and ovarian cancer, are the main cause of infertility and death of women around the world. Despite multiple attempts, there are no effective tools against these conditions; however, mesenchymal stem cell-based therapy, especially using adipose tissue, has attracted much attention in medicine in light of its advantages such as easy isolation and accessibility. Conversely, it has been suggested that MSC-conditioned medium (CM) can restore injured tissues and has high immunocompatibility. So, here, we will summarize the effects of administration of MSCs and CM derived from adipose tissue on ovarian functions and related diseases.

In this paper, with the aim to find new genes involved in mammalian spermatogenesis, we isolated, for the first time in the rat testis, a partial cDNA clone that encoded EH domain binding protein 1-like 1 (Ehbp1l1), a protein that has a single calponin homology domain (CH). Bioinformatic analysis showed that EHBP1l1 contains three domains: the N-terminal C2-like, the CH and the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domains, which are evolutionarily conserved in vertebrates. We found that Ehbp1l1 mRNA was expressed in several rat tissues, including the liver, intestine, kidney and also in the testis during its development, with a higher level in testis from 12-month-old animals. Interestingly, in situ hybridization experiments revealed that Ehbp1l1 is specifically expressed by types I and II spermatocytes, this result was validated by RT-PCR performed on total RNA obtained from enriched fractions of different testicular cell types. As EHBP1l1 has been described as linked to vesicular transport to the actin cytoskeleton and as an effector of the small GTPase Rab8, we hypothesized that it could participate both in cytoskeletal remodelling and in the regulation of vesicle sorting from the trans-Golgi network to the apical plasma membrane. Our findings provide a better understand of the molecular mechanisms of the differentiation process of spermatogenesis; Ehbp1l1 may also be used as a new marker of testicular activity.

This research aimed to examine the effects of thyme, celery and salinomycin on ovarian sex hormones, reproductive traits and antioxidant status during the estrous cycle. Seventy-five mature Barki ewes aged 2–3 years with an average weight of 40 ± 1.5 kg were assigned randomly into five groups (15 head/group). Group 1 was kept as the control; groups 2 and 3 received 20 g/head/day thyme (T) and celery (C) as dried herbs, respectively. Group 4 (T×C) received 10 g thyme + 10 g celery/head/day, and group 5 was treated with salinomycin 1 g/head/day. Blood samples were collected during follicular and luteal phases of the estrous cycle. Thyme and celery and the mixture of T×C increased (P < 0.01) estradiol-17β (E2) during the follicular phase of the estrous cycle, while only the celery group showed a marked (P < 0.001) increase in progesterone (P4) during the luteal phase compared with the control. Salinomycin supplementation decreased (P < 0.05) E2 concentrations during the follicular and luteal phases of the estrous cycle. Supplementation with thyme and celery enhanced (P < 0.001) antioxidant capacity in the luteal phase compared with the follicular stage. The salinomycin group showed increased (P < 0.01) levels of reduced glutathione (GSH) and decreased malondialdehyde (MDA) levels compared with the control group throughout luteal phase. For the interaction between estrous phases and treatments, thyme, celery, and T×C supplementation revealed an increase (P < 0.05) in superoxide dismutase (SOD), GSH, and glutathione disulfide (GSSG) levels compared with the control group during the follicular and luteal phases. Thyme and celery supplementation improved the number of services per conception and fertilization from 1st and 2nd inseminations, respectively. In conclusion, the applied treatment had significant effects on reproductive performance and antioxidant status in ewes throughout the estrous cycle.

Oocyte activation is an essential step in animal cloning to allow subsequent development of the reconstructed embryos. A special activation protocol is required for different animal species. The present study investigated low temperature, electrical pulses, ethanol, ionomycin and strontium for goat oocyte activation in order to optimize the protocols. We found, as a result, effective activation and parthenogenetic development of goat oocytes that had been derived from ionomycin, strontium and electrical pulse groups. Within each group 79.3–81.6%, 2.2–78.8% and 65.5% of the oocytes cleaved and 16.2–24.8%, 0–15.6% and 11.1% of the cleaved embryos developed into blastocysts when the oocytes were activated by ionomycin combined with 6-dimethylaminopurine, strontium plus cytochalasin B and electrical pulses combined with cytochalasin B, respectively. However, low temperature and ethanol were both unable to activate goat oocytes under our experimental conditions. When ionomycin combined with 6-dimethylaminopurine and strontium plus cytochalasin B was applied to activate somatic cell nuclear transfer embryos derived from cultured cumulus, 51.0% and 72.5% of the embryos cleaved, respectively. After transfer of 4-cell embryos into recipients, one (1/19 and 1/7) of the recipients from each group was found to be pregnant as detected by ultrasound, but both of these recipients lost the embryos between 45 and 60 days of pregnancy.

The aim of this study was to describe the effect of temperature on the fertilization, early developmental stages, and survival rate of two Neotropical catfishes Pimelodus maculatus and Pseudopimelodus mangurus. After fertilization, the eggs were incubated at 22°C, 26°C, and 30°C, which resulted in fertilization rates of 96.95 ± 1.79%, 98.74 ± 0.76%, and 98.44 ± 0.19% for P. maculatus and 96.10 ± 1.58%, 98.00 ± 0.63%, and 94.60 ± 2.09% for P. mangurus, respectively. For P. maculatus, hatching occurred after 22 h 30 min post-fertilization at 22°C, 16 h 30 min at 26°C, and 11 h 20 min at 30°C, and the hatching rates were 43.87 ± 7,46%, 57.57 ± 17.49%, and 53.63 ± 16.27%, respectively. For P. mangurus, hatching occurred after 28 h 30 min post-fertilization at 22°C and 17 h 30 min at 26°C with respective hatching rates of 45.4 ± 21.02% and 68.1 ± 12.67%. For this species, all embryos incubated at 30°C died before hatching. Additionally, for P. maculatus, the larvae from the lower (22°C) and higher temperatures (30°C) presented increased abnormality rates, as observed in the head, tail and yolk regions. The lowest abnormality rate was detected at 26°C, which was considered the optimal incubation temperature for both species. The developed protocol enables the manipulation of embryonic development, which is important for the application of reproductive biotechniques, including chimerism and chromosome-set manipulation. The data obtained here are also important for the surrogate propagation of this species as P. mangurus was recently categorized as an endangered fish species.

Although the in vitro fertilisation index is a parameter commonly employed to investigate sperm functional activity, little attention has been given to the occurrence of parthenogenesis. The purpose of this study was to study at 6 h or 22 h incubation: (a) the cleavage-related events that occur in in vitro incubated mouse oocytes, in the absence (parthenogenesis) or presence of homologous spermatozoa; (b) the effect of mineral oil, commonly used in in vitro fertilisation assays; (c) the effect of piroxicam, a prostaglandin synthesis inhibitor, on the parthenogenetic rate; and (d) the influence on parthenogenesis of spontaneous loss of the cumulus oophorus coat during incubation. Under the experimental conditions employed, there was parthenogenetic activation and activation due to fertilisation. Both increased in a time-dependent manner. The mineral oil enhanced the parthenogenetic rate at 22 h incubation. However, it did not have any effect when the oocytes were inseminated. Since we can not discriminate how much of this activation was due to fertilisation and how much to parthenogenesis we must be very careful with this comparison. Piroxicam 10-8 M did not show any effect on the mouse oocyte parthenogenetic rate at neither 6 h or 22 h incubation. Our results suggest that oocyte susceptibility to spontaneous parthenogenetic activation may be modified by the presence of the cumulus and corona radiata cells. In conclusion, we consider that further rigorous studies on these influences are necessary in order to confer more reliability on the results.

It has been established that fertilisation in the sea urchin involves binding of acrosome-reacted sperm to an egg cell surface receptor. The structure and function of receptor, as well as the possible involvement of other cell surface molecules in the binding, fusion and activation events, is discussed.

It has been shown previously that, in Drosophila oogenesis, potassium ions are important for bioelectric phenomena as well as for other physiological and development processes. In the present study we determined the spatial distribution and activity of the (Na+, K+)-pump and of ouabain-insensitive K+ pumps in plasma membranes of vitellogenic ovarian follicles (stage 10). We used that light micorscopic anthroylouabain method as well as the cytochemical lead and cerium precipitation methods in combination with electron spectroscopic imaging (ESI) and elelctronm energy-loss spectroscopy (EELS). (Na+, K+)-ATPase activity was predominantly observed on the oolemma as well as on the membranes of the columnar follicle cells covering the oocyte, whereas on the membranes of the nurse cells and of the squamous follicle cells covering the nurse cells the activity was vary low. The highset activity of the (Na+ K+)-pump was found at the anterior and posterior ends of the oocute, and this on the oolemma as well as on the membranes of the follicle cells located here. Strong activity of ouabain-insensitive K+-pumps was observed on most of the oolemma (except at the anterior of the oocyte) and on the membranes of some nurse cells located next to the oocyte, whereas less activity was found on the other nurse cell membranes and on the membranes of all follicle cells. The suitability of the differnet methods nurse cell membranes and on the membrances of all follicle cells. The suitability of th different methods used for determining the localisation as well as the activity of K+-pumps is discussed. We further discuss the nature of the ouabain-insensitive K+ pumps and the relevance of the observed distribution of K+-pumps for K+ uptake, extrafollicular ionic current flow intercelluar signalling and other developmental processes in Drosophila oogenesis.

Marine angelfish (family: Pomacanthidae) are among the most sought-after fish species in the saltwater aquarium trade. However, there is a lack of information in the literature on their early ontogeny. The objective of this study was to describe the embryonic and early larval development of two dwarf angelfish, the bicolour angelfish, Centropyge bicolor and the coral beauty angelfish, Centropyge bispinosa. The eggs of these two species were obtained from spontaneous spawning of the broodstock fish in captivity and incubated at 26.0 ± 0.2°C throughout the study. Fertilized eggs (n = 15) of both species are transparent, pelagic and spherical; the mean diameters of the eggs were measured at 703.6 ± 7.8 μm for C. bicolor and 627.6 ± 7.8 μm for C. bispinosa. The eggs of both species possessed a narrow perivitelline space, smooth and thin chorion, a homogenous and non-segmented yolk as well as a single oil globule. Overall, the observed embryonic development pattern of C. bicolor and C. bispinosa was very similar, and the main difference was the embryonic pigmentation pattern, which only became evident close to hatching. Larvae of both species started hatching at 13 h 30 min after fertilization, and the larval characteristics of both species also showed high levels of similarities. However, the mouth opening time for C. bicolor was 72 h after hatching (AH) and 96 AH for C. bispinosa. In general, the observed early ontogeny of C. bicolor and C. bispinosa also resembled that of other Centropyge species documented in the literature.

During the process of follicular atresia, cells are observed to invade the zona pellucida (invasive cells) where they presumably play an important role in eliminating degraded oocytes. Although our preliminary studies have suggested that these cells may originate from granulosa cells and not from macrophages, a detailed morphological analysis of the cells has not been conducted. The objective of this study was to characterize the cells more precisely by electron microscopy and immunohistochemistry, using sexually immature mice. The results show that the invasive cells were first observed within advanced primary (non-antral) atretic follicles. The cells frequently contained cytoplasmic lysosome-like granules after passing through the zona pellucida. F4/80 and Mac-1, reported as macrophage-specific antibodies, were reactive with the cells in most cases, but some immunonegative invasive cells were also observed. The ultrastructural features of the invasive cells were quite similar to those of granulosa cells, not macrophages. Gap junctions, which are typical cytoplasmic structures of epithelial cells, were frequently identified between neighbouring cells. Although direct evidence indicating a contribution by the cells to the elimination of degenerated oocytes was not obtained, our results strongly suggest that the invasive cells originated from granulosa cells surrounding the zona pellucida, and that they may have a macrophage-like cell function for the elimination of oocytes from atretic follicles in mice.

Medically assisted reproductive (MAR) treatments using donated oocytes are commonly applied in several countries to treat women who cannot conceive with their own gametes. Historically, in Italy, gamete donation has been prohibited but, in 2014, the law changed and gamete donation became allowed for couples undergoing MAR treatments. Consequently, in the last decade, there has been an increase in application of the oocyte donation programme. This study reports an egg-donation programme’s clinical efficacy, based on importing donated vitrified oocytes from cryo-banks located in a foreign country. For this, we conducted a retrospective analysis of data from a single reproductive unit located in Italy (Donna Salus Women’s Health and Fertility, Bozen). The study group consisted of 681 vitrified oocytes, which were warmed and culture to be replaced in 100 recipients. The survival rate after warming was 79.1% (n = 539/681), whereas the fertilization and blastulation rates were 90.2% (n = 486/539) and 47.9% (n = 233/486), respectively. Positive pregnancy test, clinical pregnancy rates, and live-birth rates per embryo transfer were 37.8%, 31.1% and 28.4%, respectively. The multiple pregnancy rate was 0.7%. This study is one of the first to report on the efficacy of a donor oocyte programme in Italy using imported vitrified oocytes. The above data may reassure women who are undertaking donation programmes using vitrified oocytes imported from commercial egg banks.

At the time of sperm-egg fusion in Discoglossus pictus, a large amount of electron-dense material of an unknown nature is liberated from the sperm. In the present work we studied this material in D. pictus sperm, using an assay utilising strips of autoradiographic film as a gelatin substrate for proteolytic enzymes. Upon treatment with A23187, D. pictus sperm produced a large halo on the gelatin substrate, indicating the presence of enzymes released by the sperm at the time of the acrosome reaction. In contrast, Xenopus laevis sperm did not produce halos upon treatment with A23187. The use of protease inhibitors such as TLCK, leupeptin, chymostatin, SBTI and EACA strongly suggests that the D. pictus whole acrosome contains trypsin and chymotrypsin activity while an SBTI-sensitive activity is absent in a small portion of the acrosome, possibly the anteriormost region. Furthermore, the material released at the acrosome reaction also contains an EACA-inhibited activity, indicating the presence of plasminogen activator. We conclude that D. pictus sperm release proteolytic enzyme(s) that may act at the egg surface at the time of gamete fusion.