Mauro Mattioli

Developmental competence of pig oocytes matured and fertilized in vitro

Pig follicles 3 to 6 mm in diameter were everted and matured for 44 h. The oocytes were then collected and exposed to capacitated boar sperm purified by centrifugation in a two step (65 and 70%) Percoll gradient. Of 110 ova fixed 14 h after in vitro fertilization, 78% were penetrated and 47% were monospermic. Next, 681 oocytes were cultured in vitro for 44 h after in vitro fertilization and the 266 embryos which had reached the two- to four-cell stage were transferred into the oviducts of 12 synchronized recipient gilts. Four days later, 211 embryos (79%) were recovered by uterine flushing. 40.7% of these were at the blastocyst stage, and 20% were at the morula stage. In a final experiment, four out of eight gilts which had received 40 to 50 two- to four-cell embryos, were diagnosed pregnant 30 and 37 d after in vitro fertilization. One sow farrowed nine live piglets and one stillborn, two pregnancies were in progress, while one sow returned to estrus 47 d after in vitro fertilization. These results demonstrate that pig oocytes matured and fertilized in vitro can develop to the blastocyst stage and establish a normal pregnancy resulting in the birth of live piglets.

Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction

Anandamide (AEA) is the endogenous ligand of cannabinoid (CB) receptors, and as such it plays several central and peripheral activities. Regulation of female fertility by AEA has attracted growing interest, yet a role for this endocannabinoid in controlling sperm function and male fertility in mammals has been scarcely investigated. In this study we report unprecedented evidence that boar sperm cells have the biochemical machinery to bind and degrade AEA, i.e. type-1 cannabinoid receptors (CB1R), vanilloid receptors (TRPV1), AEA-synthesizing phospholipase D (NAPE-PLD), AEA transporter (AMT) and AEA hydrolase (FAAH). We also show that the non-hydrolyzable AEA analogue methanandamide reduces sperm capacitation and, as a consequence, inhibits the process of acrosome reaction (AR) triggered by the zona pellucida, according to a cyclic AMP-dependent pathway triggered by CB1R activation. Furthermore, activation of TRPV1 receptors seems to play a role of stabilization of the plasma membranes in capacitated sperm, as demonstrated by the high incidence of spontaneous AR occurring during the cultural period when TRPV1 activity was antagonized by capsazepine. We show that sperm cells have a complete and efficient endocannabinoid system, and that activation of cannabinoid or vanilloid receptors controls, at different time-points, sperm functions required for fertilization. These observations open new perspectives on the understanding and treatment of male fertility problems.

Vascular Endothelial Growth Factor Production in Growing Pig Antral Follicles

Abstract Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small (<4 mm), medium (4–5 mm), or large (>5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.

Follicle Activation Involves Vascular Endothelial Growth Factor Production and Increased Blood Vessel Extension

Abstract The authors evaluated the relationship between vascular endothelial growth factor (VEGF) production, blood vessel extension, and steroidogenesis in small (<4 mm), medium (4–5 mm), and large (>5 mm) follicles isolated from gilts treated with eCG. VEGF and estradiol levels were measured in follicular fluid by an enzyme immunoassay and radioimmunoassay, respectively, and then each follicle wall was used to evaluate VEGF mRNA content and for the immunohistochemical analysis of blood vessels. VEGF production was low in small follicles (<3 ng/ml), high in large follicles (>10 ng/ml), and markedly differentiated in medium follicles; 44% exhibited values up to 15 ng/ml, whereas the levels never exceeded 3 ng/ml in the remaining aliquot. Medium follicles were then used as a model to investigate angiogenesis. Reverse transcription-polymerase chain reaction for VEGF mRNA demonstrated that granulosa cells represent the main component involved in the production of VEGF. The follicle wall, which presents two distinct concentric vessel networks, showed a vascular area (positive stained area/percent of field area) that was significantly wider in high VEGF follicles than in low VEGF follicles (2.54% ± 0.58% vs. 1.29% ± 0.58%, respectively). Medium follicles with high VEGF levels and extensive vascularization accumulated high estradiol levels (150–300 ng/ml), whereas follicles with low VEGF levels had basal estradiol levels that never exceeded 30 ng/ml. Early atretic medium-size follicles had undetectable levels of VEGF and estradiol paralleled by a marked reduction in blood vessel. The data presented propose an improved model for follicle dynamics in which the production of VEGF, stimulated by gonadotropin, creates the vascular conditions required for follicle growth and activity.

Effects of LH and FSH on the maturation of pig oocytes in vitro

This research was designed to investigate the effects of LH and FSH (50 ng/ml) on pig oocyte maturation in vitro. The following parameters were studied: a) the degree of heterologous coupling between cumulus cells and oocytes, evaluated by measuring the 3H-uridine and 3H-choline uptake in cumulus enclosed oocytes; b) meiotic maturation; c) cytoplasmatic maturation, evaluated by analyzing the ability of the oocytes to promote male pronucleus formation after in vitro fertilization. Despite the marked cumuli expansion induced by gonadotropins, uridine uptake was not influenced by LH or FSH. By contrast, choline uptake in LH-treated oocytes was significantly higher than in FSH-treated or control oocytes (3199 cpm+/-251 vs 1686 cpm+/-142, P<0.01). Gonadotropins accelerated meiotic progression, and after 30 hours of culture the percentage of oocytes at the germinal vesicle stage was significantly lower (P<0.01) in LH-(24%, 24/102) and FSH-(20%, 18/90) treated oocytes than in control oocytes (76%, 64/84). After 44 hours of culture, the percentage of oocytes reaching the MII stage was significantly higher (P<0.01) in the presence of LH (76%, 92/120) and FSH (86%, 92/108) than in the controls (35%, 40/116). The percentage of oocytes capable of sustaining male pronucleus formation was similar in the control (48.4%, 63/132) and FSH-treated oocytes (44.3%, 51/116), while it was markedly increased (P<0.01) by the addition of LH (72.7%, 143/197). The data reported indicate that in vitro pig oocytes tend to undergo meiotic maturation even in the absence of hormones. However, in our in vitro system, LH and FSH accelerated and facilitated meiotic progression, and LH selectively improved cytoplasmic maturation which is required to promote the formation of a male pronucleus.

In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue

Abstract In the present study, we compared the in vitro development of sheep preantral follicles obtained from unfrozen or frozen ovarian cortex. After thawing, follicles stored by a slow-freezing protocol with dimethyl sulfoxide (DMSO) or ethylene glycol (EG) were mechanically isolated and cultured for 10 days. After 1 day, approximately 50% and 34% of the DMSO and EG follicles, respectively, showed overt signs of degeneration, as confirmed by histological analysis. Follicles that survived thawing grew and formed antral-like cavities, without significant differences among experimental groups. However, the percentages of healthy oocyte-cumulus cell complexes (OCCs) retrieved from in vitro-grown follicles, as well as estradiol, were lower in DMSO than in EG or unfrozen follicles. Although cryopreservation did not cause appreciable differences in follicle morphological aspects, frozen OCCs showed lower metabolic cooperativity levels, as determined by [3H]uridine uptake. During culture, oocytes increased in diameter, but the percentage of germinal vesicle stage-arrested oocytes showing a rimmed chromatin configuration was significantly lower in the frozen groups. Our results indicate that cryopreserved sheep preantral follicles underwent growth in vitro but that freezing/thawing specifically affected gap junctional permeability and impaired the progression of regulative processes, such as the acquisition of a specific oocyte chromatin configuration. Moreover, because the cryoprotectant toxicity test excluded the occurrence of direct cellular damage, this method allowed us to discriminate the effects exerted by different cryoprotectants during the cryopreservation procedure on whole-follicular development.

Identification of capacitation in boar spermatozoa by chlortetracycline staining

The functional status of boar spermatozoa undergoing capacitation in vitro was investigated. Two fluorescent stains were used: chlortetracycline (CTC) and a FITC-conjugated lectin (FITC-PSA). The first has been used for the direct identification of the capacitated boar spermatozoa, while the second, based on the identification of capacitated spermatozoa by their ability to undergo zona-induced acrosome reaction (AR), was used to confirm and validate the CTC assay in this species. Spermatozoa obtained from 5 different boars was washed and incubated under capacitating conditions. Aliquots of spermatozoa were collected at 0, 90 and 180 min of incubation and then stained with CTC or FITC-PSA. After CTC staining, 3 different fluorescent patterns were observed: Pattern A with the fluorescence uniformly distributed on the sperm head, Pattern B with the fluorescence concentrated in the post-acrosomial region, and Pattern C with the fluorescence concentrated in the acrosomial region. The percentage of spermatozoa displaying fluorescent Pattern A decreased throughout the incubation while that of spermatozoa with Pattern C showed a concomitant progressive increase. Pattern B fluorescence remained unchanged throughout the maturation period. Exposure to zonae pellucidae (ZP) brought back the levels of Pattern C fluorescence to basal values. Since only the capacitated spermatozoa are believed to react to ZP, this observation together with the rising incidence of Pattern C throughout maturation suggests that fluorescence in the acrosomial region identifies capacitated spermatozoa. The analysis of acrosome integrity carried out with FITC-PSA showed that the proportion of zona-induced AR was nearly the same as that of spermatozoa displaying Pattern C, thus confirming that CTC staining is suitable for the detection of boar sperm capacitation. In the second part of this study, CTC was used to investigate the effects of sperm origin and storage on the capacitation process. Our finding demonstrates that capacitation kinetics show wide variations in sperm samples derived from different boars; moreover, capacitation is also affected by sperm storage. While fresh semen showed a progressive increase in capacitated spermatozoa, ranging from low levels at the beginning of the culture to 46% at the end of incubation, the refrigerated semen had a relatively high percentage of capacitated spermatozoa at the beginning of culture, but this proportion increased only slightly during the following 90 to 180 min of treatment. These data indicate that CTC can be used to identify capacitated boar spermatozoa, and, because of its rapid and easy execution, it can be used routinely to identify the optimal capacitation time for different sperm samples.

Calcium elevation in sheep cumulus-oocyte complexes after luteinising hormone stimulation

We investigated Ca2+ levels in intact cumulus‐oocyte complexes (COCs) on exposure to peak levels of luteinising hormone (LH). Specific preparations were used where cumulus corona cells were loaded with a membrane‐permeant Ca2+‐sensitive dye (FLUO‐3AM), whereas the oocyte was injected directly with the nonpermeant form of the dye (FLUO‐3). After exposure to LH, cumulus and corona radiata cells showed distinct rises in intracellular Ca2+ in 50–200 sec. The pattern of Ca2+ response varied in the different cells both for the duration of the transients and for their persistence. Interestingly, Ca2+ elevations were recorded in all the layers of the cumulus mass, including the innermost layer of corona cells, demonstrating the wide diffusion of LH receptors. Following the Ca2+ raise in somatic cells, an intracellular Ca2+ elevation also was recorded within the oocyte with a delay of 100–300 sec. The elevation started at the cortex of the oocyte and then spread all over the ooplasm. The addition of verapamil or manganese chloride did not prevent LH‐induced Ca2+ elevation in the COC, whereas mechanical uncoupling of cumulus cells from the oocyte prevented any Ca2+ response within the oocyte. The results indicate that cumulus‐corona cells are capable of transducing LH message by rising intracellular Ca2+ and show that this signal is rapidly transferred into the oocyte through gap junctions. This may result from the direct diffusion of Ca2+ or its putative releaser IP3 from cumulus cells to the oocyte. Mol. Reprod. Dev. 50:361–369, 1998.

Signal transduction mechanism for LH in the cumulus-oocyte complex.

The paper reviews recently described signalling mechanisms by which cumulus cells exposed to peak levels of gonadotropins, activate oocyte maturation. Cumulus cells react to LH with a prompt Ca raise which diffuses through gap junctions in a few minutes also into the oocyte where a local amplification system spreads the signal all over the cell. Few h later, still as a consequence of LH stimulation, cumulus cells undergo aprogressive depolarisation of their plasma membrane potential. Due to the electric coupling with these cells the oocyte depolarises too and this open specific voltage gated Ca channels responsible for a second wider and more sustained intracellular Ca rise. As a result of changes throughout maturation with a consequent modification of the size and charge of the molecules that can diffuse from one cell compartment to the other. This cell to cell interaction is further modified with cumulus expansion that leads to a progressive uncoupling of outer cumulus cells while the inner cell layer, corona radiata, remains in oocyte maturation by addressing to the oocyte nutrients and instructions in a well-orchestrated sequence. The identification of these mechanisms are a fundamental prerequisite for the development of in vitro systems suitable to produce oocytes matured in vitro with normal developmental competence.

Improving Delivery and Offspring Viability of In Vitro-Produced and Cloned Sheep Embryos

Abstract Recently developed, assisted reproductive technologies (e.g., in vitro embryo production and nuclear transfer) have encountered perinatal morbidity/mortality of the offspring produced, which are likely to hinder the application of these techniques. Consequently we have sought to develop a system of hormonal stimulation that will ensure the delivery of offspring more prepared for extrauterine life. Here we examine deliveries outcome in sheep carrying in vitro-produced and nuclear transfer (NT) embryos in comparison to artificially inseminated and naturally mated control ewes. All groups (excluding NT, which received one treatment) were subjected to one of two hormonal treatments for induction of delivery, whereas the third part of each group was left without any treatment. The first (commonly used for naturally mated ewes) dexamethasone treatment did not solve a majority of parturition disturbances, and actually the number of deliveries necessitating assistance was reduced (P < 0.05) by this treatment in the control group. On the other hand, combined estradiol plus betamethasone stimulation (E + B) solved a majority of complications regarding delivery performance such as lack of the preparation of the mammary gland, low myometrial contractility, insufficient cervical ripening, and impaired maternal behavior. Moreover, substantial reduction of neonatal mortality was observed following the combined treatment. In conclusion, the E + B induction of delivery overcame the majority of physiological and behavioral intrapartum failures of sheep foster mothers and increased the survival of offspring, and thus can be recommended as a safe method for inducing delivery in foster mothers carrying in vitro-generated embryos.