Maria Albrizio

Meiotic Competence of Equine Oocytes and Pronucleus Formation after Intracytoplasmic Sperm Injection (ICSI) as Related to Granulosa Cell Apoptosis

Abstract Follicle atresia and granulosa cell apoptosis may be related to oocyte meiotic and developmental competence. We analyzed the relationships among granulosa cell apoptosis, initial cumulus morphology, oocyte nuclear maturation in vitro, and pronucleus formation after intracytoplasmic sperm injection (ICSI) in the horse. For each follicle, the size was measured and granulosa cells were used for DNA laddering analysis. Oocytes were evaluated for cumulus morphology, cultured for in vitro maturation, and submitted to ICSI. Apoptosis was categorized as absent, intermediate, or advanced according to the relative concentrations of two DNA fragments at 900 and 360 base pairs (bp). In 98 oocyte-follicle pairs, 52 oocytes were classified as expanded (Exp), 39 as compact (Cp), and 7 as having a partial (P) cumulus. Advanced apoptosis was detected in 55% (54/98) of follicles; 37% (36/98) of follicles showed an intermediate level of apoptosis; and 8 follicles (8%) were nonapoptotic. Follicle size was not significantly correlated with granulosa cell apoptosis (P > 0.05). Significantly more Exp than Cp oocytes originated from follicles with advanced apoptosis (P < 0.001). The proportion of oocytes maturing in vitro was significantly higher in oocytes issuing from apoptotic follicles than in oocytes issuing from healthy follicles (P < 0.05). The proportion of normally (two pronuclei) or abnormally fertilized oocytes (one or greater than two pronuclei, or partially decondensed sperm) did not differ in relation to granulosa cell apoptosis. We conclude that, in the mare, granulosa cell apoptosis is related to cumulus expansion and an increase in oocyte meiotic competence but has no effect on the proportion of meiotically competent oocytes that activate after ICSI. These results provide selection criteria for horse oocytes used in assisted reproductive techniques so that embryo production may be maximized.

Influence of behavioural lateralization on interleukin-2 and interleukin-6 gene expression in dogs before and after immunization with rabies vaccine

Evidence in several species, including dogs, has been collected demonstrating that the brain hemispheres modulate the immune system in an asymmetrical way. To study the interactions between immune response and lateralization, three groups of mixed breed dogs were selected on the basis of their performance in a paw preference test involving removal of a piece of sticky tape from the snout. The expression of interleukin-2 (IL-2) and interleukin-6 (IL-6) genes was measured in left-pawed, right-pawed and ambidextrous dogs before and after immunization treatment with a rabies vaccine. The results revealed a relationship between the mRNA expression of IL-2 and IL-6 genes and the direction of behavioural lateralization. Under basal conditions, IL-2 and IL-6 gene expression was higher in left-pawed dogs than in right-pawed and ambidextrous dogs. After the vaccine administration, decreasing levels of IL-2 and IL-6 gene expression were observed in left-pawed and right-pawed dogs, but not in ambidextrous dogs. These findings represent the first evidence that brain lateralization may influence the immune system in dogs by the modulation of mRNA gene expression of cytokines such as IL-2 and IL-6, which have been recognized as key immune-regulatory proteins.

Expression and localization of the µ‐opioid receptor (MOR) in the equine cumulus–oocyte complex and its involvement in the seasonal regulation of oocyte meiotic competence

The µ‐opioid receptor (MOR) was identified in equine oocytes, cumulus and granulosa cells. By RT‐PCR, a 441bp fragment was observed. By immunoblotting, a 65 kDa band was detected in samples of winter anestrous whereas in cells recovered in breeding season, two bands, 65 and 50 kDa, were found. The 65 kDa band was significantly more intense in winter anestrous specimens. In samples recovered in the breeding season, this band significantly decreased with the raise of follicle size and was heavier in compact oocytes and cumulus cells. The protein was localized on the oolemma and within the cytoplasm of oocytes and cumulus cells. In vitro oocyte maturation rate (MR), analyzed by confocal microscopy for nuclear chromatin, microfilaments and microtubules, was reduced after the addition of 3 × 10−8 M β‐endorphin in medium without additional hormones. Inhibitory effects of 10−3 M Naloxone in oocytes collected in anestrous and spring transition were observed, both in presence and absence of hormones added to culture medium. Increased MRs were observed in oocytes collected in anestrous and cultured in presence of 10−8 M Naloxone. The exposure to 10−3 M Naloxone induced significant intracellular calcium increases in cumulus cells recovered all over the year. β‐Endorphin 3 × 10−8 M induced significant calcium increases only in cumulus cells recovered in fall transition and anestrous. Naloxone 10−8 M did not induce intracellular calcium modifications. We conclude that the MOR is differentially expressed in equine cumulus–oocyte complexes in the different seasons of the year and plays a role in the seasonal regulation of meiotic competence of equine oocytes. Mol. Reprod. Dev. 75: 1229–1246, 2008.

Expression of the μ-opioid receptor on Malassezia pachydermatis and its effect in modulating phospholipase production

Malassezia spp. may act as opportunistic skin pathogens in humans and animals. Malassezia pachydermatis proliferation and phospholipase production may play a pathogenic role in the occurrence of skin lesions in dogs. This study investigates the presence of mu-opioid receptor (MOR) in M. pachydermatis strains isolated from healthy dogs and dogs with skin lesions and its effects on phospholipase activity (p.a.). P.a. of 64 M. pachydermatis isolates was evaluated using different concentrations of naloxone (Nx), a MOR antagonist. Isolates were divided into Group A (i.e., 40 isolates from 26 dogs with dermatitis) and Group B (i.e., 24 isolates from 12 healthy dogs). The MOR expression was analyzed by Western blot and immunofluorescence. A statistically higher p.a. than that of the controls was found with isolates in Group A at a Nx concentration of 10(-6) M (P<0.05). No isolate in Group B displayed p.a. in either control samples or in the presence of any Nx concentration. Immunoblotting revealed two positive MOR immunoreactive bands of approximately 65 and 98 kDa. MOR expression and localization was also demonstrated by immunofluorescence in isolates from Groups A and B. This study provides the first evidence of MOR expression on M. pachydermatis cell membranes pointing to its possible role in modulating p.a. production in isolates from dogs with skin lesions.

The structure of the cytochrome oxidase subunit II gene and its use as a new character in the construction of the phylogenetic tree of Angiospermae

Abstract With the aim of correlating the structure of the intron present in the mitochondrial gene coding for subunit II of the cytochrome oxidase complex ( coxII gene) with some aspects of Angiosperm evolution, we analysed the organization of the coxII gene from 30 plants belonging to 18 different orders. In all the monocotyledons tested, the gene contains an intron with a composite structure. Among dicot plants, 16 contain a split gene, 5 a continuous gene. The presence or the absence of intervening sequences in the genes of dicot plants does not seem to be a casual event, on the contrary it seems to be a feature of specific phylogenetic lines. Our results suggest that this approach, extended to a wider number of plant species, could make a contribution to the construction of the phylogenetic tree of Angiosperms.

Stress-related hormones in horses before and after stunning by captive bolt gun.

In this work the slaughter-linked plasma modifications of some stress-related hormones in horses subject to standardized butchering procedures were investigated in order to highlight the compromised animal welfare during pre-slaughter handling. During pre-slaughter, animals show strong hardship behavioural patterns, probably due to being under life-threatening conditions. Blood samples from 12 male horses, ageing from 3 to 5 years, were collected before slaughtering in lairage, and during exsanguination after stunning. Catecholamines, cortisol and beta-endorphin concentrations were assessed in plasma samples by EIA. Results show that plasma beta-endorphin concentration did not increase significantly after stunning, while cortisol (P<0.05) and catecholamines (P<0.001) increased significantly. The ratio between the plasma level of norepinephrine and epinephrine decreased significantly (P<0.001) during the time considered for observation underlining a greater involvement of adrenal medulla in the stress response. Moreover these results suggest that, under stress, the release of beta-endorphin could be different from that of ACTH.

Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA)

BackgroundOpioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology.MethodsWe analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltrindole and DPDPE by means of a computer assisted sperm analyzer and a fluorescent confocal microscope. The evaluation of viability, capacitation and acrosome reaction was carried out by the double CTC/Hoechst staining, whereas mitochondrial activity was assessed by means of MitoTracker Orange dye.ResultsWe showed that in equine sperm cells, delta opioid receptor is expressed as a doublet of 65 and 50 kDa molecular mass and is localized in the mid piece of tail; we also demonstrated that naltrindole, a delta opioid receptor antagonist, could be utilized in modulating several physiological parameters of the equine spermatozoon in a dose-dependent way. We also found that low concentrations of the antagonist increase sperm motility whereas high concentrations show the opposite effect. Moreover low concentrations hamper capacitation, acrosome reaction and viability even if the percentage of cells with active mitochondria seems to be increased; the opposite effect is exerted at high concentrations. We have also observed that the delta opioid receptor agonist DPDPE is scarcely involved in affecting the same parameters at the employed concentrations.ConclusionsThe results described in this paper add new important details in the comprehension of the mammalian sperm physiology and suggest new insights for improving reproduction and for optimizing equine breeding.

Changes in the expression of the μ-opioid receptor in the mare oviduct during oestrus and anoestrus

The presence of the mu-opioid receptor (MOR) was investigated in the mare oviduct during oestrus and anoestrus, by means of immunoblotting and immunohistochemistry. Immunoblotting analysis showed that the MOR protein is expressed as 65, 50 and 30 kDa forms in the infundibulum and ampulla both in oestrus and anoestrus, while the 30 kDa form is absent in the isthmus. Moreover, different levels of expression were observed along the ampulla in the two periods examined. Immunohistochemistry revealed MOR in the mucosal epithelium, stromal cells, myocytes and blood vessels. Ciliated cells expressed MOR in the apical cytoplasm and, except for the isthmus of oestrous mares, also in the nucleus. Non-ciliated cells showed MOR only in the isthmus segment during oestrus. Stromal cells showed different immunoreactivity along the oviduct segments and during the oestrous and anoestrous phases. The myosalpinx displayed immunostained myocytes in the intrinsic musculature of the ampulla and in the extrinsic and intrinsic musculature of the isthmus without significant differences between anoestrus and oestrus. Blood vessels expressed MOR in endothelial cells and smooth muscle cells in the isthmus myosalpinx of oestrous mares only. In conclusion, these findings show diverse MOR expression in the three segments constituting the oviduct, as well as changes in MOR expression linked to the mares physiological condition.

Expression of the μ opioid receptor and effects of the opioid antagonist naloxone on in vitro maturation of oocytes recovered from anoestrous bitches.

The mu-opioid receptor (MOR) is expressed in bovine, human, equine and canine oocytes, and in seasonal breeders, it is expressed with higher intensity during the anoestrous phase. Supplementation of in vitro maturation (IVM) medium with opioid agents, agonists or antagonists, was shown to affect oocyte maturation in several species such as rat, bovine and equine. This study reports the effects of supplementing IVM medium with naloxone (Nx), an opioid antagonist, on nuclear and cytoplasmic maturation rate of oocytes recovered from anoestrous bitches. Cytoplasmic maturation was examined in terms of mitochondrial (mt) distribution. In order to confirm the receptor-mediated action of Nx, in oocytes of anoestrous bitches, MOR expression was analyzed by Western blot. Cumulus-oocyte complexes, recovered from the ovaries of bitches in anoestrous, were cultured in vitro and Nx was added at the concentrations of 1 x 10(-6), 1 x 10(-8) and 1 x 10(-10) M. The rate of oocytes resuming meiosis after culture in presence of 1 x 10(-6) M Nx (29%) was significantly higher than that of oocytes of control group (12%; p < 0.05). However, treatment with Nx did not affect mt distribution pattern. In denuded oocytes and in corresponding cumulus cells, a doublet of 65 and 50 kDa was observed. We conclude that, in oocytes of anoestrous bitches, MOR is expressed and Nx significantly improves nuclear maturation rate. Further studies should be performed to elucidate the expression of other opioid receptors, such as delta and kappa, and possible interactive effects of their antagonists on canine oocyte maturation.

Investigations of the coxII intron structure in the mitochondrial genes of angiosperms

Abstract The molecular structure of the intron of the mitochondrial gene coding for cytochrome oxidase susbunit II has been investigated by sequence analysis in eight angiosperm orders. Comparison of the overall primary structure of the intron, made with respect to Magnoliales, the ancestor group of all the angiosperms, reveals a high conservation among dicot plants. On the contrary, the introns of both ancient and advanced monocot orders show relevant divergences represented by insertions and deletions in conserved specific regions of the introns. As a consequence of some of these rearrangements, a structure similar to a transposable element is generated in advanced monocots. This specific structure, common in all the analyzed species of Poales, seems to be generated by a stepwise process and it is not dependent on an insertion event.