Identification of sperm antigens as a first step towards the generation of a contraceptive vaccine to decrease fossorial water vole Arvicola terrestris Scherman proliferations.

Immunocontraceptive strategies have proved to be efficient in controlling fertility of various mammalian species. In the present study we have made the first steps towards the identification of Arvicola terrestris sperm antigens that could be used as targets in the development of a contraceptive vaccine to limit the proliferations of this pest rodent. Rabbit-raised polyclonal antisera directed against complete A. terrestris spermatozoa were used to identify and characterize on 2D-gels coupled with a MALDI-TOF mass spectrometry analysis A. terrestris sperm proteins. Amongst the proteins pinpointed by this approach some were further investigated based on their tissue- and/or sperm-specific expression, and their relevance to fertility or sperm/egg interaction. In parallel, three proteins that have been already reported in the literature to be appropriate targets for the development of contraceptive vaccines in other mammalian species have also been looked for in A. terrestris. With the selected protein targets, a reverse-PCR approach using degenerate primers was employed to amplify corresponding A. terrestris cDNAs. After conceptual translation and sequence alignment, different proteins were studied to determine zones with sufficient sequence divergence and of antigenic/immunogenic nature that could be used in future assays to immunize animals.

Antinociceptive effect of morphine microinjections into the spinal trigeminal subnucleus oralis.

We analysed the effects of morphine microinjections (0.2 microliter) into the subnucleus oralis (SNO), i.e. the rostral division of the spinal trigeminal nucleus, on the formalin test adapted for orofacial pain. Duration of rubbing following an injection (50 microliters) of formalin solution (1.5%) in the right upper lip of the rat was measured. Compared to microinjections of saline (NaCl, 0.9%) used as control, 90 and 180 nmol microliter-1 of morphine solution in the SNO significantly curtailed rubbing. This effect was dose-dependent, reversed by naloxone, and also site-specific to the SNO since no effect was observed after microinjection of morphine into areas adjacent to the SNO. These results are further evidence for the involvement of the SNO in perioral nociceptive mechanisms.

Enhanced nociceptive behaviour following conditioning injection of formalin in the perioral area of the rat.

The possible existence of long-term modifications in response to a transient nociceptive conditioning stimulation was investigated in the rat in three experiments. (1) A nociceptive conditioning stimulus was delivered in the form of a s.c. formalin injection (conditioning injection) in the left upper lip. Evaluation of the nociceptive behaviour triggered by another formalin injection (testing injection) made in the controlateral right upper lip was carried out in distinct groups of rats 7, 14 or 28 days after the conditioning. An enhanced nociceptive response at day 7 and 14 and a return to the baseline at day 28 were observed. (2) A similar protocol was developed with formalin used for both conditioning and testing but an anaesthetic blockade of the infraorbital nerve was performed just before the conditioning injection to suppress the initial barrage. The change observed at day 7 was suppressed by the nerve block. (3) A conditioning nociceptive stimulus was applied either ipsilaterally to the right lower lip or to the tail. An increased nociceptive response was observed when the conditioning stimulus was applied to the same side as the test stimulus but no increase in the formalin test response was detected when the conditioning stimulus was applied to the tail. These results indicated that, after a single formalin injection in the left upper lip, a hyperexcitability developed that depended on the initial barrage, lasted for at least 2 weeks, was no longer present at 4 weeks and might rely on a segmental mechanism. The hypothesis of a central sensitization triggered by an initial barrage and maintained by an ongoing input induced from the periphery is discussed.

Effects of lesions in the trigeminal oralis and caudalis subnuclei on different orofacial nociceptive responses in the rat.

Behavioural responses to two different orofacial noxious stimulations were analysed following lesion of spinal trigeminal subnucleus oralis (Sp5O) in the rat. Lesions were obtained by intranuclear microinjections of quinolinic acid (0.4 microliter of 60 nmol/microliter solution). The control groups received microinjection of saline. Noxious stimulation was a subcutaneous injection of formalin into the upper lip or electrical stimulation of the tooth pulp. The measured behavioural responses were duration of rubbing induced by the formalin injection and thresholds of the jaw-opening reflex (JOR), head rotation (HR) and face rubbing (FR) evoked by the pulp stimulation. In addition, formalin injection was also performed in two groups of rats that had received intranuclear injection of quinolinic acid or saline into rostral subnucleus caudalis (Sp5C). Rubbing duration was not significantly modified by the lesion of Sp5O, whereas a significant decrease occurred after the lesion of rostral Sp5C. After the lesion of Sp5O, an increase in the threshold of JOR was observed whereas the thresholds of HR and FR were not significantly modified. These results suggest that Sp5O is not necessary for the processing and relay of nociceptive inputs triggered by intense stimulations of oral and perioral areas. However further experiments are needed to reconcile these results with the relevant data obtained from cell recording experiments which indicate the existence, in Sp5O, of neuronal activities related to the sensory discriminative aspect of intense nociception.

Sustained hyperalgesia can be induced in the rat by a single formalin injection and depends on the initial nociceptive inputs.

The present study was undertaken to find out if a hyperalgesia can be observed 1 week after a conditioning stimulation of the orofacial area of the rat. Sprague-Dawley rats received a left infraorbital infiltration with either bupivacaine or saline and then, 30 min thereafter, an injection of either saline or 10% formalin solution in the left upper lip. Four groups of animals were thus made up depending on their conditioning treatment. Seven days later, an algesimetric test initiated by a contralateral orofacial formalin injection was carried out. The duration of lip rubbing was significantly increased in formalin-conditioned groups. The hyperalgesia observed at 7 days was suppressed by an infraorbital nerve block carried out at the time of the conditioning stimulus. These results tend to indicate that a nociceptive message of short duration induces a trace in the central nervous system which can be retained for 1 week.

Is there a correlation between scores of nociceptive behavioral responses to formalin injections given at different body sites in the rat?

Correlations between behavioural nociceptive responses to an injection of formalin solution in the hindpaw and upper lip were sought in 28 male rats divided into two groups. One group received a first injection in the hindpaw and 7 days later, a second one in the lip (paw-lip group). The injection order was reversed in the second group (lip-paw group). After each injection, the duration of lip rubbing for the orofacial formalin test, the duration of paw licking and the number of paw flinchings for the hindpaw test were measured. The Spearman test revealed no correlation between lip rubbing and paw flinching in either group but a significant correlation between lip rubbing and paw licking occurred in both groups.

Mammalian glutathione peroxidases control acquisition and maintenance of spermatozoa integrity.

In mammals, posttesticular epididymal sperm maturation is considered an essential step in the transformation of immature testicular gametes to mature spermatozoa capable of fertilization. Reactive oxygen species (ROS) have been shown to be key actors in this maturation process, and it is now clear that ROS are central for sperm physiology in processes such as sperm maturation and capacitation. However, during epididymal maturation and storage and until the onset of fertilization, oxidative damage is a threat spermatozoa must face more than any other cells. Spermatozoa were found to be extremely sensitive to oxidative attacks correlated with lipid peroxidation, DNA damage, and impaired sperm motility, all affecting fertilization. To control the quantity of H(2)O(2) in the vicinity of male gametes, mammalian epididymis uses a panel of nonenzymatic and enzymatic scavengers, among which the glutathione peroxidase (GPx) family is largely represented. Among the various GPx proteins expressed in the mammalian epididymis, GPx4 and GPx5 occupy unique positions and functions that are reviewed in this paper. This paper underlines the importance of the GPx protein family in determining the fertilizing potential of mammalian spermatozoa. This is particularly relevant in the field of mammalian fertility and infertility as well as in the development of assisted medical procreation technologies and male gamete preservation techniques that are extensively used in human and animal reproduction programs.

Effects of prenatal maternal stress on the pituitary adrenocortical reactivity in guinea-pig pups.

Seven days before term, pregnant guinea-pigs were subjected to a psychosomatic stress. Adrenocorticotrophin (ACTH), cortisol and aldosterone concentrations increased in maternal and fetal plasma immediately after stress. In the offspring born from prenatally-stressed mothers that were resubjected to stress, the increases in plasma ACTH and cortisol level were lower than in the control offspring. Plasma aldosterone levels increased after stress in 12 days-old pups but did not change neither in 50 days-old animals nor in 12 days-old guinea-pigs born of prenatally stressed mothers. Thus prenatal stress of mother resulted in lowered response of pituitary-adrenocortical axis of offspring subjected to stress.

Effects of morphine microinjections into the trigeminal sensory complex on the formalin test in the rat.

The aim of this study was to find out whether morphine locally applied into the different subnuclei of the spinal trigeminal nucleus could impair the behavioral response evoked by a tonic nociceptive stimulus. Microinjections of morphine were performed unilaterally in rats through a chronically implanted tube. The duration of the formalin-induced biphasic rubbing activity was used as a measure of nociception. Morphine, 0, 2, 7, or 14 microg, in 0.2 microl of saline were microinjected 10 min before a subcutaneous injection of formalin (1.5%) in the ipsilateral upper lip. Morphine microinjections into subnucleus oralis (Sp5O) and subnucleus caudalis (Sp5C) induced a significant decrease in rubbing duration of either the early or late phase of the response or both. The early response to formalin was depressed only after morphine injection into Sp5O. The late response was depressed by microinjections into both Sp5O and Sp5C. No significant effect was observed in subnucleus interpolaris (Sp5I). These results are further evidence for the role played by Sp5C in orofacial nociception. They also give support to the involvement of the Sp5O in perioral nociceptive mechanisms of short duration.