Opioid Receptor Mu 1 Gene (OPRM1) A118G Polymorphism and Emotional Modulation of Pain.

Purpose: The A118G polymorphism in the opioid receptor mu 1 gene (OPRM1) is associated with decreased opioid receptor availability, altered emotion, and increased pain. Given that emotions modulate pain (positive emotions inhibit pain, negative emotions enhance pain), we predicted that G allele carriers would experience impaired emotional modulation of pain compared to non-G allele carriers. Patients and Methods: Emotional pictures (ie, erotica, neutral, attack) from the International Affective Picture System were used by permission from the authors to experimentally manipulate emotions in 64 adult participants while painful electrocutaneous stimulations were delivered in a cross-sectional study. Ratings of arousal and valence/pleasure were made in response to pictures, and pain ratings and a physiological measure of spinal nociception (ie, nociceptive flexion reflex, NFR) were collected in response to painful stimulations. Secondary analyses were conducted to examine the relationship between the A118G polymorphism and emotional modulation of pain/NFR. Results: Exposure to emotional pictures elicited similar changes in valence, but G-carriers rated erotic pictures as more arousing. In non-carriers, pain was facilitated by attack pictures and pain and NFR were inhibited by erotic pictures relative to neutral pictures. Among G-carriers, pain was facilitated by negative emotional pictures but there was no pain or NFR inhibition by positive emotional pictures. Conclusion: The altered response to pleasant stimuli further supports the role of opioids in appetitive behavior and describes how the A118G polymorphism may prevent carriers from inhibiting pain during pleasure.

The molecular mechanisms mediating mammalian fertilization.

Fertilization is a key biological process in which the egg and sperm must recognize one another and fuse to form a zygote. Although the process is a continuum, mammalian fertilization has been studied as a sequence of steps: sperm bind and penetrate through the zona pellucida of the egg, adhere to the egg plasma membrane and finally fuse with the egg. Following fusion, effective blocks to polyspermy ensure monospermic fertilization. Here, we review how recent advances obtained using genetically modified mouse lines bring new insights into the molecular mechanisms regulating mammalian fertilization. We discuss models for these processes and we include studies showing that these mechanisms may be conserved across different mammalian species.

ZP2 peptide beads select human sperm in vitro, decoy mouse sperm in vivo, and provide reversible contraception.

Gamete recognition in the female reproductive tract occurs at the surface of the zona pellucida surrounding ovulated eggs. The acellular zona matrix is composed of three (mouse) or four (human) proteins (ZP1 to ZP4), and the amino terminus of ZP2 is the primary sperm-binding ligand. Mouse and human sperm bind, respectively, to recombinant moZP2(35-149) and huZP2(39-154) peptides attached to agarose beads. Mouse ZP2 peptide beads markedly inhibited fertilization of ovulated mouse eggs inseminated in vitro and incubated overnight. Similarly, human ZP2 peptide beads prevented sperm binding and penetration of transgenic ZP2(Rescue) zonae pellucidae, in which human ZP2 replaced mouse ZP2. When mouse ZP2 peptide beads were transcervically deposited into the uterus, there was no change in mating behavior and copulatory plugs were present, but bound sperm did not progress into the oviduct and female mice were infertile. On average, contraception lasted >10 estrus cycles but was reversible with no detectable pathology in the reproductive tract. Despite the long-term contraceptive effect, initial sperm binding to the peptide beads was reversible in vitro. We exploited this observation to select human sperm that were better able to penetrate the zonae of human ZP2(Rescue) eggs, and the approach holds promise for identifying superior sperm for human assisted reproductive technologies (ART). We conclude that the amino-terminal ZP2 peptide supports sperm binding, which is initially reversible but, with time, becomes irreversible. Short-term, reversible binding may be useful in selecting sperm for ART, and long-term binding decoys sperm and results in effective contraception in mice.

A single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans.

The extracellular zona pellucida surrounds ovulated eggs and mediates gamete recognition that is essential for mammalian fertilization. Zonae matrices contain three (mouse) or four (human) glycoproteins (ZP1-4), but which protein binds sperm remains controversial. A defining characteristic of an essential zona ligand is sterility after genetic ablation. We have established transgenic mice expressing human ZP4 that form zonae pellucidae in the absence of mouse or human ZP2. Neither mouse nor human sperm bound to these ovulated eggs, and these female mice were sterile after in vivo insemination or natural mating. The same phenotype was observed with truncated ZP2 that lacks a restricted domain within ZP2(51-149). Chimeric human/mouse ZP2 isoforms expressed in transgenic mice and recombinant peptide bead assays confirmed that this region accounts for the taxon specificity observed in human-mouse gamete recognition. These observations in transgenic mice document that the ZP2(51-149) sperm-binding domain is necessary for human and mouse gamete recognition and penetration through the zona pellucida.

The molecular basis of gamete recognition in mice and humans.

Successful fertilization heralds the onset of development and requires both gamete recognition and a definitive block to polyspermy. Sperm initially bind and penetrate the extracellular zona pellucida (ZP) that surrounds ovulated eggs, but are unable to bind the zona surrounding preimplantation embryos. The ZP of humans is composed of four (ZP1-4) and that of mouse three (ZP1-3) glycoproteins. Models for gamete recognition developed in mice had proposed that sperm bind to ZP3 glycans. However, phenotypes observed in genetically engineered mice are not consistent with this widely accepted model. More recently, taking advantage of the observation that human sperm do not bind to mouse eggs, human ZP2 was defined as the zona ligand in transgenic mouse models using gain-of-function assays. The sperm-binding site is an N-terminal domain of ZP2 that is cleaved by ovastacin, a metalloendoprotease released from egg cortical granules following fertilization. Proteolysis of this docking site provides a definitive block to polyspermy as sperm bind to uncleaved, but not cleaved ZP2 even after fertilization and cortical granule exocytosis. While progress has been made in defining the ZP ligand, less headway has been made in identifying the cognate sperm receptor. Although a number of sperm receptor candidates have been documented to interact with specific proteins in the ZP in vitro, continued fertility after genetic ablation of the cognate gene indicates that none are essential for gamete recognition. These on-going investigations inform reproductive medicine and suggest new therapies to improve fertility and/or provide contraception, thus expanding reproductive choices for human couples.

Lactobacillus rhamnosus accelerates zebrafish backbone calcification and gonadal differentiation through effects on the GnRH and IGF systems.

Endogenous microbiota play essential roles in the host's immune system, physiology, reproduction and nutrient metabolism. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host's development. Thus, we treated zebrafish from birth to sexual maturation (2-months treatment) with Lactobacillus rhamnosus, a probiotic species intended for human use. We monitored for the presence of L. rhamnosus during the entire treatment. Zebrafish at 6 days post fertilization (dpf) exhibited elevated gene expression levels for Insulin-like growth factors -I and -II, Peroxisome proliferator activated receptors -α and -ß, VDR-α and RAR-γ when compared to untreated-10 days old zebrafish. Using a gonadotropin-releasing hormone 3 GFP transgenic zebrafish (GnRH3-GFP), higher GnRH3 expression was found at 6, 8 and 10 dpf upon L. rhamnosus treatment. The same larvae exhibited earlier backbone calcification and gonad maturation. Noteworthy in the gonad development was the presence of first testes differentiation at 3 weeks post fertilization in the treated zebrafish population -which normally occurs at 8 weeks- and a dramatic sex ratio modulation (93% females, 7% males in control vs. 55% females, 45% males in the treated group). We infer that administration of L. rhamnosus stimulated the IGF system, leading to a faster backbone calcification. Moreover we hypothesize a role for administration of L. rhamnosus on GnRH3 modulation during early larval development, which in turn affects gonadal development and sex differentiation. These findings suggest a significant role of the microbiota composition on the host organism development profile and open new perspectives in the study of probiotics usage and application.

Effect of dietary probiotics on clownfish: a molecular approach to define how lactic acid bacteria modulate development in a marine fish.

We set out to determine whether probiotic addition would improve larval development in the false percula clownfish Amphiprion ocellaris and to determine what molecular responses could be observed in the larvae following probiotic exposure. We supplied the probiotic bacterial strain Lactobacillus rhamnosus IMC 501 to clownfish larvae from the first day posthatch simultaneously by live prey and with addition to rearing water (group 2) and exclusively by live prey (group 3). We observed twofold higher body weight in both clownfish larvae and juveniles when probiotics were supplied via live prey and added to the rearing water. In addition, development was accelerated with metamorphosis occurring 3 days earlier in fingerlings treated with probiotic. Alteration in molecular biomarkers supported the faster growth observation. There was significantly increased gene expression of factors involved in growth and development (insulin-like growth factors I and II, myostatin, peroxisome proliferator-activated receptors alpha and beta, vitamin D receptor alpha, and retinoic acid receptor gamma) when probiotics were delivered via live prey and added to the rearing water. Moreover, probiotic treatment lessened the severity of the general stress response as exhibited by lower levels of glucocorticoid receptor and 70-kDa heat shock protein gene expression. Furthermore, an improvement of skeletal head development was observed, with a 10-20% reduction in deformities for juveniles treated with probiotic. All data suggest a potent effect on development resulting from the administration of lactic acid bacteria to larval clownfish, and this study provides a preliminary molecular entry path into the investigation of mechanisms responsible for probiotic enhancement in fish development.