Human umbilical cord perivascular cells prevent chemotherapeutic drug-induced male infertility in a mouse model.

OBJECTIVE: To study whether intratesticular (IT) administration of 2 sources of human umbilical cord perivascular cells (HUCPVC), rich and potent sources of mesenchymal stromal cells (MSC), before chemotherapy can prevent infertility in a mouse model. DESIGN: Two control groups of CD1 male mice without busulfan (BUS) administration (untreated and IT media injection groups) were included. Experimental groups included IT administration of media, first trimester (FTM) HUCPVCs or term HUCPVCs (n = 5 each) injected 3 days before BUS treatment (20 mg/kg). All groups were included in a mating time course study over 6 months. SETTING: Preclinical study in a fertility center research laboratory. PATIENTS: Not applicable. INTERVENTION: IT delivery of FTM or term HUCPVC before BUS treatment. MAIN OUTCOME MEASURES: Pregnancies, litter sizes, and gross morphology of offspring were monitored. Caudal epididymal sperm concentration, motility, and progressive motility were assessed by computer-assisted sperm analysis. Spermatogenesis was also assessed histologically in testicular tissue sections. RESULTS: FTM and term HUCPVC displayed an MSC-associated immunophenotype and expressed transcripts encoding paracrine factors known to regulate the testicular cell niche. IT administration of FTM and term HUCPVC before chemotherapy promoted the recovery of spermatogenesis and fertility compared with BUS-treated animals that received a media injection. Although the total number of pups sired over 6 months by males treated with FTM or term HUCPVC was reduced compared with untreated or media-injected controls, litter size and sperm parameters in fertile animals did not differ between control and cell-treated groups. CONCLUSION: HUCPVC represent a promising source of MSC-based therapy to prevent gonadotoxic chemotherapeutic drug-induced infertility.

Human umbilical cord perivascular cells maintain regenerative traits following exposure to cyclophosphamide.

Chemotherapies can cause germ cell depletion and gonadal failure. When injected post-chemotherapy, mesenchymal stromal cells (MSCs) from various sources have been shown to have regenerative effects in rodent models of chemotherapy-induced gonadal injury. Here, we evaluated two properties of a novel source of MSC, first trimester (FTM) human umbilical cord perivascular cells (HUCPVCs) (with increased regenerative potential compared to older sources), that may render them a promising candidate for chemotherapeutic gonadal injury prevention. Firstly, their ability to resist the cytotoxic effects of cyclophosphamide (CTX) in vitro, as compared to term HUCPVCs and bone marrow cells (BMSCs); and secondly, whether they prevent gonadal dysfunction if delivered prior to gonadotoxic therapy in vivo. BMSC, FTM HUCPVC, term HUCPVC, and control NTERA2 cells were treated with moderate (150 µmol/L) and high (300 µmol/L) doses of CTX in vitro. Viability, proliferative capacity, mesenchymal cell lineage markers and differentiation capacity, immunogenicity, and paracrine gene expression were assessed. CTX was administered to Wistar rats 2 days following an intra-ovarian injection of FTM HUCPVC. HUCPVC survival and ovarian follicle numbers were assessed using histological methods. We conclude that FTM HUCPVC maintain key regenerative properties following chemotherapy exposure and that pre-treatment with these cells may prevent CTX-induced ovarian damage in vivo. Therefore, HUCPVCs are promising candidates for fertility preservation.

A morphine-paired environment alters c-Fos expression in the forebrain of rats displaying conditioned place preference or aversion.

The question of whether a common mechanism mediates both aversive and rewarding drug-paired cues is still unclear. In this study, we used a place preference conditioning paradigm to train rats to associate 1 chamber with morphine and the other chamber with saline. On the test day, rats were divided into those displaying conditioned place preferences (CPP) versus conditioned place aversion (CPA). After the test, all rats were killed and c-Fos immunocytochemistry was performed. For the control group, rats were treated with the same procedure except that the injections of morphine or saline had no association with the chambers. Compared with the control group, the CPP and CPA groups showed a significant increase of c-Fos expression in the dorsomedial striatum, central medial nucleus of the thalamus, and the basolateral amygdala. However, we saw no difference between CPP and CPA rats in any brain region examined. These results suggest that a morphine-paired environment can elicit neural activity in brain regions that are involved in emotional learning. Morphine-conditioned place preference and aversion may share a common neural circuitry elicited by a morphine-paired environment.

Immunolocalization of PICK1 in the ascending auditory pathways of the adult rat.

Protein that interacts with C-kinase alpha (PICK1) is a PDZ domain protein that interacts with many binding partners in the central nervous system (CNS), including activated protein kinase Calpha and subunits of the AMPA subtype of glutamate receptor. Almost nothing is known about the anatomic distribution of PICK1 in the intact adult CNS. By using PICK1 antisera and peroxidase immunocytochemistry, we report on the distribution of PICK1 in the ascending pathways of the central auditory system of the adult rat. PICK1-immunoreactivity (ir) was observed in many component nuclei of the central auditory system, including the dorsal cochlear nucleus, anteroventral cochlear nucleus, posteroventral cochlear nucleus, some divisions of the superior olivary complex, inferior colliculus, medial geniculate body, and primary auditory cortex. The general staining pattern for PICK1-immunoreactivity was somatodendritic with scattered puncta in neuropil and somatodendritic regions. The distribution of PICK1 partially overlaps with PKCalpha and glutamate receptor subunits such as GluR2. These data suggest that PICK1 may function in the regulation of PKCalpha and GluR2 localization in components of the rat auditory system, which may be a fundamental mechanism of synaptic transmission and/or plasticity. J. Comp. Neurol.

Sexually dimorphic effects of morphine and MK-801: sex steroid-dependent and -independent mechanisms.

Rats show gender differences in responses to morphine and the N-methyl-D-aspartate receptor antagonist dizocilpine (MK-801); the role of sex steroids in mediating these differences is unclear. We tested the overall hypothesis that circulating gonadal steroids determine the gender differences in morphine- and MK-801-induced behavior and c-Fos expression. Morphine caused a greater expression of c-Fos in the striatum of intact males than of that females, which was independent of sex steroids. MK-801 completely inhibited morphine-induced c-Fos in intact females but only caused partial inhibition in intact males; castrated males showed complete inhibition, which was reversed by testosterone, but gonadal steroids had no effect on this response in females. In thalamus, there was a large sex difference in the response to MK-801 that was independent of gonadal steroids. Behavioral responses to morphine were greater in males, but responses to MK-801 were greater in females; both were sex steroid independent. These findings show significant sex differences in response to morphine and MK-801 that are mediated by sex steroid-dependent and -independent mechanisms, which may be important in treatment outcomes of drug addiction.

Modulation of glutamate receptor expression by gonadal steroid hormones in the rat striatum.

The non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist dizocilpine (MK-801) attenuates morphine-induced immediate-early gene expression in the rat striatum in a sexually dimorphic manner that depends in part on gonadal steroids. To determine if this effect was dependent on modulation of glutamate receptor gene expression, we studied the effect of gonadal hormones on levels of the NR1 subunit of NMDA receptor and the GluR2 subunit of the AMPA-subtype of glutamate receptor in the rat striatum, using autoradiographic immunocytochemistry. We found that ovariectomy decreased GluR2 immunoreactivity in the striatum, but no changes were seen in levels of NR1 following gonadectomy in either sex. Thus, the effects of gonadal steroids on NMDA receptor-mediated responses are not due to regulation of NR1 expression.

Morphine actions in the rat forebrain: role of protein kinase C.

Acute administration of morphine induces expression of the immediate-early gene (IEG) c-Fos in dorsomedial striatum, portions of cerebral cortex, and in several midline-intralaminar thalamic nuclei, partly via a trans-synaptic mechanism that involves activation of glutamate receptors. Because activation of protein kinase C (PKC) may occur following the activation of glutamate receptors, we determined whether pharmacological inhibition of PKC would attenuate morphine-induced c-Fos expression, and whether acute administration of morphine would induce translocation of PKC. The selective PKC antagonist NPC 15437 given 30 min prior to morphine significantly decreased morphine-induced c-Fos expression in striatum and cingulate cortex, but not in centrolateral thalamus. In another experiment, rats were given an acute dose of morphine, and immunocytochemical analysis was performed for the betaI and betaII isoforms of PKC. Morphine induced a rapid and transient translocation of PKC betaII, but not betaI, from perinuclear spots to plasma membrane in numerous cortical and striatal neurons. Prior administration of naloxone blocked this response. Ultrastructural studies confirmed translocation from Golgi apparatus to plasma membrane 15 min after morphine injection. Double immunocytochemistry at the light microscopic level demonstrated co-localization of translocated PKC betaII and c-Fos in some cortical neurons 90 min after morphine injection. These results support a role for PKC, especially PKC betaII, in the rapid effects of morphine on the brain.

Genomic convergence analysis of schizophrenia: mRNA sequencing reveals altered synaptic vesicular transport in post-mortem cerebellum.

Schizophrenia (SCZ) is a common, disabling mental illness with high heritability but complex, poorly understood genetic etiology. As the first phase of a genomic convergence analysis of SCZ, we generated 16.7 billion nucleotides of short read, shotgun sequences of cDNA from post-mortem cerebellar cortices of 14 patients and six, matched controls. A rigorous analysis pipeline was developed for analysis of digital gene expression studies. Sequences aligned to approximately 33,200 transcripts in each sample, with average coverage of 450 reads per gene. Following adjustments for confounding clinical, sample and experimental sources of variation, 215 genes differed significantly in expression between cases and controls. Golgi apparatus, vesicular transport, membrane association, Zinc binding and regulation of transcription were over-represented among differentially expressed genes. Twenty three genes with altered expression and involvement in presynaptic vesicular transport, Golgi function and GABAergic neurotransmission define a unifying molecular hypothesis for dysfunction in cerebellar cortex in SCZ.