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1.
Front Pharmacol ; 15: 1365151, 2024.
Article in English | MEDLINE | ID: mdl-38689663

ABSTRACT

Preparations of black cohosh extract are sold as dietary supplements marketed to relieve the vasomotor symptoms of menopause, and some studies suggest it may protect against postmenopausal bone loss. Postmenopausal women are also frequently prescribed bisphosphonates, such as risedronate, to prevent osteoporotic bone loss. However, the pharmacodynamic interactions between these compounds when taken together is not known. To investigate possible interactions, 6-month-old, female Sprague-Dawley rats underwent bilateral ovariectomy or sham surgery and were treated for 24 weeks with either vehicle, ethinyl estradiol, risedronate, black cohosh extract or coadministration of risedronate and black cohosh extract, at low or high doses. Bone mineral density (BMD) of the femur, tibia, and lumbar vertebrae was then measured by dual-energy X-ray absorptiometry (DEXA) at weeks 0, 8, 16, and 24. A high dose of risedronate significantly increased BMD of the femur and vertebrae, while black cohosh extract had no significant effect on BMD individually and minimal effects upon coadministration with risedronate. Under these experimental conditions, black cohosh extract alone had no effect on BMD, nor did it negatively impact the BMD-enhancing properties of risedronate.

3.
Reprod Toxicol ; 82: 111-123, 2018 12.
Article in English | MEDLINE | ID: mdl-30316929

ABSTRACT

2-hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet light-absorbing compound that is used in sunscreens, cosmetics and plastics. HMB has been reported to have weak estrogenic activity by in vivo and in vitro studies, making it a chemical with potential reproductive concern. To explore if prenatal and lactational HMB exposure alters gene expression profiles of the developing reproductive organs, we performed microarray analysis using the prostate and testis of postnatal day (PND) 30 male Sprague-Dawley rats offspring exposed to 0, 3000, or 30,000 ppm of HMB from gestational day 6 through PND 21. Gene expression profiles of the prostate and testis were differentially affected by HMB dose with significant alterations observed at the 30,000 ppm HMB group. Tissue-specific gene expression was also identified. These genes, whose expression was altered by HMB exposure, may be considered as candidate biomarker(s) for testicular or prostatic toxicity; however, further studies are necessary to explore this potential.


Subject(s)
Benzophenones/toxicity , Cosmetics/toxicity , Prostate/drug effects , Testis/drug effects , Animals , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental/drug effects , Lactation , Male , Maternal-Fetal Exchange , Mitochondria/drug effects , Mitochondria/metabolism , Pregnancy , Prenatal Exposure Delayed Effects , Prostate/metabolism , Rats, Sprague-Dawley , Testis/metabolism
4.
Birth Defects Res ; 109(7): 465-474, 2017 04 17.
Article in English | MEDLINE | ID: mdl-28398669

ABSTRACT

BACKGROUND: The complexity of spermatogenesis makes development of appropriate in vitro testis models challenging. A novel in vitro mouse testis culture system has been reported but not yet evaluated as an alternative model for male reproductive toxicity testing. We assessed the effects of media composition on sperm differentiation and testis morphology of cultured mouse testis fragments. METHODS: Testes from postnatal day 5 B6:CBA-Tg(Acrv1-EGFP)2727Redd/J male mice were cultured in knockout serum replacement (KSR) or Albumax I (Albumax) medium. Enhanced green fluorescent protein (EGFP) expression was examined on days 35, 42, 45, and 49 of culture. Histology and flow cytometry were performed for testis morphology and spermatid differentiation. RESULTS: EGFP signals were first observed in round spermatids on day 22 of culture (corresponding to postnatal day 27) and were observed until the end of culture, indicating testis-specific protein expression. A-kinase anchor protein 4 expression, a marker of elongated spermatid (step 15-16) occurred earlier in explants cultured in KSR than Albumax medium (typically day 35 and after day 42 of culture, respectively). The percentage of seminiferous tubules with elongated spermatid was higher in Albumax than KSR medium from days 45 to 49 of culture. CONCLUSION: Albumax medium may facilitate or support better morphology and spermatid production than KSR medium. Further studies need to improve spermatid production and refinement of this in vitro testis culture system that may be useful as a supplement to current male reproductive toxicity testing or an alternative model in cases where in vivo testing may be unfeasible. Birth Defects Research 109:465-474, 2017. © 2017 Wiley Periodicals, Inc.


Subject(s)
Organ Culture Techniques/methods , Testis/physiology , Animals , Cells, Cultured , Culture Media/metabolism , Genitalia, Male/physiology , Green Fluorescent Proteins , Male , Mice , Mice, Inbred CBA , Mice, Transgenic , Models, Animal , Seminiferous Tubules/metabolism , Serum/metabolism , Spermatids/cytology , Spermatogenesis/physiology , Testis/metabolism , Toxicity Tests
5.
Reprod Toxicol ; 69: 75-83, 2017 04.
Article in English | MEDLINE | ID: mdl-28189605

ABSTRACT

The mouse embryonic stem cell test (mEST) is a promising in vitro assay for predicting developmental toxicity. In the current study, early differentiation of D3 mouse embryonic stem cells (mESCs) under osteoblast culture conditions and embryotoxicity of cadmium sulfate were examined. D3 mESCs were exposed to cadmium sulfate for 24, 48 or 72h, and whole genome transcriptional profiles were determined. The results indicate a track of differentiation was identified as mESCs differentiate. Biological processes that were associated with differentiation related genes included embryonic development and, specifically, skeletal system development. Cadmium sulfate inhibited mESC differentiation at all three time points. Functional pathway analysis indicated biological pathways affected included those related to skeletal development, renal and reproductive function. In summary, our results suggest that transcriptional profiles are a sensitive indicator of early mESC differentiation. Transcriptomics may improve the predictivity of the mEST by suggesting possible modes of action for tested chemicals.


Subject(s)
Cadmium Compounds/toxicity , Cell Differentiation/drug effects , Gene Expression Regulation, Developmental/drug effects , Mouse Embryonic Stem Cells/drug effects , Osteoblasts/drug effects , Sulfates/toxicity , Animals , Cell Differentiation/genetics , Cells, Cultured , Gene Expression Profiling , Mice , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Osteoblasts/cytology
6.
Biol Reprod ; 93(6): 137, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26490841

ABSTRACT

While cyclin dependent kinase 1 (CDK1) has a critical role in controlling resumption of meiosis in oocytes, its role has not been investigated directly in spermatocytes. Unique aspects of male meiosis led us to hypothesize that its role is different in male meiosis than in female meiosis. We generated a conditional knockout (cKO) of the Cdk1 gene in mouse spermatocytes to test this hypothesis. We found that CDK1-null spermatocytes undergo synapsis, chiasmata formation, and desynapsis as is seen in oocytes. Additionally, CDK1-null spermatocytes relocalize SYCP3 to centromeric foci, express H3pSer10, and initiate chromosome condensation. However, CDK1-null spermatocytes fail to form condensed bivalent chromosomes in prophase of meiosis I and instead are arrested at prometaphase. Thus, CDK1 has an essential role in male meiosis that is consistent with what is known about the role of CDK1 in female meiosis, where it is required for formation of condensed bivalent metaphase chromosomes and progression to the first meiotic division. We found that cKO spermatocytes formed fully condensed bivalent chromosomes in the presence of okadaic acid, suggesting that cKO chromosomes are competent to condense, although they do not do so in vivo. Additionally, arrested cKO spermatocytes exhibited irregular cell shape, irregular large nuclei, and large distinctive nucleoli. These cells persist in the seminiferous epithelium through the next seminiferous epithelial cycle with a lack of stage XII checkpoint-associated cell death. This indicates that CDK1 is required upstream of a checkpoint-associated cell death as well as meiotic metaphase progression in mouse spermatocytes.


Subject(s)
CDC2 Protein Kinase/genetics , Infertility, Male/genetics , Meiosis/genetics , Spermatocytes/metabolism , Spermatogenesis/genetics , Animals , CDC2 Protein Kinase/metabolism , Cell Nucleus/metabolism , Cell Shape/genetics , Infertility, Male/metabolism , Male , Mice , Mice, Knockout , Spermatocytes/cytology
7.
Reprod Toxicol ; 53: 131-40, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25929818

ABSTRACT

The mouse Embryonic Stem cell Test (EST) using cardiomyocyte differentiation is a promising in vitro assay for detecting potential embryotoxicity; however, the addition of another differentiation endpoint, such as osteoblasts, may improve the predictive value of the test. A number of variables such as culture conditions and starting cell number were investigated. A 14 day direct plating method of D3 mouse embryonic stem cells (mESCs) was used to test the predictivity of osteoblast differentiation as an endpoint in the EST. Twelve compounds were tested using the prediction model developed in the ECVAM validation study. Eight of the compounds selected from the EST validation study served as model compounds; four additional compounds known to produce skeletal defects were also tested. Our results indicate comparable chemical classification between the validated cardiomyocyte endpoint and the osteoblast endpoint. These results suggest that differentiation to osteoblasts may provide confirmatory information in predicting embryotoxicity.


Subject(s)
Osteoblasts/drug effects , Teratogens/toxicity , Alkaline Phosphatase/metabolism , Animals , Calcium/metabolism , Cell Differentiation/drug effects , Cell Line , Hep G2 Cells , Humans , Mice , Mouse Embryonic Stem Cells , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Reproducibility of Results
8.
Birth Defects Res B Dev Reprod Toxicol ; 104(1): 35-51, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25707689

ABSTRACT

BACKGROUND: 2-Hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet (UV) absorbing compound used in many cosmetic products as a UV-protecting agent and in plastics for preventing UV-induced photodecomposition. HMB has been detected in over 95% of randomly collected human urine samples from adults and from premature infants, and it may have estrogenic potential. METHODS: To determine the effects of maternal and lactational exposure to HMB on development and reproductive organs of offspring, time-mated female Harlan Sprague-Dawley rats were dosed with 0, 1000, 3000, 10,000, 25,000, or 50,000 ppm HMB (seven to eight per group) added to chow from gestation day 6 until weaning on postnatal day (PND) 23. RESULTS AND CONCLUSION: Exposure to HMB was associated with reduced body and organ weights in female and male offspring. No significant differences were observed in the number of implantation sites/litter, mean resorptions/litter, % litters with resorptions, number and weights of live fetuses, or sex ratios between the control and HMB dose groups. Normalized anogenital distance in male pups at PND 23 was decreased in the highest dose group. Spermatocyte development was impaired in testes of male offspring in the highest dose group. In females, follicular development was delayed in the highest dose group. However, by evaluating levels of the compound in rat serum, the doses at which adverse events occurred are much higher than usual human exposure levels. Thus, exposure to less than 10,000 ppm HMB does not appear to be associated with adverse effects on the reproductive system in rats.


Subject(s)
Benzophenones/toxicity , Embryonic Development/drug effects , Lactation/drug effects , Prenatal Exposure Delayed Effects/pathology , Reproduction/drug effects , Animals , Animals, Newborn , Body Weight/drug effects , Cell Count , Female , Male , Organ Size/drug effects , Pregnancy , Prenatal Exposure Delayed Effects/blood , Rats, Sprague-Dawley , Seminiferous Tubules/drug effects , Seminiferous Tubules/pathology , Spermatocytes/drug effects , Spermatocytes/pathology , Testosterone/blood
9.
Syst Biol Reprod Med ; 58(1): 10-22, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22239077

ABSTRACT

In light of various pressures, toxicologists have been searching for alternative methods for safety testing of chemicals. According to a recent policy in the European Union (Regulation, Evaluation Authorisation and Restriction of Chemicals, REACH), it has been estimated that over the next twelve to fifteen years, approximately 30,000 chemicals may need to be tested for safety, and under current guidelines such testing would require the use of approximately 7.2 million laboratory animals [ Hofer et al. 2004 ]. It has also been estimated that over 80% of all animals used for safety testing under REACH legislation would be used for examining reproductive and developmental toxicity [Hofer et al., 2004]. In addition to REACH initiatives, it has been estimated that out of 5,000 to 10,000 new drug entities that a pharmaceutical company may start with, only one is finally approved by the Food and Drug Administration at a cost of over one billion dollars [ Garg et al. 2011 ]. A large portion of this cost is due to animal testing. Therefore, both the pharmaceutical and chemical industries are interested in using alternative models and in vitro tests for safety testing. This review will examine the current state of three alternative models - whole embryo culture (WEC), the mouse embryonic stem cell test (mEST), and zebrafish. Each of these alternatives will be reviewed, and advantages and disadvantages of each model will be discussed. These models were chosen because they are the models most commonly used and would appear to have the greatest potential for future applications in developmental toxicity screening and testing.


Subject(s)
Animal Testing Alternatives , Developmental Biology/methods , Models, Animal , Toxicity Tests/methods , Animals , Cell Line , Embryo Culture Techniques , Embryonic Development/drug effects , Embryonic Stem Cells/drug effects , Humans , Mice , Rats , Risk Assessment , Zebrafish/embryology
10.
Eur J Pharmacol ; 668 Suppl 1: S108-16, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21816149

ABSTRACT

Throughout the last century, possible effects of exposure to toxicants, nutrients or drugs were examined primarily by studies of groups or populations. Individual variation in responses was acknowledged but could not be analyzed due to lack of information or tools to analyze individual genetic make-ups and lifestyle factors such as diet and activity. The Human Genome, Haplotype Map, 1000Genomes, and Human Variome Projects are identifying and cataloging the variation found within humans. Advances in DNA sequencing technologies will soon permit the characterization of individual genomes in clinical and basic research studies, thus allowing associations to be made between an individual genotype and the response to a particular exposure. Such knowledge and tools have generated a significant challenge for scientists: to design and conduct research studies that account for individual genetic variation. However, before these studies are done in humans, they will be performed in various in vivo and in vitro models. The advantages and disadvantages of some of the model test systems that are being used or developed in relation to individual genetic make-up and responses to xenobiotics are discussed.


Subject(s)
Gene-Environment Interaction , Models, Animal , Research Design , Animals , Genome-Wide Association Study , Humans , Stem Cells/metabolism
11.
Genesis ; 48(2): 114-20, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20027617

ABSTRACT

We generated transgenic mouse line C57BL/6-Tg(Hspa2-cre)1Eddy/J (Hspa2-cre), which expresses cre-recombinase under the control of a 907-bp fragment of the heat shock protein 2 (Hspa2) gene promoter. Transgene expression was determined using Gt(ROSA)26Sor(tm1Sor)/J (ROSA26) and Tg(CAG-Bgeo/GFP)21Lbe/J (Z/EG) reporter strains and RT-PCR and immunohistochemistry assays. Hspa2-cre expression mimicked the spermatogenic cell-specific expression of endogenous HSPA2 within the testis, being first observed in leptotene/zygotene spermatocytes. Expression of the transgene also was detected at restricted sites in the brain, as occurs for endogenous HSPA2. Although the results of mating the Hspa2-cre mice to mice with a floxed Cdc2a allele indicated that some expression of the transgene occurs during embryogenesis, the Hspa2-cre mice provide a valuable new tool for assessing the roles of genes during and after meiotic prophase in pachytene spermatocytes.


Subject(s)
HSP27 Heat-Shock Proteins/genetics , Integrases/metabolism , Promoter Regions, Genetic , Spermatocytes/metabolism , Animals , Genes, Reporter , HSP27 Heat-Shock Proteins/metabolism , Immunohistochemistry , Male , Mice , Mice, Transgenic , Mutation , RNA, Messenger/metabolism , Recombination, Genetic , Testis/cytology , Transgenes
12.
Dev Biol ; 330(1): 142-52, 2009 Jun 01.
Article in English | MEDLINE | ID: mdl-19341723

ABSTRACT

Males homozygous for the repro32 ENU-induced mutation produced by the Reproductive Genomics program at The Jackson Laboratory are infertile, have low epididymal sperm concentrations, and produce sperm with abnormally shaped heads and poor motility. The purpose of the present study was to identify the mutated gene in repro32 mice and to define the structural and functional changes causing infertility and the aberrant sperm phenotype. In repro32/repro32 mice, we discovered a failure to shed excess cytoplasm and disorganization of the middle piece of the flagellum at spermiation, resulting in the outer dense fibers being wrapped around the sperm head within a bag of cytoplasm. Using a candidate-gene approach, a mutation was identified in the spermatid-specific "capping protein (actin filament) muscle Z-line, alpha 3" gene (Capza3). CAPZA3 protein localization was altered in spermatids concurrent with altered localization of a unique CAPZB variant isoform and disruption of the filamentous actin (F-actin) network. These observations strongly suggest the missense mutation in Capza3 is responsible for the mutant phenotype of repro32/repro32 sperm and regulation of F-actin dynamics by a spermatogenic cell-specific CAPZ heterodimer is essential for removal of the cytoplasm and maintenance of midpiece integrity during spermiation in the mouse.


Subject(s)
Actins/ultrastructure , CapZ Actin Capping Protein/genetics , Infertility, Male/genetics , Mutation, Missense , Spermatids/ultrastructure , Actins/metabolism , Amino Acid Sequence , Animals , CapZ Actin Capping Protein/analysis , CapZ Actin Capping Protein/metabolism , Fluorescent Antibody Technique , Infertility, Male/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Transgenic , Microscopy, Electron, Transmission , Molecular Sequence Data , Phenotype , Sperm Motility , Spermatids/metabolism
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