Application of Stem Cell Therapy for Infertility.

Infertility creates an immense impact on the psychosocial wellbeing of affected couples, leading to poor quality of life. Infertility is now considered to be a global health issue affecting approximately 15% of couples worldwide. It may arise from factors related to the male (30%), including varicocele, undescended testes, testicular cancer, and azoospermia; the female (30%), including premature ovarian failure and uterine disorders; or both partners (30%). With the recent advancement in assisted reproduction technology (ART), many affected couples (80%) could find a solution. However, a substantial number of couples cannot conceive even after ART. Stem cells are now increasingly being investigated as promising alternative therapeutics in translational research of regenerative medicine. Tremendous headway has been made to understand the biology and function of stem cells. Considering the minimum ethical concern and easily available abundant resources, extensive research is being conducted on induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSC) for their potential application in reproductive medicine, especially in cases of infertility resulting from azoospermia and premature ovarian insufficiency. However, most of these investigations have been carried out in animal models. Evolutionary divergence observed in pluripotency among animals and humans requires caution when extrapolating the data obtained from murine models to safely apply them to clinical applications in humans. Hence, more clinical trials based on larger populations need to be carried out to investigate the relevance of stem cell therapy, including its safety and efficacy, in translational infertility medicine.

Central prolactin binding site densities change seasonally in an adult male passerine bird (Junco hyemalis).

Seasonal reproduction is common across temperate zone avian species. In these species, physiological and behavioral adaptations have evolved to change according to day length (i.e., seasonally) in order to maximize reproductive output. The hormone prolactin regulates many aspects of parental care, a critical component of reproductive success. It's secretion in birds has been shown to be under photoperiodic control, with the highest levels measured in the spring and summer months, when birds breed and show parental care. However, to date, no study has tested whether the densities of central prolactin binding sites vary seasonally, which may also account for prolactin's effect on parental care. To test this, we collected brains from free-ranging adult male dark-eyed juncos, Junco hyemalis, a biparental songbird, in the spring, summer, and fall, and used quantitative in vitro autoradiography to compare the densities of specific prolactin binding sites across 20 different brain regions. Prolactin binding sites were found in regions that regulate parental behavior in other avian species. During the summer, several hypothalamic regions that regulate parental care, including the preoptic area and tuberal nucleus, contained lower densities of prolactin binding sites, suggesting exposure to higher endogenous prolactin levels, than at other times. This observation is consistent with the fact that circulating prolactin is highest during summer, when males would be providing care to young. Overall, these data suggest that prolactin binding sites are relatively conserved in the avian brain and that central prolactin activity supports parental care efforts in juncos and other avian species.

Next generation sequencing analysis of soy glyceollins and 17-ß estradiol: Effects on transcript abundance in the female mouse brain.

Glyceollins (Glys) are produced by soy plants in response to stress and are known for their anti-estrogenic activity both in vivo and in vitro in cancer cell lines as well as peripheral tissues. Glys can also exhibit non-estrogen receptor (ER) mediated effects. The effects of Glys on gene expression in the brain are still unclear. For this study, 17-ß estradiol (E2) or placebo slow-release pellets were implanted into ovariectomized CFW mice followed by 11 days of exposure to either Glys or vehicle i.p. injections. We then examined the female mouse brain transcriptome using paired-end RNA sequencing (RNA-Seq) on the Illumina GAIIx platform. The goal of this study was to compare and contrast the results obtained from RNA-Seq with the results from our previous whole brain microarray experiment, which indicated that Glys potentially act through both ER-mediated and non-ER-mediated mechanisms, exhibiting a gene expression profile distinct from E2-treated groups. Our results suggest that the transcripts regulated by both E2 and Glys alone or in combination annotated to similar pathway maps and networks in both microarray and RNA-Seq experiments. Additionally, unlike our microarray data analysis, RNA-Seq enabled the detection of treatment effects on low expression transcripts of interest (e.g., prolactin and growth hormone). Collectively, our results suggest that depending on the gene, Glys can regulate expression independently of E2 action, similarly to E2, or oppose E2's effects in the female mouse brain.

Investigation of pre-pubertal sex differences in wheel running and social behavior in three mouse strains.

Sex differences in social behaviors exist in mammals during adulthood, and further evidence suggests that sex differences in behavior are present before sexual maturity. In order to model behavioral disorders in animals, it is important to assess baseline sex-related behavioral differences, especially when studying disorders for which sex-related behavioral effects are expected. We investigated the effect of sex on behavior in 3 strains of pre-pubertal mice (C57BL/6, CFW, and CF1) using a wheel-running assay. We found no significant sex differences in latency to run on the wheel or total duration of wheel running within each strain. During the social interaction test, there were no differences between sexes in latency or total duration of contact or following between a subject and novel mouse. We also evaluated behavioral patterns of wheel running and stereotypical behaviors, such as burrowing and grooming. Both sexes showed characteristic wheel running behavior, spending the majority of each trial interacting with the wheel when it was free and more time performing other activities (e.g., stereotypical behaviors, general locomotion) when it was jammed. These results provide evidence that, among various strains of pre-pubertal mice, baseline sex-related behavioral differences are not strong enough to influence the measured behaviors.

Soy glyceollins regulate transcript abundance in the female mouse brain.

Glyceollins (Glys), produced by soy plants in response to stress, have anti-estrogenic activity in breast and ovarian cancer cell lines in vitro and in vivo. In addition to known anti-estrogenic effects, Gly exhibits mechanisms of action not involving estrogen receptor (ER) signaling. To date, effects of Gly on gene expression in the brain are unknown. For this study, we implanted 17-ß estradiol (E2) or placebo slow-release pellets into ovariectomized CFW mice followed by 11 days of exposure to Gly or vehicle i.p. injections. We then performed a microarray on total RNA extracted from whole-brain hemispheres and identified differentially expressed genes (DEGs) by a 2 × 2 factorial ANOVA with an false discovery rate (FDR) = 0.20. In total, we identified 33 DEGs with a significant E2 main effect, 5 DEGs with a significant Gly main effect, 74 DEGs with significant Gly and E2 main effects (but no significant interaction term), and 167 DEGs with significant interaction terms. Clustering across all DEGs revealed that transcript abundances were similar between the E2 + Gly and E2-only treatments. However, gene expression after Gly-only treatment was distinct from both of these treatments and was generally characterized by higher transcript abundance. Collectively, our results suggest that whether Gly acts in the brain through ER-dependent or ER-independent mechanisms depends on the target gene.

Androgen receptor location in the dark-eyed junco using a probe for in situ hybridization histochemistry generated from zebra finch cDNA.

Due to the role of sex steroids, namely testosterone (T), in the development and production of song in songbirds, androgen receptor (AR) densities in the brain regions controlling this behavior (i.e., the song control system) have long been studied. Many methods have been used to determine AR density and location to investigate the functional role of T in song development and production; however, a riboprobe developed from zebra finch (Taeniopygia guttata) cDNA was shown to be much more sensitive than previous methods. The zebra finch is a common model for song development and is sexually dimorphic, but does not breed seasonally or display seasonal changes in song control region volume. In this study, we used this riboprobe for in situ hybridization histochemistry (ISHH) to describe AR mRNA location in the brain of the dark-eyed junco (Junco hyemalis), a seasonally breeding model for which T has been shown to be important. Additionally, we provide a detailed comparison of AR mRNA location between these species. We found that this probe is indeed highly sensitive. We detected AR mRNA in four major regions of the song control system (HVC, MAN, RA and Area X). Additionally, we found that the location of AR mRNA in other regions varied only slightly between these two species. These findings suggest that this method is suitable for use across songbirds and it could be useful in the ongoing attempts to elucidate the roles of sex steroid hormones on the development of this and other sex steroid dependent behaviors in songbirds.

Opioid receptor densities analyzed across seasons in the POM and VTA of the dark-eyed junco, Junco hyemalis.

The motivation of songbirds to sing is influenced by two brain regions, the medial preoptic area (POM) and ventral tegmental area (VTA), which are located outside the song control system itself. These areas receive opioidergic innervation. Furthermore, the opioid enkephalin has been proposed to play a role in the reward for singing. In order to determine whether seasonal changes in song output relate to seasonal changes in opioid receptor (OR) densities in the POM and VTA, we measured the densities of micro, delta, and kappa subtypes in these brain regions in adult male dark-eyed juncos (Junco hyemalis) sampled in spring (singing), summer (singing and breeding), and fall (no singing). Receptor densities in the rostral and caudal portions of the POM were measured separately because these subregions are thought to predominantly influence appetitive and consumatory sexual behaviors, respectively. delta ORs were generally denser than micro or kappa in both parts of the POM and micro ORs were denser than the other subtypes in the VTA. Densities of micro ORs in the POM were higher in the summer than in spring or fall, although this difference was statistically significant only for cPOM (p=0.002). In rPOM, kappa OR densities tended to be higher in spring and summer than fall, although this pattern did not reach statistical significance (p=0.057). In contrast, kappa OR densities were lowest in the VTA during the summer compared to spring and fall, although this pattern did not reach statistical significance, either (p=0.094). Results obtained for cPOM micro ORs suggest a heightened reward potential for sexual behavior during the breeding season.

Glyceollin I, a novel antiestrogenic phytoalexin isolated from activated soy.

Glyceollins, a group of novel phytoalexins isolated from activated soy, have recently been demonstrated to be novel antiestrogens that bind to the estrogen receptor (ER) and inhibit estrogen-induced tumor progression. Our previous publications have focused specifically on inhibition of tumor formation and growth by the glyceollin mixture, which contains three glyceollin isomers (I, II, and III). Here, we show the glyceollin mixture is also effective as a potential antiestrogenic, therapeutic agent that prevents estrogen-stimulated tumorigenesis and displays a differential pattern of gene expression from tamoxifen. By isolating the individual glyceollin isomers (I, II, and III), we have identified the active antiestrogenic component by using competition binding assays with human ERalpha and in an estrogen-responsive element-based luciferase reporter assay. We identified glyceollin I as the active component of the combined glyceollin mixture. Ligand-receptor modeling (docking) of glyceollin I, II, and III within the ERalpha ligand binding cavity demonstrates a unique type II antiestrogenic confirmation adopted by glyceollin I but not isomers II and III. We further compared the effects of glyceollin I to the antiestrogens, 4-hydroxytamoxifen and ICI 182,780 (fulvestrant), in MCF-7 breast cancer cells and BG-1 ovarian cancer cells on 17beta-estradiol-stimulated expression of progesterone receptor and stromal derived factor-1alpha. Our results establish a novel inhibition of ER-mediated gene expression and cell proliferation/survival. Glyceollin I may represent an important component of a phytoalexin-enriched food (activated) diet in terms of chemoprevention as well as a novel therapeutic agent for hormone-dependent tumors.

Effects of vinclozolin, an anti-androgen, on affiliative behavior in the Dark-eyed Junco, Junco hyemalis.

Endocrine disrupting chemicals (EDCs) produce changes in physiology and behavior via diverse mechanisms including acting as hormone mimics or antagonists, affecting intracellular signaling pathways, and altering hormone production pathways. The fungicide vinclozolin acts as an anti-androgen and is known to affect affiliative behaviors in rodents, fish and amphibians. To investigate the possible effects of exposure to EDCs on reproductive behavior in a wild population of songbirds, we examined the effects of vinclozolin in wild-caught Dark-eyed Juncos (Junco hyemalis). For this and many other temperate songbird species, testosterone has powerful activational effects on affiliative behaviors in adulthood. We hypothesized that vinclozolin would affect male behaviors associated with female preference. Male juncos received daily oral gavage for 10 weeks with 2mM vinclozolin in vehicle or vehicle alone. Juncos were photostimulated (16L:8D) to induce breeding behavior. Each pair of a treated and non-treated male was presented to an estrogen-primed female to assess female preference. Seven of eight females exhibited a strong preference for a male exposed to vinclozolin over a control male (p=0.01). The only significant difference in measured male behaviors was increased beak wiping in controls (p=0.006) and there was no difference in gonad size or brain weight (p>0.05 for each). Our data suggest that estrogen-primed female juncos prefer to associate with male juncos exposed to this anti-androgen. This finding demonstrates that environmentally occurring anti-androgens can affect the social behavior of this species. To our knowledge, this is the first study to show that vinclozolin has effects on the social behavior of songbirds.

Dietary phytoestrogens and photoperiodic response in a male songbird, the Dark-eyed Junco (Junco hyemalis).

Many commercial bird diets are made with soy products that contain phytoestrogens (i.e., plant compounds that have weak agonist activity at estrogen receptors), but the effects of these compounds on bird physiology and behavior are largely unknown. The primary phytoestrogens present in soy are the isoflavones genistin and diadzin, which have been shown to affect reproductive measures in many taxa. Two groups of wild-caught male Dark-eyed Juncos (Junco hyemalis) were fed a diet either made with water-washed soy protein (soy(+)) or made with soy protein that had been alcohol-washed to extract isoflavones (soy(-)). Both groups exhibited a photoperiodic response to long days. Plasma luteinizing hormone (LH) concentrations increased within the first week of long day (LD) exposure for both groups, and over the course of the experiment LH was higher in the soy(+) group, although concentrations for both groups were lower than have been reported in free-living juncos. The rate of cloacal protuberance (CP) growth was significantly affected by diet, with the soy(-) birds beginning to increase their CPs about a week faster than soy(+) birds after exposure to LD. There was no group difference in food intake, fat score, body mass, or behavioral measures during the study or in testis weight at the end of the study. Although effects of dietary phytoestrogens detected were subtle (i.e., rate of CP growth), those investigating subtle effects of hormonally active substances (e.g., endocrine disruptors) or environmental cues affecting the reproductive axis in songbirds may want to consider eliminating phytoestrogens from their experimental diets.

Antiestrogenic glyceollins suppress human breast and ovarian carcinoma tumorigenesis.

PURPOSE: We have identified the phytoalexin compounds glyceollins I, II, and III, which exhibit marked antiestrogenic effects on estrogen receptor function and estrogen-dependent tumor growth in vivo. The purpose of this study was to investigate the interactions among the induced soy phytoalexins glyceollins I, II, and III on the growth of estrogen-dependent MCF-7 breast cancer and BG-1 ovarian cancer cells implanted in ovariectomized athymic mice. EXPERIMENTAL DESIGN: Four treatment groups for each cell line were used: vehicle control, 20 mg/kg/mouse/d glyceollin mixture injection, 0.72 mg estradiol (E2) implant, and E2 implant + 20 mg/kg/mouse/d glyceollin injection. RESULTS: Treatment with glyceollin suppressed E2-stimulated tumor growth of MCF-7 cells (-53.4%) and BG-1 cells (-73.1%) in ovariectomized athymic mice. These tumor-inhibiting effects corresponded with significantly lower E2-induced progesterone receptor expression in the tumors. In contrast to tamoxifen, the glyceollins had no estrogen-agonist effects on uterine morphology and partially antagonized the uterotropic effects of estrogen. CONCLUSIONS: These findings identify glyceollins as antiestrogenic agents that may be useful in the prevention or treatment of breast and ovarian carcinoma.

Simultaneous determination of vinclozolin and detection of its degradation products in mouse plasma, serum and urine, and from rabbit bile, by high-performance liquid chromatography.

A specific high-performance liquid chromatography method has been developed for simultaneous detection of vinclozolin and its degradation products (M1, M2, and M3). The method has been validated according to ICH guidelines and can be extended to quantitation of vinclozolin. A base-line separation of vinclozolin and its degradation products was found with symmetrical peak shapes on an XTerra MS C18 column using 10 mM ammonium bicarbonate at pH 9.2 and acetonitrile as mobile phase. The retention times of vinclozolin, M1, M2, and M3 were 12.8, 8.1, 11.6, and 11.1 min, respectively. A linear calibration curve was obtained across a range from 5 to 200 microM for vinclozolin. The intra- and inter-day relative standard deviations (%RSD) were <1%. Greater than 90% recoveries of vinclozolin from bio-fluids including mouse plasma, serum and urine, and rabbit bile, were obtained in a single step with a single solvent.

Age-related difference in size of brain regions for song learning in adult male dark-eyed Juncos (Junco hyemalis).

In seasonally breeding adult male songbirds, the volumes of several song control regions (SCRs) change seasonally in parallel with plasma testosterone (T) levels and decrease following gonadectomy. Testosterone treatment to castrates prevents this decrease, indicating T dependency. During the breeding season, second-year (SY: birds entering their first breeding season) free-ranging male Dark-eyed Juncos (Junco hyemalis) have smaller testes than older (after second-year, ASY: birds entering at least their second breeding season) birds. SY males also have lower plasma T concentrations than ASY males at the beginning of the breeding season. We investigated differences in song structure of the two age groups and the relationship between age differences in gonadal function and SCR sizes. The average number of syllables per song, syllable duration, trill rate, song duration, and variability in song duration were age-independent. Two brain regions that are thought to be involved primarily in song learning and perception were 13 and 18% larger, respectively, in SY than in ASY males, the opposite of what would be expected based solely on reproductive measures (testis mass and cloacal protuberance width). In contrast, the volumes of two regions that directly control song expression did not differ with age. The lack of age-related size differences in regions that are required for song production may indicate that male juncos of all ages have similar brain space requirements for motor production. Where there were size differences, they were restricted to regions primarily controlling vocal behavior acquisition/perception, suggesting that first time breeders need more brain space than experienced breeders to acquire crystallized song and/or acoustically perceive aspects of their environment.