Patients with polycystic ovary syndrome (PCOS) can suffer from psychological disorders, among which depression is the most commonly diagnosed. However, the pathogenesis is still unclear. The aims of the present study were to investigate the behaviors of dehydroepiandrosterone (DHEA)-induced PCOS mice, the effects of high-fat diet (HFD) on mouse behaviors, and the underlying mechanism. Prepubertal C57BL/6 mice (25 days of age) were divided into four groups and injected daily with the vehicle sesame oil or DHEA on the normal chow or a 60% HFD for 20 consecutive days. Depression-like behavior of the mice was examined using a forced swim test, tail suspension test, and open field test. Thereafter, the animals were killed and four brain regions were collected. The brain levels of monoamines and their metabolites, including norepinephrine, serotonin, 5-hydroxy-3-indolacetic acid, dopamine, and 3,4-hydroxyphenylacetic acid, were analyzed by HPLC. Our data show that DHEA-treated mice exhibited depression-like behavior according to the results from behavioral assessment. The brain contents of monoamines and/or their metabolites decreased in DHEA-treated mice compared with controls. HFD did not seem to markedly affect the behavioral responses, the brain monoamines, or their metabolites in the mice. These findings suggest that DHEA treatment induced depression-like behavior in PCOS mice, possibly through down-regulation of brain monoamines and/or their metabolites, which implies the contribution of hyperandrogenism to the psychological symptoms of women with PCOS.

The common environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or, commonly, dioxin) is a known endocrine disruptor that has been linked to the development of endometriosis in experimental models. Using a murine model, we previously demonstrated that in utero TCDD exposure promotes the transgenerational development of an “endometriosis-like” uterine phenotype consisting of reduced responsiveness to progesterone, as well as subfertility and an increased risk of preterm birth. Because adenomyosis is frequently observed as a comorbidity in women with endometriosis, herein we sought to determine the incidence of adenomyosis in nonpregnant mice with a history of direct or indirect TCDD exposure. Using histologic assessment and immunohistochemical staining, we analyzed murine uteri for adenomyosis, microvessel density, and expression of estrogen receptors alpha and beta (ESR1 and ESR2). Our studies revealed that unexposed control mice did not exhibit adenomyosis, whereas this disease was frequently observed in mice with a history of early-life TCDD exposure. A transgenerational impact of developmental TCDD exposure was demonstrated, because a subset of mice with only an indirect exposure (F3) also exhibited adenomyosis. Microvessel density within the uterus was significantly higher in all groups of TCDD-exposed mice compared with control animals, with density correlated to the severity of disease. Both ESR1 and ESR2 proteins exhibited alterations in expression in experimental mice compared with controls. Similar to women with endometriosis, we observed a significant reduction in the ratio of Esr1:Esr2 mRNA in all F1 mice compared with controls. Although this retrospective study was not designed to specifically address mechanisms associated with the development of adenomyosis, our data suggest that developmental TCDD exposure permanently alters adult steroid responses, which may contribute to the transgenerational development of adenomyosis.

Endometriosis causes severe chronic pelvic pain and infertility. Because the standard medication and surgical treatments of endometriosis show high recurrence of symptoms, it is necessary to improve the current treatment options. In the initial study, we examined whether niclosamide can be a useful drug for treating endometriosis in a preclinical setting. Endometriotic implants were induced using an established mouse model involving transimplantation of mouse endometrial fragments to the peritoneal wall of recipient mice. When the recipient mice were treated with niclosamide for 3 wk, niclosamide reduced the size of endometriotic implants with inhibition of cell proliferation and inflammatory signaling, including RELA (NFKB) and STAT3 activation, but did not alter expression of steroid hormone receptors. To identify genes whose expression is regulated by niclosamide in endometriotic implants, RNA-sequencing was performed, and several genes downregulated by niclosamide were related to inflammatory responses, WNT, and MAPK signaling. In a second study designed to assess whether niclosamide affects reproductive function, the recipient mice started receiving niclosamide after the induction of endometriosis. Then, the recipient mice were mated with wild-type males, and treatments continued until the pups were born. Niclosamide-treated recipient mice became pregnant and produced normal size and number of pups. These results suggest that niclosamide could be an effective therapeutic drug and acts as an inhibitor of inflammatory signaling without disrupting normal reproductive function.

Curcumin, a natural pigment for yellow color that originates from turmeric, is a diarylheptanoid widely studied for its anti-inflammatory, antiangiogenic, antioxidant, and anticancer effects on cells. In placental diseases, including preeclampsia and preterm birth, curcumin reduces proinflammatory cytokines. Even though curcumin is regarded as a novel chemotherapeutic agent with strong apoptotic effects based on phenolic structure, little is known about its functional effects on choriocarcinoma. Therefore, in the present study, we investigated the chemotherapeutic effects of curcumin on choriocarcinoma cells (JAR and JEG3), which are valuable placental models. The results showed that curcumin decreased viability of choriocarcinoma cells in a dose-dependent manner. In addition, proliferative and migratory characteristics of JAR and JEG3 cells were inhibited by curcumin treatment and curcumin-induced apoptotic effects, which were assessed using TUNEL and annexin V/propidium iodide staining. Moreover, curcumin increased depolarization of the mitochondrial membrane based on JC-1 staining and changed expression of apoptotic proteins. Phosphorylation of mitogen-activated protein kinases (MAPK) responsible for regulation of anticancer effects of curcumin were examined for dose- and time-dependent effects. The ERK1/2 and SAPK/JNK and their downstream molecules including P90RSK and c-Jun, respectively, were activated by curcumin. Moreover, pharmacological inhibitors of ERK1/2 (U0126) and SAPK/JNK (SP600125) suppressed ERK1/2 and SAPK/JNK activation respectively, and blockage of P38 MAPK by its inhibitor (SB203580) had a synergistic effect with curcumin. These results indicate that curcumin acts as a novel chemotherapeutic agent on human placental choriocarcinoma cells via activation of ERK1/2 and SAPK/JNK signal transduction cascades.

Many chemokines are present at the maternal-fetal interface and play important roles in the establishment and maintenance of pregnancy. Our study demonstrates that a chemokine, chemokine (C-C motif) ligand 28 (CCL28), is expressed in the uterine endometrium during early pregnancy in pigs. Thus, we investigated expression of CCL28 and its receptors, chemokine (C-C motif) receptor types 3 (CCR3) and 10 (CCR10), in the uterine endometrium during the estrous cycle and pregnancy and the function of CCL28 at the maternal-fetal interface during early pregnancy. Levels of CCL28 mRNAs were highest on Day 10 of pregnancy and decreased thereafter during pregnancy, and CCL28 was localized mainly to endometrial glandular epithelial cells. The presence of the CCL28 protein in uterine flushings was confirmed on Day 12 of the estrous cycle and pregnancy. Endometrial tissues expressed CCR3 and CCR10 during pregnancy. The CCR10 protein was localized to endometrial luminal and glandular epithelial cells, chorionic epithelial cells, and the allantoic membrane during pregnancy. Conceptuses during early pregnancy expressed CCL28 and CCR10, but not CCR3, and chorioallantoic tissues expressed CCR10 at increasing levels towards term. Treatment with recombinant CCL28 increased the proliferation and migration of a porcine trophectoderm cell line. These results indicated that the CCL28 chemokine and its receptors, CCR3 and CCR10, are expressed at the maternal-conceptus interface, and CCL28 induces the proliferation and migration of trophectoderm cells through CCR10, suggesting that CCL28 may play a critical role in the establishment and maintenance of pregnancy in pigs.

In cattle, maternal recognition of pregnancy occurs on Day 16 via secretion of interferon tau (IFNT) by the conceptus. The endometrium can distinguish between embryos with different developmental competencies. In eutherian mammals, X-chromosome inactivation (XCI) is required to ensure an equal transcriptional level of most X-linked genes for both male and female embryos in adult tissues, but this process is markedly different in cattle than mice. We examined how sexual dimorphism affected conceptus transcript abundance and amino acid composition as well as the endometrial transcriptome during the peri-implantation period of pregnancy. Of the 5132 genes that were differentially expressed on Day 19 in male compared to female conceptuses, 2.7% were located on the X chromosome. Concentrations of specific amino acids were higher in the uterine luminal fluid of male compared to female conceptuses, while female conceptuses had higher transcript abundance of specific amino acid transporters (SLC6A19 and SLC1A35). Of note, the endometrial transcriptome was not different in cattle gestating a male or a female conceptus. These data support the hypothesis that, far from being a blastocyst-specific phenomenon, XCI is incomplete before and during implantation in cattle. Despite differences in transcript abundance and amino acid utilization in male versus female conceptuses, the sex of the conceptus itself does not elicit a different transcriptomic response in the endometrium.

In mammals, many sperm that reach the oviduct are held in a reservoir by binding to epithelium. To leave the reservoir, sperm detach from the epithelium; however, they may bind and detach again as they ascend into the ampulla toward oocytes. In order to elucidate the nature of binding interactions along the oviduct, we compared the effects of bursts of strong fluid flow (as would be caused by oviductal contractions), heparin, and hyperactivation on detachment of bovine sperm bound in vitro to epithelium on intact folds of isthmic and ampullar mucosa. Intact folds of oviductal mucosa were used to represent the strong attachments of epithelial cells to each other and to underlying connective tissue that exist in vivo. Effects of heparin on binding were tested because heparin binds to the Binder of SPerm (BSP) proteins that attach sperm to oviductal epithelium. Sperm bound by their heads to beating cilia on both isthmic and ampullar epithelia and could not be detached by strong bursts of fluid flow. Addition of heparin immediately detached sperm from isthmic epithelium but not ampullar epithelium. Addition of 4-aminopyridine immediately stimulated hyperactivation of sperm but did not detach them from isthmic or ampullar epithelium unless added with heparin. These observations indicate that the nature of binding of sperm to ampullar epithelium differs from that of binding to isthmic epithelium; specifically, sperm bound to isthmic epithelium can be detached by heparin alone, while sperm bound to ampullar epithelium requires both heparin and hyperactivation to detach from the epithelium.

Because monotremes are the earliest offshoot of the mammalian lineage, the platypus and short-beaked echidna were studied as model animals to assess the origin and biological significance of adaptations considered unique to therian mammals: epididymal sperm maturation and subsequent capacitation. We show that spermatozoa from both species assemble into bundles of approximately 100 cells during passage through the epididymis and that an epididymal protein—secreted protein, acidic, cysteine-rich (osteonectin; SPARC)—is involved in bundle formation. The bundles persisted during incubation in vitro for at least 1 h under conditions that capacitate therian spermatozoa, and then underwent a time-dependent dissociation to release spermatozoa capable of fertilization. Only after this dissociation could the spermatozoa bind to the perivitelline membrane of a hen's egg, display an altered form of motility reminiscent of hyperactivation, and be induced to undergo an acrosome reaction. It is concluded that the development of sperm bundles in the monotreme epididymis mandates that they require a time-dependent process to be capable of fertilizing an ovum. However, because this functional end point was achieved without overt changes in protein tyrosine phosphorylation (a hallmark of capacitation in therians), it is concluded that the process in monotremes is distinctly different from capacitation in therian mammals.

The high serum estradiol (E₂) concentrations induced during in vitro fertilization are detrimental to endometrial receptivity and may result in lower embryo implantation rates. We have previously found that high E₂ concentrations inhibit the activation of nuclear factor kappa B (NF-κB), leading to endometrial epithelial cell (EEC) apoptosis. The objective of this study is to investigate the signaling pathways through which high E₂ results in NF-κB downregulation in EECs. Isolated human EECs were cultured in different concentrations of E₂ (10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷ M). The expression of heat shock protein 70 (Hsp70) and heat shock factor 1 (HSF-1) were upregulated under supraphysiological E₂ (10⁻⁷ M) concentration, whereas phosphorylated inhibitory kappa B-alpha (pI κB-α) and NF-κB p65 subunits were downregulated. Immunohistochemistry of C57BL/6 mouse EECs that were exposed in vivo to high serum E₂ from the administration of 20 IU of equine chorionic gonadotropin also demonstrated the same increase in HSF-1 and Hsp70 expression and a decrease in NF-κB. Immunoprecipitation of the induced Hsp70 proteins was achieved with the addition of inhibitory κB kinase gamma (IKK-γ) antibodies, and elimination of this reaction occurred after addition of hsp70 siRNA. In conclusion, high E₂ concentrations enhance HSF-1 and Hsp70 expression in EECs. The induced Hsp70 forms a complex with IKK-γ and inhibits pI κB-α, which consequently suppresses NF-κB activation.

The in vitro maturation (IVM) technique is beneficial for producing animal offspring, but the blastocyst rate is low after IVM. In this technique, cumulus-oocyte complexes (COCs) are collected from medium-size follicles. The follicles are ultimately selected as large dominant follicles or atretic follicles; therefore it is possible that the COCs collected using IVM are contaminated by follicles that will develop into large follicles and induce atresia. In the dominant follicles, estradiol-17beta and progesterone induce the differentiation of follicular somatic cells that exhibit the ability to respond to ovulation during follicular development. Thus, we hypothesized that changes in the hormonal condition of healthy follicles are essential for oocyte maturation during IVM. In this study, we performed a comparative analysis of the steroid hormone concentrations in nonvascularized follicles (NVFs) and vascularized follicles (VFs). The estradiol-17β concentration increased in medium VFs, whereas the level was low in NVFs of the same size. The progesterone concentration increased with large follicular size in VFs, but the level remained low in follicles of any size among NVFs. To improve the oocyte quality derived from NVFs, NVF COCs were cultured with follicle-stimulating hormone (FSH) alone or FSH under the VF hormonal conditions. Cultivation under the VF hormonal conditions dramatically improved the proliferation and survival of cumulus cells, meiotic maturation of oocytes, cumulus expansion, and blastocyst rate following in vitro fertilization. Thus, the cultivation of NVF COCs under VF hormonal conditions improves the developmental potential to the blastocyst stage by NVF oocytes.

Luteolin is a natural compound known for its anticancer effects on various human cancers by regulating signal transduction cascades. However, the effects of luteolin on human placental choriocarcinoma are not known. Results of the present study revealed that luteolin decreased viability of JAR and JEG-3 cells, which are valuable placental models, in a dose-dependent manner, and it induced apoptosis and loss of mitochondrial membrane potential in JAR and JEG-3 cells. The results also suggested that the PI3K/AKT pathway was inhibited by luteolin treatment of JAR and JEG-3 cells in a dose- and time-dependent manner. Next, we established effects of luteolin in the presence of pharmacological inhibitors of PI3K/AKT, ERK1/2 MAPK, and mTOR on proliferation of JAR and JEG-3 cells. In addition, these inhibitors were used to verify phosphorylation of AKT, GSK3beta, and ERK1/2 and to confirm mechanisms regulated by luteolin in JAR and JEG-3 cells. We also determined levels of SREBP1 and SREBP2 expression to investigate regulatory functions of luteolin in lipid metabolism in JAR and JEG-3 cells. Expression levels of both SREBP1 and SREBP2 mRNAs were significantly reduced, but only SREBP1 protein was influenced by luteolin. We compared viability of JAR and JEG-3 cells in response to luteolin alone or in combination with other chemotherapeutic drugs (etoposide, cisplatin, and paclitaxel) and found that luteolin has synergistic effects with the conventional chemotherapeutic drugs as an anticancer agent. Collectively, these results showed that luteolin plays an important role in the treatment of human choriocarcinoma cells by inhibiting the PI3K/AKT/mTOR/SREBP cascade and expression of lipogenic genes.

Studies support the idea that uterine epithelia and their secretions have important biological roles in conceptus survival, elongation, and implantation in sheep. The present study evaluated the transcriptome of the uterine luminal epithelium (LE) and glandular epithelium (GE) and the conceptus and proteome of uterine luminal fluid (ULF) during the peri-implantation period of pregnancy. Transcriptome (RNA-sequencing) analysis was conducted in LE and GE isolated from uteri of Day 10, 12, 14, 16, and 20 pregnant sheep by laser capture microdissection. In the LE, the total number of expressed genes increased between Days 10 and 20, whereas expressed genes in the GE increased from Days 10 to 14 and then decreased to Day 20. Most of the expressed genes in LE and GE from Days 10 to 14 are involved in cell survival and growth, whereas genes involved in cell organization and protein synthesis were most abundant on Days 16 and 20. Total expressed genes in the conceptus was greatest on Day 12, decreased to Day 16, and then increased to Day 20. Genes abundantly expressed in the elongating conceptus included IFNT, PTGS2, MGST1, FADS1, and FADS2, whereas SERPINA1, CSH1, and PLET1 were most abundant in the Day 20 conceptus. Proteins, identified by mass spectrometry, increased in the ULF from Days 10 to 16 and are involved in cellular reorganization or are proteases or chaperone proteins. These results support the idea that conceptus elongation and implantation is regulated by both extrinsic and intrinsic factors. This study provides critical information that serves as a foundation to discover new regulatory pathways governing uterine receptivity, conceptus elongation, trophectoderm differentiation, conceptus-endometrial interactions, and pregnancy establishment in ruminants.

Advanced methods of cellular purification are required to apply genome technology to the study of spermatogenesis. One approach, based on flow cytometry of murine testicular cells stained with Hoechst-33342 (Ho-FACS), has been extensively optimized and currently allows the isolation of nine germ cell types. This staining technique is straightforward to implement, is highly effective at purifying specific germ cell types, and yields sufficient cell numbers for high-throughput studies. Ho-FACS is a technique that does not require species-specific markers, but whose applicability to other species is largely unexplored. We hypothesized that, because of the similar cell physiology of spermatogenesis across mammals, Ho-FACS could be used to produce highly purified subpopulations of germ cells in mammals other than mouse. To test this hypothesis, we applied Ho-FACS to four mammalian species that are widely used in testis research: Rattus norvegicus, Cavia porcellus, Canis familiaris, and Sus scrofa domesticus. We successfully isolated four germ cell populations from these species with average purity of 79% for spermatocytes, 90% for spermatids, and 66% for spermatogonia. Additionally, we compare the performance of mechanical and chemical dissociation for each species, and propose an optimized gating strategy to better discriminate round and elongating spermatids in the mouse, which can potentially be applied to other species. Our work indicates that spermatogenesis may be uniquely accessible among mammalian developmental systems, as a single set of reagents may be sufficient to isolate germ cell populations from many different mammalian species, opening new avenues in the fields of development and male reproductive biology.

Until now, complete ex vivo spermatogenesis has been reported only in the mouse. In this species, the duration of spermatogenesis is 35 days, whereas it is 54 days in the rat and 74 days in humans. We performed long-term (until 60 days) cultures of fresh or frozen rat or human seminiferous tubule segments in a bioreactor made of a hollow cylinder of chitosan hydrogel. Testicular tissues were obtained from 8- or 20-day-old male rats or from adult human subjects who had undergone hormone treatments leading to a nearly complete regression of their spermatogenesis before bilateral orchiectomy for gender reassignment. The progression of spermatogenesis was assessed by cytological analyses of the cultures; it was related to a dramatic increase in the levels of the mRNAs specifically expressed by round spermatids, Transition protein 1, Transition protein 2, and Protamine 3 in rat cultures. From 2% to 3.8% of cells were found to be haploid cells by fluorescence in situ hybridization analysis of human cultures. In this bioreactor, long-term cultures of seminiferous tubule segments from prepubertal rats or from adult men allowed completion of the spermatogenic process leading to morphologically mature spermatozoa. Further studies will need to address the way of optimizing the yield of every step of spermatogenesis by adjusting the composition of the culture medium, the geometry, and the material properties of the chitosan hydrogel bioreactors. Another essential requirement is to assess the quality of the gametes produced ex vivo by showing their ability to produce normal offspring (rat) or their biochemical normality (human).

Retinoic acid (RA), the active metabolite of vitamin A, is known to be required for the differentiation of spermatogonia. The first round of spermatogenesis initiates in response to RA and occurs in patches along the length of the seminiferous tubule. However, very little is known about the individual differentiating spermatogonial populations and their progression through the cell cycle due to the heterogeneous nature of the onset of spermatogenesis. In this study, we utilized WIN 18,446 and RA as tools to generate testes enriched with different populations of spermatogonia to further investigate 1) the undifferentiated to differentiating spermatogonial transition, 2) the progression of the differentiating spermatogonia through the cell cycle, and 3) Sertoli cell number in response to altered RA levels. WIN 18,446/RA-treated neonatal mice were used to determine when synchronous S phases occurred in the differentiating spermatogonial population following treatment. Five differentiating spermatogonial S phase windows were identified between spermatogonial differentiation and formation of preleptotene spermatocytes. In addition, a slight increase in Sertoli cell number was observed following RA treatment, possibly implicating a role for RA in Sertoli cell cycle progression. This study has enhanced our understanding of the spermatogonial populations present in the neonatal testis during the onset of spermatogenesis by mapping the cell cycle kinetics of both the undifferentiated and the differentiating spermatogonial populations and identifying the precise timing of when specific individual differentiating spermatogonial populations are enriched within the testis following synchrony, thus providing an essential tool for further study of the differentiating spermatogonia.

Exposure to endocrine-disrupting chemicals (EDCs) during fetal and neonatal periods can have toxic effects that are irreversible and last a lifetime. However, the mechanism underlying this phenomenon is still unknown. Here, we show the effect of 17alpha-ethynyl estradiol (EE) on the development of the primordial follicle during early ovarian development in female rats. Microarray analysis revealed the down-regulation of Hrk, an activator of apoptosis, in neonatal ovaries exposed to EE. Real-time PCR analysis also showed a decrease of Hrk mRNA expression in ovaries treated with EE both in vitro and in neonatal rats. An immunostaining assay showed that HRK protein and cleaved caspase 3 colocalize in the oocytes at Postnatal Day 1 (PND1). The EE-exposed ovaries had a reduced number of oocytes positive for TUNEL staining compared to control ovaries at PND1. Abnormal follicle formation of EE-exposed ovaries was observed at PND7 and PND21. A TUNEL staining assay revealed that Hrk depletion reduced the number of apoptotic oocytes. In addition, down-regulation of Hrk mRNA expression was observed in ovaries treated with other estrogenic chemicals. We propose a model in which EE inhibits oocyte apoptosis in the neonatal ovary by suppressing the expression of Hrk, thereby disrupting follicle formation and ovary function.

Acute acrylamide exposure in male rodents results in reduced reproductive performance and dominant lethality. However, the reproductive effects of low-dose chronic exposure, which better reflects the nature of human exposure, remain far less certain. Human dietary consumption of acrylamide has been estimated at an average of 1–4 μg/kg bodyweight/day. In order to simulate this exposure, male mice were provided with acrylamide (1 μg/ml) via their drinking water continuously for 6 mo, which was equivalent to a human dose of 10.5 μg/kg bodyweight/day. This exposure regime increased DNA damage in the spermatozoa without affecting a concomitant reduction in overall fertility. The offspring of acrylamide-treated mice did not have an increased incidence of skin papilloma formation following the two-stage tumor-induction protocol. However, the male offspring of acrylamide-treated fathers had significantly increased levels of DNA damage in their spermatozoa, despite having had no direct toxicant exposure. It was also found that the acrylamide-treated mice and, most crucially, their progeny had increased levels of CYP2E1 protein in their spermatocytes. This is significant because CYP2E1 is the sole enzyme responsible for conversion of acrylamide to its harmful metabolite glycidamide. This altered expression may be due to epigenetic alterations. Additionally, the F0 and F1 mice had increased oxidative adducts in the DNA of their germ cells, which was hypothesized to arise as a byproduct of increased CYP2E1 levels. Therefore, chronic paternal acrylamide exposure in mice has consequences for their offspring and raises concerns for the effects of acrylamide exposure in the human population and the accumulated effects with multiple generations of exposure.