Single neonatal estrogen implant sterilizes female animals by decreasing hypothalamic KISS1 expression.

Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive behaviors and diseases. This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy. The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1), the neuropeptide that triggers and regulates pulsatile secretion of GnRH. Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2-3 weeks. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer hypothalamic Kisspeptin neurons, but the GnRH-LH axis remained responsive to Kisspeptin stimulation. Because it would be desirable to use a biodegradable carrier that is also easier to handle, an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule. A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat. In neonatal female Beagle dogs, implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus. None of the treatments produced any concerning health effects, other than infertility. Therefore, further development of this technology for sterilization in domestic female animals, such as dogs and cats is worthy of investigation.

Endometriosis leads to central nervous system-wide glial activation in a mouse model of endometriosis.

BACKGROUND: Chronic pelvic pain (CPP) is a common symptom of endometriosis. Women with endometriosis are also at a high risk of suffering from anxiety, depression, and other psychological disorders. Recent studies indicate that endometriosis can affect the central nervous system (CNS). Changes in the functional activity of neurons, functional magnetic resonance imaging signals, and gene expression have been reported in the brains of rat and mouse models of endometriosis. The majority of the studies thus far have focused on neuronal changes, whereas changes in the glial cells in different brain regions have not been studied. METHODS: Endometriosis was induced in female mice (45-day-old; n = 6-11/timepoint) by syngeneic transfer of donor uterine tissue into the peritoneal cavity of recipient animals. Brains, spines, and endometriotic lesions were collected for analysis at 4, 8, 16, and 32 days post-induction. Sham surgery mice were used as controls (n = 6/timepoint). The pain was assessed using behavioral tests. Using immunohistochemistry for microglia marker ionized calcium-binding adapter molecule-1 (IBA1) and machine learning "Weka trainable segmentation" plugin in Fiji, we evaluated the morphological changes in microglia in different brain regions. Changes in glial fibrillary acidic protein (GFAP) for astrocytes, tumor necrosis factor (TNF), and interleukin-6 (IL6) were also evaluated. RESULTS: We observed an increase in microglial soma size in the cortex, hippocampus, thalamus, and hypothalamus of mice with endometriosis compared to sham controls on days 8, 16, and 32. The percentage of IBA1 and GFAP-positive area was increased in the cortex, hippocampus, thalamus, and hypothalamus in mice with endometriosis compared to sham controls on day 16. The number of microglia and astrocytes did not differ between endometriosis and sham control groups. We observed increased TNF and IL6 expression when expression levels from all brain regions were combined. Mice with endometriosis displayed reduced burrowing behavior and hyperalgesia in the abdomen and hind-paw. CONCLUSION: We believe this is the first report of central nervous system-wide glial activation in a mouse model of endometriosis. These results have significant implications for understanding chronic pain associated with endometriosis and other issues such as anxiety and depression in women with endometriosis.

Subchronic exposure to environmentally relevant concentrations of di-(2-ethylhexyl) phthalate differentially affects the colon and ileum in adult female mice.

Di(2-ethylhexyl) phthalate (DEHP) is a large-molecular-weight phthalate added to plastics to impart versatile properties. DEHP can be found in medical equipment and devices, food containers, building materials, and children's toys. Although DEHP exposure occurs most commonly by ingesting contaminated foods in the majority of the population, its effects on the gastrointestinal tract have not been well studied. Therefore, we analyzed the effects of subchronic exposure to DEHP on the ileum and colon morphology, gene expression, and immune microenvironment. Adult C57BL/6 female mice were orally dosed with corn oil (control, n = 7) or DEHP (0.02, 0.2, or 30 mg/kg, n = 7/treatment dose) for 30-34 days. Mice were euthanized during diestrus, and colon and ileum tissues were collected for RT-qPCR and immunohistochemistry. Subchronic DEHP exposure in the ileum altered the expression of several immune-mediating factors (Muc1, Lyz1, Cldn1) and cell viability factors (Bcl2 and Aifm1). Similarly, DEHP exposure in the colon impacted the gene expression of factors involved in mediating immune responses (Muc3a, Zo2, Ocln, Il6, and Il17a); and also altered the expression of cell viability factors (Ki67, Bcl2, Cdk4, and Aifm1) as well as a specialized epithelial cell marker (Vil1). Immunohistochemical analysis of the ileum showed DEHP increased expression of VIL1, CLDN1, and TNF and decreased number of T-cells in the villi. Histological analysis of the colon showed DEHP altered morphology and reduced cell proliferation. Moreover, in the colon, DEHP increased the expression of MUC2, MUC1, VIL1, CLDN1, and TNF. DEHP also increased the number of T-cells and Type 2 immune cells in the colon. These data suggest that subchronic DEHP exposure differentially affects the ileum and colon and alters colonic morphology and the intestinal immune microenvironment. These results have important implications for understanding the effects of DEHP on the gastrointestinal system.

Isolation of DiNP-Degrading Microbes from the Mouse Colon and the Influence DiNP Exposure Has on the Microbiota, Intestinal Integrity, and Immune Status of the Colon.

Di-isononyl phthalate (DiNP) is a plasticizer used to impart flexibility or stability in a variety of products including polyvinyl chloride, cable coatings, artificial leather, and footwear. Previous studies have examined the impact of DiNP on gut integrity and the colonic immune microenvironment, but this study further expands the research by examining whether DiNP exposure alters the colonic microbiota and various immune markers. Previous studies have also revealed that environmental microbes degrade various phthalates, but no studies have examined whether anaerobic gut bacteria can degrade DiNP. Thus, this study tested the hypothesis that DiNP exposure alters the gut microbiota and immune-related factors, and that anaerobic bacteria in the gut can utilize DiNP as the sole carbon source. To test this hypothesis, adult female mice were orally dosed with corn oil or various doses of DiNP for 10-14 consecutive days. After the treatment period, mice were euthanized during diestrus. Colonic contents were collected for full-length 16S rRNA gene sequencing to identify the bacteria in the colon contents. Sanger sequencing of the 16S rRNA gene was used to identify bacteria that were able to grow in Bacteroides minimal media with DiNP as the sole carbon source. Colon tissues were collected for immunohistochemistry of immune(-related) factors. An environmentally relevant dose of DiNP (200 µg/kg) significantly increased a Lachnoclostridium taxon and decreased Blautia compared to the control. Collectively, minimal changes in the colonic microbiota were observed as indicated by non-significant beta-diversities between DiNP treatments and control. Furthermore, three strains of anaerobic bacteria derived from the colon were identified to use DiNP as the sole carbon source. Interestingly, DiNP exposure did not alter protein levels of interleukin-6, tumor necrosis factor alpha, claudin-1, and mucin-1 compared to the control. Collectively, these findings show that DiNP exposure alters the gut microbiota and that the gut contains DiNP-degrading microbes.

Subacute Exposure to an Environmentally Relevant Dose of Di-(2-ethylhexyl) Phthalate during Gestation Alters the Cecal Microbiome, but Not Pregnancy Outcomes in Mice.

Di-2-ethylhexyl phthalate (DEHP) is a plasticizer commonly found in polyvinyl chloride, medical equipment, and food packaging. DEHP has been shown to target the reproductive system and alter the gut microbiome in humans and experimental animals. However, very little is known about the impact of DEHP-induced microbiome changes and its effects during pregnancy. Thus, the objective of this study was to investigate the effects of DEHP exposure during pregnancy on the cecal microbiome and pregnancy outcomes. Specifically, this study tested the hypothesis that subacute exposure to DEHP during pregnancy alters the cecal microbiome in pregnant mice, leading to changes in birth outcomes. To test this hypothesis, pregnant dams were orally exposed to corn oil vehicle or 20 µg/kg/day DEHP for 10 days and euthanized 21 days after their last dose. Cecal contents were collected for 16S Illumina and shotgun metagenomic sequencing. Fertility studies were also conducted to examine whether DEHP exposure impacted birth outcomes. Subacute exposure to environmentally relevant doses of DEHP in pregnant dams significantly increased alpha diversity and significantly altered beta diversity. Furthermore, DEHP exposure during pregnancy significantly increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes and Deferribacteres compared with controls. The affected taxonomic families included Deferribacteraceae, Lachnospiraceae, and Mucisprillum. In addition to changes in the gut microbiota, DEHP exposure significantly altered 14 functional pathways compared with the control. Finally, DEHP exposure did not significantly impact the fertility and birth outcomes compared with the control. Collectively, these data indicate that DEHP exposure during pregnancy shifts the cecal microbiome, but the shifts do not impact fertility and birth outcomes.

The Impact of Di-Isononyl Phthalate Exposure on Specialized Epithelial Cells in the Colon.

Di-isononyl phthalate (DiNP) is a high-molecular-weight phthalate commonly used as a plasticizer for polyvinyl chloride and other end products, such as medical devices and construction materials. Most of our initial exposure to DiNP occurs by ingestion of DiNP-contaminated foods. However, little is known about the effects of DiNP on the colon. Therefore, the goal of this study was to test the hypothesis that DiNP exposure alters immune responses and impacts specialized epithelial cells in the colon. To test this hypothesis, adult female mice were orally dosed with corn-oil vehicle control or doses of DiNP ranging from 20 µg/kg/d to 200 mg/kg/d for 10-14 days. After the dosing period, mice were euthanized in diestrus, and colon tissues and sera were collected for histological, genomic, and proteomic analysis of various immune factors and specialized epithelial cells. Subacute exposure to DiNP significantly increased protein levels of Ki67 and MUC2, expression of a Paneth cell marker (Lyz1), and estradiol levels in sera compared with control. Gene expression of mucins (Muc1, Muc2, Muc3a, and Muc4), Toll-like receptors (Tlr4 and Tlr5), and specialized epithelial cells (ChgA, Lgr5, Cd24a, and Vil1) were not significantly different between treatment groups and control. Cytokine levels of IL-1RA and CXCL12 were also not significantly different between DiNP treatment groups and control. These data reveal that DiNP exposure increases circulating estradiol levels and gene expression in specialized epithelial cells with immune response capabilities (eg, goblet and Paneth cells) in the mouse colon, which may initiate immune responses to prevent further damage in the colon.

Loss of basigin expression in uterine cells leads to subfertility in female mice†.

Basigin (BSG) is a transmembrane glycoprotein involved in cell proliferation, angiogenesis, and tissue remodeling. BSG has been shown to be essential for male and female reproduction although little is known about its role in normal uterine function. To study the potential function of BSG in the female reproductive tract, we generated mice with conditional knockout of Bsg in uterine cells using progesterone receptor-Cre and hypothesized that BSG is required for normal pregnancy in mice. Fertility study data showed that the conditional knockout mice had significantly reduced fertility compared to controls. Ovarian function of the conditional knockout mice appeared normal with no difference in the number of superovulated oocytes collected or in serum progesterone levels between the conditional knockout and the control mice. Uterine tissues collected at various times of gestation showed increased abnormalities in implantation, decidualization, placentation, and parturition in the conditional knockout mice. Uterine cross sections on Day 5 of pregnancy showed implantation failure and abnormal uterine epithelial differentiation in a large proportion of the conditional knockout mice. There was a compromised decidual response to artificial decidualization stimuli and decreased mRNA and protein levels for decidualization genes in the uteri of the conditional knockout mice. We also observed altered protein expression of monocarboxylate transporter 1 (MCT1), as well as impaired angiogenesis in the conditional knockout uteri compared to the controls. These results support that BSG is required for successful pregnancy through its functions in implantation and decidualization.

Subacute exposure to di-isononyl phthalate alters the morphology, endocrine function, and immune system in the colon of adult female mice.

Di-isononyl phthalate (DiNP), a common plasticizer used in polyvinyl chloride products, exhibits endocrine-disrupting capabilities. It is also toxic to the brain, reproductive system, liver, and kidney. However, little is known about how DiNP impacts the gastrointestinal tract (GIT). It is crucial to understand how DiNP exposure affects the GIT because humans are primarily exposed to DiNP through the GIT. Thus, this study tested the hypothesis that subacute exposure to DiNP dysregulates cellular, endocrine, and immunological aspects in the colon of adult female mice. To test this hypothesis, adult female mice were dosed with vehicle control or DiNP doses ranging from 0.02 to 200 mg/kg for 10-14 days. After the treatment period, mice were euthanized during diestrus, and colon tissue samples were subjected to morphological, biochemical, and hormone assays. DiNP exposure significantly increased histological damage in the colon compared to control. Exposure to DiNP also significantly decreased sICAM-1 levels, increased Tnf expression, decreased a cell cycle regulator (Ccnb1), and increased apoptotic factors (Aifm1 and Bcl2l10) in the colon compared to control. Colon-extracted lipids revealed that DiNP exposure significantly decreased estradiol levels compared to control. Collectively, these data indicate that subacute exposure to DiNP alters colon morphology and physiology in adult female mice.

Progesterone Receptor Serves the Ovary as a Trigger of Ovulation and a Terminator of Inflammation.

Ovulation is triggered by the gonadotropin surge that induces the expression of two key genes, progesterone receptor (Pgr) and prostaglandin-endoperoxide synthase 2 (Ptgs2), in the granulosa cells of preovulatory follicles. Their gene products PGR and PTGS2 activate two separate pathways that are both essential for successful ovulation. Here, we show that the PGR plays an additional essential role: it attenuates ovulatory inflammation by diminishing the gonadotropin surge-induced Ptgs2 expression. PGR indirectly terminates Ptgs2 expression and PGE2 synthesis in granulosa cells by inhibiting the nuclear factor κB (NF-κB), a transcription factor required for Ptgs2 expression. When the expression of PGR is ablated in granulosa cells, the ovary undergoes a hyperinflammatory condition manifested by excessive PGE2 synthesis, immune cell infiltration, oxidative damage, and neoplastic transformation of ovarian cells. The PGR-driven termination of PTGS2 expression may protect the ovary from ovulatory inflammation.

Novel Candidates for Vaccine Development Against Mycoplasma Capricolum Subspecies Capripneumoniae (Mccp)-Current Knowledge and Future Prospects.

Exploration of novel candidates for vaccine development against Mycoplasma capricolum subspecies capripneumoniae (Mccp), the causative agent of contagious caprine pleuropneumonia (CCPP), has recently gained immense importance due to both the increased number of outbreaks and the alarming risk of transboundary spread of disease. Treatment by antibiotics as the only therapeutic strategy is not a viable option due to pathogen persistence, economic issues, and concerns of antibiotic resistance. Therefore, prophylactics or vaccines are becoming important under the current scenario. For quite some time inactivated, killed, or attenuated vaccines proved to be beneficial and provided good immunity up to a year. However, their adverse effects and requirement for larger doses led to the need for production of large quantities of Mccp. This is challenging because the required culture medium is costly and Mycoplasma growth is fastidious and slow. Furthermore, quality control is always an issue with such vaccines. Currently, novel candidate antigens including capsular polysaccharides (CPS), proteins, enzymes, and genes are being evaluated for potential use as vaccines. These have shown potential immunogenicity with promising results in eliciting protective immune responses. Being easy to produce, specific, effective and free from side effects, these novel vaccine candidates can revolutionize vaccination against CCPP. Use of novel proteomic approaches, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis, immunoblotting, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, tandem mass spectroscopy, fast protein liquid chromatography (FPLC), bioinformatics, computerized simulation and genomic approaches, including multilocus sequence analysis, next-generation sequencing, basic local alignment search tool (BLAST), gene expression, and recombinant expression, will further enable recognition of ideal antigenic proteins and virulence genes with vaccination potential.

Comparative evaluation of different therapeutic protocols for contagious caprine pleuropneumonia in Himalayan Pashmina goats.

Therapeutic management of contagious caprine pleuroneumonia (CCPP) involves mostly the use of oxytetracycline followed by enrofloxacin and rarely tylosin. In many parts of the world including India, the former antibiotics are commonly available than the latter. Therefore, prolonged use of the same leads to the development of antibiotic resistance and decreased efficacy of drug. Besides, inflammatory and allergic pathogenesis of CCPP envisages combination therapy. In this study, we evaluated the effectiveness of the combination therapy using different antibiotics (oxytetracycyline @ 10: group I, enrofloxacin @ 5 group II, and tylosin: group III, @ 10 mg/kg body weight), along with anti-inflammatory (meloxicam @ 0.5 mg/kg) and anti-allergic (pheneramine maleate @ 1.0 mg/kg) drugs. These drugs were given intramuscularly at the interval of 48 h for four times in three test groups (n = 10) of Pashmina goats, viz. groups I, II, and III, respectively, affected with CCPP. Group IV (n = 10) was kept as healthy control when group V (n = 10) treated with oxytetracycline @ 10 mg/kg alone was used as positive control. Clinical signs, clinical parameters, pro-inflammatory cytokine (tumor necrosis factor alpha (TNF-α)), and oxidative stress indices (total oxidant status (TOS), total antioxidant status (TAS)) were evaluated at hours 0, 48, 96, and 144 of experimental trial. Tylosin-based combination therapy resulted in a rapid and favorable recovery resulting in restoration of normal body temperature (102.46 ± 0.31 °F), respiration rate (16.30 ± 0.79 per minute), and heart rate (89.50 ± 2.63 per minute) compared to the oxytetracycline (102.95 ± 0.13, 21.30 ± 1.12, 86.00 ± 2.33, respectively) and enrofloxacin (102.97 ± 0.19, 21.00 ± 1.25, 90.00 ± 2.58, respectively) treated groups. By hour 144, all the groups showed restoration of clinical parameters of normal health and diminishing signs of CCPP, viz. fever, dyspnea, coughing, nasal discharge, weakness, and pleurodynia. Significant (P ≤ 0.05) decrease in levels of TNF-α and non-significant (P > 0.05) decrease in levels of TOS and an increase in levels of TAS were noted from hour 0 to 144 in all the test groups. Within the groups, no significant (P > 0.05) change was noted in TNF-α, TOS, and TAS levels; however, TNF-α levels were comparatively lower in group III. Hematological parameters did not differ significantly (P > 0.05). From these findings, it can be inferred that tylosin-based combination therapy is relatively better for early, rapid, and safe recovery besides minimizing inflammatory and oxidative cascade in CCPP affected Pashmina goats compared to oxytetracycline- and enrofloxacin-based therapies.