Vaginal Plug Research Articles

Maternal exposure to PM2.5 induces cognitive impairment in offspring via cerebellar neuroinflammation and oxidative stress

Available evidence suggest that exposure to PM2.5 during pregnancy is associated with reduced cognitive function in offspring. This study aimed to investigate the effects of maternal exposure to PM2.5 on offspring cognitive function and to elucidate the underlying mechanisms. In this work, pregnant C57BL/6 female mice were exposed to concentrated ambient PM2.5 or filtered air from day 0.5 (=vaginal plug) to day 15.5 in the Shanghai Meteorological and Environmental Animal Exposure System, and offspring cerebellar tissues were collected on embryonic day 15.5, as well as postnatal days 0, 10 and 42. The mean PM2.5 concentrations exposed to the pregnant mice were 73.06 ± 4.90 μg/m3 and 11.15 ± 2.71 μg/m3 in the concentrated ambient PM2.5 and filtered air chambers, respectively. Maternal concentrated PM2.5 exposure was negatively correlated with offspring spatial memory significantly as assessed by the Morris water maze. Compared with the filtered air group, PM2.5-exposed offspring mice had reduced cerebellar microglia. Both RNA and protein levels of IL-8 and TNF-α were elevated in the concentrated ambient PM2.5 group. PM2.5 exposure increased the level of 8-OHG in miRNA of microglia and Purkinje cells in 6-week-old offspring. The level of prostaglandin F2α (8-iso-PGF2Aα) in the cerebellum was increased at different growing stages of offspring after gestational exposure of PM2.5. These results suggested that maternal air pollution exposure might cause inflammatory damage and oxidative stress to the cerebellum, contributing to reduced cognitive performance in mice offspring.

Testicular Hypoplasia with Normal Fertility in Neudesin-Knockout Mice.

Neudesin is a secretory protein involved in the brain development during embryonic period and diet-induced development of adipose tissue. Although neudesin is also expressed in the testis, its physiological functions in the testis have not been documented. Therefore, we examined neudesin-encoding neuron-derived neurotrophic factor (Nenf) gene-knockout (Neudesin-KO) mice to clarify the functions of neudesin in the testis. The testicular size of the Neudesin-KO mice was significantly smaller than that of wild-type (WT) mice. However, histological analyses did not reveal any abnormalities in the testis, caput epididymis, and cauda epididymis. Sperm number in the cauda epididymis was comparable between WT and KO mice. Neudesin-KO male mice produced vaginal plugs on paired WT female mice, with a frequency similar to that in WT male mice. A similar number of embryos were developed in the females copulated with WT and Neudesin-KO males. Molecular analysis indicated that the ion transporters Slc19a1 and Kcnk3 were more expressed in the testis of Neudesin-KO mice than in the testis of WT mice, suggesting that the transport of ions and some nutrients in the testis has some abnormalities. Testicular size decreased on postnatal day 6, but not on the day of birth, indicating that neudesin is involved in the postnatal, but not embryonic, development of testis. These results indicate a novel role of neudesin in the development of testis.

Virgin queen behaviour and controlled mating in the stingless bee Tetragonulacarbonaria (Meliponini)

Improving knowledge of virgin queen behaviour and mating biology in stingless bees (Meliponini) is an important step towards artificial selection programmes for managed colonies. Here we study the Australian stingless bee Tetragonulacarbonaria to (1) describe the behaviour of queens from eclosion until egg-laying, and (2) assess whether young queens can be reared and mated under constrained conditions. We extracted mature virgin queen cells of T.carbonaria from large colonies and reared them in queen maturation boxes containing a limited number of workers (“micro-colonies”). Queen behaviour followed a typical pattern with three phases: a period of high activity and wing-flapping on top of the brood (5 ± 2 days of age), attempts to leave the colony for the nuptial flight (12 ± 3 days of age), and oviposition (20 ± 6 days of age). In total, 71% (68 of 95) of queen cells hatched in our maturation boxes and 57% (39 of 68) survived to mating age, with some observed to be actively killed by workers within the first week of life (8 of 68; 11%). For a subset of our queens, we performed controlled matings (either with or without CO2 narcosis) by presenting constrained females to males in mating aggregations, allowing one male to mate (deposit mating plug) and then returning queens to their box. In the absence of CO2 narcosis, 30% of queens mated this way were subsequently found to have sperm in their spermathecae, indicative of a successful mating (5 of 17). The remaining 70% however contained no sperm, despite the presence of the male mating plug. Amongst queens that were CO2 narcotized before and during the controlled mating, none had sperm in their spermathecae (0 of 12), though most went on to activate their ovaries following the mating and lay male (haploid) eggs. Together, these trials indicate that controlled matings with T.carbonaria are possible, but suggest that queens may need to actively accept mating for successful sperm transfer.

Preconception exposure to malathion and glucose homeostasis in rats: Effects on dams during pregnancy and post-term periods, and on their progeny.

Understanding the individual and global impact of pesticides on human physiology and the different stages of life is still a challenge in environmental health. We analyzed here whether administration of the organophosphate insecticide malathion before pregnancy could affect glucose homeostasis during pregnancy and, in addition, generate possible later consequences in mothers and offspring. For this, adult Wistar rats were allocated into two groups and were treated daily (intragastric) with malathion (14 or 140 mg/kg, body mass (bm)) for 21–25 days. Corn oil was used as vehicle in the Control group. Subgroups were defined based on the absence (nulliparous) or presence (pregnant) of a copulatory plug. Pregnant rats were followed by an additional period of 2 months after the term (post-term), without continuing malathion treatment. Fetuses and adult offspring of males and females were also evaluated. We ran an additional experimental design with rats exposed to malathion before pregnancy at a dose of 0.1 mg/kg bm. Malathion exposure resulted in glucose intolerance in the mothers during pregnancy and post-term period, regardless of the exposure dose. This was accompanied by increased visceral adipose tissue mass, dyslipidemia, unchanged pancreatic β-cell mass, and varying insulin responses to glucose in vivo. The number of total newborns and birthweight was not affected by malathion exposure. Adult offspring from both sexes also became glucose-intolerant, regardless of the pesticide dose their dams were exposed to. This alteration could be associated with changes at the epigenomic level, as reduced hepatic mRNA content of DNA methylases and demethylases was found. We demonstrated that periconceptional exposure to malathion with doses aiming to mimic from work environment to indirect contamination predisposes progenitors and offspring rats to glucose intolerance. Thus, we conclude that subchronic exposure to malathion is a risk factor for gestational diabetes and prediabetes later in life.

Abstract 11586: Therapeutic Potential of Alpha Lipoic Acid in a Rat Model of Superimposed Preeclampsia: Effect on Maternal Signs, Placental Development and Fetal Growth

Introduction: Superimposed preeclampsia (SPE) occurs in 25-40% of pregnant women affected by chronic hypertension and is associated with adverse maternal and neonatal outcomes. In previous studies, we have demonstrated that pregnant Stroke-prone Spontaneously Hypertensive Rat represent a spontaneous SPE model, characterized by defective trophoblast invasion and placental oxidative stress, asymmetric fetal growth restriction and worsening maternal hypertension with renal dysfunction towards term. Here, we sought to evaluate the therapeutic potential of alpha lipoic acid (ALA), a potent antioxidant, during early pregnancy in this model. Methods: Congenic timed breedings were established using SHRSP and Wistar Kyoto (WKY) females (10-12 weeks old, 200-250 g body weight, N = 10 animals/day analyzed). Upon vaginal plug detection (gestation day (GD)1), dams were assigned to two groups: ALA treatment, injected 25mg/kg body weight ALA i.p. on GD1, GD8 and GD12 and controls, receiving saline following the same protocol. Systolic blood pressure (SBP) profiles throughout pregnancy were recorded using a noninvasive volume pressure recording device. On GD20, animals were euthanized and fetoplacental specimens isolated for morphological and molecular analyzes. Statistical analysis was run on GraphPad Prism v 8.0, using 2 way ANOVA. Statistical significance was set at p˂ 0.05. Results: Treatment with ALA prevented the pregnancy-dependent SBP increase typical of the SHRSP model (SBP GD14 SHRSP: 169.3±2.1 mmHg vs. SHRSP+ALA: 146.1±3.4, p˂0.001). Furthermore, fetuses carried by ALA-treated SHRSP displayed increased weights (SHRSP: 1.75±0.05g vs. SHRSP+ALA: 1.99±0.01g, p˂0.05) and decreased cephalization indexes on GD20, indicating an improvement in the asymmetric growth restriction pattern. Treated dams also presented increased numbers of glycogen trophoblasts (PAS + cells) in the junctional zone and an enhanced vessel density on Isolectin B4 + staining, suggesting an improved placental function. Conclusions: ALA administration during early pregnancy in the SHRSP model ameliorated maternal signs and improved placental function and fetal growth, emerging as a novel therapeutic intervention in pregnancies affected by chronic hypertension

Influence of mating strategies on seminal material investment in crabs

Reproduction involves high energetic costs which are related to behaviour and gamete production. In females energy allocation to gamete production has been well documented. However, estimations of male investment in seminal material are scarce. The present study aims to assess and compare male investment in four brachyuran species by determining biochemical substrates present in the vasa deferentia to subsequently estimate energetic investment during the reproductive cycle. We identified two groups with contrasting energy investments. Two species, Homalaspis plana and Romaleon setosum, showed high investment due to significant quantities of proteins and lipids. Both species are characterised by large and complex vasa deferentia, and the formation of a remarkably large sperm plug deposited to the female after copulation as a sperm competition avoidance strategy. In contrast, Metacarcinus edwardsii and Taliepus dentatus invested little energy in their smaller-sized and simpler vasa deferentia. Morpho-functional traits may play a key role in determining the investment, which may also be influenced by mechanisms (i.e. mating tactics) to prevent sperm competition and the intensity of polygyny. This study emphasises the high amount of energy males invest in seminal material and highlights the diversity of mating strategies in Brachyura, which are reflected even on the physiological level.

Transcription factor gene Pea3 regulates erectile function during copulation in mice.

Male mice with homozygous loss of function mutations of the transcription factor gene Pea3 (Pea3 null) are infertile due to their inability to inseminate females, however the specific deficits in male sexual behaviors that drive this phenotype are unknown. Here, the copulatory behavior of male mice (Pea3 null and control) with hormonally primed ovariectomized females was monitored via high-speed and high-resolution digital videography to assess for differences in female-directed social behaviors, gross sexual behaviors (mounting, thrusting), and erectile and ejaculatory function. Pea3 null male mice exhibit greatly reduced erectile function, with 44% of males displaying no visible erections during copulation, and 0% achieving sustained erections. As such, Pea3 null males are incapable of intromission and copulatory plug deposition, despite displaying largely normal female-directed social behaviors, mounting behaviors, and ejaculatory grasping behavior. Additionally, the organization and timing of thrusting behaviors is impaired in Pea3 null males. Our results show that the transcription factor gene Pea3 regulates the ability to achieve and maintain erections during copulation in mice.

Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein.

Improved genome editing via oviductal nucleic acids delivery (i-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen. i-GONAD toward early zygotes has been recognized as difficult, because they are tightly surrounded by a cumulus cell layer, which often hampers effective transfer of nucleic acids to zygotes. However, in vivo EP three min after intraoviductal instillation of the genome-editing reagents enabled genome editing of early zygotes with an efficiency of 70%, which was in contrast with the rate of 18% when in vivo EP was performed immediately after intraoviductal instillation at Day 0.5 of pregnancy (corresponding to 13:00–13:30 p.m. on the day when vaginal plug was recognized after natural mating). We also found that addition of hyaluronidase, an enzyme capable of removing cumulus cells from a zygote, slightly enhanced the efficiency of genome editing in early zygotes. These findings suggest that cumulus cells surrounding a zygote can be a barrier for efficient generation of genome-edited mouse embryos and indicate that a three-minute interval before in vivo EP is effective for achieving i-GONAD-mediated genome editing at the early zygote stage. These results are particularly beneficial for researchers who want to perform genome editing experiments targeting early zygotes.

Effect of Xylene Oral Exposure on the Mouse Uterus – A Preliminary Study

Abstract Xylene is one of the environmental pollutants with a negative impact mainly on several organ systems. The aim of this study was to determine the effect of xylene on the uterus of mice. The study was performed on 12 adult female mice. Control mice (n = 6) were fed shredded pellets at a dose of 4 g per day. Xylene mice (n = 6) were fed the same diet at the same dose and orally administered xylene at 10 μl per day for 14 days. The mice were synchronized using the Whit-ten effect and introduced to males before the end of the procedure. Mice of both groups with no copulation plug were euthanized by cervical dislocation. The uteri were collected for routine histological and immunohistochemical analysis. The endometrial epithelium demonstrated vacuolar degeneration, mitotic cell activity, and the presence of leukocytes typical of metoestrus. Reductions of the endometrium, stroma, and myometrium were observed in the xylene mice. The xylene application did not have a significant effect on the superficial epithelium, or the size and number of uterine tubular glands. The immunohistochemical analysis of a proliferation marker PCNA revealed that the xylene increased its expression in the stroma, endometrial and myometrial cells, but did not significantly affect the superficial epithelial cells. The expression of an anti-apoptotic marker Bcl-xl in the xylene mice was stronger in the superficial epithelial, stromal, and endometrial cells. The Bcl-xl expression in the myometrial cells was similar to the controls. The results showed that the application of xylene stimulated the proliferation and exerted an anti-apoptotic effect on the uterine cells. However, the increased proliferation can lead to the malignant transformation of cells, resulting in their uncontrollable division.

Acupuncture Improves Endometrial Angiogenesis by Activating PI3K/AKT Pathway in a Rat Model with PCOS.

Background Acupuncture, a treatment derived from traditional Chinese medicine, can effectively relieve the symptoms and improve pregnancy outcome in patients with polycystic ovary syndrome (PCOS); however, its mechanism remains unclear. This study aimed at investigating whether acupuncture could improve endometrial angiogenesis and thus endometrial receptivity via activating PI3K/AKT pathway in PCOS rats. Methods We established a rat model with PCOS, which was induced by DHEA. Acupuncture was performed every other day for 15 days, and the PI3K inhibitor (LY294002) was intraperitoneal injected 30 mins before acupuncture treatment. Females rats were mated with male SPF SD rats in a ratio of 2 : 1 after treatment and sacrificed on the 5th day when the vaginal plug was identified. The number of implantation sites was observed, followed by ovarian and endometrial morphology detected with hematoxylin-eosin staining and a scanning electron microscope, estrous cycle detected with vaginal smear analysis, and sex hormones and angiogenesis-related PI3K/AKT gene/protein expression detected with enzyme-linked immunosorbent assay, western blotting, immune histochemistry, and quantitative real-time PCR. Results Acupuncture notably improved implantation sites' number, endometrial receptivity factors including endometrial morphology, pinopodes, HOX-10, and LIF protein expression, as well as angiogenesis and PI3K/AKT pathway factors such as VEGF, VEGFR2, Ang-1, PI3K, AKT, and P-AKT gene/protein expression and the level of eNOS and NO in the endometrium of rats with PCOS; PCOS-like symptoms were alleviated as well. The efficacy of acupuncture on a rat model with PCOS was counteracted by the combination with the PI3K inhibitor. Conclusion Acupuncture improves endometrial angiogenesis by activating the PI3K/AKT pathway, thus promoting endometrial receptivity and the number of implantation sites in rats with PCOS.

P-654 Arsenic Exposure During Puberty Significantly Affected Sex Maturation and Reproductive System Development of Female SD Rats

Abstract Study question Can sex maturation and reproductive hormones affected by Arsenic exposure during puberty in female rats. Summary answer Pubertal AS exposure at environmental-relevant levels significantly enhanced sexual precocity but reduced reproductive functions and capacity by adult. What is known already Arsenic (AS) is widely distributed in the earth’s surface and its toxic effects have been known for a long time. About 130 million people, mainly in the developing countries, are overly exposed to the chemical of AS through the drinking water. AS contaminations in drinking water (10 - 50 ug/L) were associated with human reproductive defects, such as spontaneous abortion, stillbirth, lower birth weight, and smaller birth size. As exposures in adult rats were have been related to inhibition of steroidogenic enzymes and decreased estradiol levels. Study design, size, duration Forty female rats of 21 days of age were exposed to AS at different doses (0, 0.02, 0.2, or 2 mg/L, n = 10/group) through drinking water for about 44 days until the rats reached adulthood. In a second experiment, 24 rats were exposed to AS with a similar protocol and doses, in order to examine the effects of AS on the pregnancy and reproductive capacity. Participants/materials, setting, methods Sex maturation day and estrus cycles were recorded. When rats reached adult, serum pituitary and ovarian sex hormones were assayed. Ovarian steroidogenic pathway and its regulatory signaling molecules were analyzed by qPCR and/or Western blots. The parameters compared include Mating Index, Days with Copulatory Vaginal Sperm Plug, Pregnancy Rate, Fertility Index, Weight of Pups, Litter Size and Ratio of Male/Female Pups. Main results and the role of chance AS exposure significantly reduced the weights of both ovary and uterus without affecting the overall body weight. AS promoted sexual precocity, disturbed estrus cycles, significantly reduced the numbers of primordial follicles while increased atretic follicles. AS also reduced serum levels of estradiol, progesterone and testosterone but increased the LH and FSH levels in dose-dependent manners. In addition, AS selectively down-regulated steroidogenic-related proteins FSHR, STAR, CYP17A1, HSD3B1 and CYP19A1 and signaling molecules PKA-ERK/JNK-cJUN, without affecting AKT and CREB. As about reproductive capacity, AS significantly reduced pup weights, litter size and the number of male pups. Limitations, reasons for caution Future works are need to examined how AS may affect the hypothalamic-pituitary functions during the prepubertal period. Further studies are required to address how AS may affect the environment of female reproductive tract and the egg qualities. Wider implications of the findings AS exposure at environmental-relevant level during puberty significantly affected female reproductive system development. Since the concentrations tested overlaps the environmental levels recorded, the results may have important public health significance. Trial registration number NA

Overweight and Fertility: What We Can Learn from an Intergenerational Mouse Obesity Model.

The aim of this study was to evaluate the effects of being overweight on the ability to conceive, fertilization rate, and in vivo development of embryos in regularly cycling, spontaneously ovulating, and naturally mated female mice. The study was based on statistical analysis of data collected during 14 experiments with identical design, performed on 319 control and 327 obese mice, developed in an intergenerational model of obesity induction which eliminates the impact of aging and high-fat feeding. Six-week-old mice with a vaginal sperm plug were slaughtered on embryonic days 2, 3, or 4, and the flushed contents of the oviducts and uteri were assessed by stereomicroscopy. The results showed no association between being overweight and the proportion of ovulating or fertilized females. On the other hand, a strong association was found between being overweight and ovulation yield. On embryonic day 2, significantly higher numbers of eggs were recovered from the oviducts of fertilized obese mice. Maternal overweight status was also associated with higher developmental capacities of preimplantation embryos. In conclusion, contrary to studies based on the high-fat-diet model, in female mice fed regular chow, being overweight was associated with an increased ovulation quota and higher developmental rate of fertilized oocytes. Being overweight did not impact ability to conceive. On the other hand, as documented in our previous studies, the quality of oocytes and blastocysts recovered from overweight mice developed in an intergenerational model of obesity was low.

Evaluation of Embryonal Protective Effect of Melatonin against Apoptotic Effect of Methadone in Embryo Tissues of Mice

Introduction: Methadone is a substance that is widely used in the substitution treatment of opiate addiction. This study aimed to evaluate the protective effects of melatonin on apoptosis induced by transfer of transplacental methadone in mice. Methods: This study was an experimental study aimed to assay apoptotic effect of placenta transferred methadone on brain, liver and kidney tissues and to study protective effect of melatonin in an embryo model in mice. Bulb-C mice weighing 25-30 g were kept in four shelves (3 females and one male) and checked daily for the sterile cycle. For mice with vaginal plugs, the first day of pregnancy was considered. After confirmation of pregnancy, female mice were divided into 5 groups of 6, which included the control group, methadone (10 mg / kg) and melatonin groups in three doses (2, 4, 6 mg / kg / day) by IP injection for half an hour before prescribing methadone. This prescription was given for 10 days from the beginning of pregnancy. After the infants were born, their liver, brain, and kidneys were removed and they underwent immunohistochemical tests for apoptotic expression of Bcl2, BAX, and Caspase3 proteins. The results were analyzed by SPSS version 22 statistical software and Chi-square (Chi 2) and Fisher tests (P <0.01). Results: This study showed that melatonin at dose 4 and 6mg/kg has decreased apoptotic protein BAX and Caspase9 and increased anti-apoptotic protein, Bcl2 expression approximately, but these results were not significant statistically (P>0.05). In addition, for dose of 0.2 mg/kg of melatonin, there was not any apoptotic effect. Conclusion: Our findings showed that melatonin has almost a protective effect against apoptotic effect induced by placental transfer of methadone especially via its increasing effects on Bcl2, but this anti-apoptotic effect was not significant and needed for more precise studies.

Silencing Transglutaminase Genes TGase2 and TGase3 Has Infection-Dependent Effects on the Heart Rate of the Mosquito Anopheles gambiae.

Simple SummaryThe immune and circulatory systems of insects are functionally integrated. An infection induces the migration of immune cells called hemocytes to the surface of the heart, where they kill pathogens around valves called ostia. In mosquitoes, a transglutaminase inhibits the infection-induced aggregation of hemocytes on the heart. Here, we studied whether transglutaminases also modify the heart contraction rate. First, we confirmed that an infection decreases the mosquito heart rate. Then, we found that disrupting transglutaminase genes has infection-dependent effects on the heart rate. Silencing TGase1 does not affect heart physiology. However, silencing TGase2 eliminates the infection-induced decrease in the heart rate, and silencing TGase3 decreases the heart rate in uninfected mosquitoes but increases the heart rate in infected mosquitoes. These experiments identify new factors that affect heart physiology in mosquitoes.Transglutaminases are pleiotropic enzymes that in mosquitoes participate in the formation of the mating plug and the wound-induced antimalarial response. Moreover, one transglutaminase, TGase3, negatively regulates the infection-induced aggregation of hemocytes on the heart. Given that TGase3 is an inhibitor of periostial hemocyte aggregation, we used RNAi-based gene silencing followed by intravital video imaging to scrutinize whether any of the three transglutaminases encoded in the genome of the mosquito, Anopheles gambiae, play a role in modulating the heart rate of uninfected and infected mosquitoes. Initially, we confirmed that an infection decreases the heart rate. Then, we uncovered that silencing TGase1 does not impact heart physiology, but silencing TGase2 results in a constant heart rate regardless of infection status, eliminating the infection-induced decrease in the heart rate. Finally, silencing TGase3 decreases the heart rate in uninfected mosquitoes but increases the heart rate in infected mosquitoes. We conclude that TGase2 and TGase3 modulate heart physiology and demonstrate that factors not classically associated with insect circulatory physiology are involved in the functional integration of the immune and circulatory systems of mosquitoes.

Pathogenesis of Brucella ovis in pregnant mice and protection induced by the candidate vaccine strain B. Ovis ΔabcBA

Ovine brucellosis caused by Brucella ovis is a major cause of reproductive failure in sheep. This study aimed to evaluate transplacental infection and pathogenicity of B.ovis wild type strain ATCC 25,840 (WT B.ovis) and the candidate vaccine strain B.ovis ΔabcBA in pregnant mice. A total of 40 BALB/c mice were equally divided into 4 groups: (i) non immunized and uninfected control mice (3/10 mice became pregnant); (ii) non immunized and challenged with WT B.ovis (5/10 pregnant); (iii) inoculated only with B.ovis ΔabcBA (6/10 pregnant); (iv) immunized with B.ovis ΔabcBA and challenged with WT B.ovis (5/10 pregnant). Female mice bred, and five days after visualization of the vaginal plug, they were inoculated intraperitoneally (ip) with 100 µL of sterile PBS, 100 µL of 1 × 106 CFU of B.ovis ΔabcBA, or 100 µL of 1 × 106 CFU of B.ovis WT, according to each group. At the 17th day of gestation, samples of spleen, liver, uterus, placenta, fetus and mammary gland were obtained for bacteriology, histopathology and immunohistochemistry. Non immunized mice challenged with B.ovis WT developed necrotizing placentitis as well as microgranulomas in the liver and spleen. These findings support the notion that B.ovis infection in pregnant mice induces lesions that are similar to those caused by B.abortus in the same animal model. B.ovis ΔabcBA was not recovered from any of the sampled organs, and it did not cause any gross or microscopic lesions, indicating that it is a safe and attenuated strain in this experimental model. In addition, B.ovis ΔabcBA was induced protective immunity as demonstrated by decreased numbers of B.ovis WT in the liver, uterus and fetuses of immunized mice after the challenge with B.ovis WT.

Comparison of Exosome Presence and Morphologic Changes Between Implantation and Inter-Implantation Areas in Rat's Utero by Confocal Microscope and SEM

Aim: In this study, it was aimed to compare the differences between the implantation and interimplantation sites in terms of the presence and release density of exosomes.
 Method: Wistar albino strains were used in this study. The rats were considered pregnant with the presence of vaginal plugs after mating. Then the rats were sacrificed on the 6th day when the embryos first attach to the uterus and implantation started. Implantation and inter-implantation sites were easily identified by staining the implantation sites with intravenous Chicago Blue dye given just before sacrification. After tissue preparation, sections were placed on slides. Exosomes detected with anti-CD63 fluoresence staining and imaged by confocal microscope. Further these sites were evaluated by Scaning electron microscopy (SEM).
 Result: When implantation and inter implantation sites compared, it was observed that amont of exosomes was higher than inter-implantation sites in confocal images. Additionally, SEM images confirmed the confocal results of these sites.
 Conclusion: Our study is the first in the literature to compare implantation and inter-implantation areas in terms of the presence of exosomes. These results probably suggested that the exosome plays an important role in implantation for the embryo to find the correct implantation site. Probably these exosomes must carry the necessary signals to find the right implantation site. However, further studies are needed to reveal the function of exosomes in implantation sites.

Maternal exposure to PM2.5 disrupting offspring spermatogenesis through induced sertoli cells apoptosis via inhibin B hypermethylation in mice

Particulate Matter 2.5 (PM2.5) disrupts endocrine functions and may negatively affect sperm quality and quantity in males; however, the long-term effects and potential mechanisms of this effect are unknown. This study aimed to investigate the epigenetic mechanism of maternal exposure to PM2.5-induced inhibin B hypermethylation in male offspring. In this experiment design, pregnant C57BL/6 mice were treated with two doses of PM2.5 (4.8 and 43.2 mg/kg bw). The membrane control group was given a sampling membrane and the control group received nothing. Following the formation of the vaginal plug, intratracheal instillation of PM2.5 was administered every three days until delivery of the pups. To assess the effect of PM2.5 in vitro, TM4 cells, a Sertoli-like cell line, was treated with different concentrations (0, 25, 50, 100 μg/mL) of PM2.5 for 24 h. The results displayed that Sperm motility, as well as the number of adult offspring, was decreased in the PM2.5 exposed group relative to the untreated controls. Increased vacuolization was observed in the Sertoli cells of mice that were exposed to PM2.5 in utero. The levels of inhibin and testosterone were reduced and the levels of LH and FSH increased in the PM2.5 groups relative to the untreated controls. In vitro, PM2.5 resulted in cell cycle inhibition as well as increased apoptosis in TM4 cells. Moreover, PM2.5-induced inhibin B hypermethylation and activation of the p21/Cleaved Caspase-3 pathway resulted in TM4 cell apoptosis that was rescued through the use of a DNA methylation inhibitor. Together, our data suggest that prenatal exposure to PM2.5 results in inhibin B hypermethylation and can activate the p21/Cleaved Caspase-3 pathway, resulting in Sertoli cell apoptosis, aberrant secretion of androgen binding protein, and decreased testosterone, thus resulting in the inhibition of spermatogenesis.

Ventro-Ventral Copulation by an Adult Male-Female Pair of Japanese Macaques at Arashiyama

Ventro-ventral (VV) mountings during copulation are most dominant in humans. In contrast, nonhuman primates have a strong bias toward dorso-ventral mountings, and only apes have been known to show VV mountings during copulation between a pair of mature individuals. Reporting VV mountings during copulation in primates other than apes is critical to discussions regarding why VV mountings have been limited to apes among primates. Here, I report VV mounting in a mature male and female pair of provisioned Japanese macaques (Macaca fuscata). This is the first report of VV mountings during copulation by adult nonhuman primates without disabilities other than apes. The pair of monkeys performed VV mountings 13 times over a week. Following the mounting series, including VV mountings, the presence of a copulatory plug was confirmed around the female’s vagina, indicating that the VV mountings were part of their copulatory behaviors. The male initiated all VV mountings, and the female was often uncooperative during these attempts. In all cases, the female was lying on her back, and the male was on top. The pair were in close contact and did not see each other’s faces during VV mountings. This report suggests that eye contact between mates and morphological characteristics are the primary reasons for the evolution of VV mountings during copulation in apes.

Comparison of Prostate‐Specific Transglutaminase Function in the Great Apes

The copulatory plug is a hardened structure formed through semen coagulation post‐ejaculation which is theorized to protect against remating, stimulate the female reproductive tract, and aid in sperm release and transport. This structure is prevalent in species that experience high levels of sperm competition including chimpanzees and several other primates. Chimpanzees experience high levels of sperm competition due to their polygynandrous mating system. In polygynandry females will mate with multiple partners during an estrus cycle leading to direct competition between the sperm of each male to fertilize the same egg. This varies distinctly from polygynous and monogamous species, such as humans, where females mate with only one male and sperm competition is diminished. Based on previous work in rats, prostate‐specific transglutaminase 4 (TGM4) is implicated as the driving force of the formation of the copulatory plug. TGM4 is a member of the transglutaminase family of enzymes which catalyze the calcium‐dependent formation of an isopeptide bond between glutamine and lysine residues. While humans do not experience a great deal of sperm competition or form a copulatory plug, human semen goes through a similar process of post‐ejaculatory coagulation which is predicted to be carried out by TGM4 under the same mechanism. Interestingly, there are only seven amino acid differences between the human and chimpanzee TGM4s and yet the degree of coagulation between the two species is vastly different. The aim of this study to characterize and compare the function of these proteins to determine the major contributing factor in semen coagulation. The primary hypothesis was that chimpanzee TGM4 would have higher enzymatic activity and more readily cross‐link artificial substrates than human TGM4. To test this hypothesis, the coding sequences were acquired for human and chimpanzee TGM4 as well as eight other primate species. Using a maximum likelihood‐based method, we generated a gene tree of primate TGM4 and calculated dN/dS ratios for each branch. Human TGM4 had a dN/dS ratio of 1.04 suggesting neutral evolution while chimpanzee TGM4 had a ratio of >1 suggesting strong positive selection. We further predicted the sequence of TGM4 of the last human‐chimp ancestor using ancestral sequence reconstruction which utilizes the known phylogenetic tree and a maximum likelihood approach with the software package Phylogenetic Analysis by Maximum Likelihood (PAML). Coding sequences representing TGM4 from humans, chimpanzees, and the last human‐chimp ancestor were cloned into a mammalian expression vector containing a C‐terminal His6tag and were transfected into HEK‐293T cells. Purified TGM4 was used in an in‐vitro colorimetric enzymatic assay to quantify enzymatic activity. Our results suggest that mating system variation has led to differences in both sequence and function of seminal proteins. These functional differences speak to the impact of even a small number of mutations on protein function as well as the influence of environmental pressures on protein evolution.

Maternal Hypertension and Offspring Cardiometabolic Disease Risk is Attenuated after Weight Loss in Obese Preeclamptic‐like BPH/5 Female Mice

Preeclampsia (PE) is a life‐threatening disorder of pregnancy that presents as late gestational hypertension and at least one other sign/symptom, such as proteinuria. It occurs in ~10% of pregnant women worldwide and the cause is unknown. Pre‐existing maternal hypertension and obesity has been associated with adverse outcomes, including PE and fetal growth restriction (FGR). To better understand the relationship of maternal PE risk (hypertension/obesity) and outcomes, we utilized the hyperphagic obese hypertensive BPH/5 mouse model of superimposed PE. We hypothesized that monitored food intake, via pair‐feeding the amount of normal chow consumed by age‐ and gestation‐matched lean normotensive C57 to BPH/5 mice, would attenuate maternal and offspring PE adverse outcomes.We have shown that adult BPH/5 female mice have an adverse cardiometabolic phenotype, hypertension, obesity with increased white adipose tissue (WAT), and dyslipidemia that is exaggerated by pregnancy. To test our hypothesis, BPH/5 and C57 females were instrumented with carotid catheters for radiotelemetric blood pressure monitoring prior to pregnancy. As previously described, pair‐feeding (PF) non‐pregnant BPH/5 female mice decreases body weight and WAT mass. When PF was performed for 10 days, we found a decrease in mean arterial pressure in BPH/5 PF versus ad libitum (AL) fed females (PF:121±2.8 vs AL:133±6 mmHg; n=3; p<0.05). When PF was performed in BPH/5 beginning at copulatory plug detection, embryonic day (e) 0.5, they failed to show a late‐gestational blood pressure rise from baseline (n=3; p>0.05) and attenuated proteinuria (PF: 84±15 vs AL: 177±16.5mg/dL; n=5; p<0.05). The PF paradigm increased median BPH/5 litter size (PF: 7, max‐min: 3‐8 vs AL:3, max‐min: 1‐6; n=26; p<0.05) and improved symmetrical FGR at e18.5 as assessed by fetal liver: body weight (PF: 13.63±0.7 vs AL:10.51±1.3; n=10; p<0.05).As BPH/5 female offspring age, they exhibit excessive catch‐up growth with FGR at birth and obesity with hyperleptinemia by adulthood (8 weeks) unlike male littermates. Adult female BPH/5 have cardiomegaly, hypertension, and bradycardia (n=7; p<0.05). Furthermore, adult female BPH/5 have hepatomegaly with microvesicular steatosis (p<0.05, n=3‐9). Importantly, adult BPH/5 female offspring born to PF BPH/5 mothers do not display these cardiometabolic risk factors: obesity (PF: 18±1.8 vs AL: 21±0.3g; n=16; p<0.05), hyperleptinemia (PF: 5.5±0.8 vs AL: 12.6±3.1ng/mL; n=6; p<0.05), hepatomegaly (PF: 1178±27 vs AL: 1475±69mg; n=16; p<0.05), and cardiomegaly (PF: 135.1±5.1 vs AL: 166.8±12.8mg; n=6; p<0.05) with age. Further investigations are necessary to understand the mechanism whereby maternal hyperphagia prevention and weight loss in BPH/5 improves offspring cardiometabolic disease as they age in a sex‐dependent manner. Finally, we seek to determine if interventions during pregnancy not only improve maternal cardiometabolic health, but also that of subsequent generations through augmented fetal programming during PE.