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Made available in DSpace on 2022-04-28T17:22:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-20 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) We evaluated the influence of the hyperglycemic intrauterine environment and postweaning consumption of a high-fat diet (HFD) on the glycemia, insulin, lipid, and immunological profile of rat offspring in adulthood. Female rats received citrate buffer (Control-C) or Streptozotocin (a beta cell-cytotoxic drug to induce diabetes-D) on postnatal day 5. In adulthood, these rats were mated to obtain female offspring, who were fed a standard diet (SD) or HFD from weaning to adulthood (n = 10 rats/group). OC/SD and OC/HFD represent female offspring of control mothers and received SD or HFD, respectively; OD/SD and OD/HFD represent female offspring of diabetic mothers and received SD or HFD, respectively. At adulthood, the oral glucose tolerance test (OGTT) was performed and, next, the rats were anesthetized and euthanized. Pancreas was collected and analyzed, and adipose tissue was weighted. Blood samples were collected to determine biochemical and immunological profiles. The food intake was lower in HFD-fed rats and visceral fat weight was increased in the OD/HFD group. OC/HFD, OD/SD, and OD/HFD groups presented glucose intolerance and lower insulin secretion during OGTT. An impaired pancreatic beta-cell function was shown in the adult offspring of diabetic rats, regardless of diet. Interleukin (IL)-6 and IL-10 concentrations were lower in the OD/HFD group and associated to a low-grade inflammatory condition. The fetal programming was responsible for impaired beta cell function in experimental animals. The association of maternal diabetes and postweaning HFD are responsible for greater glucose intolerance, impaired insulin secretion and immunological change. Summary sentence Summary Sentence The association of maternal diabetes and postweaning HFD causes glucose intolerance due to beta-cell disarrangement and impaired insulin secretion, and immunological changes in adult offspring. SJo Paulo State Univ UNESP, Botucatu Med Sch, Lab Expt Res Gynecol & Obstet, Tocogynecol Postgrad Course, Botucatu, SP, Brazil Fed Univ Mato Grosso UFMT, Inst Biol & Hlth Sci, Lab Syst Physiol & Reprod Toxicol, Barra Do Garcas, MT, Brazil Fed Univ Maranhao UFMA, Dept Physiol Sci, Lab Expt Physiol, Sao Luis, MA, Brazil Univ Estadual Paulista UNESP, Botucatu Med Sch, Res Support Off, Botucatu, SP, Brazil SJo Paulo State Univ UNESP, Botucatu Med Sch, Lab Expt Res Gynecol & Obstet, Tocogynecol Postgrad Course, Botucatu, SP, Brazil Univ Estadual Paulista UNESP, Botucatu Med Sch, Res Support Off, Botucatu, SP, Brazil FAPESP: 2016/25207-5

Sperm RNA can be modified by environmental factors and has been implicated in communicating signals about changes in a father's environment to the offspring. The small RNA composition of sperm could be changed during its final stage of maturation in the epididymis by extracellular vesicles (EVs) released by epididymal cells. We studied the effect of exposure to stress in early postnatal life on the transcriptome of epididymal EVs using a mouse model of transgenerational transmission. We found that the small RNA signature of epididymal EVs, particularly miRNAs, is altered in adult males exposed to postnatal stress. In some cases, these miRNA changes correlate with differences in the expression of their target genes in sperm and zygotes generated from that sperm. These results suggest that stressful experiences in early life can have persistent biological effects on the male reproductive tract that may in part be responsible for the transmission of the effects of exposure to the offspring.

This review summarizes key findings from the US National Cancer Institute (NCI) diethylstilbestrol (DES) Combined Cohort Study with a focus on the results of the NCI Third Generation Study, a cohort of DES-exposed and -unexposed granddaughters. Findings to date from the Third Generation Study are discussed in the context of other research efforts and case reports, suggesting an intergenerational heritability of DES-related effects. The DES story serves as a model for the influence of endocrine disrupting chemicals on human health. It also serves as a warning of the special hazards of pregnancy exposures, and more broadly, of the potential for invisible health consequences arising from new or changing exposures. Summary sentence Diethylstilbestrol is associated with adverse health outcomes in women exposed during pregnancy and in prenatally exposed offspring, particularly daughters; evidence suggests that exposure health effects may extend to the next generation.

Each year, millions of infants and children are anesthetized for medical and surgical procedures. Yet, a substantial body of preclinical evidence suggests that anesthetics are neurotoxins that cause rapid and widespread apoptotic cell death in the brains of infant rodents and nonhuman primates. These animals have persistent impairments in cognition and behavior many weeks or months after anesthesia exposure, leading us to hypothesize that anesthetics do more than simply kill brain cells. Indeed, anesthetics cause chronic neuropathology in neurons that survive the insult, which then interferes with major aspects of brain development, synaptic plasticity, and neuronal function. Understanding the phenomenon of anesthesia-induced developmental neurotoxicity is of critical public health importance because clinical studies now report that anesthesia in human infancy is associated with cognitive and behavioral deficits. In our search for mechanistic explanations for why a young and pliable brain cannot fully recover from a relatively brief period of anesthesia, we have accumulated evidence that neonatal anesthesia can dysregulate epigenetic tags that influence gene transcription such as histone acetylation and DNA methylation. In this review, we briefly summarize the phenomenon of anesthesia-induced developmental neurotoxicity. We then discuss chronic neuropathology caused by neonatal anesthesia, including disturbances in cognition, socio-affective behavior, neuronal morphology, and synaptic plasticity. Finally, we present evidence of anesthesia-induced genetic and epigenetic dysregulation within the developing brain that may be transmitted intergenerationally to anesthesia-naive offspring. Summary sentence Neonatal anesthesia can alter the epigenome, and these neuropathological changes can be inherited by anesthesia-naive offspring. Graphical Abstract

It is well established that environmental exposures can modify the profile of heritable factors in an individual's germ cells, ultimately affecting the inheritance of phenotypes in descendants. Similar to exposures, an ancestor's genotype can also affect the inheritance of phenotypes across generations, sometimes in offspring who do not inherit the genetic aberration. This can occur via a variety of prenatal, in utero, or postnatal mechanisms. In this review, we discuss the evidence for this process in mammals, with a focus on examples that are potentially mediated through the germline, while also considering alternate routes of inheritance. Noninherited ancestral genotypes may influence descendant's disease risk to a much greater extent than currently appreciated, and focused evaluation of this phenomenon may reveal novel mechanisms of inheritance. Summary sentence This is a review of the evidence that non-inherited ancestral alleles can influence the phenotypes of descendants via a variety of mechanisms including germline, in utero, and postnatal processes. Graphical Abstract

In many non-mammalian organisms, a population of germ-line stem cells supports continuing production of gametes during post-natal life, and germ-line stem cells are also present and functional in male mammals. Traditionally, however, they have been thought not to exist in female mammals, who instead generate all their germ cells during fetal life. Over the last several years, this dogma has been challenged by several reports, while being supported by others. We describe and compare these conflicting studies with the aim of understanding how they came to opposing conclusions. We first consider studies that, by examining marker-gene expression, the fate of genetically marked cells, and consequences of depleting the oocyte population, addressed whether ovaries of post-natal females contain oogonial stem cells that give rise to new oocytes. We next discuss whether ovaries contain cells that, even if inactive under physiological conditions, nonetheless possess oogonial stem cell properties that can be revealed through cell culture. We then examine studies of whether cells harvested after long-term culture of cells obtained from ovaries can, following transplantation into ovaries of recipient females, give rise to oocytes and offspring. Finally, we note studies where somatic cells have been re-programmed to acquire a female germ-cell fate. We conclude that the weight of evidence strongly supports the traditional interpretation that germ-line stem cells do not exist post-natally in female mammals. However, the ability to generate germ cells from somatic cells in vitro establishes a method to generate new gametes from cells of post-natal mammalian females.

Sex determination requires the commitment of bipotential gonads to either a testis or an ovarian fate. Gene deletion of the kinase Map3k4 results in gonadal sex reversal in XY mice, and transgenic re-expression of Map3k4 rescues the sex reversal phenotype. Map3k4 encodes a large, multi-functional protein possessing a kinase domain and several, additional protein–protein interaction domains. Although MAP3K4 plays a critical role in male gonadal sex determination, it is unknown if the kinase activity of MAP3K4 is required. Here, we use mice expressing full-length, kinase-inactive MAP3K4 from the endogenous Map3k4 locus to examine the requirement of MAP3K4 kinase activity in sex determination. Although homozygous kinase-inactivation of MAP3K4 (Map3k4KI/KI) is lethal, a small fraction survive to adulthood. We show Map3k4KI/KI adults exhibit a 4:1 female-biased sex ratio. Many adult Map3k4KI/KI phenotypic females have a Y chromosome. XY Map3k4KI/KI adults with sex reversal display female mating behavior, but do not give rise to offspring. Reproductive organs are overtly female, but there is a broad spectrum of ovarian phenotypes, including ovarian absence, primitive ovaries, reduced ovarian size, and ovaries having follicles in all stages of development. Further, XY Map3k4KI/KI adults are smaller than either male or female Map3k4WT/WT mice. Examination of the critical stage of gonadal sex determination at E11.5 shows that loss of MAP3K4 kinase activity results in the loss of Sry expression in XY Map3k4KI/KI embryos, indicating embryonic male gonadal sex reversal. Together, these findings demonstrate the essential role for kinase activity of MAP3K4 in male gonadal sex determination. Summary sentence Male gonadal sex determination requires the kinase activity of the multi-domain containing protein MAP3K4. Graphical Abstract

Exposures to adverse conditions in utero can lead to permanent changes in the structure and function of key physiological systems in the developing fetus, increasing the risk of disease and premature aging in later postnatal life. When considering the systems that could be affected by an adverse gestational environment, the reproductive system of developing female offspring may be particularly important, as changes have the potential to alter both reproductive capacity of the first generation, as well as health of the second generation through changes in the oocyte. The aim of this review is to examine the impact of different adverse intrauterine conditions on the reproductive system of the female offspring. It focuses on the effects of exposure to maternal undernutrition, overnutrition/obesity, hypoxia, smoking, steroid excess, endocrine-disrupting chemicals, and pollutants during gestation and draws on data from human and animal studies to illuminate underlying mechanisms. The available data indeed indicate that adverse gestational environments alter the reproductive physiology of female offspring with consequences for future reproductive capacity. These alterations are mediated via programmed changes in the hypothalamic–pituitary–gonadal axis and the structure and function of reproductive tissues, particularly the ovaries. Reproductive programming may be observed as a change in the timing of puberty onset and menopause/reproductive decline, altered menstrual/estrous cycles, polycystic ovaries, and elevated risk of reproductive tissue cancers. These reproductive outcomes can affect the fertility and fecundity of the female offspring; however, further work is needed to better define the possible impact of these programmed changes on subsequent generations.

Millions of women and their fetuses were exposed to the toxic pregnancy drug diethylstilbestrol (DES) from the 1940s into the 1970s, a time when the medical profession had little knowledge about potential developmental consequences of fetal drug exposures. Pathological consequences of DES exposure to the pregnant mothers and their offspring are well documented, but now generational research is finding that the grandchildren of women given DES in pregnancy are also at risk. This commentary summarizes presentations on this subject from the Beyond Genes panel “Heritable Impacts of Diethylstilbestrol (DES).”

Endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 17α-ethinylestradiol (EE2), can have far reaching health effects, including transgenerational abnormalities in offspring that never directly contacted either chemical. We previously reported reduced fertilization rates and embryo survival at F2 and F3 generations caused by 7-day embryonic exposure (F0) to 100 µg/L BPA or 0.05 µg/L EE2 in medaka. Crossbreeding of fish in F2 generation indicated subfertility in males. To further understand the mechanisms underlying BPA or EE2-induced adult onset and transgenerational reproductive defects in males, the present study examined the expression of genes regulating the brain–pituitary–testis (BPT) axis in the same F0 and F2 generation male medaka. Embryonic exposure to BPA or EE2 led to hyperactivation of brain and pituitary genes, which are actively involved in reproduction in adulthood of the F0 generation male fish, and some of these F0 effects continued to the F2 generation (transgenerational effects). Particularly, the F2 generation inherited the hyperactivated state of expression for kisspeptin (kiss1 and kiss2) and their receptors (kiss1r and kiss2r), and gnrh and gnrh receptors. At F2 generation, expression of DNA methyltransferase 1 (dnmt1) decreased in brain of the BPA treatment lineage, while EE2 treatment lineage showed increased dnmt3bb expression. Global hypomethylation pattern was observed in the testis of both F0 and F2 generation fish. Taken together, these results demonstrated that BPA or EE2-induced transgenerational reproductive impairment in the F2 generation was associated with alterations of reproductive gene expression in brain and testis and global DNA methylation in testis.Summary SentenceAncestral BPA or EE2 exposure during the first phase of primordial germ cell reprogramming in medaka fish leads to fertilization defects in male grandchildren and transcriptional alterations in male reproductive tissues.Graphical Abstract

Evidence suggests that maternal obesity (MO) can aggravate placental function causing severe pathologies during the perinatal window. However, molecular changes and mechanisms of placental dysfunction remain largely unknown. This work aimed to decipher structural and molecular alterations of the placental transfer zone associated with MO. To this end, mice were fed a high fat diet (HFD) to induce obesity before mating, and pregnant dams were sacrificed at E15.5 to receive placentas for molecular, histological, and ultrastructural analysis and to assess unidirectional materno-fetal transfer capacity. Laser-capture microdissection was used to collect specifically placental cells of the labyrinth zone for proteomics profiling. Using BeWo cells, fatty acid-mediated mechanisms of adherens junction stability, cell layer permeability, and lipid accumulation were deciphered. Proteomics profiling revealed downregulation of cell adhesion markers in the labyrinth zone of obese dams, and disturbed syncytial fusion and detachment of the basement membrane (BM) within this zone was observed, next to an increase in materno-fetal transfer in vivo across the placenta. We found that fetuses of obese dams develop a growth restriction and in those placentas, labyrinth zone volume-fraction was significantly reduced. Linoleic acid was shown to mediate beta-catenin level and increase cell layer permeability in vitro. Thus, MO causes fetal growth restriction, molecular and structural changes in the transfer zone leading to impaired trophoblast differentiation, BM disruption, and placental dysfunction despite increased materno-fetal transfer capacity. These adverse effects are probably mediated by fatty acids found in HFD demonstrating the need for obesity treatment to mitigate placental dysfunction and prevent offspring pathologies.

The enzyme heme oxygenase-1 (HO-1), encoded by the HMOX1 gene, mediates heme catabolism by cleaving free heme. We have previously revealed the importance of HO-1 in pregnancy. Here, we determined the impact of maternal or paternal HO-1 deficiency on fetal growth and placental parameters throughout gestation. We mated Hmox1-sufficient (WT), partial (HET)-, or total (KO)-deficient BALB/c female mice with Hmox1-WT or -KO BALB/c males and performed ultrasound analysis to monitor placental and fetal growth. Doppler measurements were used to determine maternal blood flow parameters. Offspring weights and feto-placental indices (FPI) were also determined. We found a significantly increased number of underdeveloped fetuses at gd10 in HET females that were mated with WT males compared with WT × WT pairings. At the same gestational age, underdeveloped placentas could be detected in HET females mated with KO males. Many fetuses from the KO × KO combination died in utero between gd12 and gd14. At gd14, abnormal placental parameters were found in surviving fetuses, which had significant reduced weights. Moreover, only 3.11% female and 5.33% male KO pups resulted from 10 HET × HET breeding pairs over 1 year. Our results show that HO-1 from both maternal and paternal origins is important for proper placental and fetal growth. Placental growth restriction and occurrence of abortions in mice that were partially or totally deficient in HO-1 were recorded in vivo from gd10 onwards. Future studies will focus on elucidating the cellular and molecular mechanisms behind these observations.

The number and quality of oocytes, as well as the decline in both of these parameters with age, determines reproductive potential in women. However, the underlying mechanisms of this diminution are incompletely understood. Previously, we identified novel roles for CHTF18 (Chromosome Transmission Fidelity Factor 18), a component of the conserved Replication Factor C-like complex, in male fertility and gametogenesis. Currently, we reveal crucial roles for CHTF18 in female meiosis and oocyte development. Chtf18–/– female mice are subfertile and have fewer offspring beginning at 6 months of age. Consistent with age-dependent subfertility, Chtf18–/– ovaries contain fewer follicles at all stages of folliculogenesis than wild type ovaries, but the decreases are more significant at 3 and 6 months of age. By 6 months of age, both primordial and growing ovarian follicle pools are markedly reduced to near depletion. Chromosomal synapsis in Chtf18–/– oocytes is complete, but meiotic recombination is impaired resulting in persistent DNA double-strand breaks, fewer crossovers, and early homolog disjunction during meiosis I. Consistent with poor oocyte quality, the majority of Chtf18–/– oocytes fail to progress to metaphase II following meiotic resumption and a significant percentage of those that do progress are aneuploid. Collectively, our findings indicate critical functions for CHTF18 in ensuring both the quantity and quality of the mammalian oocyte pool.Summary sentenceCHTF18 ensures both the quantity and quality of the mammalian oocyte pool and may play a role in delaying ovarian aging.

With approximately 131 million new genital tract infections occurring each year, Chlamydia is the most common sexually transmitted bacterial pathogen worldwide. Male and female infections occur at similar rates and both cause serious pathological sequelae. Despite this, the impact of chlamydial infection on male fertility has long been debated, and the effects of paternal chlamydial infection on offspring development are unknown. Using a male mouse chronic infection model, we show that chlamydial infection persists in the testes, adversely affecting the testicular environment. Infection increased leukocyte infiltration, disrupted the blood:testis barrier and reduced spermiogenic cell numbers and seminiferous tubule volume. Sperm from infected mice had decreased motility, increased abnormal morphology, decreased zona-binding capacity, and increased DNA damage. Serum anti-sperm antibodies were also increased. When both acutely and chronically infected male mice were bred with healthy female mice, 16.7% of pups displayed developmental abnormalities. Female offspring of chronically infected sires had smaller reproductive tracts than offspring of noninfected sires. The male pups of infected sires displayed delayed testicular development, with abnormalities in sperm vitality, motility, and sperm-oocyte binding evident at sexual maturity. These data suggest that chronic testicular Chlamydia infection can contribute to male infertility, which may have an intergenerational impact on sperm quality., Testicular C. muridarum infection results in tissue damage and poor sperm quality and gives rise to offspring with serious abnormalities and developmental delays.

Maternal high-fat (HF) diet negatively affects maternal metabolism and placental function. This study aimed to determine whether gestational exercise prevents the effect of HF diet on placental amino acid transporter expression and nutrient-sensing signaling and the fetal response. Pregnant Sprague-Dawley rats were either fed with a CHOW (13.5% fat) or HF (60% fat) diet during gestation and further divided into two subgroups: voluntary exercised and sedentary. Placentae were collected on gestational day (GD) 14 and GD20, and male placentae were used in this study. We found that gestational exercise ameliorated the detrimental effects of HF diet on dams’ adiposity, plasma leptin, and insulin concentrations. Maternal exercise did not influence fetoplacental growth but affected male fetal hypothalamic Leprb, Stat3, Insr, Agrp, and Pomc expressions on GD20. Maternal HF diet decreased placental labyrinth thickness and increased system A amino acid transporter SNAT2 expression, while these changes were normalized by exercise. The activation of placental mechanistic target of rapamycin complex 1/4E-BP1 and LepRb/STAT3 signaling might contribute to the increased placental SNAT2 expression in HF-fed dams, which were reversed by exercise on GD20. These data highlight that gestational exercise reverses HF-diet-induced placental alterations during late gestation without influencing fetal growth. However, maternal exercise altered fetal hypothalamic gene expression, which may affect long-term offspring health.

Anti-androgenic endocrine-disrupting chemicals (EDCs) can cross the placenta to modify early offspring sexual dimorphic markers. These changes are linked to anogenital distance (AGD), which is an androgen-sensitive anthropometric parameter used as a biomarker of perineal growth and caudal migration of the genital tubercle. This review aimed to summarize strength of evidence for associations of in utero exposure to EDCs with AGD and to identify gaps and limitations in the literature so as to inform future research. We performed an electronic search of English literature in September 2019 in medical literature analysis and retrieval system online (MEDLINE), Web of Science and Toxline. We included epidemiological studies that examined in utero exposure to persistent and nonpersistent EDCs and considered AGD in offspring as an outcome. Our review contained 16 investigations examining exposure to persistent EDCs (nine studies) and nonpersistent EDCs (seven studies). Some individual studies reported an inverse association between exposure to bisphenol A (BPA), dioxins, perfluoroalkyl substances, and organochlorides and AGD in both male and female offspring. Meta-analysis of three studies found a small reduction of AGD in female offspring exposed to BPA. The number of studies per chemical is small, and number of subjects examined is limited; so, replication of these results is needed. To achieve more specificity and better replication of results, future studies should establish the association of nonpersistent EDCs using multiple urine samples, evaluate the cumulative impact of exposure to a mixture of anti-androgenic chemicals, and offer adequate consideration of more maternal- and children-related confounding factors.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis in male due to their capability to multiply in numbers by self-renewal and subsequent meiotic processes. However, as SSCs are present in a very small proportion in the testis, in vitro proliferation of undifferentiated SSCs will facilitate the study of germ cell biology. In this study, we investigated the effectiveness of various cell lines as a feeder layer for rat SSCs. Germ cells enriched for SSCs were cultured on feeder layers including SIM mouse embryo-derived thioguanine and ouabain-resistant cells, C166 cells, and mouse and rat testicular endothelial cells (TECs) and their stem cell potential for generating donor-derived colonies and offspring was assessed by transplantation into recipient testes. Rat germ cells cultured on TECs showed increased mRNA and protein levels of undifferentiated spermatogonial markers. Rat SSCs derived from these germ cells underwent spermatogenesis and generated offspring when transplanted into recipients. Collectively, TECs can serve as an effective feeder layer that enhances the proliferative and self-renewal capacity of cultured rat SSCs while preserving their stemness properties.

Preeclampsia (PE) is a common pregnancy complication affecting 3–5% of women. Preeclampsia is diagnosed clinically as new-onset hypertension with associated end organ damage after 20 weeks of gestation. Despite being diagnosed as a maternal syndrome, fetal experience of PE is a developmental insult with lifelong cognitive consequences. These cognitive alterations are associated with distorted neuroanatomy and cerebrovasculature, including a higher risk of stroke. The pathophysiology of a PE pregnancy is complex, with many factors potentially able to affect fetal development. Deficient pro-angiogenic factor expression is one aspect that may impair fetal vascularization, alter brain structure, and affect future cognition. Of the pro-angiogenic growth factors, placental growth factor (PGF) is strongly linked to PE. Concentrations of PGF are inappropriately low in maternal blood both before and during a PE gestation. Fetal concentrations of PGF appear to mirror maternal circulating concentrations. Using Pgf−/− mice that may model effects of PE on offspring, we demonstrated altered central nervous system vascularization, neuroanatomy, and behavior. Overall, we propose that development of the fetal brain is impaired in PE, making the offspring of preeclamptic pregnancies a unique cohort with greater risk of altered cognition and cerebrovasculature. These individuals may benefit from early interventions, either pharmacological or environmental. The early neonatal period may be a promising window for intervention while the developing brain retains plasticity.

Appropriate remodeling of the female lower reproductive tract and pelvic floor is essential during normal mammalian pregnancy, labor, and postpartum recovery. During mouse pregnancy, in addition to reproductive tract modifications, the pubic symphysis (PS) is remodeled into a soft interpubic ligament (IpL) to provide safe delivery of the offspring and fast postpartum recovery. Although temporal changes in the phenotypes of myeloid cells, such as mononuclear phagocytes, are crucial to remodeling the lower reproductive tract organs in preparation for a safe delivery, little is known about the involvement of recruited monocytes or macrophages in mouse PS remodeling. We used combined light microscopy, electron microscopy, and qPCR analysis to investigate the profile of recruited monocytes and macrophage polarization markers in C57Bl6 mouse interpubic tissues during pregnancy (D12, D18, and D19) and early days postpartum (1 dpp and 3 dpp) to better identify their presence in proper remodeling of the mouse PS. Our morphological data show that the number of recruited monocytes is increased in interpubic tissues and that recruited monocytes differentiate into proinflammatory or anti-inflammatory macrophage phenotypes from D18 to 3 dpp, which may contribute to dynamic changes in the gene expression of specific inflammatory mediators involved in interpubic tissue remodeling at these time points. Therefore, our morphological and quantitative gene expression data suggest that both differentiated macrophages from recruited monocytes and polarized macrophages may collaborate for IpL relaxation at labor and the appropriate repair of the PS after the first pregnancy., Recruited monocytes and mature macrophages are present in the mouse pubic symphysis and may contribute to mouse pubic symphysis relaxation during late pregnancy and postpartum recovery.

An interspecific hybrid marine fish that developed a testis-like gonad without any germ cells, i.e., a germ cell-less gonad, was produced by hybridizing a female blue drum Nibea mitsukurii with a male white croaker Pennahia argentata. In this study, we evaluated the suitability of the germ cell-less fish as a recipient by transplanting donor testicular cells directly into the gonads through the urogenital papilla. The donor testicular cells were collected from hemizygous transgenic, green fluorescent protein (gfp) (+/−) blue drum, and transplanted into the germ cell-less gonads of the 6-month-old adult hybrid croakers. Fluorescent and histological observations showed the colonization, proliferation, and differentiation of transplanted spermatogonial cells in the gonads of hybrid croakers. The earliest production of spermatozoa in a hybrid recipient was observed at 7 weeks post-transplantation (pt), and 10% of the transplanted recipients produced donor-derived gfp-positive spermatozoa by 25 weeks pt. Sperm from the hybrid recipients were used to fertilize eggs from wild-type blue drums, and approximately 50% of the resulting offspring were gfp-positive, suggesting that all offspring originated from donor-derived sperm that were produced in the transplanted gfp (+/−) germ cells. To the best of our knowledge, this is the first report of successful spermatogonial transplantation using a germ cell-less adult fish as a recipient. This transplantation system has considerable advantages, such as the use of comparatively simple equipment and procedures, and rapid generation of donor-derived spermatogenesis and offspring, and presents numerous applications in commercial aquaculture.

Successful derivation and cultivation of primordial germ cells (PGCs) opened the way to efficient transgenesis and genome editing in the chicken. Furthermore, implantation of male PGCs from non-chicken galliform species into the chicken embryos resulted in cross-species germline chimeras and viable offspring. We have recently improved the PGC technology by demonstrating that chicken male PGCs transplanted into the testes of adult cockerel recipients mature into functional sperms. However, the availability of this orthotopic transplantation for cross-species transfer remains to be explored. Here we tested the capacity of genetically distant male PGCs to mature in the microenvironment of adult testes. We derived PGCs from the Chinese black-bone Silkie and transplanted them into infertile White Leghorn cockerels. Within 15–18 weeks after transplantation, we observed restoration of spermatogenesis in recipient cockerels and production of healthy progeny derived from the transplanted PGCs. Our findings also indicate the possibility of cross-species orthotopic transplantation of PGCs. Thus, our results might contribute to the preservation of endangered avian species and maintaining the genetic variability of the domestic chicken.

We previously developed a model of gestational diabetes mellitus (GDM) in which dams exhibit glucose intolerance, insulin resistance, and reduced insulin response to glucose challenge only during pregnancy, without accompanying obesity. Here, we aimed to determine how lean gestational glucose intolerance affects offspring risk of metabolic dysfunction. One cohort of offspring was sacrificed at 19 weeks, and one at 31 weeks, with half of the second cohort placed on a high-fat, high-sucrose diet (HFHS) at 23 weeks. Exposure to maternal glucose intolerance increased weights of HFHS-fed offspring. Chow-fed offspring of GDM dams exhibited higher body fat percentages at 4, 12, and 20 weeks of age. At 28 weeks, offspring of GDM dams fed the HFHS but not the chow diet (CD) also had higher body fat percentages than offspring of controls (CON). Exposure to GDM increased the respiratory quotient (Vol CO2/Vol O2) in offspring. Maternal GDM increased adipose mRNA levels of peroxisome proliferator-activated receptor gamma (Pparg) and adiponectin (Adipoq) in 31-week-old CD-fed male offspring, and increased mRNA levels of insulin receptor (Insr) and lipoprotein lipase (Lpl) in 31-week-old male offspring on both diets. In liver at 31 weeks, mRNA levels of peroxisome proliferator-activated receptor alpha (Ppara) were elevated in CD-fed male offspring of GDM dams, and male offspring of GDM dams exhibited higher mRNA levels of Insr on both diets. Neither fasting insulin nor glucose tolerance was affected by exposure to GDM. Our findings show that GDM comprising glucose intolerance only during pregnancy programs increased adiposity in offspring, and suggests increased insulin sensitivity of subcutaneous adipose tissue.

One in 54 children in the U.S. is diagnosed with Autism Spectrum Disorder (ASD). De novo germline and somatic mutations cannot account for all cases of ASD, suggesting that epigenetic alterations triggered by environmental exposures may be responsible for a subset of ASD cases. Human and animal studies have shown that exposure of the developing brain to general anesthetic (GA) agents can trigger neurodegeneration and neurobehavioral abnormalities but the effects of general anesthetics on the germ line have not been explored in detail. We exposed pregnant mice to sevoflurane during the time of embryonic development when the germ cells undergo epigenetic reprogramming and found that more than 38% of the directly exposed F1 animals exhibit impairments in anxiety and social interactions. Strikingly, 44-47% of the F2 and F3 animals, which were not directly exposed to sevoflurane, show the same behavioral problems. We performed ATAC-seq and identified more than 1,200 differentially accessible sites in the sperm of F1 animals, 69 of which are also present in the sperm of F2 animals. These sites are located in regulatory regions of genes strongly associated with ASD, including Arid1b, Ntrk2, and Stmn2. These findings suggest that epimutations caused by exposing germ cells to sevoflurane can lead to ASD in the offspring, and this effect can be transmitted through the male germline inter and trans-generationally.Summary sentencePregnant mouse F0 females exposed to sevoflurane give rise to F1 males with sociability and anxiety defects. These behaviors are transmitted to F2 and F3 males. Their sperm show changes in transcription factor occupancy in genes implicated in autism.

Throughout the scientific literature, heritable traits are routinely presumed to be genetic in origin. However, as emerging evidence from the realms of genetic toxicology and epigenomics demonstrate, heritability may be better understood as encompassing not only DNA sequence passed down through generations, but also disruptions to the parental germ cells causing de novo mutations or epigenetic alterations, with subsequent shifts in gene expression and functions in offspring. The Beyond Genes conference highlighted advances in understanding these aspects at molecular, experimental, and epidemiological levels. In this commentary I suggest that future research on this topic could be inspired by collecting parents' germ cell exposure histories, with particular attention to cases of families with multiple children suffering idiopathic disorders. In so doing I focus on the endpoint of autism spectrum disorders (ASDs). Rates of this serious neurodevelopment disability have climbed around the world, a growing crisis that cannot be explained by diagnostic shifts. ASD's strong heritability has prompted a research program largely focused on DNA sequencing to locate rare and common variants, but decades of this gene-focused research have revealed surprisingly little about the molecular origins of the disorder. Based on my experience as the mother of two children with idiopathic autism, and as a research philanthropist and autism advocate, I suggest ways researchers might probe parental germ cell exposure histories to develop new hypotheses that may ultimately reveal sources of nongenetic heritability in a subset of idiopathic heritable pathologies. Summary sentence As heritable idiopathic disorders like autism surge in prevalence, it behooves researchers to investigate parental germ cell exposure history for clues. Multiplex families offer an opportunity to spot hypotheses beyond the confines of traditional genetics.

The adverse effects on the child of maternal smoking in pregnancy is well-recognized, but little research has been carried out on the possible non-genetic effects of ancestral smoking prior to the pregnancy including parental initiation of cigarette smoking in their own childhoods or a grandmother smoking during pregnancy. Here, we summarize the studies that have been published mainly using data from the Avon Longitudinal Study of Parents and Children. We demonstrate evidence that ancestral smoking prior to or during pregnancy can often be beneficial for offspring health and both ancestor- and sex-specific. More specifically, we report evidence of (i) adverse effects of the father starting to smoke pre-puberty on his son’s development; (ii) beneficial effects on the grandson if his maternal grandmother had smoked in pregnancy; and (iii) mainly adverse effects on the granddaughter when the paternal grandmother had smoked in pregnancy. The ancestor- and sex-specificity of these results are consistent with earlier studies reporting associations of health and mortality with ancestral food supply in their parents’ and grandparents’ pre-pubertal childhoods., Review of published ALSPAC studies demonstrate ancestral smoking prior to pregnancy can be beneficial or detrimental for offspring health and development and often both ancestor- and sex-specific.

Overloaded iron can deposit in the reproductive system and impair ovarian function. But few studies have identified the exact effect of overloaded iron on the endocrine function and fertility capacity in female mice. Here, we established iron-overloaded mouse models by intraperitoneal injection of iron dextran to adult female C57BL/6J mice at 0.1 g/kg (LF group), 0.5 g/kg (MF group), and 1.0 g/kg (HF group) concentrations once a week for eight consecutive weeks. We found that overloaded iron resulted in smaller ovaries, as well as accumulated oxidative damages. The endocrine function and follicle development were also impeded in the MF and HF groups. The 10-month breeding trial indicated that (1) Low concentration of iron (0.1 g/kg) wasn’t detrimental to the ovary; (2) Middle concentration of iron (0.5 g/kg) impeded the childbearing process, though it could be recovered following the iron excretion; and (3) High concentration of iron (1.0 g/kg) damaged the fertility, even gave rise to sterility. Yet for those fertile mice, litter number and litter size were smaller and the ovarian reserve of their offspring was impaired. Transcriptome profiling results indicated that overloaded iron could compromise ovarian function by disrupting ovarian steroidogenesis, interfering with ovarian microenvironment, and inhibiting Wnt signaling. Taken together, we have demonstrated the effect that chronic concentration-dependent iron overload exerted on mouse ovarian function, which may act as a preliminary basis for further mechanism and intervention investigations.

Maternal obesity increases the risk of offspring to become obese and develop related pathologies. Exposure to maternal high-fat diet (HFD) only during lactation increases the risk of obesity-related diseases, suggesting that factors in milk affect long-term health. We hypothesized that prepregnancy obesity induced by HFD alters milk lipidome, and in turn, alterations may affect neonate serum lipidome. The objective of this study was to determine the effect of prepregnancy obesity induced by HFD on circulating lipids in dams and neonates and in milk. Female mice were fed an HFD (60% kcal fat) or control diet (CON, 10% kcal fat) beginning 4 weeks before breeding. On postnatal day 2 (PND2), pups were cross-fostered to create pup groups exposed to HFD during pregnancy, lactation, or both or exposed to CON. On PND12, dams were milked and then euthanized along with pups to collect blood. Serum and milk were processed for multiple reaction monitoring (MRM) lipidomics profiling to quantify the relative expression of lipid classes. Lipidome of HFD dam serum and milk had increased proportion of C18:2 free fatty acid and fatty acyl residues in all lipid classes. Lipidome of serum from pups exposed to maternal HFD during lactation was similarly affected. Thus, maternal HFD induced redistribution of fatty acyl residues in the dam's circulation, which was associated with modification in milk and suckling neonate's lipidome. Further studies are needed to determine if increased circulating levels of C18:2 in neonate affects development and predisposes offspring to obesity and metabolic syndrome.Graphical AbstractSummary SentenceMaternal prepregnancy obesity induced by high-fat diet significantly impacts circulating lipids and similarly modifies the lipid composition of milk and circulating lipid of suckling neonates.

Many bitterling species are facing extinction because of habitat destruction. Since cryopreservation of fish eggs is still not available to date due to their large size and high yolk content, long-term and stable storage of bitterling genetic resources is currently not possible. We recently discovered that cryopreservation of early-stage germ cells is possible in several fish species and that functional gametes derived from the frozen materials can be produced through their transplantation to embryonic recipients. However, bitterlings have uniquely shaped eggs and their embryos are extremely fragile, making it difficult to perform germ cell transplantation. Therefore, as a first step, we conducted intra-species spermatogonial transplantation using recessive albino Chinese rosy bitterling as donors and wild-type Chinese rosy bitterling as recipients to develop a system to convert freezable early-stage germ cells into functional gametes, particularly eggs. Approximately 3000 testicular cells were transplanted into the peritoneal cavity of 4-day-old germ cell-less recipient embryos produced by dead end (dnd)-knockdown. At 6 months, ten male recipients and nine female recipients produced gametes. Mating studies with the opposite sex of recessive albino control fish revealed that six males and three females produced only albino offspring, suggesting that these recipients' endogenous germ cells were completely removed by dnd-knockdown and they produced only donor-derived gametes. Thus, we successfully established a germ cell transplantation system in an iconic endangered teleost, bitterling. The technology established in this study can be directly applied to produce functional gametes of endangered bitterlings using cryopreserved donor cells.

We previously demonstrated that in the mouse only two Y chromosome genes are required for a male to produce an offspring with the help of assisted reproduction technologies (ART): testis determinant Sry and spermatogonial proliferation factor Eif2s3y. Subsequently, we have shown that the function of these genes can be replaced by transgenic overexpression of their homologs, autosomally encoded Sox9 and X-chromosome encoded Eif2s3x. Males with Y chromosome contribution limited to two (X(Eif2s3y)OSry), one (X(Eif2s3y)OSox9 and XOSry,Eif2s3x), and no genes (XOSox9,Eif2s3x) produced haploid germ cells and sired offspring after ART. However, despite successful assisted reproductive outcome, they had smaller testes and displayed abnormal development of the seminiferous epithelium and testicular interstitium. Here we explored whether these testicular defects originated from altered pro-testis and pro-ovary factor signaling in genital ridges at the time of sex determination. Timed pregnancies were generated to obtain transgenic X(Eif2s3y)OSry, X(Eif2s3y)OSox9, XOSry,Eif2s3x, XOSox9,Eif2s3x, and wild-type XX and XY fetuses at 12.5 days post coitum. Dissected genital ridges were assessed for their morphology and anatomy, and expression of pro-testis and pro-ovary transcripts. All transgenic males displayed incomplete masculinization of gonadal shape, impaired development of testicular cords and gonadal vasculature, and decreased expression of factors promoting male pathway. Fetal gonad masculinization was more effective when sex determination was driven by the Sry transgene, in the presence of Y chromosome genes, and to a lesser extent a double dosage of X genes. The study adds to the understanding of the role of Y chromosome genes and their homologs during sex determination.

A stable system for producing sterile domesticated fish is required to prevent genetic contamination to native populations caused by aquaculture escapees. The objective of this study was to develop a system to mass produce stock for aquaculture that is genetically sterile by surrogate broodstock via spermatogonial transplantation (SGTP). We previously discovered that female medaka carrying mutations on the follicle-stimulating hormone receptor (fshr) gene become sterile. In this study, we demonstrated that sterile hybrid recipient females that received spermatogonia isolated from sex-reversed XX males (fshr (-/-)) recovered their fertility and produced only donor-derived fshr (-) X eggs. Natural mating between these females and fshr (-/-) sex-reversed XX males successfully produced large numbers of sterile fshr (-/-) female offspring. In conclusion, we established a new strategy for efficient mass production of sterile fish. This system can be applied to any aquaculture species for which SGTP and methods for producing sterile recipients can be established.

Experimental evidence shows that parental psychological stress affects the long-term health of offspring in an inheritable fashion. Although epigenetic mechanisms, including DNA methylation, miRNA, and histone modifications, are involved in transgenerational programming, the underlining mechanisms of transgenerational inheritance remain unsolved. Here, we present a single-cell-based computational model for transgenerational inheritance for investigating the long-term dynamics of phenotype changes in response to parental stress. The model is based on a recent study that has identified the imprinted sperm gene Sfmbt2 as a key target, and incorporates crosstalks among drastically different time scales in mammalian development, including DNA methylation, transcription, cell division, and population dynamics. Computational analysis of the model suggests a positive feedback to DNA methylation in the promoter region of sperm Sfmbt2 gene that provides a possible mechanism to mediate the parental psychological stress reprogramming in offspring. This approach provides a modeling framework for the understanding of the roles that epigenetics play in transgenerational inheritance.

Inflammation may be a mechanism of maternal programming because it has the capacity to alter the maternal environment and can persist postnatally in offspring tissues. This study evaluated the effects of restricted- and over-feeding on maternal and offspring inflammatory gene expression using reverse transcription (RT)-PCR arrays. Pregnant ewes were fed 60% (Restricted), 100% (Control), or 140% (Over) of National Research Council requirements beginning on day 30.2 ± 0.2 of gestation. Maternal (n = 8-9 ewes per diet) circulating nonesterified fatty acid (NEFA) and expression of 84 inflammatory genes were evaluated at five stages during gestation. Offspring (n = 6 per diet per age) inflammatory gene expression was evaluated in the circulation and liver at day 135 of gestation and birth. Throughout gestation, circulating NEFA increased in Restricted mothers but not Over. Expression of different proinflammatory mediators increased in Over and Restricted mothers, but was diet-dependent. Maternal diet altered offspring systemic and hepatic expression of genes involved in chemotaxis at late gestation and cytokine production at birth, but the offspring response was distinct from the maternal. In the perinatal offspring, maternal nutrient restriction increased hepatic chemokine (CC motif) ligand 16 and tumor necrosis factor expression. Alternately, maternal overnutrition increased offspring systemic expression of factors induced by hypoxia, whereas expression of factors regulating hepatocyte proliferation and differentiation were altered in the liver. Maternal nutrient restriction and overnutrition may differentially predispose offspring to liver dysfunction through an altered hepatic inflammatory microenvironment that contributes to immune and metabolic disturbances postnatally.

Maternal obesity induces pregnancy complications and disturbs fetal development, but the specific mechanisms underlying these outcomes are unclear. Circadian rhythms are implicated in metabolic complications associated with obesity, and maternal metabolic adaptations to pregnancy. Accordingly, obesity-induced circadian dysfunction may drive adverse outcomes in obese pregnancy. This study investigated whether maternal obesity alters the rhythmic expression of clock genes and associated nuclear receptors across maternal, fetal, and placental tissues. Wistar rats were maintained on a cafeteria (CAF) diet prior to and throughout gestation to induce maternal obesity. Maternal and fetal liver and placental labyrinth zone (LZ) were collected at four-hourly time points across days 15-16 and 21-22 of gestation (term = 23 days). Gene expression was analyzed by RT-qPCR. Expression of the accessory clock gene Nr1d1 was rhythmic in the maternal and fetal liver and LZ of chow-fed controls, but in each case CAF feeding reduced peak Nr1d1 expression. Obesity resulted in a phase advance (approx. 1.5 h) in the rhythms of several clock genes and Ppar-delta in maternal liver. Aside from Nr1d1, expression of clock genes was mostly arrhythmic in LZ and fetal liver, and was unaffected by the CAF diet. In conclusion, maternal obesity suppressed Nr1d1 expression across maternal, fetal, and placental compartments and phase-advanced the rhythms of maternal hepatic clock genes. Given the key role of Nr1d1 in regulating metabolic, vascular, and inflammatory processes, our data suggest that disruptions to rhythmic Nr1d1 expression in utero may contribute to programmed health complications in offspring of obese pregnancies.

Intrauterine growth retardation (IUGR) is closely related to the later development of type 2 diabetes in adulthood. Excessive activation of N-methly-D-aspartate receptors (NMDARs) causes excitatory neurotoxicity, resulting in neuronal injury or death. Inhibition of NMDARs enhances the glucose-stimulated insulin secretion and survival of islet cells in type 2 diabetic mouse and human islets. Here, we examined whether antenatal blockade of NMDARs by Memantine could decrease the risk of diabetes induced by a high-fat (HF) diet at adulthood in IUGR rats. Pregnant SD rats were assigned to four groups: control, IUGR, Memantine, and Memantine + IUGR. The pregnant rats were exposed to hypoxic conditions (FiO2 = 0.105) for 8 h/day (IUGR group) or given a daily Memantine injection (5 mg/kg, i.p.) before hypoxia exposure from embryonic day (E) 14.5 to E 20.5 (Memantine + IUGR). The offspring were fed an HF diet with 60% of the calories from age 4 to 12 weeks. We found that NMDAR mRNAs were expressed in the fetal rat pancreas. An HF diet resulted in a high rate of diabetes at adulthood in the IUGR group. Antenatal Memantine treatment decreased the risk of diabetes at adulthood of rats with IUGR, which was associated with rescued glucose tolerance, increased insulin release, improved the insulin sensitivity, and increased expression of genes related to beta-cell function in the pancreas. Together, our results suggest that antenatal blockade of NMDARs by Memantine in pregnant rats improves fetal development and reduces the susceptibility to diabetes at adulthood in offspring.

Overnutrition during pregnancy could increase risks of cardiovascular diseases in late life. This study investigated whether and how reactive oxygen species (ROS) may influence functions of large-conductance Ca2+-activated K+ channels (BKCa) in the offspring exposed to prenatal high sucrose (HS). We found that prenatal HS diets significantly increased phenylephrine (PE)-induced vessel contractions in mesenteric arteries of the adult offspring. Pretreatment with iberiotoxin (BKCa blocker, IBTX) significantly increased PE-mediated vascular contractions in the control, not in the HS group. Electrophysiological studies demonstrated that BKCa current density and single-channel current were reduced in the vascular smooth muscle cells (VSMCs) of the HS offspring. The expression of BKCa alpha, beta1 subunits in mesenteric arteries was decreased in the HS offspring, indicating that both activity and number of BKCa channels in HS offspring were reduced. Superoxide production and NADPH oxidase (NOX)4 of the HS offspring were elevated. Following inhibiting NOX by apocynin, vasoconstriction in the HS offspring was weakened and the reduced currents in the VSMCs were improved with altered protein kinase B (AKT) pathway. The results suggested that NOX4-derived ROS might inhibit the offspring vascular BKCa channel activity via AKT pathway.

Cloning (SCNT) in avian species has proven unachievable due to the physical structure of the avian oocyte. Here, the plasticity of sexual differentiation of male primordial germ cells (PGCs) was investigated in opposite sex chimeric chicken hosts with the aim of producing uniparent clones. Male (ZZ) PGCs were expanded in culture and transplanted into the same and opposite sex chicken host embryos which were partially sterilized using γ-irradiation. As expected, all (28/28) chimeric (ZZ) roosters showed germline transmission with transmission rates of 3.2- 91.4% as detected by feather color. Unexpectedly, functional oogenesis of male chicken PGCs was found in three chimeric (ZW) hens (3/27), resulting in transmission rates of 2.3- 27.8%. Matings were conducted between the male and female germline chimeras which derived from the same male PGC line. 20 uniparent chicken clones were obtained with a transmission rate up to 28.4% and as expected, all uniparent offspring were male (ZZ). A genotype analysis was performed on 6 offspring using 13 microsatellite markers. The genotype profile of these offspring showed that uniparent offspring were 100% genetically identical to the donor male PGC line, and shared 69.2-88.5% identity with the donor bird at the 13 loci. At the 13 loci, homozygosity of the tested birds varied from 61.5-84.6%, which was higher than that of the donor bird (38.5%). These results demonstrate that male avian PGCs can differentiate into functional ova in an ovary and uniparent avian clones are possible. Uniparent clones differ from SCNT clones as the mitochondrial DNA derives from the donor and not from the host. This technology suggests novel approaches for generating genetically similar flocks of birds and for the conservation of avian genetic resources. Forming uniparent clones in bird species could be useful in extreme cases of low populations size.

Factors delivered to offspring in colostrum within 2 days of birth support neonatal porcine uterine development. The uterine mRNA transcriptome is affected by age and nursing during this period. Whether uterine microRNA (miRNA) expression is affected similarly is unknown. Objectives were to (1) determine effects of age and nursing on porcine uterine miRNA expression between birth and postnatal day (PND) 2 using miRNA sequencing (miRNAseq) and; (2) define affected miRNA–mRNA interactions and associated biological processes using integrated target prediction analysis. At birth (PND 0), gilts were euthanized, nursed ad libitum, or gavage-fed milk replacer for 48 h. Uteri were collected at birth or 50 h postnatal. MicroRNAseq data were validated using quantitative real-time PCR. Targets were predicted using an established mRNA database generated from the same tissues. For PND 2 versus PND 0 comparisons, 31 differentially expressed (DE) miRNAs were identified for nursed, and 42 DE miRNAs were identified for replacer-fed gilts. Six DE miRNAs were identified for nursed versus replacer-fed gilts on PND 2. Target prediction for inversely correlated DE miRNA–mRNA pairings indicated 20 miRNAs targeting 251 mRNAs in nursed, versus 29 miRNAs targeting 585 mRNAs in replacer-fed gilts for PND 2 versus PND 0 comparisons, and 5 miRNAs targeting 81 mRNAs for nursed versus replacer-fed gilts on PND 2. Biological processes predicted to be affected by age and nursing included cell-to-cell signaling, cell morphology, and tissue morphology. Results indicate novel age- and lactocrine-sensitive miRNA–mRNA relationships associated with porcine neonatal uterine development between birth and PND 2.

Sirolimus, also known as rapamycin, and its closely related rapamycin analog (rapalog) Everolimus inhibit “mammalian target of rapamycin complex 1” (mTORC1), whose activity is required for spermatogenesis. Everolimus is Food and Drug Administration approved for treating human patients to slow growth of aggressive cancers and preventing organ transplant rejection. Here, we test the hypothesis that rapalog inhibition of mTORC1 activity has a negative, but reversible, impact upon spermatogenesis. Juvenile (P20) or adult (P>60) mice received daily injections of sirolimus or Everolimus for 30 days, and tissues were examined at completion of treatment or following a recovery period. Rapalog treatments reduced body and testis weights, testis weight/body weight ratios, cauda epididymal sperm counts, and seminal vesicle weights in animals of both ages. Following rapalog treatment, numbers of differentiating spermatogonia were reduced, with concomitant increases in the ratio of undifferentiated spermatogonia to total number of remaining germ cells. To determine if even low doses of Everolimus can inhibit spermatogenesis, an additional group of adult mice received a dose of Everolimus ∼6-fold lower than a human clinical dose used to treat cancer. In these animals, only testis weights, testis weight/body weight ratios, and tubule diameters were reduced. Return to control values following a recovery period was variable for each of the measured parameters and was duration and dose dependent. Together, these data indicate rapalogs exerted a dose-dependent restriction on overall growth of juvenile and adult mice and negative impact upon spermatogenesis that were largely reversed; following treatment cessation, males from all treatment groups were able to sire offspring.

The tripeptide thiol antioxidant glutathione (GSH) has multiple physiological functions. Female mice lacking the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in GSH synthesis, have decreased GSH concentrations, ovarian oxidative stress, preimplantation embryonic mortality, and accelerated age-related decline in ovarian follicles. We hypothesized that supplementation with thiol antioxidants, N-acetyl cysteine (NAC), or α-lipoic acid (ALA) will rescue this phenotype. Gclm-/- and Gclm+/+ females received 0 or 80 mM NAC in drinking water from postnatal day (PND) 21–30; follicle growth was induced with equine chorionic gonadotropin (eCG) on PND 27, followed by an ovulatory dose of human CG and mating with a wild type male on PND 29 and zygote harvest 20 h after hCG. N-acetyl cysteine supplementation failed to rescue the low rate of second pronucleus formation in zygotes from Gclm-/- versus Gclm+/+ females. In the second study, Gclm-/- and Gclm+/+ females received diet containing 0, 150, or 600 mg/kg ALA beginning at weaning and were mated with wild type males from 8 to 20 weeks of age. α-Lipoic acid failed to rescue the decreased offspring production of Gclm-/- females. However, 150 mg/kg diet ALA partially rescued the accelerated decline in primordial follicles, as well as the increased recruitment of follicles into the growing pool and the increased percentages of follicles with γ H2AX positive oocytes or granulosa cells of Gclm-/- females. We conclude that ovarian oxidative stress is the cause of accelerated primordial follicle decline, while GSH deficiency per se may be responsible for preimplantation embryonic mortality in Gclm-/- females.Summary sentenceSupplementation with the antioxidant alpha-lipoic acid partially rescued accelerated age-related ovarian follicle depletion, but did not decrease offspring production, in glutathione-deficient Gclm null mice.

Gestational diabetes mellitus (GDM) is an obstetric disorder affecting approximately 10% of pregnancies. The four high-fat, high-sucrose (HFHS) mouse model emulates GDM in lean women. Dams are fed a HFHS diet 1 week prior to mating and throughout gestation resulting in inadequate insulin response to glucose in mid-late pregnancy. The offspring of HFHS dams have increased adiposity, thus, we hypothesized that maternal metabolic alterations during lean GDM would compromise ovarian function in offspring both basally and in response to a control or HFHS diet in adulthood. Briefly, DLPL were lean dams and control diet pups; DLPH were lean dams and HFHS pups; DHPL were HFHS dams and control diet pups; and DHPH were HFHS dams and HFHS pups. A HFHS challenge in the absence of maternal GDM (DLPL vs. DLPH) increased 3 and decreased 30 ovarian proteins. Maternal GDM in the absence of a dietary stress (DLPL vs. DHPL) increased abundance of 4 proteins and decreased abundance of 85 proteins in the offspring ovary. Finally, 87 proteins increased, and 4 proteins decreased in offspring ovaries due to dietary challenge and exposure to maternal GDM in utero (DLPL vs. DHPH). Canopy FGF signaling regulator 2, deleted in azoospermia-associated protein 1, septin 7, and serine/arginine-rich splicing factor 2 were altered across multiple offspring groups. Together, these findings suggest a possible impact on fertility and oocyte quality in relation to GDM exposure in utero as well as in response to a western diet in later life.

Bone morphogenetic protein 15 (BMP15), a member of the transforming growth factor beta superfamily, plays an essential role in ovarian follicular development in mono-ovulatory mammalian species. Studies using a biallelic knockout mouse model revealed that BMP15 potentially has just a minimal impact on female fertility and ovarian follicular development in polyovulatory species. In contrast, our previous study demonstrated that in vivo knockdown of BMP15 significantly affected porcine female fertility, as evidenced by the dysplastic ovaries containing significantly decreased numbers of follicles and an increased number of abnormal follicles. This finding implied that BMP15 plays an important role in the regulation of female fertility and ovarian follicular development in polyovulatory species. To further investigate the regulatory role of BMP15 in porcine ovarian and follicular development, here, we describe the efficient generation of BMP15-edited Yorkshire pigs using CRISPR/Cas9. Using artificial insemination experiments, we found that the biallelically edited gilts were all infertile, regardless of different genotypes. One monoallelically edited gilt #4 (Δ66 bp/WT) was fertile and could deliver offspring with a litter size comparable to that of wild-type gilts. Further analysis established that the infertility of biallelically edited gilts was caused by the arrest of follicular development at preantral stages, with formation of numerous structurally abnormal follicles, resulting in streaky ovaries and the absence of obvious estrous cycles. Our results strongly suggest that the role of BMP15 in nonrodent polyovulatory species may be as important as that in mono-ovulatory species.

Maternal high-fat diet (HFD) during pregnancy is linked to cardiovascular diseases in postnatal life. The current study tested the hypothesis that maternal HFD causes myocardial changes through angiotensin II receptor (AGTR) expression modulation in fetal and neonatal rat hearts. The control group of pregnant rats was fed a normal diet and the treatment group of pregnant rats was on a HFD (60% kcal fat). Hearts were isolated from embryonic day 21 fetuses (E21) and postnatal day 7 pups (PD7). Maternal HFD decreased the body weight of the offspring in both E21 and PD7. The ratio of heart weight to body weight was increased in E21, but not PD7, when compared to the control group. Transmission electron microscopy revealed disorganized myofibrils and effacement of mitochondria cristae in the treatment group. Maternal HFD decreased S-phase and increased G1-phase of the cellular cycle for fetal and neonatal cardiac cells. Molecular markers of cardiac hypertrophy, such as Nppa and Myh7, were found to be increased in the treatment group. There was an associated increase in Agtr2 mRNA and protein, whereas Agtr1a mRNA and AGTR1 protein were decreased in HFD fetal and neonatal hearts. Furthermore, maternal HFD decreased glucocorticoid receptors (GRs) binding to glucocorticoid response elements at the Agtr1a and Agtr2 promoter, which correlated with downregulation of GR in fetal and neonatal hearts. These findings suggest that maternal HFD may promote premature termination of fetal and neonatal cardiomyocyte proliferation and compensatory hypertrophy through intrauterine modulation of AGTR1 and AGTR2 expression via GR dependent mechanism.

Heterosis is the beneficial effect of genetical heterogeneity in animals and plants. Although heterosis induces changes in the cells and individual abilities, few reports have described the effect of heterosis on the female reproductive ability during aging. In this study, we investigated the reproductive capability of genetically heterogeneous (HET) mice established by the four-way crossing of C57BL/6N, BALB/c, C3H/He, and DBA/2. We found the HET females naturally and repeatedly produced offspring, even in old age (14-18 months of age). We also found that HET females showed a significantly enlarged body and organ sizes in both youth and old age. In histological analyses, the numbers of primordial follicles, primary follicles, secondary follicles, and corpora lutea were significantly increased in the old ovaries of HET females compared with those in inbred C57BL/6 mice of the same age. In vitro fertilization experiments revealed that aged HET oocytes showed identical rates of fertilization, early development, and birth compared to those of young and old C57BL/6 oocytes. We further found the significantly increased expression of sirtuin genes concomitant with the up-regulation of R-spondin2 in old HET ovaries. These results confirm the novel phenotype, characterized by fertility extension and follicular retention due to heterosis, in old HET females. The HET female will be a valuable model for clarifying the mechanism underlying the effect of heterosis in the field of reproduction.

Reproductive abnormalities are included as health complications in offspring exposed to poor prenatal nutrition. We have previously shown in a rodent model that offspring born to nutrient restriction during pregnancy are born small, enter puberty early, and display characteristics of early ovarian aging as adults. The present study investigated whether key proteins involved in follicle recruitment and growth mediate ovarian follicle loss. Pregnant rats were randomized to a standard diet throughout pregnancy and lactation (CON), or a calorie-restricted (50% of control) diet (UN) during pregnancy. Offspring reproductive phenotype was investigated at postnatal days 4, 27, and 65. Maternal UN resulted in young adult (P65) irregular estrous cyclicity due to persistent estrus, a significant loss of antral follicles, corpora lutea, and an increase in atretic follicles. This decrease in growing follicles in UN offspring appears to be due to increased apoptosis as seen by immunopositive staining of pro-apoptotic factor CASP3 (caspase 3) in ovaries of young adult offspring. UN prepubertal offspring had reduced expression levels of Fshr in antral follicles, which may contribute to a decrease in PI3K/AKT activation evident as a decrease in pAKT immunolocalization in prepubertal antral follicles. Moreover, neonatal ovaries of UN offspring show decreased levels of immunopositive staining for AMHR2 (anti-mullerian hormone receptor 2). Collectively, these data demonstrate that maternal UN during pregnancy impacts ovarian function in offspring as early as P65 and provides a model for understanding the mechanisms driving early life UN-induced follicle loss and reproductive dysfunction.

Malnutrition is a global threat to pregnancy health and impacts offspring development. Establishing an optimal pregnancy environment requires the coordination of maternal metabolic and immune pathways, which converge at the gut. Diet, metabolic, and immune dysfunctions have been associated with gut dysbiosis in the nonpregnant individual. In pregnancy, these states are associated with poor pregnancy outcomes and offspring development. However, the impact of malnutrition on maternal gut microbes, and their relationships with maternal metabolic and immune status, has been largely underexplored. To determine the impact of undernutrition and overnutrition on maternal metabolic status, inflammation, and the microbiome, and whether relationships exist between these systems, pregnant mice were fed either a normal, calorically restricted (CR), or a high fat (HF) diet. In late pregnancy, maternal inflammatory and metabolic biomarkers were measured and the cecal microbiome was characterized. Microbial richness was reduced in HF mothers although they did not gain more weight than controls. First trimester weight gain was associated with differences in the microbiome. Microbial abundance was associated with altered plasma and gut inflammatory phenotypes and peripheral leptin levels. Taxa potentially protective against elevated maternal leptin, without the requirement of a CR diet, were identified. Suboptimal dietary conditions common during pregnancy adversely impact maternal metabolic and immune status and the microbiome. HF nutrition exerts the greatest pressures on maternal microbial dynamics and inflammation. Key gut bacteria may mediate local and peripheral inflammatory events in response to maternal nutrient and metabolic status, with implications for maternal and offspring health.Summary SentenceMalnutrition alters immune and metabolic systems in pregnancy, and gut microbes may mediate the maternal response to these suboptimal dietary exposures.

Preterm birth (PTB), parturition prior to 37 weeks' gestation, is the leading cause of neonatal mortality. The causes of spontaneous PTB are poorly understood; however, recent studies suggest that this condition may arise as a consequence of the parental fetal environment. Specifically, we previously demonstrated that developmental exposure of male mice (F1 animals) to the environmental endocrine disruptor 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was associated with reduced sperm quantity/quality in adulthood and control female partners frequently delivered preterm. Reproductive defects persisted in the F2 and F3 descendants, and spontaneous PTB was common. Reproductive changes in the F3 males, the first generation without direct TCDD exposure, suggest the occurrence of epigenetic alterations in the sperm, which have the potential to impact placental development. Herein, we conducted an epigenetic microarray analysis of control and F1 male-derived placentae, which identified 2171 differentially methylated regions, including the progesterone receptor (Pgr) and insulin-like growth factor (Igf2). To assess if Pgr and Igf2 DNA methylation changes were present in sperm and persist in future generations, we assessed methylation and expression of these genes in F1/F3 sperm and F3-derived placentae. Although alterations in methylation and gene expression were observed, in most tissues, only Pgr reached statistical significance. Despite the modest gene expression changes in Igf2, offspring of F1 and F3 males consistently exhibited IUGR. Taken together, our data indicate that paternal developmental TCDD exposure is associated with transgenerational placental dysfunction, suggesting epigenetic modifications within the sperm have occurred. An evaluation of additional genes and alternative epigenetic mechanisms is warranted.Summary SentenceDevelopmental dioxin exposure of the paternal parent was associated with changes in the sperm and placental epigenome in association with impaired placental function; selected genes were also found to be differentially methylated in sperm and placentae of progeny without direct toxicant exposure.

Maternal body composition can be an important determinant for development of obesity and metabolic syndrome in adult offspring. Obesity-related outcomes in offspring may include epigenetic alterations; however, mechanisms of fetal programming remain to be fully elucidated. This study was conducted to determine the impact of maternal obesity in the absence of a high fat diet on equine endometrium and preimplantation embryos. Embryos were collected from normal and obese mares at 8 and 16 days and a uterine biopsy at 16 days (0 day = ovulation). With the exception of 8 day embryos, each sample was divided into two pieces. One piece was analyzed for gene expression markers related to carbohydrate metabolism, lipid homeostasis, inflammation, endoplasmic reticulum stress, oxidative stress, mitochondrial stress, and components of the insulin-like growth factor (IGF) system. The second piece was analyzed for lipid content using matrix-assisted laser desorption/ionization mass spectrometry. Obese mares had elevated concentrations of insulin, leptin, and total cholesterol, and they tended to have increased triglycerides and decreased insulin sensitivity. Embryos from obese mares had altered transcript abundance in genes for inflammation and lipid homeostasis, as well as endoplasmic reticulum, oxidative and mitochondrial stress and altered lipid fingerprints. Endometrium from obese mares had increased expression of inflammatory cytokines, lipid homeostasis regulation, mitochondrial stress, and the IGF2 system. This study demonstrates that increased adiposity in mares alters the uterine environment, transcript abundance of genes for cellular functions, and lipid profiles of embryos. These alterations could affect prenatal programming, with potential long-term effects in offspring.Summary SentenceExcess adiposity is sufficient to increase transcript abundance of inflammatory cytokines in the uterine endometrium and alters embryonic lipid content, and gene expression for lipid metabolism, endoplasmic reticulum stress, and mitochondrial function.

Maternal obesity is associated with an increased risk of obesity and metabolic disease in offspring. Increasing evidence suggests that the placenta plays an active role in fetal programming. In this study, we used a mouse model of diet-induced obesity to demonstrate that the abnormal metabolic milieu of maternal obesity sets the stage very early in pregnancy by altering the transcriptome of placenta progenitor cells in the preimplantation (trophectoderm [TE]) and early postimplantation (ectoplacental cone [EPC]) placenta precursors, which is associated with later changes in placenta development and function. Sphingolipid metabolism was markedly altered in the plasma of obese dams very early in pregnancy as was expression of genes related to sphingolipid processing in the early placenta. Upregulation of these pathways inhibits angiogenesis and causes endothelial dysfunction. The expression of many other genes related to angiogenesis and vascular development were disrupted in the TE and EPC. Other key changes in the maternal metabolome in obese dams that are likely to influence placenta and fetal development include a marked decrease in myo and chiro-inositol. These early metabolic and gene expression changes may contribute to phenotypic changes in the placenta, as we found that exposure to a high-fat diet decreased placenta microvessel density at both mid and late gestation. This is the first study to demonstrate that maternal obesity alters the transcriptome at the earliest stages of murine placenta development.