RhoA/ROCK signaling antagonizes bovine trophoblast stem cell self-renewal and regulates preimplantation embryo size and differentiation.

Exponential proliferation of trophoblast stem cells (TSC) is crucial in Ruminantia to maximize numerical access to caruncles, the restricted uterine sites that permit implantation. When translating systems biology of the undifferentiated bovine trophectoderm, we uncovered that inhibition of RhoA/Rock promoted self-renewing proliferation and substantially increased blastocyst size. Analysis of transcripts suppressed by Rock inhibition revealed transforming growth factor ß1 (TGFß1) as a primary upstream effector. TGFß1 treatment induced changes consistent with differentiation in bTSCs, a response that could be replicated by induced expression of the bovine ROCK2 transgene. Rocki could partially antagonize TGFß1 effects, and TGFß receptor inhibition promoted proliferation identical to Rocki, indicating an all-encompassing upstream regulation. Morphological differentiation included formation of binucleate cells and infrequent multinucleate syncytia, features we also localize in the in vivo bovine placenta. Collectively, we demonstrate a central role for TGFß1, RhoA and Rock in inducing bTSC differentiation, attenuation of which is sufficient to sustain self-renewal and proliferation linked to blastocyst size and preimplantation development. Unraveling these mechanisms augments evolutionary/comparative physiology of the trophoblast cell lineage and placental development in eutherians.

The uterine secretory cycle: recurring physiology of endometrial outputs that setup the uterine luminal microenvironment.

Conserved in female reproduction across all mammalian species is the estrous cycle and its regulation by the hypothalamic-pituitary-gonadal (HPG) axis, a collective of intersected hormonal events that are crucial for ensuring uterine fertility. Nonetheless, knowledge of the direct mediators that synchronously shape the uterine microenvironment for successive yet distinct events, such as the transit of sperm and support for progressive stages of preimplantation embryo development, remain principally deficient. Toward understanding the timed endometrial outputs that permit luminal events as directed by the estrous cycle, we used Bovidae as a model system to uniquely surface sample and study temporal shifts to in vivo endometrial transcripts that encode for proteins destined to be secreted. The results revealed the full quantitative profile of endometrial components that shape the uterine luminal microenvironment at distinct phases of the estrous cycle (estrus, metestrus, diestrus, and proestrus). In interpreting this comprehensive log of stage-specific endometrial secretions, we define the "uterine secretory cycle" and extract a predictive understanding of recurring physiological actions regulated within the uterine lumen in anticipation of sperm and preimplantation embryonic stages. This repetitive microenvironmental preparedness to sequentially provide operative support was a stable intrinsic framework, with only limited responses to sperm or embryos if encountered in the lumen within the cyclic time period. In uncovering the secretory cycle and unraveling realistic biological processes, we present novel foundational knowledge of terminal effectors controlled by the HPG axis to direct a recurring sequence of vital functions within the uterine lumen.NEW & NOTEWORTHY This study unravels the recurring sequence of changes within the uterus that supports vital functions (sperm transit and development of preimplantation embryonic stages) during the reproductive cycle in female Ruminantia. These data present new systems knowledge in uterine reproductive physiology crucial for setting up in vitro biomimicry and artificial environments for assisted reproduction technologies for a range of mammalian species.

Current knowledge on the acute regulation of steroidogenesis.

How rapid induction of steroid hormone biosynthesis occurs in response to trophic hormone stimulation of steroidogenic cells has been a subject of intensive investigation for approximately six decades. A key observation made very early was that acute regulation of steroid biosynthesis required swift and timely synthesis of a new protein whose role appeared to be involved in the delivery of the substrate for all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane where the process of steroidogenesis begins. It was quickly learned that this transfer of cholesterol to the inner mitochondrial membrane was the regulated and rate-limiting step in steroidogenesis. Following this observation, the quest for this putative regulator protein(s) began in earnest in the late 1950s. This review provides a history of this quest, the candidate proteins that arose over the years and facts surrounding their rise or decline. Only two have persisted-translocator protein (TSPO) and the steroidogenic acute regulatory protein (StAR). We present a detailed summary of the work that has been published for each of these two proteins, the specific data that has appeared in support of their role in cholesterol transport and steroidogenesis, and the ensuing observations that have arisen in recent years that have refuted the role of TSPO in this process. We believe that the only viable candidate that has been shown to be indispensable is the StAR protein. Lastly, we provide our view on what may be the most important questions concerning the acute regulation of steroidogenesis that need to be asked in future.

Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis.

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo(-/-)) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.

Overactivation of hedgehog signaling in the developing Müllerian duct interferes with duct regression in males and causes subfertility.

The influence of the hedgehog signaling pathway on reproduction was studied in transgenic mice in which a dominant active allele of the hedgehog signal transducer, smoothened (Smo), was conditionally expressed in the developing Müllerian duct and gonads through recombination mediated by anti-Müllerian hormone receptor 2-cre (Amhr2cre ). Previous studies showed that development of the oviduct and uterus are abnormal in female Amhr2cre/+SmoM2 mice. In the current study, focusing on mutant males, litter size was reduced 53% in crosses with wild-type females. An extra band of undifferentiated tissue extended along each epididymis and vas deferens, a position suggesting derivation from Müllerian ducts that failed to regress fully. Hedgehog signaling was elevated in this tissue, based on mRNA levels of target genes. Amhr2 mRNA was dramatically reduced in the uterus of mutant females and in the extra tissue in the tract of mutant males, suggesting that AMHR2 signaling was inadequate for complete Müllerian duct regression. Spermatogenesis and sperm motility were normal, but testis weight was reduced 37% and epididymal sperm number was reduced 36%. The number of sperm recovered from the uteri of wild-type females after mating with mutant males was reduced 78%. This suggested that sperm transport through the male tract was reduced, resulting in fewer sperm in the ejaculate. Consistent with this, mutant males had unusually tortuous vas deferentia with constrictions within the lumen. We concluded that persistence of a relatively undifferentiated remnant of Müllerian tissue is sufficient to cause subtle changes in the male reproductive tract that reduce fertility.

In vivo imaging of microglial activation by positron emission tomography with [(11)C]PBR28 in the 5XFAD model of Alzheimer's disease.

Microglial activation has been linked with deficits in neuronal function and synaptic plasticity in Alzheimer's disease (AD). The mitochondrial translocator protein (TSPO) is known to be upregulated in reactive microglia. Accurate visualization and quantification of microglial density by PET imaging using the TSPO tracer [(11)C]-R-PK11195 has been challenging due to the limitations of the ligand. In this study, it was aimed to evaluate the new TSPO tracer [(11)C]PBR28 as a marker for microglial activation in the 5XFAD transgenic mouse model of AD. Dynamic PET scans were acquired following intravenous administration of [(11)C]PBR28 in 6-month-old 5XFAD mice and in wild-type controls. Autoradiography with [(3)H]PBR28 was carried out in the same brains to further confirm the distribution of the radioligand. In addition, immunohistochemistry was performed on adjacent brain sections of the same mice to evaluate the co-localization of TSPO with microglia. PET imaging revealed that brain uptake of [(11)C]PBR28 in 5XFAD mice was increased compared with control mice. Moreover, binding of [(3)H]PBR28, measured by autoradiography, was enriched in cortical and hippocampal brain regions, coinciding with the positive staining of the microglial marker Iba-1 and amyloid deposits in the same areas. Furthermore, double-staining using antibodies against TSPO demonstrated co-localization of TSPO with microglia and not with astrocytes in 5XFAD mice and human post-mortem AD brains. The data provided support of the suitability of [(11)C]PBR28 as a tool for in vivo monitoring of microglial activation and assessment of treatment response in future studies using animal models of AD.

Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis.

Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is a mitochondrial outer membrane protein implicated as essential for cholesterol import to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Previous research on TSPO was based entirely on in vitro experiments, and its critical role was reinforced by an early report that claimed TSPO knock-out mice were embryonic lethal. In a previous publication, we examined Leydig cell-specific TSPO conditional knock-out mice that suggested TSPO was not required for testosterone production in vivo. This raised controversy and several questions regarding TSPO function. To examine the definitive role of TSPO in steroidogenesis and embryo development, we generated global TSPO null (Tspo(-/-)) mice. Contrary to the early report, Tspo(-/-) mice survived with no apparent phenotypic abnormalities and were fertile. Examination of adrenal and gonadal steroidogenesis showed no defects in Tspo(-/-) mice. Adrenal transcriptome comparison of gene expression profiles showed that genes involved in steroid hormone biosynthesis (Star, Cyp11a1, and Hsd3b1) were unchanged in Tspo(-/-) mice. Adrenocortical ultrastructure illustrated no morphological alterations in Tspo(-/-) mice. In an attempt to correlate our in vivo findings to previously used in vitro models, we also determined that siRNA knockdown or the absence of TSPO in different mouse and human steroidogenic cell lines had no effect on steroidogenesis. These findings directly refute the dogma that TSPO is indispensable for steroid hormone biosynthesis and viability. By amending the current model, this study advances our understanding of steroidogenesis with broad implications in biology and medicine.

Generation and characterization of spiking and nonspiking oligodendroglial progenitor cells from embryonic stem cells.

Pluripotent stem cells (PSCs) have been differentiated into oligodendroglial progenitor cells (OPCs), providing promising cell replacement therapies for many central nervous system disorders. Studies from rodents have shown that brain OPCs express a variety of ion channels, and that a subset of brain OPCs express voltage-gated sodium channel (NaV ), mediating the spiking properties of OPCs. However, it is unclear whether PSC-derived OPCs exhibit electrophysiological properties similar to brain OPCs and the role of NaV in the functional maturation of OPCs is unknown. Here, using a mouse embryonic stem cell (mESC) green fluorescent protein (GFP)-Olig2 knockin reporter line, we demonstrated that unlike brain OPCs, all the GFP(+) /Olig2(+) mESC-derived OPCs (mESC-OPCs) did not express functional NaV and failed to generate spikes (hence termed "nonspiking mESC-OPCs"), while expressing the delayed rectifier and inactivating potassium currents. By ectopically expressing NaV 1.2 α subunit via viral transduction, we successfully generated mESC-OPCs with spiking properties (termed "spiking mESC-OPCs"). After transplantation into the spinal cord and brain of myelin-deficient shiverer mice, the spiking mESC-OPCs demonstrated better capability in differentiating into myelin basic protein expressing oligodendrocytes and in myelinating axons in vivo than the nonspiking mESC-OPCs. Thus, by generating spiking and nonspiking mESC-OPCs, this study reveals a novel function of NaV in OPCs in their functional maturation and myelination, and sheds new light on ways to effectively develop PSC-derived OPCs for future clinical applications.

PEDF is a novel oligodendrogenic morphogen acting on the adult SVZ and corpus callosum.

Pigment epithelium-derived factor (PEDF) is a serine protease inhibitor (serpin) protein with well established neuroprotective and anti-angiogenic properties. Recent studies have also shown that PEDF enhances renewal of adult subventricular zone (SVZ) neural precursors. In neurosphere cultures prepared from the SVZ of adult mice, we found that addition of recombinant PEDF to the medium enhanced expressions of oligodendroglial lineage markers (NG2 and PDGFrα) and transcription factors (Olig1, Olig2, and Sox10). Similarly, continuous PEDF administration into the lateral ventricles of adult glial fibrillary acidic protein:green fluorescent protein (GFAP:GFP) transgenic mice increased the proportions of GFAP:GFP+ and GFAP:GFP- SVZ neural precursors coexpressing oligodendroglial lineage markers and transcription factors. Notably, PEDF infusion also resulted in an induction of doublecortin- and Sox10 double-positive cells in the adult SVZ. Immunoreactive PEDF receptor was detectable in multiple cell types in both adult SVZ and corpus callosum. Furthermore, PEDF intracerebral infusion enhanced survival and maturation of newly born oligodendroglial progenitor cells in the normal corpus callosum, and accelerated oligodendroglial regeneration in lysolecithin-induced corpus callosum demyelinative lesions. Western blot analysis showed a robust upregulation of endogenous PEDF in the corpus callosum upon lysolecithin-induced demyelination. Our results document previously unrecognized oligodendrotrophic effects of recombinant PEDF on the adult SVZ and corpus callosum, demonstrate induction of endogenous CNS PEDF production following demyelination, and make PEDF a strong candidate for pharmacological intervention in demyelinative diseases.

Arresting calcium-regulated sperm metabolic dynamics enables prolonged fertility in poultry liquid semen storage.

The preservation of liquid semen is pivotal for both industrial livestock production and genetic management/conservation of species with sperm that are not highly cryo-tolerant. Nevertheless, with regard to poultry semen, even brief in vitro storage periods can lead to a notable decline in fertility, despite the in vivo capacity to maintain fertility for several weeks when within the hen's sperm storage tubules. For fertility in sperm, intracellular calcium ions ([Ca2+]i) play a key role in signaling towards modifying energy metabolism. While reducing [Ca2+]i has been found to enhance the preservation of sperm fertility in some mammals, the connection between semen fertility and calcium availability in avian sperm has received limited attention. In this study, we demonstrate that the use of extracellular and intracellular calcium chelators in liquid semen extenders, specifically EGTA and EGTA-AM, has distinct effects on prolonging the fertility of chicken sperm. These results were validated through in vivo fertility tests. Mechanistically, the effects observed were linked to coordination of mitochondrial metabolism and ATP catabolism. Despite both calcium chelators inducing hypoxia, they differentially regulated mitochondrial respiration and ATP accumulation. This regulation was closely linked to a bimodal control of dynein ATPase activity; a direct initial activation with reduction in [Ca2+]i, and subsequent suppression by cytoplasmic acidification caused by lactic acid. These findings not only contribute to advancing poultry liquid semen preservation techniques, but also elucidates biologically relevant mechanisms that may underlie storage within the female reproductive tract in birds.

Functional metabolite reserves and lipid homeostasis revealed by the MA-10 Leydig cell metabolome.

In Leydig cells, intrinsic factors that determine cellular steroidogenic efficiency is of functional interest to decipher and monitor pathophysiology in many contexts. Nevertheless, beyond basic regulation of cholesterol storage and mobilization, systems biology interpretation of the metabolite networks in steroidogenic function is deficient. To reconstruct and describe the different molecular systems regulating steroidogenesis, we profiled the metabolites in resting MA-10 Leydig cells. Our results identified 283-annotated components (82 neutral lipids, 154 membrane lipids, and 47 other metabolites). Neutral lipids were represented by an abundance of triacyglycerols (97.1%), and low levels of cholesterol esters (2.0%). Membrane lipids were represented by an abundance of glycerophospholipids (77.8%), followed by sphingolipids (22.2%). Acylcarnitines, nucleosides, amino acids and their derivatives were the other metabolite classes identified. Among nonlipid metabolites, we recognized substantial reserves of aspartic acid, choline, creatine, betaine, glutamine, homoserine, isoleucine, and pantothenic acid none of which have been previously considered as a requirement in steroidogenic function. Individually limiting use of betaine, choline, or pantothenic acid, during luteinizing hormone-induced steroidogenesis in MA-10 cells resulted in substantial decreases to acute steroidogenic capacity, explained by intermediary metabolite imbalances affecting homeostasis. As such, our dataset represents the current level of baseline characterization and unravels the functional resting state of steroidogenic MA-10 Leydig cells. In identifying metabolite stockpiles and causal mechanisms, these results serve to further comprehend the cellular setup and regulation of steroid biosynthesis.

Exogenous cholesterol acquisition signaling in LH-responsive MA-10 Leydig cells and in adult mice.

MA-10 cells, established 4 decades ago from a murine Leydig cell tumor, has served as a key model system for studying steroidogenesis. Despite a precipitous loss in their innate ability to respond to luteinizing hormone (LH), the use of a cell-permeable cAMP analog for induction ensured their continued use. In parallel, a paradigm that serum-free conditions are essential for trophic steroidogenic stimulation was rationalized. Through the selection of LH-responsive single-cell MA-10Slip clones, we uncovered that Leydig cells remain responsive in the presence of serum in vitro and that exogenous cholesterol delivery by lipoproteins provided a significantly elevated steroid biosynthetic response (>2-fold). In scrutinizing the underlying regulation, systems biology of the MA-10 cell proteome identified multiple Rho-GTPase signaling pathways as highly enriched. Testing Rho function in steroidogenesis revealed that its modulation can negate the specific elevation in steroid biosynthesis observed in the presence of lipoproteins/serum. This signaling modality primarily linked to the regulation of endocytic traffic is evident only in the presence of exogenous cholesterol. Inhibiting Rho function in vivo also decreased hCG-induced testosterone production in mice. Collectively, our findings dispel a long-held view that the use of serum could confound or interfere with trophic stimulation and underscore the need for exogenous lipoproteins when dissecting physiological signaling and cholesterol trafficking for steroid biosynthesis in vitro. The LH-responsive MA-10Slip clones derived in this study present a reformed platform enabling biomimicry to study the cellular and molecular basis of mammalian steroidogenesis.

History, insights, and future perspectives on studies into luteal function in cattle.

The corpus luteum (CL) forms following ovulation from the remnant of the Graafian follicle. This transient tissue produces critical hormones to maintain pregnancy, including the steroid progesterone. In cattle and other ruminants, the presence of an embryo determines if the lifespan of the CL will be prolonged to ensure successful implantation and gestation, or if the tissue will undergo destruction in the process known as luteolysis. Infertility and subfertility in dairy and beef cattle results in substantial economic loss to producers each year. In addition, this has the potential to exacerbate climate change because more animals are needed to produce high-quality protein to feed the growing world population. Successful pregnancies require coordinated regulation of uterine and ovarian function by the developing embryo. These processes are often collectively termed "maternal recognition of pregnancy." Research into the formation, function, and destruction of the bovine CL by the Northeast Multistate Project, one of the oldest continuously funded Hatch projects by the USDA, has produced a large body of evidence increasing our knowledge of the contribution of ovarian processes to fertility in ruminants. This review presents some of the seminal research into the regulation of the ruminant CL, as well as identifying mechanisms that remain to be completely validated in the bovine CL. This review also contains a broad discussion of the roles of prostaglandins, immune cells, as well as mechanisms contributing to steroidogenesis in the ruminant CL. A triadic model of luteolysis is discussed wherein the interactions among immune cells, endothelial cells, and luteal cells dictate the ability of the ruminant CL to respond to a luteolytic stimulus, along with other novel hypotheses for future research.

The corpus luteum (CL) forms on the ovary from the cellular remnants of the follicle following ovulation. The function of the CL is to produce progesterone that is required for successful pregnancy. In the absence of an embryo or sufficient embryonic signaling, the uterus will release a prostaglandin that kills the CL in a process called luteolysis. Therefore, the CL and the embryo share a symbiotic relationship, each requiring the other to be healthy and functional for survival. The Northeast Multistate Project, one of the oldest in the nation, has produced a large body of evidence that has enhanced our understanding of how the CL functions, its regulation, and the impact of ovarian activity on fertility of cattle. This review highlights some of the important advances made in the understanding of the ruminant CL.

Mitochondrial translocator protein deficiency exacerbates pathology in acute experimental ulcerative colitis.

In human patients and animal models of ulcerative colitis (UC), upregulation of the mitochondrial translocator protein (TSPO) in the colon is consistent with inflammation. Although the molecular function for TSPO remains unclear, it has been investigated as a therapeutic target for ameliorating UC pathology. In this study, we examined the susceptibility of Tspo gene-deleted (Tspo -/- ) mice to insults as provided by the dextran sodium sulfate (DSS)-induced acute UC model. Our results show that UC clinical signs and pathology were severely exacerbated in Tspo -/- mice compared to control Tspo fl/fl cohorts. Histopathology showed extensive inflammation and epithelial loss in Tspo -/- mice that caused an aggravated disease. Colonic gene expression in UC uncovered an etiology linked to precipitous loss of epithelial integrity and disproportionate mast cell activation assessed by tryptase levels in Tspo -/- colons. Evaluation of baseline homeostatic shifts in Tspo -/- colons revealed gene expression changes noted in elevated epithelial Cdx2, mast cell Cd36 and Mcp6, with general indicators of lower proliferation capacity and elevated mitochondrial fatty acid oxidation. These findings demonstrate that intact physiological TSPO function serves to limit inflammation in acute UC, and provide a systemic basis for investigating TSPO-targeting mechanistic therapeutics.

Mice lacking FABP9/PERF15 develop sperm head abnormalities but are fertile.

The male germ cell-specific fatty acid-binding protein 9 (FABP9/PERF15) is the major component of the murine sperm perforatorium and perinuclear theca. Based on its cytoskeletal association and sequence homology to myelin P2 (FABP8), it has been suggested that FABP9 tethers sperm membranes to the underlying cytoskeleton. Furthermore, its upregulation in apoptotic testicular germ cells and its increased phosphorylation status during capacitation suggested multiple important functions for FABP9. Therefore, we investigated specific functions for FABP9 by means of targeted gene disruption in mice. FABP9(-/-) mice were viable and fertile. Phenotypic analysis showed that FABP9(-/-) mice had significant increases in sperm head abnormalities (~8% greater than their WT cohorts); in particular, we observed the reduction or absence of the characteristic structural element known as the "ventral spur" in ~10% of FABP9(-/-) sperm. However, deficiency of FABP9 affected neither membrane tethering to the perinuclear theca nor the fatty acid composition of sperm. Moreover, epididymal sperm numbers were not affected in FABP9(-/-) mice. Therefore, we conclude that FABP9 plays only a minor role in providing the murine sperm head its characteristic shape and is not absolutely required for spermatogenesis or sperm function.

Effects of body weight and season on serum lipid concentrations in sloth bears (Melursus ursinus ursinus).

Serum lipid levels were measured in 66 healthy sloth bears (Melursus ursinus ursinus) living under semicaptive conditions with access to natural food resources in the Bannerghatta Biological Park (Karnataka, India), a portion of their native habitat range in the Indian peninsula. Total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein cholesterol levels were analyzed. The effects of age, body weight, and season on these lipid parameters were statistically evaluated. There were no correlations between age and any of the serum lipid parameters analyzed. Positive correlations of body weight to both triglyceride and HDL cholesterol levels in these bears were identified. In addition, seasonal trends in physiological serum lipid values, potentially due to variations in the sloth bear diet, were identified. Serum triglyceride levels were higher during postmonsoon season and cholesterol levels were higher during winter compared to other seasons. Serum lipid values obtained from sloth bears in this study were also compared to previously published data on other members of the family Ursidae. This is the first report of serum lipid values as a reference for sloth bears. These values can be used as sensitive predictors of overall health and nutritional status to aid in the captive management and feeding of these bears.

Efficient induction and sustenance of pluripotent stem cells from bovine somatic cells.

Although derivation of naïve bovine embryonic stem cells is unachieved, the possibility for generation of bovine induced pluripotent stem cells (biPSCs) has been generally reported. However, attempts to sustain biPSCs by promoting self-renewal have not been successful. Methods established for maintaining murine and human induced pluripotent stem cells (iPSCs) do not support self-renewal of iPSCs for any bovid species. In this study, we examined methods to enhance complete reprogramming and concurrently investigated signaling relevant to pluripotency of the bovine blastocyst inner cell mass (ICM). First, we identified that forced expression of SV40 large T antigen together with the reprogramming genes (OCT4, SOX2, KLF4 and MYC) substantially enhanced the reprogramming efficacy of bovine fibroblasts to biPSCs. Second, we uncovered that TGFß signaling is actively perturbed in the ICM. Inhibition of ALK4/5/7 to block TGFß/activin/nodal signaling together with GSK3ß and MEK1/2 supported robust in vitro self-renewal of naïve biPSCs with unvarying colony morphology, steady expansion, expected pluripotency gene expression and committed differentiation plasticity. Core similarities between biPSCs and stem cells of the 16-cell-stage bovine embryo indicated a stable ground state of pluripotency; this allowed us to reliably gain predictive understanding of signaling in bovine pluripotency using systems biology approaches. Beyond defining a high-fidelity platform for advancing biPSC-based biotechnologies that have not been previously practicable, these findings also represent a significant step towards understanding corollaries and divergent aspects of bovine pluripotency. This article has an associated First Person interview with the joint first authors of the paper.

Temporal kinetics of bovine mammary IgG secretion into colostrum and transition milk.

Neonatal calf survival and health is predominantly dependent on sufficient consumption of immunoglobulin G (IgG) and the resulting transfer of passive immunity (TPI). In this study, we investigate the potential for continued IgG secretion and temporal kinetics of mammary IgG output in sequential milkings performed at 0, 4, 16, 28, 40, and 52 hr postcalving in Holstein dairy cows. For colostrum (0 hr), we also scrutinize the relationships between IgG concentration, volume, refractometer readings (˚Bx values, Brix) and concentration of sugars (lactose and glucose). Mammary transcripts postpartum (0 hr) indicated that active IgG secretion continues beyond the first milking (colostrum; n = 4 to 5). IgG measurements at the different timepoints indicated that colostrum represents only 25.1% of the total IgG produced across the 6 sequential milking timepoints, with a substantial 48.9% being secreted into transition milk over the next 3 timepoints (4-, 6-, and 28-hr) combined. The differences on the basis of IgG concentrations across 0-, 4-, and 16-hr milking timepoints were not statistically significant (P = 0.1522; n = 9). For colostrum, volume remained highly variable, even with induced let-down prior to milking (n = 27). Nonetheless, colostrum IgG secretion was significantly co-regulated with volume (R2 = 0.915; P < 0.001; n = 18), an association that was stronger than that measured for lactose (R2 = 0.803; P < 0.001; n = 18) and glucose (R2 = 0.467; P = 0.002; n = 17). Comparing colostrum ˚Bx values to absolute IgG concentrations showed no correlation (R2 = 0.127; P = 0.07; n = 27); biochemical separation of colostrum components indicated that both proteins and nonprotein solutes could affect ˚Bx values (P < 0.0001 for both; n = 5). This suggests that ˚Bx values do not reasonably indicate IgG concentration to serve as a measure of "colostrum quality." Additionally, our finding that early transition milk (4-, 6-, and 28-hr) can contribute substantially more IgG than colostrum forces a rethink of existing feeding paradigms and means to maximize TPI in calves. Collectively, our results reveal the remarkable value of early transition milk and caveats to colostrum assessments that could advance application in enhancing neonatal calf health.