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.

Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.

Mammals meet the increased nutritional demands of lactation through a combination of increased feed intake and a collection of adaptations known as adaptive metabolism (e.g., glucose sparing via insulin resistance, mobilization of endogenous reserves, and increased metabolic efficiency via reduced thyroid hormones). In the modern dairy cow, adaptive metabolism predominates over increased feed intake at the onset of lactation and develops concurrently with a reduction in plasma leptin. To address the role of leptin in the adaptive metabolism of early lactation, we asked which adaptations could be countered by a constant 96-h intravenous infusion of human leptin (hLeptin) starting on day 8 of lactation. Compared to saline infusion (Control), hLeptin did not alter energy intake or milk energy output but caused a modest increase in body weight loss. hLeptin reduced plasma glucose by 9% and hepatic glycogen content by 73%, and these effects were associated with a 17% increase in glucose disposal during an insulin tolerance test. hLeptin attenuated the accumulation of triglyceride in the liver by 28% in the absence of effects on plasma levels of the anti-lipolytic hormone insulin or plasma levels of free fatty acids, a marker of lipid mobilization from adipose tissue. Finally, hLeptin increased the plasma concentrations of T4 and T3 by nearly 50% without affecting other neurally regulated hormones (i.e., cortisol and luteinizing hormone (LH)). Overall these data implicate the periparturient reduction in plasma leptin as one of the signals promoting conservation of glucose and energy at the onset of lactation in the energy-deficient dairy cow.

Metabolic and Endocrine Differences Between Dairy Cows That Do or Do Not Ovulate First Postpartum Dominant Follicles.

Most dairy cows develop the first dominant follicle postpartum within 2 wk after calving, but only about 40% of these follicles produce sufficient estradiol to stimulate ovulation despite having normal ultrasound appearance and growth. This study aimed to characterize metabolic, endocrine, and follicular fluid profiles of cows in which the first dominant follicle postpartum will become ovulatory and those with nonovulatory follicles. Luteinizing hormone pulse frequency, follicular fluid androstenedione, and follicular fluid estradiol concentrations were lower in nonovulatory cows suggesting that the function of theca cells is impaired. In addition, nonovulatory cows had more severe negative energy balance and greater insulin resistance postpartum. This study describes for the first time the steroid hormone profile of early postpartum follicles and shows that a steroidogenic defect most likely occurs in theca cells limiting the amount of androgen precursor available for estradiol production that impairs their ovulatory outcome.

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.

Translocator protein/peripheral benzodiazepine receptor is not required for steroid hormone biosynthesis.

Molecular events that regulate cellular biosynthesis of steroid hormones have been a topic of intense research for more than half a century. It has been established that transport of cholesterol into the mitochondria forms the rate-limiting step in steroid hormone production. In current models, both the steroidogenic acute regulatory protein (StAR) and the translocator protein (TSPO) have been implicated to have a concerted and indispensable effort in this cholesterol transport. Deletion of StAR in mice resulted in a critical failure of steroid hormone production, but deletion of TSPO in mice was found to be embryonic lethal. As a result, the role of TSPO in cholesterol transport has been established only using pharmacologic and genetic tools in vitro. To allow us to explore in more detail the function of TSPO in cell type-specific experimental manipulations in vivo, we generated mice carrying TSPO floxed alleles (TSPOfl/fl). In this study we made conditional knockout mice (TSPOcΔ/Δ) with TSPO deletion in testicular Leydig cells by crossing with an anti-Mullerian hormone receptor type II cre/+ mouse line. Genetic ablation of TSPO in steroidogenic Leydig cells in mice did not affect testosterone production, gametogenesis, and reproduction. Expression of StAR, cytochrome P450 side chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase type I, and TSPO2 in TSPOcΔ/Δ testis was unaffected. These results challenge the prevailing dogma that claims an essential role for TSPO in steroid hormone biosynthesis and force reexamination of functional interpretations made for this protein. This is the first study examining conditional TSPO gene deletion in mice. The results show that TSPO function is not essential for steroid hormone biosynthesis.

Effects of conjugated linoleic acids on prostaglandin secretion by bovine endometrial epithelial cells in vitro.

OBJECTIVE: To determine the effects of 2 conjugated linoleic acid (CLA) isomers (cis-9, trans-11 and trans-10, cis-12) on synthesis of prostaglandin (PG) E(2) and F(2α) and expression of prostaglandin H synthase-2 (PGHS-2) of adult and fetal bovine endometrial epithelial cells in vitro. SAMPLE: Primary cultures of endometrial epithelial cells obtained from 4 adult cows and 4 fetal bovine carcasses. PROCEDURES: Cells were exposed to 0, 50, 100, or 200µM cis-9, trans-11 or trans-10, cis-12 CLA isomers for 24 hours. Culture media collected before and after 6 hours of stimulation of cells with phorbol 12-myristate 13-acetate were assayed to detect PGE(2) and PGF(2α) via ELISA. After stimulation, cells were collected for western blot analysis to quantify PGHS-2. RESULTS: Concentrations of PGF(2α) and PGE(2) were significantly lower in culture media of adult and fetal endometrial epithelial cells exposed to any concentration of either CLA than they were in media of cells not exposed to CLAs. The trans-10, cis-12 CLA isomer seemed to decrease PG production more markedly than did the cis-9, trans-11 CLA isomer. Most concentrations of both CLAs significantly reduced culture media PGE(2):PGF(2α) concentration ratios of cells. Exposure of cells to CLAs did not affect expression of PGHS-2 protein. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study indicated CLAs significantly decreased PGF(2α) and PGE(2) concentrations and PGE(2):PGF(2α) concentration ratios for cultures of adult and fetal endometrial epithelial cells with no apparent effect on PGHS-2 expression. Similar effects in cows could have effects on maternal recognition of pregnancy and immune function.

Insulin regulates hepatic leptin receptor expression in early lactating dairy cows.

Energy balance controls the expression of the leptin receptor (Lepr) in the ruminant hypothalamus but whether similar regulation occurs in peripheral tissues is unknown. To address this issue, we measured Lepr expression in the liver and adipose tissue of dairy cows during the transition from late pregnancy (LP) to early lactation (EL). This period is characterized by the development of a profound state of energy insufficiency and is associated with reduced plasma insulin and leptin and with increased plasma growth hormone. Hepatic expression of the short (Lepr-a) and long (Lepr-b) isoforms was 40% higher during EL (8 days postpartum) than LP (30 days prepartum). A similar effect was observed when negative energy balance was induced in nonpregnant, late-lactation dairy cows by food restriction, implicating energy insufficiency as a specific cause in EL. The stimulation of hepatic Lepr expression was reversed after a 48-h period of hyperinsulinemic euglycemia in EL. Changes in hepatic Lepr expression during chronic elevation of plasma leptin in EL or plasma growth hormone in nonpregnant, late-lactation cows did not support a role for these hormones in mediating the effects of energy insufficiency on hepatic Lepr expression. In adipose tissue, Lepr expression was increased 10-fold during the transition from LP to EL. Overall, these data indicate that hypoinsulinemia is partly responsible for the induction of Lepr expression in the liver, and perhaps adipose tissue, of energy-deficient dairy cows.