AMPK Suppression Due to Obesity Drives Oocyte mtDNA Heteroplasmy via ATF5-POLG Axis.

Due to the exclusive maternal transmission, oocyte mitochondrial dysfunction reduces fertility rates, affects embryonic development, and programs offspring to metabolic diseases. However, mitochondrial DNA (mtDNA) are vulnerable to mutations during oocyte maturation, leading to mitochondrial nucleotide variations (mtSNVs) within a single oocyte, referring to mtDNA heteroplasmy. Obesity (OB) accounts for more than 40% of women at the reproductive age in the USA, but little is known about impacts of OB on mtSNVs in mature oocytes. It is found that OB reduces mtDNA content and increases mtSNVs in mature oocytes, which impairs mitochondrial energetic functions and oocyte quality. In mature oocytes, OB suppresses AMPK activity, aligned with an increased binding affinity of the ATF5-POLG protein complex to mutated mtDNA D-loop and protein-coding regions. Similarly, AMPK knockout increases the binding affinity of ATF5-POLG proteins to mutated mtDNA, leading to the replication of heteroplasmic mtDNA and impairing oocyte quality. Consistently, AMPK activation blocks the detrimental impacts of OB by preventing ATF5-POLG protein recruitment, improving oocyte maturation and mitochondrial energetics. Overall, the data uncover key features of AMPK activation in suppressing mtSNVs, and improving mitochondrial biogenesis and oocyte maturation in obese females.

Cold exposure impacts DNA methylation patterns in cattle sperm.

DNA methylation is influenced by various exogenous factors such as nutrition, temperature, toxicants, and stress. Bulls from the Pacific Northwest region of the United States and other northern areas are exposed to extreme cold temperatures during winter. However, the effects of cold exposure on the methylation patterns of bovine sperm remain unclear. To address, DNA methylation profiles of sperm collected during late spring and winter from the same bulls were analyzed using whole genome bisulfite sequencing (WGBS). Bismark (0.22.3) were used for mapping the WGBS reads and R Bioconductor package DSS was used for differential methylation analysis. Cold exposure induced 3,163 differentially methylated cytosines (DMCs) with methylation difference ≥10% and a q-value < 0.05. We identified 438 differentially methylated regions (DMRs) with q-value < 0.05, which overlapped with 186 unique genes. We also identified eight unique differentially methylated genes (DMGs) (Pax6, Macf1, Mest, Ubqln1, Smg9, Ctnnb1, Lsm4, and Peg10) involved in embryonic development, and nine unique DMGs (Prmt6, Nipal1, C21h15orf40, Slc37a3, Fam210a, Raly, Rgs3, Lmbr1, and Gan) involved in osteogenesis. Peg10 and Mest, two paternally expressed imprinted genes, exhibited >50% higher methylation. The differential methylation patterns of six distinct DMRs: Peg10, Smg9 and Mest related to embryonic development and Lmbr1, C21h15orf40 and Prtm6 related to osteogenesis, were assessed by methylation-specific PCR (MS-PCR), which confirmed the existence of variable methylation patterns in those locations across the two seasons. In summary, cold exposure induces differential DNA methylation patterns in genes that appear to affect embryonic development and osteogenesis in the offspring. Our findings suggest the importance of replicating the results of the current study with a larger sample size and exploring the potential of these changes in affecting offspring development.

Maternal exercise intergenerationally drives muscle-based thermogenesis via activation of apelin-AMPK signaling.

BACKGROUND: Sarcolipin and uncoupling protein 3 (UCP3) mediate muscle-based non-shivering thermogenesis (NST) to improve metabolic homeostasis. The impacts of maternal obesity (MO) and maternal exercise (ME) on NST in offspring muscle remain unexamined. METHODS: Female mice were fed with a control diet or high fat diet to induce obesity. Then, obese mice were further separated into two groups: obesity only (OB) and OB plus daily exercise (OB/Ex). Fetal muscle was collected at embryonic day 18.5 and offspring mice at 3-month-old. Apelin administration during pregnancy and apelin receptor (APJ) knockout mouse were further used for investigating the mediatory role of APJ on muscle-based thermogenesis. To explore the direct effects of exercise on AMP-activated protein kinase (AMPK) downstream targets, AMPK knockout mouse was used. FINDINGS: MO inhibited while ME activated AMPK and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) in fetal muscle. AMPK activation increased sarcolipin expression, which inhibited the uptake of calcium ions into sarcoplasmic reticulum, thereby activating CaMKK2. Consistently, the expression of UCP3 and sarcolipin was suppressed due to MO but activated in ME fetal muscle. Importantly, changes of UCP3 and sarcolipin maintained in offspring muscle, showing the transgenerational effects. Furthermore, apelin administration during pregnancy mimicked the effects of ME on AMPK and CaMKK2 activation, and UCP3 and sarcolipin expression, underscoring the mediatory roles of apelin-AMPK signaling in improving fetal muscle development. INTERPRETATION: ME, via activation of apelin signaling-AMPK axis, enhances NST gene expression in fetal and offspring muscle impaired due to MO, which intergenerationally protects offspring from diet-induced obesity and metabolic disorders. FUNDING: This work was supported by National Institutes of Health Grant R01-HD067449.

Imprinted lncRNA Dio3os preprograms intergenerational brown fat development and obesity resistance.

Maternal obesity (MO) predisposes offspring to obesity and metabolic disorders but little is known about the contribution of offspring brown adipose tissue (BAT). We find that MO impairs fetal BAT development, which persistently suppresses BAT thermogenesis and primes female offspring to metabolic dysfunction. In fetal BAT, MO enhances expression of Dio3, which encodes deiodinase 3 (D3) to catabolize triiodothyronine (T3), while a maternally imprinted long noncoding RNA, Dio3 antisense RNA (Dio3os), is inhibited, leading to intracellular T3 deficiency and suppression of BAT development. Gain and loss of function shows Dio3os reduces D3 content and enhances BAT thermogenesis, rendering female offspring resistant to high fat diet-induced obesity. Attributing to Dio3os inactivation, its promoter has higher DNA methylation in obese dam oocytes which persists in fetal and adult BAT, uncovering an oocyte origin of intergenerational obesity. Overall, our data uncover key features of Dio3os activation in BAT to prevent intergenerational obesity and metabolic dysfunctions.

Obesity Impairs Embryonic Myogenesis by Enhancing BMP Signaling within the Dermomyotome.

Obesity during pregnancy leads to adverse health outcomes in offspring. However, the initial effects of maternal obesity (MO) on embryonic organogenesis have yet to be thoroughly examined. Using unbiased single-cell transcriptomic analyses (scRNA-seq), the effects of MO on the myogenic process is investigated in embryonic day 9.5 (E9.5) mouse embryos. The results suggest that MO induces systematic hypoxia, which is correlated with enhanced BMP signaling and impairs skeletal muscle differentiation within the dermomyotome (DM). The Notch-signaling effectors, HES1 and HEY1, which also act down-stream of BMP signaling, suppress myogenic differentiation through transcriptionally repressing the important myogenic regulator MEF2C. Moreover, the major hypoxia effector, HIF1A, enhances expression of HES1 and HEY1 and blocks myogenic differentiation in vitro. In summary, this data demonstrate that MO induces hypoxia and impairs myogenic differentiation by up-regulating BMP signaling within the DM, which may account for the disruptions of skeletal muscle development and function in progeny.

Embryonic exposure to hyper glucocorticoids suppresses brown fat development and thermogenesis via REDD1.

Maternal stress during pregnancy is prevailing worldwide, which exposes fetuses to intrauterine hyper glucocorticoids (GC), programming offspring to obesity and metabolic diseases. Despite the importance of brown adipose tissue (BAT) in maintaining long-term metabolic health, impacts of prenatal hyper GC on postnatal BAT thermogenesis and underlying regulations remain poorly defined. Pregnant mice were administrated with synthetic GC dexamethasone (DEX) at levels comparable to fetal GC exposure of stressed mothers. Prenatal GC exposure dose-dependently reduced BAT thermogenic activity, contributing to lower body temperature and higher mortality of neonates; such difference was abolished under thermoneutrality, underscoring BAT deficiency was the major contributor to adverse changes in postnatal thermogenesis due to excessive GC. Prenatal GC exposure highly activated Redd1 expression and reduced Ppargc1a transcription from the alternative promoter (Ppargc1a-AP) in neonatal BAT. During brown adipocyte differentiation, ectopic Redd1 expression reduced Ppargc1a-AP expression and mitochondrial biogenesis; and the inhibitory effects of GC on mitochondrial biogenesis and Ppargc1a-AP expression were blocked by Redd1 ablation. Redd1 reduced protein kinase A phosphorylation and suppressed cyclic adenosine monophosphate (cAMP) -responsive element-binding protein (CREB) binding to the cAMP regulatory element (CRE) in Ppargc1a-AP promoter, leading to Ppargc1a-AP inactivation. In summary, excessive maternal GC exposure during pregnancy dysregulates Redd1-Ppargc1a-AP axis, which impairs fetal BAT development, hampering postnatal thermogenic adaptation and metabolic health of offspring.

Maternal Inactivity Programs Skeletal Muscle Dysfunction in Offspring Mice by Attenuating Apelin Signaling and Mitochondrial Biogenesis.

Although maternal exercise (ME) becomes increasingly uncommon, the effects of ME on offspring muscle metabolic health remain largely undefined. Maternal mice are subject to daily exercise during pregnancy, which enhances mitochondrial biogenesis during fetal muscle development; this is correlated with higher mitochondrial content and oxidative muscle fibers in offspring muscle and improved endurance capacity. Apelin, an exerkine, is elevated due to ME, and maternal apelin administration mirrors the effect of ME on mitochondrial biogenesis in fetal muscle. Importantly, both ME and apelin induce DNA demethylation of the peroxisome proliferator-activated receptor γ coactivator-1α (Ppargc1a) promoter and enhance its expression and mitochondrial biogenesis in fetal muscle. Such changes in DNA methylation were maintained in offspring, with ME offspring muscle expressing higher levels of PGC-1α1/4 isoforms, explaining improved muscle function. In summary, ME enhances DNA demethylation of the Ppargc1a promoter in fetal muscle, which has positive programming effects on the exercise endurance capacity and protects offspring muscle against metabolic dysfunction.

Retinoic acid signalling in fibro/adipogenic progenitors robustly enhances muscle regeneration.

BACKGROUND: During muscle regeneration, excessive formation of adipogenic and fibrogenic tissues, from their respective fibro/adipogenic progenitors (FAPs), impairs functional recovery. Intrinsic mechanisms controlling the proliferation and differentiation of FAPs remain largely unexplored. METHODS: Here, we investigated the role of retinoic acid (RA) signalling in regulating FAPs and the subsequent effects on muscle restoration from a cardiotoxin-induced injury. Blockage of retinoic acid receptor (RAR) signalling was achieved through dominant negative retinoic acid receptor α (RARα403) expression specific in PDGFRα+ FAPs in vivo and by BMS493 treatment in vitro. Effects of RAR-signalling on FAP cellularity and muscle regeneration were also investigated in a high-fat diet-induced obese mice model. FINDINGS: Supplementation of RA increased the proliferation of FAPs during the early stages of regeneration while suppressing FAP differentiation and promoting apoptosis during the remodelling stage. Loss of RAR-signalling caused ectopic adipogenic differentiation of FAPs and impaired muscle regeneration. Furthermore, obesity disrupted the cellular transition of FAPs and attenuated muscle regeneration. Supplementation of RA to obese mice not only rescued impaired muscle fibre regeneration, but also inhibited infiltration of fat and fibrotic tissues during muscle repair. These beneficial effects were abolished after blocking RAR-signalling in FAPs of obese mice. INTERPRETATION: These data suggest that RAR-signalling in FAPs is a critical therapeutic target for suppressing differentiation of FAPs and facilitating the regeneration of muscle and other tissues. FUNDING: This study was supported by grants from the National Institutes of Health (R01-HD067449 and R21-AG049976) to M.D.

Maternal exercise via exerkine apelin enhances brown adipogenesis and prevents metabolic dysfunction in offspring mice.

The obesity rate is rapidly increasing, which has been attributed to lack of exercise and excessive energy intake. Here, we found a previously unidentified explanation, due to lack of maternal exercise. In this study, healthy maternal mice were assigned either to a sedentary lifestyle or to exercise daily, and fetal brown adipose tissue (BAT) development and offspring metabolic health were analyzed. Compared to the sedentary group, maternal exercise enhanced DNA demethylation of Prdm16 promoter and BAT development and prevented obesity of offspring when challenged with a high-energy diet. Apelin, an exerkine, was elevated in both maternal and fetal circulations due to exercise, and maternal administration of apelin mimicked the beneficial effects of exercise on fetal BAT development and offspring metabolic health. Together, maternal exercise enhances thermogenesis and the metabolic health of offspring mice, suggesting that the sedentary lifestyle during pregnancy contributes to the obesity epidemic in modern societies.

Excessive Glucocorticoids During Pregnancy Impair Fetal Brown Fat Development and Predispose Offspring to Metabolic Dysfunctions.

Maternal stress during pregnancy exposes fetuses to hyperglucocorticoids, which increases the risk of metabolic dysfunctions in offspring. Despite being a key tissue for maintaining metabolic health, the impacts of maternal excessive glucocorticoids (GC) on fetal brown adipose tissue (BAT) development and its long-term thermogenesis and energy expenditure remain unexamined. For testing, pregnant mice were administered dexamethasone (DEX), a synthetic GC, in the last trimester of gestation, when BAT development is the most active. DEX offspring had glucose, insulin resistance, and adiposity and also displayed cold sensitivity following cold exposure. In BAT of DEX offspring, Ppargc1a expression was suppressed, together with reduced mitochondrial density, and the brown progenitor cells sorted from offspring BAT demonstrated attenuated brown adipogenic capacity. Increased DNA methylation in Ppargc1a promoter had a fetal origin; elevated DNA methylation was also detected in neonatal BAT and brown progenitors. Mechanistically, fetal GC exposure increased GC receptor/DNMT3b complex in binding to the Ppargc1a promoter, potentially driving its de novo DNA methylation and transcriptional silencing, which impaired fetal BAT development. In summary, maternal GC exposure during pregnancy increases DNA methylation in the Ppargc1a promoter, which epigenetically impairs BAT thermogenesis and energy expenditure, predisposing offspring to metabolic dysfunctions.

Treadmill Running of Mouse as a Model for Studying Influence of Maternal Exercise on Offspring.

Epidemiological studies robustly show the beneficial effects of maternal exercise in reducing maternal birth complications and improving neonatal outcomes, though underlying mechanisms remain poorly understood. To facilitate mechanistic exploration, a protocol for maternal exercise of mice is established, with the regimen following the exercise guidelines for pregnant women. Compared to volunteer wheel running, treadmill running allows precise control of exercise intensity and duration, dramatically reducing variations among individual mouse within treatments and facilitating translation into maternal exercise in humans. Based on the maximal oxygen consumption rate (VO2max) before pregnancy, the treadmill exercise protocol is separated into three stages: early stage (E1.5 to E7.5 at 40% VO2max), mid stage (E8.5 to E14.5 at 65% VO2max), and late stage of pregnancy (E15.5 to birth at 50% VO2max), which demonstrated persistent beneficial effects on maternal health and fetal development. This protocol can be useful for standardizing maternal treadmill exercise using mice as an experimental model.

Even a low dose of tamoxifen profoundly induces adipose tissue browning in female mice.

BACKGROUND: Tamoxifen-inducible Cre/lox site-specific recombination technology has been widely used to generate conditional transgenic mice. As an estrogen receptor ligand, tamoxifen itself potentially affects energy metabolism, which may confound interpretation of data especially in metabolic studies. Considering sexual dimorphism, in this study, the effects of low-dose tamoxifen administration on energy metabolism, and browning of adipose tissues in female and male mice were investigated. METHODS: Female and male C57/BL6 mice were injected with tamoxifen oil solution (i.p.) and then housed at both room temperature (23 ± 2 °C) and cold environment (6 ± 1 °C). Serum, brown and white adipose tissues were obtained, and the effects of tamoxifen administration on energy metabolism and the browning of adipose tissues were evaluated. RESULTS: At 25 mg/kg body weight (BDW), tamoxifen administration for 3 alternative days decreased the percentage of inguinal and gonadal white adipose tissue weights in female mice accompanied by the up-regulation of thermogenesis in adipose tissues. In contrast, this dosage of tamoxifen did not induce noticeable changes in the energy metabolism and thermogenesis of adipose tissue in male mice under room temperature. Consistently, under cold stimulus, substantial browning of adipose tissues was observed in female mice injected with tamoxifen (50 mg/kg BDW, single injection) but not in male mice. Two-way ANOVA tests also demonstrated significant interactions between tamoxifen treatment and gender on the expression of thermogenic markers in adipose tissues. CONCLUSION: Tamoxifen, even at a low dose, remarkably increases thermogenesis in adipose tissues of female mice; meanwhile, such a low dose could be used in male mice for inducing gene recombination without confounding the interpretation of data related to metabolism and thermogenesis of adipose tissues.

Exercise prevents the adverse effects of maternal obesity on placental vascularization and fetal growth.

KEY POINTS: Maternal exercise improves the metabolic health of maternal mice challenged with a high-fat diet. Exercise intervention of obese mothers prevents fetal overgrowth. Exercise intervention reverses impaired placental vascularization in obese mice. Maternal exercise activates placental AMP-activated protein kinase, which was inhibited as a result of maternal obesity. ABSTRACT: More than one-third of pregnant women in the USA are obese and maternal obesity (MO) negatively affects fetal development, which predisposes offspring to metabolic diseases. The placenta mediates nutrient delivery to fetuses and its function is impaired as a result of MO. Exercise ameliorates metabolic dysfunction resulting from obesity, although its effect on placental function of obese mothers has not been explored. In the present study, C57BL/6J female mice were randomly assigned into two groups fed either a control or a high-fat diet (HFD) and then the mice on each diet were further divided into two subgroups with/without exercise. In HFD-induced obese mice, daily treadmill exercise during pregnancy reduced body weight gain, lowered serum glucose and lipid concentration, and improved insulin sensitivity of maternal mice. Importantly, maternal exercise prevented fetal overgrowth (macrosomia) induced by MO. To further examine the preventive effects of exercise on fetal overgrowth, placental vascularization and nutrient transporters were analysed. Vascular density and the expression of vasculogenic factors were reduced as a result of MO but were recovered by maternal exercise. On the other hand, the contents of nutrient transporters were not substantially altered by MO or exercise, suggesting that the protective effects of exercise in MO-induced fetal overgrowth were primarily a result of the alteration of placental vascularization and improved maternal metabolism. Furthermore, exercise enhanced downstream insulin signalling and activated AMP-activated protein kinase in HFD placenta. In sum, maternal exercise prevented fetal overgrowth induced by MO, which was associated with improved maternal metabolism and placental vascularization in obese mothers with exercise.

Neonatal vitamin A injection promotes cattle muscle growth and increases oxidative muscle fibers.

BACKGROUND: Vitamin A and its metabolite, retinoic acid (RA), are important regulators of cell differentiation and organ morphogenesis. Its impact on beef cattle muscle growth remains undefined. METHOD: Angus steer calves were administrated with 0 (control) or 150,000 IU vitamin A (retinyl palmitate in glycerol, i.m.) per calf at birth and 1 month of age. At 2 months of age, a biopsy of the Biceps femoris muscle was obtained to analyze the immediate effects of vitamin A injection on myogenic capacity of muscle cells. The resulting steers were harvested at 14 months of age. RESULTS: Vitamin A administration increased cattle growth at 2 months. At 2 months of age, Vitamin A increased PAX7 positive satellite cells and the expression of myogenic marker genes including PAX7, MYF5, MYOD and MYOG. Muscle derived mononuclear cells were further isolated and induced myogenesis in vitro. More myotubes and a higher degree of myogenesis was observed in vitamin A groups. Consistently, vitamin A increased Latissimus dorsi (LD) muscle fiber size at harvest. In addition, vitamin A increased the ratio of oxidative type I and type IIA fibers and reduced the glycolic type IIX fibers. Furthermore, we found that RA, a key bioactive metabolite of vitamin A, activated PPARGC1A promoter, which explains the upregulated expression of PPARGC1A in skeletal muscle. CONCLUSION: Vitamin A administration to neonatal calves enhanced postnatal muscle growth by promoting myogenesis and increasing satellite cell density, accompanied with a shift to oxidative muscle fibers.

Raspberry promotes brown and beige adipocyte development in mice fed high-fat diet through activation of AMP-activated protein kinase (AMPK) α1.

Development of brown and beige/brite adipocytes increases thermogenesis and helps to reduce obesity and metabolic syndrome. Our previous study suggests that dietary raspberry can ameliorate metabolic syndromes in diet-induced obese mice. Here, we further evaluated the effects of raspberry on energy expenditure and adaptive thermogenesis and determined whether these effects were mediated by AMP-activated protein kinase (AMPK). Mice deficient in the catalytic subunit of AMPKα1 and wild-type (WT) mice were fed a high-fat diet (HFD) or HFD supplemented with 5% raspberry (RAS) for 10 weeks. The thermogenic program and related regulatory factors in adipose tissue were assessed. RAS improved the insulin sensitivity and reduced fat mass in WT mice but not in AMPKα1-/- mice. In the absence of AMPKα1, RAS failed to increase oxygen consumption and heat production. Consistent with this, the thermogenic gene expression in brown adipose tissue and brown-like adipocyte formation in subcutaneous adipose tissue were not induced by RAS in AMPKα1-/- mice. In conclusion, AMPKα1 is indispensable for the effects of RAS on brown and beige/brite adipocyte development, and prevention of obesity and metabolic dysfunction.

Moderate alcohol intake induces thermogenic brown/beige adipocyte formation via elevating retinoic acid signaling.

Clinically, low and moderate alcohol intake improves human health with protection against metabolic syndromes, including type 2 diabetes; however, mechanisms that are associated with these effects remain to be elucidated. The aims of this study were to investigate the effects of moderate alcohol intake on thermogenic brown/beige adipocyte formation and glucose and lipid homeostasis, as well as the involvement of retinoic acid (RA) signaling in the entire process. C57BL6 male mice were supplemented with 8% (w/v) alcohol in water for 1 or 4 mo. Alcohol intake prevented body weight gain, induced the formation of uncoupling protein 1-positive beige adipocytes in white adipose tissue, and increased thermogenesis in mice, which is associated with decreased serum glucose and triacylglycerol levels. Mechanistically, alcohol intake increased RA levels in serum and adipose tissue, which was associated with increased expression of aldehyde dehydrogenase family 1 subfamily A1 (Aldh1a1). When RA receptor-α signaling was conditionally blocked in platelet-derived growth factor receptor-α-positive adipose progenitors, the effects of alcohol on beige adipogenesis were largely abolished. Finally, moderate alcohol prevented high-fat diet-induced obesity and metabolic dysfunction. In conclusion, moderate alcohol intake induces thermogenic brown/beige adipocyte formation and promotes glucose and lipid oxidation via elevation of RA signaling.-Wang, B., Wang, Z., de Avila, J. M., Zhu, M.-J., Zhang, F., Gomez, N. A., Zhao, L., Tian, Q., Zhao, J., Maricelli, J., Zhang, H., Rodgers, B. D., Du, M. Moderate alcohol intake induces thermogenic brown/beige adipocyte formation via elevating retinoic acid signaling.

Maternal Retinoids Increase PDGFRα+ Progenitor Population and Beige Adipogenesis in Progeny by Stimulating Vascular Development.

Maternal vitamin A intake varies but its impact on offspring metabolic health is unknown. Here we found that maternal vitamin A or retinoic acid (RA) administration expanded PDGFRα+ adipose progenitor population in progeny, accompanied by increased blood vessel density and enhanced brown-like (beige) phenotype in adipose tissue, protecting offspring from obesity. Blockage of retinoic acid signaling by either BMS493 or negative RA receptor (RARαDN) over-expression abolished the increase in blood vessel density, adipose progenitor population, and beige adipogenesis stimulated by RA. Furthermore, RA-induced beige adipogenesis was blocked following vascular endothelial growth factor receptor (VEGFR) 2 knock out in PDGFRα+ cells, suggesting its mediatory role. Our data reveal an intrinsic link between maternal retinoid level and offspring health via promoting beige adipogenesis. Thus, enhancing maternal retinoids is an amiable therapeutic strategy to prevent obesity in offspring, especially for those born to obese mothers which account for one third of all pregnancies.

VEGFA family isoforms regulate spermatogonial stem cell homeostasis in vivo.

The objective of the present study was to investigate vascular endothelial growth factor A (VEGFA) isoform regulation of cell fate decisions of spermatogonial stem cells (SSC) in vivo. The expression pattern and cell-specific distribution of VEGF isoforms, receptors, and coreceptors during testis development postnatal d 1-180 suggest a nonvascular function for VEGF regulation of early germ cell homeostasis. Populations of undifferentiated spermatogonia present shortly after birth were positive for VEGF receptor activation as demonstrated by immunohistochemical analysis. Thus, we hypothesized that proangiogenic isoforms of VEGF (VEGFA(164)) stimulate SSC self-renewal, whereas antiangiogenic isoforms of VEGF (VEGFA(165)b) induce differentiation of SSC. To test this hypothesis, we used transplantation to assay the stem cell activity of SSC obtained from neonatal mice treated daily from postnatal d 3-5 with 1) vehicle, 2) VEGFA(164), 3) VEGFA(165)b, 4) IgG control, 5) anti-VEGFA(164), and 6) anti-VEGFA(165)b. SSC transplantation analysis demonstrated that VEGFA(164) supports self-renewal, whereas VEGFA(165)b stimulates differentiation of mouse SSC in vivo. Gene expression analysis of SSC-associated factors and morphometric analysis of germ cell populations confirmed the effects of treatment on modulating the biological activity of SSC. These findings indicate a nonvascular role for VEGF in testis development and suggest that a delicate balance between VEGFA(164) and VEGFA(165)b isoforms orchestrates the cell fate decisions of SSC. Future in vivo and in vitro experimentation will focus on elucidating the mechanisms by which VEGFA isoforms regulate SSC homeostasis.

Analysis of gene expression in bovine testis tissue prior to ectopic testis tissue xenografting and during the grafting period.

The purpose of this study was to identify factors that contribute to bovine testis development and donor age-dependent differences in the abilities of bovine ectopic testis tissue grafts to produce elongated spermatids. We used real-time RT-PCR and microarrays to evaluate and to identify the expression of genes that are involved in Sertoli and germ cell development in bovine testis tissues. Testis tissues were obtained from 2-, 4-, and 8-wk-old bull calves and were grafted immediately. Grafted bovine testis tissue was removed from mice, RNA was isolated from the grafts, and real-time RT-PCR was used to evaluate gene expression during the grafting period. In addition, the gene expression in the donor tissue was analyzed using Affymetrix Bovine GeneChips, to identify differentially expressed genes. Examination of the testis tissue grafts indicated that Sertoli cell-specific gene expression was lower in 8-wk donor tissue grafts compared to the donors of other ages. Furthermore, the expression of KIT, which is a germ cell-specific gene, was low in testis tissue grafts. Microarray analysis of the donor tissue showed that several genes that are involved in angiogenesis or tissue growth were differentially expressed in 2-, 4-, and 8-wk-old bovine testes. The levels of expression of the genes for angiogenin, transgelin, thrombomodulin, early growth response 1, insulin-like growth factor 2, and insulin-like growth factor-binding protein 3 were lower in testis tissues from older animals. Using these data, it will be possible in the future to manipulate the testis xenograft microenvironment so as to improve the efficiency of sperm production within the graft.

Grafting period and donor age affect the potential for spermatogenesis in bovine ectopic testis xenografts.

Bovine testis tissue xenografts contain elongating spermatids 6 mo after grafting. The percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal varies depending on donor age and rarely exceeds 10%. These data indicate significant changes are occurring to bovine testicular cells during the first weeks of life. The objective of this research was to xenograft testis tissue from multiple ages of bull calves for 24 or 36 wk in order to gain a better understanding of early bovine testis development. Testis tissue from 1-, 2-, 4-, and 8-wk-old calves was grafted onto the backs of castrated immunodeficient mice. Testis tissue from all donor ages grew, differentiated, and produced testosterone and elongating spermatids. Testis tissue grafts from 1- and 8-wk-old calves had elongating spermatids in greater than 5.5% of seminiferous tubule cross sections at the time of graft removal regardless of grafting period. Four-week-old donor tissue never had more than 5.2% of seminiferous tubule cross sections with elongating spermatids. Extending the grafting period from 24 to 36 wk resulted in an increase in the percentage of seminiferous tubule cross sections with elongating spermatids from 2% to 10% in 2-wk donor tissue. These data demonstrate that both donor age and grafting period may be important factors regulating the maturation of bovine testis xenografts, indicating that intrinsic differences exist within testis tissue at these donor ages. These data provide the framework for further study of bovine spermatogenesis using ectopic testis xenografting.