Asja Wagener

Semen cryopreservation and the conservation of endangered species

Currently, more than 16,000 plant and animal species are imminently threatened by extinction, often as a direct consequence of anthropogenic influences. One of the measures to halt that process is genetic resource banking. This short review focuses on mammal sperm cryopreservation in combination with assisted reproduction techniques. It summarizes general problems, recent developments, and currently applied protocols and gives an overview of hitherto existing successes of assisted reproduction measures in wild animals in the light of conservation efforts.

High-fat diet leads to tissue-specific changes reflecting risk factors for diseases in DBA/2J mice.

The aim of this study was to characterize the responses of individual tissues to high-fat feeding as a function of mass, fat composition, and transcript abundance. We examined a panel of eight tissues [5 white adipose tissues (WAT), brown adipose tissue (BAT), liver, muscle] obtained from DBA/2J mice on either a standard breeding diet (SBD) or a high-fat diet (HFD). HFD led to weight gain, decreased insulin sensitivity, and tissue-specific responses, including inflammation, in these mice. The dietary fatty acids were partially metabolized and converted in both liver and fat tissues. Saturated fatty acids (SFA) were converted in the liver to monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA), and oleic acid (C18:1) was the preferred MUFA for storage of excess energy in all tissues of HFD-fed mice. Transcriptional changes largely reflected the tissue-specific fat deposition. SFA were negatively correlated with genes in the collagen family and processes involving the extracellular matrix. We propose a novel role of the tryptophan hydroxylase 2 (Tph2) gene in adipose tissues of diet-induced obesity. Tissue-specific responses to HFD were identified. Liver steatosis was evident in HFD-fed mice. Gonadal, retroperitoneal and subcutaneous adipose tissue and BAT exhibited severe inflammatory and immune responses. Mesenteric adipose tissue was the most metabolically active adipose tissue. Gluteal adipose tissue had the highest mass gain but was sluggish in its metabolism. In HFD conditions, BAT functioned largely like WAT in its role as a depot for excess energy, whereas WAT played a role in thermogenesis.

Detection of growth factors in the testis of roe deer (Capreolus capreolus).

Roe deer is a seasonal breeder characterised by a short rutting season in summer. Mature males show synchronised cycles of testicular involution and recrudescence. Therefore, this species is a valuable model to study seasonal regulation of spermatogenesis in ruminants. It is hypothesised that a time-dependent production of testicular growth factors is required to regulate seasonal changes in testis growth and spermatogenesis. To identify potential candidates, total RNA from roe deer testis tissue was extracted at three different seasonal periods (April, August, December), and using RT-PCR the presence of several growth factors (aFGF, bFGF, IGF-I, IGF-II, TGF-alpha, TGF-beta1, TGF-beta3 and two isoforms of VEGF) was detected. Sequencing of the growth factor PCR fragments revealed a high sequence homology between cattle and roe deer. To further explore the expression patterns of the identified growth factors in roe deer their expression levels were standardised using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression. The study demonstrates the expression of several growth factors in roe deer testis and supports the assumption of their seasonally diverse regulation. These results provide the basis to investigate the role of growth factors in the regulation of circannual changes of testicular activity.

High energy digestion efficiency and altered lipid metabolism contribute to obesity in BFMI mice.

To constitute a valuable resource to identify individual genes involved in the development of obesity, a novel mouse model, the Berlin Fat Mouse Inbred line 860 (BFMI860), was established. In order to characterize energy intake and energy expenditure in obese BFMI860 mice, we performed two independent sets of experiments in male BFMI860 and B6 control mice (10 per line). In experiment 1, we analyzed body fat content noninvasively by dual‐energy X‐ray absorptiometry and measured resting metabolic rate at thermoneutrality (RMRt) and respiratory quotient (RQ) in week 6, 10, and 18. In a second experiment, energy digested (energy intake minus fecal energy loss) was determined by bomb calorimetry from week 6 through week 12. BFMI860 mice were heavier and had higher fat mass (final body fat content was 24.7% compared with 14.6% in B6). They also showed fatty liver syndrome. High body fat accumulation in BFMI860 mice was restricted to weeks 6–10 and was accompanied by hyperphagia, higher energy digestion, higher RQs, and abnormally high blood triglyceride levels. Lean mass–adjusted RMRt was not altered between lines. These results indicate that in BFMI860 mice, the excessive accumulation of body fat is associated with altered lipid metabolism, high energy intake, and energy digestion. Assuming that BFMI860 mice and their obese phenotypes are of polygenic nature, this line is an excellent model for the study of obesity in humans, especially for juvenile obesity and hyperlipidemia.

Differential Changes in Expression of a and b FGF, IGF-1 and -2, and TGF-α During Seasonal Growth and Involution of Roe Deer Testis

Growth factors are involved in the regulation of testicular growth and involution of seasonal breeders. Therefore, we studied the seasonal expression of several growth factors in roe deer: aFGF, bFGF, IGF-1, IGF-2, and TGF- !. Total RNA from testis tissue was extracted monthly and analyzed using quantitative RT-PCR. Localization of mRNAs was examined by in situ -hybridization. Levels of expression differed by more than three orders of magnitude. Expression also showed different seasonal patterns. IGF-1, IGF-2 and bFGF were maximally transcribed during testis recrudescence in spring. In contrast, the mRNA amount of aFGF reached its maximum between July (breeding season) and January. TGF- ! mRNA-levels were very low and showed poor seasonal variation. Each growth factor showed its own typical expression localization in testicular tissues and cell types. The results suggest the specific role of different growth factors in the paracrine control of spermatogenesis and its seasonal regulation.

Changes in metabolite profiles caused by genetically determined obesity in mice

The Berlin Fat Mouse Inbred (BFMI) line harbors a major recessive gene defect on chromosome 3 (jobes1) leading to juvenile obesity and metabolic syndrome. The present study aimed at the identification of metabolites that might be linked to recessively acting genes in the obesity locus. Firstly, serum metabolites were analyzed between obese BFMI and lean B6 and BFMI × B6 F1 mice to identify metabolites that are different. In a second step, a metabolite–protein network analysis was performed linking metabolites typical for BFMI mice with genes of the jobes1 region. The levels of 22 diacyl-phosphatidylcholines (PC aa), two lyso-PC and three carnitines were found to be significantly lower in obese mice compared with lean mice, while serine, glycine, arginine and hydroxysphingomyelin were higher for the same comparison. The network analysis identified PC aa C42:1 as functionally linked with the genes Ccna2 and Trpc3 via the enzymes choline kinase alpha and phospholipase A2 group 1B (PLA2G1B), respectively. Gene expression analysis revealed elevated Ccna2 expression in adipose tissue of BFMI mice. Furthermore, unique mutations were found in the Ccna2 promoter of BFMI mice which are located in binding sites for transcription factors or micro RNAs and could cause differential Ccna2 mRNA levels between BFMI and B6 mice. Increased expression of Ccna2 was consistent with higher mitotic activity of adipose tissue in BFMI mice. Therefore, we suggest a higher demand for PC necessary for adipose tissue growth and remodeling. This study highlights the relationship between metabolite profiles and the underlying genetics of obesity in the BFMI line.

Early and late onset of voluntary exercise have differential effects on the metabolic syndrome in an obese mouse model.

In a mouse model for juvenile obesity, we investigated how the age of onset of voluntary exercise affects factors of the metabolic syndrome. One exercise group had access to running wheels from 3 weeks (representing childhood) and another one from 9 weeks on (early adulthood). Both groups were compared to mice without exercise. The investigations were performed under 2 diets (standard maintenance and high-fat diet). Average daily running activity was independent of diet and exercise. On both diets, mice with exercise from 3 weeks on gained 10 g body weight and 5 g fat mass less than mice without exercise. The highest body weight difference between mice on HFD without exercise and mice on standard maintenance diet with exercise was 24 g. Despite the higher energy expenditure during exercise, young mice did not increase their energy intake adjusted for lean mass, while mice with exercise from 9 weeks had an increased energy intake of 6 kJ per day and therefore could not reduce fat mass on both diets. However, mice with exercise from 9 weeks had better glucose tolerance at 20 weeks than mice with exercise from childhood on. Independently of the age of exercise onset, triglycerides were reduced from 2.4 to 1.7 mmol/l on both diets and insulin levels from 1.5 to 0.3 and 4.5 to 1.8 µg/ml on standard maintenance and high-fat diet, respectively, which represents a considerable improvement. Physical activity seems to have long-lasting effects on body composition and health, but they are different depending on when exercise has begun.

Features of the metabolic syndrome in the Berlin Fat Mouse as a model for human obesity.

Background: The Berlin Fat Mouse BFMI860 is a polygenic obesity mouse model which harbors a natural major gene defect resulting in early onset of obesity. To elucidate adult bodily responses in BFMI860 mice that develop juvenile obesity, we studied features of the metabolic syndrome at 20 weeks. Methods: We examined fat deposition patterns, adipokines, lipid profiles in serum, glucose homeostasis, and insulin sensitivity in mice that were fed either a standard maintenance (SMD) or a high-fat diet (HFD). Results: Like many obese humans, BFMI860 mice showed hyperleptinemia accompanied by hypoadiponectinemia already at SMD that was further unbalanced as a result of HFD. Furthermore, BFMI860 mice had high triglyceride concentrations. However, triglyceride clearance after an oral oil gavage was impaired on SMD but improved on HFD. The oral and intraperitoneal glucose as well as the insulin tolerance tests provided evidence for reduced insulin sensitivity under SMD and insulin resistance on HFD. BFMI860 mice can maintain normal glucose clearance over a wide range of feeding conditions according to an adaptation via increasing the insulin concentrations. Conclusions: BFMI860 mice show obesity, dyslipidemia, and insulin resistance as three major components of the metabolic syndrome. As these mice develop the described phenotype as a result of a major gene defect, they are a unique model for the investigation of genetic and pathophysiological mechanisms underlying the observed features of the metabolic syndrome and to search for potential strategies to revert the adverse effects under controlled conditions.

Reproductive fitness in roe bucks (Capreolus capreolus): seasonal timing of testis function

The exact seasonal timing of normal testis function is a crucial precondition for the reproductive fitness of roe bucks and for successful breeding during rut in July–August. Production of spermatozoa and testosterone requires both endocrine regulation and local testicular control by autocrine/paracrine factors. These local control mechanisms include the action of several growth factors. Our short review assigns histological organization of roe deer testis to new data on the involvement of several growth factors in its regulation. The expression of growth factors is season-specific and cell-type-specific. This suggests its functional role in the complex interaction between germinative and somatic cells for the regulation of testis growth, spermatogenesis and function of hormone-producing cells.

Circannual changes in the expression of vascular endothelial growth factor in the testis of roe deer (Capreolus capreolus).

Adult roe deer males show seasonal cycles of testicular growth and involution. The exact timing of these cycles requires endocrine regulation and local testicular control by autocrine/paracrine factors. Recent findings suggest that the vascular endothelial growth factor (VEGF) might have effects on both vascular and germinative cells in testis. Thus, we studied the expression pattern of vascular endothelial growth factor (VEGF) in roe deer testis using quantitative RT-PCR. The strength of VEGF mRNA expression depended on season. It reached its highest level at the peak of spermatogenesis during the pre-rutting period and had its nadir at the end of the rut when involution already began. The results suggested that VEGF may directly affect the regulation of spermatogenesis but may not be involved predominantly in testicular microvasculature as initially expected.