# Impact of a maternal diabetes on embryonic DNA methylation in rabbit blastocyst

K. J. Grybel, Eugénie Canon, J. Knelangen, D. Kradolfer, M. Pendzialek, M. Schindler, J. Guerke, T. Blachere, Murielle Godet, R. Obeid, et al.

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## To cite this version:

K. J. Grybel, Eugénie Canon, J. Knelangen, D. Kradolfer, M. Pendzialek, et al., Impact of a maternal diabetes on embryonic DNA methylation in rabbit blastocyst. 50. Annual Conference of Physiology and Pathology of Reproduction, Deutsche Veterinärmedizinische Gesellschaft. DEU., 2017, Munich, Germany. 64 p., 10.1111/rda.12909. hal-01606710

HAL Id: hal-01606710

https://hal.science/hal-01606710

Submitted on 2 Jun 2020

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43 | Impact of a maternal diabetes on embryonic DNA methylation in rabbit blastocyst Einfluss eines mütterlichen Diabetes mellitus auf die DNA-Methylierung des Embryos beim Kaninchen

KJ Grybel<fnref n="1" />; E Canon²; J Knelangen<fnref n="1" />; D Kradolfer³;
M Pendzialek<fnref n="1" />; M Schindler<fnref n="1" />; J Gürke<fnref n="1" />; T Blachère⁴;
M Godet⁴; R Obeid⁵; S Ulbrich³; B Fischer<fnref n="1" />; V Duranthon²;
A Navarrete Santos<fnref n="1" />

In early pregnancy maternal diabetes leads to delay in embryo devel-
opment and changes in nutritional and hormonal signals of the uterine
environment. The current study focuses on consequences of maternal
diabetes on embryonic tissue formation and its DNA methylation.
Therefore we investigated the expression and promoter methylation
of epiblast lineage specifier Oct4 in 6 day old rabbit blastocysts at
early gastrulation stage. The expression of Oct4 was higher in epi-
blast of diabetic blastocysts, accompanied by upregulation of Nanog
and Sox2. Specific methylation of the POU5F1 (Oct4) promoter
region was investigated by bisulphite sequencing. The Oct4 promoter
was hypomethylated in hypoblasts and trophoblasts of diabetic rab-
bits, implying also a mark of delay in differentiation. The global DNA
methylation of male and female blastocysts from diabetic and healthy
rabbits was examined, employing embryoblast and trophoblast tis-
sue separately, using LUminometric Methylation Assay (LUMA).
No significant changes in global DNA methylation were observed.
Furthermore we verified that concentrations of methyl group donor
S-adenosyl methionine (SAM) and the product of the methylation
reactions S-adenosylhomocysteine (SAH) were changed in diabetic
pregnancy. SAM and SAH were measured by use of a modified liquid
chromatography-tandem mass spectrometry in rabbit blood plasma
collected at the day 6 post-coitum. Our data showed that maternal
diabetes mellitus affects the Oct4 promoter methylation in a specific
way with consequences for Oct4 gene transcription and embryo
development. Alteration of Oct4 methylation was not caused by
global methylation changes.

44 | Urospermia in an Entlebucher Mountain Dog with ectopic ureters—a case report Urospermie bei einem Entlebucher Sennenhund mit ektopischen Ureteren—Ein Fallbericht

AR Günzel-Apel<fnref n="1" />,²; K Wolf<fnref n="1" />,²; A Baumeister<fnref n="1" />,²; JP Bach²;
S Hungerbühler²

Urospermia/US may result from retarded closure of the urinary blad-
der neck during semen emission due to local inflammation or insuf-
ficient •-adrenergic stimulation of the smooth muscle layers. By this
urine passes the bladder neck when pre-secretion and the sperm rich
fraction are reaching the pelvic urethra. In the present case US is sus-
pected to be the result of a bilateral ectopic ureter (EU), a congenital
malformation, with the ureteral orifice terminating intravesically near
the bladder neck or extravesically at the prostate, deferent duct, or
urethra. Case report: A 4-year-old Entlebucher Mountain Dog was
presented for breeding soundness evaluation due to an unsuccessful
mating 5 months ago. At the age of 2 years an abnormal course
of both ureters in the area of the bladder neck had been diagnosed
sonographically. As the finding was not classified as serious, the dog
had been approved for breeding. The current andrological exami-
nation revealed both testes and epididymides of adequate size and
shape. The collected ejaculate showed normal volume and total sperm
count, but severe deficiencies in sperm motility, plasma and acrosome
membrane integrity and sperm morphology. The most striking mac-
roscopic finding was a yellowish appearance showing continuously
increasing intensity in the watery prostatic secretion. Odour and pH-
value revealed admixture of urine. A repeat ultrasound examination
confirmed a bilateral intramural EU entering the urinary bladder about
1 cm cranial of the vesico-urethral transition with the opening point-
ing caudally. This finding may not per se indicate a causal connec-
tion between EU and US. Nevertheless the latter may be a functional
indicator of a urine leakage possibly existing as additional congenital
abnormality in the caudal vesical/cranial urethral area.

45 | Factors affecting semen evaluation of native and frozen-thawed goat semen Einflussfaktoren auf spermatologische Untersuchungsergebnisse und den Erfolg der Kryokonservierung von Ziegensperma

K Hahn<fnref n="1" />; A Wehrend²

Parameters collected during semen evaluation are the basis to estab-
lish the suitability of an ejaculate. Therefore, the conditions must be
kept as constant as possible. The aim of this study was to evaluate
possible factors affecting semen analysis of native and frozen-thawed
goat semen. A total of 20 ejaculates from 5 healthy peacock goat
bucks were obtained using an artificial vagina. After a macroscopic
evaluation 2 aliquots were assessed at different handling tempera-
tures (20 and 37 °C) and time points (TP) (3, 10, 15 and 30 min. after
collection). The pH of each sample was evaluated with indicator-
paper every 10 min. for 1 h. Density and progressive motility (PM)
were assessed using computer assisted sperm analysis (AndroVision®,
Minitüb, Germany). Evaluation of semen viability was performed using
bromophenol-nigrosine staining. For cryopreservation, a commercial

<footnote n="1">¹Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, Halle, Germany; ²INRA, UMR, Department of the Reproductive Biology, Jouy-en-Josas, France; ³Institute of Agricultural Sciences, Zürich, Switzerland; ⁴Stem-cell and Brain Research Institute, Bron, France and University of Lyon, Université Lyon I, Lyon, France; ⁵Department of Clinical Chemistry, University Hospital of the Saarland, Homburg, Germany</footnote>

<footnote n="1">¹Unit for Reproductive Medicine of Clinics; ²Small Animal Clinic, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany</footnote>

<footnote n="1">¹Clinic for Horses, Medicine and Reproduction, LMU Munich, Germany; ²Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals with Ambulatory Service, JLU Giessen, Germany</footnote>