Sertoli Cell Tumour and Uterine Leiomyoma in Miniature Schnauzer Dogs with Persistent Müllerian Duct Syndrome Caused by Mutation in the AMHR2 Gene.

Disorders of sex development (DSD) are a serious health problem in dogs. Different types of DSD have been described, including persistent Müllerian duct syndrome (PMDS), for which the molecular background has been identified in miniature schnauzers. Human patients with PMDS are at increased risk for cancers of the gonads (predominantly) or the Müllerian duct structures (rarely). This report describes two miniature schnauzer dogs with PMDS caused by a known nonsense mutation in the AMHR2 gene, with concurrent development of genital neoplasia. The first case (78,XY and SRY-positive) had unilateral cryptorchidism and a Sertoli cell tumour in the abdominal testicle. The second case (mosaic karyotype 77,XY,rob/78,XY and SRY-positive) had both gonads descended in the scrotum and developed an abdominal mass derived from the uterine wall, which showed histological features typical of leiomyoma.

Age and anti-Müllerian hormone levels predict the success of in vitro maturation of cat oocytes.

Up to date, in vitro maturation (IVM) rates of oocytes are highly variable between individual cats. This study was carried out to investigate the predictive value of age and anti-Müllerian hormone (AMH) concentration in relation to capacity for IVM of cat oocytes. Ovaries were collected from 33 cats, which were divided into three age groups: (i) 0-3 months (pre-pubertal); (ii) 3-12 months (peripubertal); and (iii) older than 12 months (pubertal). The cumulus-oocyte complexes (COCs) were matured and subsequently stained to check nuclear maturation status, and blood was taken for AMH analysis. Increasing age was significantly associated with decreasing AMH levels, and mean AMH levels differed significantly between all age categories: group 1: mean AMH 18.71 µg/L; group 2: mean AMH 9.27 µg/L; and group 3: mean AMH 4.13 µg/L. Moreover, the probability of maturation was more likely in groups 2 and 3 compared to group 1. Between categories 2 and 3, no significant difference in maturation probability was found (p = .31). Finally, the probability of oocyte maturation decreased significantly with increasing AMH levels. In age group 2, oocytes with a higher AMH level were less likely to mature. In age groups 1 and 3, no significant association between the AMH level and the proportion of maturated COC was found. We can conclude that if a higher probability of nuclear maturation is required, it is preferable to use cats with lower AMH levels and older than 3 months of age to improve cat IVM.

Replacing serum in culture medium with albumin and insulin, transferrin and selenium is the key to successful bovine embryo development in individual culture.

Individual culture of bovine embryos is usually associated with low blastocyst development. However, during preliminary experiments in our laboratory we observed high blastocyst development after individual embryo culture in a serum-free culture system. We therefore hypothesised that serum has a negative effect on embryos cultured individually whereas embryos in groups can counteract this. First, we determined whether the timing of removal of serum (during maturation or culture) had an influence on individual embryo development. The results clearly showed that removal of serum during embryo culture was the main contributing factor since high blastocyst development was observed after individual culture in synthetic oviductal fluid supplemented with bovine serum albumin (BSA) and insulin, transferrin and selenium (ITS), independent of the maturation medium. Second, we investigated whether an individual factor of the ITS supplement was essential for individual embryo development. We demonstrated that repeatable high blastocyst percentages were due to the synergistic effect of ITS. Finally, we investigated if a group-culture effect can still be observed under serum-free conditions. Group culture generated blastocysts with higher total cell numbers and less apoptosis. These data show that individual culture in serum-free conditions leads to high blastocyst development, but group culture still improves blastocyst quality.

The importance of adequate fixation for immunofluorescent staining of bovine embryos.

Immunofluorescent staining is often used to investigate the expression of specific proteins in pre-implantation embryos. The success of this method is determined by the specificity of the antibodies, but also by the protocol used for fixation and permeabilization of the samples. In this study, different fixatives are compared in combination with immunofluorescent staining of caudal-type homeobox 2 (CDX2), fibronectin 1 (FN1) and integrins (ITGs) on bovine blastocysts. For both CDX2 and the ITGs, the outcome of the staining was largely dependent on the fixation methods. Paraformaldehyde fixation was best for the intracellular CDX2 protein, whereas acetone fixation gave the best results for the transmembrane ITGs. No difference was observed for the FN1 staining between samples fixed with paraformaldehyde or acetone. These examples demonstrate that the choice of fixation and permeabilization agents is very important for the outcome of the experiment, and this choice is dictated by the (extra)cellular location of the protein under investigation. Inappropriate fixation and/or permeabilization methods can lead to erroneous conclusions regarding the site and amount of protein expression.