Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat

In vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms "PI3K-Akt", "transforming growth factor-β receptor", "ErbB", and "HIF-1" from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat.Peer reviewe

Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat

In vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms "PI3K-Akt", "transforming growth factor-beta receptor", "ErbB", and "HIF-1" from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat

Short-term preservation of canine preantral follicles : effects of temperature, medium and time

The use of the large pool of preantral follicles is a promising alternative to provide high numbers of fertilizable oocytes to reproductive biotechnology. This issue is particularly important to canids, since current rates of success of in vitro techniques using oocytes are very limited, and many species within this family are threatened by extinction. The aim of this study was to evaluate effects of temperature, medium and time on morphology and viability of canine preantral follicles during short-term preservation. Canine ovaries were cut into fragments which were incubated in 0.9% NaCl solution or in minimum essential medium (MEM) at 4, 20 or 38 °C for 2, 6, 12 or 24 h. Afterwards, preantral follicles were analyzed by histology, transmission electron microscopy and viability testing using trypan blue, calcein-AM and ethidium homodimer-1. Percentages of morphological normal and viable follicles were maintained similar to control (time 0 h) after incubation in 0.9% NaCl at 4 or 20 °C for up to 6 h and at 38 °C for 2 h. Using MEM, such preservation was possible for 12 h at 4 or 20 °C, and for 6 h at 38 °C. These results indicate that preservation of canine preantral follicles might be better accomplished through hypothermic (4 or 20 °C) storage in MEM, which ensures maintenance of morphology and viability for up to 12 h

Comparison of Different Materials for Self-Pressurized Vitrification of Feline Oocytes—First Results

Cryobanking is a crucial part on species conservation. Nowadays, there is no suitable protocol for vitrification of feline oocytes. Self-pressurized rapid freezing of different cell types proved to mimic the advantages of high pressure freezing. As this method could also be applied for gamete rescue under field conditions, the aim here was to analyse the impact of self-pressurized vitrification on feline cumulus-oocyte-complexes (COCs) and to determine the appropriate material. Therefore, COCs of domestic cat were randomly vitrified (n = 189) in metal tubes of different materials: Aluminium, silver, and titanium. No significant differences were found on oocytes’ competence after thawing. On average, 44% of the COCs presented normal morphology and 48.2% of them showed a polar body after in vitro maturation (IVM) and were subsequently fertilised. Aluminium tubes were positive on toxicity tests, producing the lowest cleavage rates. Silver tubes showed no toxic effect, but the cleavage rate was lower than with titanium tubes, and a previous association with embryotoxicity and biological alterations makes us aware of its indiscriminate use. Titanium seems to be the only inert material of them, presenting a slightly higher maturation (55.6%) and cleavage (20%) rates. Nevertheless, more studies should follow to increase embryo competence after warming

Luteinizing Hormone Effect on Luteal Cells Is Dependent on the Corpus Luteum Stage in Felids

The objective of this study was to investigate the effect of luteinizing hormone (LH) on steroidogenic luteal cells obtained from corpora lutea (CL) of the domestic cat and selected wild felids. Luteal cells were isolated enzymatically from CL at different developmental stages and cultured for two days in the presence and absence of 100 ng/mL LH, respectively. Functionality was assessed by progesterone (P4) accumulation in cell culture media determined by ELISA. In addition, steroidogenic function was confirmed using immunohistochemistry for 3β-hydroxysteroid dehydrogenase (HSD3B). The enzymatic method allowed for the isolation of mostly small luteal cells in all investigated felids. Treatment with LH resulted in an increase in P4 secretion of cultured luteal cells obtained from CL in the formation stage (African lion) and development/maintenance stage (domestic cat (p < 0.05), Javan leopard), whereas luteal cells from more advanced stages of luteal development (regression) responded moderately or not at all to LH stimulation (domestic cat, Asiatic golden cat, Asiatic lion). The protein signal for HSD3B on CL was visible until development/maintenance. In conclusion, this study shows that LH promotes P4 production in luteal cells only until the onset of regression, when morphological signs are visible on the CL of felids and HSD3B is no longer detectable

Animal Housing and Welfare: Effects of Housing Conditions on Body Weight and Cortisol in a Medium-Sized Rodent (Cavia aperea)

Schumann K, Günther A, Jewgenow K, Trillmich F. Animal Housing and Welfare: Effects of Housing Conditions on Body Weight and Cortisol in a Medium-Sized Rodent (Cavia aperea). Journal of Applied Animal Welfare Science. 2014;17(2):111-124.Rodents are the most abundant experimental nonhuman animals and are commonly studied under standard laboratory housing conditions. As housing conditions affect animals' physiology and behavior, this study investigated the effects of indoor and outdoor housing conditions on body weight and cortisol level of wild cavies, Cavia aperea. The changing housing condition strongly influenced both parameters, which are commonly used as indicators for animal welfare. The transfer from outdoor to indoor enclosures resulted in a body-weight loss of about 8%. In contrast, animals kept indoors showed a substantial weight gain of about 12% when they were transferred outdoors. These effects were reversible. To substantiate a connection between body-weight changes and the health states of the animals, blood basal cortisol concentrations were measured. Animals kept outdoors had significantly lower cortisol levels than did animals kept indoors. These results imply that indoor conditions have a direct effect on the animals' states. The physiological and metabolic consequences as well as potential welfare aspects should be taken into account when planning experimental work, especially on nondomestic animals

Blastocyst recovery and multifactorial gene expression analysis in the wild guinea pig (Cavia aperea)

Hribal R, Günther A, Ruebensam K, Jewgenow K. Blastocyst recovery and multifactorial gene expression analysis in the wild guinea pig (Cavia aperea). THERIOGENOLOGY. 2016;86(5):1299-1307.The expression of specific developmentally important genes in preimplantation embryos is an accepted marker for unraveling the influence of single factors in studies that are mostly related to artificial reproduction techniques. Such studies, however, often reveal high levels of heterogeneity between single embryos, independently of the influence of factors of interest. A possible explanation for this variation could be the large variety of physiological and environmental factors to which early embryos are exposed and their ability to react to them. Here, we investigated several potentially important parameters of development at the same time, in blastocysts of the wild guinea pig (Cavia aperea) generated in vivo after natural mating. The optimal time for flushing fully developed blastocysts was between 123 and 126 hours after mating. The abundance of POU5F1 (P = 0.042), BAX (P < 0.001), SLC2A1 (P = 0.017), and DNMT3A (P < 0.001) mRNA changed significantly over time after mating. The number of sibling embryos present influenced STAT3 levels significantly (P = 0.02). Levels of BAX and POU5F1 were significantly affected by season (P = 0.03 and 0.04). The temporal pattern of SLC2A1 levels was significantly altered both after feeding a protein-deficient diet (P = 0.04) and temperature treatment (P = 0.04) of the sire. In addition, the identity of the father had a significant influence on POU5F1 (P = 0.049) and STAT3 (P < 0.001) mRNA abundances. These data report that the expression of specific genes in early embryos reflects the entire heterogeneity of their surroundings and that it is a plastic reaction toward a multifactorial environment. (C) 2016 Elsevier Inc. All rights reserved

Non-Invasive Pregnancy Diagnosis in Big Cats using the PGFM (13,14-dihydro-15-keto-PGF2α) Assay.

Non-invasive monitoring of hormones using feces has become a vital tool for reproductive management and reliable pregnancy diagnosis in big cats. Previous studies described the PGF2α metabolite (PGFM) as an indicator of pregnancy in various feline species. The present study aimed to standardize pregnancy detection in big cats like the tiger (Panthera tigris), jaguar (Panthera onca) and lion (Panthera leo) using fecal samples. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS) were performed to identify PGFM in feces. An EIA developed against 9α,11α-dihydroxy-15-oxo-prost-5-en-1-oic acid-BSA was used to assay PGFM in fecal samples of the Bengal tiger, Asiatic lion and jaguar. The PGFM levels increased after 9 weeks of pregnancy and remained elevated until parturition. All animals showed elevated levels of PGFM in the last trimester of pregnancy, thus making PGFM a reliable tool for pregnancy diagnosis during this period that can be useful in captive breeding programs in these species

Hair cortisol analyses in different mammal species: choosing the wrong assay may lead to erroneous results

Wild animals are faced with a broad range of environmental stressors and research is needed to better understand their effect on populations. Hormone analysis based on enzyme immunoassays (EIAs) can provide valuable information on adrenocortical activity (stress), and assessment of cortisol in hair may allow the quantification of cortisol production. To validate hair hormone analysis, we compared two EIAs based on antibodies against cortisol-3-CMO-BSA and cortisol-21-HS-BSA for hair glucocorticoid (hGC) measurements in Egyptian mongoose, Iberian lynx, Alpine marmot, Asiatic black bear, spotted hyena and cheetah, with results obtained by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) measurements. Both EIAs were also characterized by HPLC immunograms. Our results revealed that the cortisol-21-HS EIA measured 2.3- to 12-fold higher hGC concentrations than the cortisol-3-CMO assay. In dependence of the species, high-performance liquid chromatography (HPLC) immunograms showed that up to 70% of immunoreactivities determined by the cortisol-21-HS constituted of unknown unpolar compounds leading to an overestimation of hGC. The cortisol-3-CMO EIA expressed a better specificity, with 32.1–67.4% of immunoreactivity represented by cortisol and cortisone. The LC-MS/MS analyses (gold standard) revealed that the cortisol-3-CMO EIA also resulted in an (up to 3-fold) overestimation of hGC, but EIA results were correlated with LC-MS/MS in the mongoose, the lynx, the spotted hyena and the marmot. No correlation was obtained for Asiatic black bears. As a result of our study, we strongly recommend to test any cortisol EIA for its specificity towards extracted hair components. In all analyzed species, except the Asiatic black bear, cortisone and cortisol were simultaneously present in hair extracts; consequently, an appropriate EIA should cross-react to these two glucocorticoid hormones and express negligible affinity towards substances with less polarity than corticosterone. Choosing the wrong EIA for hGC analyses may lead to overestimations of hGC or—in the worst case—to results that do not mirror real adrenocortical activity