Beate C. Braun

Non-invasive Monitoring of Hormones: A Tool to Improve Reproduction in Captive Breeding of the Eurasian Lynx

The survival of many critical endangered mammal species is often depending on successful captive breeding programmes which include the future option of reintroduction to the wild. Breeding in captivity also demands the application of modern assisted reproductive techniques to ensure maximal biodiversity, but knowledge on reproductive physiology is often limited. Therefore, non-invasive monitoring of urinary and faecal hormones has become an important tool for reproductive management. To exemplify the importance of non-invasive hormone monitoring, we choose the Eurasian lynx as a model for the worlds most endangered felid species, the Iberian lynx. We analysed faecal samples of pregnant and pseudo-pregnant female Eurasian lynxes during a 3-year study period. Compared to pre-mating levels faecal progesterone metabolite profiles revealed a tendency towards higher levels in pregnant and pseudo-pregnant females with no difference between both categories. Oestrogen levels raised in both pregnant and pseudo-pregnant females with a tendency to be more elevated and prolonged in pregnant females. Surprisingly both E2 and P4 metabolites were highly correlated (r(2) =0.8131, p < 0.0001) showing a postpartum increase both in pregnant and pseudo-pregnant females. The results from the Eurasian lynx revealed that the measurement of faecal progesterone metabolites led to profiles dissimilar to profiles shown in other felid species, but similar to those from faecal gestagen metabolite analysis in the Iberian lynx. To identify faecal gestagen and oestrogen metabolites a radio-metabolism study was performed. Using the progesterone immunoassay two major progesterone metabolites were detected demonstrating that the assay indeed tracks the relevant metabolites. The oestrogen assay measured authentic 17beta-oestradiol and oestrone, and their conjugates. The analysis of the faecal metabolite composition in samples from early and late pregnancy and lactation particularly revealed a distinct shift in the relation between 17beta-oestradiol and oestrone that changed in favour of oestrone. This might indicate different hormone sources during and after pregnancy (corpus luteum, placenta). We hypothesize, that placental steroid analysis in combination with other highly sophisticated analytical techniques, like liquid chromatography mass spectrometry or urinary relaxin analysis may led to analytical options to confirm pregnancy and to differentiate this from pseudo-pregnancy in lynx species.

Embryonic gene activation in in vitro produced embryos of the domestic cat (Felis catus)

Accurate embryonic gene activation (EGA) is essential for the embryos developmental potency and reflects the quality of in vitro produced embryos. To describe the dynamic and temporal patterns of EGA in the cat, the mRNA expression of developmentally important genes (DNA methyltransferases 1 and 3A, DNMT1 and DNMT3A; gap junction protein α 1, GJA1; transcription factor octamer 4, POU5F1 (OCT4); insulin-like growth factor (IGF) 1 and 2 receptors, IGF1R and IGF2R) was examined by RT-PCR techniques in preimplantation embryos obtained after in vitro maturation and IVF. Furthermore, influences of ICSI and sperm cryopreservation on the relative mRNA abundance in 4-5-days-old morulae were analyzed. Total RNA was obtained from immature and matured oocytes, 2-cell embryos, 4-cell embryos, and 8-16-cell embryos, morulae, and blastocysts. RNA was transcribed into single-stranded cDNA by reverse transcriptase. After amplification, a nonfelid standard RNA was used for semiquantitative analysis. Our results showed an increase in transcript abundance from the matured oocyte to the 2-cell embryo for all examined genes except for IGF2R, indicating that, in vitro, the embryonic genome is activated shortly after fertilization. However, the activation pattern varied markedly between the different genes. We also found different patterns of mRNA expression for the examined genes in morulae produced either by IVF or ICSI, and using fresh or cryopreserved sperm. Owing to high variations within the single groups of compared morulae, we were able to observe only a tendency toward higher relative mRNA expression in embryos derived by IVF with fresh sperm in comparison to all other groups.

Pregnancy diagnosis in urine of Iberian lynx (Lynx pardinus)

Diagnosis of pregnancies is an important management tool for the Iberian lynx Conservation Breeding Program, a program geared to recover the worlds most endangered felid. Non-invasive methods such as fecal hormone analyses are not applicable to the lynx, since fecal progestin does not follow the typical pregnancy pattern of felids. Therefore, we aimed to test whether urine can be used as an alternative substance for pregnancy diagnosis in the Iberian lynx. Progesterone immunoreactive metabolites were determined in urine samples of pregnant and non-pregnant females before and during breeding season. Additionally, we used the Witness Relaxin test to determine relaxin in blood and urine. No differences were found in progestin concentrations determined in urine samples collected from pregnant and non-pregnant animals between day 1 and 65 following mating. Although the Witness Relaxin test was positive in serum samples collected from animals between day 32 and 56 of pregnancy, it failed in both fresh and frozen urine samples collected from the same stage of pregnancy. A weak relaxin reaction in urine samples collected from animals between day 29 and 46 of pregnancy was detectable after urines were concentrated by ultrafiltration (>50x). Concentrated samples obtained from non-pregnant and early pregnant animals yielded negative test results. In conclusion, the Witness Relaxin test can be applied for pregnancy diagnosis in Iberian lynx in both serum and concentrated urine samples obtained during the second half of pregnancy. A positive relaxin test indicates an ongoing pregnancy, whereas negative tests must be judged carefully as hormone concentrations might be below detection thresholds.

Progesterone and estradiol in cat placenta--biosynthesis and tissue concentration.

Ovarian and placental steroids are essential for the maintenance of pregnancy. In some mammals it is evident that the placenta is responsible for the production of steroids. However, in the domestic cat, steroid secretion from the placenta has not yet been elucidated. Our study aimed to find out whether feline placentae are able to produce steroids. Placentae from different pregnancy stages were analyzed for mRNA expression of five steroidogenic enzymes (HSD3B1, CYP11A1, CYP17A1, HSD17B1 and CYP19A1) and for tissue concentrations of progesterone and estradiol. Steroidogenic enzymes responsible for the final steps of estradiol (CYP19A1) and progesterone synthesis (HSD3B) were expressed at very high levels and followed almost the same pattern over pregnancy as the intraplacental hormones themselves. By contrast, the other enzymes were found in very low quantities suggesting that biosynthesis occurs via extra-placental steroid precursors. The plasma steroid profiles measured by other groups differ from the placental hormone courses determined by us; therefore we conclude that the feline placenta can produce progesterone and estradiol.

The molecular detection of relaxin and its receptor RXFP1 in reproductive tissue of Felis catus and Lynx pardinus during pregnancy.

Relaxin acts as a pregnancy-specific signal in feline species, but specific information about protein structure and binding is essential for the improvement of pregnancy diagnosis in endangered feline species, like the Iberian lynx. To generate a felid-specific relaxin antibody, the DNA and protein sequences of lynx and cat were determined and peptides were chosen for antibody generation. In addition, relaxin and relaxin receptor (RXFP1) mRNA expressions were measured in uteri and ovaries of pregnant domestic cats and lynx placentae. Using real-time PCR and immunohistochemistry, it was established that feline placenta is the main source of relaxin during pregnancy. In other tested tissues, relaxin mRNA expression was weak. The RXFP1 mRNA expression was found mainly in cat uterine tissue and feline placentae. It was assumed that these tissues were main targets for relaxin. In the ovary, relaxin immunostaining was associated with blood vessels, signifying its role in vascularization.

Sequence analysis of feline oviductin and its expression during the estrous cycle in the domestic cat (Felis catus)

Oviductins belong to a family of oviduct-specific glycoproteins believed to play an important role in fertilization and/or early embryonic development. Oviductin cDNA between species is highly conserved and shares 58% to 98% similarity in the deduced amino acid sequences. Our objective in this study was to sequence the full open reading frame of the feline oviductin and to examine its expression during the estrous cycle on both mRNA and protein level. The obtained cDNA containing the full open reading frame was determined to be 1677 nucleotides coding for a deduced protein of 558 amino acids. Identities between species range from 74% (mouse) to 80% (human, baboon, and rhesus) within the N-terminal protein region. Major differences were localized in the carboxy terminal region, which corresponds to exon 11 of the gene. Feline oviductin contained one putative N-linked glycosylation site, six O-linked glycosylation sites, a potential heparin binding site, and two cholesterol recognition and/or interaction amino acid consensus (CRAC) domains. Oviductin expression was analyzed by real time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. Both approaches revealed an estrous cycle-dependent expression in the ampulla and isthmus. Quantitative PCR showed highest oviductin mRNA copy numbers in the early and late follicular stage and reduced mRNA expression during all other stages. With the exception of the early follicular stage, feline oviductin mRNA abundance was not significantly different in the oviductal segments ampulla and isthmus. A prominent immunolabeling was seen in the early and late follicular stage which disappeared after ovulation, indicating a function of the protein during sperm storage and fertilization.

Feasibility of sex-sorting sperm from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis)

The objective of these studies was to investigate the practicality of flow cytometric sex-sorting for spermatozoa from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis). In Experiment 1, four semen extenders were tested regarding their suitability for liquid preservation of spermatozoa before sorting. Dilution in MES-HEPES-based semen extender followed by incubation generated best sperm quality parameters (motility, viability, and acrosome integrity). In Experiment 2, the effect of staining method (15 degrees C for 4 to 6h during transport or 37 degrees C for 1 to 1.5h) on sort efficiency and sperm quality was investigated. Staining at 15 degrees C during transport resulted in a higher percentage of sperm samples showing a resolution of X- and Y-chromosome-bearing populations (60%) compared with that for staining at 37 degrees C after transport (33%) and resulted in superior sperm integrity after staining (43.8+/-11.3% vs. 19.6+/-12.1%). Sort rate was 300 to 700 cells/sec and sort purity, determined for one sorted sample, was 94% for X-chromosome-bearing spermatozoa. In Experiment 3, the highly viscous component of rhinoceros seminal plasma, which complicates the process of sperm sorting, was examined by gel electrophoresis and mass spectrometry. Results suggested a 250-kDa glycoprotein (most likely originating from the bulbourethral gland) to be responsible for the characteristic viscosity of ejaculates. In Experiment 4, viscosity of seminal plasma, as measured by electron spin resonance spectroscopy, was significantly decreased after addition of alpha-amylase or collagenase (0.5 and 3IU per 100 microL seminal plasma, respectively) by 28% and 21%, respectively, with no negative effect on sperm characteristics. The results of this study demonstrate for the first time that rhinoceros spermatozoa can be successfully sorted into high-purity X- and Y-chromosome-bearing populations. Furthermore, the successful liquefaction of viscous ejaculates provides the means to greatly improve sort-efficiency in this species.

Corpora lutea of pregnant and pseudopregnant domestic cats reveal similar steroidogenic capacities during the luteal life span

In domestic cats, luteal phases of pregnancy and pseudopregnancy (non-pregnant luteal phase) differ in the course and level of plasma progesterone (P4). Therefore, we assumed differences in luteal steroidogenic capacities. Here we present a comprehensive analysis of intraluteal steroid biogenesis in the domestic cat. We quantitatively measured relative mRNA levels of steroidogenic acute regulatory protein (STAR), cytochrome P450 oxidases (CYP), hydroxysteroid dehydrogenases (HSD), steroid reductase (SRD) and enzymes involved in sulfoconjugation of steroids, i.e. sulfotransferase (SULT) and sulfatase (STS). Protein expression was analysed by Western Blot for HSD3B. Additionally, intraluteal steroid contents were determined. During the pseudopregnant luteal phase, expression of STAR (p=0.005), HSD3B1 (p<0.0001), CYP19A1 (p<0.0001) and HSD17B7 (p=0.008) decreased from formation of the corpus luteum (CL) onwards. HSD3B protein expression was highest in the development/maintenance stage of CL and declined during the subsequent luteal phase of pregnancy and pseudopregnancy. This was in accordance with decreasing intraluteal levels of P4, oestrogens and androgens. In contrast, expression of SRD5A1 (p<0.001) increased with progression through stages of the pseudopregnant CL, being indicative of P4 metabolism via an alternate pathway to dihydrotestosterone (DHT). Compared to the formation stage, expression of SULT1E1 was higher in all other luteal stages of pseudopregnancy (p=0.004), implying a potential sulfoconjugation of oestrogens. Expression of CYP11A1 and CYP17A1 was unaffected by the luteal stage (p>0.05), suggesting a permanent capacity of cat CL to convert progestogens via androgen and oestrogen pathways. In general, mRNA expression profiles of steroidogenic enzymes during the pregnant luteal phase reflected the pseudopregnancy profiles. Intraluteal oestrogen (p<0.0001) and androgen (p=0.008) levels were higher in the formation stage compared to the following luteal stages of pseudopregnancy. Concentrations of P4 were higher in the development/maintenance compared to the regression stages (p=0.01). We conclude that cat CL of the same histomorphological stage are characterised by identical steroidogenic capacities independently of an on-going pregnancy.

The influence of recombinant feline oviductin on different aspects of domestic cat (Felis catus) IVF and embryo quality

Oviductin is known to be a key player providing a convenient environment for the process of fertilization affecting this by direct interaction with oocytes and sperm. As in vitro embryo production in the context of assisted reproduction for endangered felids is still in the process of optimization, oviductin might be used to improve IVF results. Recombinant His-tagged feline oviductin was expressed by transformed Escherichia coli BL21DE3 cells. The protein was purified by immobilized metal ion affinity chromatography. The effect of the recombinant protein was characterized in three experiments: a hemizona assay for sperm binding analysis, the IVF outcome, and the relative mRNA expression levels in blastocysts after IVF. A significant higher number of bound sperm cells were found after incubation in oviductin. No significant effect on cleavage, morula, and blastocyst rates with or without oviductin incubation during IVF could be observed. However, the relative mRNA abundance of GJA1, a gene, whose expression level is known to be a marker of embryo quality, was significantly increased (P value less than 0.05) in blastocysts after oviductin treatment. In contrast to this, expression of OCT4, HSP70, DNMT1, DNMT3A, BAX, IGF1R, and GAPDH was not significantly affected. We assume that our recombinant oviductin in its current nonglycosylated form is able to enhance sperm binding. Despite of a missing significant effect on IVF outcome, embryo quality in terms of relative GJA1 expression is influenced positively. These promising results demonstrate the value of recombinant oviductin for the IVF in cats.

Functional Role of Feline Zona Pellucida Protein 4 Trefoil Domain: A Sperm Receptor or Structural Component of the Domestic Cat Zona Pellucida?

The trefoil domain (TFD) is a protein structure characterized by six cysteines, which form a typical three-loop structure by three disulphide bridges. It is assumed that two of these loops generate a hydrophobic groove, which could be a binding site for carbohydrate residues or proteins. The zona pellucida (ZP) protein, ZP4, contains such a TFD. The carbohydrate-/protein-binding property of TFD allows us to assume a potential sperm receptor function of this domain. Additionally, gastrointestinal trefoil peptides are stable against proteases; therefore, a structural role of TFD within the ZP might also be possible. We were able to show that the synthesized and natural folded feline TFD (fTFD) expresses the typical protease resistance that vanished under reducing conditions and after substitution of cysteine residues within the peptide. Furthermore, an antibody directed against the first loop of fTFD was almost unable to bind to intact in vitro mature cat oocytes. Pre-incubation of oocytes in the reducing agent (DDT), however, improved antibody binding substantially. Therefore, we suggest structural masking of the fTFD domain within the intact ZP. An interaction between fTFD and feline sperm cells was examined using several methods, including immunocytochemistry, immunoelectron microscopy, co-immunoprecipitation and far western blot, but we found no indication for an involvement of TFD in the primary sperm binding to the ZP. To summarize, there is increasing evidence that the TFD of fZP4 has a structural rather than a sperm-binding function.