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.
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Beate C Braun, Astrid Vargas, and Katarina Jewgenow
Jennifer Ringleb, Marlies Rohleder, and Katarina Jewgenow
Although immunocontraception based on porcine zona pellucida (ZP) proteins is widely applied in many species, it is not suitable for cat contraception due to the lack of cross-reactivity. Since the first ZP gene expressed during oocyte growth in domestic cats is ZPB, we assumed that immunization with feline ZPB (fZPB)-derived synthetic peptides may cause irreversible infertility, which would be preferable in stray cats. Thus, the present study evaluated the immunogenicity and the contraceptive potential of synthetic fZPB peptides. Antigenic epitope sequences were detected via epitope mapping using specific rabbit anti-fZP antibodies. Six peptides representing the recognized epitopes were synthesized subsequently. Two out of six peptides (ZPB amino acid residue 130–149 = P3 and 175–193 = P6) cross-reacted with anti-fZP antiserum in dot blot analysis and ELISA. Coupled to BSA, both peptides were utilized to produce specific antibodies in rats. Despite several booster injections the antibody titers monitored by ELISA did not exceed 1:5000. Both rat antisera were tested for contraceptive potential in cat in vitro maturation/in vitro fertilization (IVF). Antiserum against peptide P3 significantly inhibited sperm binding and fertilization of cat oocytes in vitro (57.3% of sperm binding; 41.5% of fertilization), whereas the inhibition by anti-P6 was not significant. Pre-incubation of sperm cells with both peptides before IVF failed to affect either sperm binding or fertilization (22.3 ± 3.7 sperm/egg vs 25.5 ± 5.8 for P3 and 20.7 ± 4.0 for P6, respectively). In conclusion, antibodies directed against one of the two identified antigenic determinants of fZPB inhibited sperm binding and IVF and therefore showed promising results as a contraceptive. However, the specific immune response and anti-fertile properties of this synthetic vaccine have to be examined in vivo to verify the suitability of its components.
Kim Carnaby, Johanna Painer, Arne Söderberg, Dolores Gavier-Widèn, Frank Göritz, Martin Dehnhard, and Katarina Jewgenow
Lynx presents a unique sexual cycle with persistent corpora lutea (CLs) and elevated serum progesterone (P4) throughout parturition and lactation. In other mammals, CLs normally disintegrate after parturition, therefore the aim of our study was to characterise the annual life cycle of lynx CLs. Ovaries from Eurasian lynxes were obtained from the National Veterinary Institute in Sweden, where tissues from killed lynx were stored at −20 °C. Ovaries from 66 animals were weighed; each corpus luteum was segmented for histology and hormone analysis. Ovary and CLs weights were constant throughout the year, peaking during pregnancy. In non-pregnant lynxes, the seasonal level of intraluteal steroids was steady for P4 (3.2±1.9 s.d. μg/g, n=53) and total oestrogens (18.3±15.5 s.d. ng/g, n=53). Within histology slides, structurally intact luteal cells were found throughout the year with the highest incidence in March/April; evidence of luteal regression was predominantly found in post-breeding season. Ovaries from pregnant animals contained two types of CLs. Group A was bigger in size with large luteal cells (P4, 72.3±65.4 s.d. μg/g; oestrogen, 454.0±52.4 s.d. ng/g). In contrast, group B were smaller, with greater luteal regression and lower steroid concentrations (P4, 8.3±2.9 s.d. μg/g; oestrogen, 31.5±20.4 s.d. ng/g). Our results suggest that structural luteolysis proceeds throughout the year and into next breeding cycle, resulting in two CLs types on the same ovary.
Lina Zschockelt, Olga Amelkina, Marta J Siemieniuch, Mariusz P Kowalewski, Martin Dehnhard, Katarina Jewgenow, and Beate C Braun
Felids show different reproductive strategies related to the luteal phase. Domestic cats exhibit a seasonal polyoestrus and ovulation is followed by formation of corpora lutea (CL). Pregnant and non-pregnant cycles are reflected by diverging plasma progesterone (P4) profiles. Eurasian and Iberian lynxes show a seasonal monooestrus, in which physiologically persistent CL (perCL) support constantly elevated plasma P4 levels. Prostaglandins (PGs) represent key regulators of reproduction, and we aimed to characterise PG synthesis in feline CL to identify their contribution to the luteal lifespan. We assessed mRNA and protein expression of PG synthases (PTGS2/COX2, PTGES, PGFS/AKR1C3) and PG receptors (PTGER2, PTGER4, PTGFR), and intra-luteal levels of PGE2 and PGF2α. Therefore, CL of pregnant (pre-implantation, post-implantation, regression stages) and non-pregnant (formation, development/maintenance, early regression, late regression stages) domestic cats, and prooestrous Eurasian (perCL, pre-mating) and metoestrous Iberian (perCL, freshCL, post-mating) lynxes were investigated. Expression of PTGS2/COX2, PTGES and PTGER4 was independent of the luteal stage in the investigated species. High levels of luteotrophic PGE2 in perCL might be associated with persistence of luteal function in lynxes. Signals for PGFS/AKR1C3 expression were weak in mid and late luteal stages of cats but were absent in lynxes, concomitant with low PGF2α levels in these species. Thus, regulation of CL regression by luteal PGF2α seems negligible. In contrast, expression of PTGFR was evident in nearly all investigated CL of cat and lynxes, implying that luteal regression, e.g. at the end of pregnancy, is triggered by extra-luteal PGF2α.