Role of sex steroids and prostaglandins during the luteal life cycle in domestic cats and lynxes.

Although lynxes and domestic cats are both felids, their luteal life cycles differ. As in many species, corpora lutea (CLs) of domestic cats regress after pregnancy or pseudopregnancy. By contrast, CLs of lynxes do not functionally regress following the cycle of their formation. They stay physiologically active and persist for several years. To obtain an improved understanding of the life cycle of both species, we comparatively studied the CLs of these species in detail. In this review, we summarize the similarities and differences of their CLs regarding sex steroid and prostaglandin generation and receptors. The most evident differences were visible in the CLs of lynxes, which persist from previous cycles, compared with CLs of lynxes and domestic cats from the recent luteal cycle. We assume that these differences could indicate processes ensuring long-term luteal survival and functionality, for example, by high estrogen production/metabolism or by antioxidative effects.

Early preantral follicles of the domestic cat express gonadotropin and sex steroid signaling potential.

Key biomolecular processes, which regulate primordial ovarian follicle dormancy and early folliculogenesis in mammalian ovaries, are not fully understood. The domestic cat is a useful model to study ovarian folliculogenesis and is the most relevant for developing in vitro growth methods to be implemented in wild felid conservation breeding programs. Previously, RNA-sequencing of primordial (PrF), primary (PF), and secondary follicle (SF) samples from domestic cat implicated ovarian steroidogenesis and steroid reception during follicle development. Here, we aimed to identify which sex steroid biosynthesis and metabolism enzymes, gonadotropin receptors, and sex steroid receptors are present and may be potential regulators. Differential gene expression, functional annotation, and enrichment analyses were employed and protein localization was studied too. Gene transcripts for PGR, PGRMC1, AR (steroid receptors), CYP11A1, CYP17A1, HSD17B1 and HSD17B17 (steroidogenic enzymes), and STS (steroid metabolizing enzyme) were significantly differentially expressed (Q values of ≤0.05). Differential gene expression increased in all transcripts during follicle transitions apart from AR which decreased by the secondary stage. Immunohistochemistry localized FSHR and LHCGR to oocytes at each stage. PGRMC1 immunostaining was strongest in granulosa cells, whereas AR was strongest in oocytes throughout each stage. Protein signals for steroidogenic enzymes were only detectable in SFs. Products of these significantly differentially expressed genes may regulate domestic cat preantral folliculogenesis. In vitro growth could be optimized as all early follicles express gonadotropin and steroid receptors meaning hormone interaction and response may be possible. Protein expression analyses of early SFs supported its potential for producing sex steroids.

Analysis of gene expression in granulosa cells post-maturation to evaluate oocyte culture systems in the domestic cat.

Maturation of oocytes is a prerequisite for successful embryo development. The fertilization competence of in vivo derived oocytes is significantly higher than that of oocytes matured in vitro. Commonly evaluated morphological criteria for oocyte maturation do not reflect the complexity and quality of maturation processes. Oocytes and granulosa cells are communicating closely in a bidirectional way during follicular growth and maturation. Assessing the mRNA expression of specific genes in granulosa cells could be a non-invasive way to evaluate the conditions of in vitro oocyte maturation. The objective of this study was to elucidate the influence of two different FSH additives on the in vitro maturation rate and gene expression of cumulus-oocytes complexes in domestic cat. Feline oocytes were matured in a medium, supplemented with LH and 0.02 IU/ml porcine FSH versus 0.02 IU or 1.06 IU/ml human FSH. Granulosa cells were separated from oocytes directly after 24 hr of maturation or after additional 12 hr of in vitro fertilization. Gene expression levels were analysed by quantitative PCR for aromatase, antimullerian hormone, follicle stimulating hormone receptor (FSHR), luteinizing hormone/choriogonadotropin receptor (LHCGR) and prostaglandin E synthase. Neither oocyte maturation rate nor gene expression levels differed after 24 or 36 hr in all three groups. However, variations were discovered in correlations of expression levels, for instance for FSHR and LHCG, indicating differences in the fine-tuning of in vitro maturation processes under varying FSH supplementations. We suppose that correlation between gene expressions of selected genes suggests a superior maturation quality of feline oocytes.

Expression of steroidogenic enzymes and steroid receptors in foetal gonads of domestic cat-Sex similarities and differences.

Foetal gonads already produce steroid hormones and by this influence the further development of external and internal genitalia as well as of the brain. Beside this, foetal gonads themselves can be influenced by foetal or maternal hormones. The time course of foetal gonadal development can differ between species. As knowledge on processes in domestic cats is very limited, the steroidogenic enzyme expressions as well as these of steroid receptors were analysed in foetal gonads of domestic cats. We investigated a period from beginning of the second half of pregnancy to the beginning of the third trimester; a phase, where also gonadal development proceeds. The mRNA expression of most of the steroidogenic enzymes was remarkably higher in male gonads compared to female ones on all analysed days. The enzyme mRNA expression in female gonads shows a tendency for an increase towards the beginning of the third trimester, except that of aromatase gene CYP19A1-it shows the opposite trend. CYP19A1 was detectable just in female gonads, indicating that only female foetal gonads are capable of producing oestrogens. Gene expressions of genomically and non-genomically acting steroid receptors for progesterone, androgen and oestrogen reception were observed in gonads of both genders. Slightly higher expressions of some receptors were detected in female compared to male gonads; only for the non-genomically oestrogen receptor GPER, we observed the opposite. The protein staining for progesterone receptor membrane component 1 (PGRMC1) exposed a potential function of it on steroid-producing cell and/or cells that suppose early oogenesis.

Life cycle of feline Corpora lutea: histological and intraluteal hormone analysis.

The corpus luteum (CL) is a transient hormone gland on the ovary that produces progesterone (P4) for the maintenance of pregnancy. It develops from residual follicular granulosa and theca cells after ovulation. Very little is known about the cellular and hormonal processes within CLs obtained from pregnant and pseudopregnant felids. Therefore, our aim was to review the luteal function in feline CLs of different reproductive stages in conjunction with our data obtained in domestic cats and Eurasian lynxes. Corpus luteum function in lynxes is of particular interest, as a post-partum luteal activity was suggested based on repeated ultrasonography and endocrine examinations. Histology of CL from pregnant and pseudopregnant domestic cats clearly reflects the luteal function. The formation of the CL after ovulation is characterized by transforming of theca and granulosa cells into steroidogenic luteal cells and is accompanied by increased intraluteal and circulating P4 levels. Luteal regression is steadily progressive; the first signs (coarsed vacuolization, increased proportion of non-steroidogenic cells) are visible already in CL from the second trimester of pregnancy.

Feline gonads exhibit tissue specific alternative splicing of oestrogen receptor alpha (ESR1).

Male felids frequently show teratospermia. At least in the domestic cat model, teratospermia is accompanied by impaired regulation of testicular apoptosis. We hypothesize that this phenomenon is caused by dysregulations in oestrogen signalling pathways. Both classical oestrogen receptors (ESR1 and 2) are expressed in species and/or tissue-specific manners and display different variants, inter alia, caused by alternative splicing. In vitro studies showed that exon deleted transcripts are translated into proteins and that some of the variants modify the effects of the full-length ERs. It has been proposed that some of the functional and morphological dysregulations, for example, during spermatogenesis, could directly derive from this phenomenon. In the present basic study, we investigated the expression pattern of ESR1 splicing variants in the gonads of domestic cats. Testicular, epididymal as well as ovarian tissue samples were collected from routine castrations. ESR1 variants were detected by means of RT-PCR using primers spanning one to three exons. We detected the variants Δ4 and Δ7 in all tissue samples investigated. Additionally, the testicular parenchyma expressed the variant Δ6 and double exon deletions of ESR1 (Δ4/6 and Δ6/7). Using an antiserum recognizing all previously identified ESR1 splicing variants, we revealed ESR1 proteins being expressed in nearly all cells of the testicular and ovarian parenchyma. ESR1 Δ6 protein, however, detected by an antiserum specifically raised against the Δ6 variant, was predominantly located in Sertoli cells. As the exon deletion variants are significantly expressed and show a distinct expression pattern, they could specifically modulate the cellular responsiveness to hormonal stimuli within the gonads.

Comparative metabolism of gestagens and estrogens in the four lynx species, the Eurasian (Lynx lynx), the Iberian (L. pardinus), the Canada lynx (L. canadensis) and the bobcat (L. rufus).

With the increasing prevalence of faecal hormone metabolite analysis, it is important to develop a better understanding of the dynamics of faecal metabolite composition. The aim of this study was to compare the quantitative faecal gestagen and estrogen metabolite composition in the four lynx species: Eurasian lynx, Iberian lynx, Canada lynx and bobcats. Comparative HPLC immunograms were generated from faecal samples collected before, during, and after pregnancy from individual females of each lynx species. Gestagens and estrogens revealed three similar classes of immunoreactive faecal metabolites: (1) polar metabolites which were enzyme-hydrolysable and thus may be designated as conjugates, (2) non-hydrolysable polar metabolites, and (3) non-polar metabolites or free steroids. For both hormones, strong similarities in the HPLC immunograms across species suggests that steroid metabolism is relatively conserved among Lynx species. Gestagens were primarily excreted as polar conjugates or unknown metabolites, whereas estrogen metabolism revealed a huge proportion (approximately 50%) consisting of 17beta-estradiol and estrone. These results are consistent with patterns of steroid metabolism in other felid species. Only two minor species-specific patterns emerged. In bobcats, we observed an exceptionally high proportion of gestagen conjugates, and in Iberian lynx, there was an exceptionally high proportion of estrone. The comparison of HPLC immunograms within individuals revealed that intra-individual variations in steroid metabolite composition are considerably high. However, changes in metabolite composition did not correlate with specific reproductive stages; rather, they seemed to occur at random. We assume that these differences may reflect changes in liver metabolism and/or qualitative and quantitative variations in gut bacteria composition, resulting in differences in faecal metabolite composition.

Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation.

BACKGROUND Female cancer patients are offered 'banking' of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. METHODS Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. RESULTS Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. CONCLUSIONS Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research.

PGFM (13,14-dihydro-15-keto-PGF(2alpha)) in pregnant and pseudo-pregnant Iberian lynx: a new noninvasive pregnancy marker for felid species.

In mammals, uterine and placental prostaglandin F(2alpha) is involved in the regulation of reproduction-related processes such as embryonic development, initiation of parturition, and resumption of ovarian activity. Prostaglandin F(2alpha) (PGF(2alpha)) is rapidly metabolized to its plasma metabolite PGFM (13,14-dihydro-15-keto-PGF(2alpha)), which has also been detected in urine. Therefore, the current study aimed to develop and validate an efficient, quick, and inexpensive enzyme immunoassay (EIA) for PGFM estimation in urine of the Iberian lynx (Lynx pardinus) for pregnancy monitoring and for differentiation between pregnancy and pseudo-pregnancy. Urine samples collected from captive Iberian lynx (11 pregnant and 4 pseudo-pregnant cycles) were subjected directly to a PGFM EIA. The assay was validated for parallelism, precision, and stability of urinary PGFM. In addition, high-performance liquid chromatography (HPLC) immunograms and liquid chromatography-mass spectrometry (LCMS) were performed to identify PGFM within urine samples. Urinary PGFM levels before mating and after parturition were about 1.5 ng/mL. After Day 20 postmating, both pregnant and pseudo-pregnant females showed slight increase of hormone levels; in pseudo-pregnant females, this elevation did not exceed 7 ng/mL. A significant increase in pregnant females was observed after Day 45 postmating; urinary PGFM increased from 10 ng/mL at Day 45 toward a peak of 46.0+/-19.3 ng/mL around parturition. First results show that PGFM is detectable in feces as well and follows similar courses as shown for urine. In conclusion, the presented and validated PGFM assay is an easy and reliable method for noninvasive pregnancy diagnosis in the Iberian lynx (and probably other felids) if applied approximately 20 d prior parturition in pure urine or fecal extracts. High PGFM levels in urine or fecal samples may allow a pregnancy diagnosis without knowledge of mating time, making the PGFM test applicable to free-ranging animals.

Seasonal profiles of ovarian activity in Iberian lynx (Lynx pardinus) based on urinary hormone metabolite analyses.

The Iberian Lynx Ex-Situ Conservation Programme is an essential part of a co-ordinated action plan to conserve the most endangered felid species of the world. Successful captive breeding demands reliable methods for reproduction monitoring including reliable non-invasive pregnancy diagnosis. During a 3-year study, urine samples from six captive Iberian lynx females were obtained (one non-pregnant, one pseudo-pregnant and 11 pregnant cycles). Progesterone, pregnanediol and oestradiol were determined in urinary extracts and relevant urinary oestrogen metabolites were characterized by high-performance liquid chromatography (HPLC). Urinary progestins did not follow the typical pregnancy-related course of felids. In the lynx, we failed to demonstrate an urinary progestin elevation during pregnancy. In contrast, urinary oestrogens increased from 3.8 +/- 0.6 to 8.6 +/- 0.5 ng/mg creatinine (p < 0.001) during the pregnancy. A comparison of pseudo-pregnant with pregnant cycles revealed a further increase of oestrogens caused by implantation (p < 0.05). In one female, which refused to mate, no difference was estimated between oestrogens levels during the breeding and non-breeding seasons. Almost 10-fold higher oestrogen concentrations were measured in urines of females that shared enclosures with males. HPLC analysis of oestrogens in urine samples collected from Iberian lynx during the pregnancy revealed that lynx urine is composed of two polar oestrogen metabolites in addition to oestrone and minor amounts of oestradiol. Oestrone was detectable in all urinary extracts (8-12% of metabolites), whereas oestradiol was elevated only during late pregnancy (18%). Thus, seasonal luteal activity in Iberian lynx can be monitored by urinary oestrogens. The increase of urinary oestradiol during late pregnancy might indicate an oestradiol secretion by the lynx placenta.

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.

Localization of oestrogen receptors in the epididymis during sexual maturation of the domestic cat.

Oestrogens are involved in regulation of spermatogenesis and sperm maturation and are essential for male fertility. To study the role of oestrogens on epididymal function in the domestic cat, we analyzed the localization patterns of oestrogen receptors (ERs) within the epididymis of juvenile, pubertal and adults using immunohistochemistry. Cat epididymal tissues obtained during routine castrations were fixed in chilled Bouin's solution and processed for immunohistochemistry with ER-specific antibodies. For a certain receptor type, ER localization was influenced by donor age. In the juvenile epididymis, ERalpha was localized in the nuclei of epithelial cells of efferent ducts and undifferentiated epithelium of the ductus epididymis. During puberty, ERalpha localization in the undifferentiated epithelium of the epididymis shifted from the nuclei to the cytoplasm and plasma membrane. Oestrogen receptor-alpha level was highest in the pubertal and adult epididymis, especially within the cytoplasm and in plasma membranes of caput epithelial cells. This finding was suggestive of a role in fluid reabsorption within the efferent ducts and the epididymis. In corpus and cauda regions, ERalpha was less abundant, suggesting a minor role for oestrogens in sperm storage areas. Interestingly, localization of ERbeta was neither influenced by age nor location within the epididymis and was ubiquitous throughout. Results demonstrate that oestrogen actions within the epididymis may be predominantly mediated through ERalpha during sexual maturation in the domestic cat.

Ovarian response to gonadotropin stimulation in juvenile rhesus monkeys.

The objective of this study was to investigate juvenile rhesus monkeys responding to various gonadotropin regimen stimulations. Thirty-two prepubertal rhesus monkeys were randomly allocated into five groups for ovarian stimulation as follows: Groups I, II, and III were given 35, 18, and 9 IU recombinant human follicle-stimulating hormone (rhFSH), respectively, twice daily for 8 d; Group IV was given 18 IU rhFSH twice daily until the appearance of maximal increase in sex skin during the breeding season; and Group V was treated identically to Group II but during the nonbreeding season. In addition, nine menarchial monkeys (Group VI) were treated identically to Group II. Menarchial monkeys yielded two- to fivefold the numbers of MII oocytes (24.1) and almost twice the development potential of in vitro-fertilized oocytes (blastocyst rate: 50.0%) compared with those of the other groups. Moreover, prepubertal monkeys in Group V had approximately double the numbers of MII oocytes and in Groups IV and V twice the development potential compared with those of Groups I and II, whereas Group III did not respond to stimulation. The most prominent sex skin swelling was in association with peak serum estradiol concentrations, and good responses to stimulation were associated with reduced body temperatures. All stimulated monkeys had normal reproductive performance at adulthood, except those in Group I. In conclusion, gonadotropin stimulation of menarchial monkeys could be appropriate for addressing the high cost and limited availability of rhesus monkeys in studying reproductive biology in primates.

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 world's 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.

Effects of rhFSH regimen and time interval on ovarian responses to repeated stimulation cycles in rhesus monkeys during a physiologic breeding season.

We studied the effects of repeated stimulation by recombinant human FSH (rhFSH) at various time intervals during a physiologic breeding season in rhesus monkeys. Ovarian recovery and responses were assessed by ultrasonography, serum steroid concentrations, number of oocytes retrieved, and in vitro blastocyst development following IVF. One group underwent a single stimulation regimen with 18 IU rhFSH i.m., followed by 1000 IU hCG, and serum steroid concentrations and ovarian status were determined in the following three menses. Another group was stimulated as before and then allocated into three subgroups; each subgroup was re-stimulated once at the beginning of the ensuing first, second, or third menses. In the final experiment, one group was stimulated with 37.5 IU rhFSH, whereas another group received 18 IU rhFSH. In subsequent cycles, all were re-stimulated twice with 18 IU rhFSH at time intervals of two menstrual cycles (MCs). At the first menses after stimulation, serum progesterone concentrations were significantly higher and the ovaries larger than before stimulation. Monkeys that were re-stimulated at the first menses responded poorly; at the second menses, progesterone concentrations and ovarian size recovered, but the number of oocytes retrieved from re-stimulated monkeys was still significantly reduced. However, animals that were re-stimulated in two MCs later responded well (i.e., percentage of the animals responding, oocytes recovered, and potential for fertilization and blastocyst formation). In conclusion, rhesus monkeys were likely to have similar ovarian responses to repeated stimulation with the same regimen spaced at least two MCs apart.

Effects of rhFSH dose on ovarian follicular response, oocyte recovery and embryo development in rhesus monkeys.

The objective was to study the effects of dose of recombinant human follicular stimulating hormone (rhFSH) for ovarian stimulation in rhesus monkeys. Nineteen pubertal and 109 adult female rhesus monkeys were given 37.5, 18, or 9 IU of rhFSH twice-daily for 8 days (total of 600, 300, or 150 IU of rhFSH per cycle, respectively; designated Regimens 1, 2 and 3). Ovarian responses were assessed with ultrasonography, serum concentrations of E2 and FSH, and by in vitro developmental potential (following IVF) of retrieved oocytes. Regimen 1 had more monkeys with very large follicles (diameter>8 mm) than Regimen 2 (P<0.05), which impaired development potential. However, there were no differences between Regimens 1 and 2 in oocyte recovery, whereas Regimen 3 did not elicit superovulation. The developmental potential of embryos obtained from Regimen 2 was higher than that of Regimen 1, as determined by culture to the blastocyst stage in vitro (proportion of blastocysts relative to collected MII oocytes was 55.8% versus 36.8% in pubertal and 63.8% versus 44.2% in adult monkeys; P<0.05 for each), and the results of embryo transfer from Regimen 2 were acceptable. In conclusion, we inferred that the optimal rhFSH dose for ovarian stimulation in rhesus monkeys was a total of 300 IU; this dose should be efficacious for ovarian stimulation as the quality or recovered oocytes was higher and the risk of overstimulation was reduced.

Monitoring testicular activity of male Eurasian (Lynx lynx) and Iberian (Lynx pardinus) lynx by fecal testosterone metabolite measurement.

The aim of the present study was to identify relevant fecal testosterone metabolites in the Eurasian lynx (Lynx lynx) using HPLC analysis and to evaluate the specificity of two testosterone immunoassays against these fecal metabolites. Finally, fecal hormone analysis was used to characterize seasonal reproductive activity of captive male Eurasian and Iberian (Lynx pardinus) lynx. Fecal samples from a male Eurasian lynx who received an i.v. injection of [3H]testosterone were subjected to HPLC analysis. All HPLC fractions were analyzed for radioactivity and androgen content by two testosterone immune assays (EIA and Testosterone-Immulite kits, DPC Biermann, Germany). Furthermore, fecal samples from four Eurasian lynx males (n=174) and three Iberian lynx (n=52) were collected throughout the year and fecal testosterone metabolites were determined with Testosterone-Immulite assay. HPLC separation of radiolabeled Eurasian lynx fecal extract indicated that the majority of testosterone metabolites are substances with a higher polarity than testosterone. Only minor proportion of radioactivity co-eluted with authentic testosterone and dihydrotestosterone. Enzymatic hydrolysis and solvolysis of the fecal extract were insufficient to liberate testosterone. After solvolysis relatively more activity was eluated the position of DHT, but the majority of metabolites remained unaffected. The EIA measured substantial amount of immunoreactivity, which corresponded with two radioactive peaks. Additionally, both immunoassays recognized two metabolites, which were only minor components according to their radioactivity. The Immulite assay was able to recognize a metabolite at the position of dihydrotestosterone. HPLC separation of Iberian lynx feces extracts revealed a similar metabolite pattern determined by EIA that were typical for Eurasian lynx fecal extracts. Simultaneous analyses of fecal samples with both testosterone assays provided comparative results for both lynx species (Eurasian lynx, r2=0.488; p<0.001; Iberian lynx, r2=0.85, p<0.0001). Thus, seasonal reproductive activity of male Eurasian lynx was demonstrated also by Immulite -assay, confirming high testosterone levels during breeding season in March/April as previously documented with EIA. Preliminary results on testosterone measurements in Iberian lynx feces confirmed the suitability of the applied Immulite test in this highly endangered species.

Comparative endocrine investigations in three bear species based on urinary steroid metabolites and volatiles.

In order to improve breeding of in situ populations of bears, a comprehensive study of reproductive physiology in Brown (Ursus arctos), Spectacled (Tremarctos ornatus) and Giant panda bears (Ailuropoda melanoleuca) was performed. The objective was to perform non-invasive analyses of urinary and fecal steroid metabolites. In addition, we investigated the presence of reproduction-related urinary volatile substances of these bears that might trigger the reproductive behavior. Urinary estrogen concentrations, routinely used to monitor follicular activity in Giant panda, were inappropriate for monitoring follicular activity in Spectacled bear. In addition, no estrogen peak related to mating activity was observed in Brown bear. Further contrasting Giant panda, although urinary pregnanediol analyses failed to indicate luteal activity in either Spectacled or Brown bears, urinary (Spectacled bear) and fecal (Brown bear) concentrations of progesterone were an appropriate indicator of luteal activity. The Giant panda had volatile components (medium-chain fatty acids) in their urine that increased simultaneously with the seasonal increase of estrogens. These fatty acids were also detected in the Brown during estrus and Spectacled bear. Further studies on the behavioral relevance of these fatty acids are required to determine if they are pheromones.

Control of reproduction with anti-progestin and oestrogens in captive bears.

The aim of this study was to establish new methods for controlling reproduction in bears. Anti-progestins were used to interrupt pregnancies. In two consecutive years, the anti-progestin J956 was administered to 11 female bears (nine Ursus arctos, one Ursus tibethanus, one Tremarctos ornatus) living in zoos. The anti-progestin J956 was given orally (n = 4) or parenterally (n = 12). The anti-progestin was administered alone or in combination with ethinyloestradiol, and before or after embryo implantation. The effects of anti-progestin treatment were determined using ultrasonographic examination of the urogenital tract and by monitoring progesterone concentrations in the blood and faeces. Oral administration of anti-progestin was not successful (successful in 0 of 4); however, in contrast, none of the parenteral treated animals remained pregnant (successful in 12 of 12). Parenteral treatment with J956, with or without ethinyloestradiol, was effective in disrupting pregnancy before implantation (successful in 6 of 6) and after implantation (successful in 6 of 6), but administration one month after implantation (n = 2) resulted in incomplete resorption of the fetuses. In conclusion, the administration of anti-progestins may be a useful method for preventing embryo implantation in captive bears.

Comparative binding affinity study of progestins to the cytosol progestin receptor of endometrium in different mammals.

The relative binding affinity of 5 alpha-reduced progestins and a newly synthesized antiprogestin J912 (progesterone 100%) was determined in a competitive receptor binding assay using [3H]ORG-2058 as radiolabeled ligand for the progestin receptor. Uteri obtained from 12 different species of four mammalian orders were examined. The relative binding affinity of 75-100% and a blood prevalence of 5 alpha-pregnane-3,20-dione in horses and African elephants suggest a biological role of this particular 5 alpha-reduced progesterone. For pigs the binding affinity of 5 alpha-pregnane-3,20-dione was about 50% of progesterone, but blood levels are unknown. In all other cases the low binding affinity of investigated progestins precludes possible biological role. For 5 alpha-pregnane-3 alpha-ol-20-one, 5 alpha-pregnane-20 alpha-ol-3-one, and 5 alpha-pregnane-3 beta,20 alpha-diol the relative binding affinity was less than 1%. A rather low binding (< 15%) was observed in 5 alpha-pregnane-3,20-dione in all ruminant species investigated. The antiprogestin J912 was found to be highly efficient in displacing progesterone from its endometrial binding sites in carnivores and might therefore be used for pregnancy interruption during diapause in certain species, e.g., in captive bears.