Semen proteome and transcriptome of the endangered black-footed ferret (Mustela nigripes) show association with the environment and fertility outcome.

The ex situ population of the endangered black-footed ferret (Mustela nigripes) has been experiencing declines in reproductive success over the past 30 years of human-managed care. A potential cause may be environmental-dependent inbreeding depression with diet being one of the contributing factors since ferrets are not fed their natural diet of prairie dogs. Here, we generated and analyzed semen proteome and transcriptome data from both wild and ex situ ferrets maintained on various diets. We identified 1757 proteins across all samples, with 149 proteins unique to the semen of wild ferrets and forming a ribosomal predicted protein-protein interaction cluster. Wild ferrets also differed from ex situ ferrets in their transcriptomic profile, showing enrichment in ribosomal RNA processing and potassium ion transport. Successful fertility outcomes documented for ex situ ferrets showed the strongest association with the semen transcriptome, with enrichment in genes involved in translation initiation and focal adhesion. Fertility also synergized with the effect of diet on differentially expressed transcriptomes, mainly affecting genes enriched in mitochondrial function. Our data and functional networks are important for understanding the causes and mechanisms of declining fertility in the ex situ ferret population and can be used as a resource for future conservation efforts.

Trehalose delivered by cold-responsive nanoparticles improves tolerance of cumulus-oocyte complexes to microwave drying.

PURPOSE: Trehalose is a non-permeable protectant that is the key to preserve live cells in a dry state for potential storage at ambient temperatures. After intracellular trehalose delivery via cold-responsive nanoparticles (CRNPs), the objective was to characterize the tolerance of cat cumulus-oocyte complexes (COCs) to different levels of microwave-assisted dehydration. METHODS: Trehalose was first encapsulated in CRNPs. After exposure to trehalose-laden CRNPs, different water amounts were removed from cat COCs by microwave drying. After each dehydration level, meiotic and developmental competences were evaluated via in vitro maturation, fertilization, and embryo culture. In addition, expressions of critical genes were assessed by quantitative RT-PCR. RESULTS: CRNPs effectively transported trehalose into COCs within 4 h of co-incubation at 38.5 °C followed by a cold-triggered release at 4 °C for 15 min. Intracellular presence of trehalose enabled the maintenance of developmental competence (formation of blastocysts) as well as normal gene expression levels of HSP70 and DNMT1 at dehydration levels reaching up to 63% of water loss. CONCLUSION: Intracellular trehalose delivery through CRNPs improves dehydration tolerance of COCs, which opens new options for oocyte storage and fertility preservation at ambient temperatures.

Current knowledge in the biology of gametes and embryos from Carnivora.

In addition to companion animals and laboratory species, about 270 carnivore species play fundamental ecological roles in different ecosystems. However, almost 40% of carnivore species are now threatened or endangered in the wild because of human activities. While protection of natural habitats is critical, it is equally important to better understand carnivore reproduction, including a solid knowledge in sperm, oocyte, and embryo biology, to maintain sustainable populations in the wild and in conservation breeding centers. Characterizing gamete and embryo biology is also needed to develop cryopreservation and assisted reproductive technologies to enhance conservation efforts. The objective of this review is to provide the most recent knowledge in the biology of sperm cells, oocytes, and early embryos across all carnivore families. Overall, most data originate from populations maintained in breeding centers or zoos. Characterizations of sperm biology and cryopreservation are far more advanced than for oocytes and embryos. Currently, sperm biology is mainly studied in Canids, Felids, Ursids, and Mustelids, with more emphasis on structural than functional properties. Importantly, fundamental studies of gamete and embryo biology in domestic dogs, cats, and ferrets have paved the way for more precise characterizations in wild counterparts as well as the development of cryopreservation and assisted reproductive technologies. A striking feature of spermatozoa across a wide range of Canids and Felids is the presence of teratospermia (>60% of abnormal sperm cells), which is related to the loss of genetic diversity in some populations. Although sperm structures differ across carnivore families, sperm biology remains difficult to compare because of the small amount of data in many species. Regarding oocyte biology and embryology, data are much scarcer than in sperm cells, with too few studies going beyond structural descriptions. More carnivore species and more individuals (especially from wild populations in addition to captive ones) must be studied to improve our understanding about comparative germplasm biology and develop adequate conservation breeding strategies including the use of cryobanking and assisted reproductive technologies.

Damages and stress responses in sperm cells and other germplasms during dehydration and storage at nonfreezing temperatures for fertility preservation.

Long-term preservation of sperm, oocytes, and gonadal tissues at ambient temperatures has the potential to lower the costs and simplify biobanking in human reproductive medicine, as well as for the management of animal populations. Over the past decades, different dehydration protocols and long-term storage solutions at nonfreezing temperatures have been explored, mainly for mammalian sperm cells. Oocytes and gonadal tissues are more challenging to dehydrate so little to no progress have been made. Currently, the detrimental effects of the drying process itself are better characterized than the impact of long-term storage at nonfreezing temperatures. While structural and functional properties of germ cells can be preserved after dehydration, a long list of damages and stresses in nuclei, organelles, and cytoplasmic membranes have been reported and sometimes mitigated. Characterizing those damages and better understanding the response of germ cells and tissues to the stress of dehydration is fundamental. It will contribute to the development of optimal protocols while proving the safety of alternative storage options for fertility preservation. The objective of this review is to (1) document the types of damages and stress responses, as well as their mitigation in cells dried with different techniques, and (2) propose new research directions.

Feline microRNAome in ovary and testis: Exploration of in-silico miRNA-mRNA networks involved in gonadal function and cellular stress response.

The aim of the study was to perform the first in-depth analysis of miRNAs in ovarian and testicular tissues of the domestic cat, a critical biomedical model. Specifically, potential miRNA involvement was explored in gonadal function, testis development, and cellular stress response to preservation protocols. We performed miRNA-sequencing on 20 ovarian and 20 testicular samples from 15 cats, including different ages and tissue treatments. Using fresh tissues (n = 15), we confirmed gonadal expression of 183 miRNA precursors and discovered additional 52 novel feline candidate precursors. We integrated the mRNA data from our previous study on the same age and treatment groups to create in-silico miRNA-mRNA networks and their functional enrichment, which allows comprehensive exploration into possible miRNA functions in cat gonads. Clusters of miRNAs united by shared differentially expressed mRNA targets are potentially involved in testicular development and spermatogenesis. MicroRNAs could play a significant role in ovarian tissue response to stress from microwave-assisted dehydration, with smaller roles in cellular response to vitrification in both ovary and testis. This new list of miRNAs with potential function in cat gonads is a major step towards understanding the gonadal biology, as well as optimizing fertility preservation protocols.

Transcriptome dynamics in developing testes of domestic cats and impact of age on tissue resilience to cryopreservation.

BACKGROUND: Fundamental knowledge of cellular and molecular mechanisms in developing testicular tissues is critical to better understand gonadal biology and responses to non-physiological conditions. The objective of our study was to (1) analyze transcriptome dynamics in developing testis of the domestic cat and (2) characterize age effects on the initial response of the tissue to vitrification. Tissues from adult and juvenile cats were processed for histology, DNA integrity, and RNA sequencing analyses before and after vitrification. RESULTS: Transcriptomic findings enabled to further characterize juvenile period, distinguishing between early and late juvenile tissues. Changes in gene expression and functional pathways were extensive from early to late juvenile to adult development stages. Additionally, tissues from juvenile animals were more resilient to vitrification compared to adult counterparts, with early juvenile sample responding the least to vitrification and late juvenile sample response being closest to adult tissues. CONCLUSIONS: This is the first study reporting comprehensive datasets on transcriptomic dynamic coupled with structural analysis of the cat testis according to the age and exposure to cryopreservation. It provides a comprehensive network of functional terms and pathways that are affected by age in the domestic cat and are either enriched in adult or juvenile testicular tissues.

Initial response of ovarian tissue transcriptome to vitrification or microwave-assisted dehydration in the domestic cat model.

BACKGROUND: Long term preservation of living ovarian tissues is a critical approach in human reproductive medicine as well as in the conservation of rare animal genotypes. Compared to single cell preservation, optimization of protocols for tissues is highly complex because of the diversity of cells responding differently to non-physiological conditions. Using the prepubertal domestic cat as a model, the objective was to study immediate effects of vitrification or microwave-assisted dehydration on the global transcriptome dynamics in the ovarian cortex. RNA sequencing was performed on ovarian tissues (n = 6 individuals) from different conditions: fresh tissue after dissection (F), vitrified/warmed tissue (V), tissue dehydrated for 5 min (D5) or 10 min (D10) followed by rehydration. Differential gene expression analysis was performed for comparison pairs V vs. F, D10 vs. F, D5 vs. F and D10 vs. D5, and networks were built based on results of functional enrichment and in silico protein-protein interactions. RESULTS: The impact of the vitrification protocol was already measurable within 20 min after warming and involved upregulation of the expression of seven mitochondrial DNA genes related to mitochondrial respiration. The analysis of D10 vs. F revealed, 30 min after rehydration, major downregulation of gene expression with enrichment of in silico interacting genes in Ras, Rap1, PI3K-Akt and MAPK signaling pathways. However, comparison of D5 vs. F showed negligible effects of the shorter dehydration protocol with two genes enriched in Ras signaling. Comparison of D10 vs. D5 showed downregulation of only seven genes. Vitrification and dehydration protocols mainly changed the expression of different genes and functional terms, but some of the differentially expressed genes formed a major in silico protein-protein interaction cluster enriched for mitochondrial respiration and Ras/MAPK signaling pathways. CONCLUSIONS: Our results showed, for the first time, different effects of vitrification and microwave-assisted dehydration protocols on the global transcriptome of the ovarian cortex (using the domestic cat as a biomedical model). Acquired data and networks built on the basis of differentially expressed genes (1) can help to better understand stress responses to non-physiological stresses and (2) can be used as directions for future preservation protocol optimizations.

Influence of microwave-assisted dehydration on morphological integrity and viability of cat ovarian tissues: First steps toward long-term preservation of complex biomaterials at supra-zero temperatures.

Ovarian tissue contains large pools of immature oocytes enclosed in primordial follicles, making it an attractive target for fertility preservation in female cancer patients, livestock and wild species. Compared to cryopreservation, desiccation and long-term storage of samples at supra-zero temperatures (using strategies inspired from small organisms to resist extreme environments) would be more cost-effective and convenient. The objective of the study was to characterize the influence of microwave-assisted dehydration on structural and functional properties of living ovarian tissues. While this method allows preservation of single cells (cat oocytes and sperm cells so far) using trehalose as the xeroprotectant, it has not been developed for multicellular tissues yet. Ovarian cortex biopsies were reversibly permeabilized, exposed to various concentrations of trehalose, and dried for different times using a commercial microwave under thermal control. Effective dehydration of samples along with proper trehalose retention were reached within 30 min of microwave drying. Importantly, the process did not affect morphology and DNA integrity of follicles or stromal cells. Moreover, transcriptional activity and survival of follicles were partially maintained following 10 min of drying, which already was compatible with storage at non-cryogenic temperatures. Present data provide critical foundation to develop dry-preservation techniques for long-term storage of living multicellular tissues.

Identification of feline Kiss1 and distribution of immunoreactive kisspeptin in the hypothalamus of the domestic cat.

In recent years, the Kiss1 gene has been reported in a number of vertebrate species, and a substantial dataset has been acquired to demonstrate the critical role of kisspeptins in the reproductive system; yet limited information is available for carnivores. In the present study, we identified and characterized feline Kiss1 by isolating and cloning its full-length cDNA in the domestic cat hypothalamus and caracal testis, using the method of rapid amplification of cDNA ends. Additionally, we isolated and cloned the 3' end of Kiss1 cDNA, containing kisspeptin-10 (Kp10), from the ovaries of a clouded leopard and Siberian tiger. Nucleotide sequencing revealed that domestic cat Kiss1 cDNA is of 711 base pairs and caracal Kiss1 cDNA is of 792 base pairs, both having an open reading frame of 450 base pairs, encoding a precursor protein Kiss1 of 149 amino acids. The core sequence of the feline kisspeptin Kp10 was found to be identical in all species analyzed here and is highly conserved in other vertebrate species. Using an anti-Kp10 antibody, we found the immunoreactive kisspeptin to be localized in the periventricular and infundibular nuclei of the cat hypothalamus. The results show that kisspeptin is highly conserved among different feline families, and its immunoreactive distribution in the hypothalamus may indicate its physiological function in the domestic cat.

Progesterone, estrogen, and androgen receptors in the corpus luteum of the domestic cat, Iberian lynx (Lynx pardinus) and Eurasian lynx (Lynx lynx).

In contrast to the species studied, the corpus luteum (CL) of Iberian and Eurasian lynx physiologically persists in the ovary for more than 2 years and continues to secrete progesterone. Such persistent CL (perCL) transition into the next cycle and are present in the ovary together with the freshly formed CL (frCL) of a new ovulation. To date, the mechanisms supporting such CL persistence are not known. We analyzed the potential receptivity of feline CL to sex steroids through mRNA measurements of progesterone receptor (PGR), progesterone receptor membrane components (PGRMC) 1 and 2, estrogen receptor (ESR) 1 and ESR2, G protein-coupled estrogen receptor 1 (GPER1), and androgen receptor (AR). All receptors were present in domestic cat CL during pregnancy and the nonpregnant luteal phase, in frCL and perCL of post-mating Iberian lynx and in perCL of pre-mating Eurasian lynx. Mass spectrometry detected the presence of PGRMC1 protein in frCL and perCL of the Iberian lynx. In both domestic cat and lynx CL, PGR, PGRMC1, and ESR1 proteins were localized in luteal cells by immunohistochemistry. The mRNA levels of PGR, PGRMC1, PGRMC2, ESR1, and AR changed significantly throughout the domestic cat luteal phase. This may indicate involvement of these receptors in the processes of formation, maintenance, and regression of feline CL. In Iberian lynx, expression of PGRMC1, PGRCM2, ESR1, GPER1, and AR was significantly higher in perCL compared with frCL, whereas ESR2 was reversed. High mRNA amounts of these receptors in perCL suggest that physiological persistence of lynx CL may be partly regulated by actions of sex steroids through their nuclear and/or membrane receptors.

Synthesis and reception of prostaglandins in corpora lutea of domestic cat and lynx.

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.

Apoptosis-Related Factors in the Luteal Phase of the Domestic Cat and Their Involvement in the Persistence of Corpora Lutea in Lynx.

The corpus luteum (CL) is a transient gland formed in the ovary after ovulation and is the major source of progesterone. In the Iberian and Eurasian lynx, CL physiologically persist after parturition and retain their capacity to produce progesterone, thus suppressing the ovarian activity. This unique reproductive characteristic has a big impact on the success of assisted reproduction techniques in the endangered Iberian lynx. The mechanisms behind CL persistence are not yet understood and require extensive studies on potential luteotropic and luteolytic factors in felids. Because the apoptosis system has been shown to be involved in structural regression of CL in many species, we aimed to investigate the capacity of perCL to undergo apoptosis. In addition, we performed initial studies on the apoptosis system in the luteal phase of the domestic cat. No previous research on this system has been made in this species. Our factors of interest included agents of the intrinsic apoptosis pathway, i.e., pro-survival B-cell CLL/lymphoma 2 (BCL2) and pro-apoptotic BCL2-associated X protein (BAX), the executioner caspase-3 (CASP3), as well as of the extrinsic pathway, i.e., pro-apoptotic receptor FAS, and tumor necrosis factor (TNF) and its receptors (pro-apoptotic TNFRSF1A and pro-survival TNFRSF1B). We analyzed the relative mRNA levels of these factors, as well as protein localization of CASP3 and TNF during stages of pregnancy and the non-pregnant luteal phase in CL of domestic cats. The same factors were investigated in freshly ovulated CL (frCL) and perCL of Iberian and Eurasian lynx, which were histologically analyzed. All factors were present in the CL tissue of both domestic cat and lynx throughout all analyzed stages. The presence of pro-apoptotic factors BAX, CASP3, FAS and TNFRSF1A in perCL of the Eurasian and Iberian lynx might indicate the potential sensitivity of perCL to apoptotic signals. The expression of pro-survival factors BCL2 and TNFRSF1B was significantly higher in perCL compared to frCL of studied Iberian lynx, suggesting the potential involvement of these factors in the structural integrity of perCL. In both Iberian lynx and pregnant and non-pregnant domestic cats, the expression of TNFRSF1A was significantly higher in forming CL compared to other stages, suggesting the conserved involvement of this factor in the tissue reorganization during formation of the feline CL. The mRNA levels of CASP3 and TNFRSF1B were highest during regression stages of domestic cat CL. The current study provides initial results on the possible involvement of the apoptosis system in the structure and function of the feline CL and in its physiological persistence.

Comparative analysis of intraluteal steroidogenic enzymes emphasises the functionality of fresh and persistent corpora lutea during pro--and metoestrus in the lynx.

European lynx species demonstrate an atypical ovarian cycle compared to other felids. The physiological persistence of corpora lutea (CLs), reflected in constantly elevated progesterone (P4) concentrations in serum, is thought to ensure a seasonal monooestrus. Moreover, the coexistence of CLs from a recent ovulation (freshCLs) and persistent CLs from previous years (perCLs) on the same ovary has been proven. We assume that perCLs in lynxes occur due to fundamentally different mechanisms of luteal regression. Our study presents a detailed analysis of steroidogenic enzymes and steroids in fresh and perCLs obtained from Iberian lynxes during metoestrus, and in perCLs obtained from Eurasian lynxes during prooestrus. By quantitative PCR we measured relative mRNA amounts of steroidogenic acute regulatory protein (STAR), cytochrome P450 oxidases (CYPs), hydroxysteroid dehydrogenases (HSDs) and a steroid reductase (SRD). Protein expression in CLs was investigated for CYP11A1, CYP17A1, CYP19A1 and HSD3B. Additionally, the intraluteal and serum steroid content was determined. During metoestrus, mRNA amounts of STAR, CYP11A1, CYP19A1, HSD17B7 and SRD5A1 were significantly higher in perCLs compared to freshCLs. Protein of CYP11A1 was detected independently of the CL age in metoestrus, but expression was less evident in prooestrous perCLs. The protein signal of CYP17A1 was strong in freshCLs and perCLs of metoestrus, but weak at prooestrus. The presence of CYP19A1 protein was confirmed in each stage of the CL. These findings contribute to the hypothesis that CLs from previous years might support freshly developed CLs for pregnancy maintenance. However, initiation of ovulation might require a functional down-regulation of perCLs prior to breeding. It is noteworthy that the HSD3B1 mRNA amount was significantly elevated in fresh compared to perCLs (metoestrus). Accordingly, HSD3B protein was substantially present in freshCLs, whereas signals were literally absent in all perCLs. Elevated expression of HSD3B coincided with high intraluteal oestrogen concentrations in freshCLs; however, the enzyme pattern was less concordant with intraluteal P4 and androgen concentrations. Serum P4 concentrations of Iberian lynxes were constant between prooestrus and prolonged dioestrus. Moreover, constantly high serum oestrogen concentrations were measured during pro-, met- and prolonged dioestrus. The physiology of exceptionally high serum oestrogen concentrations outside the breeding season of lynxes merits further investigation. In conclusion our study supports the concept that the unique reproductive strategy of lynxes is directly linked to sustained intraluteal steroid biogenesis in persistent CLs.

The corpus luteum of the domestic cat: histologic classification and intraluteal hormone profile.

In the present study, we aimed to histologically stage and characterize the structural life span of the CL in the domestic cat. Moreover, the intraluteal levels of progesterone and estrogens were determined throughout the pregnant and nonpregnant (pseudopregnant, PP) luteal phases. On the basis of observed histomorphology of CL, the following stages were identified: CL formation (preimplantation period, PP1), development/maintenance (Days 10-36 of pregnancy, PP2), early regression (Days 38 and 39 of pregnancy, PP3), late regression (Day 48 of pregnancy, PP4), and corpus albicans. The main cellular markers included luteal cell shape, the type and degree of vacuolation, nucleus condition, and the ratio of nonsteroidogenic to luteal cells. Intraluteal levels of progesterone and estrogens differed significantly throughout stages of pseudopregnancy (P < 0.01, progesterone; P < 0.0001, estrogens). The progesterone level in PP2 was higher compared with PP3 and PP4 (P < 0.05). The estrogen level in PP1 was higher compared with PP2 (P < 0.05), PP3 and PP4 (P < 0.005), as well as in PP2 compared with PP3 and PP4 (P < 0.005). The staging of the domestic cat luteal phase established here provides a basis for further research on feline luteotropic and luteolytic factors. These data will contribute to comparative studies in felids.

Synthesis and biological evaluation of 11' imidazolyl antiprogestins and mesoprogestins.

Antiprogestins with a 4' para imidazolylphenyl moiety were synthesized and their biochemical interactions with the progesterone and glucocorticoid receptor were investigated. Depending on the substitution pattern at the 17 position partial progesterone receptor (PR)-agonistic derivatives like compounds EC339 and EC336 or pure antagonists like compound EC317 were obtained. EC317 was investigated in vivo and found to be significantly more potent than RU 486 in cycling and pregnant guinea pigs. For testing the biological action progesterone receptor modulators (PRM), guinea pigs appears as a specific model when compare to pregnant human uterus. This model correlates to human conditions such as softening and widening of the cervix, the elevation of the uterine responsiveness to prostaglandins and oxytocin, and finally to induction of labor. The use of non-pregnant guinea pigs permitted the simultaneous assessment of PR-agonistic and PR-antagonistic properties and their physiological interactions with uterine and vaginal environment. These can histologically be presumed from the presence of estrogen or progesterone dominance in the genital tract tissues. The ovarian histology indicated the effects on ovulation. Corpora lutea in guinea pigs further reflects inhibitory effects of the progesterone-dependent uterine prostaglandin secretion. PRMs are initially synthesized as analogues of RU 486. They represent a heterogeneous group of compounds with different ratios of PR-agonistic and-antagonistic properties. PR-agonistic properties may be essential for uterine anti-proliferative effects. In various clinical studies these were also attributed to RU 486 or Ulipristal [1,2]. Adjusted PR-agonistic PRMs (EC312, EC313) [3] may be more effective in achieving a mitotically resting endometrium and superior uterine tumor inhibition. For the use in termination of pregnancy, progesterone-inhibitory effects are essentially needed. Even minor PR-agonistic properties compromise the therapeutic goals. Pure PR-antagonists, as EC317, clearly exceeded the gold standard RU 486 with respect to labor inducing effects. Mechanistically it is surprising that both types of compound may be potent inhibitors of ovulation.

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.

Lynx reproduction--long-lasting life cycle of corpora lutea in a feline species.

A review of lynxes' reproductive biology and comparison between the reproductive cycles of the domestic cat and lynxes is presented. Three of the four lynx species (the bobcat excluded) express quite similar reproductive pattern (age at sexual maturity, estrus and pregnancy length, litter size). Similarly to the domestic cat, the bobcat is polyestric and can have more than one litter per year. Domestic cats and many other felid species are known to express anovulatory, pregnant and pseudo-pregnant reproductive cycles in dependence on ovulation induction and fertilization. The formation of corpora lutea (CLs) occurs after ovulation. In pregnant animals, luteal function ends with parturition, whereas during pseudo-pregnancy a shorter life span and lower hormone secretion are observed. The life cycle of corpora lutea in Eurasian lynxes is different from the pattern described in domestic cats. Lynx CLs produce progestagens in distinctive amounts permanently for at least two years, regardless of their origin (pregnancy or pseudo-pregnancy). It is suggested that long-lasting CLs induce a negative feedback to inactivate folliculogenesis, turning a normally polyestric cycle observed in most felids into a monoestric cycle in lynxes.

Synthesis and biological evaluation of partially fluorinated antiprogestins and mesoprogestins.

A series of antiprogestins have been synthesized by partially fluorinating the steroid molecule in positions relevant for receptor binding. By introducing fluorine at the exo-methylene of the 17 spirofuran ring, we obtained partial agonists (mesoprogestins) with significant applications for antiproliferative and antiovulatory treatment strategies in gynecological therapy such as uterine fibroids, endometriosis and heavy menstrual bleeding. Compared to the standard drug RU486, our synthesized compounds exhibited considerable dissociation between antiprogestational and antiglucocorticoid PR receptors. Furthermore, our studies have shown that pure antiprogestins can be generated by partially fluorinating the 17 propenyl and propynl group or by substituting the 4' acetyl phenyl group in the 11 position using trifluromethyl group.

Conserved methylation of the glucocorticoid receptor gene exon 1(7) promoter in rats subjected to a maternal methyl-supplemented diet.

It is well known that the early life experiences affect stress responses and other physiological and behavioral traits in adulthood. Both rat and human studies have shown that early postnatal effects are associated with methylation of the hippocampal glucocorticoid receptor gene exon 1(7) (rat) and 1-F (human) promoters. Methylation of these sites is also seen following methionine administration in adult rats. However, it remains unclear whether similar alterations in DNA methylation profiles can result from prenatal influences. To address this question, we fed pregnant rats a methyl-supplemented diet that resulted in alteration of the stress response. However, methylation analysis revealed no effect of methyl supplements on methylation patterns of the glucocorticoid receptor gene exon 1(7) promoter in offspring. These results suggest that the pre- and postnatal effects of methyl supplementation have different mechanisms.