Different expression of leptin and IGF1 in the adult and prepubertal testis in dogs.

Leptin (Lep) and insulin-like growth factor 1 (IGF1) are implicated in the regulation of testicular function, but in dogs, our knowledge is limited to the possible role of the IGF1 system in testicular tumours. In this study, we aimed to describe and compare gene expression and protein localization of Lep, IGF1 and their receptors (LepR and IGF1R, respectively) in the testis of healthy adult and prepubertal dogs. Testes were collected from sexually healthy mature (n = 7) and from 8-week-old dogs (n = 7). Relative gene expression of Lep, LepR, IGF1 and IGF1R was determined by semi-quantitative real-time (TaqMan) PCR and cellular distribution in the testis by immunohistochemistry. Statistical analysis was carried out with Student's t test. Lep and LepR mRNA concentration was similar between the two groups, but IGF1 and IGF1R gene expression was significantly higher in the 8-week-old pups. Protein localization and the intensity of signals differed by age. In adults, Lep and LepR immunoreactivity was detected in spermatocytes and spermatids. Leydig cells showed sporadic, weak Lep staining. In prepubertal animals, intense Lep signals were present in Leydig and Sertoli cells, and LepR was found in Leydig cells. IGF1 and IGF1R protein was expressed in spermatogonia of the mature testis; IGF1 signals in Leydig cells seemed stronger than IGF1R. In the pups, IGF1 and IGF1R staining was detected in Leydig cells and in gonocytes. Sertoli cells showed weak IGF1 and sporadic, weak IGF1R signals. In conclusion, Lep and IGF1 may support spermatogenesis in adult dogs and mediate Leydig cell function. In the immature testis, they may promote development of Sertoli and Leydig cells and gonocytes.

Decidualization of the canine uterus: From early until late gestational in vivo morphological observations, and functional characterization of immortalized canine uterine stromal cell lines.

The apparent lack of classical mechanisms for maternal recognition of pregnancy is one of the most intriguing features of canine reproduction. Consequently, similar levels of circulating luteal steroids are observed in pregnant and non-pregnant dogs. However, the early pre-implantation canine embryo locally modulates uterine responses to its presence, facilitating the successful onset of pregnancy. As a part of this interaction, the canine uterus undergoes a species-specific decidualization. Maternal stroma-derived decidual cells develop, the only cells of the canine placenta expressing progesterone receptor (PGR). There exists an acute need for an in vitro stable cell line model for canine decidualization. Therefore, herein our goal was to establish, immortalize and characterize such a cell line. We immortalized three monolayer dog uterine stromal (DUS) cell lines by stably transfecting them with SV40Tag oncogene. Cells retained their mesenchymal character for over 30 passages, as evidenced by VIMENTIN staining. Genomic incorporation of the SV40Tag protein was confirmed by immunofluorescence and Western blot analyses. Cells submitted to a classical in vitro decidualization protocol (N6,2'-O-dibutyryladenosine-3',5'-cyclic monophosphate) revealed upregulated gene levels of selected major decidualization markers (e.g. PRLR, PGR, IGF1, PTGES). Additionally, the basic decidualization capability of PGE2 was demonstrated, revealing increased levels of, for example, PGR and PRLR gene expression, thereby implying its involvement in the progesterone-dependent decidualization in the canine uterus. In summary, our in vitro model with immortalized DUS cell line could serve as an ideal and unique model to study the underlying molecular and endocrine mechanisms of canine decidualization.

Expression of GnRH receptor in the canine corpus luteum, and luteal function following deslorelin acetate-induced puberty delay.

The goals of this study were as follows: (Experiment 1) to examine the basic capability of canine corpora lutea (CL) to respond to GnRH by assessing expression of gonadotropin-releasing hormone receptor (GnRH-R) in luteal samples collected throughout the luteal lifespan from non-pregnant dogs, and (Experiment 2) to investigate the effects of pre-pubertal application of the GnRH agonist deslorelin acetate on luteal function following the first oestrus. Mature CL were collected during the mid-luteal phase (days 30-45) from treated and control bitches. Transcript levels of several factors were determined: estrogen receptors (ESR1/ERα, ESR2/ERß), progesterone (P4)-receptor (PGR), prolactin receptor (PRLR), PGE2-synthase (PTGES) and PGE2 receptors (PTGER2/EP2, PTGER4/EP4), vascular endothelial growth factor (VEGFA) and VEGF receptors (VEGFR1 and VEGFR2), cyclooxygenase 2 (COX2/PTGS2), steroidogenic acute regulatory protein (STAR) and 3ß-hydroxysteroid dehydrogenase (3ßHSD). Additionally, levels of Kisspeptin 1 (Kiss1) and its receptor (KISS1-R) were evaluated. Although generally low, GnRH-R expression was time dependent and was elevated during early dioestrus, with a significant decrease towards luteal regression. In deslorelin-treated and control dogs, its expression was either low or frequently below the detection limit. EP2 and VEGFR1 were higher in the treated group, which could be caused by a feedback mechanism after long-term suppression of reproductive activity. Despite large individual variations, 3ßHSD was higher in the deslorelin-treated group. This, along with unchanged STAR expression, was apparently not mirrored in increased luteal functionality, because similar P4 levels were detected in both groups. Finally, the deslorelin-mediated long-term delay of puberty does not have negative carry-over effects on subsequent ovarian functionality in bitches.

Prostaglandin F2α promotes angiogenesis and embryo-maternal interactions during implantation.

Implantation in humans and other mammals is a critical period during which high embryonic mortality rates occur. Prostaglandins (PGs) are key mediators regulating interactions between the reproductive tract and the conceptus (embryo with extraembryonic membranes). Although the significance of PGF2α as a regulator of corpus luteum regression is well established, the role of its high amounts in the uterine lumen in most mammals, regardless of placentation type, during the implantation period remains unresolved. We hypothesized that PGF2α acting as an embryonic signal mediator contributes to pregnancy establishment. Using a porcine model, we demonstrated that the conceptus and its signal (estradiol-17ß) elevated endometrial expression of PGF2α receptor (PTGFR) in vivo and in vitro PTGFR protein was expressed mainly in luminal epithelial (LE) and glandular epithelial cells and blood vessels in the endometrium. PGF2α stimulated the MAPK1/3 pathway in endometrial LE cells that coincided with elevated gene expression and secretion of endometrial vascular endothelial growth factor A (VEGFA) protein. PGF2α-PTGFR and adenylyl cyclase signaling were involved in this process. PGF2α-induced VEGFA acting through its receptors stimulated proliferation of endometrial endothelial cells. Moreover, PGF2α elevated gene expression of biglycan, matrix metalloproteinase 9, transforming growth factor ß3, and interleukin 1α in the endometrium. In summary, our study indicates that PGF2α participates in pregnancy establishment by promoting angiogenesis and expression of genes involved in tissue remodeling and conceptus-maternal interactions in porcine endometrium during early pregnancy.

LPS-mediated effects and spatio-temporal expression of TLR2 and TLR4 in the bovine corpus luteum.

When given intravenously (iv), lipopolysaccharide (LPS) transiently suppresses the structure and function of the bovine corpus luteum (CL). This is associated with increased release of prostaglandin (PG) F2α metabolite. The underlying regulatory mechanisms of this process remain, however, obscure. Therefore, the aims of this study were: i) to investigate the expression of the LPS receptor toll-like receptor 4 (TLR4) and 2 (TLR2) in the bovine CL during early, mid- and late luteal phases; and ii) to further dissect the mechanisms of LPS-mediated suppression of luteal function. As revealed by semi-quantitative qPCR and immunohistochemistry, both receptors were detectable throughout the luteal lifespan. Their mRNA levels increased from the early toward the mid-luteal phase; no further changes were observed thereafter. The TLR4 protein seemed more highly represented than TLR2. The cellular localization of TLRs was in blood vessels; weaker signals were observed in luteal cells. Additionally, cows were treated either with LPS (iv, 0.5 µg/kg BW) or with saline on Day 10 after ovulation. Samples were collected 1200 h after treatment and on Day 10 of the respective subsequent (untreated) cycle. The mRNA expression of several possible regulatory factors was investigated, revealing the suppression of PGF2α receptor (PTGFR), STAR protein and 3ß-hydroxysteroid dehydrogenase, compared with controls and subsequent cycles. The expression of TLR2 and TLR4, interleukin 1α (IL1A) and 1ß (IL1B) and of PGF2α and PGE2 synthases (HSD20A and mPTGES respectively) was increased. The results demonstrate the presence of TLR2 and TLR4 in the bovine CL, and implicate their possible involvement in the deleterious effects of LPS on its function.

Expression of genes involved in the embryo-maternal interaction in the early-pregnant canine uterus.

Although there is no acute luteolytic mechanism in the absence of pregnancy in the bitch, a precise and well-timed embryo-maternal interaction seems to be required for the initiation and maintenance of gestation. As only limited information is available about these processes in dogs, in this study, the uterine expression of possible decidualization markers was investigated during the pre-implantation stage (days 10-12) of pregnancy and in the corresponding nonpregnant controls. In addition, the expression of selected genes associated with blastocyst development and/or implantation was investigated in embryos flushed from the uteri of bitches used for this study (unhatched and hatched blastocysts). There was an upregulated expression of prolactin receptor (PRLR) and IGF2 observed pre-implantation. The expression of PRL and of IGF1 was unaffected, and neither was the expression of progesterone- or estrogen receptor ß (ESR2). In contrast, (ESR1) levels were elevated during early pregnancy. Prostaglandin (PG)-system revealed upregulated expression of PGE2-synthase and its receptors, PTGER2 and PTGER4, and of the PG-transporter. Elevated levels of AKR1C3 mRNA, but not the protein itself, were noted. Expression of prostaglandin-endoperoxide synthase 2 (PTGS2) remained unaffected. Most of the transcripts were predominantly localized to the uterine epithelial cells, myometrium and, to a lesser extent, to the uterine stroma. PGES (PTGES) mRNA was abundantly expressed in both groups of embryos and appeared higher in the hatched ones. The expression level of IGF2 mRNA appeared higher than that of IGF1 mRNA in hatched embryos. In unhatched embryos IGF1, IGF2, and PTGS2 mRNA levels were below the detection limit.

Uterine and placental expression of canine oxytocin receptor during pregnancy and normal and induced parturition.

Oxytocin (OT) plays an important role as an inducer of uterine contractility, acting together with its receptor (OTR) to increase synthesis of prostaglandins. Although OT is commonly used in the treatment for dystocia and uterine inertia in the bitch, little attention has been paid to the role of OT in mechanisms regulating parturition in the dog, so that knowledge about the expression of OTR in the canine uterus and placenta is sparse. Consequently, the expression and cellular localization of OTR were investigated in canine utero/placental compartments and interplacental sites throughout pregnancy and at normal and antigestagen-induced parturition, by real-time PCR, immunohistochemistry, western blot and in situ hybridization. The utero/placental and interplacental expression of OTR was constant from pre-implantation until mid-gestation, with a significant increase observed at prepartum luteolysis. In antigestagen-treated mid-pregnant dogs, OTR was upregulated in both interplacental and utero/placental samples. Besides clear myometrial signals, cellular localization of OTR was evident in the endometrial surface epithelial, stromal and vascular endothelial cells. Weaker signals were observed in superficial and deep uterine glandular epithelial cells. Placental OTR was localized in maternal decidual cells and capillary pericytes. Finally, OTR was colocalized with the progesterone receptor (PGR) in maternal decidual cells, coinciding with previously reported increased availability of prostaglandins in the foetal part of the placenta during normal and induced parturition. These findings suggest involvement of OTR in the signalling cascade leading to the prepartum release of prostaglandins from the pregnant canine uterus.

Influence of feeding and UVB exposition on the absorption mechanisms of calcium in the gastrointestinal tract of veiled chameleons (Chamaeleo calyptratus).

The purpose of this study was to investigate the influence of feeding and UVB exposition on the occurrence and distribution patterns of vitamin D receptors (VDR) and calbindin D28k (Cb-D28k) in the gastrointestinal tract of veiled chameleons. Thus, 56 veiled chameleon hatchlings were divided into six treatment groups: UV (with UVB exposure); No (no supplements, no UVB exposure); CaAUV (with calcium (Ca), vitamin A supplementation, UVB exposure); CaA (with Ca, vitamin A supplementation); CaADUV (with Ca, vitamin A, vitamin D supplementation, UVB exposure); and CaAD (with Ca, vitamin A, vitamin D supplementation). Animals were reared under the suspected conditions for 6 months on locust-based diets. Tissue samples of stomach, duodenum, ileum and colon were taken, and semi-quantitative immunohistochemical methods (IHC) were performed to detect Cb-D28k and VDR. VDR immunoreactions were higher in the luminal epithelium of the duodenum than in that of the ileum. VDR immunoreactions in the luminal epithelium were higher at the base of the villi of the duodenum as compared to the tip. Cb-D28k immunoreactions were mainly observed in the luminal epithelium of the duodenum. The two groups treated with all dietary supplements (CaADUV, CaAD) exhibited a higher Cb-D28k immunoreaction as those with no supplements and UVB exposure only. No immunoreaction for both proteins could be detected in the stomach. This study suggests that the duodenum plays an important role in the active transcellular absorption of Ca in veiled chameleons as shown by the immunohistochemical detection of VDR and Cb-D28k. Expression of Cb-D28k, in particular, appears to be regulated by dietary supplementation of vitamin D and vitamin A. VDRs, however, tended to be upregulated when animals were not supplemented with Ca, vitamin D and vitamin A. This may be due to the decreased Ca concentrations which caused vitamin D activation in the skin without any supplementation, but UVB exposure.

The spatio-temporal distribution of aromatase cytochrome in ovary throughout the canine oestrous cycle.

CONTEXT: New animal welfare legislation and ethical guidelines encourage alternative approaches for canine contraception, instead of surgical gonadectomy which is considered invasive and unjustified in healthy dogs. AIMS: Reversible contraception might be achieved by inhibition of aromatase (CYP19), an enzyme catalysing the conversion of androgens to oestrogens. This study provides insights into the spatio-temporal expression and distribution of aromatase in canine ovarian tissue. METHODS: Ovarian tissue was collected from 39 healthy and sexually mature bitches during different stages of the oestrous cycle: pro-oestrus (n =8), oestrus (n =12), dioestrus (n =9) (luteal phase) and anoestrus (n =10). Localisation of cytochrome P450 aromatase was determined by immunohistochemistry. KEY RESULTS: Aromatase activity in the dog is high during pro-oestrus, ovulation and early dioestrus. Comparing types of follicles and corpora lutea, the highest aromatase abundance was found in antral follicles and luteinising follicles, whereas corpora lutea and early antral follicles showed an intermediate presence of the enzyme. Interesting was the high abundance of aromatase in luteinising theca interna cells, prevailing over granulosa cells. CONCLUSIONS AND IMPLICATIONS: Understanding of cells involved in oestradiol production is important for targeted inhibition of oestradiol synthesis, possibly offering an approach for contraception and suppression of oestrus.

Status of the down-regulated canine testis using two different GNRH agonist implants in comparison with the juvenile testis.

Testicular function in the dog was down-regulated using two different GNRH agonist implants, with adult and juvenile testes serving as controls. Treatment resulted in an increased percentage of the interstitial area and decreased area of Leydig cell nuclei. Expression of StAR and the steroidogenic enzymes cytochrome P450 side-chain cleavage enzyme (P450scc, CYP11A1) and cytochrome P450 17α-hydroxylase-17,20-lyase (P450c17, CYP17A1) in Leydig cells was blocked at the mRNA and protein level, showing no differences between the two agonists. Staining for androgen receptor (AR) by immunohistochemistry was positive in Sertoli, Leydig and peritubular cells and some spermatogonia, with in situ hybridization confirming expression in Sertoli cells. At the mRNA level, expression of AR was not affected; however, translation was blocked (reduced percentage of AR-positive Sertoli cells), with the number of nuclei in basal position being decreased. In the juvenile testes, mRNA expression of StAR, CYP11A1 and CYP17A1 was higher compared with the other groups but distinctly lower for the AR. At the protein level, the expression was at the limit of detection for StAR; AR-positive Sertoli cells were not detected. Our observations show that the down-regulated testis is different from the juvenile one rather resembling the testicular status in seasonal breeders out of season.

Prostaglandin endoperoxide synthase 2 (PTGS2) and prostaglandins F2α and E2 synthases (PGFS and PGES) expression and prostaglandin F2α and E2 secretion following oestrogen and/or progesterone stimulation of the feline endometrium.

Sex steroids in synergy with prostaglandins (PG) are involved in the regulation of cyclic ovarian function. In this study, we investigated the mRNA expression of three genes involved in arachidonic acid (AA) metabolism and hence PG production in domestic cats: PG-endoperoxide synthase (PTGS2), PGF(2α) synthase (PGFS) and PGE(2) synthase (PGES). Feline endometria (n = 16) were collected at oestrus and mid and late phases of pseudopregnancy. In addition, the effects of E(2) and/or P(4) on PG secretion and gene expression on endometrial explants were studied in an in vitro culture system. Expression levels of all examined genes were up-regulated at the mid phase of pseudopregnancy. The effects of E(2) and/or P(4) treatment on both PG secretion and expression of the genes were observed after 12 h of culture. Expression of PGES was significantly up-regulated by E(2) plus P(4) at oestrus and the mid phase of pseudopregnancy and was also up-regulated by a single treatment with P(4) at late pseudopregnancy (p < 0.05). Simultaneous incubation with E(2) and P(4) up-regulated PTGS2 gene expression at oestrus and mid-luteal phase (p < 0.05). Progesterone plus E(2) significantly increased PGE(2) secretion at oestrus and the mid phase of pseudopregnancy. However, treatment with E(2) and/or P(4) affected neither PGF(2α) secretion nor PGFS expression at any phase after 12 h of culture. The overall findings indicate that genes involved in PG synthesis are up-regulated at the mid phase of pseudopregnancy. An increase in PGE(2) secretion and up-regulation of PGES and PTGS2 are the main responses of the endometrium to treatment with E(2) and P(4) at oestrus and the mid phase of pseudopregnancy in the cat. These data support the hypothesis that ovarian sex steroids via endometrial PGE(2) are involved in endocrine homoeostasis, especially at oestrus and the mid, but not the late, phase of pseudopregnancy in cats.

Endocrine and molecular control of luteal and placental function in dogs: a review.

In the domestic dog (Canis familiaris), the corpus luteum (CL) is the only source of progesterone (P4) in non-pregnant and pregnant animals. The progesterone secretion profiles are almost identical in both conditions until the last third of the luteal phase when the gradual P4 decline turns into a steep drop in pregnant bitches, indicating the onset of parturition. Consequently, the length of the CL-phase in non-pregnant dogs exceeds the luteal lifespan in pregnant animals. The canine CL-function is regulated by many species-specific regulatory mechanisms, the most intriguing of which is the reported independence of gonadotropic support during the first third of dioestrus. Recently, PGE2 has been proposed as one of the most important luteotropic factors acting locally during this time, but afterwards prolactin (PRL) appears to be the main luteotropic factor. Luteal regression/luteolysis occurs, however, in spite of an increased gonadotropic support. Lately, by demonstrating the expression of PRL-receptor (PRLr), a new insight into possible regulatory mechanisms has indicated that the supply of P4 could be controlled upstream of the steroidogenic machinery at the level of PRLr expression and/or function, subsequently leading to the functional suppression of the steroidogenic machinery. An endogenous source of a luteolytic agent is apparently lacking, implicating the luteal regression in non-pregnant bitches as a passive, degenerative process even if the PGF2α-receptor is constitutively expressed in canine CL. This is in contrast to pregnant dogs in which prepartum luteolysis seems to be an active process of CL destruction by PGF2α of utero/placental origin targeting the luteal PGF2α-receptor.

Leptin and leptin receptor gene expression in the canine corpus luteum during diestrus, pregnancy and after aglepristone-induced luteolysis.

Leptin (LEP) and leptin receptor (LEP-R) expression was shown to change throughout the luteal phase in several species and may be involved in steroid hormone production. In the bitch, leptin but not LEP-R protein was detected in the non-pregnant corpus luteum (CL). Until now, no further information has been available on their expression levels and role in CL function. Our objective was to compare time-related changes in luteal LEP and LEP-R mRNA levels during the non-pregnant luteal phase, pregnancy and after aglepristone treatment in mid-gestation. CLs were collected by ovariohysterectomy at different time points: day (d) 5, 15, 25, 35, 45, 65 after ovulation (p.o.) in non-pregnant bitches; pre-implantation, post-implantation, mid-gestation, during prepartum luteolysis; 24 and 72 h after aglepristone injection. Non-pregnant LEP expression was lowest on d5 p.o., increased thereafter and fell again on d45 (P ≤ 0.04). LEP-R expression was not altered (P = 0.07). In pregnant bitches, neither LEP nor LEP-R mRNA levels varied over time (P = 0.201 and P = 0.150, respectively). Aglepristone treatment caused substantial downregulation of luteal LEP expression by 72 h post-treatment (P ≤ 0.01). However, LEP-R expression did not follow the same course (P = 0.193). Our results indicate that both LEP and LEP-R mRNA are present in the canine CL during the non-pregnant luteal phase and pregnancy. LEP expression changes significantly over time in non-pregnant dogs and after aglepristone administration and thus, it may play a role in luteal steroidogenesis and regression.

Canine conceptus-maternal communication during maintenance and termination of pregnancy, including the role of species-specific decidualization.

Among domestic animal species, the reproductive biology of the dog belongs to the most peculiar. This includes the conceptus-maternal communication and endocrine mechanisms involved in maintenance of pregnancy. Dogs fully depend on luteal progesterone (P4) throughout pregnancy, with similar steroid secretion patterns in pregnant and non-pregnant bitches until prepartum luteolysis. Thus, dogs lack the classical recognition of pregnancy. The luteal P4 is the most important hormone regulating the onset and maintenance of pregnancy in previously estrogenized bitches. Although the canine uterus is exposed to high P4 levels, decidualization is not spontaneous but induced by the presence of embryos. Following implantation, decidualization continues, associated with development of the invasive endotheliochorial placenta, leading to establishment of maternal decidual cells expressing the nuclear P4 receptor (PGR). Consequently, although not producing steroids, the canine placenta remains highly sensitive to circulating ovarian steroids. The placental conceptus-maternal communication is responsible for the maintenance of pregnancy, with functional withdrawal of PGR evoking a luteolytic cascade with prepartum PGF2α release. The fetal trophoblast is the major source of prepartum placental prostaglandins. This conceptus-maternal communication is unique to the dog and has clinical implications. Due to luteal steroids, there is no prepartum estradiol increase. Elevated cortisol levels are observed irregularly. This emphasizes the unique character of canine reproductive physiology and the challenges in transferring translational research to the dog. Further research is needed for better understanding of canine reproduction and improving clinical protocols, including the latest results obtained from applying modern laboratory technologies such as the transcriptomic approach.

Uterine expression of smooth muscle alpha- and gamma-actin and smooth muscle myosin in bitches diagnosed with uterine inertia and obstructive dystocia.

Primary uterine inertia (PUI) is the most common type of dystocia in dogs. We hypothesized that PUI develops because of lower than normal expression of the basic contractile elements in the uterus, i.e., smooth muscle (SM) α- and γ-actin and SM-myosin, and that the expression of these proteins is influenced by the number of fetuses present in utero. Full-thickness inter-placental uterine biopsies were collected during Cesarean sections from dogs with PUI (n = 11), and from bitches with obstructive dystocia (OD) still presenting strong labor contractions (designated as the control group, n = 7). Relative gene expression was determined by semi-quantitative real-time (TaqMan) PCR, and protein localization by immunohistochemistry. Gene expression between PUI and OD bitches, and between PUI bitches carrying small, large, or average number of fetuses according to their breed, were compared. Uterine SM-γ-actin and SM-myosin mRNA levels were significantly higher in PUI than in OD dogs, while SM-α-actin did not differ. PUI bitches carrying large litters had lower uterine SM-γ-actin gene expression than those with small litters (P = 0.008). Immunostaining for SM-actin isoforms and SM-myosin was present in the myometrium, and localization pattern and staining intensity appeared similar in the PUI and OD groups. All proteins stained in blood vessels, and SM-γ-actin was also present in endometrial luminal and glandular epithelium. In conclusion, higher uterine SM-γ-actin and SM-myosin gene expression in PUI bitches, compared with OD dogs, might be an indication of abnormal progression with labor. Whether this is the cause of PUI due to an intrinsic error of the myometrium not becoming committed to labor, or the consequence of inadequate endocrine or mechanical stimuli, is not clear. Litter size was previously shown to be one of the risk factors for the development of uterine inertia in dogs, and our findings suggest possible differing uterine pathophysiology of PUI with respect to litter size.

Expression and activity of steroid sulphatase in the boar testis.

Oestrogens are essential for male fertility targeting the testicular-epididymal compartment. However, the underlying mechanisms are only vaguely known and species specificities must be considered. The boar has a remarkably high testicular-oestrogen output, with the biologically inactive oestrone-sulphate being the major oestrogen occurring in the testicular vein. In the boar testis and epididymis, activity of steroid sulphatase (StS) and oestrogen sulphotransferase has been demonstrated. Thus apart from their synthesis in Leydig cells, provision of biologically active free oestrone seems also to depend on the activity and localization of these enzymes. Our aim was to establish expression patterns and activity of StS in boar testis. Testes were randomly collected from healthy boars and allotted to five age groups, five animals in each, aged 50, 100, 150, 200 and 250 days. Three extra boars aged over 250 days were castrated to obtain fresh tissue for enzyme activity tests. Immunohistochemistry detected StS only in the cytoplasm of Leydig cells and - except for day-50 group in which 65.1 +/- 4.9% (X +/- SD) of the cells were positive - expression was constant with virtually all the cells staining positive. RT-PCR and in situ hybridization confirmed expression and localization of StS mRNA. The V(max) and K(m) value (X +/- SD) for StS was 24.05 +/- 0.3 fmol/s/microg protein and 2.15 +/- 0.12 microM. These data suggest that StS within the Leydig cells of the boar is involved in modulation of testicular oestrogen bioavailability and that the site of sulpho-conjugation is not the testis but a different compartment of the testes-epididymidis complex.

Molecular cloning and expression of StAR protein in the canine corpus luteum during dioestrus.

Expression of the steroidogenic acute regulatory protein (StAR) in the canine corpus luteum (CL) was examined throughout the luteal phase covering the periods of CL formation, early and late regression. Following a homology cloning and characterization of a cDNA fragment of the canine StAR spanning the sequence coding for the open reading frame (ORF), which encodes a 286 amino acid protein being highly conserved (86-91%) between species, quantitative RT-PCR was performed with the respective primers. Expression of StAR was demonstrated on all days examined; mRNA-levels increased gradually in developing CL from day 5 until 25; a steep, 4-fold downregulation was observed on day 35 with a further gradual decrease thereafter. Similar data were obtained by immunohistochemistry and the effect of time was highly significant (p<0.0001). These data suggest that the progesterone production during the canine CL-phase is controlled by the provision of substrate at the level of cholesterol transfer to the inner mitochondrial membrane, a step which involves the StAR-protein activity.

Placental steroids in cattle: hormones, placental growth factors or by-products of trophoblast giant cell differentiation?

The bovine placenta produces large amounts of steroids, mainly estrone (E1) and progesterone (P4). Specific features of bovine placental steroidogenesis are 1) the expression of all enzymes needed for the production of estrogens from cholesterol in the trophoblast 2) an only marginal and temporal contribution to peripheral maternal P4 levels restricted to a period between approx. days 150 - 240 of gestation 3) the predominance of sulfoconjugated over free E1 and 4) a complementary setting of steroidogenic enzymes in the two morphologically discriminable trophoblast cell types, the uninucleated trophoblast cells (UTC) and the trophoblast giant cells (TGC). In cattle so far no definite information is available on the specific biological roles of placental estrogens and P4. However, the detection of estrogen receptors and progesterone receptors in the placentomes suggests a role primarily as local regulators of caruncular growth, differentiation and functions. Inconsistent with a function as a caruncular growth factor is the strong evidence that in cattle placental estrogens enter the maternal compartment almost completely as estrone sulfate (E1S), which is not active at classical nuclear receptors. On the other hand, E1S may be converted locally to free active estrogens via the action of steroid sulfatase (StS), which has been detected in specific parts of the bovine caruncular epithelium. Alternatively or in addition, StS expression in the caruncular epithelium may serve the utilization of sulfated neutral steroid precursors (e.g. pregnenolone sulfate or cholesterol sulfate) supplied with maternal blood, thus providing free substrates for further metabolization in the adjacent trophoblast. The down-regulation of P450scc and P450c17 and the up-regulation of 3beta-HSD and aromatase during the differentiation of TGC from UTC in parallel with the up-regulation of ER beta and estrogen sulfotransferase in maturing TGC suggests a function of placental estrogens primarily as autoor intracrine regulators during this process and assigns to conjugated placental estrogens a role as inactivated by-products of TGC differentiation intended for excretion. Collectively, despite some evidence from recent studies for putative roles of placental steroids in cattle their exact functions in the bovine species remain still undefined.

Bovine placental steroid sulphatase: molecular cloning and expression pattern in placentomes during gestation and at parturition.

Apart from during the prepartal period, the main oestrogen produced by the bovine trophoblast is oestrone sulphate (E1S) which does not bind to nuclear oestrogen receptors (ER). High steroid sulphatase (StS) activities previously detected in the maternal part of bovine placentomes (caruncles) suggest the local activation of E1S ("sulphatase pathway"). Consequently, the expression pattern of StS in bovine placentomes was investigated by immunohistochemistry using an antiserum against human placental StS. Cross-reactivity for bovine StS was confirmed by Western blot yielding a single band of 62 kDa in both bovine and human placenta. Immunostaining for StS was detected in caruncular epithelial cells (CEC), which was clearly related to gestational age. In animals pregnant between 100 and 284 days (n=17), signals were restricted to CEC adjacent to the chorionic plate and basal primary and secondary chorionic villi. After the onset of prepartal luteolysis (days 273-282; n=3) and during active labour (n=5) overall staining intensity had increased substantially and signals occurred ubiquitously in the flattened and partially dismantled caruncular epithelium. A 2204 bp full-length mRNA transcript of the bovine StS exhibiting 74% and 77% sequence identity to human StS on the mRNA and protein levels, respectively, was cloned by RACE-PCR. Real-time RT-PCR detected a 2.5-fold increase of StS-mRNA in prepartal placentomes, which, however, was not statistically significant. The co-localisation of ERalpha and StS in CEC is consistent with a role of placental oestrogens as regulators of caruncular growth and differentiation, and the up-regulation of carunclar StS may be involved in the marked prepartal increase of free oestrogens.

Expression of cyclooxygenase-II (COX-II) and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD)/prostaglandin F-synthase (PGFS) in bovine placentomes: implications for the initiation of parturition in cattle.

The chain of events leading to prepartal luteolysis in cattle is not yet fully understood. Prostaglandin F(2alpha) (PGF(2alpha)) seems to be a major factor involved. However, only little information is available about the underlying regulatory mechanisms. Consequently, the expression of cyclooxygenase-II (COX-II) and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), an enzyme recently shown to be most likely responsible for the production of luteolytic PGF(2alpha) in the endometrium of cyclic cows, was investigated in bovine placentomes. Immunohistochemical methods were applied to placentomes from 17 pregnant cows between days 100 and 284, from three cows during the prepartal progesterone decrease (days 273-282) and from five parturient cows. COX-II was found in uninucleated trophoblast cells (UTC) from day 100 until parturition. However, between days 100 and 235 expression was only weak to moderate, focal and mainly restricted to the chorionic plate and adjacent basal parts of chorionic stem villi. In placentomes from a 270 and a 284 day pregnant cow, in which the prepartal decline of progesterone levels had not started yet, staining had substantially increased and extended to secondary and tertiary chorionic villi. In prepartal and parturient cows strong to intense staining was present in UTC all over the villous tree. Real time RT-PCR confirmed an extensive pre- and intrapartal rise of COX-II expression in bovine placentomes with a 70-100-fold increase of COX-II-mRNA levels. 20alpha-HSD/PGFS was widely expressed in UTC of chorionic villi. Like COX-II it was down-regulated in UTC differentiating into trophoblast giant cells. Immunostaining pattern did not change remarkably during the period under investigation, and 20alpha-HSD/PGFS-mRNA levels increased only 2.6-fold in the prepartal phase. Thus, in UTC PGF(2alpha) may be produced via COX-II and 20alpha-HSD/PGFS, but only COX-II may be substantially involved in the control of a putative prepartal placentomal output of luteolytic PGs, whereas 20alpha-HSD/PGFS seems to be expressed in a more constitutive manner.