The Molecular Signature Related to Local Inflammatory and Immune Response in Canine Cutaneous Hypersensitivity Reactions: A Preliminary Study.

Cutaneous hypersensitivity reactions (CHRs) are complex inflammatory skin disorders that affect humans and dogs. This study examined the inflammatory and immune responses leading to skin damage, inflammation, and irritation by investigating gene expression through quantitative PCR (qPCR) and protein localization through the immunohistochemistry (IHC) of specific receptors and molecules involved in CHRs. Formalin-fixed paraffin-embedded (FFPE) samples from canine CHR skin (n = 20) and healthy dog skin (n = 3) were analyzed for expression levels of eight genes, including members of the pattern recognition receptor (PRR) family, CD209 and CLEC4G, the Regakine-1-like chemokine, and acute phase proteins (APPs), LBP-like and Hp-like genes. Additionally, we examined the local involvement of IL-6, Janus Kinase 1 (JAK1), and the signal transducer activator of transcription 3 (STAT3) in the CHR cases. The study demonstrated statistically significant increases in the expression levels of CD209, Hp-like (p < 0.01), LBP-like, Regakine-1-like, and CLEC4G (p < 0.05) genes in CHRs compared to healthy controls. Conversely, IL-6, JAK1, and STAT3 showed no significant difference between the two groups (p > 0.05). Protein analysis revealed JAK1 and STAT3 expression in CHR hyperplastic epithelial cells, dermal fibroblasts, and endothelial cells of small capillaries, indicating a possible involvement in the JAK/STAT pathway in local inflammatory response regulation. Our findings suggest that the skin plays a role in the development of CHRs.

Resistin in endocrine pancreas of sheep: Presence and expression related to different diets.

Resistin (RETN), a recently discovered adipokine, is a cysteine-rich and secretory protein produced by adipocytes. RETN has been detected in several tissues, including human and laboratory animals' pancreas, wherein impairs glucose tolerance and insulin (INS) action and causes INS resistance. This study aims to evaluate the presence and expression of RETN in the pancreas of 15 adult female sheep reared on Apennine pastures, which show a decrease in their nutritional value due to the drought stress linked to the increasing summer aridity. The sheep were divided into 3 groups according to the diet they were subjected to: maximum pasture flowering (MxF) group, maximum pasture dryness (MxD) group, and experimental (Exp) group which received a feed supplementation in addition to the MxD group feeding. Immunohistochemistry and immunofluorescence were performed on formalin-fixed and paraffin-embedded sections of the pancreas to detect the RETN presence and to evaluate the co-localization of RETN with both glucagon (GCG)- and INS-producing cells. In addition, the expression of the three molecules was evaluated also in relation to different diets. RETN was observed only in the endocrine pancreas, showing a wide distribution throughout the pancreatic islets with few negative cells and the RETN producing cells colocalized with both α cells and ß cells. No differences in distribution and immunostaining intensity of RETN, GCG and INS were observed among the three groups. Quantitative PCR showed the expression of RETN, GCG and INS in all tested samples. No significant differences were observed for RETN and GCG among all three groups of sheep. Instead, a high statistically significant expression of INS was detected in the MxF group with respect to the Exp and MxD groups. These results highlight the localization of RETN in GCG- and INS-secreting cells involved in glucose homeostasis suggesting a modulatory role for RETN. Furthermore, the RETN expression is not influenced by food supplementation and thus is not affected by diet.

Interleukin-1 Beta (IL1B) and Nerve Growth Factor (NGF): Key Players in Rabbit Reproductive Regulation.

Several seminal plasma components, besides NGF, are implicated as ovulation-inducing factors in mammals. This study investigated the IL1B and its receptor IL1R1 in the testis (T), male accessory glands, prostate (P) and seminal vesicles (SV), and uterus (U) of adult rabbits using immunohistochemistry (IHC) and quantitative reverse transcription PCR (RT-qPCR). We also assessed the presence of IL1B in seminal plasma through Western blotting (WB) and examined the interaction between IL1B and NGF in vitro by measuring their production with enzyme-linked immunosorbent assay (ELISA) in the presence of NGF and IL1B alone or with their respective receptor antagonists. IHC revealed IL1B system expression in all reproductive organs studied, with IL1B and IL1R1 localized to the germinative epithelium of the T and the epithelial cells of the accessory glands and U. IL1B gene transcript levels were significantly higher (p < 0.01) in the P and SV compared to the T, while IL1R1 levels were significantly higher (p < 0.001) in the P compared to the other tissues, while IL1R1 levels were three times higher (p < 0.001) in the P. WB confirmed the presence of IL1B in seminal plasma with a 30-35 kDa band. The in vitro study demonstrated that IL1B increased (p < 0.05) basal NGF production in the U, whereas NGF had no effect on IL1B production. These findings provide evidence of the expression of the IL1B/IL1R1 system in both male and female rabbit reproductive tracts and suggest that IL1B in seminal plasma may influence uterine endocrine activity. The results propose a potential role for IL1B in ovulation, in conjunction with NGF, supporting that ovulation may involve inflammatory-like processes.

The Role of Genistein in Mammalian Reproduction.

Genistein is a natural compound belonging to flavonoids, having antioxidant, anti-inflammatory, and anti-neoplastic properties. Genistein is considered a phytoestrogen. As such, genistein can bind estrogen receptors (ERα and ERß), although with a lower affinity than that of estradiol. Despite considerable work, the effects of genistein are not well established yet. This review aims to clarify the role of genistein on female and male reproductive functions in mammals. In females, at a high dose, genistein diminishes the ovarian activity regulating several pathway molecules, such as topoisomerase isoform I and II, protein tyrosine kinases (v-src, Mek-4, ABL, PKC, Syk, EGFR, FGFR), ABC, CFTR, Glut1, Glut4, 5α-reductase, PPAR-γ, mitogen-activated protein kinase A, protein histidine kinase, and recently circulating RNA-miRNA. The effect of genistein on pregnancy is still controversial. In males, genistein exerts an estrogenic effect by inducing testosterone biosynthesis. The interaction of genistein with both natural and synthetic endocrine disruptors has a negative effect on testis function. The positive effect of genistein on sperm quality is still in debate. In conclusion, genistein has a potentially beneficial effect on the mechanisms regulating the reproduction of females and males. However, this is dependent on the dose, the species, the route, and the time of administration.

Nerve Growth Factor (NGF) and Animal Reproduction.

Stimuli that lead to the release of gonadotropin-releasing hormone (GnRH) and pituitary gonadotropins and, consequently, ovulation in mammals fall into two broad categories. In the first, high plasma oestrogen concentrations induce the events that trigger ovulation, a characteristic of spontaneous ovulators. In the second, nerve stimuli occurring during mating reach the hypothalamus and trigger the release of GnRH and ovulation with a neuroendocrine reflex that characterizes induced ovulators.In this review, we will give an overview of the distribution of NGF and its expression in the different tissues of the male accessory sex glands, the main sites of NGF production. Next, we will highlight the role of NGF in sperm function and its potential cryopreserving role in artificial insemination techniques. Finally, we will evaluate the functions of NGF in ovulation, particularly in induced ovulators. Overall, the information obtained so far indicates that NGF is widely distributed in organs that regulate the reproductive activity, in both males and females. In spontaneous ovulators, NGF exerts mainly a luteotrophic action, while, in induced ovulators it is the main ovulation-inducing factor. A better understanding of the role of NGF in reproduction would be of great interest, since it could help finding innovative therapeutic aids to improve mammalian fertility.

Effect of chelating and antioxidant agents on morphology and DNA methylation in freeze-drying rabbit (Oryctolagus cuniculus) spermatozoa.

Freeze-drying (FD) has been exhaustively tried in several mammalian species as an alternative technique to sperm cryopreservation, but few studies have been done in rabbits (Oryctolagus cuniculus). The main objective of this study was to compare the protective effect of various antioxidants added to EDTA medium on structural and functional components of FD rabbit spermatozoa and on their status of global DNA methylation. FD media used were composed of basic FD medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with either 50 mM EDTA alone (EDTA) or added with 105 µM of rosmarinic acid (RA, EDTA-RA) or 10 µM of melatonin (MLT, EDTA-MLT). The effect of each medium on the preservation of FD spermatozoon structure was evaluated with light and scanning electron microscopy (SEM). Global DNA methylation was quantified in all FD sperm samples as well as in fresh spermatozoa. Morphologically, fracture points were evidenced in the neck, mid and principal piece of the spermatozoon tail. No differences in spermatozoon fracture points were evidenced among FD treatments: intact spermatozoa were the largest (p < .01) category, whereas the most frequent (p < .01) injury was the neck fracture, resulting in tailless heads. At SEM, the head of spermatozoa showed a well-conserved shape and intact membrane in all treatments. DNA methylation status was the same in all FD treatments. In conclusion, supplementation of EDTA, EDTA-RA and EDTA-MLT during FD preserved rabbit sperm morphological integrity and methylation status as well. Therefore, the difficulty of getting viable offspring using FD semen is likely unrelated to the impact of the lyophilization process on DNA methylation and morphology of lyophilized spermatozoa.

Effect of aglepristone (RU534) administration during follicular phase on progesterone, estradiol-17ß and LH serum concentrations in bitches.

Aglepristone was administered in bitches during the follicular phase to evaluate its effects on progesterone, estradiol-17ß and LH serum concentrations. Ten German Shepherds were divided into two groups (treated n = 5; control n = 5). Treated bitches received 10 mg/kg BW of aglepristone subcutaneously during the early follicular phase, 24 hr after and then 7 days later. The control group was injected, at the same time periods, with saline solution (0.3 ml/kg BW). For the steroid evaluations, blood was collected daily from the onset of proestrus until the first day of cytological dioestrus. For LH base-line serum determination, blood was also collected every 20 min for 2 hr at the onset of proestrus. For LH surge identification, blood was collected daily (every 6 hr) starting from the day of the first administration of aglepristone or saline solution until the first day of dioestrus. All animals ovulated but the treated group presented longer ovulation-dioestrus intervals than the control group (5.2 ± 2.2 days p < .05). Serum concentrations of the evaluated hormones were similar between experimental animals except for serum LH. Indeed, no LH peaks were detected in the treated group while LH surges were clearly observed in the control group (9 ± 1 days after the beginning of proestrus. In particular, the area under the curve for LH was significantly lower in treated than control animals (12 ± 4 ng/ml x Day; p = .01). In conclusion, administrations of aglepristone during the follicular phase of the bitch does not affect the steroid hormone patterns but does prevent the occurrence of a LH surge. This work raises significant questions and opens perspectives concerning the mechanisms of ovulation in bitches.

The Role of Resveratrol in Mammalian Reproduction.

Resveratrol is one of the most investigated natural polyphenolic compounds and is contained in more than 70 types of plants and in red wine. The widespread interest in this polyphenol derives from its antioxidant, anti-inflammatory and anti-aging properties. Several studies have established that resveratrol regulates animal reproduction. However, the mechanisms of action and the potential therapeutic effects are still unclear. This review aims to clarify the role of resveratrol in male and female reproductive functions, with a focus on animals of veterinary interest. In females, resveratrol has been considered as a phytoestrogen due to its capacity to modulate ovarian function and steroidogenesis via sirtuins, SIRT1 in particular. Resveratrol has also been used to enhance aged oocyte quality and as a gametes cryo-protectant with mainly antioxidant and anti-apoptotic effects. In males, resveratrol enhances testes function and spermatogenesis through activation of the AMPK pathway. Furthermore, resveratrol has been supplemented to semen extenders, improving the preservation of sperm quality. In conclusion, resveratrol has potentially beneficial effects for ameliorating ovarian and testes function.

Intra-ovarian dynamic blood flow in pseudopregnant rabbits during prostaglandin F2α-induced luteolysis.

In the present study, we evaluated the dynamic changes of intra-ovarian blood flow, by real-time colour-coded and pulsed Doppler ultrasonography, as well as the immunopresence of prostaglandin F2α (PGF2α) receptor (FP) and peripheral plasma progesterone concentrations in pseudopregnant rabbit after PGF2α treatments at either early- (4 days) and mid-luteal (9 days) stages. During the pre-treatment observation interval of one hour, the ovarian blood flows showed a fluctuating pattern. Independently of luteal stage, PGF2α administration caused a fourfold decline in the blood flow within 40 min that was followed 50 min later by a reactive hyperaemia that lasted several hours, while the resistive index showed an opposite trend. Twenty-four hour later, the blood flow was one half that measured before PGF2α injection. At day 4 of pseudopregnancy, PGF2α did not affect peripheral plasma progesterone concentrations, but at day 9, it caused functional luteolysis as progesterone levels declined 6 hr later to reach basal values after 24 hr. The changes in the ovarian blood flows of pseudopregnant rabbits receiving PGF2α were accompanied by simultaneous changes in the resistance index. This biphasic response in the blood flow and vascular resistances likely reflects reactive hyperaemia following vasoconstriction. By immunohistochemistry, strong positive immune reaction for FP was detected in the cytoplasm of endothelial cells of ovarian arteries, veins and capillaries. In conclusion, these results suggest that PGF2α could acutely regulate the ovarian blood flow of pseudopregnant rabbits, even if there is no evidence of a blood flow reduction anticipating luteolysis.

New insights on a NGF-mediated pathway to induce ovulation in rabbits (Oryctolagus cuniculus).

To investigate the ovulatory mechanisms triggered by raw semen (RS) in rabbits, we examined the expression of nerve growth factor (NGF)-a supposed ovulation-inducing factor (OIF)-and cognate receptors in anterior pituitary, ovary, and cervix as well as plasma NGF and luteinizing hormone (LH) concentrations. Six does/group were sham-inseminated with sterile saline (PBS), naturally mated (NM), inseminated with RS alone or after lumbar anesthesia (ARS), or treatment with COX inhibitors (CIRS). Immunohistochemistry revealed positive signals for NGF and receptors in all tissues. RT-PCR confirmed the presence of the target transcripts in the same tissues, except NTRK1 in the cervix. Circulating NGF concentrations rose 3- to 6-fold (P < 0.01) 15 min after semen deposition into the genital tract of NM, RS, and ARS rabbits and remained sustained thereafter. Circulating NGF was 4-fold lower (P < 0.01) in CIRS than in RS does indicating that NGF is mainly synthesized by the uterus. A concomitant rise of LH and NGF concentrations was found in 83.3%, 50.0%, and 16.7% of NM, RS, and CIRS does, respectively, but not in ARS (despite high NGF circulating levels). Seminal plasma NGF concentration was 151.9 ± 9.25 µg/mL. The ovulatory responses were 0%, 83.3%, 66.7%, 16.7%, and 0% in PBS, NM, RS, ARS, and CIRS groups, respectively. Present data confirm that, although RS may induce ovulation via endocrine mechanisms through binding to NGF receptors in the ovary, a novel OIF-mediated neural mechanism facilitates ovulation in rabbits.

In vivo chronic and in vitro acute effects of di(2-ethylhexyl) phthalate on pseudopregnant rabbit corpora lutea: possible involvement of peroxisome proliferator-activated receptor gamma.

The in vivo chronic and in vitro acute effects of di(2-ethylhexyl) phthalate (DEHP) on the reproductive function of peroxisome proliferator-activated receptor gamma (PPARG) were studied in rabbit corpora lutea (CL) at early stage (Day 4), midstage (Day 9), and late stage (Day 13) of pseudopregnancy. The rabbits were in vivo treated with DEHP for 15 days before induction of pseudopregnancy. Immunohistochemistry provided evidence for the presence of PPARG, prostaglandin endoperoxide synthase 1 (PTGS1), PTGS2, prostaglandin E2-9-ketoreductase (PGE2-9-K), and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in all the luteal cells during pseudopregnancy. DEHP decreased progesterone plasma levels and CL production in all the luteal stages and PPARG protein and gene expressions in early and mid-CL. DEHP in vivo treatment reduced PTGS2 protein expression at the late stage and that of PGE2-9-K at all the stages, whereas PTGS1 and 3beta-HSD were not affected. In in vitro cultured CL, DEHP alone, the PPARG antagonist T0070907 alone, or DEHP plus T0070907 diminished progesterone production and 3beta-HSD activity and increased PGF2alpha and PTGS2 in early and mid-CL, whereas DEHP plus the PPARG agonist 15d-PGJ2 did not affect these hormones and enzymes. All the in vitro treatments did not affect PGE2 secretion as well as PTGS1 and PGE2-9-K enzymatic activities in all the luteal stages. These results provided evidence that DEHP favors functional luteolysis of pseudopregnant rabbit CL, with a mechanism that seems to involve PPARG expression down-regulation, an increase of PTGS2 activity and prostaglandin F2alpha secretion, 3beta-HSD down-regulation, and decrease in progesterone.

Seasonal variation of NGF in seminal plasma and expression of NGF and its cognate receptors NTRK1 and p75NTR in the sex organs of rams.

Nerve growth factor (NGF) has long been known as the main ovulation-inducing factor in induced ovulation species, however, recent studies suggested the NGF role also in those with spontaneous ovulation. The first aim of this study was to evaluate the presence and gene expression of NGF and its cognate receptors, high-affinity neurotrophic tyrosine kinase 1 receptor (NTRK1) and low-affinity p75 nerve growth factor receptor (p75NTR), in the ram genital tract. Moreover, the annual trend of NGF seminal plasma values was investigated to evaluate the possible relationship between the NGF production variations and the ram reproductive seasonality. The presence and expression of the NGF/receptors system was evaluated in the testis, epididymis, vas deferens ampullae, seminal vesicles, prostate, and bulbourethral glands through immunohistochemistry and real-time PCR (qPCR), respectively. Genital tract samples were collected from 5 adult rams, regularly slaughtered at a local abattoir. Semen was collected during the whole year weekly, from 5 different adult rams, reared in a breeding facility, with an artificial vagina. NGF seminal plasma values were assessed through the ELISA method. NGF, NTRK1 and p75NTR immunoreactivity was detected in all male organs examined. NGF-positive immunostaining was observed in the spermatozoa of the germinal epithelium, in the epididymis and the cells of the secretory epithelium of annexed glands, NTRK1 receptor showed a localization pattern like that of NGF, whereas p75NTR immunopositivity was localized in the nerve fibers and ganglia. NGF gene transcript was highest (p < 0.01) in the seminal vesicles and lowest (p < 0.01) in the testis than in the other tissues. NTRK1 gene transcript was highest (p < 0.01) in the seminal vesicles and lowest (p < 0.05) in all the other tissues examined. Gene expression of p75NTR was highest (p < 0.01) in the seminal vesicles and lowest (p < 0.01) in the testis and bulbourethral glands. NGF seminal plasma concentration was greater from January to May (p < 0.01) than in the other months. This study highlighted that the NGF system was expressed in the tissues of all the different genital tracts examined, confirming the role of NGF in ram reproduction. Sheep are short-day breeders, with an anestrus that corresponds to the highest seminal plasma NGF levels, thus suggesting the intriguing idea that this factor could participate in an inhibitory mechanism of male reproductive activity, activated during the female anestrus.

Evidence for a luteotropic role of peroxisome proliferator-activated receptor gamma: expression and in vitro effects on enzymatic and hormonal activities in corpora lutea of pseudopregnant rabbits.

The expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and its role in corpora lutea (CL) function were studied in pseudopregnant rabbits. Corpora lutea were collected at an early stage (Day 4), midstage (Day 9), and late stage (Day 13) of pseudopregnancy. Immunohistochemistry found evidence for the presence of PPARgamma in the perinuclear cytoplasm and nucleus of all the luteal cells; immunoreactivity decreased from the early to the late stage, with immunonegativity of the nuclei of late stage CL. PPARgamma mRNA transcript was expressed in all the luteal stages with the lowest level in the late stage. In CL cultured in vitro, the PPARgamma agonist (15-deoxy delta12,14 prostaglandin J2 [15d-PGJ2], 200 nM) increased and the antagonist (T0070907, 50 nM) decreased progesterone secretion at early and midluteal stages, whereas 15d-PGJ2 reduced and T0070907 increased PGF2alpha at the same stages. Prostaglandin-endoperoxide synthase 2 (PTGS2) activity was reduced by 15d-PGJ2 and increased by T0070907 in CL of early and midluteal stages. Conversely, 15d-PGJ2 increased and T0070907 reduced 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity in early and midluteal stage CL. PGE2 in vitro secretion as well as PTGS1 and 20alpha-HSD enzymatic activities were not affected by 15d-PGJ2 and T0070907 in any CL types. These results indicate that PPARgamma plays a luteotropic role in pseudopregnant rabbits, through PTGS2 down-regulation and 3beta-HSD up-regulation, with a consequent PGF2alpha decrease and progesterone increase.

Cruciferous vegetable-derived indole-3-carbinol prevents coronavirus cell egression mechanisms in tracheal and intestinal 3D in vitro models.

The potential antiviral effects of indole-3-carbinol (I3C), a phytochemical found in Cruciferous vegetables, were investigated. Fibroblasts and epithelial cells were co-cultured on Alvetex® scaffolds, to obtain ad hoc 3D in vitro platforms able to mimic the trachea and intestinal mucosae, which represent the primary structures involved in the coronavirus pathogenesis. The two barriers generated in vitro were treated with various concentrations of I3C for different incubation periods. A protective effect of I3C on both intestinal and trachea models was demonstrated. A significant reduction in the transcription of the two main genes belonging to the Homologous to E6AP C-terminus (HECT)-E3 ligase family members, namely NEDD4 E3 Ubiquitin Protein Ligase (NEDD4) and WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1), which promote virus matrix protein ubiquitination and inhibit viral egression, were detected. These findings indicate I3C potential effect in preventing coronavirus cell egression processes that inhibit viral production. Although further studies are needed to clarify the molecular mechanisms whereby HECT family members control virus life cycle, this work paves the way to the possible therapeutic use of new natural compounds that may reduce the clinical severity of future pandemics.

Leptin system is not affected by different diets in the abomasum of the sheep reared in semi-natural pastures of the Central Apennines.

The growing summer drought stress is affecting the nutritional value of pastures, no longer sufficient to support the nutritional status of sheep in extensive rearing. Adipokines affect organ and tissue functionality can be useful to evaluate animal welfare and prompt an improvement in the management of the grazing animals. Leptin (Lep) is an adipokine mainly produced by adipose tissue that regulates food intake by an anorexigenic action. Lep has also been detected in the human and rat gastrointestinal tract, where it regulates the rate of gastric emptying. In this study, Lep system was evaluated in the abomasum of 15 adult sheep reared on Apennine pastures and subjected to different diets. Until the maximum pasture flowering (MxF group), the sheep fed on fresh forage; from that moment until the maximum pasture dryness (MxD group), the experimental group (Exp group) received a feed supplementation in addition to MxD group feeding. The Lep system was investigated in the abomasum samples by immunohistochemistry (IHC) and RT-qPCR. Double-label localisation of Lep and leptin receptor (LepR) with neuroendocrine hormones was conducted to distinguish the gland cell types. The analysis performed revealed the presence of Lep and LepR in the chief and neuroendocrine cells of the fundic glands of the abomasum. RT-qPCR evidenced the transcript for Lep and LepR also identifying the long isoform (LepRb). No significant differences were observed among the three groups of sheep subjected to different diets. The abundant immunostaining observed in the fundic glands suggests that the Lep intervenes in the regulation of abomasum in sheep with a similar pattern to monogastric species while long term food supplementation seems do not influence the local function of the Lep system. A better understanding of the gastrointestinal system can contribute to improving sheep management and optimising the sustainability of livestock production.

Presence, Tissue Localization, and Gene Expression of the Adiponectin Receptor 1 in Testis and Accessory Glands of Male Rams during the Non-Breeding Season.

Adiponectin (ADIPOQ) is a member adipocytokines, and its actions are supported by two receptors, ADIPOQ receptor 1 and -2, respectively (ADIPOR1 and -R2). Our study was performed to evaluate the ADIPOR1 presence and location and its gene expression in reproductive tissues of the male ram, during its non-breading season. The different portions of the male ram reproductive system (testis, epididymis, seminal vesicle, ampoule vas deferens, bulb-urethral gland) were collected in a slaughterhouse. Immunohistochemistry showed ADIPOR1 positive signals in the cytoplasm of all the glandular epithelial cells, with a location near the nucleus; in the testes, the positive reaction was evidenced in the cytoplasm in the basal portion of the germinal epithelial cells. The immune reaction intensity was highest (p < 0.001) in the prostate and seminal vesicles glands than that of other parts of the ram reproductive tract. RT-qPCR detected the ADIPOR1 transcript in the testes, epididymis, vas deferens, bulbourethral glands, seminal vesicles, and prostate; the expression levels were high (p < 0.01) in the prostate and low (p < 0.01) in the testis, epididymis, and bulbourethral glands. The present results evidenced the possible ADIPOQ/ADIPOR1 system's role in regulating the testicular activity of male rams during the non-breading season.

Presence and localization of apelin and its cognate receptor in canine testes using immunohistochemical and RT-PCR techniques.

Apelin, a member of the adipokine family, is a novel endogenous peptide which regulates the male reproductive system of mammals by interacting with a specific receptor. Recent studies have highlighted that apelin may play a role in the regulation of reproduction by reducing testosterone production and inhibiting LH secretion. To the best of our knowledge, there is no available data on the presence of the apelin and its receptor in canine testes. Therefore, the aim of this study was to reveal the presence of apelin and evaluate its distribution in the canine testes using immunohistochemical and RT-PCR techniques. For this purpose, five fertile and healthy male dogs were subjected to elective orchiectomy. The immunohistochemical reaction revealed the presence of apelin and its receptor in the canine testes. Apelin was localized in spermatids and spermatozoa with a positive signal in the "acrosomal bodies". As regards the apelin receptor, a positive immunoreaction was detected in the cytoplasm of the cells localized near to the basal membrane of the seminiferous tubules and in the cytoplasm of Leydig cells. The RT-PCR analysis showed the presence of transcripts for apelin and apelin receptor in all of the samples under study. A 35kDa band confirmed apelin receptor protein expression in all of the samples analysed. In conclusion, the paracrine and endocrine role of apelin and its cognate receptor on male reproduction reported in humans and laboratory animals could also be hypothesized in dogs.

Gonadotropin-releasing hormone 1 directly affects corpora lutea lifespan in Mediterranean buffalo (Bubalus bubalis) during diestrus: presence and in vitro effects on enzymatic and hormonal activities.

The expression of gonadotropin-releasing hormone (GNRH) receptor (GNRHR) and the direct role of GNRH1 on corpora lutea function were studied in Mediterranean buffalo during diestrus. Immunohistochemistry evidenced at early, mid, and late luteal stages the presence of GNRHR only in large luteal cells and GNRH1 in both small and large luteal cells. Real-time PCR revealed GNRHR and GNRH1 mRNA at the three luteal stages, with lowest values in late corpora lutea. In vitro corpora lutea progesterone production was greater in mid stages and lesser in late luteal phases, whereas prostaglandin F2 alpha (PGF2alpha) increased from early to late stages, and PGE2 was greater in the earlier-luteal phase. Cyclooxygenase 1 (prostaglandin-endoperoxide synthase 1; PTGS1) activity did not change during diestrus, whereas PTGS2 increased from early to late stages, and PGE2-9-ketoreductase (PGE2-9-K) was greater in late corpora lutea. PTGS1 activity was greater than PTGS2 in early corpora lutea and lesser in late luteal phase. In corpora lutea cultured in vitro, the GNRH1 analog (buserelin) reduced progesterone secretion and increased PGF2alpha secretion as well as PTGS2 and PGE2-9-K activities at mid and late stages. PGE2 release and PTGS1 activity were increased by buserelin only in late corpora lutea. These results suggest that GNRH is expressed in all luteal cells of buffalo, whereas GNRHR is only expressed in large luteal phase. Additionally, GNRH directly down-regulates corpora lutea progesterone release, with the concomitant increases of PGF2alpha production and PTGS2 and PGE2-9-K enzymatic activities.

Carotenoids in female and male reproduction.

Carotenoids are among the best-known pigments in nature, confer color to plants and animals, and are mainly derived from photosynthetic bacteria, fungi, algae, plants. Mammals cannot synthesize carotenoids. Carotenoids' source is only alimentary and after their assumption, they are mainly converted in retinal, retinol and retinoic acid, collectively known also as pro-vitamins and vitamin A, which play an essential role in tissue growth and regulate different aspects of the reproductive functions. However, their mechanisms of action and potential therapeutic effects are still unclear. This review aims to clarify the role of carotenoids in the male and female reproductive functions in species of veterinary interest. In female, carotenoids and their derivatives regulate not only folliculogenesis and oogenesis but also steroidogenesis. Moreover, they improve fertility by decreasing the risk of embryonic mortality. In male, retinol and retinoic acids activate molecular pathways related to spermatogenesis. Deficiencies of these vitamins have been correlated with degeneration of testis parenchyma with consequent absence of the mature sperm. Carotenoids have also been considered anti-antioxidants as they ameliorate the effect of free radicals. The mechanisms of action seem to be exerted by activating Kit and Stra8 pathways in both female and male. In conclusion, carotenoids have potentially beneficial effects for ameliorating ovarian and testes function.

Current Knowledge on the Multifactorial Regulation of Corpora Lutea Lifespan: The Rabbit Model.

Our research group studied the biological regulatory mechanisms of the corpora lutea (CL), paying particular attention to the pseudopregnant rabbit model, which has the advantage that the relative luteal age following ovulation is induced by the gonadotrophin-releasing hormone (GnRH). CL are temporary endocrine structures that secrete progesterone, which is essential for maintaining a healthy pregnancy. It is now clear that, besides the classical regulatory mechanism exerted by prostaglandin E2 (luteotropic) and prostaglandin F2α (luteolytic), a considerable number of other effectors assist in the regulation of CL. The aim of this paper is to summarize our current knowledge of the multifactorial mechanisms regulating CL lifespan in rabbits. Given the essential role of CL in reproductive success, a deeper understanding of the regulatory mechanisms will provide us with valuable insights on various reproductive issues that hinder fertility in this and other mammalian species, allowing to overcome the challenges for new and more efficient breeding strategies.