ContentsIn 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.

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 mu g/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. Summary Sentence Raw semen induces ovulation in rabbits via an endocrine-and a nervous-mediated pathway by which NGF, mainly synthesized in the uterus, acts on the ovary and on uterine/cervix afferent neurons projecting LH surge hypothalamic centers, respectively.

Dopamine (DA) receptor (DR) type 1 (D1R) has been found to be expressed in luteal cells of various species, but the intrinsic role of the DA/DRs system on corpora lutea (CL) function is still unclear. Experiments were devised to characterize the expression of DR types and the presence of DA, as well as the in vitro effects of DA on hormone productions by CL in pseudopregnant rabbits. Immunoreactivity and gene expression for D1R decreased while that for D3R increased in luteal and blood vessel cells from early to late pseudopregnant stages. DA immunopositivity was evidenced only in luteal cells. The DA and D1R agonist increased in vitro release of progesterone and prostaglandin E2 (PGE2) by early CL, whereas the DA and D3R agonist decreased progesterone and increased PGF2α in vitro release by mid- and late CL. These results provide evidence that the DA/DR system exerts a dual modulatory function in the lifespan of CL: the DA/D1R is luteotropic while the DA/D3R is luteolytic. The present data shed new light on the physiological mechanisms regulating luteal activity that might improve our ability to optimize reproductive efficiency in mammal species, including humans.

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.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein, which protects biomembranes from oxidative damages, and it also accounts for almost the entire selenium content of mammalian testis. The present investigation was performed to localise PHGPx in the testis and in epididymal and ejaculated spermatozoa of the bull by using light and electron immunomicroscopy. The study also aimed to further clarify the possible functions of the protein in bull fertility. In the testis, spermatogenic cells of the adluminal tubular compartment showed cytoplasmatic immunostaining; whereas, in the epididymal and ejaculated spermatozoa immunostaining was specifically localised at the level of the head and mid-piece. Ultrastructural data revealed the presence of signals for PHGPx in different subcellular compartments of maturing and mature sperm (mitochondria, chromatin, nuclear envelope, acrosomes, cytoskeletal structures) suggesting that this enzyme plays versatile and important biological roles during spermatogenesis. The final localisation of the immunostaining at acrosomal level puts forward a new role of the protein which further emphasises its relevance in male reproduction: it is reported to anchor substrate of the sperm acrosome to the oocyte zona pellucida during the fertilisation process.

This is a review of original data concerning new extra- and intracellular regulators of rabbit ovarian functions. Effects of some hormones including leptin, ghrelin, oxytocin, arginine-vasotocin, endothelin (ET-1), gonadotropin releasing hormone (GnRH), adrenocorticotropic hormone (ACTH), growth factors such as insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), nuclear peroxisome proliferator-activated receptor gamma (PPARγ), pharmacological regulators of some protein kinases such as protein kinase A (PKA), mitogen-activated protein (MAP) kinase, cell division cycle protein 2 homolog (CDC2 kinase, CDK), tyrosine kinases), and plant molecules (resveratrol, rapamycin) on the functions of ovarian cells (proliferation, apoptosis, secretory activity, expression of some protein kinases) and reproductive end points (blood level of reproductive hormones, ovarian morphology, number of ovulations, embryo yield and quality, number and viability of offspring), and their possible interrelationships and practical application in rabbit breeding are reviewed.

Background: The ovarian blood flow provides oxygen-rich blood and nutrients which are necessary for the growth and secretory activities of follicles and corpora lutea (CL) in cycling and pregnant animals. The blood flow of the ovary is finely regulated by local and systemic mechanisms that integrate nervous, endocrine, and metabolic signals. In the rabbit ovary, administration of prostaglandin F2 alpha (PGF2 alpha) to pseudopregnant or pregnant animals affects specific vascular mechanisms regulating luteolytic process, in particular, the regulatory role of progesterone on luteal function through direct and uterine-mediated mechanisms. Thus, the main objective of the present work was to evaluate, at the level of ovarian blood vessels, the immunopresence of the receptors for progesterone (PR-R), PGF2 alpha (FP) and GnRH (GnRH-R), and that of the enzymes endothelial nitric oxide synthase (eNOS), cyclooxygenase 1 (COX1), COX2, and prostaglandin E2-9-ketoreductase (PGE2-9-K).Materials, Methods & Results: Sexually mature New Zealand White female rabbits were used for all experiments. The animals were housed individually, in an indoor facility under controlled conditions of light and temperature, and feed ad libitum. The rabbits were treated with equine chorionic gonadotropin followed 2 days later by an intramuscular injection of a GnRH analogue to induce pseudopregnancy. Five rabbits were sacrificed by cervical dislocation, at early, mid and late stages of pseudopregnancy, for the immunohistochemical detection of GnRH-R, COX1, COX2, PGE2-9-K, eNOS, FP, and PR- R. The ovaries were immersed for fixation in formaldehyde and subsequently processed for embedding in paraffin. Serial sections were cut and individually mounted on slides. Background labelling was prevented by incubating the sections with normal goat serum (for COX1, COX2, eNOS, GnRH-R, PR- R, and FP) and with normal rabbit serum (for PGE2-9-K). Subsequently, the sections were incubated overnight with the following primary antisera: mouse monoclonal anti-COX1, anti-COX2, anti-eNOS, anti-GnRH-R, and antiPR-R, rabbit polyclonal anti-FP, and goat polyclonal anti-PGE2-9-K (CBR1). The next day, the slides were treated again with normal goat or rabbit serum and then incubated with biotin goat anti-mouse (for COX1, COX2, eNOS, PR- R, and GnRH-R), biotin goat anti-rabbit (for FP) or biotin rabbit anti-goat (for PGE2-9-K) secondary antibodies diluted 1: 200 in TBS for 30 min at room temperature. The slides were exposed to the avidin-biotin complex and the peroxidase activity sites were visualized using the 3,3-diaminobenzidine tetrachloride kit as chromogen. At all stages of pseudopregnancy, the veins and arteries, within the ovary hilus and medulla, showed a variable immunoreactivity to anti-COX2, -PGE2-9-K, -eNOS and -FP antibodies, whereas anti-GnRH-R, -COX1 and -PR-R did not produce immunosignals. In particular, COX2 immunopresence was strongly localized only in the arterial endothelial cells but not in the veins. PGE2-9-K immunosignals were moderately detected in the nuclei of endothelial, smooth muscle and stromal cells of both veins and arteries. The eNOS immunopositivity was moderately evidenced in the nuclei and cytoplasm of endothelial, smooth muscle and stromal cells of both veins and arteries. Strong positive immune reaction for FP was detected in the endothelial cells of ovarian arteries and veins; the arterial muscle cells were negative.Discussion: The present results suggest that the ovarian blood flow of rabbits is dynamically regulated as a consequence of exogenous treatments for the synchronization of estrous and the induction of ovulation. These haemodynamic changes are likely due to combined actions by several intraovarian factors, such as prostaglandins and correlated enzymes, steroids, and other autocrine/paracrine factors (GnRH, PGF2 alpha, NO).

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.

________________________________________________________________________________________ PARILLO, F., G. CATONE, C. CAPEZZONE, M. ZERANI: The presence of immunoreactive cyclooxygenases in the ductuli efferentes and epididymis of prepubertal and adult alpaca (Lama pacos). Vet. arhiv 83, 677-684, 2013. ABSTRACT In this study, we report the cell specifi c expression of cyclooxygenase (COX) enzymes, COX1 and COX2, in the ductuli efferentes and epididymis of prepubertal and adult alpaca. COX1 weakly stained the cytoplasm of epithelial cells lining the ductuli efferentes and the epididymal corpus, whereas these cells were immunonegative in the epididymal caput and cauda. In adults, we observed an increase in the immunsignals for COX1 in the cytoplasm of epithelial cells lining the ductuli efferentes and all the epididymal regions. In prepubertal alpaca, immunoreactivity for COX2 was not revealed in the epithelial cells lining the ductuli efferentes and epididymal regions, whereas it was evidenced in adult animals. The apical rich mithocondria cells immunoreacted only with COX1 in the epididymis of prepubertal animals, whereas they expressed both COX1 and -2 in the adult alpaca. Our results suggest that COXs may play a role in the pubertal development of the excurrent duct system of the alpaca.

The study was designed to examine the aglepristone (RU534) mechanisms affecting the corpora lutea (CL) lifespan in pseudopregnant rabbits. Aglepristone (10 mg/kg b.w.) was injected subcutaneously twice at either early- or mid-luteal phase (Days 3 and 4, or Days 8 and 9, respectively) after induction of ovulation with GnRH (Day 0). Corpora lutea and uteri, explanted at days 6 and 11, were evaluated for immunohistochemistry and Western blotting of progesterone (PR) and estrogen (ER) receptors, cyclooxygenase 1 (COX1), COX2, and PGE2-9-ketoreductase (PGE2-9-K) enzymatic activities, and progesterone, PGF2α, and PGE2 in vitro synthesis. Independent of luteal stage, aglepristone prolonged the functional luteal phase by 3 Days over that of controls as assessed by blood progesterone profiles. Aglepristone decreased protein for ER during both luteal-stages in CL and uteri. Progesterone receptor protein was decreased by RU354 at Days 6 in the uterus and at Days 11 in CL, whereas RU534 increased PR at Days 11 in uteri. In the CL, RU534 enhanced progesterone production at Days 6 and 11, whereas it decreased PGF2α and increased PGE2 at Day 11. In the uteri, RU534 decreased PGF2α and increased PGE2 synthesis at both days. COX2 and PGE2-9K activities were decreased by RU534 in the CL at Day 11, whereas in the uteri COX2 increased and PGE2-9-K decreased at Days 6 and 11. In conclusion, these data on aglepristone effects suggest that progesterone has a regulatory role on luteal function through direct and uterine-mediated mechanisms in pseudopregnant rabbits.

The aim of this study was to evaluate the occurrence and the activity of prostaglandin-endoperoxide synthase 1 (PTGS1), PTGS2, and endothelial, neuronal, and inducible nitric oxide synthase (e-, n-, and iNOS) in early, mid, late, and regressive corpora lutea (CL) of bovines during diestrus. PTGS1 immunoreactivity was localised mainly in the cytoplasm of small luteal cells, whereas PTGS2 was detected in the cytoplasm of large luteal cells during early, mid, and late stages. The immunoexpression of all NOS isoforms was observed in the nuclei of luteal cells in the CL stages examined. PTGS1 enzyme activity was higher in late CL and lower in regressive ones; PTGS2 increased from early to late CL and lowered in regressive ones. Constitutive NOS enzymatic activity (eNOS plus nNOS) was higher in late CL and lower in regressive ones; iNOS was lower in regressive CL. These results support the idea that PTGSs and NOSs regulate the bovine CL life span mainly during the transition from the luteotrophic to the luteolytic phase.

Background: Corpora lutea (CL) are transient organs, essentials for the pregnancy to be successful, that results from the ovulatory follicle rupture. If pregnancy fails to occur, the CL undergo luteolysis, a dynamic regression process that ends with their complete functional and structural demise. The life span of CL is characterized by luteal development, maintenance, and regression regulated by complex interactions between luteotropic and luteolytic mediators. The life-span of bovine CL is regulated by a multifactorial system that includes various hormonal regulators that up to date are not yet well studied, therefore the aim of the present study was to evaluate the immunohistochemical presence of receptors for adrenocorticotropic hormone (melanocortin-2 receptor, MC2R), dopamine (DR1-5), gonadotropin-releasing hormone (GnRHR), and peroxisome proliferators-activated receptor γ (PPARγ) in early, mid, late and regressive CL during diestrous cycle of bovine. Materials, Methods & Results: Ovaries from 24 clinically healthy cyclic cows were collected from licensed abattoirs. The animals had undergone a general veterinary assessment of their reproductive tract and ovaries before slaughter to exclude any apparent abnormalities and early pregnancy. Corpora lutea were separated by blunt dissection from the surrounding ovarian tissues and classifi ed into four groups, covering the entire diestrous cycle length: early, mid, late and regressive. Tissue specimens were immediately processed for immunohistochemical investigation. The slides were incubated with the following primary antibodies: rabbit polyclonal anti-GnRHR, rabbit polyclonal anti-MC2R, rabbit polyclonal anti-DRD1-DR5, and mouse monoclonal anti-PPARγ. The slides were incubated with the specifi c biotinylated secondary antibody exposed to avidin-biotin complex and the peroxidase activity sites were visualized using the DAB kit as chromogen. Tissue sections in which the primary antibody was omitted or substituted by the specifi c IgG were used as negative controls of non-specifi c staining. The intensity of receptor immunostaining in CL was assessed and comparated microdensitometrically. The data of the densitometric analysis were examined by Levene’s test and one-way ANOVA followed by Student-Newman-Keuls t-test. Differences were considered signifi cant at P < 0.01. GnRHR, PPARγ, and DR2 immunostained the cytoplasm of luteal cells in early, mid and late stages, whereas they did not react in regressive CL. The immunostaining of MC2R was strong in the cytoplasm and nucleus of luteal cells during early, mid and late CL, and decreased (P < 0.01) in regressive ones. PPARγ immunostained the cytoplasm of luteal cells in early, mid and late stages, whereas they did not react in regressive CL. DR2 immunosignals were only found in early, mid and late stages, whereas DR5 was evidenced strongly in the cytoplasm of early and mid CL, and weakly (P < 0.01) in late and regressive ones. DR1, -3, and -4 were immunonegative in all CL types. Discussion: The present study showed that in bovine CL GnRHR, MC2R, PPARγ, DR2 and DR5 are variously present throughout the different luteal phases of the diestrous cycle. These preliminary data confi rm the multifactorial complexity of the bovine CL life span regulation, in particular the fi ne tuning of the mediators that balance the CL luteotropic and luteolytic condition.

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.

The present study sought to assess whether the receptors for adrenocorticotropic hormone (ACTH), MC2R, and for glucocorticoid (GR) are expressed in corpora lutea (CL) of pseudopregnant rabbits and whether ACTH and cortisol exert any direct action on luteal function. By immunohistochemistry, positive reaction for MC2R and GR was detectable within luteal cells of CL. The MC2R mRNA levels were five-fold less abundant in day 9 than in day 4 CL (P<0.01). At both stages, ACTH agonist (ACTH 1-24) increased progesterone and prostaglandin (PG) E(2) (PGE(2)) (P<0.01), but reduced PGF(2α) releases (P<0.01) in vitro. ACTH 1-24 injection increased plasma cortisol levels within 4h (P<0.01), but decreased (P<0.01) progesterone 24h later and for the following two days. ACTH administration to estrous rabbits caused a transitory increase in blood progesterone concentrations (P<0.01). Daily injections of ACTH did not modify progesterone profile following ovulation. In conclusion, ACTH directly up-regulates CL progesterone production in vitro via MC2R, but indirectly hampers luteal function via cortisol-GR associated mechanism.