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ABSTRACT To investigate the mechanisms by which caloric restriction affects reproductive function in female rabbits, we measured, in animals intact or ovariectomized (OVX) estrogen-primed and fed ad libitum or fasted for 48 h, the adenohypophysial expression of estrogen receptor-alpha (ESR1) and gonadotropin releasing hormone receptor (GnRHR) and the dynamic secretion of LH following GnRH stimulation. Fasting increased the number of GnRHR-immunoreactive (-IR) cells in intact animals, whereas reduced the density of ESR1-IR cells in OVX rabbits. Estrogen priming decreased the number of ESR1-IR cells in fasted and OVX animals. Ovariectomy increased the number of ESR1-IR cells in fed rabbits, but caused an opposite effect in both fed and fasted animals treated with estrogen. Fasting down regulated the mRNA levels for ESR1 and GnRHR. Estrogen-priming reduced the abundance for ESR1 mRNA in both fed and fasted rabbits, and that for GnRHR in fasted rabbits. Ovariectomy halved ESR1 mRNA levels independently of treatment and feeding condition, whereas increased ( P < 001) that for GnRHR in estrogen-primed rabbits. In all rabbits, an LH surge occurred 30 min after GnRH injection but the lowest levels were found in intact fasted rabbits and the highest in fasted, estrogen-primed animals. The LH profile was similar in intact and OVX rabbits and neither fasting nor estrogen priming modified it. In conclusion, fasting differentially modifies the ESR1 and GnRHR expression in the pituitary, depending on the presence of gonadal hormones, indicating complex interactions between metabolic signals and ovarian steroids. Microsc. Res. Tech. 77:201-210, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

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The stomach of adult shi drumUmbrina cirrosawas investigated using a battery of nine horseradish peroxidase-conjugated lectins combined with enzymatic treatment, in order to distinguish glycoconjugate sugar residues. Epithelial cells showed the presence of galactosyl(β1→4)N-acetylglucosamine, mannose, N-acetylgalactosamine, N-acetylglucosamine, fucose and sialic acid-galactosyl(β1→3)N-acetylgalactosamine residues. Gastric pits had similar sugar residues with the exception of N-acetylgalactosamine which was less diffused. Gastric glands were characterized by the presence of glycoconjugates containing galactosyl(β1→3)N-acetylgalactosamine, N-acetylglucosamine, galactosyl(β1→4) N-acetylglucosamine, N-acetylgalactosamine and a small amount of sialic acid linked to N-acetylgalactosamine. [ABSTRACT FROM AUTHOR]

Kisspeptin (KiSS) and its related receptors (KiSS1R) have a critical role in the reproduction of mammals. The KiSS/KiSS1R system is expressed in numerous reproductive organs including the ovary. Here, we studied the expression of the KiSS/KiSS1R system and its functional role in rabbit corpora lutea (CL) at days 4 (early-), 9 (mid-), and 13 (late-stage) of pseudopregnancy. In vitro progesterone, prostaglandin (PG) F2α (PGF2α) and E2 (PGE2) productions and prostaglandin-endoperoxide synthase 1 (PTGS1) and 2 (PTGS2) activities were evaluated. Immune reactivity (IR) for KiSS and KiSS1R were detected in luteal cells at nuclear and cytoplasmic level at all luteal stage for KiSS and only at early- and mid-stage for KiSS1R; IR decreased from early- to later stages of pseudopregnancy. The KiSS-10 augmented progesterone and PGE2 and diminished PGF2α secretions by early- and mid-CL; KiSS-10 reduced PTGS2 activity at early- and mid-stages, but did not affect PTGS1 at any luteal stages. The antagonist KiSS-234 counteracted all KiSS-10 effects. This study shows that the KiSS/KiSS1R system is expressed in CL of pseudopregnant rabbits and exerts a luteotropic action by down-regulating PTGS2, which decreases PGF2α and increases PGE2 and progesterone.

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., (© 2018 Blackwell Verlag GmbH.)

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.

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.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

This study presents the first evidence for differences in COXs, PGE2-9-ketoreductase and NOSs immunopresence and enzyme activity, and prostaglandin and testosterone production between the testes of adult and prepubertal alpacas. The prepubertal testis immunohistochemical data revealed that COX1 was expressed in spermatogonia and endothelial cells whereas COX2 was present only in the stromal cells. In adult animals, COX2 immunosignals were evidenced in germ cells, as well as both COX1 and -2 in Leydig and Sertoli cells. In adult testes, the spermatogonia, spermatocytes and round spermatids had expression of e- and n-NOS only, whereas elongated spermatids exhibited immunopositivity for i- and e-NOS and Sertoli cells expressed only n-NOS. In prepubertal alpacas, i-NOS was localized in spermatogonia, e-NOS in Sertoli cells and all three NOS isoforms in Leydig cells. PGE2-9-ketoreductase immunopresence was observed in spermatogonia nuclei and cytoplasm of prepubertal testis whereas they were localized in spermatid acrosomal vesicle of adult. The enzymatic data indicated that COX1 activity was higher than COX2 in adult alpaca testis whereas the activity of COX2 was greater than that of COX1 in prepubertal animals. Total NOS and PGE2-9-ketoreductase activities were more extensive in adult alpacas. In vitro hormone production results showed that prepubertal testes released lower amounts of testosterone and PGF2α while PGE2 synthesis was six times more elevated than in in vitro incubated adult testes. Taken together, the data on COX2, i-NOS and PGE2 led us to hypothesize that development in prepubertal male reproductive tissues utilizes a mechanism similar to that of inflammation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The expression of type I GNRH receptor (GNRHR-I) and the direct role of GNRH-I on corpora lutea (CL) function were studied in the pseudopregnant rabbit model. Immunohistochemistry evidenced GNRHR-I and GNRH-I in luteal cells at early (day 4 pseudopregnancy)-, mid (day 9)-, and late (day 13)-luteal stages. Real-time RT-PCR and western blotting revealed GNRHR-I mRNA and protein at the three luteal stages. Buserelin in vivo treatment at days 9 and 13 decreased plasma progesterone levels for 48 and 24  h respectively. In in vitro cultured CL, buserelin reduced progesterone secretion, increased prostaglandin F(2α) (PGF(2α)) secretion and cyclo-oxygenase-2 (COX-2) and nitric oxide synthase (NOS) activities at days 9 and 13, and decreased PGE₂ at day 13. Co-incubation with antagonists for GNRH-I (antide), inositol 1,4,5-trisphosphate (IP₃, 2-amino-ethoxydiphenylborate), and diacylglycerol (DAG, 1-hexadecyl-2-acetyl glycerol) or inhibitors for phospholipase C (PLC, compound 48/80), and protein kinase C (PKC, staurosporine) counteracted the buserelin effects. Buserelin co-incubated with COX inhibitor (acetylsalicylic acid) increased progesterone and decreased PGF(2α) and NOS activity at days 9 and 13, whereas co-incubation with NOS inhibitor (N-nitro-l-arginine methyl ester) increased progesterone at the same luteal stages. These results suggest that GNRHR-I is constitutively expressed in rabbit CL independently of luteal stage, whereas GNRH-I down-regulates directly CL progesterone production via PGF(2α) at mid- and late-luteal stages of pseudopregnancy, utilizing its cognate type I receptor with a post-receptorial mechanism that involves PLC, IP₃, DAG, PKC, COX-2, and NOS.

An immunohistochemical and lectin histochemical study of the efferent ducts was performed in the alpaca. Two types of epithelium, consisting of principal and ciliated cells, were detected on the basis of the different cytokeratins expression and lectin binding pattern. AE1/AE3 and 13 cytokeratin antibodies intensely immunostained the entire cytoplasm of type I PCs, whereas AE1/AE3, but not anti cytokeratin 13, immunoreacted in type II principal cells along the apical, lateral and basal plasma-membrane. The histochemical characterization of the epithelial cells was carried out using a battery of different lectins: Con-A, UEA-I, LTA, WGA, GSA-II, GSA-IB4, SBA, PNA, ECA, DBA, MAL-II and SNA. Sialidase digestion and deglycosilation pre-treatments were also employed. In type I principal cells, the staining of the Golgi zone was interpreted giving evidence for the synthesis and secretion of O- and N-linked oligosaccharides. In particular, alpha-Man/alpha-Glc, GlcNAc, beta-Gal-(1-4)-GlcNAc, Neu5Acalpha2,3Gal and Neu5Acalpha2,6Gal/GalNAc residues were included in both O- and N-linked glycans, whereas alpha-Fuc, beta-GalNAc and alpha-Gal were only found in O-linked oligosaccharides; alpha-GalNac and beta-Gal-(1-3)-D-GalNAc were found subterminal to sialic acid moieties and they were included in O- and N-glycans. In type II principal cells, the lectin staining was observed in the apical cytoplasmic granules and in vacuoles that were interpreted as components of an elaborate endocytotic apparatus specialized for the uptake of particulate material and fluid from the lumen. These results suggest the existence of two structurally different epithelial segments along the ductuli efferentes of the alpaca, with a high degree of compartmentalization of the secretory and absorptive activities.

The present work was undertaken to determine the glycoconjugates secreted by the epithelium of the equine ampulla ductus deferentis, using conventional (PAS, AB pH 2.5, AB pH 1.0) and lectin histochemical procedures in conjunction with enzymatic digestion and chemical treatment. The presence of abundant apical cytoplasmic blebs suggests that the equine ampulla secretes its products mainly in an apocrine manner. Glandular cells secrete neutral and acidic sialylated glycoconjugates as revealed by conventional histochemical procedures. Lectin histochemistry helped us to discover the following histological positive sites: the mucosal cells, the glandular epithelial cells, the apical cytoplasmic blebs and the basal cells. The ampullary secretions contained both glycoproteic material (revealed by Con-A-, LCA-, GSA-II-, WGA-, RCA-I- positivity) and sialomucins (evidenced by the reactivity of GSA-II, SBA, PNA and RCA-I after sialidase digestion) having different functional roles. The mucosal cells reacted with Con-A, LCA, and also with sialidase/GSA-II-, SBA-, PNA-, and RCA-I sequences, contributing to the chemical heterogeneity of ampullary secretions. DBA lectin was a specific marker for basal cells. The results obtained were compared with our previous findings regarding the differences in the lectin binding pattern of the plasma-membrane of equine sperm collected from epididymal cauda and the ampulla ductis deferentis. Our results support other studies that indicate that ampullary secretions are involved in altering the plasma-membrane glycoconjugates of spermatozoa, contributing to their maturation.

Apoptotic cell death is an essential homeostatic mechanism involved in the control of cellular turnover in a variety of adult tissues. Cytoplasmic and nuclear condensation morphologically define this process whose biochemical hallmark is extensive DNA fragmentation into discrete oligonucleosomic units. Hair follicle growth and regression has been shown to be correlated with apoptosis in humans, mice, rats and guinea pigs. The present study was carried out to evaluate its implication in canine hair biology in order to define the spatio-temporal relationship between apoptosis and the hair cycle in dogs. As assessed by terminal deoxy-nucleotidyl transferase-mediated d-UTP nick-end-labelling (TUNEL) and by basic histological and ultrastructural assays, apoptotic cells appeared both in the growing and in the regressing follicle epithelium showing the well characterized morphological features described in the previous relevant literature.