The immunohistochemical presence and distribution of ghrelin, apelin and their receptors in dog ovaries

The activity of ghrelin, apelin and their receptors has been correlated to the control of some infectious diseases, besides the hypothesis of their role in the control of some peripheral organs, among which ovaries. The aim of the present work was to highlight the presence and distribution of ghrelin, apelin and cognate receptors in the ovaries of pregnant bitches, by means of immunohistochemical techniques. Apelin, its receptor and the receptor of ghrelin were highlighted in the corpora lutea, with a particular localization in the cytoplasm of some luteal cells. Instead, a positive reaction for ghrelin was evident in the walls of small arteries in the connective tissue. These results allowed us to hypothesize that these molecules intervene in the control of ovaries in pregnant bitches, suggesting autocrine/paracrine mechanisms of regulation

Physiology and modulation factors of ovulation in rabbit reproduction management

[EN] Rabbit is an induced ovulatory species, so ovulation takes place after mating. Traditionally, exogenous and synthetic hormonal factors (administered by intramuscular and intravaginal route) such as GnRH and analogues, or different physical procedures (i.e. stimulation by intravaginal cannula) have been used to induce ovulation in females when artificial insemination is applied in rabbit farms. Restriction and public rejection of the use of hormones is leading to the study of the seminal plasma components with potential action on ovulation induction. The aim of the present review is to collect and summarise the strategies used in recent years to trigger ovulation and improve rabbit fertility management with respect to more animal-friendly manipulation methods. Furthermore, special attention has been paid to the use of a semen component (as endogen molecule) such as beta nerve growth factor (β-NGF) in male and female rabbit reproductive physiology. This neurotrophin and its receptors (TrKA and p75NTR) are abundantly distributed in both male and female rabbit reproductive tracts, and it seems to have an important physiological role in sperm maturation and behaviour (velocity, apoptosis and capacitation), as well as a modulatory factor of ovulation. Endogen β-NGF is diluted in the seminal doses with the extenders; hence it could be considered an innovative and alternative strategy to avoid the current exogenous (by intramuscular route) and stressful hormonal treatments used in ovulation induction. Their addition in seminal dose could be more physiological and improve animal welfare in rabbit farms.Mattioli, S.; Maranesi, M.; Castellini, C.; Dal Bosco, A.; Arias-Álvarez, M.; Lorenzo, PL.; García Rebollar, P.... (2021). Physiology and modulation factors of ovulation in rabbit reproduction management. World Rabbit Science. 29(4):221-229. https://doi.org/10.4995/wrs.2021.13184OJS221229294Adams G.P., Ratto M.H., Huanca W., Singh J. 2005. Ovulationinducing factor in the seminal plasma of alpacas and llamas. Biol. Reprod., 73: 452-457. https://doi.org/10.1095/biolreprod.105.040097Adams G.P., Ratto M.H., Silva M.E., Carrasco R.A. 2016. Ovulation-inducing factor (OIF/NGF) in seminal plasma: a review and update. Reprod. Dom. Anim., 51: 4-17. https://doi.org/10.1111/rda.12795Adams, C. E. 1961. Artificial insemination in the rabbit. J. Reprod. Fertil., 2: 251-254.Bakker J., Baum M.J. 2000. Neuroendocrine regulation of GnRH release in induced ovulators. Frontiers in neuroendocrinology, 21: 220-262. https://doi.org/10.1006/frne.2000.0198Benziger D.P., Edelson, J. 1983. Absorption from the vagina. Drug Metab. Rev., 14: 137-168. https://doi.org/10.3109/03602538308991387Berland M.A., Ulloa-Leal C., Barría M., Wright H., Dissen G.A., Silva M.E., Ojeda S.R., Ratto M.H. 2016. Seminal plasma induces ovulation in llamas in the absence of a copulatory stimulus: role of nerve growth factor as an ovulationinducing factor 1. Endocrinology, 29, en20161310. https://doi.org/10.1210/en.2016-1310Bogle O.A., Carrasco R.A., Ratto M.H., Singh J., Adams G.P. 2018. Source and localization of ovulation-inducing factor/nerve growth factor in male reproductive tissues among mammalian species. Biol. Reprod., 99: 1194-1204. https://doi.org/10.1093/biolre/ioy149Boiti C., Besenfelder U., Brecchia G., Theau Clement M., Zerani M. 2006. Reproductive physiopathology of the rabbit doe. In: Recent Advices in rabbit science. Ed. Maertens L. and Coudert P. Published ILVO. Merelbeke, Belgium.Boiti C., Castellini C., Canali C., Zampini D., Monaci, M. 1995. Long term effect of PMSG on rabbit does reproductive performance. World Rabbit Sci., 3: 51-56. https://doi.org/10.4995/wrs.1995.240Browning J., Reyes L. Wolf R. 1980. Comparison Evidence of Serum Progesterone, in Pregnant Rabbits: Recognition of Pregnancy and Estradiol-1713 and Pseudopregnant for Postimplantation rate. Biol. Reprod., 23: 1014-1019. https://doi.org/10.1095/biolreprod23.5.1014Cardinali R., Dal Bosco A., Bonanno A., Di Grigoli A., Rebollar P. G., Lorenzo P. L., Castellini C. 2008. Connection between body condition score, chemical characteristics of body and reproductive traits of rabbit does. Livestock Sci., 116: 209-215. https://doi.org/10.1016/j.livsci.2007.10.004Carrasco R.A., Ratto, M. H., Adams G. P. 2021. Differential Effects of Estradiol on Reproductive Function in Camelids. Front. Vet. Sci., 8: 124. https://doi.org/10.3389/fvets.2021.646700Casares-Crespo L., Vicente J. S., Talaván A. M., Viudesde-Castro M.P. 2015. Does the inclusion of protease inhibitors in the insemination extender affect rabbit reproductive performance?. Theriogenology, 85: 928-932. https://doi.org/10.1016/j.theriogenology.2015.10.044Casares-Crespo L., Fernandez-Serrano P., Vicente J.S., Marco-Marco-Jimenez F., Viudes-de-Castro M.P. 2018. Rabbit seminal plasma proteome: The importance of the genetic origin. Anim. Reprod. Sci., 189: 30e42.Castellini C. 1996. Recent advances in rabbit artificial insemination. In Proc.: 6th World Rabbit Congress, 9-12 July 1996, Toulouse, France, 2: 13-26.Castellini C., Dal Bosco A., Arias-Álvarez M., Lorenzo P. L., Cardinali R., Rebollar P.G. 2010. The main factors affecting the reproductive performance of rabbit does: a review. Animal Reprod. Sci., 122: 174-182. https://doi.org/10.1016/j.anireprosci.2010.10.003Castellini C., Mattioli S., Dal Bosco A., Collodel G., Pistilli A., Stabile A.M., Macchioni L., Mancuso F., Luca G., Rende M. 2019. In vitro effect of nerve growth factor on the main traits of rabbit sperm. Reprod. Biol. Endocrinol., 17: 93. https://doi.org/10.1186/s12958-019-0533-4Castellini C., Mattioli S., Dal Bosco A., Cartoni Mancinelli A., Rende M., Stabile A.M., Pistilli A. 2020a. NGF and sperm traits: a review. ICAR 2020, Theriogenology ICAR special issue. https://doi.org/10.1016/j.theriogenology.2020.01.039Castellini C., Mattioli S., Dal Bosco A., Cotozzolo E., Mancinelli A. C., Rende M., Pistilli A. 2020b. Nerve growth factor receptor role on rabbit sperm storage. Theriogenology, 153: 54-61. https://doi.org/10.1016/j.theriogenology.2020.04.042Cervantes M.P., Palomino J.M., Adams G.P. 2015. In vivo imaging in the rabbit as a model for the study of ovulation-inducing factors. Lab. Anim., 49: 1-9. https://doi.org/10.1177/0023677214547406Chen B.X., Yuen Z.X., Pan G. 1985. Semen-induced ovulation in the bactrian camel (Camelus bactrianus). Reproduction, 73: 335-339. https://doi.org/10.1530/jrf.0.0740335Dal Bosco A., Cardinali R., Brecchia G., Rebollar P.G., Fatnassi M., Millán P., Castellini, C. 2014. Induction of ovulation in rabbits by adding Lecirelin to the seminal dose: In vitro and in vivo effects of different excipients. Anim. Rep. Sci., 150: 44-49. https://doi.org/10.1016/j.anireprosci.2014.08.009Dal Bosco A., Rebollar P.G., Boiti C., Zerani M., Castellini C. 2011. Ovulation induction in rabbit does: current knowledge and perspectives. Anim. Reprod. Sci.; 129: 106-117. https://doi.org/10.1016/j.anireprosci.2011.11.007El Allali K., El Bousmaki N., Ainani H., Simonneaux V. 2017. Effect of the camelid's seminal plasma ovulation-inducingfactor/β-NGF: a kisspeptin target hypothesis. Front. Vet. Sci., 4: 99. https://doi.org/10.3389/fvets.2017.00099European Parliament. 2010. Directive 2010/63/EU. European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Text with EEA relevance. Available at http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=celex%3A32010L0063. Accesed September 2017.European Parliament. 2013. Report from the Commission to the Council and the European Parliament. Sixth Report on the statistics on the number of animals used for experimental and other scientific purposes in the Member States of the European Union SEC 2010, 1107. Available at http://eurlex.europa.eu/resource.html?uri=cellar:e99d2a56-32fc-4f60- ad69-61ead7e377e8.0001.03/DOC_1&format=PDF. Accesed September 2017Fernández-Serrano P., Casares-Crespo L., Viudes-de-Castro M.P. 2017. Chitosan-dextran sulphate nanoparticles for Gn RH release in rabbit insemination extenders. Reprod. Domest. Anim., 52: 72-74. https://doi.org/10.1111/rda.13062García-García R.M., Masdeu M.D. M., Sánchez Rodríguez A., Millán P., Arias-Álvarez M., Sakr,O.G., Rebollar P. G. 2018a. β-nerve growth factor identification in male rabbit genital tract and seminal plasma and its role in ovulation induction in rabbit does. It. J. Anim. Sci., 17: 442-453. https://doi.org/10.1080/1828051X.2017.1382315García-García R.M., Arias-Álvarez M, Sánchez Rodríguez A., García Rebollar P. Lorenzo P.L. 2018b. NGF systems is differentially expressed in the ovary, oviduct and uterus of rabbit does although independent of serum hormonal levels. In: 22nd Annual Conference of the European Society for Domestic Animal Reproduction (ESDAR). Reprod. Dom. Anim. 53, S2: 88.García-García R.M., Arias-Álvarez M, Sánchez Rodríguez A., García Rebollar P. Lorenzo P.L. 2020. Role of nerve growth factor in the reproductive physiology of female rabbits: a review. Theriogenology, 150: 321-328. https://doi.org/10.1016/j.theriogenology.2020.01.070Goodman S.B., Kugu K., Chen S.H., Preutthipan S, Tilly K.I., Tilly J.L., Dharmarajan A.M. 1998. Estradiol-mediated suppression of apoptosis in the rabbit corpus luteum with a shift in expression of Bcl-2 family members favouring cellular survival. Biol Reprod., 59: 820-827. https://doi.org/10.1095/biolreprod59.4.820Harcout-Brown F. 2002. Textbook of Rabbit Medicine. Elsevier Health Sciences, 3: 356-360. Available at http://www.questia.com/PM.qst?a=o&docId=26347764.Harper, M.J.K. 1970. Factors influencing sperm penetration of rabbit eggs in vivo. J. Exp. Zool., 173: 47-62. https://doi.org/10.1002/jez.1401730104Hassanein E.M., Hashem N.M., El-Azrak K.E.M., Gonzalez-Bulnes A., Hassan G.A., Salem M.H. 2021. Efficiency of GnRH-Loaded Chitosan Nanoparticles for Inducing LH Secretion and Fertile Ovulations in Protocols for Artificial Insemination in Rabbit Does. Animals, 11: 440. https://doi.org/10.3390/ani11020440Holgado-Madruga M., Moscatello D.K., Emlet D.R., Dieterich R., Wong A.J. 1997. Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor. In Proc.: National Academy of Sciences, 94: 12419-24. https://doi.org/10.1073/pnas.94.23.12419Johnston S.D., O'Callaghan P.O., Nilsson K., Tzipori G., Curlewis J.D. 2004. Semen-induced luteal phase and identification of a LH surge in the koala (Phascolarctos cinereus). Reproduction, 128: 629-634. https://doi.org/10.1530/rep.1.00300Lebas F., Theau-Clement M., Remy B., Drion P., Beckers J.F. 1996. Production of anti-PMSG antibodies and its relation to the productivity of rabbit does. World Rabbit Sci., 4: 57-62. https://doi.org/10.4995/wrs.1996.271Levi-Montalcini R., Hamburger V. 1951. Selective growth stimulating effects of mouse sarcoma on the sensory and sympathetic nervous system of the chick embryo. J. Exp. Zool., 116: 321-361. https://doi.org/10.1002/jez.1401160206Li C., Sun Y., Yi K., Ma Y., Zhang W., Zhou X. 2010. Detection of nerve growth factor (NGF) and its specific receptor (TrkA) in ejaculated bovine sperm, and the effects of NGF on sperm function. Theriogenology, 74: 1615-1622. https://doi.org/10.1016/j.theriogenology.2010.06.033Lin W.W., Ramirez V.D. 1991. Effect of mating behavior on luteinizing hormone-releasing in female rabbits as monitored with push-pull cannulae. Neuroendocrinology, 53: 229-325. https://doi.org/10.1159/000125723Lytton, F.D.C., Poyser N.L. 1982. Prostaglandin production by the rabbit and placenta in vitro uterus. J. Reprod. Fertil., 66: 591-599. https://doi.org/10.1530/jrf.0.0660591Maranesi M., Zerani M., Lilli L., Dall'Aglio C., Brecchia G., Gobbetti A., Boiti C. 2010. Expression of luteal estrogen receptor, interleukin-1, and apoptosis-associated genes after PGF2alpha administration in rabbits at different stages of pseudopregnancy. Domest. Anim. Endocrinol., 39: 116-30. https://doi.org/10.1016/j.domaniend.2010.03.001Maranesi M., Zerani M., Leonardi L., Pistilli A., Arruda-Alencar J., Stabile A.M., Rende M., Castellini C., Petrucci L., Parillo F.,Moura A., Boiti C. 2015. Gene expression and localization of NGF and its cognate receptors NTRK1 and NGFR in the sex organs of male rabbits. Reprod. Dom. Anim., 50: 918-925. https://doi.org/10.1111/rda.12609Maranesi M., Parillo F., Leonardi L., Rebollar P.G., Alonso B., Petrucci L., Zerani M. 2016. Expression of nerve growth factor and its receptors in the uterus of rabbits: functional involvement in prostaglandin synthesis. Domest. Anim. Endocrinol., 56: 20-28. https://doi.org/10.1016/j.domaniend.2016.02.001Maranesi M., Petrucci L., Leonardi L., Piro F., Rebollar P.G., Millán P., Cocci P., Vullo C., Parillo F., Moura A., Mariscal G.G., Boiti C., Zerani M. 2018. New insights on a NGF-mediated pathway to induce ovulation in rabbits (Oryctolagus cuniculus). Biol Reprod., 98: 634-643. https://doi.org/10.1093/biolre/ioy041Maranesi M., Palermo F.A., Bufalari A., Mercati F., Paoloni D., Cocci P., Moretti G., Crotti S., Zerani M., Dall'Aglio C. 2020. Seasonal Expression of NGF and Its Cognate Receptors in the Ovaries of Grey Squirrels (Sciurus carolinensis). Animals (Basel). 10:1558. https://doi.org/10.3390/ani10091558Maranesi M., Cocci P., Tomassoni D., Mercati F., Palermo F.A., Anipchenko P., Boiti C., Bufalari A., Zerani M., Dall'Aglio C. 2020a. Ovulation inducing factors in rabbits (Oryctolagus cuniculus): the role of IL1. ESDAR 2021, Reprod. Dom. Anim., submitted.Milligan, S., 1982. Induced ovulation in mammals. In: Oxford Reviews of Reproductive Biology, Vol. 4 (Ed. by C.A. Finn). Clarendon Press, Oxford, UK. pp.1-46.Molina I., Pla M., Vicente J.S., Martin A., Romeu A. 1991. Induction of ovulation in rabbits with pure urinary luteinizing hormone and human chorionic gonadotrophin: comparison of oocyte and embryo quality. Hum. Reprod., 6: 1449-1452. https://doi.org/10.1093/oxfordjournals.humrep.a137287Pei Y. 2010. Effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos in vitro. Veter. Res. Commun., 34: 11-18. https://doi.org/10.1007/s11259-009-9325-1Quintela L.A., Peña A.I., Vega M.D., Gullón J., Prieto M.C., Barrio M., Herradón P.G. 2004. Ovulation induction in rabbit does submitted to artificial insemination by adding buserelin to the seminal dose. Reprod. Nutr. Dev., 44: 79-88. https://doi.org/10.1051/rnd:2004015Ratto M.H., Leduc Y.A., Valderrama X.P., van Straaten K.E., Delbaere L.T.J., Pierson R.A., Adams G.P. 2012. The nerve of ovulation- inducing factor in semen. In Proc.: National Academy of Sciences, 109: 15042-15047. https://doi.org/10.1073/pnas.1206273109Rebollar P.G., Ubilla E., Alvariño J.M.R 1994. Grouping of rabbit reproduction management by means of artificial insemination. World Rabbit Sci., 2: 87-91. https://doi.org/10.4995/wrs.1994.222Rebollar P.G., Dal Bosco A., Millán P., Cardinali R., Brecchia G., Sylla L., Castellini C. 2012. Ovulating induction methods in rabbit does: the pituitary and ovarian responses. Theriogenology, 77: 292-298. https://doi.org/10.1016/j.theriogenology.2011.07.041Sánchez-Rodríguez A., Abad P., Arias-Álvarez M., Rebollar P.G., Bautista J.M., Lorenzo P.L. 2018. Recombinant production of rabbit β-Nerve Growth Factor and its biological effect on rabbit sperm. Bio Rxiv., 458612. https://doi.org/10.1101/458612Sánchez-Rodríguez A., Arias-Álvarez M., Timón P., Bautista J.M., Rebollar P.G., Lorenzo P.L. 2019a. Characterization of β-Nerve Growth Factor-TrkA system in male reproductive tract of rabbit and the relationship between β-NGF and testosterone levels with seminal quality during sexual maturation. Theriogenology, 126: 206-213. https://doi.org/10.1016/j.theriogenology.2018.12.013Sánchez-Rodríguez A., Abad P., Arias-Álvarez M., Rebollar P. G., Bautista J. M., Lorenzo P.L., García-García R.M. 2019b. Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits. PloS one, 14. https://doi.org/10.1371/journal.pone.0219780Sánchez-Rodríguez A., Arias-Álvarez M., Millan P., Lorenzo P. L., García-García R. M., Rebollar P. G. 2020. Physiological effects on rabbit sperm and reproductive response to recombinant rabbit beta nerve growth factor administered by intravaginal route in rabbit does. Theriogenology, 157: 327-334. https://doi.org/10.1016/j.theriogenology.2020.08.003Sari L.M., Zampini R., Arganaraz M.E., Carretero M.I., Fumuso F.G., Barraza D.E. 2018 Expression of beta-NGF and highaffinity NGF receptor (TrKA) in llama (Lama glama) male reproductive tract and spermatozoa. Mol. Reprod. Dev., 85: 934-44. https://doi.org/10.1002/mrd.23075Sari L. M., Zampini R., Del Pino F. G., Argañaraz M. E., Ratto M. H., Apichela S. A. 2020. Effects of NGF Addition on Llama (Lama glama) Sperm Traits After Cooling. Front. Vet. Sci., 7. https://doi.org/10.3389/fvets.2020.587596Schneidgenova M., Vašíček J., Cupka P., Chrenek P. 2011. Is it necessary to control seasonal quality of the rabbit ejaculate? Slovak J Anim Sci., 44: 48-51.Silva M., Niño A., Guerra M., Letelier C., Valderrama X.P., Adams G.P., Ratto M.H. 2011. Is an ovulationinducing factor (OIF) present in the seminal plasma of rabbits? Anim. Reprod. Sci., 127: 213-221. https://doi.org/10.1016/j.anireprosci.2011.08.004Silva M., Ulloa-Leal C., Norambuena C., Fernández A., Adams G.P., Ratto M.H. 2014. Ovulation-inducing factor (OIF/NGF) from seminal plasma origin enhances Corpus Luteum function in llamas regardless the preovulatory follicle diameter. Anim. Reprod. Sci., 148: 221-227. https://doi.org/10.1016/j.anireprosci.2014.05.012Silva M., Paiva L., Ratto M.H. 2020. Ovulation mechanism in South American Camelids: The active role of β-NGF as the chemical signal eliciting ovulation in llamas and alpacas. Theriogenology, 150: 280-287. https://doi.org/10.1016/j.theriogenology.2020.01.078Simmon J.A., Danfbrth D.R., Hutchinson J.S., Hodgen G.D. 1988. Characterization of recombinant DNAderived human luteinizing hormone in vitro and in vivo: efficacy in ovulation induction and corpus luteum support. J. Am. Med. Assoc., 259: 3290-3295. https://doi.org/10.1001/jama.1988.03720220036022Theau-Clément M., Maertens L., Castellini C., Besenfelder U., Boiti C. 2005. Recommendations and guidelines for applied reproduction trials with rabbit does. World Rabbit Sci., 13: 147-164. https://doi.org/10.4995/wrs.2005.521Theau-Clément M. 2007. Preparation of the rabbit doe to insemination: a review. World Rabbit Sci., 15: 61-80. https://doi.org/10.4995/wrs.2007.604Tometten M., Blois S., Arck P.C. 2005. Nerve growth factor in reproductive biology: link between the immune, endocrine and nervous system? Chem. Immunol. Allergy, 89: 135-48. https://doi.org/10.1159/000087962Ubilla E., Rebollar P.G. 1995. Influence of the postpartum day on plasma estradiol 17 beta levels, sexual behaviour and conception rate, in artificially inseminated rabbit does. Anim. Reprod. Sci., 38: 337-344. https://doi.org/10.1016/0378-4320(94)01366-TUlloa-Leal C., Bogle O.A., Adams G.P., Ratto M.H. 2014. Luteotrophic effect of ovulation-inducing factor/nerve growth factor present in the seminal plasma of llamas. Theriogenology, 8: 1101-1107. https://doi.org/10.1016/j.theriogenology.2014.01.038Viudes-de-Castro M.P., Moce E., Lavara R., Marco-Jiménez F., Vicente J.S. 2014. Aminopeptidase activity in seminal plasma and effect of dilution rate on rabbit reproductive performance after insemination with an extender supplemented with buserelin acetate. Theriogenology, 81: 1223-1228. https://doi.org/10.1016/j.theriogenology.2014.02.003Viudes-de-Castro M.P., Casares-Crespo L., Marco-Jiménez F., Vicente J.S. 2017. Physical effect of insemination cannula on ovulation induction in rabbit doe. XVII Jornadas sobre Producción Animal, Zaragoza, España, 30 y 31 de mayo de 2017: 380-382.Zerani M., Polisca A., Boiti C., Maranesi M. 2021. Current Knowledge on the Multifactorial Regulation of Corpora Lutea Lifespan: The Rabbit Model. Animals, 1: 296. https://doi.org/10.3390/ani1102029

Immunohistochemical identification of resistin in the uterus of ewes subjected to different diets: Preliminary results

Resistin is a polypeptide hormone of the adipokine-family, primarily, but not exclusively, produced by the adipose tissue. Recent studies suggested that resistin may affect the male and female reproductive activity. The study aim was to immunohistochemically evaluate the presence and distribution of resistin in the ovine uterus. Uterine samples were collected from two groups of ewes at the end of an experimental trial during which the animals of the first group (CTRL) were fed only by grazing while those of the second one (EXP) were supplemented with barley and corn. Using a monoclonal antibody against resistin, tested by Western Blot, the immunopositive reaction was identified in the cytoplasm of epithelial lining cells and uterine glands. The endogenous production of resistin seemed to be affected by different diet, as evidenced by staining differences between the CTRL and EXP groups. Our findings support the existence of a peripheral resistin system in the sheep uterus. It is possible that this system is involved in the functionality of the uterus, which is also affected by the animal’s nutritional status.

Leptin System in Obese Dog Skin: A Pilot Study

Obesity predisposes to several health problems including skin diseases. However, information on the relationship between obesity and skin disorders in pets is very scarce. Leptin (LEP) is mainly produced by adipose tissue and has a prominent role in skin biology. This study evaluated the LEP system in the skin of obese dogs compared to normal-weight animals. The investigation was carried out on 10 obese (Obese group) and 10 normal-weight (Normal-weight group) dogs through Real-time PCR and immunohistochemistry. Cells of skin associated immune system were also evaluated. No differences were evidenced between the two groups as well as skin inflammation. LEP differences were no significant, while LEPR transcript appeared 10-fold higher in obesedogs than in normal-weight ones. Immunostaining for both molecules was observed in several skin structures such as the epidermis, hair follicles, and glands. No differences appeared in the skin associated immune system composition. This study is a preliminary report showing that LEP system changes in obese dog skin. The increased LEPR expression observed in the obese group suggests that the receptor plays a modulating role in the system control. However, the exact role of LEPin the skin under obesity conditions needs further elucidation

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 F2 (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

Gonadotropin-Releasing Hormone 1 Directly Affects Corpora Lutea Life-Span in MediterraneanBuffalo (Bubalus bubalis) During Diestrus: Presence and In Vitro Effects on Enzymatic andHormonal 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 less in late luteal phases, while prostaglandin (PG) F2alpha (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, while 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 less 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 only 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 activitie

Apelin expression in the fallopian tubes of ewes subjected to different nutritional levels: preliminary results

Introduction: Adipokines have a role as a link between the energy availability and correct reproductive activity. Apelin (APLN) increases body weight but also lead to infertility modifying reproductive hormones in mouse. Expression of APLN and its receptor (APLNR) was observed in different tract of female genital system with differences among species. In this work, the expression and localization of APLN and APLNR were analyzed in the fallopian tubes of sheep. Materials and Methods: 15 Comisana x Appenninica adult female sheep in dry stage were fed with fresh hay from June to the pasture maximum flowering (MxF). From this period to maximum dryness, the control group (Cnt) was fed with fresh hay while, the experimental group (Exp) with fresh hay supplemented with 600g/day/head of barely and corn (1:1). Fallopian tubes were processed to perform RT-PCR and immunohistochemistry for APLN and APLNR. Results: RT-PCR evidenced the transcripts for both molecules. IHC showed APLN and APLNR staining in the epithelial cells of both the infundibulum and isthmus. APLN appeared stronger in Cnt and Exp groups respect to MxF. Conclusion: No studies describe APLN or its receptor in the fallopian tubes. Their identification in the sheep suggests that APLN plays a role in the salpinx activity and is involved in the reproduction. This is a preliminary report that introduces APLN investigation in the sheep female genital system however, the exact role of APLN and the influence of diet needs further elucidation

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 Rabbits1

The expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and its role in corpora lutea (CL) function were studied in pseudopregnant rabbits. CL were collected at early (Day 4), mid (Day 9), and late (Day 13) stages of pseudopregnancy. Immunohistochemistry evidenced the presence of PPARgamma in the perinuclear cytoplasm and nucleus of all luteal cells; immunoreactivity decreased from early to late stage, with immunonegativity of the nuclei of late CL. PPARgamma mRNA transcript was expressed in all luteal stages with the lowest level at late stage. In CL cultured in vitro, PPARgamma agonist (15-deoxy delta12,14 prostaglandin J2, 15d-PGJ2, 200 nM) increased and antagonist (T0070907, 50 nM) decreased progesterone secretion at early and mid luteal stages, whereas 15d-PGJ2 reduced and T0070907 increased PGF2alpha ones. Prostaglandin-endoperoxide synthase 2 (PTGS2) activity was reduced by 15d-PGJ2 and increased by T0070907 in corpora lutea of early and mid luteal stages. Conversely, 15d-PGJ2 increased and T0070907 reduced 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity in early and mid luteal 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 consequent PGF2alpha decrease and progesterone increase