Giuseppe Catone

In vitro effects of gonadotropin-releasing hormone (GnRH) on Leydig cells of adult alpaca (Lama pacos) testis: GnRH receptor immunolocalization, testosterone and prostaglandin synthesis, and cyclooxygenase activities.

The main objective of this study was to examine the modulatory in vitro effects of gonadotropin-releasing hormone (GnRH) on isolated Leydig cells of adult alpaca (Lama pacos) testis. We first evaluated the presence of GnRH receptor (GnRHR) and cyclooxygenase (COX) 1 and COX2 in alpaca testis. We then studied the in vitro effects of buserelin (GnRH analogue), antide (GnRH antagonist), and buserelin plus antide or inhibitor of phospholipase C (compound 48/80) and COXs (acetylsalicylic acid) on the production of testosterone, PGE(2), and PGF(2α) and on the enzymatic activities of COX1 and COX2. Immunoreactivity for GnRHR was detected in the cytoplasm of Leydig cells and in the acrosomal region of spermatids. COX1 and COX2 immunosignals were noted in the cytoplasm of spermatogonia, spermatocytes, spermatids, Leydig cells, and Sertoli cells. Western blot analysis confirmed the GnRHR and COX1 presence in alpaca testis. The in vitro experiments showed that buserelin alone increased (P < 0.01) and antide and buserelin plus acetylsalicylic acid decreased (P < 0.01) testosterone and PGF(2α) production and COX1 activity, whereas antide and compound 48/80 counteracted buserelin effects. Prostaglandin E(2) production and COX2 activity were not affected by buserelin or antide. These data suggest that GnRH directly up-regulates testosterone production in Leydig cells of adult alpaca testis with a postreceptorial mechanism that involves PLC, COX1, and PGF(2α).

The Ductus Epididymis of the Alpaca : Immunohistochemical and Lectin Histochemical Study

Our objective was to characterize epithelial cells lining the epididymal duct (caput, corpus, cauda) of the alpaca using AE1/AE3 cytokeratin antibodies and 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 deglycosylation pre-treatments were also employed. The principal cells (PCs) along the epididymis showed differences in immunostaining patterns toward keratin antibodies. Lectin histochemistry demonstrated variations in the content and distribution of glycosidic residues of glycoconjugates in different epididymal regions. In particular, staining of the Golgi zone in the epithelial PCs was interpreted as evidence for synthesis and secretion of O- and N-linked oligosaccharides. In the caput, the apical mitochondria-rich cells contained mainly beta-GalNAc, subterminal alpha-GalNAc, alpha-Gal and Neu5Ac alpha2,3Gal residues. Conversely, in the corpus they were particularly rich in alpha-GalNac and beta-Gal-(1-3)-d-GalNAc linked to sialic acid moieties. Basal cells mainly expressed beta-GalNAc and alpha-Gal in the caput, alpha-Gal in the corpus and alpha-Fuc and beta-GalNAc in the cauda. The differences in immunostaining patterns and in lectin histochemistry in the alpaca epididymis reported in this investigation seem to be related to regional differences in function.

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

ABSTRACT 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.

Immunopresence and enzymatic activity of nitric oxide synthases, cyclooxygenases and PGE2-9-ketoreductase and in vitro production of PGF2α, PGE2 and testosterone in the testis of adult and prepubertal alpaca (Lama pacos).

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.

Immunopresence and functional activity of prostaglandin-endoperoxide synthases and nitric oxide synthases in bovine corpora lutea during diestrus.

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.

Expression of Endothelin‐1 and Endothelin Receptor A in Canine Ovarian Tumours

Ovarian tumours have a low incidence in bitch. Endothelin (ET-1) and endothelin A receptor (ET-A) are overexpressed in human ovarian cancer. Twenty canine ovarian tumours and five normal samples were first evaluated by western blotting and then immunohistochemically for ET-1 and ET-A expression. Seventeen out of twenty tumours were ET-1 positive. Eight out of twenty tumours were ET-A immunohistochemically positive. At molecular level both proteins were proven to be expressed in normal as well as in tumour samples. Our results show that ET-1 and ET-A are overexpressed in canine ovarian tumours, suggesting a potential role of these two molecules in canine ovarian carcinogenesis.

Pregnancy detection in mice using ultrasound

DIAGNOSTIC ultrasound has been used in almost all medical fields and is recognised as an increasingly important modality in a variety of clinical situations. The mouse is currently the most widely used animal in biomedical research (Marshall 2000). Diagnosis of pregnancy in experimental mice is possible at 12 to 14 days gestation, when abdominal distension is apparent. Mice with large litters may show distension slightly earlier (Kaufman 1989). Diagnosis of pregnancy at 7·5 days in experimental mice is currently performed by direct inspection of the uterine horns after laparotomy, with no routine utilisation of imaging techniques. The embryonic mouse is a ubiquitous model of mammalian development, due to the obvious benefits of a short gestation cycle, and genetic homologies with developmental genes in other mammals. Such models should yield further insight into the mechanisms responsible for human developmental and disease processes. Most previous investigations of the gestation period in the mouse have been invasive in nature. Video microscopy has been used to monitor heart dynamics in surgically exposed embryos, magnetic resonance microscopy has been successfully utilised to visualise threedimensional anatomy and vasculature in fixed mouse embryos (Smith and others 1994), and servo-null pressure measurement and Doppler interrogation using implanted crystals have also been used (Keller and others 1996, MacLennan and Keller 1999). However, none of the previous methods used have provided real-time imaging of live embryos as early as 7·5 days. The lack of effective in utero imaging methods has been a significant limitation. This short communication describes the capabilities of a conventional ultrasound system as a tool for the noninvasive investigation and diagnosis of early pregnancy in the mouse. All the animals in the present study were maintained according to protocols approved by the Institutional Animal Research and Care Committee, University of Naples. Fifty-one pregnant CD1 mice, ranging in age from seven to nine weeks, were used in the study. The mice were divided into three groups of 17 mice at three different stages of pregnancy (7·5 days, 12·5 days and 16·5 days). In staging the embryos, gestational day 0·5 was defined as noon of the day a vaginal plug was found following overnight mating. FIG 1: Longitudinal section of the gestational sac (arrows) and placenta (arrowheads) at 7·5 days. The image to the left shows the full urinary bladder

Diagnosis of XX/XY Blood Cell Chimerism at a Low Percentage in Horses

&NA; Disorders of sexual development (DSDs) are common in horses and cause economic loss in horse breeding. Thus, it is important to develop methods for unambiguous and fast identification of affected horses shortly after birth, as well as those that may propagate the condition to the next generation. Genetic causes of DSDs are multivarious and still little known, and thus development of diagnostic tests requires accumulating knowledge about individual cases and their etiologies. In particular, it is necessary to perform clinical, ultrasound, surgical, histological, cytogenetic, and genetic analyses with close attention in all the affected individuals. This report describes the case of a XX/XY chimeric horse with reproductive apparatus abnormalities and a very low percentage of XY cell in blood highlighting that to avoid undiagnosed case of cell chimeras, above all when studying DSD cases, it is essential to perform both genetic and cytogenetic analyses possibly on more than one tissue. HighlightsDisorders of sexual development (DSDs) are still a main cause of economic loss in horse breeding.Genetic and cytogenetic analyses on various tissues are essential in horse DSDs.DNA profiling on blood alone is not enough to detect all cases of cell chimerism.

Immunolocalization of NOS isoforms in the ductuli efferentes and epididymis of prepubertal and adult alpaca (Lama pacos)

The present research used immunohistochemistry to analyse the detection and localisation of nitric oxide synthase (NOS) isoforms in the ductuli efferentes and epididymis of prepubertal and adult alpaca. In the ductuli efferentes and epididymis of prepubertal and adult animals, nNOS and eNOS were similarly expressed in epithelial lining cells, conversely differences were observed in the immunopresence of iNOS. Our data provide evidence that NOS isoforms may have roles in reproductive functions and in the developmental processes of the excurrent duct system in the alpaca.