Expression of a hepatocyte growth-factor activator protein in turkey (Meleagris gallopavo) deferent duct epithelial cells.

In previous research, we discovered that turkey deferent duct epithelial cells express a serine protease. Our experimental objective was to identify the gene that encodes this protein. A lambda phage cDNA library from duct cell mRNA was constructed. The library was screened using monoclonal antibodies previously produced against the turkey deferent-duct serine protease. Phage containing the protease cDNA was excised and re-circularized into plasmids. E. coli were transformed with plasmids containing protease cDNA, which was then isolated for sequencing. NCBI BLAST searches within the GenBank database returned 63.5 and 61.7% identity with murine and human hepatocyte growth-factor activator (HGFA) precursor, respectively. The turkey protease cDNA was then cloned into the pQE-32 expression vector and transformed into M15 cells for HIS-tagged expression of the recombinant protein, which was then purified using nickel-chelated Sepharose spin columns. Afterwards, Western blot analysis of the purified recombinant turkey protein revealed recognition by a monoclonal antibody specific to the proteolytic subunit of the turkey deferent duct protease. Therefore, these findings indicate that the recombinant HGFA precursor isolated from the deferent duct is the turkey seminal plasma protease that is secreted from the deferent duct. HGFA, a member of the Kringle-serine proteinase superfamily, can initiate diverse mitogenic, morphogenic and motogenic effects through its substrate hepatocyte growth factor. Although the presence of hepatocyte growth factor and its c-MET receptor have been reported in male mammalian reproductive tracts, our novel findings on the secretion of HGFA precursor from turkeys may help to elucidate the regulation of activated hepatocyte growth factor.

Localization of a proteolytic enzyme within the efferent and deferent duct epithelial cells of the turkey (Meleagris gallopavo) using immunohistochemistry.

Turkey seminal plasma contains a serine protease found to be distinct from the spermatozoal acrosin. However, the origin and biological roles of this enzyme are unknown. Our experimental objective was to identify the cellular source of this protease within the male reproductive tract. The enzyme was isolated from seminal plasma using benzamidine-Sepharose 6B chromatography. A synthetic substrate, Nalpha-benzoyl-DL-arginine p-nitroanilide, was used to detect fractions containing the enzyme. The affinity chromatography technique yielded a 150-fold increase in amidase activity. Analysis of the protease by SDS-PAGE revealed two protein bands with relative molecular masses of 37 000 and 61 000. Proteolytic activity was detected within the smaller band as evidenced by casein digestion. Further analysis of the purified protein revealed that the smaller protein band was glycosylated. To determine the cellular source of the protease, a panel of mouse monoclonal antibodies was then developed against the purified protease, and used in immunohistochemistry. Frozen tissue sections from the liver, testis, epididymal region, and deferent duct were fixed in 4% (w/v) paraformaldehyde, permeabilized with 0.2% (v/v) (octylphenoxy)polyethoxyethanol followed by routine immunohistochemistry procedures. Monoclonal antibodies did not bind to tissue sections from either the liver or testis, or to blood plasma proteins. Both the distal portion of the efferent duct and the deferent duct were immunoreactive. We concluded that the protease found in turkey seminal plasma is concentrated to the distal efferent duct and the deferent duct epithelial cells.

Spermiogenesis in commercial poultry species: anatomy and control.

Spermatogenesis is a complicated process dependent upon several factors. Formation of a testis requires the interaction of gene-products and hormones (androgens) on pluripotent tissue. In birds, the female is the heterogametic (ZW) sex, but W chromosomal genes do not influence gonadal development in a way similar to the SRY gene on the mammalian Y chromosome. However, autosomal genes such as SRY-like HMG box gene 9 (SOX9) may influence gonadal development. Hormones affect development; male gonads subjected to estrogen form an ovotestis, whereas ovaries exposed to aromatase inhibitors form an atypical testis. Sertoli cell numbers are set early in spermiogenesis, possibly under the influence of follicle-stimulating hormone and thyroid hormone, and this may determine the number of gonial cells that can be supported. Sertoli cells make a number of substances that affect testicular development and function, particularly anti-Müllerian hormone, which inhibits female oviduct formation from the Müllerian anlage, inhibits aromatase activity to stop estrogen production, and possibly stimulates androgen production by Leydig cells. Undifferentiated primordial germ cells (PGC) migrate to the testis and are converted to spermatogonia by factors from gonadal ridge tissue and androgens. The PGC of males in the ovary form oocytes of Z genotype, whereas the female PGC in males form mostly Z sperm (with a few of W genotype). Transmission electron microscopy micrographs of turkey testis are presented, and control of spermatogenesis by hormones and cytokines is discussed. This discussion includes follicle-stimulating hormone, luteinizing hormone, inhibin, activin, follistatin, tumor necrosis factor-alpha, growth factors such as transforming growth factor-beta, interleukins, and interferon. Although information concerning paracrine and autocrine regulation of the avian testis by these substances is sparse, much can be learned from mammalian studies, in which putative roles of each of these substances have been established. How Sertoli cells cause directed apoptosis of spermatogonia using the Fas-ligand, Fas-receptor pathway is reviewed, as well as ways to circumvent this process. A possible role for ubiquitin concerning prevention of heat-induced damage to the testis is presented.

Characterization of turkey spermiophages with regard to traits common to macrophages.

Turkey peritoneal exudate cells (PEC) and spermiophages (SMO) were assayed for characteristics of macrophages. The PEC elicited by i.p. injection of 3% Sephadex and SMO isolated from semen using Percoll were cultured in Dulbecco's Modified Eagle Medium supplemented with 20% bovine calf serum (DMEM-20) for 24 h at 41 C in 5% CO2 to provide adherent cells for assays. Most PEC and SMO showed esterase activity (99.3 +/- 0.6 and 98.8 +/- 0.9%, respectively), and exhibited nonspecific phagocytosis of carbon (89.5 +/- 3.6 and 95.3 +/- 0.6%, respectively), zymosan (26.5 +/- 7.6 and 24.3 +/- 2.5%, respectively), bacteria (11.3 +/- 0.8 and 9.3 +/- 0.3%, respectively), and opsonized and nonopsonized SRBC. Maximum uptake of SRBC was seen by 2 h for PEC but not until 4 h for SMO. At time of maximum uptake, SRBC were noted in 35 to 40% of PEC but only in 15 to 20% of SMO. Turkey IgG-FITC bound to both PEC and SMO, but goat anti-turkey IgG-FITC bound only to SMO. Increased nitrite was found in turkey semen after 24 h storage, with highest levels in samples in which SMO were added. Nitrite production was demonstrated using adhered PEC, but SMO could not be tested due to low cell numbers. This research clearly identifies SMO as having macrophage-like activities. Accordingly, these cells may possess the ability to process and present antigen via histocompatibility receptors. Such activity could lead to immune directed responses, including antibody production or activation of cytotoxic T-lymphocytes.

Effect of added spermiophages in pooled turkey semen on fertility, embryonic mortality, and hatchability.

The semen of turkeys with numerous spermiophages was used for isolating spermiophages by density gradient centrifugation. Isolated spermiophages were suspended in Beltsville Poultry Semen Extender (BPSE) and added to semen with low spermiophage numbers to give approximate spermiophage concentrations of: 2 x 10(5)/mL (medium) and 10(6) (high). Semen with no added spermiophages was the control. Samples were diluted to 1:1 with BPSE, and for each spermiophage level (treatment), semen aliquots were either immediately inseminated or stored 6 h at 4 C with agitation (150 rpm) before insemination. Hens were inseminated weekly, and fertility, embryonic mortality, and hatchability of eggs were determined for a 10-wk period. The experiment was performed twice. In Trial 1, there were no differences in fertility between treatments except that fertility for control stored semen was lower (P < or = 0.05) than that for fresh semen (89.27 vs 95.97, respectively; SEM = 2.2). Neither hatchability nor embryonic mortality was affected by spermiophage level in Trial 1. Spermiophages did not affect fertility in Trial 2; however, hatchability for unstored treatments with added spermiophages was significantly lower than for the control. For stored semen, hatchability was significantly (P < or = 0.05) greater for treatments with added spermiophages than for the control. Differences in embryonic mortality in Trial 2 did not relate to adding spermiophages to the semen. No clearly defined detrimental effect of seminal spermiophages was shown in the present experiments.

Morphological observations of turkey (Meleagris gallopavo) spermiophages maintained in tissue culture.

Spermiophages were isolated from turkey semen using Percoll gradient centrifugation, cultured in Roswell Park Memorial Institute 1640 medium at room temperature, and characterized by transmission electron microscopy. After 1 to 2 h, the cells enlarged and developed numerous motile mitochondria. Over time, the mitochondria appeared to increase in number and were released into the extracellular medium. Few mitochondria were observed in spermiophages from fresh semen. However, there was an apparent increase in the number and size of mitochondria after Percoll isolation, which was more pronounced in cultured spermiophages. Over a period of 3 h in culture, many spermiophages became engorged with mitochondria, which subsequently appeared to be released as blebs pinched off from the surface. The release of mitochondria resulted in spermiophages with large, empty vacuoles, although their remaining cytoplasm was engorged with mitochondria. Many free mitochondria were present in the medium. The results of the current research suggest that isolating and culturing turkey spermiophages elicit mitochondrial biogenesis, which proceeds unabated until the cells are engorged with and release numerous mitochondria. This may be due to conditions under which the spermiophages were cultured or to nonhistocompatibility of these cells in pooled semen.

Frequency and structure of macrophages and abnormal sperm cells in guinea fowl semen.

Semen from young and old guinea fowl was examined for macrophages and abnormal sperm cells. Large numbers of macrophages were found in the semen from both groups. However, there were significantly more abnormal sperm cells in the semen of the young males. The abnormal cells consisted of large coiled cells, bent spermatozoa, and round-headed cells. The predominant abnormality in the young males was bent sperm, although their semen also contained large round cells. The large round cells were identified as abnormal spermatids by electron microscopy and were more prominent in the older males. The abnormal spermatids were pleomorphic and exhibited various stages of intermediate and late spermatid development. The macrophages were activated cells that exhibited phagocytosis of large number of normal spermatozoa but were never observed to engulf abnormal cells. Their ultrastructure consisted of numerous lipid droplets, vesicles of ingested spermatozoa, lysosomal structures, residual bodies, and undigested remnants of spermatozoa. The presence of macrophages and abnormal spermatids in semen has been associated with lowered fertility in other species; thus, semen with exorbitant numbers of these cells should not be used for artificial insemination of the guinea fowl.

Morphology of the epididymal region and ductus deferens of the turkey (Meleagris gallopavo).

The ductal system of the reproductive tract of the male domestic turkey was studied by gross dissection and light microscopy of paraffin and Epon embedded tissues. The succession of ductules as one passes caudally from the testis was as follows: seminiferous tubules; rete testis; ductuli efferentes; connecting ductules; ductus epididymidis; ductus deferens; receptaculum ductus deferentis; papilla ductus deferentis. Non-ciliated cells of the male tract consisted of squamous and low cuboidal cells of the rete testis, granulated columnar cells lining the ductuli efferentes and connective ductules; agranulated columnar cells which formed the epithelium of the ductus epididymidis, ductus deferens, receptaculum and papilla ductus deferentis; and basal cells which were found in increasing number from the ductuli efferentes to the papilla. The basal cells had a reduced amount of cytoplasm and stained more intensely than the other cell types. Ciliated cells were apparent in the ductuli efferentes and connecting ductules, and these consistently stained lighter than the non-ciliated cells. Non-ciliated columnar cells of the ductuli efferentes and connecting ductules contained chromatophilic granules. Cytoplasmic blebbing into the ductal lumina was found associated with these non-ciliated cells as well as the agranular cells of the ductus epididymidis and deferens. Evidence obtained from this study suggests that the non-ciliated cells of the ductuli efferentes, ductus epididymidis and ductus deferens have a contribution to make to the seminal plasma by apocrine secretion.

Ultrastructural studies of semen abnormalities and Herpesvirus associated with cultured testicular cells from domestic turkeys.

Abnormal cells and macrophages found in white and yellow turkey semen were studied by electron microscopy. Yellow semen contained many abnormal cells, most of which were large and round or smaller and ellipsoidal. It was concluded that they were aberrant spermatids, with differentiation being more complete in the smaller cells. Only a few cells of the smaller type were detected in normal white semen. Macrophages were occasionally seen in white semen but were numerous in yellow semen. Phagocytic vacuoles of these cells contained structural elements of spermatozoa and abnormal spermatids. Virus particles were not detected in any of the seminal cells observed. Ultrastructure studies of cultured testicular cells obtained from several of the turkeys examined showed the presence of intranuclear Herpesvirus particles in germinal cells. Macrophages from the testicular cultures seldom were seen with intranuclear Herpesvirus, although these cells commonly were found with Herpesvirus particles and cellular debris contained within phagocytic vacuoles.