Developmental and hormonal regulation of specific proteins in mouse vas deferens and seminal vesicle.

This paper is concerned with hormonal regulation of the developmental pattern of major proteins of the mouse vas deferens (mouse vas deferens protein: MVDP, 34.5 kD) and seminal vesicle (15.5, 120 and 140 kD) whose expression is regulated by testosterone at adulthood. The ontogeny of these proteins, studied by SDS-polyacrylamide gel electrophoresis, appeared to be uncoordinated. MVDP was not accumulated until animals were 20 days old and its concentration increased sharply from 20 to 30 days of age. In seminal vesicle, the 15.5 kD protein did not accumulate before day 30 whereas 120 and 140 kD proteins appeared and accumulated between 30 and 40 days. In 30-day-old mice castrated at birth or treated with cyproterone acetate over 29 days, MVDP levels were not abolished and were similar to those measured in 20-day-old males. Testosterone administration, from 1 to 10 days of age, did not induce precocious expression of MVDP. These results suggest that the neonatal expression of MVDP is independent of androgens. In seminal vesicle, the first expression of the 3 proteins studied was dependent upon testicular androgens as shown by neonatal castration and injection experiments. The marked increase in the levels of the 4 proteins studied, during sexual maturation, was not associated with quantitative or qualitative changes in tissular androgen concentrations, suggesting that other factors may be necessary for protein expression. Whereas thyroxine may induce a precocious accumulation of MVDP, prolactin had no stimulatory effect on the accumulation of proteins from vas deferens and seminal vesicle. The results suggest that during sexual maturation gene activation by androgens was progressive.     

The occurrence of GABA in vas deferens, prostate, epididymis, seminal vesicle and testicle of the rat

The concentration of gamma-aminobutyric acid (GABA) was determined in vas deferens, prostate, epididymis, seminal vesicle and testicle of the adult rat. Among the organs examined, vas deferens was found to be the richest in GABA and the lowest concentration was measured in testicle. Although the GABA levels appear to be 10-50 times lower in the sex organs examined than in the brain tissue, even the low GABA contents are suggestive of a role of this amino acid in the reproductive organs of the male rat.     

Distribution and immunohistochemical characteristics of neurons in the porcine caudal mesenteric ganglion projecting to the vas deferens and seminal vesicle

Combined retrograde tracing (using fluorescent tracer Fast Blue) and double-labelling immunofluorescence were used to study the distribution and immunohistochemical characteristics of neurons in the porcine caudal mesenteric ganglion projecting to the vas deferens and seminal vesicle. The distribution and immunohistochemical properties of neurons projecting to both organs were similar. As revealed by retrograde tracing, Fast Blue-positive neurons were located within the left and right ganglia, with a distinct predominance in the ipsilateral one. In the ipsilateral ganglion, the majority of the neurons were located caudally, along the dorso-lateral ganglionic border, suggesting a somatotopic organization of the ganglion. Immunohistochemistry revealed four populations of retrogradely labelled neurons (from the largest to the smaller one): tyrosine hydroxylase-positive/neuropeptide Y-negative (TH⁺/NPY^-), TH⁺/NPY⁺, TH^-/NPY^-, TH^-/NPY⁺. With respect to their surrounding nerve fibres, two subpopulations of the dye-labelled neurons could be distinguished. The small one consisted of solitary neurons receiving a strong calcitonin gene-related peptide- and Leu⁵-enkephalin-, and a less intense vasoactive intestinal peptide-immunoreactive innervation. The remaining neurons were poorly supplied by singular nerve fibres containing some of the investigated peptides. We conclude that the caudal mesenteric ganglion should be considered as a prominent source of adrenergic and/or NPY-positive innervation for the porcine male reproductive tract.     

Coexistence and cooperation between neuropeptide Y and norepinephrine in nerve fibers of guinea pig vas deferens and seminal vesicle

Fluorescence immunocytochemistry of guinea pig vas deferens and seminal vesicle revealed dense networks of nerve fibers containing both neuropeptide Y (NPY) and dopamine-beta-hydroxylase (DBH), a marker for adrenergic neurons. The effects of norepinephrine (NE) and NPY on the smooth musculature of these organs were studied in vitro. NE inhibited the response to electrical nerve stimulation and increased the basic tension in the vas deferens and contracted the smooth muscle of the seminal vesicle, but had no effect on the contractile response to transmural stimulation in the latter organ. NPY had similar effects on the vas and vesicula, i.e. it inhibited the electrically induced contractions and had no effect on the basic tension. The results suggest a role for NPY as a transmitter that acts before the site of the neuromuscular junction to modulate the release of other transmitters from motor nerve fibers in the smooth musculature.     

Testosterone and dihydrotestosterone levels in epididymis, vas deferens, seminal vesicle and preputial gland of mice after hCG injection

Temporal changes of testosterone (T) and dihydrotestosterone (DHT) levels were measured by RIA in epididymis, vas deferens, seminal vesicle and preputial gland of adult male mice after a single injection of hCG. The response of circulating T to hCG stimulation was rapid and persisted over a period of 48 h. The temporal changes of androgen content of target organs paralleled the modifications of circulating T. In all organs the high androgen levels attained at 1 or 4 h plateaued until 24 h, decreased thereafter and returned to basal values at 72 h. The concentration of T by sex accessory organs was more accelerated by hCG injection than its conversion into DHT.     

Ascaris suum: Free amino acids and proteins in the pseudocoelom,seminal vesicle and glandular vas deferens

The free amino acids and proteins of the seminal vesicle and pseudocoelomic fluids in the male Ascaris suum were examined and compared. The seminal fluid contained a high concentration of lysine (lysine: glutamic acid ratio of 5:1) while the pseudocoelomic fluid contained more glutamic acid than lysine with alanine, serine, glycine and proline being the most abundant free amino acids. The proteins present in the seminal fluid differed from those in the pseudocoelomic fluid in both number and molecular weight. The sperm activating substance (SAS) present in homogenates of the glandular vas deferens of male worms is nondialyzable, heat-sensitive and can be precipitated using 45% saturated ammonium sulfate. Active moieties can be recovered following passage of the ammonium sulfate precipitates through ultrafiltration membranes or by applying gland homogenates to an ion exchange column. When subjected to SDS-polyacrylamide gel electrophoresis, the active fractions revealed both low and high molecular weight substances. During attempts to purify a single activating substance, it was noted that the more heterogeneous fractions contained the highest activating capacity. Thus, no precise relationship between the biological activity and the purity of the various fractions was determined.     

Immunohistochemical localization of prostaglandin H synthase in the epididymis and vas deferens of the mouse

Prostaglandins (PGE2, PGF2 alpha) in the excurrent ducts of the male reproductive tract appear to be both modulators of ductal contractility for transport of spermatozoa and factors involved in the regulation of sperm maturation. To identify the tissue sites for the production of prostaglandins (PGs) in the excurrent ductal system, we have employed an immunohistochemical technique to localize prostaglandin H (PGH) synthase in the epididymis and vas deferens of the mouse. A mouse monoclonal antibody to PGH synthase was used and was shown to be specific for the mouse enzyme by Western blot analysis. In sexually mature mice, PGH synthase was primarily localized to the epithelium of the epididymis and vas deferens. Within the epididymal epithelium, immunoactivity appeared in all cell types of the initial segment, in a subpopulation of cells with predominantly apically oriented nuclei in the caput and corpus, and in low levels in the cauda. PGH synthase reactivity was the most intense in the epithelial cells of the vas deferens. PGH synthase was not detected in smooth muscle cells, spermatozoa, or luminal fluid. This study suggests that the epithelium of the excurrent ductal system of the mouse is the major site for PG production. The regionalization of PGH synthase to cells in the epididymis thought to be involved in the absorption of luminal fluid suggests that PGs may play a role in fluid and ion transport.     

Identification of PDC-109-like protein(s) in buffalo seminal plasma

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.     

Immunohistochemical localization of substance P, vasoactive intestinal polypeptide and gastrin-releasing peptide in vas deferens and seminal vesicle, and the effect of these and eight other neuropeptides on resting tension and neurally evoked contractile activity

Immunohistochemical studies of the vas deferens and seminal vesicle of mouse, guinea-pig, and rabbit showed the presence of nerve fibres containing vasoactive intestinal polypeptide (VIP), substance P (SP), and gastrin-releasing peptide (GRP) supplying the smooth muscle layers as well as blood vessels. The nerve supply was better developed in the seminal vesicle than in the vas deferens. The motor activity of the vas deferens and seminal vesicle of the guinea-pig was studied in vitro. The vas deferens responded to transmural electrical stimulation with a twitch followed by a slow contraction. The twitch was blocked by guanethidine and tetrodotoxin, but not by atropine, propranolol, phenoxybenzamine, or fluphenazine. The slow contraction exhibited features of an alpha-receptor-mediated response. SP, physalaemin and eledoisin contracted the smooth muscle and also potentiated the twitch response to electrical nerve stimulation in a concentration-dependent manner. The SP blocking agent, (D-Pro2,D-Trp7,9)-SP, affected neither the resting tension nor the response to electrical stimulation. It is therefore suggested that the SP fibres act mainly prejunctionally. VIP, Leu-enkephalin, cholecystokinin octapeptide (CCK-8), angiotensin II, vasopressin, neurotensin, bombesin, and GRP had no effect on either the resting tension or the response to electrical nerve stimulation. The seminal vesicle responded to electrical stimulation with a contraction which was unimpaired by atropine, propranolol, phenoxybenzamine, and guanethidine, but abolished by tetrodotoxin. Hence, this contraction is mediated by a non-adrenergic, non-cholinergic neurotransmitter. Bombesin, GRP, SP, physalaemin and eledoisin contracted the smooth muscle and potentiated the response to electrical stimulation. VIP, Leu-enkephalin, CCK-8, angiotensin II, vasopressin, and neurotensin had no effect on the resting tension or on the response to transmural electrical stimulation. The SP antagonist abolished the contraction elicited by SP but did not influence the response to nerve stimulation. The results suggest that the SP and GRP nerves may have prejunctional and facilitating postjunctional effects in the seminal vesicle.     

Immunohistochemical demonstration of -(carboxymethyl)lysine protein adducts in normal and osteoarthritic cartilage

N(epsilon)-(carboxymethyl)lysine (CML) is an advanced glycation end product formed by non-enzymatic glycation and oxidation of proteins. The distribution pattern of CML-modified proteins in normal and osteoarthritic (OA) cartilage was investigated using specific antibodies. In healthy articular cartilage, immunoreactivity for CML was preferably found in the extracellular matrix (ECM) of the superficial layer. In OA samples, CML immunoreactivity was not restricted to the ECM of the superficial layer. Interestingly, OA chondrocytes showed a remarkable cytoplasmic immunoreactivity for CML. With the help of a western blot analysis CML-modified proteins between 68 and 39 kDa could be demonstrated in OA cartilage samples. These results suggest that the accumulation of CML adducts contributes to the matrix damage in osteoarthritis. Therefore, the inhibition of CML accumulation may represent an effective therapeutic strategy to prevent severe OA cartilage injury.     

Physiological functions and hormonal regulation of mouse vas deferens protein (AKR1B7) in steroidogenic tissues

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) highly expressed in vas deferens epithelium and zona fasciculata of the adrenal cortex. Recombinant MVDP showed kinetic properties distinct from those of aldose reductase, including its spectrum of substrates, cofactor preference and sensitivity to inhibitors. We demonstrate that in adrenocortical cells, MVDP, rather than aldose reductase, is the principal reductase for isocaproaldehyde (a product of side-chain cleavage of cholesterol) and 4-hydroxynonenal (a lipid peroxidation product). In steroidogenic tissues MVDP expression is regulated by pituitary trophic hormones, namely ACTH in adrenals, FSH in ovaries, and LH in testicular Leydig cells.     

Physiological functions and hormonal regulation of mouse vas deferens protein (AKR1B7) in steroidogenic tissues

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) highly expressed in vas deferens epithelium and zona fasciculata of the adrenal cortex. Recombinant MVDP showed kinetic properties distinct from those of aldose reductase, including its spectrum of substrates, cofactor preference and sensitivity to inhibitors. We demonstrate that in adrenocortical cells, MVDP, rather than aldose reductase, is the principal reductase for isocaproaldehyde (a product of side-chain cleavage of cholesterol) and 4-hydroxynonenal (a lipid peroxidation product). In steroidogenic tissues MVDP expression is regulated by pituitary trophic hormones, namely ACTH in adrenals, FSH in ovaries, and LH in testicular Leydig cells.     

Localisation of pancreatic polypeptide (PP)-like immunoreactive material in neurones of the brain of the blowfly,Calliphora erythrocephala (Diptera)

Summary The brain of the blowfly, Calliphora erythrocephala, has been studied by means of the peroxidase-antiperoxidase immunocytochemical method, with the use of antibodies to bovine pancreatic polypeptide (BPP). A number of immunoreactive neurones have been localised, some corresponding to neurones previously identified tentatively as neurosecretory. This finding is further evidence that biologically active peptides, previously considered to be vertebrate, also exist in invertebrates. It also supports the concept of their evolutionary origin in nervous tissue.     

Light and electron microscopic studies on the seminal secretions and the vas deferens of the penaeiodean shrimp,Sicyonia ingentis

Unlike the other penaeiodean shrimp, the ridge back shrimp, Sicyoniaingentis does not produce a spermatophore, but transfers sperm suspended in seminal plasm. This paper reports on the histomorphology and ultrastructure of the vas deferens with reference to its functional role in secreting the sperm bearing materials. The vas deferens is divisible into proximal secretory, mid storage and distal ejaculatory regions. The epithelial cells lining the proximal vas deferens are comprised of secretory and absorptive cell types. The loose sperm cells found in the lumen of this region are in an immature condition, and are agglutinated into a compact mass with signs of spermiogenesis in the mid vas deferens. The epithelial cells lining the mid vas deferens are short flattened cells. The distal vas deferens is ensheathed by muscular fibres. The inner epithelial cells are highly secretory and contain numerous microvilli at the luminal end. The sperm cord gets liquefied in this region facilitating the transfer of sperm in liquid form to the female during mating.     

Regional distribution of dopamine, 5-hydroxytryptamine,and noradrenaline in the rat vas deferens

The concentrations of dopamine (DA), 5-hydroxytryptamine (5-HT) and noradrenaline (NA) in the rat vas deferens divided in eight or four sections were determined by high performance liquid chromatography with electrochemical detection. Dopamine and NA had the same regional distribution; their concentrations were maximal near the prostatic end and decreased towards the epididymis. The concentration of 5-HT also decreased from the prostatic to the epididimal end, but 5-HT did not follow the same regional distribution as DA and NA. Reserpine (0.02 or 0.2 mg/kg, i.p., 24 hr) and 6-hydroxydopamine (2×80 mg/kg, i.v., 6 days) decreased the contents of DA and NA; the concentrations of both amines were modified to a similar extent. Reserpine also diminished the content of 5-HT. Pargyline (200 mg/kg, i.p., 2 hr) increased the concentration of 5-HT whilep-chlorophenylalanine (300 mg/kg, oral, 3 days) decreased the contents of the amine in some sections of the vas deferens. This study suggests that DA and NA co-exist in the same sympathetic neurons. Some of the 5-HT could be stored in mast cells as previously proposed, but the finding that tissue content of 5-HT changes after inhibiting the deamination or synthesis of the amine suggests that other source(s) of 5-HT distinct from mast cells exist in the rat vas deferens.