Immunohistochemical study of galectin-3 in mature and immature bull testis and epididymis

Galectin-3, a member of the β-galactoside-binding protein family, has been implicated in mammalian sperm maturation. We examined galectin-3 expression in the testis and epididymis of sexually mature and immature bulls. Western blot analysis showed varying levels of galectin-3 in the bull testis and epididymis, and galectin-3 immunoreactivity was higher in the mature testis and epididymis than in immature organs. Galectin-3 was primarily localized in interstitial cells of the immature bull testis and in the peritubular myoid and interstitial cells of the mature testis. In the immature epididymis head, galectin-3 was primarily in the principal and basal cells of the epithelium. In the mature epididymis head, moderate levels of galectin-3 were detected in the sperm, while low levels were found in the stereocilia, epithelium and connective tissue. In the immature epididymis body, moderate protein levels were detected in the principal cells, while lower levels were found in the basal cells. The mature epididymis body showed moderate levels of galectin-3 immunostaining in the stereocilia and epithelium, but low levels in the connective tissue. In the immature epididymis tail, only low levels of galectin-3 staining were found in the epithelium, whereas the mature epididymis tail showed high levels of galectin-3 in the principal cells, moderate levels in the basal cells and low levels in connective tissue. These findings suggest that galectin-3 expression plays a role in the maturation and activation of sperm in bulls.     

The expression and localization of inhibin isotypes in mouse testis during postnatal development

Inhibin, which is important for normal gonadal function, acts on the pituitary gonadotropins to suppress follicle-stimulating hormone (FSH) secretion. The level and cellular localization of the inhibin isotypes, α, β_A and β_B, in the testis of mice were examined during postnatal development in order to determine if inhibin expression is related to testicular maturation. Mouse testes were sampled on postnatal days (PNDs) 1, 3, 6, 18, 48 and 120, and analyzed by Western blotting and immunofluorescence. Western blot analysis showed very low levels of inhibin α, β_A and β_B expression in the testes at days 1 to 6 after birth. The levels then increased gradually from PND 18 to 48-120, and there were significant peaks at PND 48. Inhibin α, β_A and β_B were detected in testicular cells during postnatal development using immunohistochemistry. The immunoreactivity of inhibin α was rarely observed in testicular cells during PND 1 to 6, or in the cytoplasmic process of Sertoli cells surrounding the germ cells and interstitial cells during PND 18 to 120. Inhibin β_A and β_B immunoreactivity was rarely observed in the testis from PND 1 to 6. On the other hand, it was observed in some spermatogonial cells, as well as in the interstitial space between PND 48 and PND 120. We conclude that the expression of inhibin isotypes increases progressively in the testis of mice with increasing postnatal age, suggesting that inhibin is associated with a negative feedback signal for FSH in testicular maturation.     

Immunohistochemical localization of steroidogenic enzymes in the testis of Hokkaido Sika deer (Cervus nippon yesoensis)

The testes from 15 adult male Hokkaido Sika deer (Cervus nippon yesoensis) were collected during the rutting season (October and November). We investigated the localization of 4 kinds of steroidogenic enzymes (P450scc, 3betaHSD, P450c17 and P450arom) immunohistochemically in these testicular samples. The specific immunoreactivities to these enzymes were detected only in the cytoplasm of Leydig cells. This differs to the enzyme distributions reported previously in Japanese black bear, Japanese raccoon dog, Hokkaido brown bear and American black bear, in which the same immunoreactivities were detected in Leydig cells, Sertoli cells and/or spermatogenic cells. The current study suggests that in the testes of the Hokkaido Sika deer, testosterone and estradiol-17beta may be synthesized in the Leydig cells only.     

Histochemical localization of inhibin and activin alpha, beta A and beta B subunits in rat gonads.

The localization of alpha, beta A and beta B subunits of inhibin/activin polypeptides was studied in the ovary and testis of sexually mature, immature, and embryonic rats. Specific staining with these three subunits was also evident in the oocytes from embryonic to mature female rats. This result suggests that inhibin- and activin-like substances may be produced in the oocytes and these substances may play a role in the oocyte growth and differentiation. Judging from the intensity of immunoreaction in mature female rats, the three subunits should be produced more abundantly in luteal cells than in the granulosa cells. Immunoreactive alpha, beta A and beta B subunits were observed in the cummulus oophorous in the morning (11:00), but not in the evening (23:00) on proestrus. The results are in well agreement with the previous report that inhibin alpha and beta A subunit mRNA signals decline on proestrus evening. It is supposed that the cyclic change may be related with physiological phenomena prior to the ovulation, such as primary gonadotropin surges, loss of cummulus-oocyte gap junctions, or germinal vesicle breakdown. In both germ cells and Sertoli cells of the testis, alpha, beta A and beta B subunits were more abundant in the embryonic rat than in the mature rat. Although clear reactions with beta A and beta B subunits were detected in Leydig cells, alpha subunit was not detectable in the cells throughout the developmental stages examined.     

Tubular ectasia of the rete testis in an Angus bull

An 18-month-old Angus bull presented to Auburn University College of Veterinary Medicine for a routine breeding soundness evaluation and lameness evaluation. He was classified as deferred potential breeder due to a lameness and was donated to the university. Following treatment, the bull's lameness resolved. He passed the breeding soundness examination in accordance with the Society for Theriogenology standards. However, avascular dilated areas at the level of the mediastinum testis of the right testicle were detected via Doppler ultrasonography. A high level of vascularity is routinely seen with neoplasia, such as teratomas. Due to the lack of vascularity, a presumptive diagnosis of tubular ectasia of the rete testis was made. The bull was castrated. The right testicle was submitted for histopathology revealing a definitive diagnosis of tubular ectasia of the rete testis.     

Fibrolipoma of a cryptorchid testis in a young bull

A six-month-old Japanese Black bull was found to have no left testis in the scrotum. A fist-sized mass was palpated per rectum. Two months later, hCG was injected and blood samples were collected before and after injection. No testosterone response to hCG was observed. On the cut surface of the excised mass, most of the mass was composed of homogeneous adipose-like tissue. The rest of the surface was composed of a well-circumscribed testicular parenchyma-like tissue (18 × 16 × 15 mm). Histology revealed diffusely distributed mature adipocytes and septa of fibrous connective tissue. Neither germ cells nor spermatozoa were observed in the seminiferous tubules. A diagnosis was made of fibrolipoma and aspermatogenesis of the left cryptorchid testis.     

Immunohistochemical study of osteopontin in boar testis

The expression of osteopontin (OPN) in boar testis was studied. Western blot analysis detected 66- and 32-kDa OPN immunopositive bands in the testes of adult boars. In postnatal piglets, the 66-kDa OPN band was detected in the testes, but not the 32-kDa band. In the newborn testis, OPN immunostaining was seen in gonocytes and in some supporting cells in the seminiferous tubules, as well as in interstitial Leydig cells. In the adult boar testis, OPN immunoreactivity was detected in seminiferous tubules with varying intensities. Intense OPN immunostaining was seen in the residual bodies and acrosomes in the spermatids while, occasionally, OPN immunostaining was seen in spermatogonia and various stage of spermatocytes but in few Sertoli cells in the seminiferous tubules. In addition, Leydig cells in adult boars were weakly immunostained with OPN. These findings suggest that OPN is detected in the majority of germ cells and is involved in spermatogenesis in boar testis.     

Tubule length and Leydig cell volume in the normal bull testis.

Data are presented on the size, Leydig cell content and seminiferous tubule dimensions of the normal bull testis. A method of estimating total tubule length, requiring only the measurement of testis volume and the number of tubule cross-sections in a unit area is described, and its applicability to the bull established. The average bull testis contains about 5.2 km of tubules.     

Expression of inhibin/activin subunits in the ovaries of fetal and neonatal mice

In the present study, the expression of inhibin/activin subunits in the mouse ovary from 13 days post-coitus (dpc) to 30 days postpartum (dpp) was investigated. Circulating FSH, LH, inhibin A, and inhibin B in neonatal to 30 dpp ovaries were measured. Inhibin/activin subunits (alpha, beta(A), beta(B) ) were weakly stained in 13 dpc ovarian stromal cells and increased with age. Inhibin alpha subunit was immunolocalized in follicular granulosa cells at each developmental stage. In 30 dpp ovaries, several large antral follicles were strongly stained for inhibin alpha subunit. Inhibin beta(A) subunit was weakly immunolocalized in granulosa cells until 20 dpp. Moreover, 2 to 3 antral follicles from 20 to 30 dpp were strongly stained for inhibin beta(A) subunit. There was relatively high immunoactivity for inhibin beta(B) subunit in neonatal to 30 dpp mouse ovaries. All three inhibin subunits were stained in theca-interstitial cells from 15 dpp onward. RIA data showed that a temporal increase in circulating FSH occurred around 10 dpp, while the plasma concentrations of LH were sustained at a relatively higher level from 8 to 15 dpp. Inhibin B was detectable in circulation early at 1 dpp (day of birth), and a clear increase in inhibin B occurred around 8 dpp. Circulating inhibin B gradually increased from 20 dpp to 30 dpp, indicating a negative correlation with FSH. Inhibin A levels were only measured on 25 and 30 dpp, and the levels were low. These results suggest that inhibins play an important role in early folliculogenesis in mice. In addition, inhibin B seems to be the main functional isoform from the neonatal to prepubertal stage in the mouse ovary.     

Immunohistochemical localization of neuropeptides in bovine pancreas

The occurrence and density of distribution of nerves and endocrine cells that are immunoreactive for neuropeptides in the bovine pancreas were studied by immunohistochemistry. The six neuropeptides localized were galanin (GAL), substance P (SP), methionine-enkephalin (MENK), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP). The exocrine pancreas was shown to have an appreciable number of GAL- and SP-immunoreactive nerve fibres but few fibres showing immunoreactivity for VIP and CGRP. Numerous MENK-, GAL-, SP-, and NPY-immunoreactive nerve fibres were seen in the endocrine portion of the pancreas. Nerve cell bodies in the intrapancreatic ganglia showed immunoreactivity for all of the neuropeptides except CGRP. Endocrine cells showing immunoreactivity for GAL and SP were observed in the large islets and islets of Langerhans, respectively. The present results indicate a characteristic distribution of neuropeptides in the bovine pancreas, which may regulate both exocrine and endocrine secretions of pancreas.     

Immunohistochemical localization of haptoglobin in porcine lungs

The extravasation of erythrocytes into the lower respiratory tract occurs in numerous lung injuries and may lead to oxidative damages in lung tissues. Haptoglobin (Hp), the major haemoglobin-binding protein, is known to reduce lung injury associated with exposure to blood in mice. In pigs, Hp is a major acute phase protein and its serum concentrations are elevated in various infections of the respiratory tract. However, information on the porcine Hp response towards inflammatory stimuli is restricted to blood. We herein investigated the presence of Hp in lung tissues from pigs with acute and chronic bronchopneumonia via immunohistochemistry. Hp was localized in airway epithelial cells and immigrated leucocytes whereas in alveolar epithelial cells there was no distinct signal. Unaltered lungs showed less Hp-positive cells compared with lungs from pigs with acute or chronic bronchopneumonia.     

Production of inhibin A and inhibin B in boars: changes in testicular and circulating levels of dimeric inhibins and characterization of inhibin forms during testis growth

We investigated the production of inhibin in boars from the infantile to pubertal periods by: (1) measurement of testicular and circulating levels of inhibin, (2) characterization of inhibin forms and (3) localization of inhibin subunits in the testis. Total inhibin levels in the testis increased until 8 weeks of age but then declined to much lower values at 15 weeks. Testicular inhibin A and inhibin B were high until 8 weeks. Circulating levels of total inhibin and inhibin A were also high until 8 weeks, then declined from 10 weeks; inhibin B was not detected, because of low sensitivity of the inhibin B assay. Analyses of inhibin A and inhibin B levels in the eluted fractions obtained from testes after immunoaffinity chromatography and SDS-PAGE showed the presence of a peak of approximately 45 kDa until 10 weeks of age. As the boars aged, the levels of inhibin A and inhibin B increased in the molecular weight region of 29–31 kDa. The fractions corresponding to 29 and 30 kDa suppressed FSH release from rat pituitary cells, but the 45 kDa fraction had no FSH-suppressing activity. Total amounts of inhibin A isolated from the SDS gels were similar to those of inhibin B until 10 weeks of age, but were three times higher than those of inhibin B between 15 and 25 weeks. Further fractionation by reverse phase high-performance liquid chromatography revealed that the 29–31 kDa immunoreactive material was composed of mature forms of inhibin A and inhibin B, in addition to a 26 kDa monomer. Immunohistochemistry indicated that positive immunostaining for the subunits was observed in Sertoli cells from the infantile to pubertal periods. Elongated spermatids also showed positive signals at age 25 weeks. These results clearly indicated that: (1) the boar testis has the ability to produce inhibin A and inhibin B during the infantile period but inhibin A is the predominant form towards puberty and (2) the molecular weight forms of inhibin and the sites of production of inhibin change with testicular development.     

Detection and localization of two constitutive NOS isoforms in bull spermatozoa

Bull spermatozoa were examined for the presence and localization of constitutive Nitric Oxide Synthase (NOS), as nitric oxide (NO) is involved in calcium-dependent capacitation. In bull spermatozoa, NO generation is enhanced by l-arginine (3 microm) and abolished by the NOS-inhibitor N-nitro-l-arginine methyl ester (l-NAME). In addition, presence of NOS in bull spermatozoa was verified by immunohistochemistry, revealing the existence of both neuronal NOS (nNOS) and endothelial NOS (eNOS) immunoreaction. These findings were confirmed by Western blot technique, showing immunoreactive bands at 161 kDa (nNOS) and 133 kDa (eNOS). Confocal laser microscopy localized nNOS related immunofluorescence at the acrosome cap of sperms and their flagellum-mainpart. This technique also identified eNOS staining spread over the spermatozoan head. In conclusion, immunohistochemistry, Western blot technique, and NO generation suggest the presence of n- and eNOS in bull spermatozoa.     

Immunohistochemical localization of inhibin and steroidogenic enzymes in the ovary of common tree shrew (Tupaia glis) and northern smooth-tailed tree shrew (Dendrogale murina)

To study the ovarian function of the Order Scandentia, the localization of inhibin and steroidogenic enzymes (3 beta-hydroxysteroid dehydrogenase/isomerase and aromatase) in the ovaries of common tree shrew (Tupaia glis) and northern smooth-tailed tree shrew (Dendrogale murina) was immunohistochemically analysed. As in the results reported for other mammals, inhibin alpha-chain was localized in the follicular epithelium of secondary or Graafian follicles in the two species. The localization of aromatase in the ovary of these two species, however, was different. In the common tree shrew, the aromatase was localized in the thecal cells, whilst in other mammals it is localized in the granulosa cells. These results indicate that in the ovary of the common tree shrew, the oestradiol may be synthesized in the thecal cells.     

Immunohistochemical localization of S-100 protein in the pig pituitary gland

Immunohistochemical localization of S-100 protein was studied in anterior, intermediate and posterior lobe of the pig pituitary gland. Two immunopositive cells for S-100 protein were identified: the folliculo-stellate cells (FSc) in the glandular lobes and the pituicytes in the neural lobe. In the anterior lobe, immunoreactive folliculo-stellate cells were scattered among secretory cells. In the area where the secretory cells form strands and follicle-like groups the positive cells were concentrated in groups. In the intermediate lobe, S-100 protein-positive cells were located sparsely among secretory cells and next to secretory follicles and the pituitary cleft. These FSc were more voluminous and displayed fewer cytoplasmic processes. In the neurohypophysis, positive reaction for S-100 protein was seen in the pituicytes. These cells were distributed singly or concentrated in groups. The distribution, and morphologic characteristics of the FSc in the glandular lobes and the pituicytes in the neural lobe in the pig indicate different origin of both S-100 protein-positive cells.     

Immunolocalization of inhibin/activin subunits in the shiba goat fetal, neonatal, and adult testes

The objective of this study was to investigate the cellular immunolocalization of inhibin alpha and inhibin/activin (betaA and betaB) subunits in the fetal, neonatal and adult testes of Shiba goats. The testes were obtained from a fetus at 90 days, a neonate at 15 days, and two adult Shiba goats (both of 3 years old). The sections of testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA, and inhibin/activin betaB. Inhibin alpha and inhibin/activin (betaA and betaB) subunits were expressed in Leydig cells, but not in the Sertoli cells of the fetus with a weak immunostaining. An increase in the number of positive cells and a more intense immunohistochemical signal for inhibin alpha and inhibin/activin (betaA and betaB) subunits were observed in the Leydig cells of neonatal testes. Moreover, inhibin alpha, betaA, and betaB subunits were expressed in the Sertoli cells and Leydig cells of adult testes, respectively. These results suggest that Shiba goats testes have the ability to synthesize inhibins in the fetus, neonate, and adult, and the cellular localization of inhibin/activin subunits showed age-related changes in fetal, neonatal, and adult testes of Shiba goats.