Ultrastructural Studies of Acrosomal Formation in the Testis of Male Greater Cane Rat (Thryonomys swinderianus).

PURPOSE: In furthering the understanding of the process of spermatogenesis in the greater cane rat, this study describes the ultrastructural spermiogenic transformation and acrosomal formation in the testes of this hystricomorphic rodent that is currently undergoing domestication in parts of West Africa. MATERIALS AND METHODS: Testicular samples were obtained from ten sexually mature cane rats that were perfused-fixed using Karnovsky's fixative (phosphate buffered 2% paraformaldehyde - 2.5% glutaraldehyde fixative at pH 7.4). The samples were processed for ultrastructural analysis and examined under the transmission electron microscope. RESULTS: The testes of the cane rat showed uniqueness in its cellular associations and the ultrastructure of the spermatogenic cells especially in the formation of the acrosome. The spermatid differentiation and acrosomal formation occurred in 12 steps with the first three steps being the Golgi phase and the next three steps making up the cap phase. While the three steps that follow constitute the acrosomal phase, the last 3 steps make up the maturation phase. At the cap and acrosomal phases, the entire acrosomal system comprising the vesicle and granule covers the head of the spermatids with no clear indentation of the nuclear surface by the formed acrosome. Furthermore, elongated spermatids at the maturation phase contained abundance of nuclear vacuoles. CONCLUSION: This work has not only provided information that will further the understanding of spermatogenesis but also aid the understanding of acrosomal reaction in the reproduction of the greater cane rat.

Immunolocalization And Distribution Pattern of Estrogen (ERα and ERß) and Progesterone (PR) Receptors along the Excurrent Duct of Male Greater Cane Rat (Thryonomys swinderianus).

The excurrent duct, which plays vital roles in the reproductive biology of all male mammals, shows some structural variations among different species. Some hormones such as testosterone, estrogen and progesterone, through their different receptors, have been known to be involved in the normal functioning of the excurrent duct. Here we evaluated the presence, localization and patterns of distribution of three hormone receptors, estrogen alpha (ERα), estrogen beta (ERß) receptors and progesterone receptors (PR) along the excurrent duct of sexually matured male greater cane rats. Immunohistochemistry revealed presence of ERα in epididymal stroma but not epithelium, selective ERß staining in narrow & apical cells as well as unique presence of PR in caudal epididymis, which to the best of our knowledge, is the first report on the cellular localization of progesterone receptor in the cauda epididymis. The result suggests the possible involvement of not only estrogen but also progesterone in the modulation of epididymal function in greater cane rat.

The West African ebola virus disease epidemic 2014-2015: A commissioned review.

The first epidemic of Ebola haemorrhagic disease in West Africa is the largest and longest Ebola epidemic till date, where the outbreak notably involved three countries with distant spread to other countries. It has caused significant mortality, with reported case fatality rates of up to 70%. Data and relevant information were extracted from the review of majorly relevant publications/papers about the Ebola epidemic in West Africa and other previous outbreaks of Ebola virus (EBOV). As of 2016, with the epidemic under control, the World Health Organization has warned that flare-ups of the disease are likely to continue for some time as recently occurred in Sierra Leone and the on-going in Guinea. As this may not be the last outbreak of Ebola virus disease (EVD) in West Africa, there is a need to focus on diagnostic and research capacity required to curtail EVD with adequate measures for emergency preparedness and policies for innovative treatment strategies.

Histology and ultrastructure of the uterus of African giant rat (Cricetomys Gambianus, Waterhouse) during oestrous cycle.

This study describes histology and ultrastructure of uterus in the African giant rat during oestrous cycle. Endometrial histology displayed glandular mucosa consisting of lamina epithelialis and lamina propria mucosae. Its epithelium varied between simple and pseudostratified columnar. The myometrium consisted of inner circular and outer longitudinal smooth muscles with medium sized arteries and veins in-between. The perimetrium contained simple squamous epithelium. Endometrial ultrastructures were variable during oestrous cycle. At mid oestrus, hemidesmosomes anchored undulating basement membrane of the mucosal epithelium. Preponderance spherical mitochondria, lipofuscin granules concentration, flocculent homogenous materials and indented nuclei were displayed. At mid metoestrus, late metoestrus/early dioestrus and mid dioestrus, the base of the mucosal columnar epithelium lain on relatively straight basement membrane and their cytoplasmic ultrastructure displayed variation to mid oestrus. Epithelial apex showed intermediate filament, microvilli and junctional complexes. The uterine glands occurred in variable numbers and sizes during oestrous cycle and shared similar ultrastructure. Mid dioestrus showed cell ultrastructure of uterine glands having apical accumulation of secretory vesicles. Some actively secreting uterine glands were lined by simple ciliated columnar epithelium mingled with pseudostratified epithelium. The findings of the study indicate that giant rat endometrial ultrastructure varies during oestrous cycle and glandular secretion is merocrine.

The coagulating gland in the male greater cane rat (Thryonomys swinderianus): morphological and immunohistochemical features.

This study reveals the structure, ultrastructure and immunoexpression of oestrogen alpha and beta receptors (ERα and ERß) in the coagulating glands of the greater cane rat. Gland samples from 15 adult male cane rats were processed for histological and ultrastructural studies while immunohistochemistry was also carried out. Coagulating gland in the cane rat is a paired, triangularly shaped, transparent gland weighing about 1 ± 0.48 g. Histologically, each secretory acinus is composed of folded mucosa surrounded by fibromuscular stroma. The simple columnar epithelium consists of principal cells at different stages of secretion evidenced by their apical blebs of various heights and occasional basal cells. Fine structure of the principal cells revealed the presence of apical blebs that contained secretory granules of varying electron-density, secretory vesicles and vacuoles on both their luminal surfaces and the lumen. While supranuclear cytoplasm contained Golgi apparatus with different cisternal arrangements, the infranuclear part is covered with dilated rough endoplasmic reticulum cisternae. Nuclei, apical bleb and stroma of secretory epithelium all showed positive immunostaining for ERα and ERß. These findings revealed the prominence of apocrine secretion with no structural evidence of merocrine secretion and the uncommon ERα and ERß distribution pattern in the coagulating gland of the cane rat.

Gross, histological and ultrastructural features of the bulbourethral gland in the greater cane rat (Thryonomys swinderianus).

The present study examines the structure and ultrastructure of the bulbourethral glands in 10 sexually matured male greater cane rats raised in captivity. Following anaesthesia, the rats were perfusion-fixed transcardially and the bulbourethral glands dissected out. Upon morphologic and morphometric analysis, the Cowper's glands were observed to have an average volume of 0.24±0.08 ml, a diameter of 6.3±0.6 mm and weighs 0.199±0.06 g. The paired, gourd-shaped tubuloalveolar glands were surrounded by dense connective tissues and separated into lobules by capsular septae. Each lobule consists of endpiece/secretory units and excretory ducts lined by simple glandular epithelium and pseudo-stratified epithelium, respectively. The round end pieces consisted of 8-10 pyramidal to columnar epithelial cells with flattened, basally located nuclei and granule-filled cytoplasm that bounded a narrow glandular lumen. The striking ultrastructural features of these secretory cells were the presence of some granules with uniform electron density and those with regions of lesser density as well as the absence of secretory vacuoles. Another unique characteristic of these secretory granules is the presence of electron dense strands radiating from their surfaces. The apical surfaces of the cells were also studded with abundant microvilli. From the findings, the structure of bulbourethral glands in the greater cane rat shows more resemblances to that of humans than to its rodent phylogeny. These findings serve as additional knowledge in the structural interpretation of the bulbourethral gland and its secretory products.

Anatomical and immunohistochemical characteristics of the prostate gland in the greater cane rat (Thryonomys swinderianus).

This study examined the morphology and immunohistochemical features of the prostate gland in 15 captive-reared male greater cane rat of known reproductive and medical history. Samples of the glands were taken after gross examination and routinely prepared for both histological and ultrastructural analysis. Immunohistochemistry was also carried out on paraffin-embedded sections of the glands using rabbit polyclonal antibodies against oestrogen receptors (ERα and ERß) and mouse monoclonal antibody for the progesterone receptor (PR). The prostate, which constitutes 0.04% of the body weight, was a paired, lobulated, brownish gland having three left and four right lobes that partly cover the pelvic urethra. Based on the amount and arrangement of the secretory epithelial folding and relative to their distances to the urethra, two histological zones, the central and peripheral, were identified. However, the epithelium of both zones was lined by predominantly simple cuboidal cells with occasional basal cells. The main ultrastructural features of these cuboidal cells were the presence of several nuclear pores on the nucleus, moderately well-developed, short microvilli and bleb-like apical projections, as well as inter-cellular lacunae seen between these cells and the basal cells. The cuboidal epithelial cells also showed positive nuclear staining for ERα and ERß but not for PR. It is however interesting that the ERα-positive staining was more at the epithelial cells, which is uncommon. These findings highlight the peculiarities in the structure and ultrastructure as well as the unique expression of the oestrogen receptors in the prostate gland of the greater cane rat.

Pregnancy, obstetric and neonatal outcomes in HIV positive Nigerian women.

While the effect of HIV infection on some maternal outcomes is well established, for some others there is conflicting information on possible association with HIV. In this study we investigated pregnancy and neonatal outcome of HIV positive women in large HIV treatment centre over a period of 84 months. They were managed according to the Nigerian PMTCT protocol. Adverse obstetric and neonatal outcome were observed in 48.3% HIV positives compared 30.3% to the negatives (OR: 2.08; CI: 1.84-2.34). Low birth weight ( OR:2.95; CI:1.95-3.1), preterm delivery (OR:2.05; CI:1.3-3.1), perinatal death (OR:1.9;CI:1.3-3.2), and spontaneous abortion (OR:1.37; CI:1.1-2.3) were factors found to be independently associated with HIV. Low CD4 count (OR: 2.45; CI: 1.34- 4.56) and opportunistic infections (OR: 2.11; CI: 1.56-3.45) were to be associated with adverse obstetric and neonatal outcome. This study confirms the association of HIV, severe immunosuppression and opportunistic infection and adverse obstetric and neonatal outcome.

The structure of the penis with the associated baculum in the male greater cane rat (Thryonomys swinderianus).

In this study, the structures of penises of eight sexually mature male greater cane rats were examined at both macroscopic and histological levels. Each animal was sacrificed after anaesthesia with ether and then dissected open with the penis exposed from its root. The penises were first grossly examined, measured, and then prepared for histological examination. From this study it was observed that the body size has no allometry with penile size, but the testicular weight correlated with Os penis length in the greater cane rat. Grossly, the penis which was whitish in colour, with a mean length of 5.46 ± 0.36 cm, has no obvious collum penis but a flexura that turns it caudo-ventral and separates the corpus and glans penis. There was the presence of cornified papillae covering parts of the corpus and glans penis as well as a blind sac sacculus urethralis under the urethra on the glans penis. Histologically, the corpora cavernosa penis were completely separated by a connective tissue septum which sent the trabeculae network into the cavernous tissues and replaced the caverns as it moves from corpus to glans penis. The Os penis formed through endochondral ossification after 42 months of age in this animal. Therefore, from a histological standpoint, the cane rat penis belongs to the intermediate type. In conclusion, these findings provide vital information on the penile anatomy of the greater cane rat, which will serve as a basis for comparing penile morphology among the suborder hystricomorpha and expand knowledge of the reproductive biology in this animal.

The immunohistochemical localization of desmin and smooth muscle actin in the ovary of the African giant rat (Cricetomys gambianus) during the oestrous cycle.

The aim of this study was to describe the distribution of smooth muscle actin and desmin immunopositive cells in the ovary of the giant rat. In addition, the study describes the morphological changes in the ovary of this species during the oestrous cycle. Healthy secondary and tertiary follicles dominated the ovary during pro-oestrus and oestrus. The theca externa of the tertiary follicles was immunopositive for smooth muscle actin, but immunonegative for desmin. Oestrus was also characterized by the presence of corpora haemorrhagica, which had an outer layer of smooth muscle actin immunopositive cells. Differentiating corpora lutea were observed during metoestrus. A further notable feature of the ovary during metoestrus was the presence of numerous atretic secondary and tertiary follicles. In the later stages of atresia, the follicles were infiltrated by desmin and smooth muscle actin immunopositive cells. Dioestrus was characterized by the presence of non-regressing and regressing corpora lutea. Immunostaining for smooth muscle actin was demonstrated in the enclosing layer of the corpora lutea, as well as in the tunica media of blood vessels within the corpora lutea. The results of this study have shown that morphological changes in the ovary of the giant rat during the oestrus cycle are similar to those of laboratory rodents. Furthermore, the results of the immunohistochemical study indicate that the perifollicular distribution of desmin and smooth muscle actin cells changes during follicular development and atresia.

Immunohistochemical localization of the progesterone and oestrogen alpha receptors in the uterine horns of the African giant rat (Cricetomys gambianus).

The present study investigated the immunolocalization of the progesterone and oestrogen alpha receptors in the uterine horns of the African giant rat during the oestrous cycle. The progesterone and oestrogen alpha receptors were demonstrated in various cellular constituents of the endometrium, myometrium and perimetrium. The intensity of progesterone and oestrogen alpha receptor immunostaining in the endometrial and myometrial layers of the uterine horns varied during the oestrous cycle. The intensity of oestrogen alpha receptor immunoreactivity in the luminal epithelium was high during pro-oestrus, oestrus and dioestrus. Progesterone and oestrogen alpha receptor immunoreactivity in the endometrial epithelia was absent during metoestrus. Moderate to strong immunostaining for the progesterone and oestrogen alpha receptors was demonstrated in the myometrial smooth muscle cells during pro-oestrus, oestrus and dioestrus. The intensity of progesterone and oestrogen alpha receptor immunostaining in the myometrial smooth muscle cells was low during metoestrus. Stromal cells in the perimetrium consistently expressed progesterone and oestrogen alpha receptor immunoreactivity throughout the oestrous cycle. The findings of the study indicate that in the giant rat the immunolocalization of the progesterone and oestrogen alpha receptors, in endometrial and myometrial regions of the uterine horns, varies during the oestrous cycle.

The influence of protein malnutrition on the antifertility action of gossypol in the Trypanosoma brucei-infected rat: some ultrastructural observations from the testis.

The testes of adult male Wistar rats that were variously protein malnourished, gossypol treated, and/or trypanosome infected (Trypanosoma brucei) were evaluated ultrastructurally. The findings included several necrotic germ cells in the seminiferous epithelium and focal degeneration of Sertoli cells. Leydig cells showed a remarkable paucity of smooth endoplasmic reticulum, and aggregation of mitochondria. These observations were prevalent in those animals that were simultaneously protein-malnourished/gossypol treated/ trypanosome infected, and were either absent or markedly reduced in the other groups. These structural alterations are probably related to increased gossypol availability to the testis and/or represent the additive effects of gossypol and trypanosomes. Such tissue changes could compromise spermatogenesis in affected animals, and suggest that trypanosome infections may exacerbate testicular lesions in the gossypol-treated rat.

The epithelium of the vesicular gland of the African giant rat (Cricetomys gambianus, Waterhouse): histology and ultrastructure.

The epithelium of the vesicular gland of the African giant rat was studied by the light and electron microscopes. The gland is compound tubuloalveolar, lined by a columnar epithelium, the apical surface of which is covered by a few short microvilli. The epithelial cells are the principal and basal cells. The principal cells have an abundance of rough surfaced endoplasmic reticulum (rER), which are often arranged in whorls. Basal cells are few, being wedged between adjacent principal cells in the basal region. They are fairly electron lucent and contain fewer organelles. Intraepithelial lymphocytes, which also occur mostly in the basal region are also present in the epithelium. The presence of these latter cells is noteworthy in that they are not a known feature of the epithelium of the vesicular gland of other species.

Gossypol toxicosis in the rat associated with protein malnutrition and experimental infection with Trypanosoma brucei.

The toxicity of gossypol, a compound occurring naturally in the cotton plant, was investigated in Trypanosoma brucei-infected, gossypol-treated rats, with and without protein malnutrition. The liver, heart, lungs, spleen and adrenal glands were enlarged in all gossypol-treated rats. Gossypol treatment or trypanosome infection, either alone or together, invariably caused significant reductions in the serum activity of creatine phosphokinase and amylase and in the serum concentration of cortisol. The serum biochemical changes, together with histopathological findings in various organs, indicated that the toxicity of gossypol and pathology of trypanosome infection, either alone or in concert, could be exacerbated by protein malnutrition. This finding suggests that the previously reported antiparasitic properties of gossypol may be of little ultimate benefit due to these serious side effects. The spleen in the protein-malnourished, trypanosome-infected and gossypol-treated animals exhibited only a slight decrease in the number of lymphatic nodules, but a marked cellular depletion, especially of cortical tissue, was observed in the thymus. These observations would seem to justify further study of the immune status of trypanosome-infected, gossypol-treated animals.

Infection with Trypanosoma brucei potentiates the antifertility effect of gossypol, especially in the protein-malnourished male rat.

Reproductive parameters were investigated in gossypol-treated male rats that had been infected experimentally with Trypanosoma brucei and then place on one of two diets of differing (low and normal) protein content. The endpoints assessed were reproductive organ weights, semen epididymal sperm counts, serum testosterone and histological, stereological and histomorphometric evaluation of the testis and accessory reproductive organs. Most of the parameters studied were lowest in the protein-malnourished, gossypol-treated, trypanosome-infected animals, when compared to those obtained from the corresponding animals that were only gossypol-treated or trypanosome-infected. Mean testicular size and the diameter and the total length of seminiferous tubules were especially reduced, indicating that the overall volume of the seminiferous epithelium in these animals was smaller. These findings suggest that reproductive capacity could be impaired in protein-malnourished, trypanosome-infected animals fed on gossypol-containing products, even when there are no obvious clinical signs of disease. This could translate into increased production costs in a farm enterprise. Screening for haemoparasitism would also seem to be worthwhile prior to evaluation and/or use of gossypol (or perhaps other potential contraceptives) in human subjects, especially in communities that are confronted with severe food shortages.

The effects of protein malnutrition and experimental infection with Trypanosoma brucei on gossypol treatment in the rat: haematological and serum biochemical changes.

This study was designed to evaluate the interaction between protein malnutrition, gossypol treatment and blood parasitosis (Trypanosoma brucei) in the Wistar rat. Haematological and serum biochemical changes were evaluated in the rats, which were placed on two planes of nutrition--low protein (LP) and normal protein (NP)--and either treated with gossypol or infected with Trypanosoma brucei, or both. Higher parasitaemia occurred in gossypol-treated NP rats than in the corresponding LP group. Gossypol treatment and trypanosomal infection, either alone or in concert, caused an anaemia that was both macrocytic and hypochromic. Both treatments together also caused increases in serum alkaline phosphatase and alanine aminotransferase activities, which were accompanied by depressed serum albumin concentrations, suggestive of hepatic dysfunction in affected rats. These results suggest that, with adequate protein intake, the growth and infectivity of trypanosomes is not inhibited by gossypol but that protein malnutrition has a beneficial effect of reduced parasitaemia. Unfortunately, this beneficial effect is counteracted by gossypol enhancement of hepatic dysfunction caused by trypanosomes.

Relationship between submandibular gland epidermal growth factor and spermatogenesis in C3H mice.

Epidermal growth factor (EGF), a potent mitogen produced primarily in the submandibular gland of adult male mice, has been implicated in modulating processes known to be of vital importance in the regulation of spermatogenesis. In the present investigation we demonstrate that submandibular gland EGF from adult male mice is indeed capable of displacing radiolabeled EGF from testicular membranes. Scatchard analysis of this binding site reveals that it is of high affinity (Kd = 0.77 nM) and low capacity (Bmax = 8.15 fmol/mg protein). Cross-linking of 125I-EGF to the identical membrane preparation resulted in the SDS-PAGE/autoradiography identification of a single band at approximately 170 kDa. Next, we examined the cellular distribution of the EGF receptor in the testis using biotin-streptavidin immunoperoxidase and employing different antisera probes generated to a conserved sequence of the EGF receptor. The Scatchard and cross-linking data described above, along with the immunocytochemistry results, suggest strongly that there is only one functional binding site for EGF in the adult testis and that this receptor is present in Sertoli and Leydig cells.

Localization of secretory, membrane-associated and cytoskeletal proteins in rat testis using an improved immunocytochemical protocol that employs polyester wax.

Immunocytochemistry is a compromise between maintaining antigenicity and preserving tissue morphology. In the testis, successful immunostaining results at the level of resolution provided by the light microscope have been obtained through use of either frozen or paraffin sections, although both techniques are fraught with limitations. With freezing, tissue preservation is not optimum, whereas with paraffin embedding, antigenicity is often destroyed. These limitations are not trivial and have led to numerous ambiguous results in the literature. In the present study we wish to report the results of immunocytochemical localization of various proteins in testis fixed by perfusion with Bouin's fluid and embedded in polyester wax, a ribboning embedding medium for histology. The advantages of this medium are that it does not require clearing of tissues in xylene solvents before embedding and that unlike paraffin, it liquifies at 38 degrees C. Because of these two properties, the polyester was appears to adequately maintain antigenicity as compared to that observed in frozen sections, yet because it is a ribboning wax, it preserves detailed structure as well as paraffin does. Proteins that were immunolocalized included cytoskeletal proteins (tubulin, actin, vinculin, vimentin) and cell-specific markers: 1) androgen-binding protein (ABP) for Sertoli cells; 2) peripheral type benzodiazepine receptor (PBR) for Leydig cells; and 3) nuclear lamins for germ cells. Biotin-streptavidin peroxidase immunocytochemistry was employed to determine the specific distribution of the various proteins, and both rabbit antisera and mouse monoclonal antibodies were used with equal success. In addition, fluorochrome-labeled second antibodies combined with confocal microscopy were used to examine the disposition of the antigens in the testis. Results revealed maintenance of antigenicity and morphology far superior to that obtained with paraffin and frozen sections, respectively, they also showed that within the seminiferous epithelium, germ cell or Sertoli cell-specific proteins were unambiguously immunolocalized to their respective cells. Specific observations made possible through use of this protocol suggest that neither tubulin or vimentin immunostaining patterns in Sertoli cells are altered during the cycle of the seminiferous epithelium. Similarly, ABP staining appeared constant throughout the cycle. Further, we wish to report that anti-PBR is a specific probe for Leydig cells in vivo and that an anti-nuclear lamin antibody appears to serve as a specific probe for spermatogonia and pachytene spermatocytes, but that the commercially available anti-smooth muscle alpha-actin monoclonal antibody immunostains both the myoid and lymphatic endothelial cells forming the peritubular cells layer of the seminiferous tubule.(ABSTRACT TRUNCATED AT 400 WORDS)

Diazepam binding inhibitor is a paracrine/autocrine regulator of Leydig cell proliferation and steroidogenesis: action via peripheral-type benzodiazepine receptor and independent mechanisms.

Previous studies demonstrated that the polypeptide diazepam binding inhibitor (DBI) and its receptor, the peripheral-type benzodiazepine receptor (PBR), are involved in the regulation of steroid biosynthesis and that one site of PBR action resides in mitochondria. In the present investigation, evidence is presented that a functional form of PBR is also present at the cell surface. First, PBR was immunolocalized in the rat testis using biotin-streptavidin peroxidase immunocytochemistry, and results revealed that PBR was present exclusively in the interstitial Leydig cells. Next, the distribution of PBR in MA-10 Leydig cells was further examined using confocal microscopy. MA-10 cells were either fixed and immunostained or fixed/permeabilized and immunostained for PBR, followed by generation of confocal microscope optical sections, three-dimensional reconstructions of these sections, and then generation of vertical confocal sections of the three-dimensional reconstruction. In the fixed/unpermeabilized cells, PBR immunostaining at the cell surface was clearly evident, whereas in the fixed/permeabilized cells, intracellular PBR distribution was more robust. These results suggest that the plasma membrane fraction of the receptor could mediate the action of extracellular PBR ligands on Leydig cell function. Next, we examined whether DBI, the naturally occurring PBR ligand, is secreted by testicular cells and whether it could activate the cell surface PBR. Immunolocalization of DBI demonstrated that it was present in both Leydig and Sertoli cells. Further, using an immunoblot assay, we demonstrated that DBI is present in rat testicular interstitial fluid. Metabolic labeling of cultured immature rat Sertoli cells and MA-10 mouse tumor Leydig cells, followed by immunoprecipitation of the secreted proteins with an anti-DBI antiserum, demonstrated that both Leydig and Sertoli cells secrete DBI and could serve as a cell source for the interstitial fluid DBI. Then, we partially purified the DBI present in conditioned medium and interstitial fluid by reverse phase chromatography and demonstrated it to be bioactive, based on displacement of a radiolabeled benzodiazepine (Ro5-4864)-specific ligand for PBR; pronase treatment of different preparations eliminated all bioactivity. We then examined the effects of DBI on Leydig cell function. DBI added to MA-10 cells affected DNA synthesis and cell growth in a biphasic manner; at low concentrations (1 nM), DBI was mitogenic, increasing [3H]thymidine incorporation and cell numbers by 30-40%, while at high concentrations (1 microM), DBI inhibited cell growth (30-40%). Similar effects on cell growth were obtained using the benzodiazepine Ro5-4864.

Characterization of epidermal growth factor in mouse testis.

Considerable evidence exists to suggest that epidermal growth factor (EGF) influences spermatogenesis directly. The tissue source of this EGF, however, is not yet clear. In this study we examine whether the testis itself can serve as a source of EGF. Gel filtration fractions of acid extracted testes exhibited the ability to displace 125I-EGF from testis membranes. The testicular fractions containing the 125I-EGF displacement activity coeluted within the same range as those of submandibular gland (SG) fractions containing mature EGF and prepared in an identical fashion. Next, we employed specific antisera probes to investigate first, whether the testis synthesizes this EGF displacement activity and second, to determine the cell distribution of the testicular EGF. Two types of antisera probes were employed: 1) commercially available antisera to mature EGF (EGFm), i.e. the 6,000 M(r) peptide, and 2) polypeptide specific antisera to the C-terminus of the EGF precursor (EGFp), i.e. the 140,000 M(r) integral membrane molecule which exhibits seven EGF-like repeats in addition to the EGFm. Metabolic labeling of testis with 35S-methionine was performed, followed by immunoprecipitation with the anti-EGFm antisera. Parallel studies using kidney and SG were used as positive controls. Fluorograms exhibited a prominent band at M(r) 140,000 for testis and kidney, corresponding to the EGFp. There was, in addition, a M(r) 50,000 band present for the testis. In SG, a band at M(r) 6,000, corresponding to EGFm, in addition to bands at M(r) 21,000 and 46,000 were observed also. Immunoblotting of testis, kidney, and SG membrane preparations with the specific antisera to either the EGFm or EGFp also resulted in identifying the EGFp at M(r) 140,000, as well as other lower mol wt bands. Preadsorption of anti-EGFm antisera with excess EGFm eliminated all of the specific bands that were immunoblotted. Peroxidase immunocytochemistry of testis, kidney, and SG was also performed using the specific antisera to either EGFm or EGFp. EGFp and EGFm staining in SG and kidney was identical to previously published results in which the distribution of EGFm in these tissues was established. In testis, EGFm immunostaining showed positive results in Sertoli cells, pachytene spermatocytes and round spermatids. In contrast, EGFp immunostaining was limited to pachytene spermatocytes and round spermatids. These results suggest that the testis must now be included in the list of tissues capable of synthesizing EGFp. Specifically, EGFp synthesis appears limited to the post meiotic germ cells.