Secretory cell types and cell proliferation of human bronchial epithelial cells in an organ-culture system

We examined the localization of steroidogenic cells in rainbow trout (Oncorhynchus mykiss) testis during spermatogenesis by using polyclonal antibodies generated against rainbow trout cholesterol side-chain cleavage enzyme cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17α-hydroxylase/C17,21 lyase (P450c17), and aromatase cytochrome P450 (P450arom) as markers of steroid production. Since we had previously produced specific antibodies against 3β-HSD and P450arom, antibodies against oligopeptides corresponding to C-terminal sequences of P450scc and P450c17, predicted from rainbow trout P450scc and P450c17 cDNAs, were produced in this study. These two antibodies recognized 54-kDa (P450scc) and 59-kDa (P450c17) bands specifically in several steroidogenic organs, i.e., testis, ovary, and interrenal tissue (head kidney) in Western blots. Immunohistochemically, immunoreactive P450scc, P450c17, and 3β-HSD, but not P450arom, were found only in interstitial Leydig cells of immature and mature testes. Immunoreactive P450arom was not detected in either testis. This study suggests that Sertoli cells and germ cells of rainbow trout testis do not contain P450scc, P450c17, P450arom, or 3β-HSD.     

FORMATION OF GERM CELL-LIKE CELLS IN THE PERITONEAL EPITHELIUM OF THE TESTES OF ESTROGEN-TREATED ADULT MALES OF THE JAPANESE RED-BELLIED NEWT, CYNOPS PYRRHOGASTER PYRRHOGASTER

Columnar cells of the peritoneal epithelium in slender cords of the testes were examined in normal and estradiol benzoate-treated Japanese red-bellied newt, Cynops pyrrhogaster pyrrhogaster, by light and electron microscopy. In normal newts, the peritoneal epithelium covering the slender cord consists of columnar cells, which contain extraordinarily large, oval or spindle-shaped nuclei with conspicuous indentations. The nucleus contains chromatin granules and the cytoplasm is filled with numerous tonofilaments. The primordial germ cells are scattered throughout the slender cord, and each cell is surrounded by a few follicle cells. Between the germ cells and follicle cells there are microvilli-like processes. The nucleus of primordial germ cells is multilobate and has electron lucent areas, dispersed chromatin and several electron-dense nucleoli. In the lighter cytoplasm, the nuage material is found very near to nuclear pores, and is frequently seen among the mitochondria. The nucleolus-like body is not associated with other organelles. The primary spermatogonia have bilobate nuclei. It is remarkable that most of the cytoplasmic organelles are found in the deep nuclear indentations. The nuage material and nucleolus-like body are well developed in the cytoplasm. After treatment of newts with estradiol benzoate for one year, four types of cells can be distinguished in the peritoneal epithelium. One type is quite different from the columnar cells. These newly appeared cells are large and light in appearance. Their nucleus is highly lobate, and contains dispersed chromatin and several nucleoli with compact electron dense material in its periphery. The cells are characterized by the presence of nuage material and nucleolus-like bodies in the cytoplasm. There are microvilli-like processes between these cells and adjacent elongated cells. These ultrastructural characteristics of the light cells are very similar to those of primordial germ cells and/or primary spermatogonia in normal testes. These findings suggest that the light cells which appear in the peritoneal epithelium of the testes on administration of estrogen may be germ line cells.     

Seasonal Changes in the Newt,Trituroides hongkongensis,Testis. I. A Histological and Histochemical Study

The testis of the newt, Trituroides hongkongensis is found to be made up of a chain of lobes each being divided into spermatogenetic zones which display seasonal cycles. Seasonal changes in the histology of the testis are studied. Lobule boundary cells are found present at the same time of the year when the secondary sex character, a lateral blue line along the tail, is best expressed. Both 5-ene-3β- and 17β-HSD activities have been located in the lobule boundary cells which are suggested to be steroidogenic and their secretion may probably be responsible for the maintenance of the secondary sex character. The Sertoli cell, which originates from the follicle cell, shows accumulation of cholesterol-positive lipid at certain stages of development but its capability in hormone production is uncertain.     

Expression of Cytochrome P450 Side-Chain Cleavage Enzyme and 3β-Hydroxysteroid Dehydrogenase in the Rat Central Nervous System: A Study by Polymerase Chain Reaction and In Situ Hybridization

Abstract: In examining steroid synthesis in the CNS, expression of the mRNAs encoding for cytochrome P450 side-chain cleavage enzyme (P450_SCC) and 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase (3β-HSD) has been studied in the rat brain. P450_SCC transforms cholesterol into pregnenolone and 3β-HSD transforms pregnenolone into progesterone. PCR was used to amplify cDNA sequences from total RNA extracts. Classical steroidogenic tissues, like adrenal and testis, as well as the non-steroidogenic tissue lung have been used as controls. The expression of P450_SCC and 3β-HSD have been demonstrated by PCR in cortex, cerebellum, and spinal cord. In addition, primary cultures of rat cerebellar glial cells and rat cerebellar granule cells were found to express P450_SCC and 3β-HSD at comparable levels. Furthermore, three of the four known isoenzymes of 3β-HSD were identified, as determined using selective PCR primers coupled with discriminative restriction enzymes and sequencing analysis of the amplified brain products. Using RNA probes, in situ hybridization indicated that P450_SCC and 3β-HSD are expressed throughout the brain at a low level and mainly in white matter. Enrichment of glial cell cultures in oligodendrocytes, however, does not increase the relative abundance of P450_SCC and 3β-HSD mRNA detected by PCR. This discrepancy suggests that the developmental state of cultured cells and their intercellular environment may be critical for regulating the expression of these enzymes. These findings support the proposal that the brain apparently has the capacity to synthesize progesterone from cholesterol, through pregnenolone, but that the expression of these enzymes appears to be quite low. Furthermore, the identification of these messages in cerebellar granule cell cultures implies that certain neurons, in addition to glial cells, may express these steroidogenic enzymes.     

Cloning of cDNA for newt WT1 and the differential expression during spermatogenesis of the Japanese newt, Cynops pyrrhogaster

To investigate the function of Wilms' tumor 1 (WT1) during spermatogenesis, cDNA for newt WT1 homolog was cloned and the expression of WT1 in newt testes was examined. The cDNA is 2089 bp in length and encodes 426 amino acid (aa) residues. The deduced aa sequence shares 76 and 79% homology with human and Xenopus WT1, respectively. Northern blot analysis shows that WT1 mRNA, 3.2 and 4.5kb in length, are expressed in the testis and kidney. Both WT1 mRNA species are detected in various stages of spermatogenesis, but the 3.2kb mRNA is highly expressed in spermatogonia and mature sperm stages, while the amount of 4.5kb mRNA is almost constant throughout spermatogenesis. In situ hybridization reveals that WT1 mRNA is localized in Sertoli cells. Moreover, immunohistochemical analysis shows that WT1 protein is highly expressed in the nuclei of Sertoli cells in early spermatogonia and mature sperm stages, but not in pericystic cells or germ cells. These results suggest that WT1 is involved in the regulation of gene expression in Sertoli cells, depending on the spermatogenic stage.     

Testicular steroidogenic enzymes in the lizard Podarcis sicula during the spermatogenic cycle

Steroidogenic Acute Regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), 5α-Reductase (5α-Red), P450 aromatase are key enzymes involved in steroidogenesis. Recently, we showed the expression and the localization of P450 aromatase in Podarcis sicula testis during the different phases of the reproductive cycle, showing its involvement in the control of steroidogenesis, particularly in 17β-estradiol synthesis. Now, we have investigated the presence and distribution of the other enzymes involved in steroidogenesis, i.e. StAR, 3β-HSD, 17β-HSD and 5α-Red, during three significant periods of the reproductive cycle: summer stasis (July–August), autumnal resumption (November) and reproductive period (May–June). We demonstrated for the first time that all these enzymes are always present in somatic cells (Leydig and Sertoli) and germ cells (spermatogonia, spermatocytes I and II, spermatids and spermatozoa) of Podarcis testis, mainly in spermatids and spermatozoa. The present results strongly suggest that in Podarcis testis both somatic and germ cells could be involved in local sex hormone synthesis and that 5α-Red and P450 could carry out a pivot role.     

Mono-(2-ethylhexyl) phthalate (MEHP) regulates glucocorticoid metabolism through 11β-hydroxysteroid dehydrogenase 2 in murine gonadotrope cells

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolite mono-(2-ethylhexyl) phthalate (MEHP) have been classified as toxicants to the reproductive system at the testis level and DEHP may also impair reproductive axis function at the pituitary levels. However, MEHP is 10-fold more potent than DEHP in toxicity and little is known about the toxicological effect of MEHP on pituitary. In this study, we demonstrated that 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), not 11β-HSD1, is strongly expressed in murine gonadotrope LβT2 cells. Interestingly, MEHP inhibited Hsd11b2 mRNA level and 11β-HSD2 enzyme activity in LβT2 cells at as low as 10⁻⁷ M. Corticosterone (CORT) at a concentration of 10⁻⁶ M significantly inhibited LβT2 cell proliferation after 2-day culture, and 10⁻⁶ M RU486, an antagonist of glucocorticoid receptor (GR), reversed this inhibition. However, in the presence of 10⁻⁵ or 10⁻⁴ M MEHP, the minimal concentration of CORT to inhibit the proliferation of LβT2 cells was lowered to 10⁻⁷ M, and 10⁻⁶ M RU486 was not able to completely reverse the CORT effect. In conclusion, along with the regulation of GR, 11β-HSD2 may have a key role in glucocorticoid metabolism in LβT2 cells. MEHP may participate in the glucocorticoid metabolism in LβT2 cells through inhibition of 11β-HSD2 enzyme activity. Such perturbation may be of pathological significance as MEHP may interfere with the reproductive system at pituitary level through regulation of glucocorticoid metabolism, especially in neonates with higher risk of phthalates exposure.     

Androgen regulation of progestin biosynthetic enzymes in FSH-treated rat granulosa cells in vitro

The influence of androgens on the FSH modulation of progestin biosynthetic enzymes was studied $\text{in vitro}$. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 3 days in a serum-free medium containing FSH (20 ng/ml) with or without increasing concentrations (10^?9?10^?6 M) of 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone; DHT), 5α-androstane-3α, 17β-diol (3α-diol), or the synthetic androgen 17β-hydroxy-17-methyl-4,9,11-estratrien-3-one (methyltrienolone; R1881). FSH treatment increased progesterone and 20α-hydroxy-4-pregnen-3-one(20α-OH-P) production by 10.2- and 11-fold, respectively. Concurrent androgen treatment augmented FSH-stimulated progesterone and 20α-OH-P production in a dose-related manner (R1881 > 3α-diol > DHT). In the presence of an inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), the FSH-stimulated pregnenolone (3β-hydroxy-5-pregnen-20-one) production (a 20-fold increase) was further enhanced by co-treatment with R1881, 3α-diol or DHT. Furthermore, FSH treatment increased 4.4-fold the activity of 3β-HSD, which converts pregnenolone to progesterone. This stimulatory action of FSH was further augmented by concurrent androgen treatment. In contrast, androgen treatment did not affect FSH-stimulated activity of a progesterone breakdown enzyme, 20α-hydroxysteroid dehydrogenase(20α-HSD). These results demonstrate that the augmenting effect of androgens upon FSH-stimulated progesterone biosynthesis is not due to changes in the conversion of progesterone to 20α-OH-P, but involves an enhancing action upon 3β-HSDΔ⁵, Δ⁴-isomerase complexes and additional enzymes prior to pregnenolone biosynthesis.     

Immunohistochemical detection of the retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) in the marbled newt along the annual cycle

Retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) expression in the testis of the marbled newt were investigated with special attention to the changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. The annual testicular cycle of the marbled newt (Triturus marmoratus marmoratus) comprises three periods: (a) proliferative period (germ cell proliferation from primordial germ cells to round spermatids, April-June); (b) spermiogenesis period (July-September); and (c) quiescence period (interstitial and follicular cells form the glandular tissue, October-April). In the proliferative period, primordial germ cells and primary spermatogonia immunostained intensely to the three types of RXRs and also to FXR. In the other periods, immunostaining to these antibodies was weak or absent. Secondary spermatogonia stained weakly to the four antibodies in the proliferative period, and only to FXR, also weakly, in the spermiogenesis period. Immunoreactive primary spermatocytes were weakly labeled with the RXR antibodies in the proliferative period. Spermatids and spermatozoa did not stain to any antibody in any period. Follicular cells only immunostained to RXR-gamma and only in the quiescence period when they are forming the glandular tissue, together with the interstitial cells. As follicular cells, interstitial cells only immunostained in the quiescence period; however, they immunoreacted to the three types of RXRs. These findings suggest that in the newt, RXRs and FXR are involved in spermatogenesis control by regulating the proliferation of primordial germ cells and spermatogonia. In addition, RXR-gamma seems to be also involved in the development of the glandular (steroidogenic) tissue.     

Distribution of 7B2 (secretogranin V)-like immunoreactivity in the Japanese red-bellied newt (Cynops pyrrhogaster) pituitary

In this study, we examined 7B2 (secretogranin V)-like immunoreactivity (IR) in the Japanese red-bellied newt (Cynops pyrrhogaster) pituitary. Results showed that the pars nervosa was filled with immunoreactive granules. In the pars intermedia, all melanotrophs showed 7B2-IR. In the pars distalis, immunoreactive cells were dispersed, and the 7B2-immunoreactive cells were also immunopositive for the β-subunit of bullfrog luteinizing hormone (fLHβ). 7B2-IR co-localized with fLHβ-IR in the same secretory granules. Our results suggest that 7B2 may participate in the secretion processes of gonadotropins in the pars distalis.     

Inhibition of 3β- and 17β-hydroxysteroid dehydrogenase activities in rat Leydig cells by perfluorooctane acid

Perfluorooctane acid (PFOA) is classified as a persistent organic pollutant and as an endocrine disruptor. The mechanism by which PFOA causes reduced testosterone production in males is not known. We tested our hypothesis that PFOA interferes with Leydig cell steroidogenic enzymes by measuring its effect on 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) activities in rat testis microsomes and Leydig cells. The IC₅₀s of PFOA and mode of inhibition were assayed. PFOA inhibited microsomal 3β-HSD with an IC₅₀ of 53.2 ± 25.9 μM and 17β-HSD3 with an IC₅₀ 17.7 ± 6.8 μM. PFOA inhibited intact Leydig cell 3β-HSD with an IC₅₀ of 146.1 ± 0.9 μM and 17β-HSD3 with an IC₅₀ of 194.8 ± 1.0 μM. The inhibitions of 3β-HSD and 17β-HSD3 by PFOA were competitive for the substrates. In conclusion, PFOA inhibits 3β-HSD and 17β-HSD3 in rat Leydig cells.     

DNA SYNTHESIS AND MITOSIS IN ERYTHROPOIETIC CELLS

Studies of newt (Triturus or Diemictylus viridescens) erythropoietic cells showed that DNA synthesis and mitosis normally occur throughout most of the developmental process. Mitotic divisions were found in all immature precursor stages from the proerythroblast to the highly hemoglobinized reticulocyte. Mitoses were absent in mature erythrocytes. Radioautographic examination of thymidine-³H incorporation into DNA revealed that all erythroid cells except the mature erythrocyte were labeled. Microphotometric measurements of Feulgen-stained smears showed that all immature stages were undergoing DNA synthesis whereas the mature erythrocyte was inactive. The results obtained from three independent methods clearly demonstrate that (a) no loss of DNA or of chromosomes occurs during erythrocytic development and (b) highly hemoglobinized and, therefore, well-differentiated cells normally do undergo DNA synthesis and mitosis.     

The biosynthesis of progesterone by cultured mouse midgestation trophoblast cells.

The ability of cultured midgestation mouse trophoblast cells to synthesize progesterone from pregnenolone has been monitored by radioimmunoassay or chromatography and crystallization. The conversion of pregnenolone to progesterone is almost completely blocked by cyanoketone, a known inhibitor of Δ⁵,3β-hydroxysteroid dehydrogenase (3β-HSD) activity. Since there is little or no further metabolism of the progesterone formed, the ability of trophoblast cells to convert pregnenolone to progesterone in vitro is an accurate reflection of the activity of 3β-HSD in these cells.Midgestation cultures of giant trophoblast cells have a substantially higher 3β-HSD specific activity than the smaller ectoplacental cone cells. Neither giant trophoblast nor ectoplacental cone cell cultures show an increased 3β-HSD specific activity in response to a variety of hormones, including gonadotrophins. In fact, regardless of the gestation age at which the trophoblast cultures are initiated, 3β-HSD activity inevitably follows the same temporal pattern observed in vivo. Taken together, these facts suggest that the levels of 3β-HSD in trophoblast cells are intrinsically controlled and that, unlike the ovary, progesterone production by trophoblast cells in vivo is not influenced by gonadotrophic hormone levels.     

Localization of testosterone and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase in cynomolgus monkey (Macaca fascicularis) testes

The enzyme 3β-hydroxysteroid dehydrogenase /Δ⁵-Δ⁴-isomerase (3β-HSD) is essential for the biosynthesis of all classes of steroid hormones, including androgens. We localized testosterone and 3β-HSD by light microscopic immunocytochemistry in the testes of adult cynomolgus monkeys. Immunoreactive testosterone was located as intense deposits in the labeled cytoplasm of Leydig cells, and located weakly in the interstitial tissues, basement membranes, and the regions near tubular walls within tubules. Immunoreactive 3β-HSD was located in the cytoplasm of all Sertoli cells and was especially intense in the parts near tubular walls and located weakly to intensely in the cytoplasm of some Leydig cells. This is the first immunocytochemical evidence that Sertoli cells of cynomolgus monkeys, as well as Leydig cells, are involved in biosynthesis of androgens.     

THE OCCURRENCE OF HISTOCHEMICAL ACTIVITY OF 3β-HYDROXYSTEROID DEHYDROGENASE IN THE DEVELOPING TESTES OF POECILIA RETICULATA

In the mature testes of the guppy, Poecilia reticulata , some groups of cells, distributed sparsely in the interspace between the peripheral germ cell layer and the hilar duct system, show evident histochemical response for Δ⁵-3β-hydroxysteroid dehydrogenase (3β-HSD). In the testis of newly delivered guppies, somatic cells are present in the testicular hilus as a compact mass without revealing any structural differentiation. In the testis of juvenile fish of the 8mm stage about 7 days after birth, interstitial cells resembling histologically those of adult testes become differentiated from the somatic cell mass and, though only in some specimens, coincidentally begin to display weak but obvious histochemical response for 3β-HSD. Thereafter the occurrence of enzyme activity becomes increasingly regular in the developing testes, and attains the adult pattern of distribution in testes of all specimens after the 11 13mm stage or 17 ∽ 20 days of age.
The appearance and enhancement of 3β-HSD activity in the testis is concurrent with the differentiation and development of the testicular duct system. Treatments of newly delivered fish with methyltestosterone (30 ∽ 50 μg/g diet) distinctly stimulate the development of the duct system, which suggests a possible role of androgen secretion occurring in the early phase of the testicular development in the control of testicular organogenesis in the guppy.     

11β-Hydroxysteroid dehydrogenase 1 contributes to the pro-inflammatory response of keratinocytes

The endogenous glucocorticoid, cortisol, is released from the adrenal gland in response to various stress stimuli. Extra-adrenal cortisol production has recently been reported to occur in various tissues. Skin is known to synthesize cortisol through a de novo pathway and through an activating enzyme. The enzyme that catalyzes the intracellular conversion of hormonally-inactive cortisone into active cortisol is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). We recently reported that 11β-HSD1 is expressed in normal human epidermal keratinocytes (NHEKs) and negatively regulates proliferation of NHEKs. In this study, we investigated the role of 11β-HSD1 in skin inflammation. Expression of 11β-HSD1 was induced by UV-B irradiation and in response to the pro-inflammatory cytokines, IL-1β and TNFα. Increased cortisol concentrations in culture media also increased in response to these stimuli. To investigate the function of increased 11β-HSD1 in response to pro-inflammatory cytokines, we knocked down 11β-HSD1 by transfecting siRNA. Production of IL-6 and IL-8 in response to IL-1β or TNFα stimulation was attenuated in NHEKs transfected with si11β-HSD1 compared with control cells. In addition, IL-1β-induced IL-6 production was enhanced in cultures containing 1 × 10⁻¹³ M cortisol, whereas 1 × 10⁻⁵ M cortisol attenuated production of IL-6. Thus, cortisol showed immunostimulatory and immunosuppressive activities depending on its concentration. Our results indicate that 11β-HSD1 expression is increased by various stimuli. Thus, regulation of cytosolic cortisol concentrations by 11β-HSD1 appears to modulate expression of inflammatory cytokines in NHEKs.     

Regulation of 11β-hydroxysteroid dehydrogenase 1 and 2 by IGF-1 in mice

Two isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD1 and 11β-HSD2) play an important role in regulation of glucocorticoid corticosterone (CORT, the active form in rodents) by the interconversion between CORT and 11-dehydrocorticosterone (11DHC, the biologically inert form). 11β-HSD1 is an NADP+/NADPH-dependent oxidoreductase which is mainly expressed in liver and kidney, while 11β-HSD2 is an NAD+-dependent oxidase which is predominantly expressed in kidney. The regulation of 11β-HSD1 and 11β-HSD2 mRNA (Hsd11b1 and Hsd11b2) levels and their activities by IGF-1 was performed in liver, kidney, and testis of IGF-1 knockout male mice. Real-time PCR showed that Hsd11b1 in liver was decreased while Hsd11b2 mRNA level was decreased in kidney of IGF-1 null mice. 11β-HSD1 and 11β-HSD2 activities fluctuated with the changes of their respective Hsd11b1 or Hsd11b2 mRNA levels. In conclusion, IGF-I tissue-specifically regulates Hsd11b1 and Hsd11b2 expression.     

Purification,partial characterization,and localization of Sak57, an acidic intermediate filament keratin present in rat spermatocytes,spermatids, and sperm

We have purified a 57 kDa protein (designated Sak57, for spermatogenic cell/sperm-associated keratin) from sodium dodecyl sulfate-β-mercaptoethanol(SDS-βME)-dissociated outer dense fibers isolated from rat sperm tails. Internal protein sequence analysis of Sak57 yielded two 15-mer and 10-mer fragments with 70–100% homology to human, rat, and mouse keratins and corresponding to the 1A and 2A regions of the α-helical rod domain of keratins. A multiple antigenic peptide (MAP) was constructed using the 10-mer amino acid sequence KAQYEDIAQK (corresponding to the 2A region) and used as antigen for the production of polyclonal antibodies in rabbit. Anti-MAP sera were used for further analysis of the biochemical characteristics of Sak57 in testis and sperm tails using chromatofocusing, immunobloting, and [³²P]orthophosphate-labeling. We have found that rat testis displays two immunoreactive proteins: a soluble 83 kDa protein with pl range 5.9–6.3, regarded as a precursor, and both detergent-insoluble and soluble 57 kDa protein with pl range 5.0–5.9, corresponding to the mature form Sak57. The testicular soluble form was phosphorylated. Rat sperm tail samples displayed only the Sak57 detergent-insoluble form and its pl was more acidic (4.7–4.8). Whole-mount electron microscopy of negatively stained preparations of sperm-derived Sak57 resuspended in SDS-βME revealed a rod-shaped pattern. A decrease in the concentration of SDS-βME resulted in the side-by-side aggregation of rod-shaped Sak57 forming thick bundles. Indirect immunofluorescence was used to determine the localization of Sak57 in isolated outer dense fibers, epididymal sperm, spermatids, and pachytene spermatocytes. Confocal laser scanning microscopy was used to analyze the three-dimensional arrangement of Sak57 in pachytene spermatocytes. Isolated outer dense fiber and sperm tails displayed an immunoreactive product in the form of linear clusters. In elongating spermatids (steps 10–11), Sak57 immunoreactivity was predominant in the head region whereas pachytene spermatocytes displayed a cortical cytoplasmic distribution. Results of this study demonstrate that Sak57 has the characteristics of a keratin intermediate filament and is present during meiotic and postmeiotic stages of spermatogenesis. © 1996 Wiley-Liss, Inc.     

Distribution and Structure of the Adrenocortical Homolog in Polypterus palmas Ayres

The identity, distribution and structure of the adrenocortical homolog (AH) was made in Polypterus palmas Ayres using routine light and electron microscopy and histochemistry for the enzyme δ⁵-3β-hydroxysteroid dehydrogenase (3β-HSD). The AH is confined to yellow corpuscles which are positioned near the posterior cardinal and renal veins in the anterior two-thirds of each kidney. The 46–57 spherial to cigar-shaped corpuscles are placed end to end and are equally distributed in the kidneys. The tortuous cords of epithelial cells of each corpuscle are surrounded by sinusoids and are completely delimited from the haemopoietic and renal tissue of the kidneys. The steroidogenic nature of the cells is demonstrated by their 3β-HSD activity and their ultrastructure, namely lipid droplets, tubular smooth endoplasmic reticulum and mitochondria with tubulovesicular cristae. Giant mitochondria and gap junctions are notable features of AH cells in this fish. The yellow corpuscles of the kidneys in P. palmas represent the AH and this tissue has a distribution which is in accordance with the taxonomic position of Polypteriformes.