Expression of beta-nerve growth factor and its receptor in rat seminiferous epithelium: specific function at the onset of meiosis.

beta-Nerve growth factor (NGF) is expressed in spermatogenic cells and has testosterone-downregulated low-affinity receptors on Sertoli cells suggesting a paracrine role in the regulation of spermatogenesis. An analysis of the stage-specific expression of NGF and its low affinity receptor during the cycle of the seminiferous epithelium in the rat revealed NGF mRNA and protein at all stages of the cycle. Tyrosine kinase receptor (trk) mRNA encoding an essential component of the high-affinity NGF receptor was also present at all stages. In contrast, expression of low affinity NGF receptor mRNA was only found in stages VIIcd and VIII of the cycle, the sites of onset of meiosis. The low-affinity NGF receptor protein was present in the plasma membrane of the apical Sertoli cell processes as well as in the basal plasma membrane of these cells at stages VIIcd to XI. NGF was shown to stimulate in vitro DNA synthesis of seminiferous tubule segments with preleptotene spermatocytes at the onset of meiosis while other segments remained nonresponsive. We conclude that NGF is a meiotic growth factor that acts through Sertoli cells.

Systematic review of hospital-wide complication registries.

BACKGROUND: An institutional registry covering all surgical specialties could be an implementation tool in quality benchmarking between hospitals and aid determination of their cost-effectiveness. The objective of this systematic literature review was to evaluate original articles on existing prospective surgical registries that can be used by single institutions across surgical specialties. METHOD: A systematic review of the literature using PRISMA guidelines was conducted for articles focusing on hospital-wide surgical registries. Single-specialty retrospective registries, non-defined outcome measures or system protocols, and studies not in English were excluded. RESULTS: Five articles were included for analysis. Evaluation of the articles revealed wide methodological heterogeneity in the classification and categorization of complications and data collection methods. CONCLUSION: Ideal surgical quality monitoring systems should be real-time, contain patient-related risk factors, and encompass all surgical specialties. At present, such institutional registries are rarely reported and no consensus exists on their standard definitions and methodology.

Derivation of functional antagonists using N-terminal extracellular domain of gonadotropin and thyrotropin receptors.

Receptors for the glycoprotein hormones, LH/CG, FSH, and TSH, are a unique subclass of the seven-transmembrane, G protein-coupled proteins with a large N-terminal extracellular (ecto-) domain. Although ecto-domains of gonadotropin receptors confer ligand binding, expression of soluble binding proteins has been difficult. We fused the ecto-domains of LH or FSH receptors to the single-transmembrane domain of CD8 and found that hybrid proteins anchored on the cell surface retained high-affinity ligand binding. Inclusion of a junctional thrombin cleavage site in the hybrids allowed generation of soluble receptor fragments that interfered with gonadotropin binding to their receptors and blocked cAMP production stimulated by gonadotropins. Cross-linking analyses confirmed the formation of high molecular weight complexes between receptor ecto-domains and their specific ligands. A similar approach also generated a soluble TSH receptor fragment capable of blocking TSH-induced signal transduction. When administered to rats, the soluble FSH receptor fragment retarded testis growth and induced testis cell apoptosis. These findings demonstrate the feasibility of generating ligand-binding regions of glycoprotein hormone receptors to selectively neutralize actions of gonadotropins and TSH, thus allowing future design of novel contraceptives and management of different gonadal and thyroid dysfunction. The present study represents the first successful derivation of soluble, ligand-binding domains from glycoprotein hormone receptors as functional antagonists. Similar approaches could allow generation of ecto-domains of related receptors to neutralize actions of ligands or receptor antibodies and to facilitate structural-functional analysis.

Interference of BAD (Bcl-xL/Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11.

Apoptosis and survival of diverse cell types are under hormonal control, but intracellular mechanisms regulating cell death are unclear. The Bcl-2/Ced-9 family of proteins contains conserved Bcl-2 homology regions that mediate the formation of homo- or heterodimers important for enhancing or suppressing apoptosis. Unlike most other members of the Bcl-2 family, BAD (Bcl-xL/Bcl-2 associated death promoter), a death enhancer, has no C-terminal transmembrane domain for targeting to the outer mitochondrial membrane and nuclear envelope. We hypothesized that BAD, in addition to binding Bcl-xL and Bcl-2, may interact with proteins outside the Bcl-2 family. Using the yeast two-hybrid system to search for BAD-binding proteins in an ovarian fusion cDNA library, we identified multiple cDNA clones encoding different isoforms of 14-3-3, a group of evolutionally conserved proteins essential for signal transduction and cell cycle progression. Point mutation of BAD in one (S137A), but not the other (S113A), putative binding site found in diverse 14-3-3 interacting proteins abolished the interaction between BAD and 14-3-3 without affecting interactions between BAD and Bcl-2. Because the S137A BAD mutant presumably resembles an underphosphorylated form of BAD, we used this mutant to screen for additional BAD-interacting proteins in the yeast two-hybrid system. P11, a nerve growth factor-induced neurite extension factor and member of the calcium-binding S-100 protein family, interacted strongly with the mutant BAD but less effectively with the wild type protein. In Chinese hamster ovary (CHO) cells, transient expression of wild type BAD or its mutants increased apoptotic cell death, which was blocked by cotransfection with the baculovirus-derived cysteine protease inhibitor, P35. Cotransfection with 14-3-3 suppressed apoptosis induced by wild type or the S113A mutant BAD but not by the S137A mutant incapable of binding 14-3-3. Furthermore, cotransfection with P11 attenuated the proapoptotic effect of both wild type BAD and the S137A mutant. For both 14-3-3 and P11, direct binding to BAD was also demonstrated in vitro. These results suggest that both 14-3-3 and P11 may function as BAD-binding proteins to dampen its apoptotic activity. Because the 14-3-3 family of proteins could interact with key signaling proteins including Raf-1 kinase, protein kinase C, and phosphatidyl inositol 3 kinase, whereas P11 is an early response gene induced by the neuronal survival factor, nerve growth factor, the present findings suggest that BAD plays an important role in mediating communication between different signal transduction pathways regulated by hormonal signals and the apoptotic mechanism controlled by Bcl-2 family members.

Localization of activin receptor (ActR-IIB2) mRNA in the rat seminiferous epithelium.

In situ hybridization was used to localize the mRNA expression of the high affinity activin receptor (ActR-IIB2) in the rat seminiferous epithelium. ActR-IIB2 mRNA was expressed maximally in stages IX-XI of the seminiferous epithelial cycle. The mRNA signal was detected basally in the epithelium in type A1 and A2 spermatogonia and in Sertoli cells. In the pubertal rat testis the expression was localized in Sertoli cells around primary spermatocytes and around meiotically dividing cells. The localization of ActR-IIB2 mRNA in spermatogonia lends support to the hypothesis that activin is a spermatogonial growth factor. The expression of activin receptor mRNA in pubertal rat testis suggests that activin may have a function during meiotic maturation.

Expression and function of a proapoptotic Bcl-2 family member Bcl-XL/Bcl-2-associated death promoter (BAD) in rat ovary.

Bcl-2-related anti- and proapoptotic proteins are important in the decision step of the intracellular death program upstream from the caspase proteases. Targeted overexpression of Bcl-2 in ovarian somatic cells of transgenic mice leads to decreased apoptosis of granulosa cells and is associated with higher ovulation rate, increased litter size, and ovarian teratoma formation. The ability of exogenous Bcl-2 proteins to promote follicle cell survival suggests that the transgene can bind to endogenous ovarian Bcl-2 family members and modulate the intracellular apoptosis process in favor of cell survival. We used the yeast two-hybrid system to search for ovarian Bcl-2 interacting proteins. The screening of an ovarian fusion complementary DNA library yielded several clones encoding for the death agonist Bcl-XL/Bcl-2-associated death promoter (BAD). Dimerization of Bcl-2-related proteins mediated by the consensus Bcl-2 homology (BH) domains is essential for their apoptosis-regulating function. Consistent with these observations, yeast two-hybrid assays indicated that the interaction of Bcl-2 with BAD is dependent on both BH4 and BH2 domains of Bcl-2. Northern blot analysis showed a wide distribution of BAD messenger RNA (mRNA) in diverse tissues with highest levels in the lung, ovary, uterus, and brain. In situ hybridization analysis indicated BAD mRNA expression in granulosa cells of different sizes of follicles and also in the theca and interstitial cells. BAD mRNA was expressed in the ovaries between postnatal 15-27 days and did not alter during the developmentally occurring apoptosis found about postnatal day 18 when the first group of early antral follicles were formed. Similarly, BAD mRNA levels did not change during follicle atresia induced by estrogen withdrawal in immature rats. To study the role of BAD in the ovary, BAD complementary DNA was transfected into primary cultures of granulosa cells and in a gonadal tumor cell line. Overexpression of BAD induced apoptosis in both cell types, and the effect of BAD was reversed by a membrane-permeable caspase inhibitor, indicating that BAD induces apoptosis via the activation of caspase cysteine proteases. In summary, the death agonist BAD was identified as a Bcl-2-interacting protein in the ovary, and BAD mRNA is constitutively expressed in granulosa cells, suggesting that BAD is an essential part of the ovarian cell death process. Because BAD overexpression in granulosa cells leads to apoptosis, future studies on ovarian BAD binding proteins and hormonal regulation of the interactions among different Bcl-2 family members could provide a better understanding of the cellular mechanism of ovarian follicle atresia.

Effects of activin-A, inhibin-A, and transforming growth factor-beta 1 on stage-specific deoxyribonucleic acid synthesis during rat seminiferous epithelial cycle.

Activin and inhibin are members of the transforming growth factor-beta (TGF beta) gene family. They are expressed in various organ systems, where they possess regulatory functions. Inhibin, activin, and TGF beta have been reported to also be expressed in the adult rat testis. We studied in vitro the action of these growth factors on premitotic and premeiotic DNA synthesis during the rat seminiferous epithelial cycle. Two-millimeter rat seminiferous tubule segments were isolated by transillumination-assisted microdissection from stages V, VIIa, VIII-IX, and I of the cycle and incubated in vitro in the presence of activin-A, inhibin-A, or TGF beta 1. During 24-, 48-, and 72-h incubation spontaneous progression of spermatogenesis was noted. The staged samples allowed us to selectively quantitate DNA synthetic activity of specific germ cell types. At the end of the culture, the tubules were pulse labeled with [3H]thymidine, and DNA synthesis was quantified by liquid scintillation counting, and the activated cells were detected by autoradiography. Activin-A stimulated preleptotene spermatocyte DNA synthesis in a dose-dependent manner. DNA synthesis of intermediate spermatogonia was also stimulated by activin-A, whereas inhibin-A inhibited DNA synthesis of these cells. TGF beta 1 had a small, but significant, stimulatory effect on DNA synthetic activity at stage VII. These results support the view that activin-A, inhibin-A, and TGF beta 1 take part in the regulation of DNA synthesis during rat spermatogenesis.

Sex difference in the action of activin-A on cell proliferation of differentiating rat gonad.

The effect of recombinant bovine activin-A on DNA synthesis in fetal rat gonads and mesonephroi was studied in vitro by using [3H]thymidine autoradiography to evaluate the role of activin in the regulation of gonadal development. mRNA levels of inhibin-beta A and -beta B, activin receptor II and IIB (ActRII and ActRIIB), and follistatin were studied by Northern hybridization in the fetal rat gonads and mesonephroi. Activin stimulated thymidine incorporation in ovaries and female mesonephroi on days 14 and 15 postcoitum (pc) in a dose-dependent manner, as assessed by autoradiography. In contrast, activin inhibited thymidine incorporation in testes and male mesonephroi on day 14 pc in a dose-dependent way. On day 18 pc, the stimulatory effect of activin in ovary was nearly lost, whereas that in mesonephros was still pronounced. Activin had no effect on thymidine incorporation in testes and male mesonephroi on day 15 or 18 pc. Inhibin-beta A mRNA was seen in testes and mesonephroi in the male and in mesonephroi in the female of 15 and 18 day pc embryos. Inhibin-beta A mRNA expression was also seen in the testes and epididymes of newborn male rats and in the ovaries of 11-day-old postnatal rats. Inhibin-beta B mRNA was present in both testes and ovaries from 15 day pc onward. ActRIIB mRNA was most abundant in the testes, ovaries, and mesonephroi of 15 day pc embryos. The expression of ActRIIB message diminished during aging. ActRII mRNA was ubiquitously expressed during development. Follistatin mRNA was seen in the ovaries from 15 day pc onward and in the oviducts at 11 days postnatally. The present novel findings of activin subunit and receptor mRNA expression and activin action on gonadal and mesonephric cell proliferation suggest an important, sexually dimorphic role for this substance in fetal gonadal differentiation.

Tumor necrosis factor-alpha and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles.

In the mammalian ovary, only a small fraction of follicles fully mature and ovulate, while most of them die via apoptosis. Multiple factors promoting follicle survival have been identified, but intraovarian mediators of apoptosis are poorly known. Tumor necrosis factor-alpha (TNF alpha) is a cytokine capable of inducing apoptosis in diverse cell types, and the apoptotic effect of TNF alpha is, partially, coupled to the sphingomyelin signaling pathway with ceramide as a second messenger. Because TNF alpha has been localized in the rat ovary, and TNF alpha treatment increases granulosa cell ceramide production, we studied the effect of treatment with TNF alpha and ceramide on follicle apoptosis. Immature rats were implanted with diethylstilbestrol to stimulate the development of early antral follicles. Follicles were isolated and cultured in a serum-free medium for 24 h with or without hormone treatments. During culture, spontaneous follicle apoptosis occurred (10-fold increase in DNA fragmentation), which was partially blocked by 100 ng/ml FSH (60% suppression). The effect of FSH was counteracted by TNF alpha in a dose-dependent manner, with the maximal effect at 100 ng/ml TNF alpha (90% reversal of FSH action). In situ analysis indicated that the granulosa cell is the follicle cell type undergoing DNA fragmentation. A membrane-permeable ceramide analog, C2-ceramide N-acetyl sphingosine, mimicked the effect of TNF alpha and was able to completely abolish the action of FSH at 50 microM. In contrast, another ceramide analog, C2-dihydroceramide N-acetyl dihydrosphingosine, did not alter the effect of FSH, verifying the specificity of ceramide action. To study the mechanism of TNF alpha and ceramide action, the effect of sodium aurathiomalate (ATM), an inhibitor of interleukin-1 beta-converting enzyme/ced-3-related cystine proteases known to be essential in the execution of mammalian cell apoptosis, was studied. Treatment with ATM (1 mM) prevented the apoptosis-inducing effect of both TNF alpha and ceramide, suggesting a role for cysteine proteases in mediating follicle apoptosis. Treatment with either TNF alpha or ceramide increased both basal and FSH-stimulated progesterone production by cultured follicles. Concomitant treatment by ATM did not alter the stimulatory effect of TNF alpha or ceramide on progesterone production, ruling out nonspecific toxic effect of the inhibitor and indicating that the apoptotic and steroidogenic pathways are independent. In summary, treatment with TNF alpha or its second messenger, ceramide, stimulates apoptosis of early antral follicles in culture, suggesting a potential role for TNF alpha as an intraovarian regulator of follicle atresia by acting through the ceramide signaling pathway.

Ontogeny of luteinizing hormone receptor gene expression in the rat testis.

The ontogeny of expression of the LH receptor (LHR) gene was studied in rat testis between day 12.5 of fetal life and adulthood. Specific hybridization of testicular mRNA with a LHR cRNA probe encoding the extracellular domain of the receptor was found from day 16.5 of fetal life onward in Northern hybridization. Transcripts of 6.8, 4.2, and 2.7 kilobases were present at all ages, and a 1.8-kilobase species was present mainly in the adult testes. Hybridization was most intensive in day 21.5 fetuses, decreased after birth, and increased again by adulthood. The LHR mRNA was also analyzed by the reverse transcriptase-polymerase chain reaction technique, with primers multiplying either the full-length LHR mRNA or its extracellular domain. The specificity of the DNA species generated was verified by Southern hybridization using a nested 32P-labeled oligonucleotide. The results indicated that a truncated mRNA form, encoding the extracellular part of LHR, appears 1 day before the full-length LHR mRNA, i.e. on fetal days 14.5 and 15.5, respectively. This is in striking contrast to the rat fetal ovary, in which a difference of more than 10 days is found in the appearance of these two LHR mRNAs (17.5 days of fetal and 7 days of postnatal age, respectively). The appearance of the full-length LHR mRNA coincides in both sexes with the developmental onset of LHR binding observed in earlier studies. In situ hybridization using an antisense cRNA probe demonstrated that the LHR mRNA was confined to Leydig cells at all fetal and postnatal ages studied. In conclusion, there is good correlation in the developing rat testes between the onset of LHR gene expression and LHR binding, as observed in earlier studies. The findings in the fetal testis are at striking variance with those in the ovary, which starts expressing the extracellular domain of the LHR mRNA at roughly the same age as the testis. However, the appearance of full-length LHR mRNA and the functional receptor are delayed until day 7 postpartum.

In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion.

The effects of FSH on stage-specific apoptosis and DNA synthesis in the adult rat seminiferous epithelium were studied in vitro. Seminiferous tubular segments from stages I, V, VIIa, and VIII-IX were cultured for 24, 48, and 72 h in different concentrations of FSH. Apoptotic cells were detected by in situ end labeling of DNA strands and quantified from squash preparations. After 48 h of culture, a FSH concentration of 2 ng/ml prevented apoptosis of early (steps 1-3) spermatids. In stage VIII-IX tubules cultured for 72 h, FSH decreased the apoptosis of pachytene spermatocytes. An apoptotic type of cell death of germ cells was confirmed by DNA laddering, electron microscopy, supravital acridine orange staining, and phase contrast microscopy of unstained living cells. The effects of FSH on stage-specific DNA synthesis were studied using the same culture system. FSH increased [3H]thymidine incorporation specifically at stages I and VIII-IX, and autoradiography confirmed stimulation of mitotic and meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes, respectively. Increased thymidine incorporation also suggested that FSH stimulated DNA synthesis of type A and intermediate spermatogonia. Most effects exerted by FSH were seen in stages containing high levels of FSH receptors and FSH-stimulated cAMP production. In conclusion, the results suggest that FSH, probably acting via Sertoli cells, has a regulatory function in spermatogenic apoptosis and DNA synthesis in stages previously demonstrated to be preferentially dependent on FSH stimulation.

Expression of inhibin beta A and beta B, follistatin and activin-A receptor messenger ribonucleic acids in the rat seminiferous epithelium.

The expression of inhibin beta A and beta B subunits, follistatin, and activin-A receptor messenger RNA (mRNAs) in different stages of rat seminiferous epithelial cycle was analyzed by in situ hybridization in order to understand their role in the regulation of spermatogenesis. Inhibin beta A mRNA was expressed in Sertoli cells in a highly stage-specific manner. The mRNA levels started to accumulate in Sertoli cells at stage VIII of the cycle and were highly expressed during stages IX-XI. Follistatin mRNA expression was identical to that of inhibin beta A, while inhibin beta B mRNA was maximally expressed in Sertoli cells at stages XIII-III. Low expression was found in stages VII-VIII. Activin-A receptor mRNA was localized mainly in spermatogenic cells. Maximal expression was seen in late primary spermatocytes at stages XIII-XIV and in early round spermatids at stages I-IV. A low even expression by Sertoli cells was also seen. Inhibin beta A and follistatin mRNAs were coexpressed in stage IX-XI Sertoli cells, suggesting close interplay between these molecules. The pattern of inhibin beta B mRNA expression was similar to that of inhibin alpha-mRNA. Localization of activin-A receptor mRNA in spermatogenic cells suggests that activin may influence meiotic divisions and early spermiogenesis.

Identification, ontogeny, and regulation of an interleukin-6-like factor in the rat seminiferous tubule.

The present study's aims are to search for the presence of interleukin-6 bioactivity (IL-6) in medium conditioned by various testicular cell types and to investigate the cellular and hormonal regulation of testicular IL-6 production. Sertoli cells prepared from rats of increasing ages (20, 35, and 45 days) secreted IL-6 in vitro, whereas medium conditioned by pachytene spermatocytes, early spermatids, and peritubular cells showed no activity. Lipopolysaccharide (LPS) and latex beads, two known stimulators of monocyte/macrophage IL-6 production, markedly stimulated IL-6 secretion by Sertoli cells at all the ages investigated. Maximum levels of IL-6 were reached after 6 h of culture of Sertoli cells with LPS and after 24 h with latex beads. When Sertoli cells were cocultured with pachytene spermatocytes, early spermatids, or fractions containing residual bodies and cytoplasts from elongated spermatids, only the latter significantly stimulated IL-6 levels. Maximum levels of IL-6 were attained by adding 2 x 10(6) residual bodies to Sertoli cells; a significant increase in IL-6 secretion was seen after 6 h, and maximum levels were observed after 24 h. The levels of IL-6 varied throughout different stages of the seminiferous epithelium cycle; highest levels were observed in stages II-VI and lowest in stages VII-VIII. IL-6 bioactivities induced by LPS and residual bodies and cytoplasts from elongated spermatids could be totally neutralized with a specific monoclonal antibody at all of the ages studied. FSH, phorbol myristate acetate, and IL-1 alpha augmented Sertoli cell IL-6 secretion in a dose-dependent manner. Furthermore, FSH and (Bu)2cAMP differentially stimulated IL-6 secretion during the seminiferous epithelial cycle. It is concluded that the release of IL-6 from Sertoli cells is regulated by a complex interplay between residual bodies and humoral factors.

Expression of inhibin alpha, beta A and beta B messenger ribonucleic acids in the normal human ovary and in polycystic ovarian syndrome.

We studied the cellular distribution of inhibin alpha, beta A and beta B mRNAs in the normal human ovary and in polycystic ovarian syndrome (PCOS) by in situ hybridization. Our results show that human granulosa cells express inhibin alpha, beta A and beta B subunit mRNAs, and theca cells express inhibin alpha and beta A subunit mRNAs. The co-localization of alpha and beta A mRNAs in theca cells supports the hypothesis that inhibin also has an autocrine function in these cells. We did not detect any inhibin subunit mRNA in the granulosa cells of atretic follicles, while theca cells also expressed alpha subunit mRNA in those follicles. The present findings suggest that the expression of inhibin subunits is regulated differently in human follicular granulosa and theca cells. It has been speculated that inhibin may be involved in the development of PCOS. Our results show that the cellular localization of inhibin subunit mRNAs is not disturbed in PCOS ovaries.

Stage-specific cellular regulation of inhibin alpha-subunit mRNA expression in the rat seminiferous epithelium.

To find out the local regulation of inhibin production and its possible paracrine role in the seminiferous epithelium, inhibin alpha mRNA levels were measured in sequential 1 mm segments of rat seminiferous tubules accurately staged by transillumination technique. Highest levels were found at stages XIV-I-IV of the cycle, and lowest at stages VI-VIIb of the cycle. When dividing spermatogonia were selectively destroyed by 3 Gy of high-energy X-irradiation, stage-specific inhibin alpha mRNA levels remained unchanged until 26 and 38 days after irradiation when stages VII and VIII of the cycle showed 6- and 4-fold increases during a selective reduction of pachytene spermatocyte and round spermatid numbers, respectively. The results suggest that these cells at a strictly stage-specific fashion have a paracrine inhibitory effect on Sertoli cell inhibin alpha gene expression. Inhibin alpha mRNA level also correlates closely to the follicle-stimulating hormone-stimulated cAMP production during the cycle of the seminiferous epithelium, but does not seem to have a correlation to spermatogonial DNA synthesis.

Cell death and survival during ovarian follicle development.

Mammalian germ cells arise in the yolk sac endoderm at the caudal aspect of the embryo and migrate to the mesodermally-derived gonadal ridge early in development. After the oogonia reach the gonadal ridge, the process of meiosis begins which coincides with the first major wave of apoptosis of female germ cells (Coucouvanis et al., 1993). Subsequently, oocytes progress to the dictyate stage of prophase I where they remain arrested until ovulation.

Localization of urokinase- and tissue-type plasminogen activator mRNAs in rat testes.

The expressions of urokinase (uPA) and tissue-type plasminogen activators (tPA) in different stages of the rat seminiferous epithelial cycle were analyzed by in situ and Northern hybridizations combined with zymographic analysis. Irradiated rat testes were used to assess the cell localization. Both of the plasminogen activators were expressed in a strictly stage specific manner. Maximal expression of uPA mRNA was seen in Sertoli cells during stages VII-VIII of the cycle. The same expression in the basal compartment of the tubules was detected at 7 days post-irradiation (p-i), during a selective reduction of spermatogonia and preleptotene spermatocytes. Levels of tPA mRNA started to accumulate in Sertoli cells at stage VIII and were high during stages IX-XII and detectable during stages XIII-XIV. At 26 days p-i, reduction of pachytene spermatocytes, which are shown to be immunoreactive for tPA, did not have an effect on tPA mRNA expression. Catalytic activities of uPA and tPA changed concomitantly to their RNA levels in different stages of the cycle. However, at 7 days p-i, uPA activity was decreased at stages VII-VIII of the cycle suggesting that germ cell Sertoli cell interaction is important for uPA activity.

Stage- and cell-specific expression of the ornithine decarboxylase gene during rat and mouse spermatogenesis.

Ornithine decarboxylase (ODC) is an enzyme that has been shown to be induced in the growth, differentiation and proliferation of cells. We have used a cDNA probe to determine ODC mRNA levels in different stages of the cycle of rat and mouse seminiferous epithelium. For Northern and slot-blot hybridizations, RNA was isolated from microdissected staged seminiferous tubules. Cell-specific localization of ODC mRNA was studied by in situ hybridization. In the rat, in situ hybridization showed increasing mRNA levels during prophase of meiosis with the highest mRNA levels seen in late pachytene spermatocytes and step 3-5 spermatids. In the mouse, the mRNA levels increased in a similar fashion and the highest mRNA levels were found in step 1-8 spermatids. In the rat, Northern blot hybridizations revealed three molecular sizes of ODC mRNA: 2.2, 2.7 and 1.6 kb. The levels of all molecular sizes were highest in stages VII-VIII, and the lowest mRNA levels were seen in stage I of the seminiferous epithelial cycle. The level of the 2.2 kb transcript was low during stages XIII-I. In the mouse, the Northern blot hybridizations also showed three molecular sizes of ODC mRNA: 2.2 and 2.7 kb and very low levels of 1.6 kb transcript. The levels of the transcripts were steady throughout the cycle. In the mouse, the 2.2 kb transcript was more abundant than the 2.7 kb transcript indicating a species difference between rat and mouse in the usage of the two polyadenylation signals within the ODC gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Follicle-stimulating hormone regulation of inhibin alpha-subunit mRNA in staged rat seminiferous tubules.

Steady-state levels of inhibin-alpha and -beta B mRNAs are higher in stages II-VI of the seminiferous epithelial cycle than in stages VII-VIII. We investigated follicle-stimulating hormone (FSH) regulation of inhibin alpha-subunit mRNA in stages II-VI and VII-VIII to study whether the stage specificity is due to differential hormonal regulation by FSH. Follicle-stimulating hormone caused a significant increase of inhibin-alpha mRNA levels in both stages during a 20-h incubation. The mechanism of the FSH effect was studied further in stages VII-VIII. Maximal stimulation of the inhibin-alpha mRNA level was achieved with 100 micrograms/l FSH, dibutyryl-3',5'-cyclic adenosine monophosphate (db-cAMP, 0.2 mmol/l) and Sp-adenosine-3',5'-monophosphothionate (Sp-cAMPS, 10 mumol/l) (a cAMP agonist). The presence of Rp-cAMPS (200 mumol/l) (a cAMP antagonist) abolished the stimulation, Rp-cAMPS alone had no effect. Inhibin-beta B mRNA levels in stages VII-VIII were not affected by FSH, db-cAMP, Sp-cAMPS or Rp-cAMPS. Phorbol 12-myristate 13-acetate (100 nmol/l) had no effect on inhibin-alpha or -beta B mRNA levels. Actinomycin D abolished the stimulatory effect of FSH on inhibin-alpha mRNA expression. In conclusion, FSH stimulated inhibin-alpha mRNA expression similarly both in stages II-VI and VII-VIII of the seminiferous epithelial cycle and the stimulation in stages VII-VIII was cAMP-mediated.

The expression and regulation of Bcl-2-related ovarian killer (Bok) mRNA in the developing and adult rat testis.

OBJECTIVE: To study the role of Bcl-2-related ovarian killer (Bok) in the regulation of apoptosis in the testis of developing and adult rat. METHODS: Bok mRNA expression was analyzed by Northern hybridization before and after culturing rat seminiferous tubules in vitro. Seminiferous tubules were cultured with different hormones and growth factors. Changes in the expression level of Bok mRNA during testicular development was analyzed by Northern hybridization. Localization of Bok mRNA was verified by in situ hybridization. RESULTS: Bok mRNA was highly expressed in the rat testis, varying during development. Highest expression levels were found in immature rats. Highest hybridization intensity appeared to be in spermatogonia, pachytene spermatocytes and Sertoli cells. Treatment with FSH was able to inhibit spontaneous increase of Bok mRNA expression that occurred in the defined stages of the rat seminiferous epithelium. CONCLUSIONS: FSH protects germ cells from apoptosis and this protective effect may at least partly be due to the inhibition of Bok gene expression. The amount of apoptosis varies during testicular development and highest expression of Bok mRNA occurs at the time of apoptosis, suggesting a possible role for Bok in its regulation.