Biomedical subjects

M Parvinen

Publications and source records attributed to M Parvinen.

At least 55 records · Page 3Linked to original sources

PubMed1996

Spermiogenesis deficiency and germ-cell apoptosis in CREM-mutant mice.

Spermiogenesis is a complex process by which postmeiotic male germ cells differentiate into mature spermatozoa. This process involves remarkable structural and biochemical changes including nuclear DNA compaction and acrosome formation. Transcription activator CREM (cyclic AMP-responsive element modulator) is highly expressed in postmeiotic cells, and CREM may be responsible for the activation of several haploid germ cell-specific genes involved in the structuring of the spermatozoon. The specific role of CREM in spermiogenesis was addressed using CREM-mutant mice generated by homologous recombination. Analysis of the seminiferous epithelium in mutant male mice reveals postmeiotic arrest at the first step of spermiogenesis. Late spermatids are completely absent, and there is a significant increase in apoptotic germ cells. We show that CREM deficiency results in the lack of postmeiotic cell-specific gene expression. The complete lack of spermatozoa in the mutant mice is reminiscent of cases of human infertility.

Animals↗

PubMed1996

Microtubule-associated protein-2 in the rat testis: a novel site of expression.

The testis is one of the most abundant sources of microtubule networks. These networks include mitotic and meiotic spindles, the spermatid manchette and axoneme, and the Sertoli cell cytoskeleton. Microtubules are composed of alpha- and beta-tubulin subunits that are polymerized and stabilized by a variety of microtubule-associated proteins (MAPs). One of these, MAP2, has been extensively characterized as a brain-specific protein with the capacity to bind tubulin, cAMP-dependent kinase, and calmodulin. MAP2 mRNA is processed into at least two variants encoding proteins designated MAP2a, MAP2b, and MAP2c. Of the 5.7 kb of coding sequence in the 9-kb mRNA that encodes MAP2a and MAP2b, a deletion of approximately 4 kb produces mRNA encoding MAP2c, which consists of only the N- and C- terminal regions of MAP2b. To determine whether MAP2 was present in the rat testis, microtubule preparations were isolated from adult rat testis and brain by means of taxol-mediated polymerization and analyzed by gel filtration, ELISA, and Western blotting using polyclonal and monoclonal antibodies reactive with MAP2. A 74-kDa protein corresponding to MAP2c was detected in the testis. These results were confirmed by Northern blot analysis of total RNA from adult rat brain and testis with cDNA probes that distinguish between the known MAP2 splice variants. The predominant mRNAs in testis of 6 kb and 2.5-3.5 kb corresponded to MAP2c. A single 6-kb mRNA with the potential to encode MAP2c was detected in enriched preparations of immature Sertoli cells and adult Leydig cells. Round spermatids contained at least two MAP2 mRNAs between approximately 2.5 and 3.5 kb in size that displayed a stage-specific pattern of expression. Immunohistochemistry showed a MAP2-like protein in both somatic and germ cells, with a particularly distinct localization within the cytoplasm of primary and secondary spermatocytes at stage XIV of the seminiferous cycle during meiotic metaphase. In addition to cytoplasmic staining, a novel localization of this protein was observed in the nucleus of many testicular cells.

Animals↗

PubMed1996

Variation in expression of hsp27 messenger ribonucleic acid during the cycle of the seminiferous epithelium and co-localization of hsp27 and microfilaments in Sertoli cells of the rat.

The purpose of these studies was to define expression of hsp27 mRNA during the cycle of the seminiferous epithelium and to determine the distribution of hsp27 protein in the rat testis. To study hsp27 mRNA expression, a rat hsp27 cDNA was isolated and sequenced (GenBank no. M86389). The cDNA was used in Northern blot analysis to estimate the relative levels of hsp27 mRNA in rat seminiferous tubule segments selected for different stages of the cycle of the seminiferous epithelium. The level of hsp27 mRNA was low during stages IX-XII of the cycle of the seminiferous epithelium. Approximately 15-fold higher levels of hsp27 mRNA were expressed during stages II-VI, with intermediate levels being expressed during stages XIII-I and VII-VIII. No effect of FSH on hsp27 mRNA expression was detected in cultured Sertoli cells, suggesting that hsp27 synthesis in Sertoli cells is not directly regulated by FSH. The distribution of hsp27 was also studied by use of immunofluorescence in frozen sections of rat testis, in isolated seminiferous tubules, in primary cultures of Sertoli cells isolated from 19-26-day-old rats, in peritubular myoid cells from 26-day-old rats, and in several cell lines. Microfilaments were localized in similar preparations by using rhodamine-phalloidin or BODIPY-phallicidin (Molecular Probes, Eugene, OR). The hsp27 was co-localized with micro-filaments in Sertoli cells from 20-day-old and older rats, but not in Sertoli cells from younger rats. In other cell types, hsp27 was diffusely distributed throughout the cytoplasm. These results demonstrate that hsp27 expression varies with the cycle of the seminiferous epithelium and provide the first direct morphological evidence that hsp27 is associated with microfilaments in a normal, intact tissue. They also suggest that Sertoli cell micro-filaments, by virtue of their associated hsp27, may be different in composition and function from microfilaments of peritubular myoid cells and many other cell types.

Actin Cytoskeleton↗

PubMed1996

Lipopolysaccharide induced apoptosis of rat pancreatic acinar cells.

BACKGROUND: Bacterial lipopolysaccharide (LPS) has been proposed to participate in the pathogenesis of pancreatic inflammatory disease. AIMS: This study investigated the role of endotoxaemia in the pathogenesis of pancreatic acinar cell injury. METHODS: Sixty eight male Spraque-Dawley rats were used in the study. Escherichia coli LPS (5 mg/kg) was injected into the peritoneal cavity of the rats. The concentration of pancreatic phospholipase A2 (PLA2) in plasma was measured and pancreatic tissue examined by histology, in situ detection of free DNA 3'-ends, and electrophoretic DNA analysis. RESULTS: The concentration of pancreatic PLA2 increased in plasma and the catalytic activity of PLA2 increased in pancreatic tissue after an LPS injection. Apoptosis in pancreatic acinar cells and fragmentation of DNA typical of apoptosis in pancreatic tissue was seen 24 hours after an LPS injection. Pancreatic acinar atrophy was seen 72 hours after the LPS injection. CONCLUSIONS: These data show that LPS causes release of pancreatic PLA2 into blood plasma, activation of PLA2 in pancreatic tissue, and apoptosis of acinar cells.

Animals↗

PubMed1996

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.

Acridine Orange↗

PubMed1996

Hormonal and developmental regulation of DAX-1 expression in Sertoli cells.

Mutations in the human DAX-1 gene lead to X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. DAX-1 has been proposed to play a role in steroidogenesis because it is highly expressed in adrenocortical and testicular Leydig cells and because loss-of-function mutations lead to low serum levels of steroid hormones. Recent reports of DAX-1 expression in hypothalamus and pituitary, however, suggest additional functions for this protein. Here we demonstrate that DAX-1 is expressed in Sertoli cells of rat testis. This expression is regulated during spermatogenesis and peaks during the androgen-sensitive phase of the spermatogenic cycle. In addition, we show that DAX-1 expression in Sertoli cells is regulated developmentally. Maximum levels are present in the rat between postnatal days 20 and 30, during the first spermatogenic wave. Moreover, we show that activation of the cAMP-signaling pathway by the pituitary hormone FSH leads to a potent down-regulation of DAX-1 expression in cultured Sertoli cells. This down-regulation requires transcription and de novo protein synthesis. Taken together, these data indicate that DAX-1 expression in Sertoli cells may influence the development of spermatogenic cells in response to steroid and pituitary hormones.

Animals↗

PubMed1996

Stage-specific expression of the FSH receptor gene in the prepubertal and adult rat seminiferous epithelium.

Stage-specific expression of the FSH receptor (FSHR) gene in the rat seminiferous epithelium was studied. Using transillumination-assisted microdissection for sample preparation and Northern hybridization for analysis of total RNA, we first reassessed the stage specificity of the FSHR gene expression in the adult rat testis. Sixfold higher FSHR mRNA levels were found in stages XIII-I compared with stage VI of the seminiferous epithelial cycle, which had the lowest signal level (P < 0.01). The other stages had intermediate signal levels. In situ hybridization showed distribution of grains which confirmed the data obtained by Northern analysis. Prepubertal stage-specific FSHR gene expression was studied using in situ hybridization. Stage specificity could first be demonstrated at the age of 16 days when the average grain counts in stages I-IV were threefold higher than in stages VI-VII (P < 0.01). The present data are in agreement with earlier findings on stage-specific FSH binding and FSHR gene expression using both microdissected and stage-synchronized seminiferous tubules. The onset of stage-specific FSHR gene expression is concomitant with maturation of the Sertoli cell population and completion of the first generation of spermatocytes. This supports the hypothesis that spermatogonia and spermatocytes may be involved in the regulation of FSHR gene expression.

Animals↗

PubMed1995

Follicle-stimulating hormone regulates the expression of cyclic protein-2/cathepsin L messenger ribonucleic acid in rat Sertoli cells in a stage-specific manner.

Cyclic protein-2/cathepsin L (CP-2) is secreted by Sertoli cells in a highly stage-specific manner, maximally during stages VI-VII of the rat seminiferous epithelial cycle. We investigated FSH regulation of CP-2 mRNA expression of its cellular localization in isolated staged seminiferous tubular segments. FSH induced a significant increase of CP-2 mRNA expression and its cellular localization in isolated staged seminiferous tubular segments. FSH induced a significant increase of CP-2 mRNA levels in stages IX-I, whereas in stages II-VIII, the levels of CP-2 mRNA were reduced. A similar effect was produced by two cAMP analogs, dbcAMP (0.2 mM) and Sp cAMP (20 microM). FSH and cAMP did not affect on the levels of SGP-2 mRNA during the seminiferous epithelial cycle. The magnitude of the response was time- and dose-dependent; the maximum was obtained with 100 ng/ml of FSH. It is likely that FSH regulates Cp-2 gene transcription, since de novo RNA synthesis was required for the stimulatory FSH effect on CP-2 mRNA levels, while ongoing protein synthesis was not. In conclusion, the data suggest that FSH, via cAMP-mediated pathway, regulates CP-2/cathepsin L gene transcription in rat Sertoli cells and modulated the stage-specific expression pattern.

Animals↗

PubMed1995

Function of interleukin-6 as an inhibitor of meiotic DNA synthesis in the rat seminiferous epithelium.

Interleukin-6 bioactivity (IL-6) has been shown to be present in Sertoli cells. To further characterize the IL-6 in the seminiferous epithelium, the IL-6 like-antigen was detected, stage-specific basal distribution of IL-6-like bioactivity and its regulation by FSH, cAMP and TPA was characterized in isolated, rat seminiferous tubule segments. In addition, the effects of human recombinant IL-6 on stage-specific DNA synthesis was investigated. Both monoclonal and polyclonal antibodies recognized M(r) 22 and 23 kDa of IL-6 like immunoreactivity in the seminiferous epithelium. The basal IL-6 production showed high levels during stages XIII-XIV-I-V, low during VII and VIII. FSH stimulated IL-6 production at nearly all stages and most significantly at stage VII of the cycle. Human recombinant IL-6 dose-dependently inhibited the onset of meiotic DNA synthesis of preleptotene spermatocytes, and a minor inhibition was found on advanced (A3-type B) spermatogonia. These results support the hypothesis that IL-6 is a stage-specific paracrine regulator of the seminiferous epithelium exerting a specific inhibitory action on meiotic DNA synthesis.

Animals↗

PubMed1995

Germ cell-Sertoli cell interactions: regulation by germ cells of the stage-specific expression of CP-2/cathepsin L mRNA by Sertoli cells.

CP-2/cathepsin L mRNA is expressed primarily by rat Sertoli cells within stage VI-VIII seminiferous tubules. To test whether germ cells regulated this expression, we examined if separating Sertoli cells from specific germ cells affected expression of this transcript in Sertoli cells. First, Sertoli cells were isolated from adult (90-day-old) and immature (25-day-old) rats and levels of this transcript measured immediately or after 1, 3 and 5 days in culture. Results demonstrated that immediately upon isolation, CP-2/cathepsin L mRNA levels were significantly higher in mature cells. However, after 1 day in culture, the levels of this transcript increased in immature cells and remained high in mature cells. We therefore conclude that in vivo, a subset of germ cells inhibit the expression of CP-2/cathepsin L mRNA by immature Sertoli cells. Second, to examine the effect of specific germ cells on CP-2/cathepsin L mRNA expression, we exposed the testes of mature rats to 3 Gy of gamma-radiation and analyzed stage-specific expression of this transcript at varying times during maturation depletion and subsequent germ cell restoration. Loss of spermatogonia or spermatocytes was without effect. However, when pachytene spermatocytes through step 14 spermatids were depleted, expression at stages VI-VIII was reduced by half and expression at stages IX-I was increased 14-fold. These changes resulted in the loss of stage-specific expression of CP-2/cathepsin L mRNA by Sertoli cells. Finally, stage VI-VIII tubules, depleted primarily in step 15-19 spermatids, had levels of CP-2/cathepsin L mRNA that were 60% of control. However, stage-specific expression of this transcript was detected in these tubules. In contrast to what we noted with CP-2/cathepsin L mRNA, loss and restoration of germ cells had no effect on Sertoli cell levels of SGP-2 mRNA, indicating that testicular irradiation had no overall effect on Sertoli cell function. Taken together, these data suggest that the stage-specific expression of the CP-2/cathepsin L gene results from the sequential stimulation and inhibition of Sertoli cells by germ cells, that pachytene spermatocytes through step 14 spermatids are required for this stage-specific expression and that step 18 and 19 spermatids amplify this expression at stages VI-VIII.

Animals↗

PubMed1995

Stage-specific DNA synthesis of rat spermatogenesis as an indicator of genotoxic effects of vinblastine, mitomycin C and ionizing radiation on rat spermatogonia and spermatocytes.

We have studied the effects of three known mutagens: vinblastine sulphate, mitomycin C and local irradiation of testes on the stage-specific DNA synthesis in the rat testis by using transillumination assisted microdissection of rat seminiferous tubules. It enables us to investigate the sensitivity of different types of spermatogonia and preleptotene spermatocytes to the genotoxic effects of these agents. According to our results, spermatogonia and preleptotene spermatocytes are quite resistant to the action of vinblastine at the treatment times and the doses used. After treatment with mitomycin C, type A2, A3 and A4 spermatogonia seem to be the first cell types affected, which shows itself as a reduction in the DNA synthesis at stages I, II-III, XIII-XIV of the epithelial cycle two and/or three days after the treatment. It also seems that they are mostly affected during the S-phase of their cell cycles. In addition, preleptotene spermatocytes are also sensitive to the action of mitomycin C when they are treated in the G1 phase of the cell cycle. The local irradiation of 3 Gy has severe effects on the spermatogonia of rat testis which can be seen already 18 h after the treatment and becomes more evident 42 and 66 h after the treatment as a reduction of DNA synthesis at stages XII-V. Type A spermatogonia (A1-A4) seem to be the most sensitive cell types to the action of irradiation. This study indicates that the novel method of stage-specific DNA synthesis in rat spermatogenesis allows detailed studies of sensitivities in differentiating spermatogonia to genotoxic agents.

Animals↗

PubMed1995

Expression of the mad gene during cell differentiation in vivo and its inhibition of cell growth in vitro.

Mad is a basic region helix-loop-helix leucine zipper transcription factor which can dimerize with the Max protein and antagonize transcriptional activation by the Myc-Max transcription factor heterodimer. While the expression of Myc is necessary for cell proliferation, the expression of Mad is induced upon differentiation of at least some leukemia cell lines. Here, the expression of the mad gene has been explored in developing mouse tissues. During organogenesis in mouse embryos mad mRNA was predominantly expressed in the liver and in the mantle layer of the developing brain. At later stages mad expression was detected in neuroretina, epidermis, and whisker follicles, and in adult mice mad was expressed at variable levels in most organs analyzed. Interestingly, in the skin mad was highly expressed in the differentiating epidermal keratinocytes, but not in the underlying proliferating basal keratinocyte layer. Also, in the gut mad mRNA was abundant in the intestinal villi, where cells cease proliferation and differentiate, but not in the crypts, where the intestinal epithelial cells proliferate. In the testis, mad expression was associated with the completion of meiosis and early development of haploid cells. In cell culture, Mad inhibited colony formation of a mouse keratinocyte cell line and rat embryo fibroblast transformation by Myc and Ras. The pattern of mad expression in tissues and its ability to inhibit cell growth in vitro suggests that Mad can cause the cessation of cell proliferation associated with cell differentiation in vivo.

Amino Acid Sequence↗

PubMed1995

Expression of mitochondrial heat shock protein 60 in distinct cell types and defined stages of rat seminiferous epithelium.

Changes in the level of the gene transcript of heat shock protein (hsp)60, a mitochondrial chaperonin, during the cycle of rat seminiferous epithelium and its cellular localization were studied. The seminiferous epithelium showed a cell type-specific expression of hsp60. Immunostaining of adult rat testis revealed localization in Sertoli and Leydig cells. In germ cells, mitochondria of spermatogonia and early primary spermatocytes were immunoreactive for hsp60. Mitochondria of all other germ cell types were completely negative for hsp60. Stage-specific expression of hsp60 was determined from pooled segments of stage-specific microdissected tubules by a combination of Western blotting and polymerase chain reaction (PCR). High concentrations of hsp60 were found in stages I-V and IX-XIV, and low levels were detected in the other stages, i.e., VI-VIII. In stages with high hsp60 expression, spermatogonia divide mitotically, whereas in stages lacking mitosis, the hsp60 level was much weaker. In seminiferous epithelium, two different types of mitochondria are present. Therefore, immunoelectron microscopy was used to differentiate these two morphologically distinct types of mitochondria. The crista type of mitochondria (e.g., in Sertoli cells and spermatogonia) reacted with the antibody against hsp60, whereas hsp60 was negative in so-called "condensed"-type mitochondria found in midpachytene spermatocytes and more advanced germ cells. It could be shown for the first time that expression of the hsp60 gene is regulated during the cycle of the seminiferous epithelium. The results indicate that the gene product is primarily needed during the initial steps of spermatogenesis in which most of the cell divisions occur, while its expression during the differentiation of spermatids and sperm is obviously not necessary. The presence of hsp60 in stages with mitotic activity suggests a very active mitochondrial protein import and protein assembly machinery that generates further mitochondria for the dividing cells.

Animals↗

PubMed1995

Expression of immediate early genes in tubular cells of rat testis.

In this study we investigated the expression of the immediate early genes (IEGs) c-fos, c-jun, and junD on mRNA and protein levels during the spermatogenic cycle of the rat using Northern blotting, in situ hybridization, and immunocytochemistry. The expression of these IEG mRNAs and proteins exhibited stage-specific variations. The results suggest that IEGs take part in transcriptional events that are involved in regulating the proliferation and differentiation of spermatogenic cells during specific stages of the cycle of the seminiferous epithelium.

Animals↗

PubMed1995

Haploid gene expression: temporal onset and storage patterns of 13 novel transcripts during rat and mouse spermiogenesis.

The temporal and spatial expression of thirteen novel spermatid-specific genes corresponding to cDNA clones isolated from an adult mouse testis library was analyzed. Northern analysis of RNA from seminiferous tubules at defined stages of the rat and mouse seminiferous epithelial cycle and in situ hybridization of testis sections revealed that these mRNAs were expressed in a stage-specific manner. The expression of all mRNAs was first detected in early round spermatids, and it increased to abundance during stages VII-VIII of the epithelial cycle. Twelve out of thirteen mRNAs were found not only in round spermatids but also in transcriptionally inactive elongated spermatids, suggesting that they are stored and regulated at the translational level. The variation in the length of the poly(A) tail was detected for four of the transcripts, represented by cDNA clones MTEST70, MTEST627, MTEST641, and MTEST643 at defined stages of the cycle. Similarity in the stage-specific expression pattern displayed by this group of haploid-specific genes strongly suggests the presence of common regulatory mechanisms that act during spermiogenesis, and these genes also provide a means for further studies of these mechanisms.

Animals↗

PubMed1995

Apoptosis in testis germ cells: developmental changes in gonadotropin dependence and localization to selective tubule stages.

Recent studies have demonstrated apoptotic DNA fragmentation in the testis of immature rats deprived of gonadotropins. However, the exact cell type undergoing apoptosis during testis development and the age differences of gonadotropin dependence of testis cell apoptosis are unclear. The present study used gel fractionation and in situ methods to quantitate developmental changes of testis cell DNA fragmentation and to localize the specific cell type affected in developing rats with and without treatment with a GnRH antagonist. Apoptotic DNA fragmentation in whole testis was measured in rats between 8-70 days of age. A gradual increase (1.8- to 2.0-fold) in testis apoptotic DNA fragmentation was seen in rats between 16-28 days of age, compared with 8-day-old animals, followed by a decrease in adult animals. To study gonadotropin dependence of testicular apoptosis, serum FSH and, to a lesser extent, LH were suppressed by treatment with a long-acting GnRH antagonist (azaline-B, 250 micrograms/kg body wt, two injections at 2-day intervals). Pretreatment with the GnRH antagonist increased apoptotic DNA fragmentation in rats between 16-32 days of age but not in younger and adult animals demonstrating an age-related change in gonadotropin dependence. To identify the exact testis cell type undergoing apoptosis, in situ analysis of DNA fragmentation was performed. In rats at 16-24 days of age, spermatocytes in selected tubules were found to have increased DNA fragmentation. In contrast, neither Leydig cells nor Sertoli cells were affected. In 32-day-old and adult animals, increased DNA fragmentation was seen in early primary spermatocytes of some tubules. Treatment with GnRH antagonist increased the number of cells with DNA fragmentation as well as percentage of tubules affected. In animals between 16-32 days of age, meiotic spermatocytes were labeled, whereas early spermatids were also labeled in 24- and 32-day-old animals. In adult animals, the level of apoptotic DNA fragmentation was not affected by GnRH antagonist treatment. However, DNA isolated from specific stages of the seminiferous tubules of adult animals showed stage-specific changes of apoptotic DNA fragmentation with 2-fold higher levels found in stages I and XII-XIV compared with stage VIII. In situ analysis of adult testis demonstrated that spermatocytes were the major cell type affected. In conclusion, the present study demonstrated that at least three factors determine the onset of apoptosis of the male germ cells: 1) the developmental stage of the animal; 2) serum levels of gonadotropins, especially FSH; and 3) specific stage of the seminiferous epithelial cycle. The present approach provides the basis for future analysis of the role of gonadotropins and other factors in the regulation of testis cell degeneration in normal and pathological states.

Animals↗

PubMed1995

Anti-müllerian hormone and anti-müllerian hormone type II receptor messenger ribonucleic acid expression during postnatal testis development and in the adult testis of the rat.

Anti-müllerian hormone (AMH) induces degeneration of the müllerian ducts during male sex differentiation and may have additional functions concerning gonadal development. In the immature rat testis, there is a marked developmental increase in AMH type II receptor (AMHRII) messenger RNA (mRNA) expression in Sertoli cells, concomitant with the initiation of spermatogenesis. AMHRII mRNA is also expressed at a high level in Sertoli cells in adult rats. To obtain information about the possible functions of AMH in the testis, we investigated the postnatal expression patterns of the genes encoding AMH and AMHRII in the rat testis in more detail. Using RNase protection assays, AMH and AMHRII mRNA expression was measured in total RNA preparations from testes or testicular tubule segments isolated from control rats and from rats that had received various treatments. The testicular level of AMHRII mRNA was found to be much higher than that of AMH mRNA in adult rats. AMH mRNA was detected at a maximal level at stage VII of the spermatogenic cycle and at a low level at the other stages. AMHRII mRNA increases from stage XIII, is highest at stages VI and VII, and then rapidly declines at stage VIII to almost undetectable levels at stages IX-XII. It was found that the increase in testicular AMHRII mRNA expression during the first 3 weeks of postnatal development also occurs in sterile rats (prenatally irradiated), and hence, is independent of the presence or absence of germ cells. Yet, the total testicular level of AMHRII mRNA was decreased in sterile adult rats (prenatally irradiated or experimental cryptorchidism), as compared with intact control rats. However, treatment of adult rats with methoxyacetic acid or hydroxyurea, which resulted in partial germ cell depletion, had no effect on total testicular AMHRII mRNA expression. We conclude that a combination of multiple spermatogenic cycle events, possibly involving changes of Sertoli cell structure and/or Sertoli cell-basal membrane interactions, regulate autocrine AMH action on Sertoli cells, in particular at stage VII of the spermatogenic cycle.

Age Factors↗

PubMed1995

Residual bodies activate Sertoli cell interleukin-1 alpha (IL-1 alpha) release, which triggers IL-6 production by an autocrine mechanism, through the lipoxygenase pathway.

Interleukin-1 (IL-1) and IL-6 are produced by Sertoli cells. As IL-1 stimulates IL-6 production in some tissues, the cascade of events that results in IL-6 secretion by Sertoli cells was studied. The addition of IL-1 alpha to Sertoli cells resulted in a time-dependent increase in IL-6 secretion. Incubation of Sertoli cells with two known stimulators of IL-1 production, lipopolysaccharide (LPS) and residual bodies, resulted in a significant increase in IL-1 release into the medium several hours before IL-6 release. That IL-1 is essential for IL-6 production from Sertoli cells was established by blocking the actions of LPS and residual bodies with an anti-IL-1 alpha antibody. An increase in the release of IL-1 before IL-6 was also observed in medium obtained from staged segments of intact seminiferous tubules; IL-1 reached a maximum level at stage VIII, when mature spermatozoa are released and residual bodies are formed and phagocytosed. The secretion of IL-6 was low during this stage and then increased progressively from stage IX onward, consistent with IL-1 stimulation of IL-6. The pathway of IL-1 alpha-induced release of IL-6 was studied in the presence of agents that influence arachidonic acid release and metabolism. IL-1 alpha was found to stimulate arachidonic acid release by Sertoli cells. Furthermore, a phospholipase A2 inhibitor, aristolochic acid, significantly decreased IL-1-, LPS-, and pyrularia pubera thionin-induced IL-6 secretion from Sertoli cells. Indomethacin, a specific inhibitor of the cyclooxygenase pathway, had no significant effect on basal, but enhanced IL-1- and LPS-stimulated IL-6 production. The involvement of arachidonic acid metabolites produced in the lipoxygenase pathway on the release of IL-6 was investigated indirectly, using nordihydroguaiaretic acid. This inhibitor reduced basal and IL-1 alpha- and LPS-stimulated IL-6 production. Ethacrynic acid, an inhibitor of peptido-leukotriene synthesis, also reduced basal IL-6 levels and blocked IL-1 alpha- as well as LPS-induced IL-6 secretion. It is concluded that IL-1 produced by Sertoli cells in response to LPS or residual bodies induces IL-6 through the lipoxygenase pathway.

Animals↗