Martti Parvinen

Apoptosis in Human Acute Myocardial Infarction

BACKGROUND After reopening of the infarct-related coronary artery, cardiomyocytes continue to die during reperfusion. The mechanisms of cell death have been subject to debate. We studied whether an apoptotic type of cell death occurs in human acute myocardial infarction (AMI). METHODS AND RESULTS We studied myocardial samples of eight patients who died of AMI and had patent infarct-related arteries at autopsy. Six of the patients had received initially successful thrombolysis. Extensive formation of DNA strand breaks, the typical biochemical feature of apoptosis, was detected with the use of the in situ DNA end-labeling method. Apoptotic cardiomyocytes were observed particularly in the border zones of histologically infarcted myocardium, whereas very few apoptotic cells were present in the remote noninfarcted myocardium. Internucleosomal fragmentation was confirmed by agarose gel electrophoresis of DNA isolated from the representative myocardial areas. CONCLUSIONS This study provides evidence that in addition to overt necrosis, a subset of myocytes undergo apoptosis during ischemia-reperfusion injury. Apoptosis may provide a new target for cardioprotection during evolving AMI in humans.

Late Arrest of Spermiogenesis and Germ Cell Apoptosis in Mice Lacking the TBP-like TLF/TRF2 Gene

Metazoan genomes encode two related proteins, TBP and the TBP-like factor (TLF/TRF2), sharing a highly conserved saddle-like domain. TLF is highly expressed in a finely regulated pattern in the mouse testis during spermatogenesis. The murine TLF gene has been inactivated using homologous recombination. TLF-/- mice are viable, but mutant male mice are sterile due to a late, complete arrest of spermiogenesis. In mutant animals, spermatogonia and spermatocytes develop normally, but round spermatids undergo apoptosis at step 7. Although the expression of the transcriptional activator CREM and many other postmeiotic genes was unaltered in TLF null mice, several spermiogenesis genes transcribed in late round spermatids appeared to be under TLF control. Hence, TLF is not required for embryonic development in the mouse but is essential for spermiogenesis.

Cell Interactions during the Seminiferous Epithelial Cycle

Publisher Summary This chapter discusses the cell interactions during the seminiferous epithelial cycle. Spermatozoa belong to the most differentiated cells of the body. Their special features are a haploid number of chromosomes, a tightly packed inactive form of the chromatin, a small amount of cytoplasm, and an ability of independent movement by a flagellum. The development of spermatozoa in the seminiferous epithelium includes three main phases: (1) spermatogonial multiplication, (2) meiosis, and (3) spermiogenesis. Cells in these phases are called spermatogonia, spermatocytes, and spermatids, respectively. The stage-dependent variation of the hormone responses in the seminiferous epithelium strongly suggests an existence of local paracrine regulation and cell interaction mechanisms in the seminiferous epithelium, that are dependent on spermatogenic cells associated with the Sertoli cells at each stage of the cycle of the seminiferous epithelium. The nature of this interaction is obscure; however some advances have been made. The secretion of a proteolytic enzyme, urokinase-type plasminogen activator, seems to be dependent on both cellular and hormonal regulation in the seminiferous epithelium. Testicular GnRH-like factors and proopiomelanocortin-derived peptides may play a role in seminiferous tubule-Leydig cell interaction. In the seminiferous epithelium, a Sertoli cell-derived growth factor is suggested to have a role in local regulation together with other factors, such as meiosis-inducing and -preventing substances, a somatomedin-like compound, and the spermatogonial chalone.

Real‐time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein

We produced transgenic mouse lines that accumulate mutated green fluorescent protein (EGFP) in sperm acrosome, a membrane limited organelle overlying the nucleus. The sperm showed normal fertilizing ability and the integrity of their acrosome was easily examined in a non‐invasive manner by tracing the GFP in individual ‘live’ sperm with fluorescent microscopy. The time required for the dispersal of acrosomal contents was demonstrated to be approximately 3 s after the onset of acrosome reaction.

Preparation, isolation and characterization of stage-specific spermatogenic cells for cellular and molecular analysis

Preparation, isolation and characterization of stage-specific spermatogenic cells for cellular and molecular analysis

Proliferation and Differentiation of Bovine Type A Spermatogonia During Long-Term Culture

Abstract The present study was aimed at developing a method for long-term culture of bovine type A spermatogonia. Testes from 5-mo-old calves were used, and pure populations of type A spermatogonia were isolated. Cells were cultured in minimal essential medium (MEM) or KSOM (potassium-rich medium prepared according to the simplex optimization method) and different concentrations of fetal calf serum (FCS) for 2–4 wk at 32°C or 37°C. Culture in MEM resulted in more viable cells and more proliferation than culture in KSOM, and better results were obtained at 37°C than at 32°C. After 1 wk of culture in the absence of serum, only 20% of the cells were alive. However, in the presence of 2.5% FCS, approximately 80% of cells were alive and proliferating. Higher concentrations of FCS only enhanced numbers of somatic cells. In long-term culture, spermatogonia continued to proliferate, and eventually, type A spermatogonial colonies were formed. The majority of colonies consisted mostly of groups of cells connected by intercellular bridges. Most of the cells in these colonies underwent differentiation because they were c-kit positive, and ultimately, cells with morphological and molecular characteristics of spermatocytes and spermatids were formed. Occasionally, large round colonies consisting of single, c-kit-negative, type A spermatogonia (presumably spermatogonial stem cells) were observed. For the first time to our knowledge, a method has been developed to allow proliferation and differentiation of highly purified type A spermatogonia, including spermatogonial stem cells during long-term culture.

Interplay of PIWI/Argonaute protein MIWI and kinesin KIF17b in chromatoid bodies of male germ cells.

Chromatoid bodies are thought to act as male-germ-cell-specific platforms for the storing and processing of haploid transcripts. The molecular mechanisms governing the formation and function of these germ-cell-specific structures have remained elusive. In this study, we show that the kinesin motor protein KIF17b, which is involved in the nucleocytoplasmic transport of RNA and of a transcriptional coactivator, localizes in chromatoid bodies. The chromatoid body moves actively and non-randomly in the cytoplasm of round spermatids, making frequent contacts with the nuclear envelope. The localization of KIF17b thereby offers a potential mechanism for microtubule-dependent mobility of chromatoid bodies, as well as for the transport of the specific components in and out of the chromatoid body. Interestingly, we demonstrate that KIF17b physically interacts with a testis-specific member of the PIWI/Argonaute family, MIWI, a component of chromatoid bodies implicated in RNA metabolism. A functional interplay between KIF17b and MIWI might be needed for the loading of haploid RNAs in the chromatoid body. Importantly, chromatoid bodies from round spermatids of miwi-null mice are not fully compacted and remain as a diffuse chromatoid material, revealing the essential role played by MIWI in the formation of chromatoid bodies. These results shed new light on the function of chromatoid bodies in the post-transcriptional regulation of gene expression in haploid germ cells.

Doxorubicin Induces Apoptosis in Germ Line Stem Cells in the Immature Rat Testis and Amifostine Cannot Protect against This Cytotoxicity

The underlying primary damage to the seminiferous epithelium caused by chemotherapeutic regimens at childhood is largely unknown. The present investigation was designed to identify acute cytotoxic events in the testis caused by a single dose of doxorubicin. Male rats at 6, 16, and 24 days of age were injected with doxorubicin (3 mg/kg, i.p.) or vehicle (saline) alone and 24 and 48 hours later, the germ cell types and apoptotic cells in the seminiferous epithelium were examined. As indicated by microscopy and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining, an 8-fold increase in the number of apoptotic germ cells in the testes of 6-day-old rats was observed 48 hours after doxorubicin treatment. Spermatogonia migrating to the basement membrane were the primary cell type undergoing this induced apoptosis. A single dose of amifostine (200 mg/kg) administered i.p. 15 minutes before injection of doxorubicin provided no protection against this enhanced apoptosis. Under the same conditions, testicular levels of p53 and activated caspase 8 were elevated, whereas the level of murine double minute-2 was lowered. In contrast, doxorubicin treatment did not result in any significant change in the physiologic, stage-specific germ cell apoptosis occurring in the testes of 16- and 24-day-old rats. These observations suggest that the initiation phase of spermatogenesis is highly sensitive to doxorubicin-induced apoptosis. Gonocytes and early spermatogonia are the cell types that are vulnerable to this p53-trigged apoptosis, which results in a decrease in the size of the pool of germ-line stem cells. Amifostine fails to protect the germ cells against this cytotoxic insult.

Stage-dependent secretion of ABP by rat seminiferous tubules

The secretion rate of the Sertoli-cell-specific androgen-binding protein (ABP), has been studied in isolated rate seminiferous tubules, where the stages of the spermatogenic cycle have been identified by a transillumination method. The secretion of ABP was highest in stages VII-XII, as determined by steady-state polyacrylamide-gel electrophoresis as well as by radioimmunoassay. More specifically, when the sensitive RIA technique permitted the assay of ABP secretion from a total amount of 10-30 mm of isolated tubules, it was found that maximal ABP secretion occurred at stages VIII-XI and minimal at stages IV-V. The present results show that the secretory activity of the Sertoli cells (ABP) is influenced by the type of germ cell at each cell association. It is postulated that the variation is Sertoli-cell secretory activity is of importance for the normal maintenance of spermatogenesis.

Influence of rat seminiferous tubules on Leydig cell testosterone production in vitro.

The interaction of seminiferous tubules and Leydig cells was investigated in the rat in vitro. Crude collagenase-dispersed, or Percoll-purified, Leydig cells were incubated together with seminiferous tubules at different stages of the cycle, isolated by transillumination-assisted microdissection. The testosterone production was measured. Seminiferous tubules inhibited testosterone production of crude Leydig cell preparations, but induced a clear stimulation (30-100%) in Percoll-purified cells. The stimulation was maximal at stages VII and VIII of the cycle, significantly higher than at stages II-VI (P less than 0.05). The stimulation by seminiferous tubules was observed both in basal and hCG-stimulated testosterone production. The effect was independent of FSH or GnRH action. These results demonstrate the presence of paracrine regulatory interaction between seminiferous tubules and Leydig cells, and are in agreement with the concept of a preferential androgen requirement of stages VII and VIII of the cycle.