Molecular genetics of the immotile short tail sperm defect

Hedelmättömyyttä aiheuttavan siittiöiden puolihäntävian molekyyligenetiikka Suomalaisissa Yorkshire karjuissa yleistyi 1990-luvun lopulla autosomaalisesti ja resessiivisesti periytyvä hedelmättömyyttä aiheuttava siittiöiden puolihäntävika (ISTS, immotile short tail sperm). Sairaus aiheuttaa normaalia lyhyemmän ja täysin liikkumattoman siittiön hännän muodostuksen. Muita oireita sairailla karjuilla ei ole havaittu ja emakot ovat oireettomia. Tämän tutkimuksen tarkoituksena oli kartoittaa siittiöiden puolihäntävian aiheuttava geenivirhe ja kehittää DNA-testi markkeri- ja geeniavusteiseen valintaan. Koko genomin kartoituksessa vian aiheuttava alue paikannettiin sian kromosomiin 16. Paikannuksen perusteella kahden geenimerkin haplotyyppi kehitettiin käytettäväksi markkeri-avusteisessa valinnassa. Sairauteen kytkeytyneen alueen hienokartoitusta jatkettiin geenitestin kehittämiseksi kantajadiagnostiikkaan. Vertailevalla kartoituksella oireeseen kytkeytynyt alue paikannettiin 2 cM:n alueelle ihmisen kromosomiin viisi (5p13.2). Tällä alueella sijaitsevia geenejä vastaavista sian sekvensseistä löydetyn muuntelun perusteella voitiin tarkentaa sairauteen kytkeytyneitä haplotyyppejä. Haplotyyppien perusteella puolihäntäoireeseen kytkeytynyt alue rajattiin kahdeksan geenin alueelle ihmisen geenikartalla. Alueelle paikannetun kandidaattigeenin (KPL2) sekvensointi paljasti introniin liittyneen liikkuvan DNA-sekvenssin, Line-1 retroposonin. Tämä retroposoni muuttaa geenin silmikointia siten, että sitä edeltävä eksoni jätetään pois tai myös osa introni- ja inserttisekvenssiä liitetään geenin mRNA tuotteeseen. Molemmissa tapauksissa tuloksena on lyhentynyt KPL2 proteiini. Tähän retroposoni-inserttiin perustuva geenitesti on ollut sianjalostajien käytössä vuodesta 2006. KPL2 geenin ilmenemisen tarkastelu sialla ja hiirellä paljasti useita kudosspesifisiä silmikointimuotoja. KPL2 geenin pitkä muoto ilmenee pääasiassa vain kiveksessä, mikä selittää geenivirheen aiheuttamat erityisesti siittiön kehitykseen liittyvät oireet. KPL2 proteiinin ilmeneminen hiiren siittiön hännän kehityksen aikana ja mahdollinen yhteistoiminta IFT20 proteiinin kanssa viittaavat tehtävään proteiinien kuljetuksessa siittiön häntään. Mahdollisen kuljetustehtävän lisäksi KPL2 saattaa toimia myös siittiön hännän rakenneosana, koska se paikannettiin valmiin siittiön hännän keskiosaan. Lisäksi KPL2 proteiini saattaa myös toimia Golgin laitteessa sekä Sertolin solujen ja spermatidien liitoksissa, mutta nämä havainnot kuitenkin vaativat lisätutkimuksia. Tämän tutkimuksen tulokset osoittavat, että KPL2 geeni on tärkeä siittiön hännän kehitykselle ja sen rakennemuutos aiheuttaa siittiöiden puolihäntäoireen suomalaisilla Yorkshire karjuilla. KPL2 proteiinin ilmeneminen ja paikannus siittiön kehityksen aikana antaa viitteitä proteiinin toiminnasta. Koska KPL2 geenisekvenssi on erittäin konservoitunut, nämä tulokset tuovat uutta tietoa kaikkien nisäkkäiden siittiöiden kehitykseen ja urosten hedelmättömyyteen syihin.

Maintenance of male sterile germplasm in Brassica rapa in vitro propagation

Selostus: Isästeriilin kevätrypsin geeniaineksen ylläpito in vitro mikroviljelyllä

Sweet and low and Schubert songs for male chorus

Digitoituna vain levyn B-puoli, "Schubert Songs for Male Chorus".

The Effects of Endocrine Disrupters on Fetal Male Rat Testicular and Adrenal Development

Many of the reproductive disorders that emerge in adulthood have their origin during fetal development. Numerous studies have demonstrated that exposure to endocrine disrupting chemicals can permanently affect the reproductive health of experimental animals. In mammals, male sexual differentiation and development are androgen-dependent processes. In rat, the critical programming window for masculinization occurs between embryonic days (EDs) 15.5 and 19.5. Disorders in sex steroid balance during fetal life can disturb the development of the male reproductive tract. In addition to the fetal testis, the adrenal cortex starts to produce steroid hormones before birth. Glucocorticoids produced by the adrenal cortex are essential for preparing the fetus for birth. In the present study, the effects of exposure to endocrine disrupters on fetal male rat testicular and adrenal development were investigated. To differentiate the systemic and direct testicular effects of endocrine disrupters, both in vivo and in vitro experiments were performed. The present study also clarified the role of desert hedgehog signalling (Dhh) in the development of the testis. The results indicate that endocrine disrupters, diethylstilbestrol (DES) and flutamide, are able to induce rapid steroidogenic changes in fetal rat testis under in vitro conditions. Although in utero exposure to these chemicals did not show overt effects in fetal testis, they can induce permanent changes in the developing testis and accessory sex organs later in life. We also reported that exposure to antiandrogens can interfere with testicular Dhh signalling and result in impaired differentiation of the fetal Leydig cells and subsequently lead to abnormal testicular development and sexual differentiation. In utero exposure to tetrachlorodibenzo-p-dioxin (TCDD) caused direct testicular and pituitary effects on the fetal male rat but with different dose responses. In a study in which the effects of developmental exposure to environmental antiandrogens, di-isononylphthalate and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p^’-DDE), on fetal male rat steroidogenesis were investigated, chemicals did not down-regulate testicular or adrenal steroid hormone synthesis or production in 19.5-day-old fetal rats. However, p,p^’-DDE-treatment caused clear histological and ultrastructural changes in the prenatal testis and adrenal gland. These structural alterations can disturb the development and function of fetal testis and adrenal gland that may become evident later in life. Exposure to endocrine disrupters during fetal life can cause morphological abnormalities and alter steroid hormone production by fetal rat Leydig cells and adrenocortical cells. These changes may contribute to the maldevelopment of the testis and the adrenal gland. The present study highlights the importance of the fetal period as a sensitive window for endocrine disruption.

De Christo a Judaeis male intellecto Joh. X. 30-38

Arkit: 1 arkintunnukseton lehti, A4 [B1].

The Effects of Anesthetic Induced Loss of Consciousness on Quantitative Electroen Cephalogram, and Bispectral and Spectral Entropy Indices. Studies on Healthy Male

Changes in the electroencephalography (EEG) signal have been used to study the effects of anesthetic agents on the brain function. Several commercial EEG based anesthesia depth monitors have been developed to measure the level of the hypnotic component of anesthesia. Specific anesthetic related changes can be seen in the EEG, but still it remains difficult to determine whether the subject is consciousness or not during anesthesia. EEG reactivity to external stimuli may be seen in unconsciousness subjects, in anesthesia or even in coma. Changes in regional cerebral blood flow, which can be measured with positron emission tomography (PET), can be used as a surrogate for changes in neuronal activity. The aim of this study was to investigate the effects of dexmedetomidine, propofol, sevoflurane and xenon on the EEG and the behavior of two commercial anesthesia depth monitors, Bispectral Index (BIS) and Entropy. Slowly escalating drug concentrations were used with dexmedetomidine, propofol and sevoflurane. EEG reactivity at clinically determined similar level of consciousness was studied and the performance of BIS and Entropy in differentiating consciousness form unconsciousness was evaluated. Changes in brain activity during emergence from dexmedetomidine and propofol induced unconsciousness were studied using PET imaging. Additionally, the effects of normobaric hyperoxia, induced during denitrogenation prior to xenon anesthesia induction, on the EEG were studied. Dexmedetomidine and propofol caused increases in the low frequency, high amplitude (delta 0.5-4 Hz and theta 4.1-8 Hz) EEG activity during stepwise increased drug concentrations from the awake state to unconsciousness. With sevoflurane, an increase in delta activity was also seen, and an increase in alpha- slow beta (8.1-15 Hz) band power was seen in both propofol and sevoflurane. EEG reactivity to a verbal command in the unconsciousness state was best retained with propofol, and almost disappeared with sevoflurane. The ability of BIS and Entropy to differentiate consciousness from unconsciousness was poor. At the emergence from dexmedetomidine and propofol induced unconsciousness, activation was detected in deep brain structures, but not within the cortex. In xenon anesthesia, EEG band powers increased in delta, theta and alpha (8-12Hz) frequencies. In steady state xenon anesthesia, BIS and Entropy indices were low and these monitors seemed to work well in xenon anesthesia. Normobaric hyperoxia alone did not cause changes in the EEG. All of these results are based on studies in healthy volunteers and their application to clinical practice should be considered carefully.

Chromatoid body mediated RNA regulation in mouse male germline

Male germ cell differentiation, spermatogenesis is an exceptional developmental process that produces a massive amount of genetically unique spermatozoa. The complexity of this process along with the technical limitations in the germline research has left many aspects of spermatogenesis poorly understood. Post-meiotic haploid round spermatids possess the most complex transcriptomes of the whole body. Correspondingly, efficient and accurate control mechanisms are necessary to deal with the huge diversity of transcribed RNAs in these cells. The high transcriptional activity in round spermatids is accompanied by the presence of an uncommonly large cytoplasmic ribonucleoprotein granule, called the chromatoid body (CB) that is conjectured to participate in the RNA post-transcriptional regulation. However, very little is known about the possible mechanisms of the CB function. The development of a procedure to isolate CBs from mouse testes was this study’s objective. Anti-MVH immunoprecipitation of cross-linked CBs from a fractionated testicular cell lysate was optimized to yield considerable quantities of pure and intact CBs from mice testes. This protocol produced reliable and reproducible data from the subsequent analysis of CB’s protein and RNA components. We found that the majority of the CB’s proteome consists of RNA-binding proteins that associate functionally with different pathways. We also demonstrated notable localization patterns of one of the CB transient components, SAM68 and showed that its ablation does not change the general composition or structure of the CB. CB-associated RNA analysis revealed a strong accumulation of PIWI-interacting RNAs (piRNAs), mRNAs and long non-coding RNAs (lncRNAs) in the CB. When the CB transcriptome and proteome analysis results were combined, the most pronounced molecular functions in the CB were related to piRNA pathway, RNA post-transcriptional processing and CB structural scaffolding. In addition, we demonstrated that the CB is a target for the main RNA flux from the nucleus throughout all steps of round spermatid development. Moreover, we provided preliminary evidence that those isolated CBs slice target RNAs in vitro in an ATPdependent manner. Altogether, these results make a strong suggestion that the CB functions involve RNA-related and RNA-mediated mechanisms. All the existing data supports the hypothesis that the CB coordinates the highly complex haploid transcriptome during the preparation of the male gametes for fertilization. Thereby, this study provides a fundamental basis for the future functional analyses of ribonucleoprotein granules and offers also important insights into the mechanisms governing male fertility.

Family rivalry in the testis: Retinoblastoma protein and E2F transcription factors in testicular development and adult spermatogenesis

Disorders of male reproductive health are becoming increasingly prevalent globally. These defects, ranging from decreasing sperm counts to an increasing rate of infertility and testicular cancer, have a common origin in the early phases of testicular development, but the exact mechanisms that cause them remain unknown. Testicular development and adult spermatogenesis are complex processes in which different cell types undergo mitosis, meiosis, differentiation and apoptosis. The retinoblastoma protein family and its associated E2F transcription factors are key regulators of these cellular events. In the present study, the functions of these factors in postnatal testicular development and adult spermatogenesis were explored using different animal models. In addition, a new application of flow cytometry to study testicular cell dynamics was developed. An ablation of retinoblastoma protein in mouse Sertoli cells resulted in their cell cycle re-entry in adult testes, dedifferentiation and a severe spermatogenic defect. We showed that deregulated E2F3 contributed to these changes. Our results indicated that the E2F1 transcription factor is critical for the control of apoptosis in the developing postnatal testis. In the adult testis, E2F1 controls the maintenance of the spermatogonial stem cell pool, in addition to inhibiting apoptosis of spermatocytes. In summary, this study elucidated the complex interdependencies of the RB and E2F transcription factor families in the control of postnatal testicular development and adult spermatogenesis. Furthermore, this study provided a new methodology for the analysis of testicular cells.