Hormad1 mutation disrupts synaptonemal complex formation, recombination, and chromosome segregation in mammalian meiosis

Meiosis is unique to germ cells and essential for reproduction. During the first meiotic division, homologous chromosomes pair, recombine, and form chiasmata. The homologues connect via axial elements and numerous transverse filaments to form the synaptonemal complex. The synaptonemal complex is a critical component for chromosome pairing, segregation, and recombination. We previously identified a novel germ cell-specific HORMA domain encoding gene, Hormad1, a member of the synaptonemal complex and a mammalian counterpart to the yeast meiotic HORMA domain protein Hop1. Hormad1 is essential for mammalian gametogenesis as knockout male and female mice are infertile. Hormad1 deficient (Hormad1-/-) testes exhibit meiotic arrest in the early pachytene stage, and synaptonemal complexes cannot be visualized by electron microscopy. Hormad1 deficiency does not affect localization of other synaptonemal complex proteins, SYCP2 and SYCP3, but disrupts homologous chromosome pairing. Double stranded break formation and early recombination events are disrupted in Hormad1-/- testes and ovaries as shown by the drastic decrease in the γH2AX, DMC1, RAD51, and RPA foci. HORMAD1 co-localizes with cH2AX to the sex body during pachytene. BRCA1, ATR, and γH2AX co-localize to the sex body and participate in meiotic sex chromosome inactivation and transcriptional silencing. Hormad1 deficiency abolishes γH2AX, ATR, and BRCA1 localization to the sex chromosomes and causes transcriptional de-repression on the X chromosome. Unlike testes, Hormad1-/- ovaries have seemingly normal ovarian folliculogenesis after puberty. However, embryos generated from Hormad1-/- oocytes are hyper- and hypodiploid at the 2 cell and 8 cell stage, and they arrest at the blastocyst stage. HORMAD1 is therefore a critical component of the synaptonemal complex that affects synapsis, recombination, and meiotic sex chromosome inactivation and transcriptional silencing. © 2010 Shin et al

Ultra-Fast and Optimized Method for the Preparation of Rodent Testicular Cells for Flow Cytometric Analysis

Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson). This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use

Amifostine reduces the seminiferous epithelium damage in doxorubicin-treated prepubertal rats without improving the fertility status

<p>Abstract</p> <p>Background</p> <p>Amifostine is an efficient cytoprotector against toxicity caused by some chemotherapeutic drugs. Doxorubicin, a potent anticancer anthracycline, is known to produce spermatogenic damage even in low doses. Although some studies have suggested that amifostine does not confer protection to doxorubicin-induced testicular damage, schedules and age of treatment have different approach depending on the protocol. Thus, we proposed to investigate the potential cytoprotective action of amifostine against the damage provoked by doxorubicin to prepubertal rat testes (30-day-old) by assessing some macro and microscopic morphometric parameters 15, 30 and 60 days after the treatment; for fertility evaluation, quantitative analyses of sperm parameters and reproductive competence in the adult phase were also carried out.</p> <p>Methods</p> <p>Thirty-day-old male rats were distributed into four groups: Doxorubicin (5 mg/kg), Amifostine (400 mg/kg), Amifostine/Doxorubicin (amifostine 15 minutes before doxorubicin) and Sham Control (0.9% saline solution). "Standard One Way Anova" parametric and "Anova on Ranks" non-parametric tests were applied according to the behavior of the obtained data; significant differences were considered when p < 0.05.</p> <p>Results</p> <p>The rats killed 30 and 60 days after doxorubicin treatment showed diminution of seminiferous epithelium height and reduction on the frequency of tubular sections containing at least one type of differentiated spermatogonia; reduction of sperm concentration and motility and an increase of sperm anomalous forms where observed in doxorubicin-treated animals. All these parameters were improved in the Amifostine/Doxorubicin group only when compared to Doxorubicin group. Such reduction, however, still remained below the values obtained from the Sham Control group. Nevertheless, the reproductive competence of doxorubicin-treated rats was not improved by amifostine pre-administration.</p> <p>Conclusions</p> <p>These results suggest that amifostine promotes a significant reduction of the doxorubicin long-term side effects on the seminiferous epithelium of prepubertal rats, which is reflected in the epidydimal fluid parameters in the adult phase. However, fertility status results suggest that such protection may not be effective against sperm DNA content damage. Further investigation of sperm DNA integrity must be carried out using amifostine and doxorubicin-treated experimental models.</p

Offspring sex ratio and gonadal irradiation in the British Childhood Cancer Survivor Study

We investigated offspring sex ratio among 6232 offspring born to 3218 survivors of childhood cancer in relation to therapeutic irradiation, and pooled our data with those from two other large-scale studies giving a total of 9685 offspring. Exposure to high-dose gonadal irradiation was not associated with a significant alteration in offspring sex ratio compared to low doses (men: P=0.58, women: P=0.66). There was also no evidence that the ratio varied with time since cancer diagnosis when comparing survivors treated with radiotherapy vs those without (men: P=0.51; women: P=0.46). This, the largest study to date, finds no evidence that exposure to radiation affects the offspring sex ratio among survivors of childhood cancer

Acid Glycohydrolases in Rat Spermatocytes, Spermatids and Spermatozoa: Enzyme Activities, Biosynthesis and Immunolocalization

Mammalian sperm acrosome contains several glycohydrolases thought to aid in the dispersion and digestion of vestments surrounding the egg. In this study, we have used multiple approaches to examine the origin of acrosome-associated glycohdyrdolases. Mixed spermatogenic cells, prepared from rat testis, were separated by unit gravity sedimentation. The purified germ cells (spermatocytes [SP], round spermatids [RS], and elongated/condensed spermatids [E/CS]) contained several glycohydrolase activities. Metabolic labeling in the cell culture, immunoprecipitation, and autoradiographic approaches revealed that β-D-galactosidase was synthesized in SP and RS in 88/90 kDa forms which undergo processing in a cell-specific manner. Immunohistochemical approaches demonstrated that the enzyme was localized in Golgi membranes/vesicles, and lysosome-like structures in SP and RS, and forming/formed acrosome of E/CS

Semen cryopreservation, utilisation and reproductive outcome in men treated for Hodgkin's disease

Between 1978 and 1990, 122 men underwent semen analysis before starting sterilising chemotherapy for Hodgkin's disease. Eighty-one (66%) had semen quality within the normal range, 25 were oligospermic (<20×106 sperm per ml) and five were azoospermic (no sperm in the ejaculate). Semen from 115 men was cryopreserved and after a median follow-up time of 10.1 years, 33 men have utilised stored semen (actuarial rate 27%) and nine partners have become pregnant resulting in 11 live births and one termination for foetal malformation. Actuarial 10 year rates of destruction of semen before death or utilisation and death before utilisation are 19% and 13% respectively. This retrospective cohort study demonstrates that approximately one-quarter of men utilising cryopreserved semen after treatment for Hodgkin's disease obtain a live birth. The high non-utilisation rate is intriguing and warrants further investigation

PP1 Forms an Active Complex with TLRR (lrrc67), a Putative PP1 Regulatory Subunit, during the Early Stages of Spermiogenesis in Mice

Mammalian spermatogenesis is a highly regulated developmental pathway that demands dramatic rearrangement of the cytoskeleton of the male germ cell. We have described previously a leucine rich repeat protein, TLRR (also known as lrrc67), which is associated with the spermatid cytoskeleton in mouse testis and is a binding partner of protein phosphatase-1 (PP1), an extremely well conserved signaling molecule. The activity of PP1 is modulated by numerous specific regulators of which TLRR is a candidate. In this study we measured the phosphatase activity of the TLRR-PP1 complex in the adult and the developing mouse testis, which contains varying populations of developing germ cell types, in order to determine whether TLRR acts as an activator or an inhibitor of PP1 and whether the phosphatase activity of this complex is developmentally regulated during spermatogenesis. Additionally, we assayed the ability of bacterially expressed TLRR to affect the enzymatic activity of PP1. Furthermore, we examined phosphorylation of TLRR, and elements of the spermatid cytoskeleton during the first wave of spermatogenesis in the developing testis. We demonstrate here that the TLRR complex is associated with a phosphatase activity in adult mouse testis. The relative phosphatase activity of this complex appears to reach a peak at about 21 days after birth, when pachytene spermatocytes and round spermatids are abundant in the seminiferous epithelium of the mouse testis. TLRR, in addition to tubulin and kinesin-1B, is phosphorylated during the first wave of spermatogenesis. These findings indicate that the TLRR-PP1 complex is active prior to translocation of TLRR toward the sperm flagella and that TLRR, and constituents of the spermatid cytoskeleton, may be subject to regulation by reversible phosphorylation during spermatogenesis in murine testis

Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids

<p>Abstract</p> <p>Background</p> <p>Spermatogenesis is comprised of a series of highly regulated developmental changes that transform the precursor germ cell into a highly specialized spermatozoon. The last phase of spermatogenesis, termed spermiogenesis, involves dramatic morphological change including formation of the acrosome, elongation and condensation of the nucleus, formation of the flagella, and disposal of unnecessary cytoplasm. A prominent cytoskeletal component of the developing spermatid is the manchette, a unique microtubular structure that surrounds the nucleus of the developing spermatid and is thought to assist in both the reshaping of the nucleus and redistribution of spermatid cytoplasm. Although the molecular motor KIFC1 has been shown to associate with the manchette, its precise role in function of the manchette and the identity of its testis specific protein partners are unknown. The purpose of this study was to identify proteins in the testis that interact with KIFC1 using a yeast 2 hybrid screen of a testis cDNA library.</p> <p>Results</p> <p>Thirty percent of the interacting clones identified in our screen contain an identical cDNA encoding a 40 kD protein. This interacting protein has 4 leucine-rich repeats in its amino terminal half and is expressed primarily in the testis; therefore we have named this protein testis leucine-rich repeat protein or TLRR. TLRR was also found to associate tightly with the KIFC1 targeting domain using affinity chromatography. In addition to the leucine-rich repeats, TLRR contains a consensus-binding site for protein phosphatase-1 (PP1). Immunocytochemistry using a TLRR specific antibody demonstrates that this protein is found near the manchette of developing spermatids.</p> <p>Conclusion</p> <p>We have identified a previously uncharacterized leucine-rich repeat protein that is expressed abundantly in the testis and associates with the manchette of developing spermatids, possibly through its interaction with the KIFC1 molecular motor. TLRR is homologous to a class of regulatory subunits for PP1, a central phosphatase in the reversible phosphorylation of proteins that is key to modulation of many intracellular processes. TLRR may serve to target this important signaling molecule near the nucleus of developing spermatids in order to control the cellular rearrangements of spermiogenesis.</p

A transcriptome analysis of mitten crab testes (Eriocheir sinensis)

The identification of expressed genes involved in sexual precocity of the mitten crab (Eriocheir sinensis) is critical for a better understanding of its reproductive development. To this end, we constructed a cDNA library from the rapid developmental stage of testis of E. sinensis and sequenced 3,388 randomly picked clones. After processing, 2,990 high-quality expressed sequence tags (ESTs) were clustered into 2,415 unigenes including 307 contigs and 2,108 singlets, which were then compared to the NCBI non-redundant (nr) protein and nucleotide (nt) database for annotation with Blastx and Blastn, respectively. After further analysis, 922 unigenes were obtained with concrete annotations and 30 unigenes were found to have functions possibly related to the process of reproduction in male crabs – six transcripts relevant to spermatogenesis (especially Cyclin K and RecA homolog DMC1), two transcripts involved in nuclear protein transformation, two heat-shock protein genes, eleven transcription factor genes (a series of zinc-finger proteins), and nine cytoskeleton protein-related genes. Our results, besides providing valuable information related to crustacean reproduction, can also serve as a base for future studies of reproductive and developmental biology