Histone H4 acetylation is dysregulated in active seminiferous tubules adjacent to testicular tumours.

STUDY QUESTION: Is histone H4 acetylation (H4ac) altered in the seminiferous tubules of patients affected by testicular tumours? SUMMARY ANSWER: A considerable dysregulation of H4ac was detected in the cells of the seminiferous tubules adjacent to testicular tumours of different aetiology and prior to any treatment, while no comparable alterations were observed in patients with disrupted spermatogenesis. WHAT IS KNOWN ALREADY: Altered H4ac levels have been associated with a variety of testicular pathological conditions. However, no information has been available regarding potential alterations in the spermatogenic cells adjacent to the neoplasia in testicular tumour patients. STUDY DESIGN, SIZE, DURATION: A retrospective analysis using testicular sections from 33 men aged between 21 and 74 years old was performed. Three study groups were defined and subjected to double-blind evaluation: a control group with normal spermatogenesis (n = 6), patients with testicular tumours (n = 18) and patients with spermatogenic impairments (n = 8). One additional sample with normal spermatogenesis was used as a technical internal control in all evaluations. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunohistochemistry against H4ac and, when needed, Placental-like alkaline phosphatase and CD117, was performed on testicular sections. The H4ac H-score, based on the percentage of detection and signal intensity, was used as the scoring method for statistical analyses. Protein expression data from the Human Protein Atlas were used to compare the expression levels of predicted secreted proteins from testicular tumours with those present in the normal tissue. MAIN RESULTS AND THE ROLE OF CHANCE: We revealed, for the first time, a dramatic disruption of the spermatogenic H4ac pattern in unaffected seminiferous tubule cells from different testicular tumour patients prior to any antineoplastic treatment, as compared to controls (P < 0.05). Since no similar alterations were associated with spermatogenic impairments and the in silico analysis revealed proteins potentially secreted by the tumour to the testicular stroma, we propose a potential paracrine effect of the neoplasia as a mechanistic hypothesis for this dysregulation. LIMITATIONS, REASONS FOR CAUTION: Statistical analyses were not performed on the hypospermatogenesis and Leydig cell tumour groups due to limited availability of samples. WIDER IMPLICATIONS OF THE FINDINGS: To the best of our knowledge, this is the first report showing an epigenetic alteration in cells from active seminiferous tubules adjacent to tumour cells in testicular tumour patients. Our results suggest that, despite presenting spermatogenic activity, the global epigenetic dysregulation found in the testicular tumour patients could lead to molecular alterations of the male germ cells. Since testicular tumours are normally diagnosed in men at reproductive age, H4ac alterations might have an impact when these testicular tumour patients express a desire for fatherhood. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the European Union Marie Curie European Training Network actions and by grants to R.O. from the 'Ministerio de Economía y Competividad (Spain)' (fondos FEDER 'una manera de hacer Europa', PI13/00699, PI16/00346 and PI20/00936) and from EU-FP7-PEOPLE-2011-ITN289880. J.C. was supported by the Sara Borrell Postdoctoral Fellowship, Acción Estratégica en Salud, CD17/00109. J.C. is a Serra Húnter fellow (Universitat de Barcelona, Generalitat de Catalunya). F.B. has received grants from the Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario (Spain) (FPU15/02306). A.d.l.I. is supported by a fellowship of the Ministerio de Economía, Industria y Competitividad (Spain) (PFIS, FI17/00224). M.J. is supported by the Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016-2020, SLT002/16/00337). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Age-related changes in interstitial fibrosis and germ cell degeneration of the canine testis.

Fifty-five healthy medium-sized dogs were divided into four groups; young (1-3 years old, n = 14), adult (>3 to 6 years old, n = 12), old (>6 to 9 years old, n = 14) and senile (>9 years old, n = 15). After routine orchiectomy, testes were collected, and the degree of white streak areas on cut surfaces was subjectively assessed. Later, testicular tissue sections were stained with haematoxylin and eosin and Masson's trichrome for evaluation of germ cell degeneration and the proportion of interstitial connective tissue, respectively. Semiquantitative severity scoring of germ cell degeneration and quantitative analysis of spermatogenic cells for spermatic index (SI) and Sertoli cell index (SEI) was performed. The score of white streak on cut surface area of the testes increased with age, being higher (p < 0.05) in senile dogs than other age groups; no difference was found between adult and old dogs. The proportion of testicular interstitial fibrosis was highest (p < 0.05) in senile dogs. Positive correlations between age and white streak area (rho = 0.77, p < 0.01) as well as age and interstitial fibrosis (rho = 0.63, p < 0.01) were observed. The severity of germ cell degeneration gradually increased with age and differed among age groups (p < 0.05). Age positively correlated with atrophy of seminiferous tubules (rho = 0.93, p < 0.01). The SI was lower (p < 0.05) in senile dogs compared to other age groups, and SI was not different among young, adult and old dogs. Conversely, SEI was significantly higher in senile dogs compared to young, adult and old dogs. A negative correlation between age and SI (rho = -0.69) and a positive correlation between age and SEI (rho = 0.68) were significant (p < 0.01). In conclusion, influence of age on testicular interstitial fibrosis and germ cell degeneration/depletion were pronounced in dogs over 9 years old.

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model.

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

Leydig cell insufficiency in hypospermatogenesis: a paracrine effect of activin-inhibin signaling?

Clinical findings and a variety of experimental models indicate that Leydig cell dysfunction accompanies damage to the seminiferous tubules with increasing severity. Most studies support the idea that intratesticular signaling from the seminiferous tubules to Leydig cells regulates steroidogenesis, which is disrupted when hypospermatogenesis occurs. Sertoli cells seem to play a pivotal role in this process. In this review, we summarize relevant clinical and experimental observations and present evidence to support the hypothesis that testicular activin signaling and its regulation by testicular inhibin may link seminiferous tubular dysfunction to reduced testosterone biosynthesis.

Gradual decrease in spermatogenesis caused by chronic stress.

Chronic stress induces decreased sperm motility, viability and concentration in stressed males. Also, stress modifies oxidative status and causes apoptosis in testes, as well as a decrease in the epithelial area of seminiferous tubules. However, there are no studies that analyze the alterations caused by stress in testicular cells. Thus, in this study, alterations in the morphology of testicular germ cells caused by different days of chronic stress were assessed. Adult male rats were exposed to stress by immersion in cold water (ICW) daily for 3, 8, 20 or 50 consecutive days. Plasma testosterone and corticosterone were also assessed. Results showed that chronic stress causes loss of germ cells, and alteration of spermatogenesis. Seminiferous tubules from stressed males showed several degenerative signs, such as vacuoles in the basal epithelium, with picnotic indicia; moderate to severe exfoliation of degenerative germinal cells in the tubule lumen was also observed. These alterations were observed in all days of stress in a gradual way, from day 3-50. Testosterone levels were decreased at all those times, and corticosterone concentrations were increased on the same days. These results show that chronic stress causes severe damage to germ cells, which can account for infertility problems in males. These alterations are related to a decrease in testosterone as well as an increase in corticosterone caused by stress.

Manchette-acrosome disorders during spermiogenesis and low efficiency of seminiferous tubules in hypercholesterolemic rabbit model.

Hypercholesterolemia is a marker for several adult chronic diseases. Recently we demonstrated that sub/infertility is also associated to Hypercholesterolemia in rabbits. Seminal alterations included: abnormal sperm morphology, decreased sperm number and declined percentage of motile sperm, among others. In this work, our objective was to evaluate the effects of hypercholesterolemia on testicular efficiency and spermiogenesis, as the latter are directly related to sperm number and morphology respectively. Tubular efficiency was determined by comparing total number of spermatogenic cells with each cell type within the proliferation/differentiation compartments. We found lower testicular efficiency related to both a decrease in spermatogonial cells and an increase in germ cell apoptosis in hypercholesterolemic rabbits. On the other hand, spermiogenesis-the last step of spermatogenesis involved in sperm shaping-was detaily analyzed, particularly the acrosome-nucleus-manchette complex. The manchette is a microtubular-based temporary structure responsible in sperm cell elongation. We analyzed the contribution of actin filaments and raft microdomains in the arrangement of the manchette. Under fluorescence microscopy, spermatocyte to sperm cell development was followed in cells isolated from V to VIII tubular stages. In cells from hypercholesterolemic rabbits, abnormal development of acrosome, nucleus and inaccurate tail implantation were associated with actin-alpha-tubulin-GM1 sphingolipid altered distribution. Morphological alterations were also observed at electron microscopy. We demonstrated for the first time that GM1-enriched microdomains together with actin filaments and microtubules are involved in allowing the correct anchoring of the manchette complex. In conclusion, cholesterol enriched diets promote male fertility alterations by affecting critical steps in sperm development: spermatogenesis and spermiogenesis. It was also demonstrated that hypercholesterolemic rabbit model is a useful tool to study serum cholesterol increment linked to sub/infertility.

[FACTORS AFFECTING SPERMATOGENESIS PRESERVATION IN RATS WITH SEMINIFEROUS TRACT OBSTRUCTION].

The aim of the study was to examine the effect of the seminal tract obstruction of different degree and duration on the morphological and functional condition of testicular tissue. The study was conducted in 50 male Wistar rats. Three experimental models of seminiferous tract obstruction were set up: obstruction of the proximal part of the ductus deferens, obstruction of the distal part of the ductus deferens and obstruction of at the epididymis level. Morphological evaluation of testicular tissue was performed at 3 and 6 months after the obstruction. It was found that obstruction at the epididymis level caused the most severe impairment of spermatogenesis.

The histology and the proapoptotic control in the ipsilateral and the contralateral testes following unilateral vasectomy.

OBJECTIVE: The aim of this study was to enlighten both the testicular histology and the genetic aspects of the apoptotic process. Thus an experimental study was designed with a model of unilateral vasectomy. METHODS: Twenty-two adult male rats were used and 4 main groups were formed. The first (A), the second (B), the third (C), and the fourth group (D) consisted of 4, 4, 4 and 10 rats respectively. Rats in group A had sham operation while rats in other groups (B, C, D) underwent left vasectomy operation including binding of ductus deferens with a 3/0 silk and cutting a minimum of 1 cm part while preserving the vascular structure under 9x magnification. Rats undergoing unilateral vasectomy were sacrificed at the 1(st), 2(nd) and 8(th) weeks and their testicular structure and proapoptotic gene proteins were compared with that of the control group undergoing sham operation. RESULTS: We found that vasectomy gradually caused destruction and both ipsilateral and contralateral testicles were affected showing initial apoptosis. CONCLUSION: The procedure causes destruction in the testicular structure by causing bilateral intratubular germ cell necrosis, unilateral obstruction, increase in the tubular pressure and processes that are aggravated by some probable autoimmune reactions.

Androgen Receptor Coactivator ARID4B Is Required for the Function of Sertoli Cells in Spermatogenesis.

Defects in spermatogenesis, a process that produces spermatozoa inside seminiferous tubules of the testis, result in male infertility. Spermatogenic progression is highly dependent on a microenvironment provided by Sertoli cells, the only somatic cells and epithelium of seminiferous tubules. However, genes that regulate such an important activity of Sertoli cells are poorly understood. Here, we found that AT-rich interactive domain 4B (ARID4B), is essential for the function of Sertoli cells to regulate spermatogenesis. Specifically, we generated Sertoli cell-specific Arid4b knockout (Arid4bSCKO) mice, and showed that the Arid4bSCKO male mice were completely infertile with impaired testis development and significantly reduced testis size. Importantly, severe structural defects accompanied by loss of germ cells and Sertoli cell-only phenotype were found in many seminiferous tubules of the Arid4bSCKO testes. In addition, maturation of Sertoli cells was significantly delayed in the Arid4bSCKO mice, associated with delayed onset of spermatogenesis. Spermatogenic progression was also defective, showing an arrest at the round spermatid stage in the Arid4bSCKO testes. Interestingly, we showed that ARID4B functions as a "coactivator" of androgen receptor and is required for optimal transcriptional activation of reproductive homeobox 5, an androgen receptor target gene specifically expressed in Sertoli cells and critical for spermatogenesis. Together, our study identified ARID4B to be a key regulator of Sertoli cell function important for male germ cell development.

Mechanisms of reparative regeneration of rat testis after injection of paclitaxel.

Population of spermatogonia was reduced in 2, 3, and 6 months after single intravenous injection of antitumor drug paclitaxel in maximum tolerated dose (MTD). The count of Sertoli cell increased in 3 months after the start of the experiment. The maturity of the seminiferous tubule epithelium was lower than in intact rats. Spermatogenesis productivity did not differ from that in intact animals 6 months after start of the experiment. These data indicate that regeneration of the spermatogenous tissue after paclitaxel treatment is realized via renewal of the spermatogenic epithelium, but considering the amount of spermatogonial cell population, the recovery rate would be low.

Raman spectroscopy as an ex vivo noninvasive approach to distinguish complete and incomplete spermatogenesis within human seminiferous tubules.

OBJECTIVE: To evaluate the potential clinical application of Raman spectroscopy (RS) as a tool that may identify spermatogenesis within human seminiferous tubules. DESIGN: RS scanning of human testicular tissue at different maturational stages; immunohistochemistry study and metabolomic analysis of nonobstructive azoospermic/obstructive azoospermic testes. SETTING: State-owned hospital. PATIENT(S): Fifty-two patients with clinical indications of nonobstructive azoospermia (NOA) and obstructive azoospermia (OA) who underwent infertility evaluation and treatment. INTERVENTION(S): None. MAIN OUTCOME MEASUREMENT(S): Raman spectra of seminiferous tubules, thickness of lamina propria (LP), immunohistochemistry of type I, III, and IV collagens and laminin, metabolites of human testes. RESULT(S): Tubules of OA patients had spectral intensities below 2,000 (au), while tubules of NOA patients had higher intensities, depending on the degree of spermatogenesis. RS was able to separate samples of NOA and OA testicular tissue with a sensitivity of 90% and specificity of 85.71%. The LP of NOA tubules were thickened and had increased deposition of type I and type III collagens. Gas chromatography-mass spectrometer (GC-MS) detected 12 metabolites that showed significant differences between NOA and OA testes. CONCLUSION(S): RS can noninvasively distinguish seminiferous tubules with complete and incomplete spermatogenesis and may serve as a novel and potentially useful tool to guide surgeons performing micro-testicular sperm extraction to improve sperm retrieval.

Intratesticular pressure after testicular torsion as a predictor of subsequent spermatogenesis: a rat model.

OBJECTIVE: To assess the correlation between intratesticular pressure (ITP) after testicular torsion and subsequent testicular function using a rat model and to show that ITP at surgery is a useful predictor of future spermatogenesis. MATERIALS AND METHODS: Fourteen rats were divided into a torsion group (n= 7) and a control group with sham operation (n= 7). Torsion was created by 720° rotation of the left testis in a counter-clockwise direction. Using a handheld compartment monitor, the ITP of the torsed testes was measured three times: before torsion (pre-torsion), just before torsion repair (pre-detorsion) and 5 min after torsion repair (post-detorsion). We evaluated the correlation between ITP and testicular weight, epididymal sperm count or pathological findings, such as the seminiferous tubule diameter (STD) and the modified Johnsen's score, 4 weeks after surgery. RESULTS: Mean (se) pre-torsion, pre-detorsion and post-detorsion ITP values in the torsion group were 5.9 (2.5), 19.7 (10.7) and 8.2 (4.8) cm H(2) O, respectively. The ITP in torsed testes significantly increased after torsion (P < 0.01) and decreased after detorsion (P < 0.01). Strong correlations were observed between the reduction of ITP after detorsion and testicular weight (r= 0.87, P < 0.05), epididymal sperm count (r= 0.94, P < 0.05), STD (r= 0.87, P < 0.05) or the Johnsen's score (r= 0.99, P < 0.001). CONCLUSION: A smaller reduction in ITP after detorsion can be a risk factor for subsequent disturbance of spermatogenesis, suggesting that ITP can be an index for determining whether the affected testis should be removed after testicular torsion.

Testicular development evaluation in rats exposed to 60 Hz and 1 mT electromagnetic field.

Society has been increasingly exposed to low-frequency electromagnetic fields (EMF), mainly from electricity distribution networks and electro-electronic devices. Aiming to clarify the extension of possible interactions between EMF and testicular development, this study evaluated the effects of exposure to 60 Hz and 1 mT EMF in the maturation of testicular components. Wistar rats were exposed to EMF three times per day for 30 min, between the 13th day of gestation and the 21st postnatal day. Results showed a decrease in the following parameters: tubular diameter and seminiferous tubules area; seminiferous epithelium height; total volume of seminiferous tubule; tubular lumen; seminiferous epithelium; and Leydig cells. On the other hand, an increase was observed in connective tissue cells and blood vessels volume. Plasma testosterone, Sertoli cells population, tubular length and gonadosomatic index did not change when exposed to EMF. Histomorphometric analysis showed that exposure to EMF can promote a delay in testicular development.

Single administration of di(n-butyl) phthalate delays spermatogenesis in prepubertal rats.

Morphological alterations in seminiferous tubules caused by single administration of di(n-butyl) phthalate (DBP) in 3-week-old rats were investigated throughout the first wave of spermatogenesis. Single administration of DBP (500 mg/kg) showed progressive detachment and displacement of spermatogenic cells and disappearance of tubular lumen at 3h after treatment, and then showed thin seminiferous epithelia and wide tubular lumen at day 1 (D1). At D1, quite significant numbers of apoptotic spermatogenic cells were detected, and then they gradually decreased in accordance with the passage of time. In contrast, the testes revealed lower weight gain, even after completion of first wave of spermatogenesis in the DBP-treated group, compared to the control. In order to clarify whether spermatogenic cells differentiate into mature spermatids in the DBP-treated rats, immunohistochemical staining for Hsc 70t, a specific marker for elongate spermatids, was carried out. As a result, the decrease in mature spermatids in the DBP-treated testes, compared to the control, was demonstrated. For example, at D20 (41-day-old) after treatment, the most advanced spermatids in the tubules from rats in the DBP-treated groups were steps 2-4, while those of the control were steps 12-13. Moreover, in some tubules, pachytene spermatocytes were the most advanced spermatogenic cell. At D30 (51-day-old) after treatment, maturation of spermatogenic cells in the DBP-treated rats proceeded further, and the most advanced spermatids in tubules were steps 8-9, while those of the control were steps 15-19. These results lead us to the postulation that a single administration of DBP to prepubertal rats delays maturation of spermatogenic cells, even after completion of first wave of spermatogenesis.

[Protective effects of inhibition of tissue nitric oxide in the initial stage against tardive injury of testicular spermatogenic function: experiment with rats].

OBJECTIVE: To study the protective effects of inhibition of tissue nitric oxide in the initial stage against the tardive injury of contralateral testicular spermatogenic function after unilateral testicular torsion. METHODS: 56 prepubertal Sprague-Dawley rats were randomly divided into 4 equal groups: placebo group undergoing rotation of left testis 720 degrees clockwise for 4 h, fixing thereof to the scrotum, and then intravenous injection of normal saline; cyclosporine group, undergoing intraperitoneal injection of cyclosporine once daily for 1 month after the testicular rotation and fixation; NG-methyl L-arginine (L-NMMA) group, undergoing intravenous injection of L-NMMA 30 mg/kg 30 min before the testicular rotation; and sham-operation group, undergoing isolation and suture of testis only. The right (untwisted) testes were removed from 7 rats from each group 1 week and 2 months after surgery. The malondialdehyde (MDA) level and nitrous oxide (NO)/nitrogen oxide synthase (NOS) content were evaluated. Histological examination was conducted. The mean seminiferous tubule diameter (MSTD) was assessed. The level of MHC peptide-tetramer complex was determined by flow cytometry. RESULTS: The levels of MDA, NO, NOS, and MHC peptide-tetramer complex of the L-NMMA, placebo, and cyclosporine groups one week after surgery were all significantly higher than those of the sham operation group (all P < 0.05). The levels of MDA, NO, NOS, and MHC peptide-tetramer complex of the L-NMMA group were all significantly lower then those of the placebo group (all P < 0.05). The pathological damage of the contralateral testis in early and late stages in the L-NMMA group was lighter than in the placebo group. CONCLUSION: By inhibiting tissue NO production in focal organizations during the early period, L-NMMA reduces the damages inthe testis contralateral to the testis undergoing unilateral torsion.

Study of spermatogenesis and thickening of lamina propria in the human seminiferous tubules.

The negative correlation between the thickness of the lamina propria and Johnsen's score count in individual seminiferous tubules was observed and the tubules with thin lamina propria indicated almost complete spermatogenesis. It is suggested that the thin lamina propria may be one of the good indicators of complete spermatogenesis.

Seminiferous tubule function in delayed-onset X-linked adrenal hypoplasia congenita associated with incomplete hypogonadotrophic hypogonadism.

OBJECTIVE: X-linked adrenal hypoplasia congenita (AHC, OMIM 300200) due to mutations in the DAX-1 gene is frequently associated to hypogonadotrophic hypogonadism (HHG, OMIM 238320). Clinical variants with delayed-onset have been recognized. The objective of this study is to assess Sertoli cell function throughout pubertal development in patients with childhood-onset AHC due to stop mutations in the DAX-1 gene. DESIGN: Observational follow-up study of gonadotrophin pulsatility pattern, and serum levels of antimüllerian hormone and inhibin B through pubertal development in these patients. PATIENTS: Three patients belonging to two families with AHC were included in this study. MEASUREMENTS: The gonadotrophic pattern, serum inhibin B and antimüllerian hormone were determined in relation to clinical Tanner stage of pubertal development. RESULTS: One patient showed a marked elevation in serum FSH concomitantly with low inhibin B and antimüllerian hormone levels, indicating a primary testicular dysfunction. The other two patients showed a gonadotrophic pattern of HHG, and their serum levels of inhibin B and antimüllerian hormone also reflected a moderate primary testicular dysfunction. The three patients were azoospermic. CONCLUSIONS: These cases give further insight into the clinical spectrum of phenotypes of the hypothalamic-pituitary-gonadal axis in patients with variants in hypogonadism associated with childhood-onset X-linked AHC due to DAX-1 mutations.

Testicular degeneration in Huntington disease.

Huntington disease (HD) is an adult onset, neurodegenerative disorder that results from CAG expansion in the HD gene. Recent work has demonstrated testicular degeneration in mouse models of HD and alterations in the hypothalamic-pituitary-gonadal (HPG) axis in HD patients. Here, we show that HD patients have specific testicular pathology with reduced numbers of germ cells and abnormal seminiferous tubule morphology. In the YAC128 mouse model, testicular degeneration develops prior to 12 months of age, but at 12 months, there is no evidence for decreased testosterone levels or loss of GnRH neurons in the hypothalamus. This suggests that testicular pathology results from a direct toxic effect of mutant huntingtin in the testis and is supported by the fact that huntingtin is highly expressed in the affected cell populations in the testis. Understanding the pathogenesis of HD in the testis may reveal common critical pathways which lead to degeneration in both the brain and testis.

Cold shock domain family members YB-1 and MSY4 share essential functions during murine embryogenesis.

Three cold shock domain (CSD) family members (YB-1, MSY2, and MSY4) exist in vertebrate species ranging from frogs to humans. YB-1 is expressed throughout embryogenesis and is ubiquitously expressed in adult animals; it protects cells from senescence during periods of proliferative stress. YB-1-deficient embryos die unexpectedly late in embryogenesis (embryonic day 18.5 [E18.5] to postnatal day 1) with a runting phenotype. We have now determined that MSY4, but not MSY2, is also expressed during embryogenesis; its abundance declines substantially from E9.5 to E17.5 and is undetectable on postnatal day 1(adult mice express MSY4 in testes only). Whole-mount analysis revealed similar patterns of YB-1 and MSY4 RNA expression in E11.5 embryos. To determine whether MSY4 delays the death of YB-1-deficient embryos, we created and analyzed MSY4-deficient mice and then generated YB-1 and MSY4 double-knockout embryos. MSY4 is dispensable for normal development and survival, but the testes of adult mice have excessive spermatocyte apoptosis and seminiferous tubule degeneration. Embryos doubly deficient for YB-1 and MSY4 are severely runted and die much earlier (E8.5 to E11.5) than YB-1-deficient embryos, suggesting that MSY4 indeed shares critical cellular functions with YB-1 in the embryonic tissues where they are coexpressed.