Number Of Germ Cells Research Articles

MORPHOLOGICAL CRITERIA FOR ESTIMATION OF THE DEGREE OF TESTICULAR HYPOPLASIA IN CHILDREN WITH CRYPTORCHIDISM

The possible risk of the development of hypogonadism, infertility, and malignant neoplasm of the testicles in adults dictates the need to develop and implement diagnostic criteria. The identification of such morphological signs as a decrease in the number and diameter of the tubules, the number of germ cells, the development and progression of fibrosis of interstitium may have prognostic significance. The aim of the study is to develop morphological criteria for assessing the severity of testicular hypoplasia in cryptorchidism in children of different age groups. Material and methods. In the study there were identified three groups, taking into account the age of children with cryptorchidism. Group, I included 12 (34.3%) children aged from 1 to 3 years. Group II was presented by 14 (40%) patients aged from 4 to 10 years. Group III included 9 (25.7%) patients aged from 11-14 years. Results and discussion. Morphological changes in the testicle in cryptorchidism children of different age groups vary: in Group I moderate and pronounced structural changes were detected in 53% of cases, in group II - in 50% and in group III - in 72% of cases. Taking into account the morphological criteria of testicular tissue hypoplasia, in our study in cryptorchidism children, hypoplasia of the mild degree was revealed in 17.1% of cases, a moderate degree - in 31.4%, a severe degree - in 31.4% of cases. Conclusion. Morphological criteria for estimating hypoplasia and grading of the severity of hypoplasia of testicular tissue were determined on the basis of a scoring of morphological characters and morphometric indices.

Regulation of anti-Müllerian hormone (AMH) in males and the associations of serum AMH with the disorders of male fertility.

Anti-Müllerian hormone (AMH) is a functional marker of fetal Sertoli cells. The germ cell number in adults depends on the number of Sertoli cells produced during perinatal development. Recently, AMH has received increasing attention in research of disorders related to male fertility. This paper reviews and summarizes the articles on the regulation of AMH in males and the serum levels of AMH in male fertility-related disorders. We have determined that follicle-stimulating hormone (FSH) promotes AMH transcription in the absence of androgen signaling. Testosterone inhibits the transcriptional activation of AMH. The undetectable levels of serum AMH and testosterone levels indicate a lack of functional testicular tissue, for example, that in patients with anorchia or severe Klinefelter syndrome suffering from impaired spermatogenesis. The normal serum testosterone level and undetectable AMH are highly suggestive of persistent Müllerian duct syndrome (PMDS), combined with clinical manifestations. The levels of both AMH and testosterone are always subnormal in patients with mixed disorders of sex development (DSD). Mixed DSD is an early-onset complete type of disorder with fetal hypogonadism resulting from the dysfunction of both Leydig and Sertoli cells. Serum AMH levels are varying in patients with male fertility-related disorders, including pubertal delay, severe congenital hypogonadotropic hypogonadism, nonobstructive azoospermia, Klinefelter syndrome, varicocele, McCune-Albright syndrome, and male senescence.

Apoptosis in Spermiogenesis of Multiple Robertsonian Heterozygotes of Mus musculus domesticus

Mus musculus domesticus is a species that is characterized by a diploid number of 40 chromosomes, all telocentrics (acrocentrics). In natural populations of Mus with high frequency Robertsonian chromosome translocations (RBs), a fusion at centromere-level between two autosomal telocentric, producing metacentric Rb chromosomes and a variety of natural subspecies with diploid chromosome numbers below 40. Rb chromosomes do not affect the viability of individuals but mainly fertility of Rb heterozygotes. In their meiosis the metacentric Rb and the homologous telocentrics form trivalents that have problems in synapsis and segregation. This paper presents a comparative analysis of spermatogenesis considering the stages of the epithelium and the number of germ cells loss by apoptosis comparing heterozygote males 2n=32, carriers 8 trivalents, and homozygotes 2n=40 and 2n=24. It was found that the number of spermatocytes in first prophase was similar in the different seminiferous epithelium stages in all the chromosome constitutions. In the 2n=32 heterozygotes a significant decreased number of spermatids was reflected in the proportion of spermatocytes and spermatids that was 1:1.7. In the homozygote males rare germ cell in apoptosis (positive for caspase 3) were observed, which were mainly concentrated in the XII stage of the seminiferous epithelium. In heterozygote spermatocytes apoptotic germ cell number was significantly higher than that registered in homozygote males, and generally correspond to spermatocytes in meiotic divisions. This selective removal of cells possibly carrying anomalies, either in chromosome alignment or segregation, is consistent with the smaller number of spermatids and the relative sub-fertility observed in 2n=32 Rb heterozygotes.

Necrosis and necroptosis in germ cell depletion from mammalian ovary.

The maximum number of germ cells is present during the fetal life in mammals. Follicular atresia results in rapid depletion of germ cells from the cohort of the ovary. At the time of puberty, only a few hundred (<1%) germ cells are either culminated into oocytes or further get eliminated during the reproductive life. Although apoptosis plays a major role, necrosis as well as necroptosis, might also be involved in germ cell elimination from the mammalian ovary. Both necrosis and necroptosis show similar morphological features and are characterized by an increase in cell volume, cell membrane permeabilization, and rupture that lead to cellular demise. Necroptosis is initiated by tumor necrosis factor and operated through receptor interacting protein kinase as well as mixed lineage kinase domain-like protein. The acetylcholinesterase, cytokines, starvation, and oxidative stress play important roles in necroptosis-mediated granulosa cell death. The granulosa cell necroptosis directly or indirectly induces susceptibility toward necroptotic or apoptotic cell death in oocytes. Indeed, prevention of necrosis and necroptosis pathways using their specific inhibitors could enhance growth/differentiation factor-9 expression, improve survivability as well as the meiotic competency of oocytes, and prevent decline of reproductive potential in several mammalian species and early onset of menopause in women. This study updates the information and focuses on the possible involvement of necrosis and necroptosis in germ cell depletion from the mammalian ovary.

All-Trans Retinoic Acid Disrupts Development in Ex Vivo Cultured Fetal Rat Testes. I: Altered Seminiferous Cord Maturation and Testicular Cell Fate.

Exposure to excess retinoic acid (RA) disrupts the development of the mammalian testicular seminiferous cord. However, the molecular events surrounding RA-driven loss of cord structure have not previously been examined. To investigate the mechanisms associated with this adverse developmental effect, fetal rat testes were isolated on gestational day 15, after testis determination and the initiation of cord development, and cultured in media containing all-trans RA (ATRA; 10-8 to 10-6 M) or vehicle for 3 days. ATRA exposure resulted in a concentration-dependent decrease in the number of seminiferous cords per testis section and number of germ cells, assessed by histopathology and immunohistochemistry. Following 1 day of culture, genome-wide expression profiling by microarray demonstrated that ATRA exposure altered biological processes related to retinoid metabolism and gonadal sex determination. Real-time RT-PCR analysis confirmed that ATRA enhanced the expression of the key ovarian development gene Wnt4 and the antitestis gene Nr0b1 in a concentration-dependent manner. After 3 days of culture, ATRA-treated testes contained both immunohistochemically DMRT1-positive and FOXL2-positive somatic cells, providing evidence of disrupted testicular cell fate maintenance following ATRA exposure. We conclude that exogenous RA disrupts seminiferous cord development in ex vivo cultured fetal rat testes, resulting in a reduction in seminiferous cord number, and interferes with maintenance of somatic cell fate by enhancing expression of factors that promote ovarian development.

Effect of Fe and Cd Co-Exposure on Testicular Steroid Metabolism, Morphometry, and Spermatogenesis in Mice.

The mechanism of testicular toxicity of simultaneous multiple exposures to metals is poorly understood. Previous studies reported that the toxic effect of cadmium (Cd) is modified by tissue concentration of iron (Fe). Using the mice (Mus musculus) model in the present study, we demonstrated that combined Cd (25mgkg-1 bw) and Fe (100mgkg-1 bw) treatment increased both Cd and Fe testicular concentrations much more than separate exposures to either of the metals. Intratesticular Cd and Fe concentrations were inversely correlated (r = - 0.731, p < 0.05) on administration of Fe but not on combined exposure to both metals when they were positively correlated (versus Cd; r = 0.793, versus Fe; r = 0.779, p < 0.05). Additionally, Cd + Fe treatment increased testicular lipid peroxidation and depleted intratestesticular testosterone, cholesterol and glutathione concentrations much more than their separate treatment. This was also associated with decreased activity of the germ cell marker, testicular lactate dehydrogenase, and increased testicular myeloperoxidase activity. These changes resulted in decreased seminiferous epithelial height, tubular diameter, germ cell (spermatogonia, spermatocytes, and spermatids) numbers, and severe tissue damage. In conclusion, Cd + Fe intake have synergistic toxic effects on testicular steroid formation and spermatogenesis due to the high testicular concentrations of both metals.

Formation of ovarian reserve

The review of the literature is devoted to modern data on the formation of the ovarian reserve of the female sexual organ. The relationship between the size of the ovarian reserve and length of reproductive capacity emphasizes the importance of understanding the regulatory factors and processes that determine its creation. We described ovarian reserve markers and regulators such as oocyte phosphotidylinositol-3-kinase, a stem-cell factor (kit ligand) that promote the survival of follicles during neonatal development, synaptonemic complex (SCP3), which is the marker of the first division of meiosis, as well as genes DMC1 and PTEN, involved in meiotic transformations and recruitment of primordial follicles. Changes in the expression of some genes and factors in the human fetal ovaries during primary follicular assembly now give an idea of the ways controlling early folliculogenesis. Aberrant production of these factors can cause dysfunction, the development of ovarian disorders and a defective follicular reserve. In particular, the degree of change in the number of germ cells at each of the stages leading to the creation of an ovarian reserve should be noted. This change can affect the final size of the follicular stock, and, consequently, the reproductive longevity of a person and health in the postproductive period. In particular, the number of primary follicles during puberty is positively correlated with the number of growing follicles and their response to gonadotropin treatment. The size of the ovarian reserve depends on the genes involved in proliferation and differentiation of germ cells, sexual differentiation, meiosis, germ cell degeneration, the formation of primary follicles, and the potential mechanism for self-renewal of embryonic stem cells. For example, a possible molecular mechanism has been established leading to a meiotic process in oocytes involving the above genes and factors, as well as apoptotic and antiapoptical signals: Bax, Bcl-2, p53, CDK1, Lsd1, Notch, Stra8, Dazl, Dmc1, Rec8, XIAP , PUMA. Therefore, understanding all the subtleties and molecular mechanisms at each stage of laying down and developing the ovaries, sex cells and their environment, and the death of gametes, can help to search for possible regulators and prevent pathological depletion of the follicular stock.

Loss of connexin 43 in Sertoli cells provokes postnatal spermatogonial arrest, reduced germ cell numbers and impaired spermatogenesis

For the reason that adult Sertoli cell specific connexin 43 knockout (SCCx43KO) mice show arrested spermatogenesis at spermatogonial level or Sertoli cell only tubules and significantly reduced germ cell (GC) numbers, the aims of the present study were (1) to characterize the remaining GC population and (2) to elucidate possible mechanisms of their fading. Apoptosis was analyzed in both, KO and wild type (WT) male littermates during postnatal development and in adulthood using TUNEL. Although GC numbers were significantly reduced in KO at 2 and 8 days postpartum (dpp) when compared to WT, no differences were found concerning apoptotic incidence between genotypes. From 10 dpp, the substantial GC deficiency became more obvious. However, significantly higher apoptotic GC numbers were seen in WT during this period, possibly related to the first wave of spermatogenesis, a known phenomenon in normal pubertal testes associated with increased apoptosis. Characterization of residual spermatogonia in postnatal to adult KO and WT mice was performed by immunohistochemical reaction against VASA (marker of GCs in general), Lin28 and Fox01 (markers for undifferentiated spermatogonia) and Stra8 (marker for differentiating spermatogonia and early spermatocytes). During puberty, the GC component in SCCx43KO mice consisted likely of undifferentiated spermatogonia, few differentiating spermatogonia and very few early spermatocytes, which seemed to be rapidly cleared by apoptosis. In adult KOs, spermatogenesis was arrested at the level of undifferentiated spermatogonia. Overall, our data indicate that Cx43 gap junctions in SCs influence male GC development and differentiation rather than their survival.

Spermatogonia survival in young ram lambs following irradiation, Busulfan or thermal treatment

Many alternatives to surgical castration have been explored to induce short or long term infertility in male animals. Comparatively few have been carefully evaluated in very young production animals. A comparison of four treatments known to deplete testicular cells in rodents (heating, cooling, chemotherapy, radiation) was undertaken using ram lambs.Neither testicular cooling (0 °C) nor heating (45 °C) affected testis weights, tubule diameters or germ cell numbers. Low dose chemotherapy (Busulfan 4 mg/kg) treatment caused dramatic falls in white blood cells and platelets numbers which recovered within 3 weeks. Spermatogonia numbers were not significantly reduced (27% change).The impact of irradiation doses (0–15 Gy) delivered with high precision to the testis by a 6 MV photon beam was assessed by serial biopsies. Sertoli cell numbers were depleted by 90% at 3 weeks in 15 Gy treated testes. Spermatogonia were depleted 8 weeks after irradiation with 9 Gy, 12 Gy and 15 Gy. By 13 weeks, only in the 15 Gy treated testes were spermatogonia and Sertoli cell numbers lower. At 13 weeks testis atrophy resulted in 3/6 lambs irradiated with 12 Gy or 15 Gy. Irradiation of very young lambs clearly compromised testis function, thermal treatment was ineffective and busulfan treatment resulted in minimal effects.

Selecting Infants With Cryptorchidism and High Risk of Infertility for Optional Adjuvant Hormonal Therapy and Cryopreservation of Germ Cells: Experience From a Pilot Study.

Orchiopexy for congenital cryptorchid testes is recommended between ½ and 1 year of age to preserve testicular germ cell maturation. Early operation is not enough to preserve fertility in 22 and 36% of cases. Aim of this study was to set up a protocol for optional adjuvant hormonal therapy after orchiopexy and thereafter cryopreservation of testicular biopsies from infants with bilateral cryptorchidism and high infertility risk. We included 17 boys with bilateral cryptorchidism, normal FSH, and impaired germ cell number per tubular transverse section (G/T) in testicular biopsies at orchiopexy, 7 months to 3½ years old. Postoperatively, optional adjuvant LHRH (kryptocur®) 0.2 mg/0.1 mL 2× every second day in 16 weeks were offered. Ten boys were applicable for age matching according to parent's choice of treatment regime and G/T. Five of them had kryptocur®, and five were controls. Repeat bilateral testicular biopsy evaluation and cryopreservation were offered to all boys 12 months after primary orchiopexy. For cryopreservation, tissue pieces were incubated with a cryoprotectant with a slow program freezing. Two out of five kryptorcur®-treated boys normalized both the average G/T and the number of adult dark spermatogonia (Ad-S). Another kryptocur®-treated boy with initial low G/T and no Ad-S increased the G/T and achieved normal number of Ad-S at time of cryopreservation. In the control group, two patients reached only normal lower range regarding the G/T and the number of Ad-S. None of boys with less than average 0.2 G/T improved significantly, whether they were kryptocur®-treated or not. Based on literature and the present results, we recommend adjuvant LHRH treatment to boys with cryptorchidism and insufficient genuine gonadotropin stimulation at time of surgery, as these patients have high infertility risk. Cryopreservation should be an option in case of treatment failure of adjuvant LHRH. However, to avoid repeat surgery with biopsy, some parents may choose biopsy for cryopreservation at time of the initial bilateral orchiopexy, well informed that the procedure may only be truly indicated in 22 and 36% of the cases.

The Expression of Markers for Intratubular Germ Cell Neoplasia in Normal Infantile Testes

Positive immunohistochemical expression of testicular cancer markers is often reported beyond 12 months of age in cryptorchid testes, which is assumed to indicate delayed maturation of the fetal germ cells, or neoplastic changes. These findings allowed for questions as to the extent of positive reaction in normal testes. The aim of the study was to clarify the expression of these markers in a normal material up to 2 years. Testicular material from 69 boys aged 1-690 days, who died of causes with no association of testicular pathology. Histology sections were incubated with primary antibodies including anti-placental-like alkaline phosphatase (PLAP), anti-C-Kit, anti-D2-40, and anti-Oct3/4. The mean germ cell number per tubular transverse section (G/T) was calculated based on the G/T of both testes of every boy. The mean G/T declined through the 690 days. PLAP appeared stably expressed throughout the ages studied. The likelihood of a positive reaction for C-Kit waned with increasing age within the study period. Positive staining for D2-40 and Oct3/4 was demonstrated up to 6 and 9 months respectively. Up to 1 or 2 years of age, normal infantile testes contain germ cells positive for the immunohistochemical markers commonly utilized to aid in the detection of testicular cancer. This finding supports the concept of germ cells undergoing a continuous maturational process in a heterogeneous fashion, and that this process is not complete by 2 years of age.

Glutathione deficiency sensitizes cultured embryonic mouse ovaries to benzo[a]pyrene-induced germ cell apoptosis

Mice lacking the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in glutathione (GSH) synthesis, have decreased tissue GSH. We previously showed that Gclm−/− embryos have increased sensitivity to the prenatal in vivo ovarian toxicity of the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) compared with Gclm+/+ littermates. We also showed that BaP-induced germ cell death in cultured wild type embryonic ovaries is caspase-dependent. Here, we hypothesized that GSH deficiency increases sensitivity of cultured embryonic ovaries to BaP-induced germ cell death. 13.5 days post coitum (dpc) embryonic ovaries of all Gclm genotypes were fixed immediately or cultured for 24 h in media supplemented with DMSO vehicle or 500 ng/ml BaP. The percentage of activated caspase-3 positive germ cells varied significantly among groups. Within each genotype, DMSO and BaP-treated groups had increased germ cell caspase-3 activation compared to uncultured. Gclm+/− ovaries had significantly increased caspase-3 activation with BaP treatment compared to DMSO, and caspase-3 activation increased non-significantly in Gclm−/− ovaries treated with BaP compared to DMSO. There was no statistically significant effect of BaP treatment on germ cell numbers at 24 h, consistent with our prior observations in wild type ovaries, but Gclm−/− ovaries in both cultured groups had lower germ cell numbers than Gclm+/+ ovaries. There were no statistically significant BaP-treatment or genotype-related differences among groups in lipid peroxidation and germ cell proliferation. These data indicate that Gclm heterozygous or homozygous deletion sensitizes embryonic ovaries to BaP- and tissue culture-induced germ cell apoptosis.

Interspecific germ cell transplantation: a new light in the conservation of valuable Balkan trout genetic resources?

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia-27%; grayling spermatogonia-28%; grayling oogonia-23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.

When does germ cell loss and fibrosis occur in patients with Klinefelter syndrome?

When does germ cell loss and fibrosis occur in patients with Klinefelter syndrome (KS)? In KS, germ cell loss is not observed in testicular tissue from fetuses in the second semester of pregnancy but present at a prepubertal age when the testicular architecture is still normal, while fibrosis is highly present at an adolescent age. Most KS patients are azoospermic at adult age because of a massive germ cell loss. However, the timing when this germ cell loss starts is not known. It is assumed that germ cell loss increases at puberty. Therefore, testicular sperm extraction (TESE) at an adolescent age has been suggested to increase the chances of sperm retrieval at onset of spermatogenesis. However, recent data indicate that testicular biopsies from peripubertal KS patients contain only a few germ cells. In this study, we give an update on fertility preservation in adolescent KS patients and evaluate whether fertility preservation would be beneficial at prepubertal age. The possibility of retrieving testicular spermatozoa by TESE was evaluated in adolescent and adult KS men. The presence of spermatogonia and the degree of fibrosis were also analysed in testicular biopsies from KS patients at different ages. The patients were divided into four age groups: foetal (n = 5), prepubertal (aged 4-7 years; n = 4), peripubertal (aged 12-16 years; n = 20) and adult (aged 18-41 years; n = 27) KS patients. In peripubertal and adult KS patients, retrieval of spermatozoa was attempted by semen analysis after masturbation, vibrostimulation, electroejaculation or by TESE. MAGE-A4 immunohistochemistry was performed to evaluate the presence of germ cells in testicular biopsies from foetal, prepubertal, peripubertal and adult KS patients. Tissue morphology was evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Testicular spermatozoa were collected by TESE in 48.1% of the adult KS patients, while spermatozoa were recovered after TESE in only one peripubertal patient (5.0%). Germ cells were detectable in testicular biopsies from 21% of adult men for whom no spermatozoa could be retrieved by TESE and in 31.5% of peripubertal KS boys. Very small numbers of spermatogonia (0.03-0.06 spermatogonia/tubule) were detected in three out of four (75%) prepubertal patients. At a foetal age, the number of germ cells was similar for KS and control samples. Increased signs of fibrosis were not present at foetal and prepubertal ages, but peripubertal and adult KS patients showed high levels of fibrosis. N/A. Only four prepubertal biopsies were included in this study, but they all showed a very low germ cell number. A high variability in the number of spermatogonia per mm2 was observed in the limited (n = 5) number of foetal biopsies. However, testicular biopsies from prepubertal and foetal Klinefelter patients are difficult to obtain. Testicular tissue banking at a prepubertal age has been suggested as a potential method for fertility preservation in early diagnosed KS boys. However, our results show that a reduction in germ cell number has already taken place in childhood. Therefore, offering testicular tissue banking in young KS boys to prevent subsequent sterility might be a questionable strategy. However, this should be confirmed in a larger study population. This project was funded by the scientific Fund Willy Gepts from the UZ Brussel (D.V.S., J.D.S.), grants from the Vrije Universiteit Brussel (E.G.) and a Methusalem grant (K.S.). D.V.S is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

Impaired serum inhibin-B and number of germ cells in boys with cryptorchidism following heavily gestational maternal smoking

PurposeA meta-analysis including 11,900 cases showed that maternal gestational smoking was associated with increased risk of cryptorchidism. The aim of study was to investigate whether a hormone profile of cryptorchid boys and a supplementing histopathological evaluation of testicular biopsies could add detailed knowledge to the impact of maternal gestational smoking on pathogenesis of cryptorchidism. Methods601 cryptorchid boys aged 4 months to 14 years old were included. Because normal hormones have a pronounced age dependency, we compared results from boys whose mothers had smoked heavily (>10 cigarettes/day) during pregnancy with age matched cryptorchid controls of nonsmoking mothers (1:6). We studied: birthweight, germ-cell number/tubular cross section, frequency of germ cells positive for placental-like alkaline phosphatase (PLAP), gonadotropins and inhibin-B. Results501 boys were sons of nonsmokers, 72 boys of intermittent smokers and 28 boys of heavy smokers. 39%, 44% and 61% respectively had bilateral cryptorchidism. Compared to age-matched cryptorchid controls of nonsmoking mothers, sons of heavy smokers had lower birthweight (p = 0.006), germ-cell number/tubular cross section (p = 0.009), frequency of germ cells positive for PLAP (p = 0.037) and inhibin-B (p = 0.042). ConclusionsAll findings could be associated with placental dysfunction with altered human chorionic gonadotropin production well described in women smoking during pregnancy. Type of studyPrognosis study (prospective cohort study with >80% follow-up). Level of evidenceLevel 1.

In Vivo Genetic Manipulation of Spermatogonial Stem Cells and Their Microenvironment by Adeno-Associated Viruses

SummaryAdeno-associated virus (AAV) penetrates the blood-brain barrier, but it is unknown whether AAV penetrates other tight junctions. Genetic manipulation of testis has been hampered by the basement membrane of seminiferous tubules and the blood-testis barrier (BTB), which forms between Sertoli cells and divides the tubules into basal and adluminal compartments. Here, we demonstrate in vivo genetic manipulation of spermatogonial stem cells (SSCs) and their microenvironment via AAV1/9. AAV1/9 microinjected into the seminiferous tubules penetrated both the basement membrane and BTB, thereby transducing not only Sertoli cells and SSCs but also peritubular cells and Leydig cells. Moreover, when congenitally infertile KitlSl/KitlSl-d mouse testes with defective Sertoli cells received Kitl-expressing AAVs, spermatogenesis regenerated and offspring were produced. None of the offspring contained the AAV genome. Thus, AAV1/9 allows efficient germline and niche manipulation by penetrating the BTB and basement membrane, providing a promising strategy for the development of gene therapies for reproductive defects.

Sensitivity of spermatogonia to irradiation varies with age in pre-pubertal ram lambs

Although germ cells from donor rams transplanted into irradiated recipient testes have produced donor derived offspring, efficiency is low. Further optimization of recipient irradiation protocols will add precision to the depletion of recipient spermatogonia prior to germ cell transplant. Three irradiation doses (9,12,15 Gy) were administered to ram lambs aged 14 weeks (Group 1) and 20 weeks (Group 2), then testicular biopsies were collected 1, 2 and 3 months after irradiation. At 1 month after irradiation of Group 1, only the largest dose (15 Gy) reduced spermatogonia numbers below 10% of non-irradiated controls, whereas in Group 2 lambs, each irradiation dose reduced spermatogonia below 10% of controls. In both Groups, fewer differentiated germ cells were present in seminiferous tubules compared to controls. At 2 months after irradiation, spermatogonia numbers in both Groups increased more than sixfold to be similar to controls, whereas fewer differentiated germ cells were present in the tubules of both Groups. At 3 months in Group 1, each irradiation dose reduced spermatogonia numbers to <30% of controls and fewer tubules contained differentiated germ cells. Lesser expression of spermatogonial genes, VASA and UCHL-1, was observed in the 15 Gy group. In Group 2, only 12 Gy treated tubules contained fewer spermatogonia. Knowledge of these subtle differences between age groups in the effect of irradiation doses on spermatogonia or differentiated germ cell numbers and the duration of recovery of spermatogonia numbers after irradiation will aid the timing of germ cell transplants into prepubertal recipient lambs.

Testicular development in male lambs prenatally exposed to a high-starch diet.

Maternal nutrition during critical gestation periods impacts on offspring in later life; effects of high-starch maternal diet on testicular development in lambs were addressed. Dairy ewes were fed diets providing either 27% (Starch, S) or 11% (Fiber, F) of starch from mating to lambing (∼147 days; S147, F147) or for the last 75 days of gestation (S75, F75). Testes of single male lambs were measured and then sampled for histological and gene expression analyses at selected ages. Testicular dimensions and weight were similar among groups, but the total area of seminiferous tubules increased with age and tended to be higher (p = 0.057) in lambs from starch- than fiber-fed ewes. Sertoli and germ cells number increased with age, but was not influenced by maternal diet. Transcript abundances of androgen receptor (AR), insulin-like growth factor 1 (IGF1), and hydroxysteroid (17-beta) dehydrogenase 3 (HSD17B3) was similar between S147 and F147 lambs (p > 0.1). Abundance of luteinizing hormone/choriogonadotropin receptor (LHCGR) and steroidogenic acute regulatory protein (STAR) was higher in young vs older lambs, whereas insulin-like growth factor 2 (IGF2) levels increased with age. The expression of vascular endothelial growth factor A (VEGFA), Anti-Müllerian hormone (AMH), IGF1, follicle stimulating hormone receptor (FSHR), and insulin-like growth factor 2 receptor (IGF2R) was not influenced by maternal diet or lamb age (p > 0.1). In conclusion, a high-starch maternal diet did not influence gene expression, but may have affected testicular structure in infant offspring, as seen by an increase in the total area of seminiferous tubules.

Age-related and photoperiodic variation of the DAZ gene family in the testis of the Syrian hamster (Mesocricetus auratus).

SummaryThe Deleted in AZoospermia (DAZ) gene family regulates the development, maturation and maintenance of germ cells and spermatogenesis in mammals. The DAZ family consists of two autosomal genes, Boule and Dazl (Daz-like), and the Daz gene on chromosome Y. The aim of this study was to analyze the localization of DAZL and BOULE during testicular ontogeny of the seasonal-breeding Syrian hamster, Mesocricetus auratus. We also evaluated the testicular expression of DAZ family genes under short- or long-photoperiod conditions. In the pre-pubertal and adult testis, DAZL protein was found mainly in spermatogonia. BOULE was found in the spermatogonia from 20 days of age and during the pre-pubertal and adult period it was also detected in spermatocytes and round spermatids. DAZL and BOULE expression in spermatogonia was strictly nuclear only in 20-day-old hamsters. We also detected the novel mRNA and protein expression of BOULE in Leydig cells. In adult hamsters, Dazl expression was increased in regressed testis compared with non-regressed testis and DAZL protein expression was restricted to primary spermatocytes in regressed testis. These results show that DAZL and BOULE are expressed in spermatogonia at early stages in the Syrian hamster, then both proteins translocate to the cytoplasm when meiosis starts. In the adult regressed testis, the absence of DAZL in spermatogonia might be related to the decrease in germ cell number, suggesting that DAZ gene family expression is involved in changes in seminiferous epithelium during photoregression.

Endocrine-disrupting chemicals and male reproductive health: a review

Balanced functioning of the endocrine system is essential for preservation of human species by providing normal growth and development, reproduction, and normal functioning of all other organ systems. In the last decades, emerging area of interest is the impact of environmental exposures to human health. Important environmental pollutants are endocrine-disrupting che- micals (EDCs), which can have adverse e ects on the living organism due to their interference with the endocrine system. The group of known EDCs embraces ubiquitous synthetic substan- ces used as industrial lubricants and solvents, with their by-products, incomplete combustion remains, pharmaceuticals and personal care products, pesticides and plasticizers. Natural com- pounds such as genistein, a phytoestrogen, and heavy metals can also have endocrine e ects. Endocrine disruption is a serious public health problem. EDCs among other health problems ge- nerate reproductive disorders in males, such as decreases in sperm count and quality, increases in testicular germ cell numbers, prostate and breast cancers, cryptorchidism and hypospadias, impaired fertility, and infertility. This paper critically reviews the current knowledge of the impa- ct of EDCs on reproductive disorders in human males.