Male Rat Germ Cells Display Age-Dependent and Cell-Specific Susceptibility in Response to Oxidative Stress Challenges.

For decades male germ cells were considered unaffected by aging, due to the fact that males continue to generate sperm into old age; however, evidence indicates that germ cells from aged males are of lower quality than those of young males. The current study examines the effects of aging on pachytene spermatocytes and round spermatids, and is the first study to culture these cells in isolation for an extended period. Our objective is to determine the cell-specific responses germ cells have to aging and oxidative insult. Culturing isolated germ cells from young and aged Brown Norway rats revealed that germ cells from aged males displayed an earlier decline in viability, elevated levels of reactive oxygen species (ROS), and increased spermatocyte DNA damage, compared to young males. Furthermore, oxidative insult by prooxidant 3-morpholinosydnonimine provides insight into how spermatocytes and spermatids manage excess ROS. Genome-wide microarray analyses revealed that several transcripts for antioxidants, Sod1, Cat, and Prdxs, were up-regulated in response to ROS in germ cells from young males while being expressed at lower levels in the aged. In contrast, the expression of DNA damage repair genes Rad50 and Atm were increased in the germ cells from aged animals. Our data indicate that as germ cells undergo spermatogenesis, they adapt and respond to oxidative stress differently, depending on their phase of development, and the process of aging results in redox dysfunction. Thus, even at early stages of spermatogenesis, germ cells from aged males are unable to mount an appropriate response to manage oxidative stress.

Aging results in molecular changes in an enriched population of undifferentiated rat spermatogonia.

A strong correlation exists between increasing paternal age and a decline in reproductive function. Testis aging is associated with testicular atrophy, increased DNA damage, and de novo mutations. It is unclear whether these problems arise from the spermatogonial stem cells (SSCs), a buildup of anomalies as older germ cells progress through spermatogenesis, or both. We hypothesize that with the continual divisions of SSCs that maintain the germ cell population, an alteration of these cells occurs over time. To test this, we utilized young (4-mo-old) and aged (18- and 21-mo-old) transgenic rats that express GFP in germ cells only. We first examined the number and activity of SSCs from the different age groups by transplantation. Aged rats had numerically fewer SSCs than young rats (<50%; not significant) despite the lack of testicular atrophy, and 21-mo-old rats show a significant reduction in colony length, suggesting that the quality of SSCs also deteriorates. To evaluate any molecular changes occurring in the early cells of spermatogenesis with age, we isolated an SSC-enriched population of CD9-positive (CD9(+)) cells using fluorescence-activated cell sorting (confirmed by transplantation studies) and extracted RNA for microarray analysis. In the aged CD9(+) cells, 60 transcripts were upregulated and more than 500 downregulated compared to the young cells. An altered expression was found for transcripts involved in mitosis and in DNA damage response. These results suggest molecular alterations in the SSC-enriched population of aged CD9(+) cells, implying that reproductive aging originates in the undifferentiated cells of spermatogenesis.

Aging results in differential regulation of DNA repair pathways in pachytene spermatocytes in the Brown Norway rat.

The present trend of increasing paternal age is accompanied by concerns for the development of complex multigene diseases (e.g., autism and schizophrenia) in progeny. Recent studies have established strong correlations between male age, increased oxidative stress, decreased sperm quality, and structural aberrations of chromatin and DNA in spermatozoa. We tested the hypothesis that increasing age would result in altered gene expression relating to oxidative stress and DNA damage/repair in germ cells. To test this hypothesis, pachytene spermatocytes and round spermatids were isolated from Brown Norway (BN) rats at 4 (young) and 18 (aged) mo of age. Microarray analysis was used to compare gene expression between the groups. The probe sets with significantly altered expression were linked to DNA damage/repair and oxidative stress in pachytene spermatocytes but not in round spermatids. Further analysis of pachytene spermatocytes demonstrated that genes involved in the base excision repair (BER) and nucleotide excision repair (NER) pathways were specifically altered. Quantitative RT-PCR confirmed that NER genes were upregulated (>1.5-fold), whereas BER genes were downregulated (>1.5-fold). At the protein level the members of the BER pathway were also altered by up to 2.3-fold; levels of NER proteins remained unchanged. Furthermore, there was an increase in 8-oxo-2'-deoxyguanosine (8-oxodG) immunoreactivity in testes from aged males and in the number of spermatozoa positive for 8-oxodG. In conclusion, aging is associated with differential regulation of DNA repair pathways with a decrease in the BER pathway leading to deficient repair of 8-oxo-dG lesions in germ cells and spermatozoa.

Histopathological characterisation of effects of the mouse Pax6(Leca4) missense mutation on eye development.

Mutations in PAX6/Pax6 lead to a variety of ocular anomalies in humans and mice. The aim of the study was to characterise the ocular abnormalities caused by the missense Pax6(Leca4) mutation and compare them to published observations on Pax6 alleles that are functionally equivalent to Pax6(-) null alleles (such as Pax6(Sey) and Pax6(Sey-Neu)) and human inherited eye diseases. Ocular features of homozygous Pax6(Leca4)(/Leca4) and heterozygous Pax6(Leca4)(/+) embryos at E12.5-E18.5, heterozygous Pax6(Leca4)(/+) young mice at P18 and heterozygous Pax6(Leca4)(/+) adults at 12 weeks were analysed histologically with their wild-type Pax6(+/+) littermates. Homozygous Pax6(Leca4)(/Leca4) fetuses died perinatally with no eyes although an optic cup rudiment with pigmented cells developed. Pax6(Leca4)(/+) mice were microphthalmic and a range of other severe ocular phenotypes affected both the anterior and the posterior segments. In contrast to Pax6(+/-), the Pax6(Leca4)(/+) eyes had no goblet cells in the corneal epithelium, the iris was not hypoplastic and there was no lens-corneal epithelial plug. However, microphthalmia was more severe, corneal vascularisation occurred earlier (during fetal stages), pigmented cells were present in the vitreous and corneal stroma and the ciliary body was malformed or abnormal. These results show that, although Pax6(Leca4)(/+) lacked some eye abnormalities commonly seen in Pax6(Sey)(/+) and Pax6(Sey-Neu)(/+) eyes, in most respects their eyes were more severely affected. These differences probably reflect both differences between the Pax6(Leca4) and the Pax6(Sey-Neu) mutations and differences in modifier gene expression in different genetic backgrounds. The presence of pigmented cells in the cornea is a novel observation. Some Pax6(Leca4)(/+) ocular abnormalities were similar to those present in human Peters' anomaly and persistent hyperplastic primary vitreous (PHPV) so Pax6(Leca4)(/+) mice provide a useful model for some inherited eye diseases.

Do heat stress and deficits in DNA repair pathways have a negative impact on male fertility?

In Europe up to one in four couples experience difficulty conceiving and in half of these cases the problem has been attributed to sub or infertility in the male partner. The development of assisted reproductive technologies (ART) such as in vitro fertilization and intra-cytoplasmic spermatozoa injection has allowed some such couples to achieve a pregnancy. Concerns have been raised over the increasing use of ART not least because of the discovery of elevated levels of DNA damage in sperm from subfertile men. The impact of damaged DNA originating in the male germ line is poorly understood, but is thought to contribute to early pregnancy loss (recurrent miscarriage), placental problems and have a long-term impact on the health of the offspring. DNA repair is essential for meiotic recombination and correction of DNA damage in germ cells and proteins involved in all the major repair pathways are expressed in the testis. In this review, we will consider evidence that the production of sperm containing damaged DNA can be the result of suboptimal DNA repair and/or a mild environmental insult, such as heat stress, and how studies in mice may give us insight into the origins and consequences of DNA damage in human sperm.

A single, mild, transient scrotal heat stress causes DNA damage, subfertility and impairs formation of blastocysts in mice.

Infertility represents a major clinical problem and 50% of cases are attributable to the male partner. Testicular function is temperature dependent, and in both man and mouse the position of the testes in the scrotum ensures that they are kept at between 2 and 8 degrees C below core body temperature. We used a mouse model to investigate the impact of a single, transient, mild, scrotal heat stress (38, 40 or 42 degrees C for 30 min) on testicular function, sperm DNA integrity and embryo survival. We detected temperature-dependent changes in testicular architecture, number of apoptotic cells and a significant reduction in testis weight 7 and 14 days after heat stress at 42 degrees C. We report for the first time that DNA strand breaks (gamma-H2AX-positive foci) were present in spermatocytes recovered from testes subjected to 40 or 42 degrees C. Fertility of heat-stressed males was tested 23-28 d after treatment (sperm at this time would have been spermatocytes at time of heating). Paternal heat stress at 42 degrees C resulted in reduced pregnancy rate, placental weight and litter size; pregnancies from the 40 degrees C group had increased resorptions at e14.5. Abnormalities in embryonic development were detected at e3.5 and in vitro fertilisation with sperm recovered 16 h or 23 d after scrotal stress at 42 degrees C revealed a block in development between the 4-cell and blastocyst stages. This study has provided evidence of temperature-dependent effects on germ cell DNA integrity and highlighted the importance of an intact paternal genome for normal embryo development.

Cellular and hormonal disruption of fetal testis development in sheep reared on pasture treated with sewage sludge.

The purpose of this study was to evaluate whether experimental exposure of pregnant sheep to a mixture of environmental chemicals added to pasture as sewage sludge (n = 9 treated animals) exerted effects on fetal testis development or function; application of sewage sludge was undertaken so as to maximize exposure of the ewes to its contents. Control ewes (n = 9) were reared on pasture treated with an equivalent amount of inorganic nitrogenous fertilizer. Treatment had no effect on body weight of ewes, but it reduced body weight by 12-15% in male (n = 12) and female (n = 8) fetuses on gestation day 110. In treated male fetuses (n = 11), testis weight was significantly reduced (32%), as were the numbers of Sertoli cells (34% reduction), Leydig cells (37% reduction), and gonocytes (44% reduction), compared with control fetuses (n = 8). Fetal blood levels of testosterone and inhibin A were also reduced (36% and 38%, respectively) in treated compared with control fetuses, whereas blood levels of luteinizing hormone and follicle-stimulating hormone were unchanged. Based on immunoexpression of anti-Müllerian hormone, cytochrome P450 side chain cleavage enzyme, and Leydig cell cytoplasmic volume, we conclude that the hormone changes in treated male fetuses probably result from the reduction in somatic cell numbers. This reduction could result from fetal growth restriction in male fetuses and/or from the lowered testosterone action; reduced immunoexpression of alpha-smooth muscle actin in peritubular cells and of androgen receptor in testes of treated animals supports the latter possibility. These findings indicate that exposure of the developing male sheep fetus to real-world mixtures of environmental chemicals can result in major attenuation of testicular development and hormonal function, which may have consequences in adulthood.

Ageing of the male germ line.

Several studies have demonstrated a decline in the male reproductive system, sperm quality, and fertility with advancing paternal age, yet many of the biological mechanisms that underlie this process remain poorly understood. It is unclear whether the problem arises from the progenitor spermatogonial stem cells (for example, from an accumulation of DNA damage and mutations), from the somatic niche present in the testis (consisting of Sertoli and peritubular myoid cells), or from a combination of the two. Current data, albeit from a small number of studies, suggest that both factors have a role in age-associated germ cell loss. What is clear, on the other hand, is that mounting evidence links paternal age to chromosomal damage and genetic problems in the children of older fathers. The frequency of de novo mutations increases markedly with age, leading to increased risk of breast cancer, cardiac defects, developmental disorders, behavioural disorders, and neurological disease in the children of older men. The current trend towards fathering children at a later age raises concerns regarding the risk of offspring developing complex multigene diseases.

Impaired function of the blood-testis barrier during aging is preceded by a decline in cell adhesion proteins and GTPases.

With increasing age comes many changes in the testis, including germ cell loss. Cell junctions in the testis tether both seminiferous epithelial and germ cells together and assist in the formation of the blood-testis barrier (BTB), which limits transport of biomolecules, ions and electrolytes from the basal to the adluminal compartment and protects post-meiotic germ cells. We hypothesize that as male rats age the proteins involved in forming the junctions decrease and that this alters the ability of the BTB to protect the germ cells. Pachytene spermatocytes were isolated from Brown Norway rat testes at 4 (young) and 18 (aged) months of age using STA-PUT velocity sedimentation technique. RNA was extracted and gene expression was assessed using Affymetrix rat 230 2.0 whole rat genome microarrays. Microarray data were confirmed by q-RT-PCR and protein expression by Western blotting. Of the genes that were significantly decreased by at least 1.5 fold, 70 were involved in cell adhesion; of these, at least 20 are known to be specifically involved in junction dynamics within the seminiferous epithelium. The mRNA and protein levels of Jam2, Ocln, cdh2 (N-cadherin), ctnna (α-catenin), and cldn11 (involved in adherens junctions), among others, were decreased by approximately 50% in aged spermatocytes. In addition, the GTPases Rac1 and cdc42, involved in the recruitment of cadherins to the adherens junctions, were similarly decreased. It is therefore not surprising that with lower expression of these proteins that the BTB becomes diminished with age. We saw, using a FITC tracer, a gradual collapse of the BTB between 18 and 24 months. This provides the opportunity for harmful substances and immune cells to cross the BTB and cause the disruption of spermatogenesis that is observed with increasing age.

A single, mild, transient scrotal heat stress causes hypoxia and oxidative stress in mouse testes, which induces germ cell death.

Spermatogenesis is a temperature-dependent process, and increases in scrotal temperature can disrupt its progression. We previously showed that heat stress causes DNA damage in germ cells, an increase in germ cell death (as seen on TUNEL staining), and subfertility. The present study evaluated the stress response in mouse testes following a single mild transient scrotal heat exposure (40 degrees C or 42 degrees C for 30 min). We investigated markers of three types of stress response, namely, hypoxia, oxidative stress, and apoptosis. Heat stress caused an increase in expression of hypoxia-inducible factor 1 alpha (Hif1a) mRNA expression and translocation of HIF1A protein to the germ cell nucleus, consistent with hypoxic stress. Increased expression of heme oxygenase 1 (Hmox1) and the antioxidant enzymes glutathione peroxidase 1 (GPX1) and glutathione S-transferase alpha (GSTA) was consistent with a robust oxidative stress response. Germ cell death was associated with an increase in expression of the effector caspase cleaved caspase 3 and a decrease in expression of the protein inhibitor of caspase-activated DNase (ICAD). Reduced expression of ICAD contributes to increased activity of caspase-activated DNase and is consistent with the increased rates of DNA fragmentation that have been detected previously using TUNEL staining. These studies confirmed that transient mild testicular hyperthermia results in temperature-dependent germ cell death and demonstrated that elevated temperature results in a complex stress response, including induction of genes associated with oxidative stress and hypoxia.

Deletion of genes implicated in protecting the integrity of male germ cells has differential effects on the incidence of DNA breaks and germ cell loss.

BACKGROUND: Infertility affects approximately 20% of couples in Europe and in 50% of cases the problem lies with the male partner. The impact of damaged DNA originating in the male germ line on infertility is poorly understood but may increase miscarriage. Mouse models allow us to investigate how deficiencies in DNA repair/damage response pathways impact on formation and function of male germ cells. We have investigated mice with deletions of ERCC1 (excision repair cross-complementing gene 1), MSH2 (MutS homolog 2, involved in mismatch repair pathway), and p53 (tumour suppressor gene implicated in elimination of germ cells with DNA damage). PRINCIPAL FINDINGS: We demonstrate for the first time that depletion of ERCC1 or p53 from germ cells results in an increased incidence of unrepaired DNA breaks in pachytene spermatocytes and increased numbers of caspase-3 positive (apoptotic) germ cells. Sertoli cell-only tubules were detected in testes from mice lacking expression of ERCC1 or MSH2 but not p53. The number of sperm recovered from epididymes was significantly reduced in mice lacking testicular ERCC1 and 40% of sperm contained DNA breaks whereas the numbers of sperm were not different to controls in adult Msh2 -/- or p53 -/- mice nor did they have significantly compromised DNA. CONCLUSIONS: These data have demonstrated that deletion of Ercc1, Msh2 and p53 can have differential but overlapping affects on germ cell function and sperm production. These findings increase our understanding of the ways in which gene mutations can have an impact on male fertility.