The growing follicles develop from a reserve of primordial follicles constituted early in life. From this pre-established reserve, a second ovarian reserve is formed, which consists of gonadotropin-responsive small antral growing follicles and is a dynamic reserve for ovulation. Its size, evaluated by direct antral follicular count or endocrine markers, determines the success of assisted reproductive technologies in humans and embryo production biotechnologies in animals. Strong evidence indicates that these two reserves are functionally related. The size of both reserves appears to be highly variable between individuals of similar age, but the equilibrium size of the dynamic reserve in adults seems to be specific to each individual. The dynamics of both follicular reserves appears to result from the fine tuning of regulations involving two main pathways, the phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDPK1)/v-akt murine thymoma viral oncogene homolog 1 (AKT1) and the bone morphogenetic protein (BMP)/anti-Müllerian hormone (AMH)/SMAD signaling pathways. Mutations in genes encoding the ligands, receptors, or signaling effectors of these pathways can accelerate or modulate the exhaustion rate of the ovarian reserves, causing premature ovarian insufficiency (POI) or increase in reproductive longevity, respectively. With female aging, the decline in primordial follicle numbers parallels the decrease in the size of the dynamic reserve of small antral follicles and the deterioration of oocyte quality. Recent progress in our knowledge of signaling pathways and their environmental and hormonal control during adult and fetal life opens new perspectives to improve the management of the ovarian reserves.

The preimplantation period is a time of reprogramming that may be vulnerable to disruption. This question has wide clinical relevance since the number of children conceived by in vitro fertilization (IVF) is rising. To examine this question, outbred mice (CF1 × B6D2F1) conceived by IVF and cultured using Whitten medium and 20% O₂ (IVF_WM group, less optimal) or K simplex optimized medium with amino acids and 5% O₂ (IVF_KAA group, more optimal and similar to conditions used in human IVF) were studied postnatally. We found that flushed blastocysts transferred to recipient mice provided the best control group (FB group), as this accounted for the effects of superovulation, embryo transfer, and litter size. We observed that many physiological parameters were normal. Reassuringly, IVF_KAA offspring did not differ significantly from FB offspring. However, male IVF_WM mice (but not females) were larger during the first 19 wk of life and exhibited glucose intolerance. Male IVF_WM mice also showed enlarged left heart despite normal blood pressure. Expression of candidate imprinted genes (H19, Igf2, and Slc38a4) in multiple adult tissues did not show differences among the groups; only Slc38a4 was down-regulated following IVF (in both culture conditions) in female adipose tissue. These studies demonstrate that adult metabolism is affected by the type of conditions encountered during the preimplantation stage. Further, the postnatal growth trajectory and glucose homeostasis following ex vivo manipulation may be sexual dimorphic. Future work on the long-term effects of IVF offspring should focus on glucose metabolism and the cardiovascular system.

Zinc is an essential nutrient for optimal fertility, but the effects of preconception zinc deficiency on postimplantation development are not known. Female mice were fed a control or a zinc-deficient diet (ZDD) for 4–5 days before ovulation (preconception). Embryonic and/or placental development were evaluated on Days 3.5, 6.5, 10.5, 12.5, and 16.5 of pregnancy. The findings show a decrease in embryo length (31%, Day 10.5; 13%, Day 12.5; 10%, Day 16.5) and weight (23%, Day 16.5) in embryos from mothers fed a ZDD preconception. Zinc deficiency also caused a high incidence of pregnancy loss (46%, Day 10.5; 34%, Day 12.5; 51%, Day 16.5) compared to control (2%, Day 10.5; 7%, Day 12.5; 9%, Day 16.5). ZDD embryos transferred to normal recipients were 38% smaller and implantation rate was only 10% compared to 40% for controls. Trophoblast cell differentiation and implantation on Day 6.5 of pregnancy were compromised by preconception zinc deficiency. On Day 12.5 of pregnancy, placenta weight and area of fetal placenta were decreased 37% and 31%, respectively, by preconception zinc deficiency. Consistent with a smaller fetal placenta, expression of key placental transcripts, including Ar, Esx1, Syna, Tfeb, Dlx3, and Gcm1 mRNA, but not Ctsq mRNA, were decreased 30%–70% in the ZDD group. Preconception zinc deficiency caused 41%–57% of embryos to exhibit delayed or aberrant neural tube development, as examined by light microscopy and magnetic resonance imaging. Collectively, the findings provide evidence for the importance of preconception zinc in promoting optimal fertility and oocyte developmental potential.

Embryo implantation is a complex interaction between maternal endometrium and embryonic structures. Failure to implant is highly recurrent and impossible to diagnose. Inflammation and infections in the female reproductive tract are common causes of infertility, embryo loss, and preterm labor. The current work describes how the activation of endometrial Toll-like receptor (TLR) 2 and 2/6 reduces embryo implantation chances. We developed a morphometric index to evaluate the effects of the TLR 2/6 activation along the uterine horn (UH). TLR 2/6 ligation reduced the endometrial myometrial and glandular indexes and increased the luminal index. Furthermore, TLR 2/6 activation increased the proinflammatory cytokines such as interleukin (IL)-1beta and monocyte chemotactic protein (MCP)-1 in UH lavages in the preimplantation day and IL-1 receptor antagonist in the implantation day. The engagement of TLR 2/6 with its ligand in the UH during embryo transfer severely affected the rate of embryonic implantation (45.00% ± 6.49% vs. 16.69% ± 5.01%, P < 0.05, control vs. test, respectively). Furthermore, this interference with the embryo implantation process was verified using an in vitro model of human embryo implantation where trophoblast spheroids failed to adhere to a monolayer of TLR 2- and TLR 2/6-activated endometrial cells. The inhibition of TLR receptors 2 and 6 in the presence of their specific ligands restored the ability of the spheroids to bind to the endometrial cells. In conclusion, the activation of the innate immune system in the uterus at the time of implantation interfered with the endometrial receptivity and reduced the chances of implantation success.

Previous reports have demonstrated that embryonic stem cells were capable of differentiating into primordial germ cells through the formation of embryoid bodies that subsequently generated oocyte-like cells (OLCs). Such a process could facilitate studies of primordial follicle oocyte development in vitro and regenerative medicine. To investigate the pluripotency of human amniotic fluid stem cells (hAFSCs) and their ability to differentiate into germ cells, we isolated a CD117 /CD44 hAFSC line that showed fibroblastoid morphology and intrinsically expressed both stem cell markers (OCT4, NANOG, SOX2) and germ cell markers (DAZL, STELLA). To encourage differentiation into OLCs, the hAFSCs were first cultured in a medium supplemented with 5% porcine follicular fluid for 10 days. During the induction period, cell aggregates formed and syntheses of steroid hormones were detected; some OLCs and granulosa cell-like cells could be loosened from the surface of the culture dish. Cell aggregates were collected and replated in oocyte culture medium for an additional 7–10 days. OLCs ranging from 50 to 120 μm presenting zona pellucida were observed in cumulus-oocyte complexes; some OLCs developed spontaneously into multicell structures similar to preimplantation embryos. Approximately 2% of the hAFSCs differentiated to meiotic germ cells that expressed folliculogenesis- and oogenesis-associated markers. Although the in vitro maturation and fertilization potentials are as yet unproven, short-term (<25 days) and high-efficiency (>2%) derivation of OLCs from hAFSCs might provide a new approach to the study of human germ cell development in vitro.

In various studies, chronic elevation of corticosterone levels in female birds under natural or experimental conditions resulted in female biased offspring sex ratios. In chicken, one study with injected corticosterone resulted in a male sex ratio bias. In the current study, we chronically elevated blood plasma corticosterone levels through corticosterone feeding (20 mg/kg feed) for 14 days using 30 chicken hens in each of treatment and control groups and studied the primary offspring sex ratio (here defined as the proportion of male fertile eggs determined in freshly laid eggs, i.e., without egg incubation). Mean plasma corticosterone concentrations were significantly higher in the treatment group but were not associated with sex ratio, laying rate, and fertility rate. Corticosterone treatment by itself did not affect egg sex but affected sex ratio as well as laying rate and fertility rate in interaction with hen body mass. Body mass had a negative association with sex ratio, laying rate, and fertility rate per hen in the corticosterone group, but a positive association with sex ratio in untreated hens. These interactions were already seen when taking the body mass at the beginning of the experiment, indicating intrinsic differences between light and heavy hens with regard to their reaction to corticosterone treatment. The effects on laying rate, fertility rate, and sex ratio suggest that some factor related to body mass act together with corticosterone to modulate ovarian functions. We propose that corticosterone treatment in conjunction with hen body mass can interfere with meiosis, which can lead to meiotic drive and to chromosomal aberrations resulting in postponed ovulation or infertile ova.

Betaine (N,N,N-trimethylglycine) has previously been shown to function in cell volume homeostasis in early mouse embryos and also to be a key donor to the methyl pool in the blastocyst. A betaine transporter (SLC6A20A or SIT1) has been shown to be activated after fertilization, but there is no saturable betaine uptake in mouse oocytes or eggs. Unexpectedly, the same high level of betaine is present in mature metaphase II (MII) eggs as is found in one-cell embryos despite the lack of transport in oocytes or eggs. Significant saturable betaine transport is, however, present in intact cumulus-oocyte complexes (COCs). This transport system has an affinity for betaine of ∼227 μM. The inhibition profile indicates that betaine transport by COCs could be completely blocked by methionine, proline, leucine, lysine, and arginine, and transport is dependent on Na but not Cl⁻. This is consistent with transport by a y L-type amino acid transport system. Both transcripts and protein of one y L isoform, SLC7A6 (y LAT2), are present in COCs, with little or no expression in isolated germinal vesicle (GV)-stage oocytes, MII eggs, or one-cell embryos. Betaine accumulated by COCs is transferred into the enclosed GV oocyte, which requires functional gap junctions. Thus, at least a portion of the endogenous betaine in MII eggs could be derived from transport into cumulus cells and subsequent transfer into the enclosed oocyte before gap junction closure during meiotic maturation. The oocyte-derived betaine then could be regulated and supplemented by the SIT1 transporter that arises in the embryo after fertilization.

This study examined the influences of epidermal growth factor (EGF) and growth differentiation factor 9 (GDF9) on in vitro viability and activation of primordial follicles in the ovarian tissue of prepubertal (age, <6 mo) versus adult (age, >8 mo) cats. Ovarian cortical slices were cultured in medium containing EGF and/or GDF9 for 14 days. EGF, but not GDF9, improved (P < 0.05) follicle viability in prepubertal donors in a dose-dependent fashion. Neither EGF nor GDF9 enhanced follicle viability in ovarian tissue from adults, and neither factor activated primordial follicles regardless of age group. We then explored how EGF influenced primordial follicles in the prepubertal donors by coincubation with an inhibitor of EGF receptor (AG1478), mitogen-activated protein kinase (MAPK; U0126), or phosphoinositide 3-kinase (PI3K; LY294002). EGF enhanced (P < 0.05) MAPK and AKT phosphorylation, follicle viability, and stromal cell proliferation. These effects were suppressed (P < 0.05) when the tissue was cultured with this growth factor combined with each inhibitor. To identify the underlying influence of age in response to EGF, we assessed cell proliferation and discovered a greater thriving stromal cell population in prepubertal compared to adult tissue. We conclude that EGF plays a significant role in maintaining intraovarian primordial follicle viability (but without promoting activation) in the prepubertal cat. The mechanism of action is via stimulation of MAPK and PI3K signaling pathways that, in turn, promote ovarian cell proliferation. Particularly intriguing is that the ability of cat ovarian cells to multiply in reaction to EGF is age-dependent and highly responsive in prepubertal females.

Inflammation dysregulation in placenta is implicated in the pathogenesis of numerous pregnancy complications. Glucocorticoids (GCs), universally considered anti-inflammatory, can also exert proinflammatory actions under some conditions, whereas whether and how GCs promote placental inflammation have not been intensively investigated. In this paper we report the opposing regulation of rat placental inflammation by synthetic GC dexamethasone (Dex). When Dex was subcutaneously injected 1 h after we administered an intraperitoneal lipopolysaccharide (LPS) challenge, neutrophil infiltration and proinflammatory Il1b, Il6, and Tnfa expression in rat placenta were significantly reduced. In contrast, Dex pretreatment for 24 h potentiated rat placental proinflammatory response to LPS and delayed inflammation resolution, which involved MAPKs and NF-kappaB activation. Mechanically, Dex pretreatment promoted 5-lipoxygenase (ALOX5) activation and increased leukotriene B₄ production, whereas it inhibited the anti-inflammatory and proresolving lipid mediator lipoxin A₄ (LXA₄) biosynthesis in rat placenta via downregulating ALOX15 and ALOX15B expression. Moreover, LXA₄ supplementation dampened Dex-potentiated placental inflammation and suppressed Dex-mediated ALOX5 activation in vivo and in vitro. Taken together, these findings suggest that GCs exposure could promote placental inflammation initiation and delay resolution via disrupting LXA₄ biosynthesis.

The overexpression of leptin is a crucial feature for the maintenance of pregnancy. The effects of leptin on trophoblast invasion are important to its reproductive function, but the underlying mechanisms remain poorly understood. MMP14 is a member of matrix metalloproteinase (MMP) family that is closely involved in the invasion process. Here, we characterized the importance of MMP14 in the proinvasion effect of leptin on EVT cells and elucidated its molecular mechanisms. Transwell assay revealed that leptin promoted invasion of the immortalized EVT cell line HTR-8/SVneo in a dose- and time-related fashion. Further studies suggested that leptin enhanced HTR-8/SVneo cell invasion by up-regulating MMP14 expression and that knockdown of MMP14 by small interference RNA (siRNA) blocked the proinvasion effect of leptin. Notably, leptin promoted the expression of Notch1 receptor and activated its signaling in HTR-8/SVneo cells, and blocking this pathway by siRNA inhibited both leptin-enhanced MMP14 expression and invasiveness of HTR-8/SVneo cells. Such effects of Notch1 signaling were related with the activation of the PI3K/Akt pathway, which was significantly activated after leptin stimulation and was interfered by Notch1 signaling perturbation. Taken together, our observations suggest that leptin is an effective regulator of MMP14 expression, which consequently plays critical roles in invasion of EVT cells. The promoting effects of leptin on MMP14 require the cross talk between Notch1 and PI3K/Akt signaling pathways.

The duration of the female fertile life span is influenced by the number of oocytes stored in the ovary as primordial follicles. Cell death, both during ovarian development in the embryo and in the postnatal ovary, plays a critical role in determining how many primordial follicles are established and maintained within the ovary. However, the roles of individual apoptotic regulators in mediating cell death within the ovary have not yet been characterized. In this study, gene targeted mice were used to investigate the role of BCL-2-modifying factor (BMF), a proapoptotic protein belonging to the BH3-only subgroup of the BCL-2 family, in determining the number of primordial follicles maintained in the adult ovary and the length of the fertile life span. Stereological analysis of ovaries showed that Bmf^−/− mice had significantly more primordial follicles than wild-type (WT) control animals at Postnatal Days 100, 200, 300, and 400 but not at Day 20. No differences were observed between WT and Bmf^−/− mice in the number of ova shed following ovulatory stimulation with exogenous gonadotropins. Bmf^−/− females were fertile and produced the same number pups/litter as WT females, but Bmf^−/− females produced litters more frequently and consequently more offspring than WT females over a 6-mo period. Furthermore, the fertile life span of Bmf^−/− females was significantly extended compared to WT females. Our findings support an important role for BMF in determining the number of primordial follicles maintained in the ovary throughout adult reproductive life and thus indicate that the length of female fertility may be extended by increasing the number of primordial follicles maintained within the ovary through inhibition of BMF.

Recent data from human and mouse studies strongly support an indispensable role for steroid receptor coactivator-2 (SRC-2)—a member of the p160/SRC family of coregulators—in progesterone-dependent endometrial stromal cell decidualization, an essential cellular transformation process that regulates invasion of the developing embryo into the maternal compartment. To identify the key progesterone-induced transcriptional changes that are dependent on SRC-2 and required for endometrial decidualization, we performed comparative genome-wide transcriptional profiling of endometrial tissue RNA from ovariectomized SRC-2^flox/flox (SRC-2^f/f [control]) and PR^cre/ /SRC-2^flox/flox (SRC-2^d/d [SRC-2-depleted]) mice, acutely treated with vehicle or progesterone. Although data mining revealed that only a small subset of the total progesterone-dependent transcriptional changes is dependent on SRC-2 (∼13%), key genes previously reported to mediate progesterone-driven endometrial stromal cell decidualization are present within this subset. Along with providing a more detailed molecular portrait of the decidual transcriptional program governed by SRC-2, the degree of functional diversity of these progesterone mediators underscores the pleiotropic regulatory role of SRC-2 in this tissue. To showcase the utility of this powerful informational resource to uncover novel signaling paradigms, we stratified the total SRC-2-dependent subset of progesterone-induced transcriptional changes in terms of novel gene expression and identified transcription factor 23 (Tcf23), a basic-helix-loop-helix transcription factor, as a new progesterone-induced target gene that requires SRC-2 for full induction. Importantly, using primary human endometrial stromal cells in culture, we demonstrate that TCF23 function is essential for progesterone-dependent decidualization, providing crucial translational support for this transcription factor as a new decidual mediator of progesterone action.

The invasion of placental trophoblast cells into the maternal decidua is an essential aspect of placental embedment. The process of placentation bears several striking similarities to tumor cell metastasis. However, trophoblastic migration during implantation and placentation is stringently controlled both in space and time. RhoGDI2 belongs to a family of Rho guanosine diphosphate dissociation inhibitors (RhoGDIs), and RhoGDI2 is a metastasis suppressor gene and a marker of aggressive human cancer. We evaluated whether RhoGDI2 has a physiological role in embryo implantation, particularly trophoblast migration. The mRNA and protein expression levels of RhoGDI2 were higher in term placentas compared with first-trimester placentas as detected by real-time PCR and Western blot. Immunohistochemical studies indicated that RhoGDI2 localized to the cytotrophoblast layer and extravillous trophoblast in first-trimester placentas and was distributed in the syncytiotrophoblast layers of term placentas. Overexpression of RhoGDI2 in HTR-8/SVneo cells was associated with reduced RAC1-guanosine triphosphate (GTP) levels and inhibited cell migration. Conversely, small interfering RNA-mediated downregulation of RhoGDI2 resulted in significantly increased RAC1-GTP levels. Altered RhoGDI2 expression had no significant effects on cell proliferation. In conclusion, RhoGDI2 inhibits trophoblast cell migration, and this function may involve suppression of RAC1 activation.

Ornithine decarboxylase (ODC1) is considered the rate-controlling enzyme for the classical de novo biosynthesis of polyamines (putrescine, spermidine, and spermine) in mammals. However, metabolism of arginine to agmatine via arginine decarboxylase (ADC) and conversion of agmatine to polyamines via agmatinase (AGMAT) is an alternative pathway long recognized in lower organisms, but only recently suggested for neurons and liver cells of mammals. We now provide evidence for a functional ADC/AGMAT pathway for the synthesis of polyamines in mammalian reproductive tissue for embryonic survival and development. We first investigated cellular functions of polyamines by in vivo knockdown of translation of mRNA for ODC1 in ovine conceptus trophectoderm using morpholino antisense oligonucleotides (MAOs) and found that one-half of the conceptuses were morphologically and functionally either normal or abnormal. Furthermore, we found that increases in ADC/AGMAT mRNA levels and in the translation of AGMAT mRNA among conceptuses in MAO-ODC1 knockdown compensated for the loss of ODC1, supporting polyamine synthesis from arginine and accounting for the normal and abnormal phenotypes of conceptuses. We conclude that the majority of polyamine synthesis is by the conventional ODC1-dependent pathway (arginine-ornithine-putrescine) and that deficiencies in ODC1 result in increased activity of the rescue ADC/AGMAT-dependent pathway (arginine-agmatine-putrescine) for production of polyamines. The presence of an alternative ADC/AGMAT pathway for converting arginine into putrescine is functionally important for supporting survival and development of mammalian conceptuses.

Wilms tumor gene, Wt1, is abundantly expressed in testis Sertoli cells. Our recent study demonstrated that Wt1 is involved in spermatogenesis by regulating Sertoli cell polarity. In the present study, we found that Wt1 is also required for steroidogenesis in Leydig cells and that deletion of the Wt1 gene resulted in defects in testosterone biosynthesis and downregulation of steroidogenic gene expression, including cytochrome P450 side-chain cleavage (P450scc), steroidogenic acute regulatory protein (StAR), 3beta-hydroxysteroid dehydrogenase I (3beta-HSD), and cytochrome P450 17A1 (Cyp17a1). The expression of LHR was significantly decreased in Wt1^−/flox; Cre-ER^TM testes after tamoxifen induction, whereas the luteinizing hormone level in serum was unchanged. Further studies revealed that desert hedgehog (Dhh) expression was regulated by Wt1 in Sertoli cells and that its expression was significantly reduced in Wt1-deficient testes. In vitro study demonstrated that the defect in testosterone production and decreased expression of several steroidogenic genes in Wt1-deficient testis explants was partially rescued by smoothened agonist (SAG), a hedgehog pathway agonist. These results indicate that Wt1 is most likely involved in Leydig cell steroidogenesis by regulating the expression of paracrine factors in seminiferous tubules. Dhh probably had important roles in this process, but we could not exclude the possibility that other factors were also required for Leydig cell steroidogenesis. Loss of Wt1 leads to downregulation of paracrine factors, which in turn causes a decrease in steroidogenic enzyme expression and reduces testosterone production in Leydig cells. The results of this study further confirm that the cross talk between Sertoli cells and Leydig cells has important roles in Leydig cell steroidogenesis.

Continuous or cyclic production of spermatozoa throughout life in adult male vertebrates depends on a subpopulation of undifferentiated germ cells acting as spermatogonial stem cells (SSCs). What makes these cells self-renew or differentiate is barely understood, in particular in nonmammalian species, including fish. In the highly seasonal rainbow trout, at the end of the annual spermatogenetic cycle, tubules of the spawning testis contain only spermatozoa, with the exception of scarce undifferentiated spermatogonia that remain on the tubular wall and that will support the next round of spermatogenesis. Taking advantage of this model, we identified putative SSCs in fish testis using morphological, molecular, and functional approaches. In all stages, large spermatogonia with ultrastructural characteristics of germinal stem cells were found, isolated or in doublet. Trout homologues of SSC and/or immature progenitor markers in mammals—nanos2 and nanos3, pou2, plzf, and piwil2—were preferentially expressed in the prepubertal testis and in the undifferentiated A spermatogonia populations purified by centrifugal elutriation. This expression profile strongly suggests that these genes are functionally conserved between fish and mammals. Moreover, transplantation into embryonic recipients of the undifferentiated spermatogonial cells demonstrated their high “stemness” efficiency in terms of migration into gonads and the ability to give functional gametes. Interestingly, we show that nanos2 expression was restricted to a subpopulation of undifferentiated spermatogonia (less than 20%) present as isolated cells or in doublet in the juvenile and in the maturing trout testis. In contrast, nanos2 transcript was detected in all the undifferentiated spermatogonia remaining in the spawning testis. Plzf was also immunodetected in A-Spg from spawning testis, reinforcing the idea that these cells are stem cells. From those results, we hypothesize that the subset of undifferentiated A spermatogonia expressing nanos2 transcript are putative SSC in trout.

DEAD-box polypeptide 4 (DDX4) is an evolutionally conserved ATP-dependent RNA helicase that is exclusively expressed in germ cell lineage. Although DDX4 is believed to reside and function in the cytoplasm, recent studies in mice and humans suggest that its epitope is expressed on the cell surface of a small subpopulation in the ovary, putative oogonial stem cells. No study has examined whether such cell-surface DDX4 cells exist in the testes of any species. In this study, we explored cell-surface DDX4 cells in postnatal porcine testes before the onset of spermatogenesis, where gonocytes, which are the precursors of spermatogonial stem cells, are the only germ cell population. Transfection experiments demonstrated that recombinant porcine DDX4 can be expressed on the cell surface, and cell-surface DDX4-immunoreactive cells were identified in the testis by flow cytometry. Although the DDX4-expressing cells identified in the testis were indeed gonocytes, the cell-surface DDX4-immunoreactive cells expressed negligible DDX4 mRNA and protein levels. Furthermore, they did not express other germ cell markers, such as ZBTB16, NANOS2, and DAZL, but prominently expressed early primordial germ cell markers, such as PRDM1, IFITM3, and EPCAM. Nonetheless, the cell-surface DDX4-immunoreactive cells generated neither germ cell colonies nor teratomas following transplantation into immunocompromised mouse testes. Taken together, these results demonstrate that testicular cell-surface DDX4-immunoreactive cells are not germ cells and constitute a distinct subpopulation that is different from gonocytes. Moreover, the subpopulation in porcine testes might be species specific because no DDX4-immunoreactive cells were found in postnatal mouse testes.

Treatment with chemotherapeutics agents may induce persistent DNA damage in male germ cells with the possibility of long-term consequences on fertility and progeny outcome. Telomeres, specialized structures at the physical ends of chromosomes, play an important role in the maintenance of genetic stability and in the response of somatic cells to anticancer drugs. Our objective was to test the hypothesis that exposure to bleomycin, etoposide, or cisplatin (the drugs used to treat testicular cancer) or cyclophosphamide (an anticancer agent and immunosuppressant) targets telomeres in the male germ line. C18-4 spermatogonial cells were exposed to bleomycin, etoposide, cisplatin, or 4-hydroperoxycyclophosphamide (4OOH-CPA, a preactivated analog of cyclophosphamide). All four anticancer drugs induced a significant increase in DNA damage in C18-4 cells, as assessed by gamma-H2AX immunofluorescence. Interestingly, the gamma-H2AX signal was localized to telomeres after treatment with bleomycin, cisplatin, and 4OOH-CPA, but not etoposide. Mean telomere lengths, the intensity of the telomere fluorescence in situ hybridization signal, telomerase activity, and the expression of the telomerase enzyme mRNA components, Tert and Terc, were reduced by exposure to cisplatin and 4OOH-CPA, but not by bleomycin or etoposide. Thus, although all four anticancer drugs induced DNA damage in this spermatogonial cell line, telomeres were not specifically affected by etoposide and only the two alkylating agents, cisplatin and 4OOH-CPA, induced telomere dysfunction. This telomere dysfunction may contribute to infertility and developmental defects in the offspring.