Environmental conditions during perinatal development such as maternal undernutrition, maternal glucocorticoids, placental insufficiency, and maternal sodium overload can program changes in renal Na excretion leading to hypertension. Experimental studies indicate that fetal exposure to an adverse maternal environment may reduce glomerular filtration rate by decreasing the surface area of the glomerular capillaries. Moreover, fetal responses to environmental insults during early life that contribute to the development of hypertension may include increased expression of tubular apical or basolateral membrane Na transporters and increased production of renal superoxide leading to enhanced Na reabsorption. This review will address the role of these potential renal mechanisms in the fetal programming of hypertension in experimental models induced by maternal undernutrition, fetal exposure to glucocorticoids, placental insufficiency, and maternal sodium overload in the rat.

Colony-stimulating factor 2 (CSF2) enhances competence of the bovine embryo to establish and maintain pregnancy after the embryo is transferred into a recipient. Mechanisms involved could include regulation of lineage commitment, growth, or differentiation of the inner cell mass (ICM) and trophectoderm (TE). Experiments were conducted to evaluate regulation by CSF2 of pluripotency of the ICM and differentiation and growth of the TE. Embryos were cultured with 10 ng/ml recombinant bovine CSF2 or a vehicle control from Days 5 to 7 or 6 to 8 postinsemination. CSF2 increased the number of putative zygotes that developed to blastocysts when the percent of embryos becoming blastocysts in the control group was low but decreased blastocyst yield when blastocyst development in controls was high. ICM isolated from blastocysts by lysing the trophectoderm using antibody and complement via immunosurgery were more likely to survive passage when cultured on mitomycin C-treated fetal fibroblasts if derived from blastocysts treated with CSF2 than if from control blastocysts. There was little effect of CSF2 on characteristics of TE outgrowths from blastocysts. The exception was a decrease in outgrowth size for embryos treated with CSF2 from Days 5 to 7 and an increase in expression of CDX2 when treatment was from Days 6 to 8. Expression of the receptor subunit gene CSF2RA increased from the zygote stage to the 9–16 cell stage before decreasing to the blastocyst stage. In contrast, CSF2RB was undetectable at all stages. In conclusion, CSF2 improves competence of the ICM to survive in a pluripotent state and alters TE outgrowths. Actions of CSF2 occur through a signaling pathway that is likely to be independent of CSF2RB.

The global chromatin configuration is dramatically remodeled during fertilization and early preimplantation development. Although the chromocenters, which are pericentromeric heterochromatin clusters, are observed in the nuclei of oocytes, they disappear after fertilization and then reappear at the four-cell stage. To elucidate the mechanism of this reorganization of heterochromatin, we investigated the expression and nuclear localization of DOT1L, which is involved in the regulation of heterochromatin structure through histone H3 lysine 79 (H3K79) methyltransferase activity, during preimplantation development. The Dot1L mRNA level was low at the two-cell stage. In the analysis by the immunocytochemistry, DOT1L protein was not observed in the nuclei at this stage. Microinjection of Flag-tagged Dot1L cRNA revealed that the DOT1L protein was localized in the nucleus of the embryos at the one-cell and four-cell stages but not at the two-cell stage. However, C-terminus-truncated DOT1L was localized in the nucleus of two-cell-stage embryos. Expression of the truncated DOT1L caused hypermethylation on H3K79 and the formation of chromocenter-like structures at the two-cell stage. Intriguingly, the expression of catalytically inactive truncated DOT1L also caused the formation of chromocenter-like structures without an increase in H3K79 methylation. Most embryos expressing the truncated DOT1L or its inactive form were arrested at the two-cell stage. These results suggest that the absence of DOT1L, which is involved in the formation of a specific configuration of heterochromatin at the two-cell stage, is essential for early preimplantation development.

Loss of pelvic organ support (i.e., pelvic organ prolapse) is common in menopausal women. Surgical reconstruction of pelvic organ prolapse is plagued with high failure rates. The objective of this study was to determine the effects of estrogen on biomechanical properties, lysyl oxidase (LOX), collagen content, and histomorphology of the vagina with or without surgical injury. Nulliparous ovariectomized guinea pigs were treated systemically with either 50 μg/kg/day estradiol (E2,) or vehicle. After 2 wk, vaginal surgery was performed, and animals were treated with either beta-aminopropionitrile (BAPN, an irreversible LOX inhibitor), or vehicle to determine the role of LOX in recovery of the vaginal wall from injury with or without E2. Estradiol resulted in (i) significant growth, increased smooth muscle, and increased thickness of the vagina, (ii) increased distensibility without compromise of maximal force at failure, and (iii) increased total and cross-linked collagen. In the absence of E2, BAPN resulted in decreased collagen and vaginal wall strength in the area of the injury. In contrast, in E2-treated animals, increased distensibility, maximal forces, and total collagen were maintained despite BAPN. Interestingly, LOX mRNA was induced dramatically (9.5-fold) in the injured vagina with or without E2 at 4 days. By 21 days, however, LOX levels declined to near baseline in E2-deprived animals. LOX mRNA levels remained strikingly elevated (12-fold) at 21 days in the estrogenized vagina. The results suggest that prolonged E2 induced increases in LOX, and collagen cross-links may act to sustain a matrix environment that optimizes long-term surgical wound healing in the vagina.

Most animal sperm are quiescent in the male reproductive tract and become activated after mixing with accessory secretions from the male and/or female reproductive tract. Sperm from the mosquito Culex quinquefasciatus initiate flagellar motility after mixing with male accessory gland components, and the sperm flagellum displays three distinct motility patterns over time: a low amplitude, a long wavelength form (Wave A), a double waveform consisting of two superimposed waveforms over the length of the flagellum (Wave B), and finally, a single helical waveform that propels the sperm at high velocity (Wave C). This flagellar behavior is replicated by treating quiescent sperm with trypsin. When exposed to either broad spectrum or tyrosine kinase inhibitors, sperm activated by accessory gland secretions exhibited motility through Wave B but were unable to progress to Wave C. The MEK1/2 inhibitor UO126 and the ERK1/2 inhibitor FR180204 each blocked the transition from Wave B to Wave C, indicating a role for MAPK activity in the control of waveform and, accordingly, progressive movement. Furthermore, a MAPK substrate antibody stained the flagellum of activated sperm. In the absence of extracellular Ca² , a small fraction of sperm swam backwards, whereas most could not be activated by either accessory glands or trypsin and were immotile. However, the phosphatase inhibitor okadaic acid in the absence of extracellular Ca² induced all sperm to swim backwards with a flagellar waveform similar to Wave A. These results indicate that flagellar waveform generation and direction of motility are controlled by protein phosphorylation and Ca² levels, respectively.

The PIWI-interacting RNA (piRNA) pathway is essential for germline development and transposable element repression. Key elements of this pathway are members of the piRNA-binding PIWI/Argonaute protein family and associated factors (e.g., VASA, MAELSTROM, and TUDOR domain proteins). PIWI-interacting RNAs have been identified in mouse testis and oocytes, but information about the expression of the different piRNA pathway genes, in particular in the mammalian ovary, remains incomplete. We investigated the evolution and expression of piRNA pathway genes in gonads of amniote species (chicken, platypus, and mouse). Database searches confirm a high level of conservation and revealed lineage-specific gain and loss of Piwi genes in vertebrates. Expression analysis in mammals shows that orthologs of Piwi-like (Piwil) genes, Mael (Maelstrom), Mvh (mouse vasa homolog), and Tdrd1 (Tudor domain-containing protein 1) are expressed in platypus adult testis. In contrast to mouse, Piwil4 is expressed in platypus and human adult testis. We found evidence for Mael and Piwil2 expression in mouse Sertoli cells. Importantly, we show mRNA expression of Piwil2, Piwil4, and Mael in oocytes and supporting cells of human, mouse, and platypus ovary. We found no Piwil1 expression in mouse and chicken ovary. The conservation of gene expression in somatic parts of the gonad and germ cells of species that diverged over 800 million yr ago indicates an important role in adult male and female gonad.

Inhibition of progesterone (P4) synthesis by cumulus cells during bovine in vitro oocyte maturation (IVM) causes a decrease in subsequent embryo development, indicating that P4 intracellular signaling within the cumulus oocyte complex (COC) is important for oocyte developmental competence. The aim of the present study was to further elucidate, on a protein level, the downstream signaling pathway involved in P4 regulation of oocyte developmental competence. COCs were subjected to IVM for 24 h in the presence or absence of trilostane, aglepristone, or promegestone (R5020). These altered IVM conditions resulted in dynamic changes in protein expression of the progesterone receptors and the cell death-regulated proteins AVEN, BCL-xL, and active caspase 3. In addition, AVEN protein localization, caspase 3 activation, and mitochondrial distribution were studied by immunofluorescence. Inhibition of progesterone synthesis (trilostane treatment) resulted in changes in AVEN localization within the COC, corresponding to caspase 3 activation and altered mitochondrial distribution. AVEN was also found to bind BCL-xL in COCs, but this interaction was lost following treatment with trilostane.

Normal development of germ cells is essential for fertility and mammalian reproduction. Although abnormal development of oocytes or follicles may lead to primary ovarian insufficiency (POI), a disorder that causes infertility in 1% of women less than 40 yr of age, the genes and signaling pathways activated in POI are not as yet fully elucidated. Tbx4, a member of the T-box family of transcription factors, is expressed in embryonic germ cells and postnatal oocytes at all stages of folliculogenesis. To investigate the requirement for Tbx4 in the germline, we analyzed germ cell development in the absence of Tbx4. We show that primordial germ cells (PGCs) are reduced in Tbx4 homozygous null (Tbx4^−/−) embryos at Embryonic Day (E) 10.0. Tbx4^−/− embryos die by E10.5; to study later time points in vitro, a tamoxifen-inducible estrogen receptor Cre recombinase was used to delete Tbx4 conditional mutant alleles. In addition, Gdf9cre and Zp3cre, two oocyte-specific Cre recombinases, were used to delete Tbx4 from postnatal primordial and primary follicles, respectively. We show that in vitro differentiation of the gonad into morphologically distinct testes and ovaries occurs normally starting at E11.5 when Tbx4 is deleted. In Gdf9cre; Tbx4^fl/− and Zp3cre; Tbx4^fl/− adult females, primordial, primary, secondary, and antral follicles form, ovulation occurs, corpus luteum formation is normal, and the mice are fertile without any evidence of diminished ovarian reserve. Although postnatal deletion of Tbx4 in oocytes does not obviously impair fertility, it is possible that the reduction in PGCs observed in Tbx4 homozygous null mutant embryos could affect long-term fertility in adults.

Androgens and insulin-like 3 (INSL3) are required for development of the fetal gubernaculum and testicular descent. Previous studies suggested that the INSL3-exposed fetal gubernacular transcriptome is enriched for genes involved in neural pathways. In the present study, we profiled the transcriptome of fetal gubernaculum explants exposed to dihydrotestosterone (DHT) and compared this response to that with INSL3. We exposed fetal (Embryonic Day 17) rat gubernacula to DHT for 24 h (10 and 30 nM) or 6 h (1 and 10 nM) in organ culture and analyzed gene expression relative to that of vehicle-treated controls using Affymetrix arrays. Results were annotated using functional, pathway, and promoter analyses and independently validated for selected transcripts using quantitative RT-PCR (qRT-PCR). Transcripts were differentially expressed after 24 h but not 6 h. Most highly overrepresented functional categories included those related to gene expression, skeletal and muscular development and function, and Wnt signaling. Promoter response elements enriched in the DHT-specific transcriptome included consensus sequences for c-ETS1, ELK1, CREB, CRE-BP1/c-June, NRF2, and USF. We observed that 55% of DHT probe sets were also differentially expressed after INSL3 exposure and that the direction of change was the same in 96%. The qRT-PCR results confirmed that DHT increased expression of the INSL3-responsive genes Crlf1 and Chrdl2 but reduced expression of Wnt4. We also validated reduced Tgfb2 and Cxcl12 and increased Slit3 expression following DHT exposure. These data suggest a robust overlap in the DHT- and INSL3-regulated transcriptome that may be mediated in part by CREB signaling and a common Wnt pathway response for both hormones in the fetal gubernaculum.

Uterine fibroids (leiomyomas) are the most common benign tumors associated with excessive deposition of extracellular matrix (ECM)-associated proteins that increase fibroid tumorigenicity. Herein, we determined the expression levels of vitamin D receptor (VDR) protein in human uterine fibroids and compared these levels to those in adjacent normal myometrium. Using Western blot analysis, we found that more than 60% of uterine fibroids analyzed (25 of 40) expressed low levels of VDR. We also found that the biologically active 1,25-dihydroxyvitamin D3 (1,25[OH]₂D3), which functions via binding to its nuclear VDR, induced VDR in a concentration-dependent manner and reduced ECM-associated fibrotic and proteoglycans expression in immortalized human uterine fibroid cell line (HuLM). At 1–10 nM concentrations, 1,25(OH)₂D3 significantly induced (P < 0.05) nuclear VDR, which was further stimulated by higher concentrations of 1,25(OH)₂D3 in HuLM cells. 1,25(OH)₂D3 at 10 nM also significantly reduced (P < 0.05) the protein expression of ECM-associated collagen type 1, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) in HuLM cells. We also found that 1,25(OH)₂D3 reduced mRNA and protein expressions of proteoglycans such as fibromodulin, biglycan, and versican in HuLM cells. Moreover, the aberrant expression of structural smooth muscle actin fibers was reduced by 1,25(OH)₂D3 treatment in a concentration-dependent manner in HuLM cells. Taken together, our results suggest that human uterine fibroids express reduced levels of VDR compared to the adjacent normal myometrium and that treatment with 1,25(OH)₂D3 can potentially reduce the aberrant expression of major ECM-associated proteins in HuLM cells. Thus, 1,25(OH)₂D3 might be an effective, safe, nonsurgical treatment option for human uterine fibroids.

Using the baboon as a model for studies of human reproductive biology, we previously showed that placental estrogen regulates fetal ovarian follicle development. In this study, offspring of baboons untreated or treated in utero with the aromatase inhibitor letrozole (estradiol reduced >95%) or letrozole and estradiol were reared to adulthood to determine whether estrogen programming of the fetal ovary impacted puberty and reproduction in adulthood. All offspring exhibited normal growth and blood pressure/chemistries. Puberty onset in untreated baboons (43.2 ± 1.4 mo) was delayed (P < 0.01) in animals of letrozole-treated mothers (49.0 ± 1.2 mo) and normal in offspring of mothers treated with letrozole and estradiol (42.7 ± 0.8 mo). During the first 2 yr postmenarche, menstrual cycles in estrogen-suppressed animals (43.2 ± 1.3 days) were longer (P < 0.05) than in untreated baboons (38.3 ± 0.5 days) or those treated with letrozole and estrogen (39.6 ± 0.8 days). Moreover, in estrogen-suppressed offspring, serum levels of estradiol were lower and follicle-stimulating hormone greater (P < 0.05) in the follicular and luteal phases, and the elevation in luteal-phase progesterone extended (P < 0.02). Thus, puberty onset was delayed and menstrual cycles prolonged and associated with altered serum hormone levels in baboon offspring that developed in an intrauterine environment in which estradiol levels were suppressed. Because puberty and follicle development, as shown previously, were normal in baboons treated in utero with letrozole and estradiol, we propose that fetal ovarian development and timely onset of puberty in the primate is programmed by fetal exposure to placental estrogen.

We previously established a potential role for cocaine and amphetamine regulated transcript (CARTPT) in dominant follicle selection in cattle. CARTPT expression is elevated in subordinate versus dominant follicles, and treatment with the mature form of the CARTPT peptide (CART) decreases follicle-stimulating hormone (FSH)-stimulated granulosa cell estradiol production in vitro and follicular fluid estradiol and granulosa cell CYP19A1 mRNA in vivo. However, mechanisms that regulate granulosa cell CART responsiveness are not understood. In this study, we investigated hormonal regulation of granulosa cell CART-binding sites in vitro and temporal regulation of granulosa cell CART-binding sites in bovine follicles collected at specific stages of a follicular wave. We also determined the effect of inhibition of CART receptor signaling in vivo on estradiol production in future subordinate follicles. Granulosa cell CART binding in vitro was increased by FSH, and this induction was blocked by estrogen receptor antagonist treatment. In follicles collected in vivo at specific stages of a follicular wave, granulosa cell CART binding in the F2 (second largest), future subordinate follicle increased during dominant follicle selection. Injection into the F2 follicle (at onset of diameter deviation) of an inhibitor of the o/i subclass of G proteins (previously shown to block CART actions in vitro) resulted in increased follicular fluid estradiol concentrations in vivo. Collectively, results demonstrate hormonal regulation of granulosa cell CART binding in vitro and temporal regulation of CART binding in subordinate follicles during dominant follicle selection. Results also suggest that CART signaling may help suppress estradiol-producing capacity of the F2 (subordinate) follicle during this time period.

Fetoplacental endothelial cells are exposed to oxygen levels ranging from 2% to 8% in vivo. However, little is known regarding endothelial function within this range of oxygen because most laboratories use ambient air (21% O₂) as a standard culture condition (SCN). We asked whether human umbilical artery endothelial cells (HUAECs) that were steadily exposed to the physiological chronic normoxia (PCN, 3% O₂) for ∼20–25 days differed in their proliferative and migratory responses to FGF2 and VEGFA as well as in their global gene expression compared with those in the SCN. We observed that PCN enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. In oxygen reversal experiments (i.e., when PCN cells were exposed to SCN for 24 h and vice versa), we found that preexposure to 21% O₂ decreased the migratory ability, but not the proliferative ability, of the PCN-HUAECs in response to FGF2 and VEGFA. These PCN-enhanced cellular responses were associated with increased protein levels of HIF1A and NOS3, but not FGFR1, VEGFR1, and VEGFR2. Microarray analysis demonstrated that PCN up-regulated 74 genes and down-regulated 86, 14 of which were directly regulated by hypoxia-inducible factors as evaluated using in silico analysis. Gene function analysis further indicated that the PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from our functional assays. Given that PCN significantly alters cellular responses to FGF2 and VEGFA as well as transcription in HUAECs, it is likely that we may need to reexamine the current cellular and molecular mechanisms controlling fetoplacental endothelial functions, which were largely derived from endothelial models established under ambient O₂.

The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Days 11 and 12 of gestation. Concomitant with trophoblast elongation, production of conceptus estrogen, the porcine embryonic pregnancy recognition signal, increases. Conceptus attachment to the uterine surface epithelium starts after Day 13, initiating epitheliochorial placentation. To analyze the transcriptome changes in the endometrium in the course of maternal recognition of pregnancy, deep sequencing of endometrial RNA samples of Day 12 pregnant animals (n = 4) and corresponding nonpregnant controls (n = 4) was performed using RNA sequencing (RNA-Seq). Between 30 000 000 and 35 000 000 sequence reads per sample were produced and mapped to the porcine genome (Sscrofa10.2). Analysis of read counts revealed 2593 differentially expressed genes (DEGs). Expression of selected genes was validated by the use of quantitative real-time RT-PCR. Bioinformatics analysis identified several functional terms specifically overrepresented for up-regulated or down-regulated genes. Comparison of the RNA-Seq data from Days 12 and 14 of pregnancy was performed at the level of all expressed genes, the level of the DEG, and the level of functional categories. This revealed specific gene expression patterns reflecting the different functions of the endometrium during these stages (i.e., recognition of pregnancy and preparation for conceptus attachment). Genes related to mitosis, immune response, epithelial cell differentiation and development, proteolysis, and prostaglandin signaling and metabolism are discussed in detail. This study identified comprehensive transcriptome changes in porcine endometrium associated with establishment of pregnancy and could be a resource for targeted studies of genes and pathways potentially involved in regulation of this process.

Dynamic control of maternal blood flow to the placenta is critical for healthy pregnancy. In many tissues, microvasculature arteries control the flow. The uterine/endometrial vascular bed changes during pregnancy include physiological remodeling of spiral arteries from constricted artery-like structures to dilated vein-like structures between Gestation Day 8 (gd8) and gd12 in mice and wk 12–16 in humans. These changes occur, in part, due to local environmental changes such as decidualization, recruitment of maternal uterine natural killer cells, and invasion of conceptus-derived trophoblasts. No current preparations permit in vivo testing of decidual microvascular reactivity. We report an in vivo intravital fluorescence microscopy model that permits functional study of the entire uterine microvascular bed (uterine, arcuate, radial, basal, and spiral arteries) in gravid C57BL/6 mice. Vascular reactivities were measured at gd8 prespiral arterial remodeling and gd12 (postremodeling) to a range of concentrations of adenosine (10⁻⁸–10⁻⁶ M), acetylcholine (10⁻⁷–10⁻⁵ M), phenylephrine (10⁻⁷–10⁻⁵ M), and angiotensin II (10⁻⁸–10⁻⁶ M). At baseline, each arterial branch order was significantly more dilated on gd12 than gd8. Each microvascular level responded to each agonist on gd8 and gd12. At gd12, vasodilation to adenosine was attenuated in uterine, arcuate, and basal arteries, while constrictor activity to angiotensin II was enhanced in uterine and arcuate arteries. The tendency for increasing vasoconstriction between gd8 to gd12 and the constrictor responses of modified spiral arteries were unexpected findings that may reflect influences of the intact in vivo environment rather than inherent properties of the vessels and may be relevant to ongoing human pregnancies.

Excessive release of syncytiotrophoblast extracellular vesicles (STBMs) from the placenta into the maternal circulation may contribute to the systemic inflammation that is characteristic of pre-eclampsia (PE). Other intravascular cells types (platelets, leukocytes, red blood cells [RBCs], and endothelium) may also be activated and release extracellular vesicles (EVs). We developed a multicolor flow cytometry antibody panel to enumerate and phenotype STBMs in relation to other EVs in plasma from nonpregnant (NonP) and normal pregnant (NormP) women, and women with late-onset PE. Nanoparticle tracking analysis (NTA) was used to determine EV size and concentration. In vitro-derived STBMs and EVs from platelets, leukocytes, RBCs, and endothelial cells were examined to select suitable antibodies to analyze the corresponding plasma EVs. Flow cytometry analysis of plasma from NonP, NormP, and PE showed that STBMs comprised the smallest group of circulating EVs, whereas most were derived from platelets. The next most abundant group comprised unidentified orphan EVs (which did not label with any of the antibodies in the panel), followed by EVs from RBCs and leukocytes. NTA showed that the total number of EVs in plasma was significantly elevated in NormP and late-onset PE women compared to NonP controls, and that EVs were smaller in size. In general, EVs were elevated in pregnancy plasma apart from platelet EVs, which were reduced. These studies did not show any differences in EVs between NormP and PE, probably because late-onset PE was studied.

Several studies have linked assisted reproductive technologies to aberrant imprinting. We previously showed that 12-day in vitro follicle culture supports normal imprinting establishment in mouse oocytes. The aim of the present study was to assess whether shortened in vitro follicle growth (8 days of culture compared with 12 days, as a model for human in vitro maturation) or preovulatory intrafollicular oocyte “aging” in culture (14 days of culture) leads to imprinting mutations in oocytes. Limiting-dilution bisulphite sequencing showed that shortened in vitro follicle growth (8 days) does not induce oocyte epimutations at the imprinted Snrpn and Mest genes. In contrast, extension of oocyte residence in large unluteinized follicles in vitro was associated with a low level (1 of 54 alleles) of epimutations for Mest but not for Snrpn. The latter condition may occur during controlled ovarian stimulation where the oocyte growth phase may be extended for several days. Furthermore, we studied the dynamics during follicle culture of transcript levels for genes previously shown to be essential for imprinting establishment in oocytes, including Dnmt3a, Dnmt3L, and Zfp57. Oocyte total mRNA levels during in vitro follicle culture showed the timely shutdown in transcription at the antral follicle stage, and total mRNA levels were comparable to those of in vivo grown equine chorionic gonadotropin-stimulated oocytes.

Spermatogonial stem cells (SSCs) comprise a small population of germ cells that have self-renewal potential. However, studies on SSCs are hampered by the lack of SSC-specific markers. Although the cryptorchid operation is often used to obtain an enriched SSC population by destroying differentiating germ cells using high body temperature, SSCs in cryptorchid testes have different biological characteristics than those in a normal environment. Therefore, it is necessary to develop new methods for SSC selection. In this study, we report a method of isolating SSCs from wild-type mouse testes based on their functional characteristics using aldehyde dehydrogenase (ALDH) activity levels, which have been successfully used for stem cell isolation from many self-renewing tissues. Testis cells selected using CD9 or CDH1, both of which are expressed by SSCs, exhibit ALDH activity in flow cytometric analyses. However, spermatogonial transplantation revealed that SSCs do not show ALDH activity, whereas somatic stem cells have high ALDH activity levels. Nevertheless, SSCs could be enriched 183.7-fold based on CDH1 preselection and transplantation of cells that lacked ALDH activity. In contrast, we failed to enrich SSCs from cultured spermatogonia, which exhibited ALDH upregulation in vitro. These results suggest that SSCs are unique among tissue-specific stem cells in their regulation of ALDH activity. Development of a new technique for SSC isolation from wild-type testes based on their functional properties will facilitate investigation of SSCs in a normal testicular environment.

Deregulated expression of protein tyrosine phosphorylation has been implicated in testicular response to different stimuli. Herein, YES1, a nonreceptor protein tyrosine kinase, was found to be significantly up-regulated in pachytene spermatocytes (PS) during early recovery from a transient testicular heat stress. Coculture of PS with Sertoli cells (SCs) could enhance the hyperthermia-induced YES1 activation, indicative of a positive regulation of the paracrine signaling. Moreover, SU6656, a selective YES1 inhibitor, was shown to effectively block YES1 activity, thereafter resulting in a dramatic increase of heat stress-induced apoptosis in primary cultured PS. Mechanistically, the antiapoptotic effect of YES1 activation in response to testicular heat insult may mediate via the regulation of extracellular signal-regulated kinase (ERK)/metastasis-associated 1 (MTA1) cascade. From a clinical standpoint, a notably higher level of YES1 expression was observed in the pathological testis from varicocele patients as compared to a negligible staining in the control group. Taken together, our present results provide the first evidence that the YES1/ERK/MTA1/p53 cascade may serve as a naturally occurring, indispensable self-defensive mechanism maintaining apoptotic balance during meiotic heat stress. Our study may have also partially answered the question of how activation of signal pathways at the cell membrane surface interacts with the key regulatory events occurring in the nucleus during testicular heat shock.

Many studies have addressed the role of cigarette smoking on semen quality, but the exact mechanisms remain inconclusive. To evaluate the detrimental effects of smoking on the spermatogenesis process, we initially screened and investigated 31 differentially expressed proteins extracted from the testes of mice exposed daily to cigarette smoke using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analysis. Data mining analysis showed that these 31 proteins were categorized into five functional clustering groups: metabolic process, cell growth and/or maintenance, RNA and protein processing, stress response, and spermatogenesis. Additionally, 23 of 31 proteins were involved in a main pathway network, including Pkc (s), ERK1/2, Akt, and NF-kappaB, which are known to be involved in cell communication, proliferation, and differentiation. Interestingly, among the 31 proteins, a spermatogenesis-associated protein, phosphatidylethanolamine-binding protein 1 (PEBP1), was especially expressed in serial sections of spermatids of spermiogenesis and interacted with ERKs. The bisulfite sequencing result showed four CpGs near the Pebp1 transcriptional start site were largely methylated in the treated group. A 5-aza-2′-deoxycytidine treatment on GC-1 spg cells reversed the hypermethylation status and elevated both Pebp1 mRNA and protein expression levels. ERK1/2 phosphorylation levels were also increased with upregulation of Pebp1 expression in GC-1 spg cells. In conclusion, protein profile in testes could be altered by cigarette smoking. Moreover, we also suggest that epigenetic Pebp1 inactivation may affect activation of ERK, and it could impair spermatogenesis of mice. Our data could provide further insight into the mechanisms of spermatogenesis.

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.