Activin and BMP Signalling in Human Testicular Cancer Cell Lines, and a Role for the Nucleocytoplasmic Transport Protein Importin-5 in Their Crosstalk.

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-ß, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-ß, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-ß superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline. IPO5 knockdown in TCam-2 cells using siRNA blunted BMP4-induced transcript changes, indicating that IPO5 levels could determine TGF-ß signalling pathway outcomes in TGCTs.

MiMIC analysis reveals an isoform specific role for Drosophila Musashi in follicle stem cell maintenance and escort cell function.

The Drosophila ovary is regenerated from germline and somatic stem cell populations that have provided fundamental conceptual understanding on how adult stem cells are regulated within their niches. Recent ovarian transcriptomic studies have failed to identify mRNAs that are specific to follicle stem cells (FSCs), suggesting that their fate may be regulated post-transcriptionally. We have identified that the RNA-binding protein, Musashi (Msi) is required for maintaining the stem cell state of FSCs. Loss of msi function results in stem cell loss, due to a change in differentiation state, indicated by upregulation of Lamin C in the stem cell population. In msi mutant ovaries, Lamin C upregulation was also observed in posterior escort cells that interact with newly formed germ cell cysts. Mutant somatic cells within this region were dysfunctional, as evidenced by the presence of germline cyst collisions, fused egg chambers and an increase in germ cell cyst apoptosis. The msi locus produces two classes of mRNAs (long and short). We show that FSC maintenance and escort cell function specifically requires the long transcripts, thus providing the first evidence of isoform-specific regulation in a population of Drosophila epithelial cells. We further demonstrate that although male germline stem cells have previously been shown to require Msi function to prevent differentiation this is not the case for female germline stem cells, indicating that these similar stem cell types have different requirements for Msi, in addition to the differential use of Msi isoforms between soma and germline. In summary, we show that different isoforms of the Msi RNA-binding protein are expressed in specific cell populations of the ovarian stem cell niche where Msi regulates stem cell differentiation, niche cell function and subsequent germ cell survival and differentiation.

The association between reproductive health smartphone applications and fertility knowledge of Australian women.

BACKGROUND: Previous studies have identified that women living in developed countries have insufficient knowledge of factors which may be contributing to the increasingly high global infertility rates such as maternal age and assisted reproductive technologies. There is a large market of reproductive health smartphone applications, yet little is known about the advantages these apps may confer to users in regards to reproductive health knowledge. METHODS: An anonymous, online survey of women living in Australia aged 18 and above was open March-June 2018, until ≥200 responses were acquired for statistical power. Respondents answered questions regarding knowledge about general fertility and related factors (age, cyclic fertility, smoking, obesity, miscarriage rate, and success of assisted reproductive technologies). Fertility knowledge was compared in respondents who did or did not use apps relating to female reproductive health. Additionally the functions preferred in reproductive health apps was described by app using respondents. Sociodemographic information was also collected, and relevant data within the dataset was subject to multivariable modelling for the outcome of the fertility knowledge questions. RESULTS: Of the 673 respondents that completed the survey, 43.09% reported using mobile phone applications relating to female reproductive health. On average, respondents answered only three of the six fertility knowledge questions correctly. App using respondents were more likely to score better on one question, related to fertility during the menstrual cycle (p < 0.001). App users most commonly reported using the menstrual tracking function in apps (82.4%), which may account for the increased knowledge of cyclic fertility. CONCLUSIONS: This data provides preliminary evidence toward the usefulness of smartphone applications as a medium for providing information about fertility to women. A limited understanding of one's own fertility was demonstrated despite being essential for the decision-making of women throughout their reproductive years.

Detection of chlamydia infection within human testicular biopsies.

STUDY QUESTION: Can Chlamydia be found in the testes of infertile men? SUMMARY ANSWER: Chlamydia can be found in 16.7% of fresh testicular biopsies and 45.3% of fixed testicular biopsies taken from a selection of infertile men. WHAT IS KNOWN ALREADY: Male chlamydial infection has been understudied despite male and female infections occurring at similar rates. This is particularly true of asymptomatic infections, which occur in 50% of cases. Chlamydial infection has also been associated with increased sperm DNA damage and reduced male fertility. STUDY DESIGN, SIZE, DURATION: We collected diagnostic (fixed, n = 100) and therapeutic (fresh, n = 18) human testicular biopsies during sperm recovery procedures from moderately to severely infertile men in a cross-sectional approach to sampling. PARTICIPANTS/MATERIALS, SETTING, METHODS: The diagnostic and therapeutic biopsies were tested for Chlamydia-specific DNA and protein, using real-time PCR and immunohistochemical approaches, respectively. Serum samples matched to the fresh biopsies were also assayed for the presence of Chlamydia-specific antibodies using immunoblotting techniques. MAIN RESULTS AND THE ROLE OF CHANCE: Chlamydial major outer membrane protein was detected in fixed biopsies at a rate of 45.3%. This was confirmed by detection of chlamydial DNA and TC0500 protein (replication marker). C. trachomatis DNA was detected in fresh biopsies at a rate of 16.7%, and the sera from each of these three positive patients contained C. trachomatis-specific antibodies. Overall, C. trachomatis-specific antibodies were detected in 72.2% of the serum samples from the patients providing fresh biopsies, although none of the patients were symptomatic nor had they reported a previous sexually transmitted infection diagnosis including Chlamydia. LIMITATIONS, REASONS FOR CAUTION: No reproductively healthy male testicular biopsies were tested for the presence of Chlamydia DNA or proteins or Chlamydia-specific antibodies due to the unavailability of these samples. WIDER IMPLICATIONS FOR THE FINDINGS: Application of Chlamydia-specific PCR and immunohistochemistry in this human male infertility context of testicular biopsies reveals evidence of a high prevalence of previously unrecognised infection, which may potentially have a pathogenic role in spermatogenic failure. STUDY FUNDING/COMPETING INTEREST(S): Funding for this project was provided by the Australian NHMRC under project grant number APP1062198. We also acknowledge assistance from the Monash IVF Group and Queensland Fertility Group in the collection of fresh biopsies, and the Monash Health and co-author McLachlan (declared equity interest) in retrieval and sectioning of fixed biopsies. E.M. declares an equity interest in the study due to financing of fixed biopsy sectioning. All other authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Hematogenous dissemination of Chlamydia muridarum from the urethra in macrophages causes testicular infection and sperm DNA damage†.

The incidence of Chlamydia infection, in both females and males, is increasing worldwide. Male infections have been associated clinically with urethritis, epididymitis, and orchitis, believed to be caused by ascending infection, although the impact of infection on male fertility remains controversial. Using a mouse model of male chlamydial infection, we show that all the major testicular cell populations, germ cells, Sertoli cells, Leydig cells, and testicular macrophages can be productively infected. Furthermore, sperm isolated from vas deferens of infected mice also had increased levels of DNA damage as early as 4 weeks post-infection. Bilateral vasectomy, prior to infection, did not affect the chlamydial load recovered from testes at 2, 4, and 8 weeks post-infection, and Chlamydia-infected macrophages were detectable in blood and the testes as soon as 3 days post-infection. Partial depletion of macrophages with clodronate liposomes significantly reduced the testicular chlamydial burden, consistent with a hematogenous route of infection, with Chlamydia transported to the testes in infected macrophages. These data suggest that macrophages serve as Trojan horses, transporting Chlamydia from the penile urethra to the testes within 3 days of infection, bypassing the entire male reproductive tract. In the testes, infected macrophages likely transfer infection to Leydig, Sertoli, and germ cells, causing sperm DNA damage and impaired spermatogenesis.

Esrp1 is a marker of mouse fetal germ cells and differentially expressed during spermatogenesis.

ESRP1 regulates alternative splicing, producing multiple transcripts from its target genes in epithelial tissues. It is upregulated during mesenchymal to epithelial transition associated with reprogramming of fibroblasts to iPS cells and has been linked to pluripotency. Mouse fetal germ cells are the founders of the adult gonadal lineages and we found that Esrp1 mRNA was expressed in both male and female germ cells but not in gonadal somatic cells at various stages of gonadal development (E12.5-E15.5). In the postnatal testis, Esrp1 mRNA was highly expressed in isolated cell preparations enriched for spermatogonia but expressed at lower levels in those enriched for pachytene spermatocytes and round spermatids. Co-labelling experiments with PLZF and c-KIT showed that ESRP1 was localized to nuclei of both Type A and B spermatogonia in a speckled pattern, but was not detected in SOX9+ somatic Sertoli cells. No co-localization with the nuclear speckle marker, SC35, which has been associated with post-transcriptional splicing, was observed, suggesting that ESRP1 may be associated with co-transcriptional splicing or have other functions. RNA interference mediated knockdown of Esrp1 expression in the seminoma-derived Tcam-2 cell line demonstrated that ESRP1 regulates alternative splicing of mRNAs in a non-epithelial cell germ cell tumour cell line.

Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) follicular signalling is conserved in the mare ovary.

The mare ovary is unique in its anatomical structure; however, the signalling pathways responsible for physiological processes, such as follicular activation, remain uncharacterised. This provided us with the impetus to explore whether signalling molecules from important folliculogenesis pathways, phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT), are conserved in the mare ovary. Messenger RNA expression of six genes important in follicle development was measured using quantitative polymerase chain reaction and protein localisation of key pathway members (PI3K, AKT1, phosphatase and tensin homologue (PTEN), JAK1, STAT3 and suppressor of cytokine signalling 4 (SOCS4)) was compared in tissue from fetal and adult mare ovaries. Tissue from adult ovaries exhibited significantly increased levels of mRNA expression of PI3K, AKT1, PTEN, JAK1, STAT3 and SOCS4 compared with tissue from fetal ovaries. PI3K, AKT1, JAK1 and STAT3 demonstrated redistributed localisation, from pregranulosa cells in fetal development, to both the oocyte and granulosa cells of follicles in the adult ovary, whilst negative feedback molecules PTEN and SOCS4 were only localised to the granulosa cells in the adult ovary. These findings suggest that the PI3K/AKT and JAK/STAT signalling pathways are utilised during folliculogenesis in the mare, similarly to previously studied mammalian species, and may serve as useful biomarkers for assessment of ovary development in the horse.

Characterization of a novel role for the dynamin mechanoenzymes in the regulation of human sperm acrosomal exocytosis.

STUDY QUESTION: Does dynamin regulate human sperm acrosomal exocytosis? SUMMARY ANSWER: Our studies of dynamin localization and function have implicated this family of mechanoenzymes in the regulation of progesterone-induced acrosomal exocytosis in human spermatozoa. WHAT IS KNOWN ALREADY: Completion of an acrosome reaction is a prerequisite for successful fertilization in all studied mammalian species. It follows that failure to complete this unique exocytotic event represents a common aetiology in the defective spermatozoa of male infertility patients that have failed IVF in a clinical setting. Recent studies have implicated the dynamin family of mechanoenzymes as important regulators of the acrosome reaction in murine spermatozoa. The biological basis of this activity appears to rest with the ability of dynamin to polymerize around newly formed membrane vesicles and subsequently regulate the rate of fusion pore expansion. To date, however, the dynamin family of GTPases have not been studied in the spermatozoa of non-rodent species. Here, we have sought to examine the presence and functional significance of dynamin in human spermatozoa. STUDY DESIGN, SIZE, DURATION: Dynamin expression was characterized in the testis and spermatozoa of several healthy normozoospermic individuals. In addition, we assessed the influence of selective dynamin inhibition on the competence of human spermatozoa to undergo a progesterone-induced acrosome reaction. A minimum of five biological and technical replicates were performed to investigate both inter- and intra-donor variability in dynamin expression and establish statistical significance in terms of the impact of dynamin inhibition. PARTICIPANTS/MATERIALS, SETTING, METHODS: The expression and the localization of dynamin in the human testis, epididymis and mature spermatozoa were determined through the application of immunofluorescence, immunoblotting and/or electron microscopy. Human semen samples were fractionated via density gradient centrifugation and the resultant populations of good and poor quality spermatozoa were induced to capacitate and acrosome react in the presence or absence of selective dynamin inhibitors. The acrosome integrity of live spermatozoa was subsequently assessed via the use of fluorescently conjugated Arachis hypogea lectin (PNA). The influence of dynamin phosphorylation and the regulatory kinase(s) responsible for this modification in human spermatozoa were also assessed via the use of in situ proximity ligation assays and pharmacological inhibition. In all experiments, ≥100 spermatozoa were assessed/treatment group and all graphical data are presented as the mean values ± SEM, with statistical significance being determined by ANOVA. MAIN RESULTS AND THE ROLE OF CHANCE: Dynamin 1 (DNM1) and DNM2, but not DNM3, were specifically localized to the acrosomal region of the head of human spermatozoa, an ideal position from which to regulate acrosomal exocytosis. In keeping with this notion, pharmacological inhibition of DNM1 and DNM2 was able to significantly suppress the rates of acrosomal exocytosis stimulated by progesterone. Furthermore, our comparison of dynamin expression in good and poor quality spermatozoa recovered from the same ejaculate, revealed a significant reduction in the amount of DNM2 in the latter subpopulation of cells. In contrast, DNM1 was detected at equivalent levels in both subpopulations of spermatozoa. Such findings are of potential significance given that the poor quality spermatozoa proved refractory to the induction of a progesterone stimulated acrosome reaction. In seeking to identify the regulatory influence of progesterone on DNM2 function, we were able to establish that the protein is a substrate for CDK1-dependent phosphorylation. The functional significance of DNM2 phosphorylation was illustrated by the fact that pharmacological inhibition of CDK1 elicited a concomitant suppression of both DNM2-Ser764 phosphorylation and the overall rates of progesterone-induced acrosomal exocytosis. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This was an in vitro study performed mainly on ejaculated human spermatozoa. This experimental paradigm necessarily eliminates the physiological contributions of the female reproductive tract that would normally support capacitation and acrosomal responsiveness. WIDER IMPLICATIONS OF THE FINDINGS: This study identifies a novel causative link between dynamin activity and the ability of human spermatozoa to complete a progesterone-induced acrosome reaction. Such findings encourage a more detailed analysis of the contribution of dynamin dysregulation as an underlying aetiology in infertile males whose spermatozoa are unable to penetrate the zona pellucida. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by a National Health and Medical Research Council of Australia Project Grant (APP1103176) awarded to B.N. and E.A.M. The authors report no conflict of interest.

Neonatal immune activation depletes the ovarian follicle reserve and alters ovarian acute inflammatory mediators in neonatal rats.

Normal ovarian development is crucial for female reproductive success and longevity. Interruptions to the delicate process of initial folliculogenesis may lead to ovarian dysfunction. We have previously demonstrated that an early life immune challenge in the rat, induced by administration of lipopolysaccharide (LPS) on postnatal day (PND) 3 and 5, depletes ovarian follicle reserve long term. Here, we hypothesized that this neonatal immune challenge leads to an increase in peripheral and ovarian inflammatory signaling, contributing to an acute depletion of ovarian follicles. Morphological analysis of neonatal ovaries indicated that LPS administration significantly depleted PND 5 primordial follicle populations and accelerated follicle maturation. LPS exposure upregulated circulating interleukin 6, tumor necrosis factor alpha (TNFa), and C-reactive protein on PND 5, and upregulated ovarian mRNA expression of Tnfa, mitogen-activated protein kinase 8 (Mapk8/Jnk1), and growth differentiation factor 9 (Gdf9) (P < 0.05). Mass spectrometry and cell signaling pathway analysis indicated upregulation of cellular pathways associated with acute phase signaling, and cellular survival and assembly. Apoptosis assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling indicated significantly increased positive staining in the ovaries of LPS-treated neonates. These findings suggest that increased proinflammatory signaling within the neonatal ovary may be responsible for the LPS-induced depletion of the primordial follicle pool. These findings also have implications for female reproductive health, as the ovarian reserve is a major determinate of female reproductive longevity.

Linking Stress and Infertility: A Novel Role for Ghrelin.

Infertility affects a remarkable one in four couples in developing countries. Psychological stress is a ubiquitous facet of life, and although stress affects us all at some point, prolonged or unmanageable stress may become harmful for some individuals, negatively impacting on their health, including fertility. For instance, women who struggle to conceive are twice as likely to suffer from emotional distress than fertile women. Assisted reproductive technology treatments place an additional physical, emotional, and financial burden of stress, particularly on women, who are often exposed to invasive techniques associated with treatment. Stress-reduction interventions can reduce negative affect and in some cases to improve in vitro fertilization outcomes. Although it has been well-established that stress negatively affects fertility in animal models, human research remains inconsistent due to individual differences and methodological flaws. Attempts to isolate single causal links between stress and infertility have not yet been successful due to their multifaceted etiologies. In this review, we will discuss the current literature in the field of stress-induced reproductive dysfunction based on animal and human models, and introduce a recently unexplored link between stress and infertility, the gut-derived hormone, ghrelin. We also present evidence from recent seminal studies demonstrating that ghrelin has a principal role in the stress response and reward processing, as well as in regulating reproductive function, and that these roles are tightly interlinked. Collectively, these data support the hypothesis that stress may negatively impact upon fertility at least in part by stimulating a dysregulation in ghrelin signaling.

Analysis of the small non-protein-coding RNA profile of mouse spermatozoa reveals specific enrichment of piRNAs within mature spermatozoa.

Post-testicular sperm maturation and storage within the epididymis is a key determinant of gamete quality and fertilization competence. Here we demonstrate that mouse spermatozoa possess a complex small non-protein-coding RNA (sRNA) profile, the composition of which is markedly influenced by their epididymal transit. Thus, although microRNAs (miRNAs) are highly represented in the spermatozoa of the proximal epididymis, this sRNA class is largely diminished in mature spermatozoa of the distal epididymis. Coincident with this, a substantial enrichment in Piwi-interacting RNA (piRNA) abundance in cauda spermatozoa was detected. Further, features of cauda piRNAs, including; predominantly 29-31 nts in length; preference for uracil at their 5' terminus; no adenine enrichment at piRNA nt 10, and; predominantly mapping to intergenic regions of the mouse genome, indicate that these piRNAs are generated by the PIWIL1-directed primary piRNA production pathway. Accordingly, PIWIL1 was detected via immunoblotting and mass spectrometry in epididymal spermatozoa. These data provide insight into the complexity and dynamic nature of the sRNA profile of spermatozoa and raise the intriguing prospect that piRNAs are generated in situ in maturing spermatozoa. Such information is of particular interest in view of the potential role for paternal sRNAs in influencing conception, embryo development and intergenerational inheritance.

Motoring through: the role of kinesin superfamily proteins in female meiosis.

BACKGROUND: The kinesin motor protein family consists of 14 distinct subclasses and 45 kinesin proteins in humans. A large number of these proteins, or their orthologues, have been shown to possess essential function(s) in both the mitotic and the meiotic cell cycle. Kinesins have important roles in chromosome separation, microtubule dynamics, spindle formation, cytokinesis and cell cycle progression. This article contains a review of the literature with respect to the role of kinesin motor proteins in female meiosis in model species. Throughout, we discuss the function of each class of kinesin proteins during oocyte meiosis, and where such data are not available their role in mitosis is considered. Finally, the review highlights the potential clinical importance of this family of proteins for human oocyte quality. OBJECTIVE AND RATIONALE: To examine the role of kinesin motor proteins in oocyte meiosis. SEARCH METHODS: A search was performed on the Pubmed database for journal articles published between January 1970 and February 2017. Search terms included 'oocyte kinesin' and 'meiosis kinesin' in addition to individual kinesin names with the terms oocyte or meiosis. OUTCOMES: Within human cells 45 kinesin motor proteins have been discovered, with the role of only 13 of these proteins, or their orthologues, investigated in female meiosis. Furthermore, of these kinesins only half have been examined in mammalian oocytes, despite alterations occurring in gene transcripts or protein expression with maternal ageing, cryopreservation or behavioral conditions, such as binge drinking, for many of them. WIDER IMPLICATIONS: Kinesin motor proteins have distinct and important roles throughout oocyte meiosis in many non-mammalian model species. However, the functions these proteins have in mammalian meiosis, particularly in humans, are less clear owing to lack of research. This review brings to light the need for more experimental investigation of kinesin motor proteins, particularly those associated with maternal ageing, cryopreservation or exposure to environmental toxicants.

Quinolone-1-(2H)-ones as hedgehog signalling pathway inhibitors.

A series of quinolone-2-(1H)-ones derived from the Ugi-Knoevenagel three- and four-component reaction were prepared exhibiting low micromolar cytotoxicity against a panel of eight human cancer cell lines known to possess the Hedgehog Signalling Pathway (HSP) components, as well as the seminoma TCAM-2 cell line. A focused SAR study was conducted and revealed core characteristics of the quinolone-2-(1H)-ones required for cytotoxicity. These requirements included a C3-tethered indole moiety, an indole C5-methyl moiety, an aliphatic tail or an ester, as well as an additional aromatic moiety. Further investigation in the SAG-activated Shh-LIGHT2 cell line with the most active analogues: 2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(1-methyl-1H-indol-3-yl)-N-(pentan-2-yl)acetamide (5), 2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(5-methyl-1H-indol-3-yl)-N-(pentan-2-yl)acetamide (23) and ethyl (2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(5-methyl-1H-indol-3-yl)acetyl)glycinate (24) demonstrated a down regulation of the HSP via a reduction in Gli expression, and in the mRNA levels of Ptch1 and Gli2. Analogues 5, 23 and 24 returned in cell inhibition values of 11.6, 2.9 and 3.1 µM, respectively, making this new HSP-inhibitor pharmacophore amongst the most potent non-Smo targeted inhibitors thus far reported.

A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation.

Spermatozoa require the process of capacitation to enable them to fertilize an egg. PKA is crucial to capacitation and the development of hyperactivated motility. Sperm PKA is activated by cAMP generated by the germ cell-enriched adenylyl cyclase encoded by Adcy10 Male mice lacking Adcy10 are sterile, because their spermatozoa are immotile. The current study was designed to identify binding partners of the sperm-specific (Cα2) catalytic subunit of PKA (PRKACA) by using it as the "bait" in a yeast 2-hybrid system. This approach was used to identify a novel germ cell-enriched protein, sperm PKA interacting factor (SPIF), in 25% of the positive clones. Homozygous Spif-null mice were embryonically lethal. SPIF was coexpressed and coregulated with PRKACA and with t-complex protein (TCP)-11, a protein associated with PKA signaling. We established that these 3 proteins form part of a novel complex in mouse spermatozoa. Upon capacitation, the SPIF protein becomes tyrosine phosphorylated in >95% of sperm. An apparent molecular rearrangement in the complex occurs, bringing PRKACA and TCP11 into proximity. Taken together, these results suggest a role for the novel complex of SPIF, PRKACA, and TCP11 during sperm capacitation, fertilization, and embryogenesis.-Stanger, S. J., Law, E. A., Jamsai, D., O'Bryan, M. K., Nixon, B., McLaughlin, E. A., Aitken, R. J., Roman, S. D. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation.

Maternal Smoke Exposure Impairs the Long-Term Fertility of Female Offspring in a Murine Model.

The theory of fetal origins of adult disease was first proposed in 1989, and in the decades since, a wide range of other diseases from obesity to asthma have been found to originate in early development. Because mammalian oocyte development begins in fetal life it has been suggested that environmental and lifestyle factors of the mother could directly impact the fertility of subsequent generations. Cigarette smoke is a known ovotoxicant in active smokers, yet disturbingly 13% of Australian and 12% of US women continue to smoke throughout pregnancy. The focus of our investigation was to characterize the adverse effects of smoking on ovary and oocyte quality in female offspring exposed in utero. Pregnant mice were nasally exposed to cigarette smoke for 12 wk throughout pregnancy/lactation, and ovary and oocyte quality of the F1 (maternal smoke exposed) generation was examined. Neonatal ovaries displayed abnormal somatic cell proliferation and increased apoptosis, leading to a reduction in follicle numbers. Further investigation found that altered somatic cell proliferation and reduced follicle number continued into adulthood; however, apoptosis did not. This reduction in follicles resulted in decreased oocyte numbers, with these oocytes found to have elevated levels of oxidative stress, altered metaphase II spindle, and reduced sperm-egg interaction. These ovarian and oocyte changes ultimately lead to subfertility, with maternal smoke-exposed animals having smaller litters and also taking longer to conceive. In conclusion, our results demonstrate that in utero and lactational exposure to cigarette smoke can have long-lasting effects on the fertility of the next generation of females.

Next Generation Sequencing Analysis Reveals Segmental Patterns of microRNA Expression in Mouse Epididymal Epithelial Cells.

The functional maturation of mammalian spermatozoa is accomplished as the cells descend through the highly specialized microenvironment of the epididymis. This dynamic environment is, in turn, created by the combined secretory and absorptive activity of the surrounding epithelium and displays an extraordinary level of regionalization. Although the regulatory network responsible for spatial coordination of epididymal function remains unclear, recent evidence has highlighted a novel role for the RNA interference pathway. Indeed, as noncanonical regulators of gene expression, small noncoding RNAs have emerged as key elements of the circuitry involved in regulating epididymal function and hence sperm maturation. Herein we have employed next generation sequencing technology to profile the genome-wide miRNA signatures of mouse epididymal cells and characterize segmental patterns of expression. An impressive profile of some 370 miRNAs were detected in the mouse epididymis, with a subset of these specifically identified within the epithelial cells that line the tubule (218). A majority of the latter miRNAs (75%) were detected at equivalent levels along the entire length of the mouse epididymis. We did however identify a small cohort of miRNAs that displayed highly regionalized patterns of expression, including miR-204-5p and miR-196b-5p, which were down- and up-regulated by approximately 39- and 45-fold between the caput/caudal regions, respectively. In addition we identified 79 miRNAs (representing ~ 21% of all miRNAs) as displaying conserved expression within all regions of the mouse, rat and human epididymal tissue. These included 8/14 members of let-7 family of miRNAs that have been widely implicated in the control of androgen signaling and the repression of cell proliferation and oncogenic pathways. Overall these data provide novel insights into the sophistication of the miRNA network that regulates the function of the male reproductive tract.

Glycogen synthase kinase 3 regulates acrosomal exocytosis in mouse spermatozoa via dynamin phosphorylation.

The dynamin family of GTPases has been implicated as novel regulators of the acrosome reaction, a unique exocytotic event that is essential for fertilization. Dynamin activity during the acrosome reaction is accompanied by phosphorylation of key serine residues. We now tested the hypothesis that glycogen synthase kinase 3 (GSK3) is the protein kinase responsible for dynamin phosphorylation at these phosphosites in mouse spermatozoa. Pharmacologic inhibition of GSK3 in mature mouse spermatozoa (CHIR99021: IC50 = 6.7 nM) led to a significant reduction in dynamin phosphorylation (10.3% vs. 27.3%; P < 0.001), acrosomal exocytosis (9.7% vs. 25.7%; P < 0.01), and in vitro fertilization (53% vs. 100%; P < 0.01). GSK3 was shown to be present in developing germ cells where it colocalized with dynamin in the peri-acrosomal domain. However, additional GSK3 was acquired by maturing mouse spermatozoa within the male reproductive tract, via a novel mechanism involving direct interaction of sperm heads with extracellular structures known as epididymal dense bodies. These data reveal a novel mode for the cellular acquisition of a protein kinase and identify a key role for GSK3 in the regulation of sperm maturation and acrosomal exocytosis.

Chlamydia muridarum infection-induced destruction of male germ cells and sertoli cells is partially prevented by Chlamydia major outer membrane protein-specific immune CD4 cells.

Chlamydia trachomatis infections are increasingly prevalent worldwide. Male chlamydial infections are associated with urethritis, epididymitis, and orchitis; however, the role of Chlamydia in prostatitis and male factor infertility remains controversial. Using a model of Chlamydia muridarum infection in male C57BL/6 mice, we investigated the effects of chlamydial infection on spermatogenesis and determined the potential of immune T cells to prevent infection-induced outcomes. Antigen-specific CD4 T cells significantly reduced the infectious burden in the penile urethra, epididymis, and vas deferens. Infection disrupted seminiferous tubules, causing loss of germ cells at 4 and 8 wk after infection, with the most severely affected tubules containing only Sertoli cells. Increased mitotic proliferation, DNA repair, and apoptosis in spermatogonial cells and damaged germ cells were evident in atrophic tubules. Activated caspase 3 (casp3) staining revealed increased (6-fold) numbers of Sertoli cells with abnormal morphology that were casp3 positive in tubules of infected mice, indicating increased levels of apoptosis. Sperm count and motility were both decreased in infected mice, and there was a significant decrease in morphologically normal spermatozoa. Assessment of the spermatogonial stem cell population revealed a decrease in promyelocytic leukemia zinc finger (PLZF)-positive cells in the seminiferous tubules. Interestingly, adoptive transfer of antigen-specific CD4 cells, particularly T-helper 2-like cells, prior to infection prevented these effects in spermatogenesis and Sertoli cells. These data suggest that chlamydial infection adversely affects spermatogenesis and male fertility, and that vaccination can potentially prevent the spread of infection and these adverse outcomes.

Through the smoke: use of in vivo and in vitro cigarette smoking models to elucidate its effect on female fertility.

A finite number of oocytes are established within the mammalian ovary prior to birth to form a precious ovarian reserve. Damage to this limited pool of gametes by environmental factors such as cigarette smoke and its constituents therefore represents a significant risk to a woman's reproductive capacity. Although evidence from human studies to date implicates a detrimental effect of cigarette smoking on female fertility, these retrospective studies are limited and present conflicting results. In an effort to more clearly understand the effect of cigarette smoke, and its chemical constituents, on female fertility, a variety of in vivo and in vitro animal models have been developed. This article represents a systematic review of the literature regarding four of experimental model types: 1) direct exposure of ovarian cells and follicles to smoking constituents' in vitro, 2) direct exposure of whole ovarian tissue with smoking constituents in vitro, 3) whole body exposure of animals to smoking constituents and 4) whole body exposure of animals to cigarette smoke. We summarise key findings and highlight the strengths and weaknesses of each model system, and link these to the molecular mechanisms identified in smoke-induced fertility changes.

The Role of the Immune Response in Chlamydia trachomatis Infection of the Male Genital Tract: A Double-Edged Sword.

Chlamydia trachomatis (CT) is the most prevalent bacterial sexually transmitted infection in the world, with more than 100 million cases reported annually. While there have been extensive studies into the adverse effects that CT infection has on the female genital tract, and on the subsequent ability of these women to conceive, studies into the consequences on male fertility have been limited and controversial. This is in part due to the asymptomatic nature of the infection, where it is estimated that 50% of men with Chlamydia fail to show any symptoms. It is accepted, however, that acute and/or persistent CT infection is the causative agent for conditions such as urethritis, epididymitis, epididymo-orchitis, and potentially prostatitis. As with most infections, the immune system plays a fundamental role in the body's attempts to eradicate the infection. The first and most important immune response to Chlamydia infection is a local one, whereby immune cells such as leukocytes are recruited to the site of infections, and subsequently secrete pro-inflammatory cytokines and chemokines such as interferon gamma. Immune cells also work to initiate and potentiate chronic inflammation through the production of reactive oxygen species (ROS), and the release of molecules with degradative properties including defensins, elastase, collagenase, cathespins, and lysozyme. This long-term inflammation can lead to cell proliferation (a possible precursor to cancer), tissue remodeling, and scarring, as well as being linked to the onset of autoimmune responses in genetically disposed individuals. This review will focus on the ability of the immune system to recognize and clear acute and persistent chlamydial infections in the male genital tract, and on the paradoxical damage that chronic inflammation resulting from the infection can cause on the reproductive health of the individual.