Reproduction

Aalberts, Marian; Stout, Tom A E; Stoorvogel, Willem

doi: 10.1530/rep-13-0358pmid: 24149515

The term ‘prostasomes’ is generally used to classify the extracellular vesicles (EVs) released into prostatic fluid by prostate epithelial cells. However, other epithelia within the male reproductive tract also release EVs that mix with ‘true’ prostasomes during semen emission or ejaculation. Prostasomes have been proposed to regulate the timing of sperm cell capacitation and induction of the acrosome reaction, as well as to stimulate sperm motility where all three are prerequisite processes for spermatozoa to attain fertilising capacity. Other proposed functions of prostasomes include interfering with the destruction of spermatozoa by immune cells within the female reproductive tract. On the other hand, it is unclear whether the distinct presumed functions are performed collectively by a single type of prostasome or by separate distinct sub-populations of EVs. Moreover, the exact molecular mechanisms through which prostasomes exert their functions have not been fully resolved. Besides their physiological functions, prostasomes produced by prostate tumour cells have been suggested to support prostate cancer spread development, and prostasomes in peripheral blood plasma may prove to be valuable biomarkers for prostate cancer.

Obata, Yayoi; Wakai, Takuya; Hara, Satoshi; Kono, Tomohiro

doi: 10.1530/rep-13-0359pmid: 24123131

DNA methylation imprints that are established in spermatogenesis and oogenesis are essential for functional gametes. However, the mechanisms underlying gamete-specific imprinting remain unclear. In this study, we investigated whether male and female gametes derived from newborn mice are epigenetically plastic and whether DNA methylation imprints are influenced by the niche surrounding the nuclei of the gametes. When prospermatogonia possessing sperm-specific DNA methylation imprints were fused with enucleated fully grown oocytes and exposed to the ooplasm for 5–6 days, the DNA methylation status of the reconstituted oocytes remained identical to that of prospermatogonia for all the imprinted regions analysed. These results suggest that the imprinting status of prospermatogonia is stable and that the epigenome of prospermatogonia loses sexual plasticity. By contrast, when non-growing oocytes lacking oocyte-specific DNA methylation imprints were fused with enucleated fully grown oocytes and the reconstituted oocytes were then cultured for 5–6 days, the Igf2r, Kcnq1ot1 and, unexpectedly, H19/Igf2 differentially methylated regions (DMRs) were methylated. Methylation imprints were entirely absent in oocytes derived from 5-day-old mice, and H19/Igf2 DMR is usually methylated only in spermatogenesis. These findings indicate that in the nuclei of non-growing oocytes the chromatin conformation changes and becomes permissive to DNA methyltransferases in some DMRs and that mechanisms for maintaining non-methylated status at the H19/Igf2 DMR are lost upon long exposure to mature ooplasm.

Dyke, James U Van; Brandley, Matthew C; Thompson, Michael B

doi: 10.1530/REP-13-0309pmid: 24129151

Squamate reptiles (lizards and snakes) are an ideal model system for testing hypotheses regarding the evolution of viviparity(live birth) in amniote vertebrates. Viviparity has evolved over 100 times in squamates, resulting in major changes in reproductivephysiology. At a minimum, all viviparous squamates exhibit placentae formed by the appositions of maternal and embryonic tissues,which are homologous in origin with the tissues that form the placenta in therian mammals. These placentae facilitate adhesionof the conceptus to the uterus as well as exchange of oxygen, carbon dioxide, water, sodium, and calcium. However, most viviparoussquamates continue to rely on yolk for nearly all of their organic nutrition. In contrast, some species, which rely on theplacenta for at least a portion of organic nutrition, exhibit complex placental specializations associated with the transportof amino acids and fatty acids. Some viviparous squamates also exhibit reduced immunocompetence during pregnancy, which couldbe the result of immunosuppression to protect developing embryos. Recent molecular studies using both candidate-gene and next-generationsequencing approaches have suggested that at least some of the genes and gene families underlying these phenomena play similarroles in the uterus and placenta of viviparous mammals and squamates. Therefore, studies of the evolution of viviparity insquamates should inform hypotheses of the evolution of viviparity in all amniotes, including mammals.

Aalberts, Marian; Stout, Tom A E; Stoorvogel, Willem

doi: 10.1530/REP-13-0358pmid: 24149515

The term ‘prostasomes’ is generally used to classify the extracellular vesicles (EVs) released into prostatic fluid by prostateepithelial cells. However, other epithelia within the male reproductive tract also release EVs that mix with ‘true’ prostasomesduring semen emission or ejaculation. Prostasomes have been proposed to regulate the timing of sperm cell capacitation andinduction of the acrosome reaction, as well as to stimulate sperm motility where all three are prerequisite processes forspermatozoa to attain fertilising capacity. Other proposed functions of prostasomes include interfering with the destructionof spermatozoa by immune cells within the female reproductive tract. On the other hand, it is unclear whether the distinctpresumed functions are performed collectively by a single type of prostasome or by separate distinct sub-populations of EVs.Moreover, the exact molecular mechanisms through which prostasomes exert their functions have not been fully resolved. Besidestheir physiological functions, prostasomes produced by prostate tumour cells have been suggested to support prostate cancerspread development, and prostasomes in peripheral blood plasma may prove to be valuable biomarkers for prostate cancer.

Obata, Yayoi; Wakai, Takuya; Hara, Satoshi; Kono, Tomohiro

doi: 10.1530/REP-13-0359pmid: 24123131

DNA methylation imprints that are established in spermatogenesis and oogenesis are essential for functional gametes. However,the mechanisms underlying gamete-specific imprinting remain unclear. In this study, we investigated whether male and femalegametes derived from newborn mice are epigenetically plastic and whether DNA methylation imprints are influenced by the nichesurrounding the nuclei of the gametes. When prospermatogonia possessing sperm-specific DNA methylation imprints were fusedwith enucleated fully grown oocytes and exposed to the ooplasm for 5–6 days, the DNA methylation status of the reconstitutedoocytes remained identical to that of prospermatogonia for all the imprinted regions analysed. These results suggest thatthe imprinting status of prospermatogonia is stable and that the epigenome of prospermatogonia loses sexual plasticity. Bycontrast, when non-growing oocytes lacking oocyte-specific DNA methylation imprints were fused with enucleated fully grownoocytes and the reconstituted oocytes were then cultured for 5–6 days, the Igf2r, Kcnq1ot1 and, unexpectedly, H19/Igf2 differentially methylated regions (DMRs) were methylated. Methylation imprints were entirely absent in oocytes derived from5-day-old mice, and H19/Igf2 DMR is usually methylated only in spermatogenesis. These findings indicate that in the nuclei of non-growing oocytes thechromatin conformation changes and becomes permissive to DNA methyltransferases in some DMRs and that mechanisms for maintainingnon-methylated status at the H19/Igf2 DMR are lost upon long exposure to mature ooplasm.

Van Dyke, James U; Brandley, Matthew C; Thompson, Michael B

doi: 10.1530/rep-13-0309pmid: 24129151

Squamate reptiles (lizards and snakes) are an ideal model system for testing hypotheses regarding the evolution of viviparity (live birth) in amniote vertebrates. Viviparity has evolved over 100 times in squamates, resulting in major changes in reproductive physiology. At a minimum, all viviparous squamates exhibit placentae formed by the appositions of maternal and embryonic tissues, which are homologous in origin with the tissues that form the placenta in therian mammals. These placentae facilitate adhesion of the conceptus to the uterus as well as exchange of oxygen, carbon dioxide, water, sodium, and calcium. However, most viviparous squamates continue to rely on yolk for nearly all of their organic nutrition. In contrast, some species, which rely on the placenta for at least a portion of organic nutrition, exhibit complex placental specializations associated with the transport of amino acids and fatty acids. Some viviparous squamates also exhibit reduced immunocompetence during pregnancy, which could be the result of immunosuppression to protect developing embryos. Recent molecular studies using both candidate-gene and next-generation sequencing approaches have suggested that at least some of the genes and gene families underlying these phenomena play similar roles in the uterus and placenta of viviparous mammals and squamates. Therefore, studies of the evolution of viviparity in squamates should inform hypotheses of the evolution of viviparity in all amniotes, including mammals.

doi: 10.1530/rep-147-a1pmid: N/A

MacLeod, Graham; Shang, Peng; Booth, Gregory T; Mastropaolo, Lucas A; Manafpoursakha, Niloufar; Vogl, A Wayne; Varmuza, Susannah

doi: 10.1530/REP-13-0224pmid: 24088291

The mouse protein phosphatase gene Ppp1cc is essential for male fertility, with mutants displaying a failure in spermatogenesis including a widespread loss of post-meioticgerm cells and abnormalities in the mitochondrial sheath. This phenotype is hypothesized to be responsible for the loss ofthe testis-specific isoform PPP1CC2. To identify PPP1CC2-interacting proteins with a function in spermatogenesis, we carriedout GST pull-down assays in mouse testis lysates. Amongst the identified candidate interactors was the testis-specific proteinkinase TSSK1, which is also essential for male fertility. Subsequent interaction experiments confirmed the capability of PPP1CC2to form a complex with TSSK1 mediated by the direct interaction of each with the kinase substrate protein TSKS. Interactionbetween PPP1CC2 and TSKS is mediated through an RVxF docking motif on the TSKS surface. Phosphoproteomic analysis of the mousetestis identified a novel serine phosphorylation site within the TSKS RVxF motif that appears to negatively regulate bindingto PPP1CC2. Immunohistochemical analysis of TSSK1 and TSKS in the Ppp1cc mutant testis showed reduced accumulation to distinct cytoplasmic foci and other abnormalities in their distribution consistentwith the loss of germ cells and seminiferous tubule disorganization observed in the Ppp1cc mutant phenotype. A comparison of Ppp1cc and Tssk1/2 knockout phenotypes via electron microscopy revealed similar abnormalities in the morphology of the mitochondrial sheath.These data demonstrate a novel kinase/phosphatase complex in the testis that could play a critical role in the completionof spermatogenesis.

MacLeod, Graham; Shang, Peng; Booth, Gregory T; Mastropaolo, Lucas A; Manafpoursakha, Niloufar; Vogl, A Wayne; Varmuza, Susannah

doi: 10.1530/rep-13-0224pmid: 24088291

The mouse protein phosphatase gene Ppp1cc is essential for male fertility, with mutants displaying a failure in spermatogenesis including a widespread loss of post-meiotic germ cells and abnormalities in the mitochondrial sheath. This phenotype is hypothesized to be responsible for the loss of the testis-specific isoform PPP1CC2. To identify PPP1CC2-interacting proteins with a function in spermatogenesis, we carried out GST pull-down assays in mouse testis lysates. Amongst the identified candidate interactors was the testis-specific protein kinase TSSK1, which is also essential for male fertility. Subsequent interaction experiments confirmed the capability of PPP1CC2 to form a complex with TSSK1 mediated by the direct interaction of each with the kinase substrate protein TSKS. Interaction between PPP1CC2 and TSKS is mediated through an RVxF docking motif on the TSKS surface. Phosphoproteomic analysis of the mouse testis identified a novel serine phosphorylation site within the TSKS RVxF motif that appears to negatively regulate binding to PPP1CC2. Immunohistochemical analysis of TSSK1 and TSKS in the Ppp1cc mutant testis showed reduced accumulation to distinct cytoplasmic foci and other abnormalities in their distribution consistent with the loss of germ cells and seminiferous tubule disorganization observed in the Ppp1cc mutant phenotype. A comparison of Ppp1cc and Tssk1/2 knockout phenotypes via electron microscopy revealed similar abnormalities in the morphology of the mitochondrial sheath. These data demonstrate a novel kinase/phosphatase complex in the testis that could play a critical role in the completion of spermatogenesis.

Santos, P R S; Oliveira, M F; Arroyo, M A M; Silva, A R; Rici, R E G; Miglino, M A; Assis Neto, A C

doi: 10.1530/rep-13-0452pmid: 24101585

This was a pioneer study of the spermatogenic process from the onset of puberty in Spix's yellow-toothed cavies (SYC, Galea spixii) bred in captivity. The study aimed to characterize fine structure of spermatogenesis. Twelve testes from pubertal and post-pubertal SYC males were studied using transmission electron microscopy. Spermatogenesis can be divided into three phases: proliferation, meiosis, and spermiogenesis. In proliferation phase, three types of spermatogonia were identified and characterized as Adark, Apale, and B. In the second phase, spermatocytes (2n) undergo meiotic divisions that generate spermatids (n); the process begins in spermatocytes in the preleptotene stage when they increase their nuclear size, differentiating into spermatocytes in the leptotene stage when cell division is initiated. In addition, we found chromatin condensation, and formation of a structure composed of proteins that formed a central shaft and two lateral bars associated with pairing of homologous chromosomes. During spermiogenesis, the following main events occurred: condensation of nuclear chromatin, formation of acrosome with perfuratorium, elimination of residual cytoplasm, and development of the flagellum. The sperm head is different from that of other rodents. The endoplasmic reticulum and the Golgi complex are the two main organelles demonstrated during this process. These organelles collaborate through synthesis of proteins and hormones for the development of germ cells during spermatogenesis in SYC.