Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicles or interactions with epithelial cells, in the epididymis, in the seminal fluid and in the female genital tract. Two different ways of oocyte membrane transfer to spermatozoa have been described: trogocytosis and exosomes. We here report an analysis of in vitro exchanges between the membranes of unfertilised oocytes and capacitated spermatozoa. We showed that optimum conditions are fulfilled when unfertilised oocytes interact with acrosome-reacted spermatozoa, a scenario mimicking the events occurring when the fertilising spermatozoon is inside the perivitelline space. Although CD9 tetraspanin is an essential molecule for fertilisation, exosome and trogocytosis transfer persists in Cd9-null oocytes in spite of their dramatic fusion failure. These exchanges are CD9 tetraspanin independent. We also confirm that mice sperm express CD9 tetraspanin and that when Cd9-null oocytes were inseminated with sperm covered with oocyte membrane materials, including CD9 tetraspanin, no rescue of the oocytes' fertilisability could be obtained. Thus, the existence of two ways of exchange between gametes during fertilisation suggests that these events could be of a physiological importance in this process.
Virginie Barraud-Lange, Céline Chalas Boissonnas, Catherine Serres, Jana Auer, Alain Schmitt, Brigitte Lefèvre, Jean-Philippe Wolf, and Ahmed Ziyyat
Kathrein von Kopylow, Hannah Staege, Andrej-Nikolai Spiess, Wolfgang Schulze, Hans Will, Michael Primig, and Christiane Kirchhoff
It is unclear whether the distinct nuclear morphologies of human Adark (Ad) and Apale (Ap) spermatogonia are manifestations of different stages of germ cell development or phases of the mitotic cycle, or whether they may reflect still unknown molecular differences. According to the classical description by Clermont, human dark type A spermatogonium (Ad) may contain one, sometimes two or three nuclear ‘vacuolar spaces’ representing chromatin rarefaction zones. These structures were readily discerned in paraffin sections of human testis tissue during immunohistochemical and immunofluorescence analyses and thus represented robust morphological markers for our study. While a majority of the marker proteins tested did not discriminate between spermatogonia with and without chromatin rarefaction zones, doublesex- and mab-3-related transcription factor (DMRT1), tyrosine kinase receptor c-Kit/CD117 (KIT) and proliferation-associated antigen Ki-67 (KI-67) appeared to be restricted to subtypes which lacked the rarefaction zones. Conversely, exosome component 10 (EXOSC10) was found to accumulate within the rarefaction zones, which points to a possible role of this nuclear domain in RNA processing.
J G Nicoletti, B G White, E I Miskiewicz, and D J MacPhee
During pregnancy the myometrium undergoes a programme of differentiation induced by endocrine, cellular, and biophysical inputs. Small heat shock proteins (HSPs) are a family of ten (B1–B10) small-molecular-weight proteins that not only act as chaperones, but also assist in processes such as cytoskeleton rearrangements and immune system activation. Thus, it was hypothesized that HSPB5 (CRYAB) would be highly expressed in the rat myometrium during the contractile and labour phases of myometrial differentiation when such processes are prominent. Immunoblot analysis revealed that myometrial CRYAB protein expression significantly increased from day (D) 15 to D23 (labour; P<0.05). In correlation with these findings, serine 59-phosphorylated (pSer59) CRYAB protein expression significantly increased from D15 to D23, and was also elevated 1-day post-partum (P<0.05). pSer59-CRYAB was detected in the cytoplasm of myocytes within both uterine muscle layers mid- to late-pregnancy. In unilaterally pregnant rats, pSer59-CRYAB protein expression was significantly elevated in the gravid uterine horns at both D19 and D23 of gestation compared with non-gravid horns. Co-immunolocalization experiments using the hTERT-human myometrial cell line and confocal microscopy demonstrated that pSer59-CRYAB co-localized with the focal adhesion protein FERMT2 at the ends of actin filaments as well as with the exosomal marker CD63. Overall, pSer59-CRYAB is highly expressed in myometrium during late pregnancy and labour and its expression appears to be regulated by uterine distension. CRYAB may be involved in the regulation of actin filament dynamics at focal adhesions and could be secreted by exosomes as a prelude to involvement in immune activation in the myometrium.
Veronica Maillo, Maria Jesus Sánchez-Calabuig, Ricaurte Lopera-Vasquez, Meriem Hamdi, Alfonso Gutierrez-Adan, Patrick Lonergan, and Dimitrios Rizos
The oviduct is a complex and organized thin tubular structure connecting the ovary with the uterus. It is the site of final sperm capacitation, oocyte fertilization and, in most species, the first 3–4days of early embryo development. The oviductal epithelium is made up of ciliary and secretory cells responsible for the secretion of proteins and other factors which contribute to the formation of the oviductal fluid. Despite significant research, most of the pathways and oviductal factors implicated in the crosstalk between gametes/early embryo and the oviduct remain unknown. Therefore, studying the oviductal environment is crucial to improve our understanding of the regulatory mechanisms controlling fertilization and embryo development. In vitro systems are a valuable tool to study in vivo pathways and mechanisms, particularly those in the oviducts which in livestock species are challenging to access. In studies of gamete and embryo interaction with the reproductive tract, oviductal epithelial cells, oviductal fluid and microvesicles co-cultured with gametes/embryos represent the most appropriate in vitro models to mimic the physiological conditions in vivo.
Anna Lange-Consiglio, Claudia Perrini, Giulia Albini, Silvia Modina, Valentina Lodde, Eleonora Orsini, Paola Esposti, and Fausto Cremonesi
The effect of conditioned medium (CM) or microvesicles (MVs), secreted by multicellular spheroids of oviductal cells, and the involvement of some microRNAs (miRNAs) were investigated in canine oocyte maturation. To generate CM, spheroids were cultured for 3 days. MVs were obtained by ultracentrifugation of CM at 100,000 g and measured for size and concentration by NanoSight instrument. Cumulus-oocyte complexes (COCs) were matured at 38.5°C with 5% CO2 and 5% of O2 in synthetic oviductal fluid (SOF) in biphasic systems: for 24 h, with 5.0 μg/mL of LH and for other 48 h with 10% oestrous bitch serum. SOF was used as control (CTR) or supplemented with 10% CM or 25–50–75–100–150 × 106 MVs/mL labeled with PKH-26. Results show that multicellular aggregates secreted shedding vesicles. By fluorescence microscopy, the incorporation of labeled MVs was visible only at 72 h in oocyte cytoplasm. These MVs had a positive effect (P < 0.05) on maturation rate (MII) at the concentration of 75 and 100 × 106 MVs/mL compared to CM and CTR (20.34% and 21.82% vs 9.09% and 8.66% respectively). The concentration of 150 × 106 MVs/mL provided only 9.26% of MII. The expression of three specific miRNAs (miR-30b, miR-375 and miR-503) was studied. The lower rate of MII with the higher concentration of MVs is possibly due to the high level of miR-375. In conclusion, the oviductal MVs could be involved in cellular trafficking during oocyte maturation and their possible use in vitro could facilitate the exploitment of canine reproductive biotechnologies.
A. D. Franek, L. A. Salamonsen, and A. Lopata
Marmoset monkey blastocysts maintained in culture produced trophoblastic vesicles up to 4 mm in diameter that were subdivided into fragments and subcultured to produce new vesicles. These tissues are composed of an outer layer of trophoblast-like cells and an inner layer of endoderm-like cells, and resemble a blastocyst wall. When such vesicles were cultured in serum-free medium for 14 days, they increased in size but there was no significant difference in their protein content at the end of culture. The proliferation index, measured by BrdU incorporation, varied considerably within and between vesicles. The purpose of this investigation was to determine which matrix metalloproteinases are secreted by marmoset monkey trophoblastic tissue in vitro, and the effect of extracellular laminin on this secretion. It was determined by zymography that the vesicles secreted matrix metalloproteinase 2, but not matrix metalloproteinase 9, and that matrix metalloproteinase 2 was secreted as the proenzyme (72 kDa). Matrix metalloproteinases 1, 3 and 7 were not detectable in the culture medium. The addition of laminin (5–20 μg ml−1), either as a substrate or in solution in the medium, did not have a consistent effect on matrix metalloproteinase 2 secretion during the culture period. The vesicles were found to express both matrix metalloproteinases 2 and 9 in both types of cell when examined by immunohistochemistry. The expression of matrix metalloproteinase 9 in the vesicles, but the absence of its secretion, indicates that specific factors, possibly of endometrial origin, may be required for inducing secretion.
Chunjin Li and Xu Zhou
Neurotrophins are a family of polypeptide growth factors that are required for the proliferation, differentiation, survival, and death of neuronal cells. A growing body of evidence suggests that they may have broader physiological roles in various non-neuronal tissues. The testicles are complex non-neuronal organs in which diverse cell types interact to achieve correct spermatogenesis. Both neurotrophins and their receptors have been detected in various cell types from mammalian testes, suggesting that neurotrophins may regulate or mediate intercellular communication within this organ. This review summarizes the existing data on the cellular distribution and possible biological roles of neurotrophins in the testes. The data reported in the literature indicate that neurotrophins affect somatic cell growth and spermatogenesis and imply that they play a role in regulating testicular development and male reproduction.
Michael K G Stewart, Jamie Simek, and Dale W Laird
Gap junctions formed of connexin subunits link adjacent cells by direct intercellular communication that is essential for normal tissue homeostasis in the mammary gland. The mammary gland undergoes immense remodeling and requires exquisite regulation to control the proliferative, differentiating, and cell death mechanisms regulating gland development and function. The generation of novel genetically modified mice with reduced or ablated connexin function within the mammary gland has advanced our understanding of the role of gap junctions during the complex and dynamic process of mammary gland development. These studies have revealed an important stage-specific role for Cx26 (GJA1) and Cx43 (GJB2), while Cx30 (GJB6) and Cx32 (Gjb1) can be eliminated without compromising the gland. Yet, there remain gaps in our understanding of the role of mammary gland gap junctions.
Robert Sullivan and Fabrice Saez
Mammalian spermatozoa are unique cells in many ways, and the acquisition of their main function, i.e. fertilization capacity, is a multistep process starting in the male gonad and ending near the female egg for the few cells reaching this point. Owing to the unique character of this cell, the molecular pathways necessary to achieve its maturation also show some specific characteristics. One of the most striking specificities of the spermatozoon is that its DNA is highly compacted after the replacement of histones by protamines, making the classical processes of transcription and translation impossible. The sperm cells are thus totally dependent on their extracellular environment for their protection against oxidative stress, for example, or for the molecular changes occurring during the transit of the epididymis; the first organ in which post-testicular maturation takes place. The molecular mechanisms underlying sperm maturation are still largely unknown, but it has been shown in the past three decades that extracellular vesicles secreted by the male reproductive tract are involved in this process. This review will examine the roles played by two types of naturally occurring extracellular vesicles, epididymosomes and prostasomes, secreted by the epididymis and the prostate respectively. We will also describe how the use of artificial vesicles, liposomes, contributed to the study of male reproductive physiology.
Hadas Grossman, Efrat Har-Paz, Natalie Gindi, Irit Miller, and Ruth Shalgi
miR-125a-3p, a post-transcription regulator of Fyn kinase, is expressed in mouse pre-ovulatory follicles; its expression within the follicle decreases toward ovulation. Our aim was to follow the synthesis of miR-125a-3p and regulation of its expression in all follicular compartments, focusing on intercellular communication. Mural granulosa cells (GCs) or cumulus cells (CCs) were transfected with either scrambled-miR (negative control) or miR-125a-3p mimic. Freshly isolated GCs or CCs were incubated overnight in culture media conditioned by transfected cells. To examine a possible role of gap junctions in the regulation of miR-125a-3p, we incubated large antral follicles in the presence of carbenoxolone, a gap-junction inhibitor, and triggered them to mature with hGC. Levels of miR-125a family members in GCs, CCs, oocytes, and culture media were measured by qPCR. We showed that miR-125a-3p is synthesized by all follicular components, but is regulated within the follicle as a whole. It is secreted by mural-GCs and taken up by CCs, where it remains functional, and vice versa, mural-GCs can take up miR-125a-3p secreted by CCs. miR-125a-3p is transcribed and accumulated in oocytes throughout oogenesis. Transcriptionally quiescent GV oocytes utilize their accompanying follicular cells to monitor the level of miR-125a-3p within them, as indicated in an ex vivo follicle culture. Our study reveals that miR-125a-3p expression is modulated by a network of intercellular communications within pre-ovulatory follicles, thus enabling a coordinated decrease of miR-125a-3p toward ovulation.