Gene expression and in vitro nuclear maturation in bovine cumulus oocyte complexes maturated in a medium supplemented with bovine fetal serum or bovine serum albumin

Background: The expansion and mucifi cation of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be infl uenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and β-actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplifi ed using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplifi cation intensity was quantifi ed to obtain the relative mRNA abundance. Part of oocytes submitted to IVM medium supplemented with FBS (G2) or BSA (G3) was stained with Hoechst 33342 to assess the nuclear maturation rate by fl uorescence microscopy. The results revealed that relative abundances of HAS (P = 0.000), link protein 1 (P = 0.001), connexin 43 (P =0.007) and β-actin (P = 0.011) transcripts differed between COCs submitted to IVM in FBS medium (G2) and COCs not submitted to IVM (G1) or COCs maturated in BSA medium (G3). When COCs submitted to IVM in FBS or BSA media are compared, no statistically signifi cant differences (P > 0.05) were observed in meiosis resumption (86.7% and 91.5%, respectively) or in nuclear maturation rates (56.1% and 58.5%). Discussion: HAS2 is involved in the synthesis of hyaluronic acid (HA) by cumulus cells, and plays an important role in ECM expansion and in oocyte competence development. This protein organization of the ECM, formed by the aggregation of HA and proteoglycans, depends on link protein 1; it is also produced by cumulus cells and is implicated in COC expansion. Connexin 43 belongs to a protein family that establishes gap junctions that play an important role in the cellular communication and coordinated response processes. The role of gap junctions in bovine oocytes during IVM has been associated with maturation rates and cumulus expansion; this expansion of cumulus cells is accompanied by changes in the transmembrane channels formed by connexin 43. The higher mRNA expression of the HAS2, link protein 1, connexin 43 and β-actin genes in bovine COCs submitted to IVM in FBS medium, in comparison with COCs before IVM or COCs maturated in BSA medium may be associated with FBS constituents, which would act as transcription factors for these genes during ECM expansion. Although the results obtained allow associating the differential expression of transcripts to the presence of FBS in the IVM medium, the data reveal that meiosis resumption and nuclear maturation apparently were not infl uenced by the protein supplementation regimens in the IVM medium, supplemented either with FBS or BSA

Semen amyloids participate in spermatozoa selection and clearance

Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens

Semen inhibits Zika virus infection of cells and tissues from the anogenital region

Zika virus (ZIKV) causes severe birth defects and can be transmitted via sexual intercourse. Semen from ZIKV-infected individuals contains high viral loads and may therefore serve as an important vector for virus transmission. Here we analyze the effect of semen on ZIKV infection of cells and tissues derived from the anogenital region. ZIKV replicates in all analyzed cell lines, primary cells, and endometrial or vaginal tissues. However, in the presence of semen, infection by ZIKV and other flaviviruses is potently inhibited. We show that semen prevents ZIKV attachment to target cells, and that an extracellular vesicle preparation from semen is responsible for this anti-ZIKV activity. Our findings suggest that ZIKV transmission is limited by semen. As such, semen appears to serve as a protector against sexual ZIKV transmission, despite the availability of highly susceptible cells in the anogenital tract and high viral loads in this bodily fluid.Peer reviewe

Physiological functions of seminal amyloids in reproduction and its involvement in sperm clearance

Seminal amyloids derived from two abundant semen proteins have been identified due to their ability to enhance HIV-1 (human immunodeficiency virus) infection in vitro. However, their physiological role remained to be studied. As seminal amyloids inhibit fertilization in vitro by entrapping motile sperm, one of the aims of this work was to elucidate their involvement in male infertility. Furthermore, this work aimed to elucidate which semen characteristics are important for the fibril-dependent enhancement of HIV-1 infection. Therefore, we detected and purified them from fresh semen. Additionally, this work sought to clarify which sperm population becomes entrapped, how the fibrils can be degraded and which physiological role this may present in vivo. In a multiparameter analysis of more than 100 donors, opposed to the expected results, we found that seminal amyloids do not correlate with male infertility. On the contrary, infertile donors tended to present lower concentrations of seminal amyloids than fertile/subfertile donors. On the other hand, amyloid content in semen positively correlated with infectivity enhancement, strengthening the fact that semen´s potential to enhance HIV-1 infection depends directly on seminal amyloids. Amyloid content also positively correlated with age, as shown for other forms of amyloidosis. Moreover, increased concentrations of clusterin, an extracellular chaperone, do not prevent amyloid formation. Interestingly, amyloid content negatively correlated with luteinizing hormone (LH) and follicle-stimulating hormone (FSH) but not with testosterone, suggesting that the presence of amyloids in semen is not hormone-regulated as for other amyloid-related diseases. In addition, semen viscosity and pH negatively correlated with infection enhancement but not with amyloid content, indicating that not only the presence and total concentration of amyloids but also the physical properties determine their infectivity enhancing potential. Endogenous seminal amyloids could be successfully detected and quantified in unmodified semen by confocal microscopy using an amyloid-sensitive dye, ProteoStat®. With this method, semen samples from selected donors were analyzed but no correlation between fertile and infertile donors regarding amyloid content could be observed. In fact, the presence of large fibril aggregates was a rather rare finding. Furthermore, seminal fluid itself increased the percentage of immotile spermatozoa and decreased the mean amplitude of lateral head displacement. These findings may be related to the presence of amyloids or other motility-inhibiting molecules in semen. Moreover, synthetic seminal fibrils were not degraded by abundant seminal or vaginal proteases but by seminal fluid itself, suggesting that a mixture of different enzymes is responsible for degradation of seminal amyloids and probably also the release of entrapped spermatozoa within hours after ejaculation in vivo. Additionally, seminal amyloids did efficiently entrap apoptotic spermatozoa and avoid that sperm cells swim-up. However, the preferential entrapment of apoptotic cells did not result in an efficient selection of defective spermatozoa in the swim-up assay. These results suggest that spermatozoa entrapment by seminal fibrils is not the only factor in selecting the fittest sperm to continue its journey in vivo. In this rationale, our collaborators tested whether seminal amyloids are involved in spermatozoa clearance. They showed that healthy and damaged spermatozoa can be promptly phagocytosed by macrophages and that semenogelin (SEM) fibrils are able to increase phagocytosis kinetics. Besides, macrophages phagocytosed preferentially damaged sperm cells, whose uptake was 3 fold increased in the presence of SEM fibrils. In order to verify the results produced with fibrils formed by synthetic peptide, endogenous seminal amyloids were purified and shown to behave in a similar manner in entrapment of human spermatozoa compared to their synthetic counterparts. In conclusion, seminal amyloids exert a unique quality control function. The results presented herein sustain their role as functional amyloid, as its presence in semen does not correlate with male infertility and its effects on spermatozoa movement facilitate sperm clearance in the lower female reproductive tract. Finally, spermatozoa entrapment in vivo may be a well-regulated mechanism, with viable, healthy cells being quickly released from entrapment, due to the action of seminal and vaginal proteases, and apoptotic, defective sperm retained for macrophage clearance. These results give the first insights into the physiological role of seminal amyloids

Semen amyloids participate in spermatozoa selection and clearance.

Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens

Semen inhibits Zika virus infection of cells and tissues from the anogenital region.

Zika virus (ZIKV) causes severe birth defects and can be transmitted via sexual intercourse. Semen from ZIKV-infected individuals contains high viral loads and may therefore serve as an important vector for virus transmission. Here we analyze the effect of semen on ZIKV infection of cells and tissues derived from the anogenital region. ZIKV replicates in all analyzed cell lines, primary cells, and endometrial or vaginal tissues. However, in the presence of semen, infection by ZIKV and other flaviviruses is potently inhibited. We show that semen prevents ZIKV attachment to target cells, and that an extracellular vesicle preparation from semen is responsible for this anti-ZIKV activity. Our findings suggest that ZIKV transmission is limited by semen. As such, semen appears to serve as a protector against sexual ZIKV transmission, despite the availability of highly susceptible cells in the anogenital tract and high viral loads in this bodily fluid