Ram sperm and seminal plasma alter immune transcriptome and cytokine secretion in ovine cervical explants ex vivo.

Frozen-thawed ram spermatozoa show reduced fertility following cervical artificial insemination, likely attributed to an elevated immune response in the ewe. This paper identifies the immunoprotective effect of ram seminal plasma (SP) against polymorphonuclear neutrophil binding, highlighting potential for fertility improvement. Following cervical artificial insemination, frozen-thawed (FT) ram spermatozoa display reduced fertility compared to fresh spermatozoa, irrespective of sperm motility and viability, likely attributed to an elevated immune response in the ovine cervix. SP has previously been shown to be beneficial to sperm transport in the ovine cervix, yet the components responsible for this protective effect remain unknown. As such, the present study examined the immune dynamics of frozen-thawed ram spermatozoa with polymorphonuclear neutrophils (PMNs), utilising a neutrophil binding assay. The effect of 25% v/v SP supplementation on binding susceptibility was also investigated. A portion of SP was heat-treated before incubation (HTSP) to isolate the impact of SP proteins. The presence of SP significantly reduced sperm-PMN binding (37.15 ± 0.02%, 46.59 ± 0.02%, 38.83 ± 0.02%) compared to FT alone (62.83 ± 0.02%, 75.74 ± 0.02%, 56.0 ± 0.03%) across all serum groups (no serum, serum and heat-treated serum, respectively). HTSP showed comparable binding susceptibility to the FT treatments, indicating that the protective effect of SP is attributed to a heat-labile factor. Serum significantly increased sperm-neutrophil binding irrespective of SP treatment. However, this was reduced to serum-free levels following heat-treatment, suggesting sperm-neutrophil dynamics are further mediated by protein components within ewe serum, likely complement proteins. The viability of sperm or the presence of freezing medium did not influence PMN binding. Furthermore, PMN viability and therefore binding were not influenced by the presence of serum or SP. Together, the current study presents new evidence for the immunoprotective properties of SP in sheep, which could be leveraged to aid transit through the cervix.

Cryopreservation and egg yolk extender components modify the interaction between seminal plasma proteins and the sperm surface

Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Preservation of equine sperm stored in the epididymis at 4 °C for 24, 48, 72 and 96 hours

Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process

Spermatozoa deposited vaginally must navigate the physical, chemical and immune barriers of the cervix to reach the site of fertilisation. Characteristics that favour successful cervical transit remain largely unknown beyond the obvious factors of motility and viability. Epididymal and cryopreserved ram spermatozoa demonstrate poor cervical transit, for unknown reasons. We hypothesised that seminal plasma exposure and cryopreservation alter the surface sugars of these sperm populations and, consequently, their interaction with immune cells, both potential factors for successful cervical transit. The carbohydrate profiles of epididymal, ejaculated and frozen-thawed ram spermatozoa were assessed by flow cytometry and western blotting using lectins for galactose, sialic acid, N-acetylglucosamine and mannose. Seminal plasma exposure and cryopreservation caused significant changes to the relative amounts of surface sugars detected by flow cytometry and lectin blotting. Immune cell interaction was characterised using a neutrophil-binding assay. Seminal plasma acted as a robust protective mechanism, limiting binding of spermatozoa, whereas the media used for cryopreservation caused a significant disruption to opsonin-mediated binding. We were unable to demonstrate a link between changes to surface sugars and neutrophil susceptibility. Seminal plasma and cryopreservation clearly alter the sperm glycocalyx, as well as the interaction of spermatozoa with immune cells.

Seminal plasma purportedly plays a critical role in reproduction, but epididymal spermatozoa are capable of fertilisation following deposition in the uterus, calling into question the biological requirement of this substance. Through a combination of direct observation of spermatozoa in utero using probe-based Confocal Laser Endomicroscopy, in vivo assessment of sperm fertility and in vitro analysis of various sperm functional parameters, this study investigated the role of seminal plasma in spermatozoa transit through the cervix of the ewe. Following deposition in the cervical os, epididymal spermatozoa previously exposed to seminal plasma displayed an enhanced ability to traverse the cervix as evidenced by both significantly higher pregnancy rates and numbers of spermatozoa observed at the utero-tubal junction when compared with epididymal spermatozoa not previously exposed to seminal plasma. The beneficial effect of seminal plasma on sperm transport was clearly localised to transit through the cervix as pregnancy rates of spermatozoa deposited directly into the uterus were unaffected by exposure to seminal plasma. This phenomenon was not explained by changes to sperm motion characteristics, as seminal plasma had no effect on the motility, kinematic parameters or mitochondrial membrane potential of spermatozoa. Rather, in vitro testing revealed that seminal plasma improved the ability of epididymal spermatozoa to penetrate cervical mucus recovered from ewes in oestrus. These results demonstrate that the survival and transport of ram spermatozoa through the cervix of the ewe is not linked to their motility or velocity but rather the presence of some cervical penetration trait conferred by exposure to seminal plasma.

Seminal plasma purportedly plays a critical role in reproduction, but epididymal spermatozoa are capable of fertilisation following deposition in the uterus, calling into question the biological requirement of this substance. Through a combination of direct observation of spermatozoa in utero using probe-based Confocal Laser Endomicroscopy, in vivo assessment of sperm fertility and in vitro analysis of various sperm functional parameters, this study investigated the role of seminal plasma in spermatozoa transit through the cervix of the ewe. Following deposition in the cervical os, epididymal spermatozoa previously exposed to seminal plasma displayed an enhanced ability to traverse the cervix as evidenced by both significantly higher pregnancy rates and numbers of spermatozoa observed at the utero-tubal junction when compared with epididymal spermatozoa not previously exposed to seminal plasma. The beneficial effect of seminal plasma on sperm transport was clearly localised to transit through the cervix as pregnancy rates of spermatozoa deposited directly into the uterus were unaffected by exposure to seminal plasma. This phenomenon was not explained by changes to sperm motion characteristics, as seminal plasma had no effect on the motility, kinematic parameters or mitochondrial membrane potential of spermatozoa. Rather, in vitro testing revealed that seminal plasma improved the ability of epididymal spermatozoa to penetrate cervical mucus recovered from ewes in oestrus. These results demonstrate that the survival and transport of ram spermatozoa through the cervix of the ewe is not linked to their motility or velocity but rather the presence of some cervical penetration trait conferred by exposure to seminal plasma.

Study question What is the effect of seminal plasma (SP) from fertile males on the transcriptome of endometrial epithelial organoids of fertile and subfertile women? Summary answer Transcriptomic changes in fertile and subfertile endometrial organoids are observed upon treatment with SP, related to hormone- and immune-regulation, reproduction and cellular organisation. What is known already Clinical studies have demonstrated the potential beneficial effect of intrauterine SP (liquid fraction of semen) insemination during assisted reproductive technology (ART), resulting in higher implantation- and pregnancy rates. However, the molecular mechanism by which SP exerts its function remains unclear. SP components such as TGF-β1 or IL-8 have the potential capacity of conditioning the uterus for pregnancy. SP has shown to induce significant transcriptional changes in 2D endometrial epithelial cells. 3D self-organising human endometrial organoids have been established over the last years, mimicking a more in vivo-like physiology, and therefore may better represent the interaction of the endometrium with SP. Study design, size, duration Endometrial tissue biopsies from fertile (n = 5) and subfertile (n = 5; subfertile defined as women not having reached clinical pregnancy after at least 12 months of regular unprotected intercourse) were used to establish endometrial epithelial organoids. Subsequently, SP from fertile males (n = 5) was collected and pooled. Endometrial organoids were cultured in complex media supplemented with 1 µM 17β-estradiol (E2) and incubated with or without (control) 1% SP for 6 h. Participants/materials, setting, methods RNA sequencing was performed using NextSeq600 (Illumina). Significant DEGs upon SP exposure were identified using DESeq2 (Fold Change (FC) of ≥ 1.5 or ≤ -1.5). Eight selected DEGs were validated by qPCR in one cell line. Principal Component Analysis (PCA) was performed to compare endometrium donor correlation. Gene Set Enrichment Analysis (GSEA; v4.3.2) was then used to identify overrepresented pathways and gene ontology (GO) gene sets (P < 0.05; False Discovery Rate (FDR) < 0.25). Main results and the role of chance High heterogeneity between donors was observed, regardless of their fertility phenotype. PCA analysis indicated clustering based on donor rather than SP treatment or phenotype. Moreover, the number of significantly DEGs ranged from 629 to 2332. Treatment with SP resulted in identification of 105 significant DEGs (p < 0.05) among all donors (fertile and subfertile) from which 80 DEGs were upregulated and 25 DEGs were downregulated. Several of these DEGs were also reported to be affected by SP in other 2D epithelial cultures studies. Interesting DEGs include progesterone-associated endometrial protein (PAEP; log2FC 2.0 ± 1.0), interleukin-6 (IL6; log2FC 4.5 ± 1.5), insulin-like growth factor-binding protein 3 (IGFBP3; log2FC 1.1 ± 0.2) and mucin 6 (MUC6; log2FC 3.7 ± 1.6). GSEA analysis identified gene set enrichment of pathways and biological processes related to hormone- and immune regulation, reproduction, and cellular organisation in all endometrial organoids treated with SP. Preliminary RNAseq validation with qPCR showed comparable expression patterns for 8 genes (e.g. PAEP and IL6). Moreover, expression of these genes was uniform when using a distinct SP batch (n = 6; fertile males). No clear differential response between fertile and subfertile organoids upon SP exposure was observed. Limitations, reasons for caution The apical-inside organisation of the organoids (i.e. SP interaction from the basolateral side) should be taken into account, since in vivo interaction will be apically. Moreover, high heterogeneity between donors was observed between and within the fertility phenotypes. Therefore, conclusions based on fertility phenotypes should be drawn with caution. Wider implications of the findings Understanding the effect of SP on the endometrium will shed light on male-female reproductive tract crosstalk, which is omitted during ART. Future research should focus on identifying key regulatory SP molecules and functional studies should include apical SP interaction. Potentially providing therapeutic options for improving success rates in ART. Trial registration number Not applicable

Changes to sperm surface carbohydrates following exposure to seminal plasma and freezing

Melatonin is a ubiquitous molecule, present in a wide range of organisms, and involved in multiple functions. Melatonin relays the information about the photoperiod to the tissues that express melatonin-binding sites in both central and peripheral nervous systems. This hormone has a complex mechanism of action. It can cross the cell plasma membrane and exert its actions in all cells of the body. Certain melatonin actions are mediated by receptors that belong to the superfamily of G-protein-coupled receptors (GPCRs), the MT1 and MT2 membrane. Melatonin can also bind to calmodulin as well as to nuclear receptors of the retinoic acid receptor family, RORα1, RORα2 and RZRβ. The purpose of this review is to report on recent developments in the physiological role of melatonin and its receptors. Specific issues concerning the biological function of melatonin in mammalian seasonal reproduction and spermatozoa are considered. The significance of the continuous presence of melatonin in seminal plasma with a fairly constant concentration is also discussed.

Seminal plasma has conventionally been viewed as a transport and survival medium for mammalian sperm; however, its role now extends beyond this process to actively targeting female tissues. Studies in rodents, swine, and humans demonstrate that seminal plasma induces molecular and cellular changes within the endometrium or cervix following insemination. Seminal-plasma-induced alterations to the maternal environment have been theorized to facilitate embryo development, modulate maternal immunity toward the conceptus, and potentially improve pregnancy success. It is unknown if bovine seminal plasma modulates the uterine environment following insemination in the cow, where routine use of artificial insemination reduces maternal exposure to seminal plasma. We hypothesize that seminal plasma modulates the expression of inflammatory mediators in the endometrium, altering the maternal environment of early pregnancy. In vitro, seminal plasma altered intact endometrial explant expression of CSF2, IL1B, IL6, IL17A, TGFB1, IFNE, PTGS2, and AKR1C4. Furthermore, endometrial epithelial cell CSF2, CXCL8, TGFB1, PTGS2, and AKR1C4 expression were increased after seminal plasma exposure, while endometrial stromal cell CSF2, IL1B, IL6, CXCL8, IL17A, TGFB1, PTGS2, and AKR1C4 expression were increased following seminal plasma exposure. Endometrial expression of IL1B was increased in the cow 24 h after uterine infusion of seminal plasma, while other evaluated inflammatory mediators remained unchanged. These data indicate that seminal plasma may induce changes in the bovine endometrium in a temporal manner. Understanding the role of seminal plasma in modulating the maternal environment may aid in improving pregnancy success in cattle.

To improve the fertility of cervical artificial insemination (AI) in sheep, we investigated isoxsuprine HCl usage on the cervical passage during cervical AI. We also compared cervical and laparoscopic AI fertility results of using chilled semen at different durations. Semen was collected from rams and diluted as 20 × 106 or 400 × 106 spermatozoa/straw for laparoscopic and cervical AI, respectively, and chilled to 4°C within 2 h. Sheep were inseminated with chilled semen for 8 or 24 h via the laparoscopic or cervical AI method. Moreover, some of the cervical inseminated sheep were injected intramuscularly with 0.5 mg/kg of isoxsuprine HCl 15 min before AI. As a result, the use of isoxsuprine HCl did not affect cervical transit and fertility. In addition, fertility was affected by the storage duration of the semen; laparoscopic AI was more successful than cervical AI in terms of fertility; if cervical AI is performed, the duration between semen collection and AI should be less than 8 h after chilling the semen at 4°C, and if laparoscopic AI is performed, the time between semen collection and insemination can be up to 24 h after chilling the semen at 4°C. Longer storage periods should be studied.

This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C (n = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 (n = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination.

Avaliou-se o efeito da adição de plasma seminal ovino ao sêmen descongelado sobre a taxa de prenhez de ovelhas em rebanho comercial. Cento e setenta e quatro ovelhas cruza Texel foram distribuídas em quatro tratamentos: T1) inseminação artificial cervical (IAC) com sêmen descongelado (SD) diluído em solução tampão fosfato salino (PBS); T2) IAC com SD e adição de plasma seminal ovino; T3) grupo-controle I: IAC com sêmen fresco diluído em PBS; T4) grupo-controle II: inseminação artificial por laparoscopia com SD diluído em PBS. Para indução de cio, utilizaram-se esponjas impregnadas com acetato de medroxiprogesterona (MAP) por 12 dias, com aplicação intramuscular de 400 UI de eCG (Novormon®) e de 37,5µg de cloprostenol sódico (Sincrocio®), no dia da retirada das esponjas. O aparecimento de cio foi monitorado com rufiões vasectomizados a partir da retirada das esponjas até a inseminação artificial em tempo fixo - 54 a 60 horas. A taxa de prenhez do tratamento com adição de plasma seminal ao sêmen descongelado (7,0%) não diferiu (P>0,05) do tratamento sem adição de plasma (4,3%), entretanto foi menor (P<0,05) se comparada à taxa de prenhez dos grupos-controle I inseminação via cervical superficial com sêmen fresco diluído (50,0%) e II inseminação via laparoscopia com sêmen descongelado (39,4%). A inseminação artificial por via cervical superficial com adição de plasma seminal ao sêmen descongelado não elevou o percentual de prenhez em valores que justifiquem a indicação desta biotecnologia em rebanhos comerciais de ovinos.