Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0026
E. Behboodi, S. Mohan, J. Rodriguez-Sosa, Y. Li, S. Megee, I. Dobrinski
Undifferentiated spermatogonia are a potential source of pluripotent cells and could be used for targeted genetic alteration in pigs. Our understanding of mechanisms maintaining porcine spermatogonial stem cells (SSCs)in vivo and of conditions to propagate SSCs in vitro remains limited. This is largely due to the small number of SSCs present in the testis and the lack of specific morphological and cell-surface markers to isolate a purified population. The goal of this study was to establish a modified differential culture system to effectively enrich SSCsfrom prepubertal porcine testes for subsequent culture. Germ cell enrichment was quantified by immunocytochemistry and RT-PCR analysis of proteins and genes known to be specifically expressed in spermatogonia (PGP 9.5, VASA and DBA). Testes were collected from 10 week-old pigs and washed with PBS and transported on ice within 24 h to the laboratory in PBS that was supplemented with antibiotics. Testes were pooled for the isolation of germ cells (2-4 testes per trial). Cells were isolated by a two step enzymatic digestion (Honaramooz et al, 2002). The cells were incubated in DMEm containing 0.1 mM 1-mercaptoethanol, 0.1mM MEM non-essential amino-acids, 200mM LGlutamine and 5% FCSsupplemented with 100 IU penicillin streptomycin, in tissue culture dishes coated with 0.010/0 gelatin 12 h prior to use, at a concentration of 50x106cells per dish (60x15 mm) for 1 h at 37 °C in 50/0CO, in air. By counting cells in the supernatant it was determined that 50% of the total cells attached to the culture plates after 1 h, most of which were somatic cells. Germ cells largely remained in suspension and were transferred to new culture dishes. After an additional 14 h of incubation, unattached cells were collected, concentrated by centrifugation for 5 min and counted before use for long term culture. Enrichment of germ cells at each time point (0, 14 h) was determined by immunocytochemistry for alkaline phosphatase activity, and expression of DBA, PGP 9.5 and VASA. Counterstaining for vimentin was employed to identify somatic cells. mRNA was isolated for RT-PCRanalysis to confirm expression of PGP 9.5 and VASA. Isolated cells were seeded at a density of 5 x 105 cells in 6-well plates in DMEM medium as above, supplemented with glial cell-derived neurotrophic factor (GDNF, 20 ng/ ml), epidermal growth factor (EGF; 200 ng/ml), and basic fibroblast growth factor (bFGF; 200 ng/ml). Comparison between groups was by Student's t-test. The two-step differential culture increased the concentration of germ cells from 5.4 ± 3% in the initial cell suspension to 46.6± 22% in the non-adherent population at 14 h of culture (Table 1). Enriched germ cells formed more (21 ± 7 versus 10 ± 3.4; P <0.01) and larger colonies at day 7-10 post culture than those arising from control cells not subjected to differential culture (Table 2). The cultured cells grew vigorously in medium that was supplemented with growth factors as mentioned abo
{"title":"Enrichment of porcine spermatogonia by differential culture.","authors":"E. Behboodi, S. Mohan, J. Rodriguez-Sosa, Y. Li, S. Megee, I. Dobrinski","doi":"10.1530/biosciprocs.18.0026","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0026","url":null,"abstract":"Undifferentiated spermatogonia are a potential source of pluripotent cells and could be used for targeted genetic alteration in pigs. Our understanding of mechanisms maintaining porcine spermatogonial stem cells (SSCs)in vivo and of conditions to propagate SSCs in vitro remains limited. This is largely due to the small number of SSCs present in the testis and the lack of specific morphological and cell-surface markers to isolate a purified population. The goal of this study was to establish a modified differential culture system to effectively enrich SSCsfrom prepubertal porcine testes for subsequent culture. Germ cell enrichment was quantified by immunocytochemistry and RT-PCR analysis of proteins and genes known to be specifically expressed in spermatogonia (PGP 9.5, VASA and DBA). Testes were collected from 10 week-old pigs and washed with PBS and transported on ice within 24 h to the laboratory in PBS that was supplemented with antibiotics. Testes were pooled for the isolation of germ cells (2-4 testes per trial). Cells were isolated by a two step enzymatic digestion (Honaramooz et al, 2002). The cells were incubated in DMEm containing 0.1 mM 1-mercaptoethanol, 0.1mM MEM non-essential amino-acids, 200mM LGlutamine and 5% FCSsupplemented with 100 IU penicillin streptomycin, in tissue culture dishes coated with 0.010/0 gelatin 12 h prior to use, at a concentration of 50x106cells per dish (60x15 mm) for 1 h at 37 °C in 50/0CO, in air. By counting cells in the supernatant it was determined that 50% of the total cells attached to the culture plates after 1 h, most of which were somatic cells. Germ cells largely remained in suspension and were transferred to new culture dishes. After an additional 14 h of incubation, unattached cells were collected, concentrated by centrifugation for 5 min and counted before use for long term culture. Enrichment of germ cells at each time point (0, 14 h) was determined by immunocytochemistry for alkaline phosphatase activity, and expression of DBA, PGP 9.5 and VASA. Counterstaining for vimentin was employed to identify somatic cells. mRNA was isolated for RT-PCRanalysis to confirm expression of PGP 9.5 and VASA. Isolated cells were seeded at a density of 5 x 105 cells in 6-well plates in DMEM medium as above, supplemented with glial cell-derived neurotrophic factor (GDNF, 20 ng/ ml), epidermal growth factor (EGF; 200 ng/ml), and basic fibroblast growth factor (bFGF; 200 ng/ml). Comparison between groups was by Student's t-test. The two-step differential culture increased the concentration of germ cells from 5.4 ± 3% in the initial cell suspension to 46.6± 22% in the non-adherent population at 14 h of culture (Table 1). Enriched germ cells formed more (21 ± 7 versus 10 ± 3.4; P <0.01) and larger colonies at day 7-10 post culture than those arising from control cells not subjected to differential culture (Table 2). The cultured cells grew vigorously in medium that was supplemented with growth factors as mentioned abo","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"209-10"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44455521","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0015
K. Kikuchi, T. Somfai, M. Nakai, T. Nagai
In vitro production (IVP) including in vitro maturation (IVM) and fertilization (IVF) is now an important technology for obtaining live piglets. However, there are still two significant obstacles to the efficient production of viable porcine embryos: (1) polyspermy and (2) fertilization of oocytes arrested at the immature stage. These phenomena relate to production of embryos with abnormal ploidy (polyploidy). To avoid these problems, careful selection of mature oocytes for IVF, and regular monitoring of normal and abnormal fertilization (polyspermy and/or lack of male pronucleus formation) are very important. In our recent studies, however, we have confirmed that some oocytes with abnormal ploidy after polyspermy can develop into diploid embryos with potentially normal developmental ability. The mechanism by which such fertilized polyploid oocytes develop to a normal state during embryo development is still not well understood. Attempts to clarify this mechanism would hopefully reveal data that are very useful for not only IVP but also other technologies such as the production of transgenic or cloned animals using IVM oocytes, including other species, also for human reproductive manipulation. In this review, we focus on studies of normality of IVM oocytes and ploidy of IVP embryos, and try to suggest practical ways of solving the problems mentioned above in pigs.
{"title":"Appearance, fate and utilization of abnormal porcine embryos produced by in vitro maturation and fertilization.","authors":"K. Kikuchi, T. Somfai, M. Nakai, T. Nagai","doi":"10.1530/biosciprocs.18.0015","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0015","url":null,"abstract":"In vitro production (IVP) including in vitro maturation (IVM) and fertilization (IVF) is now an important technology for obtaining live piglets. However, there are still two significant obstacles to the efficient production of viable porcine embryos: (1) polyspermy and (2) fertilization of oocytes arrested at the immature stage. These phenomena relate to production of embryos with abnormal ploidy (polyploidy). To avoid these problems, careful selection of mature oocytes for IVF, and regular monitoring of normal and abnormal fertilization (polyspermy and/or lack of male pronucleus formation) are very important. In our recent studies, however, we have confirmed that some oocytes with abnormal ploidy after polyspermy can develop into diploid embryos with potentially normal developmental ability. The mechanism by which such fertilized polyploid oocytes develop to a normal state during embryo development is still not well understood. Attempts to clarify this mechanism would hopefully reveal data that are very useful for not only IVP but also other technologies such as the production of transgenic or cloned animals using IVM oocytes, including other species, also for human reproductive manipulation. In this review, we focus on studies of normality of IVM oocytes and ploidy of IVP embryos, and try to suggest practical ways of solving the problems mentioned above in pigs.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"135-47"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47072129","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0038
D. Mathew, E. Sellner, C. Okamura, R. Geisert, L. Anderson, M. Lucy
Porcine peri-implantation development and maternal recognition of pregnancy is temporally associated with down-regulation of progesterone receptor (PGR) in the endometrial epithelium on days 10 to 12 (Geisert et al. 2006). One theory for down-regulation of uterine epithelial PGR is progesterone stimulates epithelial PGRto induce expression of RANKL [receptor activator for nuclear factor-kappa B (NF-KB) ligand or TNESF11]. RANKL binds to its receptor, RANK (TNERSF11A) to activate NF-KB. NE-KB and PGR are mutually antagonistic to one another. Activation of NE-KB, therefore, may inhibit PGR expression and induce the increase in endometrial prostaglandinendoperoxide synthase 2 (PTGS2 or COX2) expression that occurs in the endometrium of cyclic and pregnant pigs on days 10 to 12. The PGRantagonist, RU486, could be usedto determine if blocking PGRdown-regulation in the uterine epithelium prevents RANKL expression and NE-KB activation. To test this hypothesis, gilts were inseminated at estrus (d 0) and assigned to one of three treatments: RU486 (400 mg/d) on d 3, 4, and 5 (T1; n = 10); RU486 on d 6 and 7 (T2; n = 9); or control (n = 9). Blood was collected daily for plasma progesterone analysis, and the uterus and ovaries were harvested after slaughter on d 8 or d 12. Endometrial total RNA was isolated and analyzed with specific primers for RANKL, PTGS2, PGR isoform B (PGR-B) or the region common to PGR isoforms A and B (PGR-AB)by real-time reverse transcriptase PCR (RTPCR).NF-KBactivation was measured by immunohistochemistry and scored objectively by three independent individuals. Gilts treated with RU486 (T1 and 12) had heavier ovaries (17.9, 19.8 and 16.1 g [SEM = 1.1]; T1, 12 and control; P < 0.05), greater average follicular diameters (5.6, 4.9 and 3.6 mm [SEM = 0.5]; P < 0.01), a tendency for a greater number of corpora lutea (16.8, 15.0 and 13.7 [SEM = 1.0]; P < 0.07) and greater mid-cycle plasma progesterone concentration (25.2, 28.0 and 20.6 ng/mL; P < 0.05; d 9 to 11). Uterine weight (g) was reduced (P < 0.05) for T1 (608 ± 46) compared with T2 (780 ± 49) or control (785 ± 44). Treatment of giRswith RU486 affected early embryonic development. The proportion of gilts with normal early embryonic development was lowest for gilts in T1 (chi-square= 11.2; P < 0.01; Table 1). There was a treatment effect (P < 0.01) on log-transformed RANKL mRNA expression (fold change relative to internal assaycontrol) because compared with the control gilts, RANKL expression was greater in T1 (d 8 and d 12) and greater in T2 on d 12. Treatment affected both endometrial PGR-B (P < 0.0W) and PGR-AB (P < 0.001) mRNA abundance. The PGR-BmRNA was more abundant in T1 (9.1 ± 1.0) compared with control (3.1+ 1.0) with mRNA expression intermediate (6.0 ± 1.0) for T2 pigs. Likewise, the
{"title":"Effect of progesterone antagonist RU486 on uterine progesterone receptor mRNA expression, embryonic development and ovarian function during early pregnancy in pigs.","authors":"D. Mathew, E. Sellner, C. Okamura, R. Geisert, L. Anderson, M. Lucy","doi":"10.1530/biosciprocs.18.0038","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0038","url":null,"abstract":"Porcine peri-implantation development and maternal recognition of pregnancy is temporally associated with down-regulation of progesterone receptor (PGR) in the endometrial epithelium on days 10 to 12 (Geisert et al. 2006). One theory for down-regulation of uterine epithelial PGR is progesterone stimulates epithelial PGRto induce expression of RANKL [receptor activator for nuclear factor-kappa B (NF-KB) ligand or TNESF11]. RANKL binds to its receptor, RANK (TNERSF11A) to activate NF-KB. NE-KB and PGR are mutually antagonistic to one another. Activation of NE-KB, therefore, may inhibit PGR expression and induce the increase in endometrial prostaglandinendoperoxide synthase 2 (PTGS2 or COX2) expression that occurs in the endometrium of cyclic and pregnant pigs on days 10 to 12. The PGRantagonist, RU486, could be usedto determine if blocking PGRdown-regulation in the uterine epithelium prevents RANKL expression and NE-KB activation. To test this hypothesis, gilts were inseminated at estrus (d 0) and assigned to one of three treatments: RU486 (400 mg/d) on d 3, 4, and 5 (T1; n = 10); RU486 on d 6 and 7 (T2; n = 9); or control (n = 9). Blood was collected daily for plasma progesterone analysis, and the uterus and ovaries were harvested after slaughter on d 8 or d 12. Endometrial total RNA was isolated and analyzed with specific primers for RANKL, PTGS2, PGR isoform B (PGR-B) or the region common to PGR isoforms A and B (PGR-AB)by real-time reverse transcriptase PCR (RTPCR).NF-KBactivation was measured by immunohistochemistry and scored objectively by three independent individuals. Gilts treated with RU486 (T1 and 12) had heavier ovaries (17.9, 19.8 and 16.1 g [SEM = 1.1]; T1, 12 and control; P < 0.05), greater average follicular diameters (5.6, 4.9 and 3.6 mm [SEM = 0.5]; P < 0.01), a tendency for a greater number of corpora lutea (16.8, 15.0 and 13.7 [SEM = 1.0]; P < 0.07) and greater mid-cycle plasma progesterone concentration (25.2, 28.0 and 20.6 ng/mL; P < 0.05; d 9 to 11). Uterine weight (g) was reduced (P < 0.05) for T1 (608 ± 46) compared with T2 (780 ± 49) or control (785 ± 44). Treatment of giRswith RU486 affected early embryonic development. The proportion of gilts with normal early embryonic development was lowest for gilts in T1 (chi-square= 11.2; P < 0.01; Table 1). There was a treatment effect (P < 0.01) on log-transformed RANKL mRNA expression (fold change relative to internal assaycontrol) because compared with the control gilts, RANKL expression was greater in T1 (d 8 and d 12) and greater in T2 on d 12. Treatment affected both endometrial PGR-B (P < 0.0W) and PGR-AB (P < 0.001) mRNA abundance. The PGR-BmRNA was more abundant in T1 (9.1 ± 1.0) compared with control (3.1+ 1.0) with mRNA expression intermediate (6.0 ± 1.0) for T2 pigs. Likewise, the","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"333-4"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44619350","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0029
B. Croy, J. M. Wessels, N. Linton, M. J. van den Heuvel, A. Edwards, C. Tayade
Commercial, North American pork breeds (Sus scrofa) experience significant loss of genetically-normal conceptuses during the peri-implantation (attachment) period and at mid-gestation (day 50 to 90 of the 114 day porcine gestation interval). Although exact causes for these losses are not defined, asynchronous in-utero development and deficits in vascularization of the endometrium and placenta appear to be involved. Understanding of normal maternal-fetal dialogue is critical to develop breeding or therapeutic strategies that improve fetal health and overall litter size in commercial pigs. The non-invasive, epitheliochorial porcine placenta permits investigation of maternal or fetal compartments without cross contaminating cells. We developed and use protocols to capture single, homogenous populations of porcine cells (endometrial lymphocytes, dendritic or endothelial cells) from histological sections using laser capture microdissection (LCM), a powerful tool for study of gene expression that reflects the in vivo environment. These data are compared with gene expression in biopsies of endometrium and of trophoblast from the same, attachment sites. Here we review justifications for selection of the genes we have studied and our published and in progress work. These data provide new insights into the roles of the endometrial immune environment in the regulation of the success and failure of porcine conceptuses.
{"title":"Cellular and molecular events in early and mid gestation porcine implantation sites: a review.","authors":"B. Croy, J. M. Wessels, N. Linton, M. J. van den Heuvel, A. Edwards, C. Tayade","doi":"10.1530/biosciprocs.18.0029","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0029","url":null,"abstract":"Commercial, North American pork breeds (Sus scrofa) experience significant loss of genetically-normal conceptuses during the peri-implantation (attachment) period and at mid-gestation (day 50 to 90 of the 114 day porcine gestation interval). Although exact causes for these losses are not defined, asynchronous in-utero development and deficits in vascularization of the endometrium and placenta appear to be involved. Understanding of normal maternal-fetal dialogue is critical to develop breeding or therapeutic strategies that improve fetal health and overall litter size in commercial pigs. The non-invasive, epitheliochorial porcine placenta permits investigation of maternal or fetal compartments without cross contaminating cells. We developed and use protocols to capture single, homogenous populations of porcine cells (endometrial lymphocytes, dendritic or endothelial cells) from histological sections using laser capture microdissection (LCM), a powerful tool for study of gene expression that reflects the in vivo environment. These data are compared with gene expression in biopsies of endometrium and of trophoblast from the same, attachment sites. Here we review justifications for selection of the genes we have studied and our published and in progress work. These data provide new insights into the roles of the endometrial immune environment in the regulation of the success and failure of porcine conceptuses.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"233-44"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42880036","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0020
J. V. van Leeuwen, S. Williams, B. Kemp, N. Soede
{"title":"Post-weaning altrenogest treatment in primiparous sows; the effect of duration and dosage on follicular development and consequences for early pregnancy.","authors":"J. V. van Leeuwen, S. Williams, B. Kemp, N. Soede","doi":"10.1530/biosciprocs.18.0020","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0020","url":null,"abstract":"","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"197-8"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47753661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0004
D. Rath, R. Bathgate, H. Rodríguez-Martínez, J. Roca, J. Strzeżek, D. Waberski
Since 35 years ago boar semen has been frozen and used for artificial insemination (AI). However, fertility of cryopreserved porcine sperm has consistently been low as boar sperm are more sensitive to cellular stress imposed by changing osmotic balance, oxidative stress, low-temperature exposure, cryo-protectant intoxication etc. and are less able to compensate for these deficiencies at commercially applicable dosages. Additionally, differences in sperm freezability among individuals are well known. Here we review current advances on tests to screen sperm quality post-thaw, on ways of diminishing individual boar effects, on improvement of cryo-protection by novel extender components, on packaging and freezing protocols and freezing and thawing methods, and on the handling of sexed boar sperm. Major advances have been registered, which have improved cryo-survival and the capacity to process boar semen for commercial AI.
{"title":"Recent advances in boar semen cryopreservation.","authors":"D. Rath, R. Bathgate, H. Rodríguez-Martínez, J. Roca, J. Strzeżek, D. Waberski","doi":"10.1530/biosciprocs.18.0004","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0004","url":null,"abstract":"Since 35 years ago boar semen has been frozen and used for artificial insemination (AI). However, fertility of cryopreserved porcine sperm has consistently been low as boar sperm are more sensitive to cellular stress imposed by changing osmotic balance, oxidative stress, low-temperature exposure, cryo-protectant intoxication etc. and are less able to compensate for these deficiencies at commercially applicable dosages. Additionally, differences in sperm freezability among individuals are well known. Here we review current advances on tests to screen sperm quality post-thaw, on ways of diminishing individual boar effects, on improvement of cryo-protection by novel extender components, on packaging and freezing protocols and freezing and thawing methods, and on the handling of sexed boar sperm. Major advances have been registered, which have improved cryo-survival and the capacity to process boar semen for commercial AI.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"51-66"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49083381","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-10-12DOI: 10.1530/biosciprocs.17.0018
William V. Holt, R. M. Elliott, Alireza Fazeli, E. Sostaric, A. S. Georgiou, N. Satake, N. Prathalingam, Paul F. Watson
Spermatozoa fulfil a single role, namely achieving syngamy by transporting the haploid genome to their counterpart gamete, the oocyte. Simple as this may seem, it is fraught with many difficulties, especially in the face of biological processes that enable females to select spermatozoa after they have mated multiply with several males. Conversely, the female reproductive tract sequesters a privileged sperm subpopulation in the oviductal isthmus for variable periods of time, releasing them when the time is opportune for fertilisation. Recent studies of sperm transport in the female reproductive tract suggest that these phenomena involve signalling dialogues between spermatozoa and the female reproductive tract environment. Opportunities for mutual signalling are immense but have received relatively little attention. The oviduct is an organ of crucial significance in modulating sperm function and may be one of the most important sites for determining many aspects of sperm selection and competition. The oviductal environment possesses the potential for enhancing sperm survival, suppressing and activating sperm motility as required, and responds to the arrival of spermatozoa by producing novel proteins. While the biological nature of the sperm-oviduct dialogue is interesting for its own sake, the mechanisms that govern these processes offer opportunities for the improvement of artificial insemination procedures. If oviductal proteins enhance sperm survival, they offer opportunities for the development of long-life semen diluents. Conversely, if we understood the basis of sperm selection we may be able to concentrate on identifying and using only the best sperm subpopulations for improved animal breeding efficiency.
{"title":"Harnessing the biology of the oviduct for the benefit of artificial insemination.","authors":"William V. Holt, R. M. Elliott, Alireza Fazeli, E. Sostaric, A. S. Georgiou, N. Satake, N. Prathalingam, Paul F. Watson","doi":"10.1530/biosciprocs.17.0018","DOIUrl":"https://doi.org/10.1530/biosciprocs.17.0018","url":null,"abstract":"Spermatozoa fulfil a single role, namely achieving syngamy by transporting the haploid genome to their counterpart gamete, the oocyte. Simple as this may seem, it is fraught with many difficulties, especially in the face of biological processes that enable females to select spermatozoa after they have mated multiply with several males. Conversely, the female reproductive tract sequesters a privileged sperm subpopulation in the oviductal isthmus for variable periods of time, releasing them when the time is opportune for fertilisation. Recent studies of sperm transport in the female reproductive tract suggest that these phenomena involve signalling dialogues between spermatozoa and the female reproductive tract environment. Opportunities for mutual signalling are immense but have received relatively little attention. The oviduct is an organ of crucial significance in modulating sperm function and may be one of the most important sites for determining many aspects of sperm selection and competition. The oviductal environment possesses the potential for enhancing sperm survival, suppressing and activating sperm motility as required, and responds to the arrival of spermatozoa by producing novel proteins. While the biological nature of the sperm-oviduct dialogue is interesting for its own sake, the mechanisms that govern these processes offer opportunities for the improvement of artificial insemination procedures. If oviductal proteins enhance sperm survival, they offer opportunities for the development of long-life semen diluents. Conversely, if we understood the basis of sperm selection we may be able to concentrate on identifying and using only the best sperm subpopulations for improved animal breeding efficiency.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"62 1","pages":"247-59"},"PeriodicalIF":0.0,"publicationDate":"2019-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43076745","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-10-12DOI: 10.1530/biosciprocs.17.0012
J. Green, J. G. Kim, K. Whitworth, C. Agca, R. Prather
In swine and other livestock, the uterine endometrium exhibits dramatic morphological and secretory changes throughout the oestrous cycle and during pregnancy. Such physiological changes are a reflection of extremely complex interactions between gene products (RNA and protein). The recent development of genomics and proteomics methods, as well as associated bioinformatics tools, has provided the means to begin characterising such interactions. Indeed, the analysis of the transcriptome and proteome of cells and tissues now comprises a new field of study known as 'systems biology'. Currently, the most powerful technique available to the systems biologist is the microarray. These platforms represent oligonucleotide or cDNA fragments spotted in a specified high-density pattern on a solid support. Hybridisation of fluorescently-tagged cDNAs from different tissue sources permits the measurement of thousands of RNAs in parallel. The method permits the identification of genes that are present at different amounts between the two tissues and, more importantly, it permits the identification of groups of genes (clusters) that are expressed in comparable patterns. Results from a recent expression profiling experiment are described. The goal of the profiling experiment was to define genes that are differentially expressed in endometrium during the oestrous cycle. The experiment used an in-house cDNA microarray with > 14,000 distinct cDNAs cloned from reproductive tissues. Total RNAs from cyclic endometrium (Days 0, 3, 6, 10, 12, 14 and 18 post-oestrus) were reverse transcribed into cDNAs, labelled with fluorescent dye and hybridised to the arrays along with cDNAs derived from a reference RNA pool. A total of 4,827 genes were found to differ significantly at some time during the oestrous cycle. Clustering methods were able to define numerous groups of similarly expressed genes. These data will help to define the complex patterns of endometrial genes acting in concert to create the environments required for fertilisation, embryo growth and conceptus development in swine.
{"title":"The use of microarrays to define functionally-related genes that are differentially expressed in the cycling pig uterus.","authors":"J. Green, J. G. Kim, K. Whitworth, C. Agca, R. Prather","doi":"10.1530/biosciprocs.17.0012","DOIUrl":"https://doi.org/10.1530/biosciprocs.17.0012","url":null,"abstract":"In swine and other livestock, the uterine endometrium exhibits dramatic morphological and secretory changes throughout the oestrous cycle and during pregnancy. Such physiological changes are a reflection of extremely complex interactions between gene products (RNA and protein). The recent development of genomics and proteomics methods, as well as associated bioinformatics tools, has provided the means to begin characterising such interactions. Indeed, the analysis of the transcriptome and proteome of cells and tissues now comprises a new field of study known as 'systems biology'. Currently, the most powerful technique available to the systems biologist is the microarray. These platforms represent oligonucleotide or cDNA fragments spotted in a specified high-density pattern on a solid support. Hybridisation of fluorescently-tagged cDNAs from different tissue sources permits the measurement of thousands of RNAs in parallel. The method permits the identification of genes that are present at different amounts between the two tissues and, more importantly, it permits the identification of groups of genes (clusters) that are expressed in comparable patterns. Results from a recent expression profiling experiment are described. The goal of the profiling experiment was to define genes that are differentially expressed in endometrium during the oestrous cycle. The experiment used an in-house cDNA microarray with > 14,000 distinct cDNAs cloned from reproductive tissues. Total RNAs from cyclic endometrium (Days 0, 3, 6, 10, 12, 14 and 18 post-oestrus) were reverse transcribed into cDNAs, labelled with fluorescent dye and hybridised to the arrays along with cDNAs derived from a reference RNA pool. A total of 4,827 genes were found to differ significantly at some time during the oestrous cycle. Clustering methods were able to define numerous groups of similarly expressed genes. These data will help to define the complex patterns of endometrial genes acting in concert to create the environments required for fertilisation, embryo growth and conceptus development in swine.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"62 1","pages":"163-76"},"PeriodicalIF":0.0,"publicationDate":"2019-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44237981","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-10-12DOI: 10.1530/biosciprocs.17.0001
W. Sienkiewicz
Neuropeptides and catecholamines are biologically active substances which play the roles of neurotransmitters, neuromodulators and cotransmitters in the central nervous system (CNS). These substances are known to regulate and influence a wide spectrum of functions; such as food and water intake, thermoregulation, growth and maturation, sexual behaviour, reproduction, function of the hypothalamic-hypophysial-organ axes, and many others. This large family of neuropeptides is comprised of around 40 fundamental polypeptides, which does not include their derivatives and related peptides. Localization and development of several of them was intensively studied in the brain of a wide range of species, including the rat and the pig. In this present paper, data regarding distribution of catecholamine synthesising enzymes and some neuropeptides, as well as their gene expression during development in the rodent and porcine brain are summarised and related to development of LHRH-containing structures.
{"title":"Distribution and gene expression of neuropeptides during brain development.","authors":"W. Sienkiewicz","doi":"10.1530/biosciprocs.17.0001","DOIUrl":"https://doi.org/10.1530/biosciprocs.17.0001","url":null,"abstract":"Neuropeptides and catecholamines are biologically active substances which play the roles of neurotransmitters, neuromodulators and cotransmitters in the central nervous system (CNS). These substances are known to regulate and influence a wide spectrum of functions; such as food and water intake, thermoregulation, growth and maturation, sexual behaviour, reproduction, function of the hypothalamic-hypophysial-organ axes, and many others. This large family of neuropeptides is comprised of around 40 fundamental polypeptides, which does not include their derivatives and related peptides. Localization and development of several of them was intensively studied in the brain of a wide range of species, including the rat and the pig. In this present paper, data regarding distribution of catecholamine synthesising enzymes and some neuropeptides, as well as their gene expression during development in the rodent and porcine brain are summarised and related to development of LHRH-containing structures.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"62 1","pages":"1-17"},"PeriodicalIF":0.0,"publicationDate":"2019-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44876676","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-10-12DOI: 10.1530/biosciprocs.17.0011
A. Ziecik, A. Blitek, M. Kaczmarek, A. Waclawik, M. Bogacki
Inhibition of luteolysis and establishment of pregnancy in pigs results from oestrogen secretion by the conceptuses and requires progesterone produced by the corpus luteum (CL). An integral part of maternal recognition of pregnancy in the pig is the redirection of prostaglandin (PG) F2alpha secretion from endocrine (blood) to exocrine (uterus) direction and an increase of PGE2 synthesis in both the endometrium and conceptus. Uterine and conceptus PGE2 synthases play an integrated role in establishing the PGE2:PGF2alpha ratio necessary for luteal maintenance. The luteolytic or luteotrophic changes in the CL are synchronised with the release of maternal pituitary and ovarian hormones. The presence of uterine oxytocin (OT) and luteinising hormone (LH) receptors are important for the luteolytic effect of PGF2alpha. Conceptus oestrogen secretion coincides with autocrine and paracrine dialogue between the multiple conceptuses and uterine biological compounds and their receptors in trophoblast and endometrium.
{"title":"Inhibition of luteolysis and embryo-uterine interactions during the peri-implantation period in pigs.","authors":"A. Ziecik, A. Blitek, M. Kaczmarek, A. Waclawik, M. Bogacki","doi":"10.1530/biosciprocs.17.0011","DOIUrl":"https://doi.org/10.1530/biosciprocs.17.0011","url":null,"abstract":"Inhibition of luteolysis and establishment of pregnancy in pigs results from oestrogen secretion by the conceptuses and requires progesterone produced by the corpus luteum (CL). An integral part of maternal recognition of pregnancy in the pig is the redirection of prostaglandin (PG) F2alpha secretion from endocrine (blood) to exocrine (uterus) direction and an increase of PGE2 synthesis in both the endometrium and conceptus. Uterine and conceptus PGE2 synthases play an integrated role in establishing the PGE2:PGF2alpha ratio necessary for luteal maintenance. The luteolytic or luteotrophic changes in the CL are synchronised with the release of maternal pituitary and ovarian hormones. The presence of uterine oxytocin (OT) and luteinising hormone (LH) receptors are important for the luteolytic effect of PGF2alpha. Conceptus oestrogen secretion coincides with autocrine and paracrine dialogue between the multiple conceptuses and uterine biological compounds and their receptors in trophoblast and endometrium.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"62 1","pages":"147-61"},"PeriodicalIF":0.0,"publicationDate":"2019-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49633942","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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