Deciphering the voids in determining male infertility
Male fertility is one of the crucial factors required for the continuity of the particular species. The sire has an impact not just on fertilization but also on embryo growth and conception. Hence it is important to study the various factors associated with male ejaculate to determine the causes associated with infertility. To decipher the cause of male infertility the spermatozoa present in the ejaculate is given the prime importance while disregarding the other contents of the ejaculate. Spermatozoa being the carrier of the genetic information is crucial, however the other contents of the ejaculate play important role in maintaining vital functions of spermatozoa. The male ejaculate is a rich source of seminal plasma derived EVs and studies have shown that these EVs impacts spermatozoa positively. During artificial insemination (AI) in bovine the whole ejaculate including EVs are deposited in maternal tract. Hence apart from spermatozoa the EVs also contribute to the genome by delivering cargo in form of nucleic acids (miRNA, DNA, proteins) to the female reproductive tract. This project is set to study the differences in RNA derived from spermatozoa versus EVs isolated from cryopreserved semen straw and seminal plasma from fresh ejaculates. In order to study the effects of freezing, this project will also investigate the differences in the number and EVs miRNAs derived from seminal plasma that were prepared from fresh ejaculates versus EVs in cryopreserved semen straw. Apart from being used in AI, the bovine spermatozoa is also utilized to produce in vitro (IVP) embryos. Hence, this project is also aimed to study the relationship between spermatozoa quality traits and spermatozoa ability to promote oocyte in vitro fertilization (IVF) and embryo development, as well as the male effect on expression of developmentally important genes in embryos. As a novel aspect, we will characterize EV from the seminal plasma in relation to fertility and identify whether addition of EVs from the highly fertile bull has an effect on IVP outcome when using bull with low IVP fertility. The findings of this project are readily applicable to different aspects of andrology and can help devising new methods of diagnosis and treatment of male infertility in human.
Project background
According to a number of studies, abnormalities in spermatozoa owing to insufficient numbers (azoospermia/oligospermia), poor motility, and abnormal morphology are the most common reasons of male infertility. In addition, male infertility has been defined as not very responsive to the primary treatments. According to various reports, only a very small percentage of cases with male factor infertility can be cured using the conventional treatments available. Male factor infertility is often treated using secondary methods, such as artificial insemination (AI), intra-uterine insemination (IUI), in vitro fertilization (IVF) and embryo transfer, intra-cytoplasmic sperm transfer. In cattle, AI has been used to increase the rate of successful reproduction and desirable genetic traits in farm animals, and it has had a tremendous global influence on the development of many species, especially dairy cattle. The cryopreserved semen used to inseminate the cow contains spermatozoa, cryodiluent (to retain the fertilizing ability of the spermatozoa) and seminal plasma. Multiple reports suggests that seminal plasma contains an enormous number of EVs, a class of small sized heterogeneous cell-derived nano scaled vesicles playing an important role in cell-cell communications. EVs have emerged as important agents of intercellular communication by the transferring biomolecules (i.e., mRNA fragments, miRNAs, DNA, proteins) to recipient cells along with modulating its activity. Although, a plethora of studies have reported regarding EVs but their role in AI-based technology is not evolved. Seminal plasma EVs have been shown to be vital for maintaining sperm viability, motility as well as fertility. A study has also revealed that seminal EVs could infiltrate the sperm membrane and addition of 4-16 times EVs to the storage diluent compared to the control, had better plasma membrane integrity, higher survival as well as motile sperms. This study indicated that EVs help the sperms to remain viable under long durations of cryopeservation or storage. Addition of external EVs to the storage liquid also improved the sperm’s total-antioxidant capacity along with decreased rate of pre-mature capacitation of the stored spermatozoa.
In addition to chromosomal DNA, the spermatozoon contributes structural components, transcription factors, and many RNA species to the zygote. The genomic contribution of EVs derived from the contents of semen straw (except spermatozoa) and the impact of these EVs in IVF have not been studied thoroughly and remains unexplored. The variations in the cargo of EVs derived from seminal plasma of low performing (LP) and high performing (HP) bull is also not very well studied. Multiple variables, which includes spermatozoa fertilizing capacity, embryo and placenta development, contribute to the lower ability of embryos from low fertility bulls to establish pregnancy.
While accessing the male fertility, only the parameters of spermatozoa are being analyzed, and the other contents present in the male ejaculate are ignored. Male ejaculate has a high concentration of EVs that enhance sperm function in a variety of ways, including enhancing sperm motility, controlling acrosome activity. Exosomes may impact sperm quality and enter sperm, hence improving sperm motility and capacitation following ejaculation, as shown by related investigations. Exosomes in seminal plasma have been found to affect sperm motility and mitochondrial metabolism in mammals. In male reproductive tract, the epididymis plays a crucial role in spermatozoa maturation which relies heavily on interactions with epididymal epithelial secretions. The epididymal and prostate exosomes are closely associated with the final phases involved with the maturation of spermatozoa. Therefore, it is reasonable to think that EVs analysis may be used to assess a patient's semen quality or that EVs can be used in the treatment of male infertility. Furthermore, a study conducted in bovine revealed that 65 genes were upregulated in embryos generated derived from high fertility bulls and 33 genes were upregulated in embryos derived from bulls with low fertility. Recent research indicates that EVs from fertile males have an effect on the sperm viability of non-fertile male chickens. EVs have the potential to find noninvasive biomarkers and create innovative treatments for enhancing reproductive success. In conclusion, producing embryos in vitro has become a financially feasible method. However, there is still room for improvement, not just by optimizing the cell growth environment, but also by gaining a better knowledge of spermatozoa characteristics, sperm and seminal biomarkers associated with embryo development.
According to a number of studies, abnormalities in spermatozoa owing to insufficient numbers (azoospermia/oligospermia), poor motility, and abnormal morphology are the most common reasons of male infertility. In addition, male infertility has been defined as not very responsive to the primary treatments. According to various reports, only a very small percentage of cases with male factor infertility can be cured using the conventional treatments available. Male factor infertility is often treated using secondary methods, such as artificial insemination (AI), intra-uterine insemination (IUI), in vitro fertilization (IVF) and embryo transfer, intra-cytoplasmic sperm transfer. In cattle, AI has been used to increase the rate of successful reproduction and desirable genetic traits in farm animals, and it has had a tremendous global influence on the development of many species, especially dairy cattle. The cryopreserved semen used to inseminate the cow contains spermatozoa, cryodiluent (to retain the fertilizing ability of the spermatozoa) and seminal plasma. Multiple reports suggests that seminal plasma contains an enormous number of EVs, a class of small sized heterogeneous cell-derived nano scaled vesicles playing an important role in cell-cell communications. EVs have emerged as important agents of intercellular communication by the transferring biomolecules (i.e., mRNA fragments, miRNAs, DNA, proteins) to recipient cells along with modulating its activity. Although, a plethora of studies have reported regarding EVs but their role in AI-based technology is not evolved. Seminal plasma EVs have been shown to be vital for maintaining sperm viability, motility as well as fertility. A study has also revealed that seminal EVs could infiltrate the sperm membrane and addition of 4-16 times EVs to the storage diluent compared to the control, had better plasma membrane integrity, higher survival as well as motile sperms. This study indicated that EVs help the sperms to remain viable under long durations of cryopeservation or storage. Addition of external EVs to the storage liquid also improved the sperm’s total-antioxidant capacity along with decreased rate of pre-mature capacitation of the stored spermatozoa.
In addition to chromosomal DNA, the spermatozoon contributes structural components, transcription factors, and many RNA species to the zygote. The genomic contribution of EVs derived from the contents of semen straw (except spermatozoa) and the impact of these EVs in IVF have not been studied thoroughly and remains unexplored. The variations in the cargo of EVs derived from seminal plasma of low performing (LP) and high performing (HP) bull is also not very well studied. Multiple variables, which includes spermatozoa fertilizing capacity, embryo and placenta development, contribute to the lower ability of embryos from low fertility bulls to establish pregnancy.
While accessing the male fertility, only the parameters of spermatozoa are being analyzed, and the other contents present in the male ejaculate are ignored. Male ejaculate has a high concentration of EVs that enhance sperm function in a variety of ways, including enhancing sperm motility, controlling acrosome activity. Exosomes may impact sperm quality and enter sperm, hence improving sperm motility and capacitation following ejaculation, as shown by related investigations. Exosomes in seminal plasma have been found to affect sperm motility and mitochondrial metabolism in mammals. In male reproductive tract, the epididymis plays a crucial role in spermatozoa maturation which relies heavily on interactions with epididymal epithelial secretions. The epididymal and prostate exosomes are closely associated with the final phases involved with the maturation of spermatozoa. Therefore, it is reasonable to think that EVs analysis may be used to assess a patient's semen quality or that EVs can be used in the treatment of male infertility. Furthermore, a study conducted in bovine revealed that 65 genes were upregulated in embryos generated derived from high fertility bulls and 33 genes were upregulated in embryos derived from bulls with low fertility. Recent research indicates that EVs from fertile males have an effect on the sperm viability of non-fertile male chickens. EVs have the potential to find noninvasive biomarkers and create innovative treatments for enhancing reproductive success. In conclusion, producing embryos in vitro has become a financially feasible method. However, there is still room for improvement, not just by optimizing the cell growth environment, but also by gaining a better knowledge of spermatozoa characteristics, sperm and seminal biomarkers associated with embryo development.
Project Objectives
- To quantify the number of EVs and amount of RNA contributed by a cryopreserved semen straw during artificial insemination in bovine.
- To study the differences in the EV cargo of seminal plasma versus the EVs present in the cryopreserved semen straw.
- To characterize the EVs derived from the seminal plasma of HP and LP bulls.
- To identify whether addition of EVs from the HP bull during sperm preparation or IVF has an effect on in vitro production (IVP) outcome when using spermatozoa from LP bulls.
- To differentiate between LP and HP bulls in in vitro embryo production system by comparing the embryo yields, quality and expression of developmentally important genes in blastocysts.
Integration with EMU expertise, research strategy and development
Long roots of reproductive research existed in EMU. The history of the Department of Reproductive Biology dates back to 1956 when the Laboratory of Artificial Insemination was founded as one of the seven subunits established at the Estonian Research Institute of Animal Breeding and Veterinary Science by a decree of the Ministry of Agriculture of the Estonian SSR. Currently The Chair of Animal Breeding and Biotechnology is Estonia’s only competence centre in its field. It offers learning opportunities at all levels of higher education. Basic and applied research involving breeding of livestock, preservation of animal genetic resources, animal genetics, genomics, transgenic technology, reproduction and reproductive disorders, and reproduction biotechnology.
Understanding the Causes of male infertility in human as well as livestock is an important comparative medicine question. There is excellent opportunities for anthologists to collaborate and cooperate to help find new diagnostics as well as therapies in this field. Treating infertility and subfertility in animals is a very important aspect of comparative medicine and one health approach too. The current research project is well in line with aims of the Combivet project to support research in human and animal based common diseases as well as understanding the mechanism of disease to help Human and animals. All these points are very important in upgrading the quality of research currently performed in EMU.
Long roots of reproductive research existed in EMU. The history of the Department of Reproductive Biology dates back to 1956 when the Laboratory of Artificial Insemination was founded as one of the seven subunits established at the Estonian Research Institute of Animal Breeding and Veterinary Science by a decree of the Ministry of Agriculture of the Estonian SSR. Currently The Chair of Animal Breeding and Biotechnology is Estonia’s only competence centre in its field. It offers learning opportunities at all levels of higher education. Basic and applied research involving breeding of livestock, preservation of animal genetic resources, animal genetics, genomics, transgenic technology, reproduction and reproductive disorders, and reproduction biotechnology.
Understanding the Causes of male infertility in human as well as livestock is an important comparative medicine question. There is excellent opportunities for anthologists to collaborate and cooperate to help find new diagnostics as well as therapies in this field. Treating infertility and subfertility in animals is a very important aspect of comparative medicine and one health approach too. The current research project is well in line with aims of the Combivet project to support research in human and animal based common diseases as well as understanding the mechanism of disease to help Human and animals. All these points are very important in upgrading the quality of research currently performed in EMU.
Project team
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Qurat Ul Ain Reshi
Institute of Veterinary Medicine and Animal Sciences Investigator |
Alireza Fazeli
Institute of Veterinary Medicine and Animal Sciences Supervisor |
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Mohammad Mehedi Hasan
Institute of Veterinary Medicine and Animal Sciences Investigator |
Kasun Godakumara
Institute of Veterinary Medicine and Animal Sciences Supervisor |
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Anni Viljaste
Institute of Veterinary Medicine and Animal Sciences Supervisor |
Ülle Jaakma
Institute of Veterinary Medicine and Animal Sciences Supervisor |
