Cryopreservation and its effects on motility and gene expression patterns and fertilizing potential of bovine epididymal sperm

Abstract

Despite encountering new challenges in using epididymal sperm recovered from cauda epididymides, this accessible and, in some species, worthwhile sample makes inevitable the further development of a suitable cryopreservation protocol. In this study, sperm was recovered from the epididymis of 4°C overnight stored slaughtered bulls' testes and the effects of cryopreservation on the bovine epididymal sperm motility (with CASA) and gene expression patterns (with quantitative Real time‐PCR) were evaluated. Moreover the fertilizing potential of cryopreserved epididymal sperm was used in in vitro fertilization (IVF). After freezing and thawing of epididymal sperm, total and slow progressive sperm motility, VCL, VAP, MAD, ALH and BCF were significantly decreased (p < .05), while in the parameters of fast progressive motility, VSL, LIN, WOB and STR there were not any significant variations in the frozen sperm compared to fresh (non‐frozen) counterpart. The assessment of abundance of transcripts encoding motility (TSSK6) and fertility (PRM1 and PRM2)‐related genes in epididymal sperm, showed that these transcripts were affected by freezing especially in slow progressive motility status (p < .01). Furthermore, cleavage and blastocyst rate did not present any significant differences between bovine embryos produced in vitro by fresh or frozen‐thawed epididymal sperm. It can be concluded that epididymal sperm has enough freezability after overnight testes storage, and cryopreservation could not affect the percentage of in vitro produced embryos in spite of the changes of relative abundance of some transcripts and direction progressive motility pattern of sperm.

Molecular Markers: A New Paradigm in the Prediction of Sperm Freezability

“…Nazari et al [ 55 ] studied the impact of cryopreservation on the motility and gene expression of testis-specific serine kinase 6 (TSSK6) and both protamine 1 (PRM1) and 2 (PRM2) in epididymal bovine spermatozoa. As a part of AMP-activated protein kinases, TSSK6 is postmeiotically expressed in spermatids and mature cells, which is associated with a high motility status and late gamete fusion, while PRMs are related to protamine/histone exchange, initiation of transcription, and DNA methylation.…”

“…Due to freezing/thawing, decreased levels of TSSK6, PRM1, and PRM2 encoding transcripts were present in cells with slow progressive motility in comparison to fresh samples where the relative expression was higher. Moreover, the family of TSSKs had the ability to interact with heat shock proteins (HSPs), which worked as a regulator of their levels in the germ cells and provided them protection against protein instability, ubiquitination, and proteasome-dependent degradation [ 55 , 65 , 66 ].…”

Current perspectives on ruminant sperm freezability: Harnessing molecular changes related to semen quality through omics technologies

“…The sperm's contribution to the oocyte during fertilization includes an RNA pool composed of messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), mitochondrial RNAs (mt-RNAs), transfer RNAs (tRNAs), and noncoding RNAs (ncRNAs) including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) (Capra et al, 2017;Card et al, 2013;Sellappan et al, 2017). Nevertheless, successive freeze-thaw cycles result in the differential expressions of spermatozoal transcripts from fresh and frozen-thawed samples, such as the ribosomal protein L31 (RPL31), protein kinase C epsilon (PRKCE), 3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2), proteolipid protein 1 (PLP1), serine/threonine testis-specific protein kinase (TSSK6), protamine 1 (PRM1) and protamine 2 (PRM2) (Chen et al, 2015;Nazari et al, 2020;Shangguan et al, 2020). Expression of mRNAs and miRNAs like PRM1, TSSK6, metalloproteinase non-coding RNA (ADAM5P), cytochrome P450 aromatase (aromP450), and cytochrome oxidase subunit 7C (COX7C) also varied between higher and lower fertility and motility sperm populations (Bissonnette et al, 2009;Capra et al, 2017;Card et al, 2017;Ganguly et al, 2013;Govindaraju et al, 2012;Tiwari et al, 2008), implying RNAs' influence on sperm vitality and over-all quality for later embryogenesis.…”