Testis-Specific Isoform of Na+-K+ ATPase and Regulation of Bull Fertility.

An advanced understanding of sperm function is relevant for evidence-based male fertility prediction and addressing male infertility. A standard breeding soundness evaluation (BSE) merely identifies gross abnormalities in bulls, whereas selection based on single nucleotide polymorphisms and genomic estimated breeding values overlooks sub-microscopic differences in sperm. Molecular tools are important for validating genomic selection and advancing knowledge on the regulation of male fertility at an interdisciplinary level. Therefore, research in this field is now focused on developing a combination of in vitro sperm function tests and identifying biomarkers such as sperm proteins with critical roles in fertility. The Na+-K+ ATPase is a ubiquitous transmembrane protein and its α4 isoform (ATP1A4) is exclusively expressed in germ cells and sperm. Furthermore, ATP1A4 is essential for male fertility, as it interacts with signaling molecules in both raft and non-raft fractions of the sperm plasma membrane to regulate capacitation-associated signaling, hyperactivation, sperm-oocyte interactions, and activation. Interestingly, ATP1A4 activity and expression increase during capacitation, challenging the widely accepted dogma of sperm translational quiescence. This review discusses the literature on the role of ATP1A4 during capacitation and fertilization events and its prospective use in improving male fertility prediction.

Characterization of the Testis-Specific Angiotensin Converting Enzyme (tACE)-Interactome during Bovine Sperm Capacitation.

A comprehensive understanding of molecular and biochemical changes during sperm capacitation is critical to the success of assisted reproductive technologies. We reported involvement of the testis-specific isoform of Angiotensin Converting Enzyme (tACE) in bovine sperm capacitation. The objective of this study was to characterize the tACE interactome in fresh and heparin-capacitated bovine sperm through immunoprecipitation coupled with mass spectrometry. These interactions were validated by co-localization of tACE with beta-tubulin as an identified interactome constituent. Although interactions between tACE and several proteins remained unchanged in fresh and capacitated sperm, mitochondrial aldehyde dehydrogenase 2 (ALDH2), inactive serine/threonine protein-kinase 3 (VRK3), tubulin-beta-4B chain (TUBB4B), and tubulin-alpha-8 chain (TUBA8) were recruited during capacitation, with implications for cytoskeletal and membrane reorganization, vesicle-mediated transport, GTP-binding, and redox regulation. A proposed tACE interactional network with identified interactome constituents was generated. Despite tACE function being integral to capacitation, the relevance of interactions with its binding partners during capacitation and subsequent events leading to fertilization remains to be elucidated.

Testis-specific isoform of angiotensin-converting enzyme (tACE) as a candidate marker for bull fertility.

Although a traditional bull breeding soundness evaluation is designed to identify bulls that are grossly abnormal, bulls classified as satisfactory potential breeders still vary in fertility, implying submicroscopic differences in sperm characteristics. Testis-specific isozyme of angiotensin-converting enzyme (tACE) is involved in the regulation of sperm function. Therefore, the aim of the present study was to determine tACE content, activity and localisation in bull spermatozoa and their associations with fertility. Semen from low-fertility (LF) and high-fertility (HF) Holstein bulls (n=20) with known FERTSOL rates, which represents the 56-day non-return rate, were used. There was greater tACE content (P<0.05) and tACE activity (P<0.01) in HF versus LF spermatozoa. Based on immunolocalisation, tACE was either in the acrosomal or postacrosomal region of the sperm head, with HF bulls having a higher proportion of spermatozoa with tACE in the acrosomal region than LF bulls (P<0.05). tACE content, activity, localisation to the acrosomal region and progressive motility were significantly correlated with fertility and, based on regression analysis, tACE content was predictive of fertility. tACE content and activity in semen were similar between yearling (10-13 months old) and mature (3-4 years old) bulls. Therefore, tACE has potential as a marker of field fertility in bulls at their earliest possible age.

Content and activity of the testis-specific isoform of angiotensin-converting enzyme are reduced in frozen-thawed bull spermatozoa.

Sperm cryopreservation and thawing reduces fertility and alters the content and function of various sperm proteins. Previously, we reported that a testes-specific isoform of angiotensin-converting enzyme (tACE) was required for capacitation of bovine spermatozoa. The aim of the present study was to determine effects of sperm cryopreservation and thawing on the content, activity and localisation of tACE in bovine spermatozoa. Relative median fluorescence intensity (flow cytometry) was greater (P<0.01), tACE content (110 kDa protein) in sperm proteins was higher (P<0.01) and there was greater tACE enzyme activity (mean (±s.e.m.) 0.16±0.01 vs 0.06±0.02UmL-1; P<0.01) in fresh versus frozen-thawed spermatozoa (n=6 bulls). In fresh spermatozoa, tACE was immunolocalised in the acrosomal and principal piece regions of the sperm head and tail respectively. However, in frozen-thawed spermatozoa, there were four patterns of localisation: most frozen-thawed spermatozoa (64%) had fluorescence in the acrosomal ridge, whereas in 17% and 9% of spermatozoa the signal was limited to the post-acrosomal region and the equatorial segment respectively; in the remainder (10%), there was no signal. We conclude that cryopreservation and thawing decrease the content and activity of tACE and cause it to be translocated to other parts of the sperm head.

Testis-specific isoform of angiotensin-converting enzyme (tACE) is involved in the regulation of bovine sperm capacitation.

We hypothesized that the testis-specific isoform of angiotensin-converting enzyme (tACE) is released during bovine sperm capacitation, and its peptidase activity is required for capacitation. Specific objectives of this study were to (i) develop an anti-tACE antibody; (ii) characterize expression of tACE in bovine testes and sperm; and (iii) determine the role of tACE in capacitation. A 110-kDa protein, consistent with the mass of tACE, was detected in sperm extract by our anti-tACE immunoserum. This immunotarget localized at the acrosomal region and principal piece, but was only expressed in testis of mature bulls. When bull sperm were incubated in Sp-TALP (0 and 4 hr) plus 10 µg/ml heparin (capacitation group) or 10 µg/ml heparin + 10 µM captopril (an ACE inhibitor) for 4 hr, the number of acrosome-reacted (40.1 vs. 24.0%, respectively) and hyperactivated (15.0 vs. 9.7%) sperm increased, and tyrosine phosphoprotein content were higher (p < 0.05) for sperm in heparin alone. tACE activity was also higher (0.04 U/ml; p < 0.01) in incubation medium of sperm exposed to heparin compared to 0- and 4-hr incubation controls or heparin + captopril conditions (0, 0.005, and 0.009 U/ml, respectively). Furthermore, capacitation-associated shedding of a portion of tACE into the medium decreased sperm content of the 110-kDa tACE, but concurrently increased the abundance of a 60-kDa tACE variant. Thus, a portion of the extracellular region of tACE (containing its catalytic site) is released from bovine sperm during capacitation, and tACE activity may be required for sperm capacitation.