Protein fraction isolated from epididymal fluid re‐associates sperm in vitro: Possible role of serpins in rat rosettes assembly

Abstract

In many mammalian species, sperm associate as a consequence of the epididymal transit. From the classic Rouleaux in guinea pig to the most recent work in mouse and echidna, authors have focused mainly on a detailed morphological description of this phenomenon. Some of these articles have also begun to describe the nature of the material present between sperm heads. Here, we try to better understand the factor/s involved in rat sperm association (Rosette). Based on previous work describing the appearance of Rosettes in the distal segments of the rat epididymis, we consider that sperm during their transit must be in contact with factor/s present in the caudal lumen in order to associate with each other. By an in vitro sperm re-associating assay, we try to determine the in vivo phenomenon observed in the lumen. The assay consists of co-incubating non-associated sperm with several protein fractions obtained from epididymal caudal fluid. After establishing the most active fraction, the proteins were characterized by MALDI-TOF mass spectrometry. Among the proteins we found two members of the serine protease inhibitors family; an alpha-1 antitrypsin and a new protein with an alpha-1 antitrypsin like domain which includes a sequence compatible with the serpins' reactive center loop. These serpins may play a role in the assembly/disassembly process of Rosettes by modulating lumenal protease activity. Finally, a biochemical-morphological model which explains the sperm-proteases interaction was proposed.

Sperm conjugation in mammal reproductive function: Different names for the same phenomenon?

“…The fractions that promoted in vitro sperm re‐association into rosettes contained proteins with molecular weights ranging from 40 to 80 kDa; two of the identified proteins, alpha‐1 antitrypsin and Serpin 1F, a protein with an alpha‐1 antitrypsin‐like domain, are members of the serine protease inhibitor family. The Serpin 1F gene is expressed in the epididymis, prostate, and seminal vesicles, but not in the testis (Monclus et al, ), and was sensitive to flutamide administration (unpublished data). These serpins were hypothesized to participate in the assembly/disassembly of rosettes by modulating luminal protease activity (Cesari et al, ; Monclus et al, ).…”

“…Many similarities between mouse and rat rosettes were observed, particularly in their morphology and their presence at the distal regions of the epididymal lumen. While analyzing the mechanism of rosette assembly in vitro (see below) using sperm from the Wistar rat ( R. norvegicus ), we could not determine if rosettes move faster than isolated sperm (Monclus et al, ); therefore, we could not establish if sperm conjugation provided an advantage in sperm competition. We instead proposed that sperm rosettes play a protective role that prevents a premature acrosome reaction during epididymal sperm storage (Monclus et al, ).…”

“…An in vitro sperm re‐association assay was performed to study the mechanism of sperm rosette arrangement in the rat (Monclus et al, ). The most motile fraction of individual caudal epididymis sperm, based on swim‐up selection, were incubated with several protein fractions obtained from the epididymal cauda fluid.…”

“…Immler () summarized different forms of sperm cooperation, and indicated which sperm form pairs among rodents and American marsupials. Higginson and Pitnick () also provided a table summarizing examples of mammalian sperm conjugation, such as “rouleaux,” “pairs,” “bundles,” and “trains.” Despite the update, however, Higginson and Pitnick were not comprehensive, omitting or overlooking examples such as from flying squirrels (Martan and Hruban, ), primates (Phillips and Bedford, ), and rodents (Fornes and Burgos, , ; Monclus et al, ; Monclus et al, ).…”

Oviduct binding ability of porcine spermatozoa develops in the epididymis and can be advanced by incubation with caudal fluid

“…These include different secretory proteins either native to caudal fluid or transported from proximal regions and accumulated in this fluid [12,[16][17][18], enzymes [19], Ca 2+ concentrations and signalling mechanisms [20], sperm association or formation [21], and chemical characteristics of the fluid itself [22]. Given the above factors and the amount of time that spermatozoa spend in the cauda prior to ejaculation, it is logical to assume that the cauda and caudal fluid may play a significant role in developing the ability of spermatozoa to bind to oviduct epithelium.…”