Xavier Druart

Mammalian epididymal proteome.

In all mammalian species, the final differentiation of the male germ cell occurs in the epididymal duct where the spermatozoa develop the ability to be motile and fertilize an ovum. Understanding of these biological processes is the key to understanding and controlling male fertility. Comparative studies between several mammals could be an informative approach to finding common sperm modifications which are not species-specific. The new global biological approaches such as transcriptomes and proteomes provide considerable information which can be used for such comparative approaches. This report summarizes our proteomic studies of the epididymis of several mammals, including humans.

Proteomic characterization and cross species comparison of mammalian seminal plasma

UNLABELLED Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammalian species. Nucleobindin 1 and RSVP14, a member of the BSP (binder of sperm protein) family, were identified in all species. Beta nerve growth factor (bNGF), previously identified as an ovulation inducing factor in alpacas and llamas, was identified in this study in alpaca and camel (induced ovulators), cattle, sheep and horse (spontaneous ovulators) seminal plasma. These findings indicate that while the mammalian species studied have common ancestry as ungulates, their seminal plasma is divergent in protein composition, which may explain variation in reproductive capacity and function. The identification of major specific proteins within seminal plasma facilitates future investigation of the role of each protein in mammalian reproduction. BIOLOGICAL SIGNIFICANCE This proteomic study is the first study to compare the protein composition of seminal plasma from seven mammalian species including two camelid species. Beta nerve growth factor, previously described as the ovulation inducing factor in camelids is shown to be the major protein in alpaca and camel seminal plasma and also present in small amounts in bull, ram, and horse seminal plasma.

The contribution of proteomics to understanding epididymal maturation of mammalian spermatozoa

The acquisition of the ability of the male gamete to fertilize an ovum is the result of numerous and sequential steps of differentiation of spermatozoa that occur as they transit from the testis to the end of the epididymal tubule. The post gonadal sperm modifications are mostly related to motility, egg binding, and penetration processes. As the activity of the epididymis and its luminal fluid composition are believed to be directly related to ‘sperm maturation’, a review on epididymal proteins is presented. Comparative studies have shown that the epididymal activities are species specific. Nevertheless, for all mammalian species studied, similarities exist in the sequential proteomic changes of the luminal composition of the epididymal tubule and proteins on the sperm surface. The potential roles of these modifications are discussed.

Estimation of genetic parameters and genome scan for 15 semen characteristics traits of Holstein bulls

A QTL detection experiment was performed in French dairy cattle to search for QTL related to male fertility. Ten families, involving a total of 515 bulls, were phenotyped for ejaculated volume and sperm concentration, number of spermatozoa, motility, velocity, percentage of motile spermatozoa after thawing and abnormal spermatozoa. A set of 148 microsatellite markers were used to realize a genome scan. First, genetic parameters were estimated for all traits. Semen production traits were found to have moderate heritabilities (from 0.15 to 0.30) while some of the semen quality traits such as motility had high heritabilities (close to 0.60). Genetic correlations among traits showed negative relationships between volume and concentration and between volume and most quality traits such as motility or abnormal sperm while correlations between concentration and these traits were rather favourable. Percentages of abnormal sperm were negatively related to quality traits, especially with motility and velocity of spermatozoa. Three QTL related to abnormal sperm frequencies were significant at p < 0.01. In total, 11 QTL (p < 0.05) were detected. However, the number of QTL detected was within the range of expected false positives. Because of the lack of power to find QTL in this design further analyses are required to confirm these QTL.

In vivo imaging of in situ motility of fresh and liquid stored ram spermatozoa in the ewe genital tract

The fertility of ram semen after cervical insemination is substantially reduced by 24 h of storage in liquid form. The effects of liquid storage on the transit of ram spermatozoa in the ewe genital tract was investigated using a new procedure allowing direct observation of the spermatozoa in the genital tract. Ejaculated ram spermatozoa were double labeled with R18 and MitoTracker Green FM, and used to inseminate ewes in estrus either cervically through the vagina or laparoscopically into the base of the uterine horns. Four hours after insemination, the spermatozoa were directly observed in situ using fibered confocal fluorescence microscopy in the base, middle and tip of the uterine horns, the utero-tubal junction (UTJ) and the oviduct. The high resolution video images obtained with this technique allowed determination of the distribution of spermatozoa and individual motility in the lumen of the ewes genital tract. The results showed a gradient of increasing concentration of spermatozoa from the base of the uterus to the UTJ 4 h after intra-uterine insemination into the base of the horns. The UTJ was shown to be a storage region for spermatozoa before their transfer to the oviduct. The in vitro storage of spermatozoa in liquid form decreased their migration through the cervix and reduced the proportion of motile spermatozoa and their straight line velocity at the UTJ and their transit into the oviduct.

Hypotonic resistance of boar spermatozoa: sperm subpopulations and relationship with epididymal maturation and fertility.

Hypotonic resistance of boar spermatozoa was investigated by measuring the ratio of live/dead spermatozoa (SYBR-14/propidium iodide) by flow cytometry after hypotonic stress. The survival rate of ejaculated spermatozoa incubated in hypotonic solutions ranging from 3 to 330 mmol/kg followed a sigmoid curve that fitted a simple logistic model. The critical osmolality value (Osm(crit)) at which 50% of spermatozoa died was determined with this model. Hypotonic resistance of spermatozoa increased with temperature between 15 and 39 degrees C and decreased after hydrogen superoxide treatment, but was not modified during 8 days of preservation in Beltsville thawing solution. Hypotonic resistance markedly decreased during epididymal maturation and after ejaculation as Osm(crit) at 15 degrees C was 54.7+/-3.2, 68.5+/-10.6, 116.7+/-2.1 and 194.3+/-3.7 mmol/kg for the caput, corpus, cauda and ejaculated spermatozoa respectively. Hypo-osmotic stress of 100 mmol/kg revealed a sperm subpopulation exhibiting increased hypotonic resistance compared with the whole ejaculate (Osm(crit)=67.8+/-2.1 mmol/kg). Consistent differences were observed between lean and standard breeds (Pietrain versus Large White) and between boars within the same breed. According to data collected by artificial insemination centers during a large-scale field trial, hypotonic resistance of ejaculates was found to be positively correlated with in vivo fertility.

Ram seminal plasma proteome and its impact on liquid preservation of spermatozoa

UNLABELLED Seminal plasma is composed of secretions from the epididymis and the accessory sex glands and plays a critical role in the fertilising ability of spermatozoa. In rams, analysis of seminal plasma by GeLC-MS/MS has allowed the identification of more than 700 proteins, including a high abundance of Binder of Sperm family proteins (BSP1, BSP5, SPADH1, SPADH2), the spermadhesin family (bodhesin2), lactoferrin and newly identified proteins like UPF0762 (C6orf58 gene). When spermatogenesis was stopped by scrotal insulation, changes in the proteome profile revealed the sperm origin of 40 seminal proteins, such as glycolysis pathway enzymes, the chaperonin containing TCP1 (CCT) complex and the 26S proteasome complex. Sperm mobility after liquid preservation (24h in milk at 15°C) is male dependent and can be correlated to differences in the seminal plasma proteome, detected by spectral counting. The negative association of zinc alpha-2 glycoprotein (ZAG) with semen preservation was confirmed by the use of recombinant human ZAG, which induced an increase in mobility of fresh sperm, but then decreased sperm mobility after 24h of incubation. Several sperm membrane proteins interacting with the cytoskeleton, glycolysis enzymes and sperm-associated proteins involved in capacitation correlated with better liquid storage and can be considered as seminal biomarkers of sperm preservation. BIOLOGICAL SIGNIFICANCE Extensive analysis of the ram seminal plasma proteome reveals a complex and diverse protein composition. This composition varies between males with different sperm preservation abilities. Several proteins were shown to originate from the spermatozoa and positively correlate with sperm liquid preservation, indicating that these proteins can be traced as sperm biomarkers within the seminal plasma. The zinc alpha-2 glycoprotein (ZAG) was found to have a biphasic effect on sperm mobility, with a short-term stimulation followed by a long-term exhaustion of sperm mobility after a 24h preservation period.

Seminal plasma aids the survival and cervical transit of epididymal ram spermatozoa

Seminal plasma purportedly plays a critical role in reproduction, but epididymal spermatozoa are capable of fertilisation following deposition in the uterus, calling into question the biological requirement of this substance. Through a combination of direct observation of spermatozoa in utero using probe-based Confocal Laser Endomicroscopy, in vivo assessment of sperm fertility and in vitro analysis of various sperm functional parameters, this study investigated the role of seminal plasma in spermatozoa transit through the cervix of the ewe. Following deposition in the cervical os, epididymal spermatozoa previously exposed to seminal plasma displayed an enhanced ability to traverse the cervix as evidenced by both significantly higher pregnancy rates and numbers of spermatozoa observed at the utero-tubal junction when compared with epididymal spermatozoa not previously exposed to seminal plasma. The beneficial effect of seminal plasma on sperm transport was clearly localised to transit through the cervix as pregnancy rates of spermatozoa deposited directly into the uterus were unaffected by exposure to seminal plasma. This phenomenon was not explained by changes to sperm motion characteristics, as seminal plasma had no effect on the motility, kinematic parameters or mitochondrial membrane potential of spermatozoa. Rather, in vitro testing revealed that seminal plasma improved the ability of epididymal spermatozoa to penetrate cervical mucus recovered from ewes in oestrus. These results demonstrate that the survival and transport of ram spermatozoa through the cervix of the ewe is not linked to their motility or velocity but rather the presence of some cervical penetration trait conferred by exposure to seminal plasma.

Biochemical Characterization of Two Ram Cauda Epididymal Maturation-Dependent Sperm Glycoproteins

Abstract Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-β-d-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.

Proteomes of the Female Genital Tract During the Oestrous Cycle

The female genital tract includes several anatomical regions whose luminal fluids successively interact with gametes and embryos and are involved in the fertilisation and development processes. The luminal fluids from the inner cervix, the uterus and the oviduct were collected along the oestrous cycle at oestrus (Day 0 of the cycle) and during the luteal phase (Day 10) from adult cyclic ewes. The proteomes were assessed by GeLC-MS/MS and quantified by spectral counting. A set of 940 proteins were identified including 291 proteins differentially present along the cycle in one or several regions. The global analysis of the fluid proteomes revealed a general pattern of endocrine regulation of the tract, with the cervix and the oviduct showing an increased differential proteins abundance mainly at oestrus while the uterus showed an increased abundance mainly during the luteal phase. The proteins more abundant at oestrus included several families such as the heat shock proteins (HSP), the mucins, the complement cascade proteins and several redox enzymes. Other proteins known for their interaction with gametes such as oviductin (OVGP), osteopontin, HSPA8, and the spermadhesin AWN were also overexpressed at oestrus. The proteins more abundant during the luteal phase were associated with the immune system such as ceruloplasmin, lactoferrin, DMBT1, or PIGR, and also with tissue remodeling such as galectin 3 binding protein, alkaline phosphatase, CD9, or fibulin. Several proteins differentially abundant between estrus and the luteal phase, such as myosin 9 and fibronectin, were also validated by immunohistochemistry. The potential roles in sperm transit and uterine receptivity of the proteins differentially regulated along the cycle in the female genital tract are discussed.