Human immature germ cells and ejaculated spermatozoa contain aromatase and oestrogen receptors

Abstract

It is now well established that oestrogens play a part in germ cell function. These hormones are synthesised by the cytochrome P450 aromatase (P450 arom) and act via two kinds of receptor (ER and ER ). Although the presence of aromatase and oestrogen receptors in mammalian testis is now well documented, the localisation of these proteins in human germ cells is not yet clear. The primary purpose of the current study was to look for the expression of aromatase and oestrogen receptors in human germ cells. Human immature germ cells were collected from semen samples with an excess of rounds cells (>20%) and purified spermatozoa were obtained after sedimentation on a discontinuous PureSperm gradient. Expression of aromatase and oestrogen receptors was determined by RT-PCR with specific primers, and by Western blot using monoclonal antibodies. RT-PCR products for aromatase, ER and ER were amplified from total RNA isolated from human germ cells and spermatozoa. We identified an ER isoform variant that lacks exon 4 in human germ cells and visualised P450 arom as a single band of 49 kDa in germ cells, as we have already reported for human ejaculated spermatozoa. By Western blot, we identified two proteins for ER at ∼ 50 and ∼ 60 kDa, which could correspond to the long and short forms of ER formed from the use of alternative start sites. In human ejaculated spermatozoa, ER protein was not detected, even though we could amplify mRNA. Using Western blot analysis and a monoclonal antibody specific for ER , we detected two proteins in human immature germ cells: one of the expected size (66 kDa) and a second one of 46 kDa. In mature spermatozoa, only the 46 kDa band was observed and we speculate it may be related to the ER isoform lacking exon I. In conclusion, we have identified P450 arom and ER proteins (full-length and variant) in human germ cells. Further studies are now required to elucidate the mechanism of action of oestrogens on human male germ cells, in terms of both genomic and 'non-genomic' pathways.

Presence, localization, and origin of clusterin in normal human spermatozoa

“…This sperm separation technique could effectively acquire sperm populations with high motility and normal morphology free of seminal plasma, debris, microbial contamination, and residual cells (immature germ cells or polynuclear cells). This was confirmed by our microscopy inspection and has been shown in previous reports [16,17,19,21,22]. Sperm motility was monitored Fig.…”

“…Routine semen analysis monitored by a computer-assisted semen analyzer (IVOS; Hamilton-Thorne, Beverly, MA, U.S.) revealed that spermatozoa were within the normal range stipulated by WHO1999 guidelines. To separate high motile and morphologically normal spermatozoa from semen, the liquefied ejaculate was washed on a two-layer (90 % and 45 %) discontinuous Percoll (GE Healthcare; Piscataway, NJ, U.S.) gradient, a modified protocol described previously [16,17,19]. After centrifugation at 400 g for 18 min, the cell pellets from the 90 % layer were washed twice in Earle's balanced salts (Sigma; St. Louis, MO, U.S.).…”

Analysis and difference of voltage-dependent anion channel mRNA in ejaculated spermatozoa from normozoospermic fertile donors and infertile patients with idiopathic asthenozoospermia

“…For removing the round cells (immature germ cells and leukocytes) and debris, and collecting high motile and low motile spermatozoa respectively from normal and asthenozoospermic semen, the liquefied semen samples were washed in a discontinuous Percoll (GE Healthcare, USA) gradient consisting of four successive layers (90%, 76%, 57% and 45%), which was a modification of the protocol described previously [24][25][26]. After centrifugation at 300 g for 20 min at room temperature, high motile spermatozoa in donors' semen and low motile spermatozoa in patients' semen were separated respectively from the 90% layer and from the interface 76%-57%.…”

“…In order to confirm the separated sperm fractions free from round cells, the c-kit (as the marker of immature germ cells) and CD45 (as the marker of leukocytes) were amplified from cDNA samples as previously reported [25,26]. GAPDH was amplified as an internal control.…”

“…Negative controls (ddH 2 O as the template) were included. PCR cycle profiles were performed according to the previous protocols [25,26] (see Table 1). The PCR products were analyzed on a 2.5% agarose gel stained with ethidium bromide and visualized under UV transillumination.…”