The thyroid-stimulating hormone receptor (TSHR), activated by either TSH or the newly discovered glycoprotein hormone thyrostimulin, plays a central role in the control of body metabolism. Interestingly, in addition to its thyroid expression, we discovered that the mRNA level of TSHR is periodically regulated in rat ovary by gonadotropins. Ovarian microdissection followed by real-time PCR analysis indicated that granulosa cells show the highest level of TSHR expression. Cultures of follicles and primary granulosa cells demonstrated that the level of TSHR is up-regulated and decreased by the gonadotropindriven cAMP cascade and estradiol production, respectively. Furthermore, in contrast to the negligible expression of TSH in the ovary, we also found by real-time PCR and immunohistochemical analysis that thyrostimulin is expressed mainly in oocytes. Evolving before the appearance of gonadotropins, thyrostimulin is considered the most ancestral glycoprotein hormone. Therefore, the presence of thyrostimulin in the ovary suggests that it may have a primitive function in reproduction when it activates ovarian TSHR. Next, we generated recombinant thyrostimulin protein and characterized its non-covalent heterodimeric nature. Using purified recombinant thyrostimulin, we show that the human ovarian cell line NIH:OVCAR-3 also expresses endogenous and functional TSHR. Using cultured rat granulosa cells isolated from different ovarian stages, we found that treatments with thyrostimulin significantly increase cAMP production and the c-fos gene response in the presence of gonadotropins. Thus, this study demonstrates that oocyte-derived thyrostimulin and granulosa cell-expressed TSHR compose a novel paracrine system in the ovary, where the activity is tightly controlled by gonadotropins.

SERPINE2, one of the potent serine protease inhibitors that modulates the activity of plasminogen activator and thrombin, is implicated in many biological processes. In the present study, we purified SERPINE2 from mouse seminal vesicle secretion (SVS), using liquid chromatography and identified it by liquid chromatography/tandem mass spectrometry, and it showed potent inhibitory activity against the urokinase-type plasminogen activator. SERPINE2 was expressed predominantly in seminal vesicles among murine male reproductive tissues. It was immunolocalized to the SVS and mucosal epithelium of the seminal vesicle, epididymis, coagulating gland, and vas deferens. In the testes, SERPINE2 was immunostained in spermatogonia, spermatocytes, spermatids, Leydig cells, and spermatozoa. SERPINE2 was also detected on the acrosomal cap of testicular and epididymal sperm and was suggested to be an intrinsic sperm surface protein. The purified SERPINE2 protein could bind to epididymal sperm. A prominent amount of SERPINE2 was detected on ejaculated and oviductal spermatozoa. Nevertheless, SERPINE2 was detected predominantly on uncapacitated sperm, indicating that SERPINE2 is lost before initiation of the capacitation process. Moreover, SERPINE2 could inhibit in vitro bovine serum albumin-induced sperm capacitation and prevent sperm binding to the egg, thus blocking fertilization. It acts through preventing cholesterol efflux, one of the initiation events of capacitation, from the sperm. These findings suggest that the SERPINE2 protein may play a role as a sperm decapacitation factor.

We report a secreted serine protease inhibitor Kazal-type-like (SPINKL) protein. The SPINKL protein was purified from mouse seminal vesicle secretions through a series of steps, including ion-exchange chromatography on a diethylaminoethyl-Sephacel column, gel filtration on a Sephadex G-75 column, and ion-exchange HPLC on a Q strong anion exchange column. Further analysis identified several SPINKL proteins with various N-linked carbohydrates. The SPINKL protein has six conserved cysteine residues that are nearly identical to those of members of the SPINK protein family. It was noted that the SPINKL protein showed no inhibitory activities against common serine proteases such as trypsin, chymotrypsin, subtilisin, or elastase. Spinkl mRNA and SPINKL proteins were found to be primarily expressed in seminal vesicles. Immunohistochemistry revealed that the SPINKL protein occurred in the luminal fluid and mucosal epithelium of the seminal vesicles and was regulated by testosterone. The SPINKL protein was able to bind onto sperm and enhance sperm motility. Also, it was able to suppress BSAstimulated sperm capacitation and block sperm-oocyte interactions in vitro, suggesting that SPINKL may be a decapacitation factor.

The Ly-6 protein family refers to a group of glycophosphatidyl inositol-anchored membrane proteins with ten conserved cysteines. They are thought to be involved in cellular adhesion and signaling. Recently, a subfamily of secreted Ly-6 proteins has been identified. In the present study, we report a secreted Ly-6 protein, secreted seminal vesicle Ly-6 protein 1 (SSLP-1) purified from mouse seminal vesicles using a series of steps including ion-exchange chromatography on a diethylaminoethyl (DEAE)-Sephacel column, gel filtration on a Sephadex G-75 column, and ion-exchange HPLC on a sulfopropyl column. Further analysis demonstrated it to be a novel, previously unnamed, 17 kDa glycoprotein. N-glycosidase F treatment revealed a core protein with a molecular mass of 8720 Da. By Basic Local Alignment Search Tool Protein analysis, we found that SSLP-1 had ten conserved cysteine residues identical with other secreted Ly-6 proteins. The gene Gm191, which is located on chromosome 9, encodes SSLP-1. By Northern blotting with 21 different mouse tissues, we found that Sslp-1 mRNA was predominantly expressed in the seminal vesicle. Immunohistochemistry revealed SSLP-1 protein in the luminal fluid and mucosal epithelium of the seminal vesicles. The amount of Sslp-1 mRNA and SSLP-1 protein in the seminal vesicle was regulated by testosterone and correlated with the stage of animal maturation. The tissue-specific expression pattern suggests that SSLP-1 may play a physiological role in male mouse reproduction.

Several miRNAs are expressed in human gestational tissue, and some have been shown to be associated with placental dysfunction and complicated pregnancy outcomes. To investigate the roles of miR-346 and miR-582-3p in adverse obstetric events, we analyzed these 2 miRNAs in three samples (maternal blood, umbilical cord blood and placenta) obtained from pregnant women in four groups, including healthy control (n = 60), preeclampsia (n = 31), preterm delivery (n = 29) and small for gestational age (n = 19) patients. The expression levels of miR-346 and miR-582-3p in all included adverse obstetric outcome groups were significantly higher in the maternal plasma samples but lower in the placenta samples (all p value < 0.05). In addition, the miR-346 expression levels in fetal cord blood were also significantly lower in all of the included adverse obstetric outcome groups (all p < 0.05). Multivariate analysis of the three specimens after adjusting for maternal age and gestational age at delivery gave the same results. In conclusion, aberrant miR-346 and miR-582-3p expression level in pregnancy was associated with multiple maternal and fetal complications. Their differential expression in maternal blood, umbilical cord blood and placenta could be potential biomarkers or therapeutic targets for adverse obstetric outcomes

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Lysozyme (LYZ) c‐like proteins are primarily present in the testis and epididymis of male reproductive tissues. Here, we report a novel member of the c‐type LYZ family, the seminal vesicle‐secreted LYZ c‐like protein (SVLLP). Three forms of SVLLP were purified from mouse seminal vesicle secretions and characterized as glycoproteins with the same protein core but different N‐linked glycans. SVLLP is structurally similar to c‐type LYZ proteins. Only one of the 20 invariant residues was altered in the consensus sequence of c‐type LYZs; however, the changed residue (N53S) is one of two essential catalytic residues. LYZ activity assays demonstrated that the three glycoforms of SVLLP lacked enzyme activity. SVLLP is primarily expressed in seminal vesicles. Immunohistochemistry revealed that it occurs in the luminal fluid and mucosal epithelium of the seminal vesicles. Testosterone is not the primary regulator for its expression in the seminal vesicle. SVLLP binds to sperm and suppresses bovine serum albumin‐induced sperm capacitation, inhibits the acrosome reaction, and blocks sperm–oocyte interactions in vitro, suggesting that SVLLP is a sperm capacitation inhibitor.

BackgroundTotal motile sperm count (TMSC) and curvilinear velocity (VCL) are two important parameters in preliminary semen analysis for male infertility. Traditionally, both parameters are evaluated manually by embryologists or automatically using an expensive computer-assisted sperm analysis (CASA) instrument. The latter applies a point-tracking method using an image processing technique to detect, recognize and classify each of the target objects, individually, which is complicated. However, as semen is dense, manual counting is exhausting while CASA suffers from severe overlapping and heavy computation.MethodsWe proposed a simple frame-differencing method that tracks motile sperms collectively and treats their overlapping with a statistical occupation probability without heavy computation. The proposed method leads to an overall image of all of the differential footprint trajectories (DFTs) of all motile sperms and thus the overall area of the DFTs in a real-time manner. Accordingly, a theoretical DFT model was also developed to formulate the overall DFT area of a group of moving beads as a function of time as well as the total number and average speed of the beads. Then, using the least square fitting method, we obtained the optimal values of the TMSC and the average VCL that yielded the best fit for the theoretical DFT area to the measured DFT area.ResultsThe proposed method was used to evaluate the TMSC and the VCL of 20 semen samples. The maximum TMSC evaluated using the method is more than 980 sperms per video frame. The Pearson correlation coefficient (PCC) between the two series of TMSC obtained using the method and the CASA instrument is 0.946. The PCC between the two series of VCL obtained using the method and CASA is 0.771. As a consequence, the proposed method is as accurate as the CASA method in TMSC and VCL evaluations.ConclusionIn comparison with the individual point-tracking techniques, the collective DFT tracking method is relatively simple in computation without complicated image processing. Therefore, incorporating the proposed method into a cell phone equipped with a microscopic lens can facilitate the design of a simple sperm analyzer for clinical or household use without advance dilution.