A sperm component, HSD-3.8 (SPAG1), interacts with G-protein beta 1 subunit and activates extracellular signal-regulated kinases (ERK).

HSD-3.8 cDNA (accession number AF311312) encodes a human sperm component. A 0.7 kb fragment (HSD-0.7) containing three immunological epitopes of HSD-3.8 cDNA was prepared and expressed in E. coli. Immunization of female rats with the recombinant HSD-0.7 proteins induced infertility. A cDNA fragment encoding the C-terminal 144 amino acids of human G-protein beta l subunit (Gbeta1-C144) was screened by yeast two-hybrid, when HSD-0.7 segment was used as a bait. Recombinant His6-tagged-Gbeta1-C144 protein was expressed in E. coli BL21 and Anti-Gbeta1 serum was raised with purified Gbeta1-C144. HA-tagged HSD-0.7 and FLAG-tagged Gbeta1 plasmids were constructed and co-transfected into human embryonal kidney 293 cells. Two proteins were localized at superimposable sites in the cytoplasm, and they formed a complex when 500 micromol/L GDP existed. Overexpression of HSD-0.7 activated the G-protein-mediated extracellular signal-regulated kinases (ERK1/2); however, the truncated fragments of HSD-0.7, which lacked either TPR domain or P-loop, lost the ability to activate the ERK1/2 pathway. Further study revealed that the activation of ERK1/2 was protein kinase C (PKC) rather than Ras dependent. These results provide evidence that HSD-3.8 present in spermatocytes and sperm may participate in spermatogenesis and fertilization process by activating the PKC-dependent ERK1/2 signal transduction pathway.

Identification of GABABR2 in rat testis and sperm.

GABA is capable of mimicking and potentiating the action of progesterone in initiating the acrosome reaction (AR) of mammalian sperm. The GABA-initiated AR is mediated by GABA(A)R; whereas GABA(B)R1 protein found in rat testis and sperm tends to modify this process. Moreover, the occurrence of GABA(B)R2, a subunit essential for the formation of a functionally active GABA (B)R, in rat testis and sperm has not been established. In the present study, rat testis and sperm were analyzed for the presence of GABA(B)R2 transcript and protein by RT-PCR, Northern blot, Western blot and an indirect immunofluorescence technique. Northern blot shows that the transcript of testis GABA(B)R2 is shorter (~3.0 Kb) than that of the brain (~5.6 Kb). The full length testis GABA(B)R2 cDNA was prepared by RACE-PCR and found to be shorter by 2.2 Kb in the segment at the extreme terminus of 3'UTR of rat brain GABA(B)R2 but, the sequences corresponding to the open reading frame and 5'-UTR of rat testis GABA(B)R2 were found to be identical to those of rat brain. GABA(B)R2 protein isolated from rat epididymal sperm was slighter larger than those of rat testis and brain. It was principally localized in the acrosome region of the head of rat sperm by an indirect immunofluorescence technique. The present results establish that GABA(B)R2 protein is produced in rat testis and sperm and may play a role in GABA triggering of AR.

Biphasic effect of GABA on rat sperm acrosome reaction: involvement of GABA(A) and GABA(B) receptors.

The functional relationship between GABA(A) and GABA(B) receptors in regulating acrosome reaction (AR) of rat spermatozoa was demonstrated by studying the differential effects of a GABA(B) agonist and an antagonist on the process. AR rates were determined using the chlortetracycline staining assay. The induction of AR in rat sperm by GABA was found to be a biphasic phenomenon; i.e., AR rates increased with increasing GABA concentrations up to <5 micro M and at higher concentrations of the neurotransmitter (>5 micro M), there was a reductionin the AR rates. This biphasic phenomenon is apparently due to the differential interaction of the neurotransmitter with GABA receptor subtypes in a dose-dependent manner; i.e., GABA(A) receptors (stimulatory) are primarily activated at low concentration of GABA, while GABA(B) receptors (inhibitory) become activated at higher concentrations. This hypothesis is supported by the present findings that treatment with saclofen, a GABA(B) receptor antagonist, did not influence the AR rates effected by GABA at low concentrations; while the AR rates were maintained at the maximum level at higher concentrations of GABA, resulting in the elimination of the biphasic phenomenon. Baclofen, a GABA(B) receptor agonist, blocks the AR activating action of GABA at both low and high concentrations. It would appear that the induction of AR in rat sperm by GABA is regulated by the proportionality of activated GABA(A) and GABA(B) receptors acting as a yin-yang control.

Expression and function of the HSD-3.8 gene encoding a testis-specific protein.

The nucleotide sequence of the full length HSD-3.8 cDNA (accession number AF311312), encoding a human sperm component, was determined to consist of 3818 bp with a reading frame of 2778 bp encoding a deduced polypeptide composed of 926 amino acids. A 0.7 kb fragment containing three immunological epitopes of HSD-3.8 cDNA was prepared and used to construct recombinant expression vectors. The constructs were transformed into E.coli BL-21, and the fusion proteins were expressed, isolated and purified. Using the polyclonal antibodies raised against the purified expressed fusion proteins, positive immunostaining occurred over the surface of the postacrosomal zone of human spermatozoa and of germ cells within the seminiferous epithelium of human testis. Intense staining of large pachytene primary spermatocytes occurred. The capacity of the recombinant protein to reduce fertility as an immunogen in adult female rats was assessed. Immunized animals were infertile or exhibited marked reduction in their fertility. Analysis of the deduced HSD-3.8 polypeptide revealed the presence of a tetratricopeptide repeat (TPR) motif, a P-loop sequence that acts as a binding site for ATP/GTP and phosphorylation sites for PKC, CK2 and cAMP/cGMP-dependent protein kinases. A blot overlay assay with [alpha-(32)P]GTP showed that the polypeptide encoded by the 0.7 kb fragment of HSD-3.8 is a GTP binding protein. It was also shown to possess GTPase activity and to be phosphorylated by PKC in vitro. In conclusion, HSD-3.8 is a GTP binding protein and its activity may be regulated by phosphorylation.

[A case of Chinese herbs nephropathy in which the progression of renal dysfunction was slowed by steroid therapy].

The patients was a 43-year-old woman whose chief complaints were nausea and heaviness of the heads. There was a history of toxemia of pregnancy. The patient had previously taken Tenshin Tokishigyaku-ka-goshuyu-shokyo-to for two years because of cold sensitivity. Fever, thirst, and loss of appetite developed from approximately 18 months after she started treatment with the Chinese herbal preparation, and she presented at our outpatient clinic 2.5 years later. On initial examination, deterioration of renal function was evident and the serum creatinine level was 3.4 mg/dl. A renal biopsy specimen showed marked interstitial fibrosis without inflammatory cell infiltration, leading to the diagnosis of Chinese herbs nephropathy (CHN). Steroid therapy was started on the 36th hospital day after a sharp rise in the serum creatinine level to 5.1 mg/dl. This resulted in the rapid improvement of renal function and reduction of the serum creatinine to 2.6 mg/dl by 8 weeks after the initiation of treatment. In a study on the use of steroids for patients with progressive moderate renal dysfunction caused by Chinese herbs, Vanherweghem et al. reported that the progression of renal failure was appreciably slowed in patients given steroids when compared with the control group. We were also able to slow the progression of renal dysfunction in our patient by steroid therapy, although the prognosis of CHN is generally considered to be very poor.

Glutamine protects function and improves preservation of small bowel segments.

BACKGROUND: Improved organ preservation is essential for the success of small bowel transplantation. Small bowel is usually preserved in UW (University of Wisconsin) solution which does not contain glutamine (Gln), the principal fuel for the enterocyte. We hypothesized that Gln-supplemented UW would improve mucosal function and structure of cold preserved small intestine. MATERIALS AND METHODS: Jejunum (40 cm) was harvested from Lewis rats and preserved for 18 hr at 4 degrees C in saline; UW solution only; UW with 1, 2, or 4% Gln; and UW containing 1, 2, or 4% isonitrogenous balanced nonessential amino acids (NEAA). 14C glucose transport, mucosal protein, mucosal maltase and alkaline phosphatase, jejunal villous height, and histologic damage were measured. RESULTS: UW with 2% Gln significantly increased glucose transport and mucosal protein when compared to the 2% NEAA and UW-only groups. Two percent Gln significantly decreased histologic damage of jejunum following cold preservation. Increasing Gln to 4% did not significantly increase its efficacy when compared to the UW with 2% Gln group. There were no significant differences in the activities of mucosal maltase and alkaline phosphatase among the various treatment groups. CONCLUSIONS: The addition of Gln, optimally provided at a concentration of 2%, to UW solution may protect the preserved small bowel segments from cold ischemic injury and improve mucosal function.

Expression of the calpastatin gene segment during spermiogenesis in human testis: an in situ hybridization study.

Serum obtained from an infertile woman contained antibodies that agglutinate human sperm. The antibodies interacted with a sperm protein with an estimated M(r) of 17.5 kD. The cDNA coding the 17.5-kD protein was isolated from a human testis lambda gt11 expression library and identified as a segment of the calpastatin gene. Single-stranded 35S-labeled RNA probes were prepared from the calpastatin cDNA segment. Using the techniques of in situ hybridization, the calpastatin mRNA was located in spermatids of human testis. The results support a previous observation that the calpastatin segment is produced during spermiogenesis and suggest that transcription of the calpastatin gene occurred during the postmeiotic haploid stage of spermatogenesis.

Calpastatin gene in human testis.

Antibodies present in a serum obtained from an infertile woman interacted with a 17.5 kD glycoprotein (BS-17 component) extracted from human sperm by Western blot. Polyclonal antibodies were raised against the BS-17 component and used to identify positive staining clones from a human testis lambda gt11 expression library. The cDNA encoding the BS-17 component was isolated and its nucleotide sequence determined. The BS-17 cDNA contained 758 nucleotides with an open reading frame of 558 nucleotides encoding a polypeptide consisting of 186 amino acids. The BS-17 cDNA showed 99.7% homology in 758 nucleotides overlap with the 3' terminus of the gene coding calpastatin and 99.5% identity in 186 amino acid overlap with the carboxyl terminus of calpastatin. The BS-17 component of human sperm corresponds to the carboxyl terminus of calpastatin. This conclusion is supported by the finding that the polyclonal antibodies also interacted with a 84 kD protein corresponding to the M(r) of calpastatin.

Characterization of a folding intermediate of apoplastocyanin trapped by proline isomerization.

The unfolding and refolding transitions of French bean apoplastocyanin (apo-Pc), a beta-sandwich protein, have been characterized. The apoprotein is stabilized by sodium sulfate and can be reversibly unfolded by guanidine hydrochloride (GuHCl). However, in contrast to holo-Pc, apo-Pc is unstable at low ionic strength, suggesting that the copper ion stabilizes the holoprotein. The equilibrium unfolding transition monitored by peptide circular dichroism (CD) and tyrosine fluorescence is described by a two-state model. The kinetics of the unfolding transition were monitored using a manual mixing technique and are consistent with a single two-state transition. In contrast, the kinetics of the refolding reaction measured by fluorescence and CD show two transitions with different rates. The relaxation time of the slower phase (800-1000 s) is almost independent of GuHCl concentration. The faster phase was observed only under strongly native conditions, and its relaxation time is GuHCl-dependent. Double-jump experiments and acceleration by cyclophilin demonstrate that both phases involve cis-trans isomerization of proline residues. The changes in fluorescence associated with the two phases are more than 150% of the total change expected from equilibrium experiments, indicating the presence of intermediate(s) with fluorescence greater than the unfolded state. Amide hydrogen-exchange experiments coupled with two-dimensional NMR spectroscopy demonstrate the formation of an intermediate in the very low refolding reaction in which amide protons in the beta-sheets are weakly protected from exchange. No CD evidence for nativelike beta-sheet formation was found for this intermediate. The NMR experiments suggest that the intermediate is compact with flexible beta-sheets and altered packing of the hydrophobic core. It has many of the characteristics of a molten globule. However, the 1H NMR spectrum of the intermediate exhibits a small number of shifted resonances that indicate the presence of specific tertiary interactions in a localized region. A mechanism for refolding of apoplastocyanin is proposed that includes two slow steps corresponding to trans-->cis isomerization of two prolines.

Purification and characterization of an incompletely glycosylated form of human chorionic gonadotropin from human placenta.

A small form of hCG (SP-hCG) was purified from an acetone powder preparation of human first trimester placenta by repeated gel filtration and ion exchange chromatography on a Q-Sepharose or FPLC Mono Q column. The estimated mol wt (Mr) of the small hCG by gel filtration is 43K compared to 58K for authentic hCG. The pI of SP-hCG is 10.0, suggesting deficiency of sialic acids. SP-hCG dissociates into subunits when treated with 6 M guanidine-HCl or analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two beta-subunits of SP-hCG were found with estimated Mr of 23K and 20K. Both are distinctly smaller than authentic hCG beta. A single alpha-subunit was found, with an estimated Mr of 21 K. The immunoactivity (8,900-10,000 IU/mg) of highly purified SP-hCG was comparable to that of reference hCG (CR119) determined by a RIA method using anti-hCG antibodies. The hCG/LH receptor-binding activity of SP-hCG is equivalent to that of reference hCG (CR119). Its biological activity is lower than that of reference hCG (approximately 30% or more) assayed by the in vitro stimulation of rat Leydig cells to produce testosterone and cAMP. A high dose is required to attain the same level of stimulation as reference hCG. The amino acid composition of SP-hCG is similar to that of reference hCG, whereas its hexsamine content is significantly lower. Its glucosamine content is about half that in reference hCG, while it completely lacks galactosamine. These findings suggest that SP-hCG is deficient in O-linked oligosaccharide chain in the beta-subunit, and that the N-linked oligosaccharide chains of both subunits are shortened. SP-hCG is one of the principal forms of the hormone present in first trimester placenta and may be a key intermediate in the posttranslational biosynthesis of hCG. Although it lacks O-linked sugar chains and shortened N-linked sugar chains, it possesses substantial biological activity. To have full biological activity, the hCG molecule must contain the complete complement of sugar chains.

Isolation and characterization of a rabbit sperm tail protein.

Rabbit sperm tails were obtained by a nitrogen cavitation procedure and separated by discontinuous sucrose gradient centrifugation. Proteins were extracted from sperm tails with 3[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) and separative disk electrophoresis and DEAE-trisacryl column chromatography. A protein with an estimated mol wt of 20.1 +/- 1.1 kD was isolated and found to be homogeneous by SDS-PAGE and designated as rSMP-B. The isoelectric point of rSMP-B was in the range of pH 4.4-4.7. The amino acid composition was determined, and glycine was identified as the N-terminal residue. Antisera were raised against purified rSMP-B. Using a peroxidase-antiperoxidase method, the rSMP-B antigen was located on the surface of the midpiece and tail of the sperm. Testis sections showed intense staining of late spermatids located within the seminiferous tubules. Adult male rabbits were inoculated with rSMP-B protein and Freund's adjuvant. The testis and epididymis of the immunized animals showed absence of sperm. The immunolocalization findings and the immunization data suggest that rSMP-B is formed in late spermatid and that it is an essential structural component of sperm.