Summaries of new peer-reviewed publications
RISUG
“Contact mode atomic force microscopy (AFM) has been used to analyze quantitatively the micro-structural properties of RISUG and its precipitate in various systems… [RISUG] has a tendency to form a complex with ionic biomolecules present in the seminal plasma. This is supported by the experimental observations using AFM. This RISUG-biomolecule complex possibly acts as an ionic trap for spermatozoa passing through the vas deferens… Significant alterations in the surface charge distribution of the sperm cell is observed on exposure to RISUG.”
Study of the micro-structural properties of RISUG--a newly developed male contraceptive.
Kumar S, Roy S, Chaudhury K, Sen P, Guha SK.
J Biomed Mater Res B Appl Biomater. 2008 Jul;86(1):154-61.
PMID: 18161821
Endocrinological approaches
Incomplete suppression of spermatogenesis has been an ongoing mystery in the quest for a globally efficacious hormonal male contraceptive. This is a new epidemiological analysis of the factors associated with incomplete suppression in men participating in the Phase III WHO-sponsored testosterone undecanoate trial in China. The study was designed as a case control series, comparing 43 “partial suppressors” dropped from the trial’s efficacy phase to 855 “complete suppressors” who entered the efficacy phase after 6 months of monthly depot TU injections (500 mg). “Baseline serum LH level was higher and serum LH as well as FSH level during the suppression phase was less suppressed in partial suppressors. Additionally, in a logistic regression analysis larger testis volume, higher serum FSH concentrations alone, or interaction of serum LH, FSH, testosterone and sperm concentrations were associated with degree of suppression. The distribution of polymorphisms of AR or FSH receptor genes did not differ between partial and complete suppressors.” This analysis does not support pharmacogenetics as a possible mechanism of action for non-response within an ethnically homogeneous population.
Predictors for partial suppression of spermatogenesis of hormonal male contraception.
Li JW, Gu YQ.
Asian J Androl. 2008 Sep;10(5):723-30.
PMID: 18645675
Proteomic analysis of testicular biopsies before and 2 weeks after treatment with either testosterone undecanoate or TU + LNG revealed distinct mechanisms of action. TU only treatment altered protein expression associated with assembly and induced cell death. TU + LNG treatment resulted in protein expression linked to “‘proliferation/cell survival’ and ‘apoptosis/death’… hnRNP K, PSMF1, SOD2, P4HB, Annexin II, and Pvalb are key proteins that may be early molecular targets responsible for spermatogenesis suppression induced by hormone treatment.”
Proteomic Analysis of Testis Biopsies in Men Treated with Injectable Testosterone Undecanoate Alone or in Combination with Oral Levonorgestrel as Potential Male Contraceptive.
Cui Y, Zhu H, Zhu Y, Guo X, Huo R, Wang X, Tong J, Qian L, Zhou Z, Jia Y, Lue YH, Hikim AS, Wang C, Swerdloff RS, Sha J.
J Proteome Res. 2008 Aug 15. [Epub ahead of print]
PMID: 18702538
Supporting endocrinological research
Combined hormonal treatment and testicular heating in cynomolgus monkey showed “spatiotemporal expression changes of 16 reported or novel genes in [the] epididymis… It was demonstrated that these region-specific genes, some of which were not regionally fixed, changed greatly with these treatments. The expression levels of these epididymal genes fluctuated, and the expression of most of the genes returned to nearly normal level at the end of treatments.” The spatiotemporal expression changes of 16 epididymis-specific genes induced by testosterone, heat, and combination treatment in cynomolgus monkey.
Li X, Liu Q, Liu S, Zhang X, Liu Y, Zhang Y.
Acta Biochim Biophys Sin (Shanghai). 2008 Aug;40(8):721-8.
PMID: 18685788
Testicular biopsies from 18 infertile men were “assessed by in-situ hybridization and immunohistochemistry” for progesterone receptor (PR) expression. “Semi-quantitative assessment of PR expression and Johnsen scores in the testicular biopsies of infertile men demonstrating different phenotypes indicated a direct relationship between PR expression and extent of spermatogenesis.”
Altered expression of progesterone receptors in testis of infertile men.
Abid S, Gokral J, Maitra A, Meherji P, Kadam S, Pires E, Modi D.
Reprod Biomed Online. 2008 Aug;17(2):175-84.
PMID: 18681990
Epididymal targets
Additional information about the role of Eppin, a possible male contraceptive target. “Human semenogelin (Sg) cDNA (nucleotides 82-849) and Eppin cDNA (nucleotides 70-723) were generated by polymerase chain reaction (PCR) and cloned… In vitro the digestion of recombinant Sg by PSA in the presence or absence of recombinant Eppin was studied… Eppin binds Sg on the surface of human spermatozoa with the C-terminal of Eppin (amino acids 75-133). rSg was digested with PSA and many low molecular weight fragments were produced. When rEppin is bound to rSg, then digested by PSA, incomplete digestion and a 15-kDa fragment results”
Molecular mechanism of epididymal protease inhibitor modulating the liquefaction of human semen.
Wang ZJ, Zhang W, Feng NH, Song NH, Wu HF, Sui YG.
Asian J Androl. 2008 Sep;10(5):770-5.
PMID: 18645681
Supporting motility research
Spindlin 1 (Spin 1) isoform2 was identified in mouse testis by RT-PCR and immunoblot. “[The] isoform was more highly expressed in adult testes compared with newborn testes. Interestingly, Spin1 isoform2 did not show up in the cytoplasm of primary spermatocytes until day 14… Spin1 isoform2 is a protein expressed highly in adult testis, which might be involved in spermatogenesis and could be necessary for normal sperm motility.”
Characterization of Spindlin1 isoform2 in mouse testis.
Zhang KM, Wang YF, Huo R, Bi Y, Lin M, Sha JH, Zhou ZM.
Asian J Androl. 2008 Sep;10(5):741-8.
PMID: 18645677
Cell adhesion targets
“Somatic, targeted, inactivation of Rxrb in Sertoli cells (yielding RxrbSer-/- mutants) leads to failure of spermatid release, accumulation of cholesterol esters and, subsequently, testis degeneration… The failure of spermiation was also reported in mice lacking the retinoic acid (RA) receptor alpha in Sertoli cells (RaraSer-/- mutants) and represents, in addition, a feature of vitamin A deficiency that can be readily induced in mice lacking the lecithin:retinol acyl transferase (Lrat-/- mutants). Altogether, these findings support the conclusion that RXRbeta heterodimerized with a RA-liganded RARalpha transduces signals required in Sertoli cells for spermatid release.”
RXR beta expression in Sertoli cells controls cholesterol homeostasis and spermiation.
Vernet N, Dennefeld C, Kloppfenstein M, Ruiz A, Bok D, Ghyselinck N, Mark M.
Reproduction. 2008 Aug 19. [Epub ahead of print]
PMID: 18713813
Gamete binding / fusion targets
“Sperm-egg interactions have been studied for many years using biochemical approaches such as the employment of antibodies and ligands that interact with sperm or with eggs and their vestments. As a result, various factors that participate in fertilization have emerged. However, when animals were genetically manipulated to examine the roles of those factors, most of them were found, to our surprise, to be "not essential"… In this review, information about sperm-egg interactions obtained from genetically manipulated animals is mainly revisited in order to propose a new vision.”
Mechanisms of sperm-egg interactions emerging from gene-manipulated animals.
Ikawa M, Inoue N, Okabe M.
Int J Dev Biol. 2008;52(5-6):657-64.
PMID: 18649279
“A combination of biochemical and proteomic membrane protein techniques have enabled us to dissect and highly purify the apical sperm plasma membrane area from control and capacitated sperm cells. The actual ZP-binding proteins identified predominantly belonged to the sperm membrane-associated family members of spermadhesins (AQN-3) and were present in the aggregating lipid ordered membrane microdomains (lipid rafts) that emerged during in vitro capacitation in the apical ridge area of the sperm head plasma membrane… We inferred that the capacitation-dependent formation of an aggregated lipid ordered apical ridge surface area in the sperm head plasma membrane was not only relevant for ZP-binding, but also for the ZP-induced acrosome reaction.
Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: Functional relationship with zona binding and the zona-induced acrosome reaction.
Boerke A, Tsai PS, Garcia-Gil N, Brewis IA, Gadella BM.
Theriogenology. 2008 Jul 18. [Epub ahead of print]
PMID: 18640708
“CRISP1 (cysteine-rich secretory protein 1) is an epididymal protein thought to participate in gamete fusion through its binding to egg-complementary sites. Structure-function studies using recombinant fragments of CRISP1 as well as synthetic peptides reveal that its egg-binding ability resides in a 12 amino acid region corresponding to an evolutionary conserved motif of the CRISP family, named Signature 2 (S2)… [Our] results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization.”
Participation of cysteine-rich secretory proteins (CRISP) in mammalian sperm-egg interaction.
Cohen DJ, Busso D, Da Ros V, Ellerman DA, Maldera JA, Goldweic N, Cuasnicu PS.
Int J Dev Biol. 2008;52(5-6):737-42.
PMID: 18649285
“[We] demonstrate that [rat epididymal epithelium CRISP1’s] Protein D associates transiently with the sperm surface in a concentration dependent manner, exhibiting saturable binding to both caput and cauda sperm in a concentration range that is consistent with its capacitation inhibiting activity. In contrast, Protein E persists on the sperm surface after all exogenous Protein D has been dissociated… These studies also confirm a tightly bound population of Protein E that could act in the female tract.”
Association of the Protein D and Protein E Forms of Rat CRISP1 with Epididymal Sperm.
Roberts KP, Ensrud-Bowlin KM, Piehl LB, Parent KR, Bernhardt ML, Hamilton DW.
Biol Reprod. 2008 Aug 13. [Epub ahead of print]
PMID: 18703418
“Crisp proteins appear to play multiple roles in the life history of sperm. One of these roles is to act as a sperm chemoattractant. Allurin, a 21 kDa Crisp protein rapidly released from the egg jelly of at least two frogs, X. laevis and X. tropicalis, elicits directed motility in both homospecific and heterospecific sperm… [The] observation that allurin can also elicit chemotaxis in mouse sperm raises the question of whether allurin-like proteins might act as sperm chemoattractants in mammals.”
Crisp proteins and sperm chemotaxis: discovery in amphibians and explorations in mammals.
Burnett LA, Xiang X, Bieber AL, Chandler DE.
Int J Dev Biol. 2008;52(5-6):489-501.
PMID: 18649262
Gossypol
An in vitro study identifies a possible mechanism of action for gossypol. “The present study was designed to investigate the effects of gossypol on the gap junctional intercellular communication (GJIC) and the expression of connexin43 (Cx43) in the cultured cells… The scrape loading and dye transfer (SLDT) assay showed gossypol significantly decreased GJIC between adjacent cells. RT-PCR, immunofluorescence and Western blot analyses demonstrated the expression of Cx43 in TM4 cells. The expression of Cx43 was gradually decreased with the increasing concentrations of gossypol, and the effect occurred as early as 6h after the treatment and continued until 48h. These results suggested that gossypol impaired GJIC by decrease of Cx43 expression in the cells, which is important for Sertoli cells to regulate spermatogenesis.”
Gossypol repressed the gap junctional intercellular communication between Sertoli cells by decreasing the expression of Connexin43.
Zhou DR, Zhou YC, Cui GH, Guo X, Qin J, Gui YT, Cai ZM.
Toxicol In Vitro. 2008 Jul 29. [Epub ahead of print]
PMID: 18706992
“In this study, we compared the toxicity and efficacy in mice of natural product gossypol and its semisynthetic derivative apo-gossypol, compounds that bind and inhibit antiapoptotic Bcl-2 family proteins. Daily oral dosing studies showed that mice tolerate doses of apogossypol 2- to 4-times higher than gossypol. Hepatotoxicity and gastrointestinal toxicity represented the major adverse activities of gossypol, with apogossypol far less toxic.”
Bcl-2 antagonist apogossypol (NSC736630) displays single-agent activity in Bcl-2-transgenic mice and has superior efficacy with less toxicity compared with gossypol (NSC19048).
Kitada S, Kress CL, Krajewska M, Jia L, Pellecchia M, Reed JC.
Blood. 2008 Mar 15;111(6):3211-9.
PMID: 18202226
Novel compounds
A study comparing the “antifertility effects of 50% ethanolic extract of the root bark of Cananga odorata with gossypol” after oral dosing for 60 days. “Differences were not observed in the sperm count and fertility index of the gossypol group in comparison with the C. odorata group. But statistically significant alterations were noted in the sperm morphology as well as in the activity of HMG CoA reductase, 3beta-hydroxy steroid dehydrogenase, glucose-6-phosphate dehydrogenase, cholesterol, and protein of the testis and in serum testosterone. On withdrawal of the drugs, sperms in C. odorata group became completely motile but not in the gossypol group. The active component is a 52 kd protein.”
Spermatotoxic effects of Cananga odorata (Lam): a comparison with gossypol.
Pankajakshy A, Madambath I.
Fertil Steril. 2008 Aug 8. [Epub ahead of print]
PMID: 18692834
Proteomic / genomic supporting research
“Despite the importance of [gametogenesis], progress in elucidating the molecular mechanisms underpinning mammalian germ cell development has been retarded by the lack of an efficient and reproducible system of in vitro culture for the expansion and trans-meiotic differentiation of germline cells. The dearth of such a culture system has rendered the study of germ cell biology refractory to the application of new high-throughput technologies such as RNA interference (RNAi), leaving in vivo gene-targeting approaches as the only option to determine the function of genes believed to be involved in gametogenesis… This review considers the developments made in modelling germ cell development using stem cells, and some of the challenges that need to be overcome to make this a useful tool for studying gametogenesis and to realise any future clinical application.
New research horizons: modelling germ cell development in vitro.
Childs AJ, Saunders PT, Anderson RA.
Mol Hum Reprod. 2008 Aug 1. [Epub ahead of print]
PMID: 18676971
“A significant proportion of [vasectomized men who undergo vasovasostomy] remain infertile, despite the reestablishment of patent ducts, possibly due to epididymal damage caused by vasectomy… To complete the list of epididymal genes affected by vasectomy, we analyzed the epididymal gene expression pattern of three vasectomized donors using the Affymetrix human GeneChip U133 Plus 2. These results were compared with the gene expression pattern of three "normal" donors… Quantitative (Qt)-PCR and Western blot analysis of six selected genes known to be expressed in specific epididymal segments were performed… These results contribute to an understanding of the reasons why fertility is not recovered in vasovasostomized men.”
Effects of vasectomy on gene expression profiling along the human epididymis.
Thimon V, Calvo E, Koukoui O, Légaré C, Sullivan R.
Biol Reprod. 2008 Aug;79(2):262-73. Epub 2008 Apr 23.
PMID: 18434627
“We investigated eight proteins encoded by novel spermatogenic cell-specific genes previously identified from the mouse round spermatid UniGene library… The authenticity of the eight novel proteins and their specificity to spermatogenic cells were confirmed… [Four] proteins [were] only in testicular germ cells, a single protein [was] in testicular germ cells and testicular sperm, and three proteins [were] in the testicular stages and mature sperm from the epididymis.” Of the three present in sperm, “one is located at the surface of the acrosomal region and the other two are associated with cytoskeletal structures in the sperm flagellum. We name the genes for these sperm proteins Shsp1 (Sperm head surface protein 1), Sfap1 (Sperm flagellum associated protein 1) and Sfap2 (Sperm flagellum associated protein 2).”
Characterization of eight novel proteins with male germ cell-specific expression in mouse.
Baek N, Woo JM, Han C, Choi E, Park I, Kim do H, Eddy EM, Cho C.
Reprod Biol Endocrinol. 2008 Jul 24;6:32.
PMID: 18652659
A report of a putative mechanism for the transformation of “guinea pig heterodimeric membrane protein ADAM1/ADAM2 (fertilin)… from a stationary state to one of rapid diffusion within the lipid bilayer… [These] data suggest that the release of ADAM1/ADAM2 from its diffusion constraints results from a cAMP-induced signaling pathway that, like others of capacitation, is established during epididymal sperm maturation.”
Cyclic 3', 5'-AMP Causes ADAM1/ADAM2 to Rapidly Diffuse Within the Plasma Membrane of Guinea Pig Sperm.
Hunnicutt GR, Koppel DE, Kwitny S, Cowan AE.
Biol Reprod. 2008 Jul 30. [Epub ahead of print]
PMID: 18667756
“Here we report the identification and characterization of a novel homeobox gene of the paired-like class on the X chromosome distal to the reproductive homeobox (Rhox) cluster in mice. Transcripts are found in the testis and ovary as early as 13.5 days post coitum (dpc). Transcription ceases in the ovary by 3 days post partum (dpp), but continues in the testis through adulthood. The Rhox13 gene encodes a 25.3 kDa protein expressed in the adult testis in germ cells at the basal aspect of the seminiferous epithelium.”
Identification and characterization of Rhox13, a novel X-linked mouse homeobox gene.
Geyer CB, Eddy EM.
Gene. 2008 Jul 15. [Epub ahead of print]
PMID: 18675325
“To gain insight into the physiological function of nephrocystin, Nphp1-targeted mutant mice were generated by homologous recombination. Interestingly, homozygous Nphp1 mutant mice were viable without renal manifestations of nephronophthisis. They appeared normal, but males were infertile… Electron microscopic analysis revealed detachment of early elongating spermatids from Sertoli cells, and a failure of sperm head and tail morphogenesis… These novel findings indicate that nephrocystin is critically required for the differentiation of early elongating spermatids into spermatozoa in mice. The possible roles of nephrocystin in the formation and maintenance of Sertoli-spermatid junctions are still under investigation.
Targeted disruption of Nphp1 causes male infertility due to defects in the later steps of sperm morphogenesis in mice.
Jiang ST, Chiou YY, Wang E, Lin HK, Lee SP, Lu HY, Wang CK, Tang MJ, Li H.
Hum Mol Genet. 2008 Aug 5. [Epub ahead of print]
PMID: 18684731
“We set out to test the hypothesis that HSP27 protein is expressed in the human testes and its expression varies with the state of spermatogenesis. HSP27 expression was examined in 30 human testicular biopsy specimens (normal spermatogenesis, maturation arrest and Sertoli cell only syndrome, 10 cases each) using immunofluorescent methods… We report for the first time the expression patterns of HSP27 in the human testes and show differential expression during normal spermatogenesis, indicating a possible role in this process. The altered expression of this protein in testes showing abnormal spermatogenesis may be related to the pathogenesis of male infertility.”
Heat shock protein 27 expression in the human testis showing normal and abnormal spermatogenesis.
Adly MA, Assaf HA, Hussein MR.
Cell Biol Int. 2008 Jul 23. [Epub ahead of print]
PMID: 18692580
“To study the behavior of Sertoli cells as a first approach to the molecular and functional characterization of the vitamin C transporters in [the blood-testis] barrier, we used the 42GPA9 cell line immortalized from mouse Sertoli cells… This work describe[s] the molecular identity of the transporters involved in vitamin C transport in these cells, which we hope will improve our understanding of how germ cells obtain vitamin C, transported from the plasma into the adluminal compartment of the seminiferous tubules.”
Molecular identification and functional characterization of the vitamin C transporters expressed by Sertoli cells.
Angulo C, Castro MA, Rivas CI, Segretain D, Maldonado R, Yañez AJ, Slebe JC, Vera JC, Concha II.
J Cell Physiol. 2008 Jul 30. [Epub ahead of print]
PMID: 18668520
The “epididymis is responsible for the sperm maturation and storage, and its gene expression is highly region-specific, so it maybe an ideal model for proteomics, especially for differential proteomics study. This paper gives a brief introduction [to] the application of proteomics in the epididymis and the differential proteins expressed in different epididymal regions. These epididymis-specific/selective proteins identified by proteomics technology may prove to be targets for contraception or may provide insight to the unique responsibilities of the epididymis in supporting male fertility.”
The application of proteomics in epididymis research [Chinese]
Zou M, Wang HF, Hu JM.
Sheng Li Ke Xue Jin Zhan. 2008 Apr;39(2):139-44.
PMID: 18664178
Blood-testis barrier elucidation
“The objective of the current study was to examine the immunoprotective properties of a mouse Sertoli cell line (MSC-1) in order to identify a Sertoli cell line that could be used to aid in investigation of the immunoprotective abilities of Sertoli cells… MSC-1 cells were found to express known Sertoli cell-expressed, immunoprotective factors, clusterin, Fas ligand, and transforming growth factor-beta1, suggesting additional factors may be involved in Sertoli cell immune privilege. These data indicate the MSC-1 cell line lacks the immunoprotective properties associated with primary Sertoli cells. Further study of this cell line could be useful in examining the mechanisms that enable Sertoli cells to provide immune privilege.”
Sertoli cell line lacks the immunoprotective properties associated with primary Sertoli cells.
Dufour JM, Dass B, Halley KR, Korbutt GS, Dixon DE, Rajotte RV.
Cell Transplant. 2008;17(5):525-34.
PMID: 18714671
“To investigate the correlation of exogenous estrogens with the expression of FasL in Sertoli cells and the blood-testis barrier, … super-physiological doses of exogenous estrogenic compounds (diethylstilbestrol and estradiol) were administered to pubertal Sprague-Dawley rats in vitro and in vivo… FasL expression was markedly up-regulated in the immature Sertoli cells (P < 0.05) as well as in the Sertoli cell membrane and the blood-testis barrier of the epithelium. The tracer lanthanum passed through the blood-testis barrier and reached the whole layer of the epithelium at 18 days.”
Estrogens affect Sertoli cells and the blood-testis barrier in pubertal rats. [Article in Chinese]
Pan YQ, Guo QS, Wang RY, Xu C.
Zhonghua Nan Ke Xue. 2008 Jul;14(7):590-6.
PMID: 18686377
“Adult male BALB/c mice were exposed to EMP at 200 kV/m for 200 pulses with 2 seconds interval… The permeability of the blood-testis barrier (BTB) in mice was observed by using Evans Blue tracer and lanthanum nitrate tracer. After exposure, cloudy Evans Blue was found in the testicle convoluted seminiferous tubule of mice. Lanthanum nitrate was observed not only between testicle spermatogonia near seminiferous tubule wall and Sertoli cells, but also between Sertoli cells and primary spermatocyte or secondary spermatocyte.”
Effect of electromagnetic pulse exposure on permeability of blood-testicle barrier in mice.
Wang XW, Ding GR, Shi CH, Zhao T, Zhang J, Zeng LH, Guo GZ.
Biomed Environ Sci. 2008 Jun;21(3):218-21.
PMID: 18714819
