Huai L. Feng

Decreased expression of the c-kit receptor is associated with increased apoptosis in subfertile human testes

OBJECTIVE To examine the expression of the c-kit receptor and its ligand, stem cell factor, and their possible relation with apoptosis in infertile men. DESIGN Prospective laboratory study. SETTING Urology laboratory in a university hospital. PATIENT(S) Men undergoing testicular biopsy during an investigation of subfertility. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Expression of the c-kit receptor protein, stem cell factor, and apoptosis in the testes. RESULT(S) The c-kit receptor was strongly present in Leydig cells and type A spermatogonia of normal testes, with decreased staining in Leydig cells and type A spermatogonia of testes with maturational arrest, and staining in only Leydig cells of Sertoli cell-only specimens. Stem cell factor was demonstrated in Leydig cells and Sertoli cells in all specimens. Western blotting demonstrated the 150-kd c-kit protein in the normal testes and the testes with maturational arrest, but not in the testes with the Sertoli cell-only pattern. Stem cell factor was expressed in all specimens, with a protein size of 45 kd. Increased apoptosis was demonstrated in type A spermatogonia and spermatocytes of tissue with maturational arrest compared with normal testicular tissue. CONCLUSION(S) C-kit receptor expression is decreased in subfertile testicular tissue compared with normal testicular tissue. Stem cell factor expression is present in Leydig cells and Sertoli cells. Increased apoptosis is seen in tissue with maturational arrest compared with normal tissue.

Decreased expression of the heat shock protein hsp70-2 is associated with the pathogenesis of male infertility.

OBJECTIVE To examine the expression of the heat shock protein hsp70-2, and the possible relationship with the pathogenesis of male infertility. DESIGN Prospective study. SETTING Reproductive testing laboratory in a university hospital. PATIENT(S) Men undergoing testicular biopsy during an investigation of subfertility. INTERVENTION(S) Testicular tissues were obtained from biopsies of men undergoing infertility evaluation and subdivided into three groups: normal testes, maturational arrest and Sertoli cell-only syndrome. Immunostaining and Western blotting techniques determined expression of the heat shock protein hsp70-2 MAIN OUTCOME MEASURE(S) Expression of the heat shock protein hsp70-2 in the testes. RESULT(S) The experimental data demonstrated that the heat shock protein hsp70-2 was expressed in the normal and maturation arrest testicular specimens. The heat shock protein hsp70-2 was strongly present in the cytoplasm of spermatocytes and spermatides in the adluminal compartment of the seminiferous epithelium in normal testis. However, maturation arrest testis tissue demonstrated light staining in spermatocytes and spermatides, and Sertoli-only specimens demonstrated no staining for the heat shock protein hsp70-2. The Western blotting data showed a 70-kDa heat shock protein in the normal and maturation arrest testicular tissues, but not in the Sertoli-only tissues. CONCLUSIONS These results suggest that the heat shock protein hsp70-2 is expressed in spermatocytes and spermatides in normal and maturation arrest tissues. However, the expression of the heat shock protein hsp70-2 was low in maturation arrest, and no heat shock protein hsp70-2 was demonstrated in Sertoli-only specimens. Therefore the decreased expression of the heat shock protein hsp70-2 is associated with the pathogenesis of male infertility.

MOLECULAR BIOLOGY OF MALE INFERTILITY

About 15% of couples have reduced fertility and in approximately one-half of all cases the reason is male infertility, usually of genetic origin. Thus, in the context of research in genes involved in reproduction and sex determination, genetic anomalies in gametogenesis are being extensively studied. The most frequent pathogenic causes of male infertility are Y-chromosomal microdeletions (8-15%) in the long arm of the Y chromosome, which, by loss of specific DNA segments, leads to loss of vital genes for sperm production. Infertile men, who attend infertility clinics, rise to 15% among those with azoospermia or spermatogenesis problem. The new technique of intracytoplasmic sperm injection has allowed many infertile men to achieve their dreams of fatherhood. However, the spermatogenic defect is genetic anomalies, which might be a potential risk of transmitting this defect to future offspring. Therefore, genetic counseling of all couples with the diagnosis of male infertility is recommended before their enrolment in intrauterine insemination, in vitro fertilization, and intracytoplasmic sperm injection. The important role of genetic abnormalities in the causation of human male infertility is increasingly recognized. While much remains to be learned in this fast-moving field, considerable progress has been made in the clinical delineation of genetic forms of male infertility and in the characterization of the responsible genes and their mutations or deletions. This review should provide insight into the understanding of parthenogenesis of male infertility in the human.

The effect of semen processing on sperm DNA integrity: comparison of two techniques using the novel Toluidine Blue Assay

OBJECTIVE The first goal of this study was to determine the effect that semen processing has on sperm DNA integrity. The second goal was to assess which processing technique (modified swim-up versus density gradient centrifugation) results in a superior sample. DNA integrity was measured using a novel Toluidine Blue Assay. STUDY DESIGN Side-by-side comparison. MATERIALS AND METHODS Raw semen samples were collected from thirty-two male individuals and scored for routine semen analysis. Prior to discarding the specimens identical aliquots were divided and processed by density gradient centrifugation and a modified swim-up technique. The Toluidine Blue Assay was used to analyze raw and processed samples. RESULTS Both density gradient centrifugation and the modified swim-up improved DNA quality compared to the unprocessed sample. However, the modified swim-up technique proved superior. CONCLUSIONS The swim-up technique generates a sperm sample with better DNA integrity. Should DNA integrity correlate with better pregnancy rates in IUI and IVF, respectively, the swim-up may be the sperm processing technique of choice for these procedures.

Role(s) of the Serine/Threonine Protein Phosphatase 1 on Mammalian Sperm Motility

Mammalian spermatozoa acquire the capacity for motility and fertilization during the transit through the epididymis under the control of different factors, such as cAMP, intracellular pH, intracellular calcium and phosphorylation of sperm proteins. As the acquisition of functional competence including gaining motility during epididymal transit occurs in the complete absence of contemporaneous gene transcription and translation on the part of the spermatozoa, it is widely accepted that post-translational modifications are the only means by which spermatozoa can acquire functionality. Serine-threonine protein phosphatase 1 (PP1) together with their testis/sperm-specific interacting proteins might be involved in this regulatory mechanism. PP1α, PP1β/δ, PP1γ1 and PP1γ2 are all expressed in the testis whereas PP1γ2 is the only isoform expressed on spermatozoa. I2, I3, sds22, 14-3-3 and hsp90 are associated with PP1γ2 in spermatozoa located on the sperm head and tail. Activity of PP1γ2 and the binding pattern to these regulatory proteins changes in spermatozoa recruited from the caput and those from the cauda part of the epididymis. In this review, we summarize the possible roles of PP1 on spermatozoa during spermatogenesis and flagellar motility control. We suggest that PP1 might take part in the inhibition of the sperm motility activation by interacting with AKAPs and CAMKII. A hypothesized signaling pathway of mammalian sperm motility activation and PP1s function has been proposed.

Comparing Expression of Progesterone and Estrogen Receptors in Testicular Tissue From Men With Obstructive and Nonobstructive Azoospermia

The objective of this study was to identify and compare the expression profiles of progesterone receptor (PR) and estrogen receptor alpha (ERalpha) in the testes of men with obstructive azoospermia (OA), maturation arrest (MA), and Sertoli cell-only (SCO) histology. Testicular biopsies were obtained from 10 patients with OA, 10 patients with MA (either early or late arrest), and 8 patients with SCO who did not have hormonal abnormalities and varicoceles. Expression of PR and ERalpha was detected by immunofluorescence and Western blot. PR was expressed in the spermatogenic, Leydig, and Sertoli cells in the testes of OA patients. In the MA and SCO patients, the expression of PR was reduced in all cell types as compared with that in the OA patients. Western blot demonstrated that both the full-size (120 KDa) and the truncated (52 KDa) isoforms of the PR were expressed in the OA and MA testes. However, in the SCO testes, only the truncated isoform of PR (52 KDa) was expressed. ERalpha (66 KDa) was expressed principally in the spermatogenic and Leydig cells in the OA testes. By immunohistochemistry staining, expression of ERalpha was decreased in the spermatogenic and Leydig cells of the MA testes, whereas its expression was enhanced in the Leydig cells of the SCO testes. However, by Western blot, expression of ERalpha was significantly reduced in the SCO testes as compared with that in the OA and MA testes. We conclude that PR and ERalpha may play a role in the pathogenesis of the MA and SCO phenotype in patients with infertility.

A retroprospective study comparing three different assisted hatching techniques

The purpose of this study was to determine the effect of four different assisted hatching techniques on pregnancy rates in women with prior IVF failure in fresh IVF cycles. The results suggested that assisted hatching utilizing laser, chemical, or microsurgical techniques increases both implantation and pregnancy rates.

Effect of slow freeze versus vitrification on the oocyte: an animal model

OBJECTIVE To determine whether there is a deleterious effect on dynamic events in the nucleus and cytoplasm of oocytes by using different cryopreservation protocols in an animal model. DESIGN Prospective study. SETTING University hospitals. PATIENT(S) Not applicable. INTERVENTION(S) Immunostaining and confocal laser scanning microscope techniques were used. MAIN OUTCOME MEASURE(S) The spindle and chromosomal configurations, as well as dynamic changes of the cortical granules (CGs) and mitochondria in different cryogroups. RESULT(S) After thawing/warming of bovine oocytes, CGs became more dispersed in the cytoplasm, particularly in the DMSO group. A significant reduction in normal spindle and chromosomal configurations was observed in all three cryogroups, particularly in the propylene glycol (PROH) group, when compared with the fresh group. Global DNA methylation levels were significantly reduced in the slow and DMSO groups, as compared with the fresh group; however, methylation levels were significantly increased in the PROH group. The proportion of severely apoptotic oocytes was dramatically increased in all three cryogroups, compared with the fresh group. CONCLUSION(S) Overall, results demonstrate that using DMSO as the cryoprotectant is better for preserving the cellular and nuclear integrity of the oocyte. The PROH method makes the oocyte more vulnerable to increased DNA methylation, which may be associated with imprinting gene alteration. This study adds to the increasing body of evidence that cryopreservation protocols vary in their impact upon the oocyte.

Current assessment of sperm DNA integrity.

Conventional semen analysis is rapidly losing its place as the gold standard of diagnosis and the cornerstone of treating the infertile male in modern times. Recent technology allows scientists to analyze sperm fertilizing potential and subsequent embryonic growth by studying factors that have previously escaped traditional parameters. It has become increasingly evident that nuclear DNA arrangement is essential to the fertilizing potential of sperm. A vast array of tests are now available to examine the genetic makeup of individual spermatozoa, ranging the entire gamut from simple bench top assays performed routinely to complex flow cytometric assays requiring highly-skilled technologists. Future research to compare these new tests to those more commonly in use, correlating them with reproductive outcome promises to fill the current void in the field of male infertility, paring innovative diagnostic (and prognostic) technological standards to the already existing sophisticated assortment of successful treatment modalities.

NEW HOPE FOR INFERTILITY THERAPY: FABRICATING GAMETES FROM STEM CELLS

The field of reproductive and developmental biology has been revolutionized by recent advanced studies. These studies indicate that stem cells are capable of forming gametes in vivo and in vitro in both mouse and human. This has provided powerful tools for undertaking new types of reproductive studies, and particularly might provide new technology and novel approaches in assisted reproductive medicine.