The Regulation of Spermatogonial Stem Cells in an Adult Testis by Glial Cell Line-Derived Neurotrophic Factor.

This review focuses on the in vivo regulation of spermatogonial stem cells (SSCs) in adult testes by glial cell line-derived neurotrophic factor (GDNF). To study adult mouse testes, we reversibly inhibited GDNF stimulation of SSCs via a chemical-genetic approach. This inhibition diminishes replication and increases differentiation of SSCs, and inhibition for 9 days reduces transplantable SSC numbers by 90%. With more sustained inhibition, all SSCs are lost, and testes eventually resemble human testes with Sertoli cell-only (SCO) syndrome. This resemblance prompted us to ask if GDNF expression is abnormally low in these infertile human testes. It is. Expression of FGF2 and FGF8 is also reduced, but some SCO testes contain SSCs. To evaluate the possible rebuilding of an SSC pool depleted due to inadequate GDNF signaling, we inhibited and then restored signaling to mouse SSCs. Partial rebuilding occurred, suggesting GDNF as therapy for men with SCO syndrome.

Spermatogonial Stem Cell Numbers Are Reduced by Transient Inhibition of GDNF Signaling but Restored by Self-Renewing Replication when Signaling Resumes.

One cause of human male infertility is a scarcity of spermatogonial stem cells (SSCs) in testes with Sertoli cells that neither produce adequate amounts of GDNF nor form the Sertoli-Sertoli junctions that form the blood-testis barrier (BTB). These patients raise the issue of whether a pool of SSCs, depleted due to inadequate GDNF stimulation, will expand if normal signaling is restored. Here, we reduce adult mouse SSC numbers by 90% using a chemical-genetic approach that reversibly inhibits GDNF signaling. Signal resumption causes all remaining SSCs to replicate immediately, but they primarily form differentiating progenitor spermatogonia. Subsequently, self-renewing replication restores SSC numbers. Testicular GDNF levels are not increased during restoration. However, SSC replication decreases as numbers of SSCs and progenitors increase, suggesting important regulatory interactions among these cells. Finally, sequential loss of SSCs and then pachytene spermatocytes causes dissolution of the BTB, thereby recapitulating another important characteristic of some infertile men.

Aberrant gene expression by Sertoli cells in infertile men with Sertoli cell-only syndrome.

Sertoli cell-only (SCO) syndrome is a severe form of human male infertility seemingly characterized by the lack all spermatogenic cells. However, tubules of some SCO testes contain small patches of active spermatogenesis and thus spermatogonial stem cells. We hypothesized that these stem cells cannot replicate and seed spermatogenesis in barren areas of tubule because as-of-yet unrecognized deficits in Sertoli cell gene expression disable most stem cell niches. Performing the first thorough comparison of the transcriptomes of human testes exhibiting complete spermatogenesis with the transcriptomes of testes with SCO syndrome, we defined transcripts that are both predominantly expressed by Sertoli cells and expressed at aberrant levels in SCO testes. Some of these transcripts encode proteins required for the proper assembly of adherent and gap junctions at sites of contact with other cells, including spermatogonial stem cells (SSCs). Other transcripts encode GDNF, FGF8 and BMP4, known regulators of mouse SSCs. Thus, most SCO Sertoli cells can neither organize junctions at normal sites of cell-cell contact nor stimulate SSCs with adequate levels of growth factors. We propose that the critical deficits in Sertoli cell gene expression we have identified contribute to the inability of spermatogonial stem cells within small patches of spermatogenesis in some SCO testes to seed spermatogenesis to adjacent areas of tubule that are barren of spermatogenesis. Furthermore, we predict that one or more of these deficits in gene expression are primary causes of human SCO syndrome.

Multiple aspects of male germ cell development and interactions with Sertoli cells require inositol hexakisphosphate kinase-1.

Inositol hexakisphosphate kinase-1 (IP6K1) is required for male fertility, but the underlying mechanisms have been elusive. Here, we report that IP6K1 is required for multiple aspects of male germ cell development. This development requires selective interactions between germ cells and Sertoli cells, namely apical ectoplasmic specialization. Spermiation (sperm release) requires tubulobulbar complexes. We found that the apical ectoplasmic specialization and tubulobulbar complexes were poorly formed or disrupted in IP6K1 KOs. Deletion of IP6K1 elicited several aberrations, including: 1, sloughing off of round germ cells; 2, disorientation and malformation of elongating/elongated spermatids; 3, degeneration of acrosomes; 4, defects in germ-Sertoli cell interactions and 5, failure of spermiation. Eventually the sperm cells were not released but phagocytosed by Sertoli cells leading to an absence of sperm in the epididymis.

Responses to glial cell line-derived neurotrophic factor change in mice as spermatogonial stem cells form progenitor spermatogonia which replicate and give rise to more differentiated progeny.

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis. These cells are classically defined as a subset of morphologically defined A single (As) spermatogonia, which can produce more SSCs or they can give rise to nonstem As cells that, upon replication, generate A paired (Apr) and then A aligned (Aal) spermatogonia. These latter two cell types, along with the nonstem As cells, function as transit-amplifying progenitor cells. It is known that glial cell line-derived neurotrophic factor (GDNF) is essential for maintaining all of these cells, but it is unknown if or how the responses of these cells change as they progress down the pathway to differentiated type A1 spermatogonia. We address this issue by using a chemical-genetic approach to inhibit GDNF signaling in vivo and an in vitro approach to increase GDNF stimulation. We show that inhibition for 2 days suppresses replication of As, Apr, and Aal spermatogonia to an equal extent, whereas stimulation by GDNF preferentially increases replication of As and Apr spermatogonia. We also test if inhibiting GDNF signaling causes As, Apr, and Aal spermatogonia to express Kit, an essential step in their differentiation into type A1 spermatogonia. Inhibition for 3 or 7 days produces a progressive increase in the percentages of As, Apr, and Aal undergoing differentiation, with the largest increase observed in Aal spermatogonia. Finally, we demonstrate that numbers of SSCs decrease more slowly than numbers of progenitor spermatogonia when GDNF signaling is inhibited. Taken together, these data suggest that there are significant changes in the responses to GDNF as SSCs give rise to progenitor spermatogonia, which replicate and gradually differentiate into type A1 spermatogonia.

The in vivo response of stem and other undifferentiated spermatogonia to the reversible inhibition of glial cell line-derived neurotrophic factor signaling in the adult.

Maintaining adequate numbers of spermatogonial stem cells is required for the production of the millions of sperm required for male fertility. To date, however, the mechanisms that regulate the size of this pool in the adult are poorly defined. Glial cell line-derived neurotrophic factor (GDNF) is required for establishing this pool in the prepubertal animal, but its in vivo function in the normal adult testis has never been examined directly. We used a chemical-genetic approach to address this issue. We generated mice carrying a single amino acid mutation (V805A) in Ret, the kinase subunit of the GDNF receptor. This mutation does not affect normal GDNF signaling but renders it susceptible to inhibition by the ATP competitive inhibitor, NA-PP1. When GDNF signaling was blocked in adults for 11 days, only a few cells remained that expressed the stem spermatogonial markers, Gfrα1 and Zbtb16, and testicular Ret mRNA content was reduced markedly. These decreases were associated with depletion of functional stem spermatogonia; some were lost when GDNF signaling was inhibited for only 2 days while others survived for up to 11 days. However, when signaling was restored, the remaining stem cells proliferated, initiating tissue restoration. In conclusion, these results provide the first direct proof that GDNF acutely regulates the number of spermatogonial stem cells in the normal adult testis. Additionally, these results demonstrate different sensitivities among subpopulation of these stem cells to inhibition of GDNF signaling.

Simultaneous quantification of steroids in rat intratesticular fluid by HPLC-isotope dilution tandem mass spectrometry.

An isotope dilution mass spectrometry method has been developed for the simultaneous measurement of picolinoyl derivatives of testosterone (T), dihydrotestosterone (DHT), 17ß-estradiol (E(2)), and 5α-androstan-3α,17ß-diol (3α-diol) in rat intratesticular fluid. The method uses reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Following derivatization of 10-µL samples of testicular fluid with picolinoyl chloride hydrochloride, the samples were purified by solid phase extraction before analysis. The accuracy of the method was satisfactory for the 4 analytes at 3 concentrations, and both inter- and intraday reproducibility were satisfactory for T, DHT, and E(2). Measurements of intratesticular T concentrations in a group of 8 untreated adult rats by this method correlated well with measurements of the same samples by radioimmunoassay. As in men, there was considerable rat-to-rat variability in T concentration, despite the fact that the rats were inbred. Although its levels were more than an order of magnitude lower than those of T, DHT was measured reliably in all 8 intratesticular fluid samples. DHT concentration also varied from rat to rat and was highly correlated with T levels. The levels of E(2) and 3α-diol also were measurable. The availability of a sensitive method by which to measure steroids accurately and rapidly in the small volumes of intratesticular fluid obtainable from individual rats will make it possible to examine the effects, over time, of such perturbations as hormone and drug administration and environmental toxicant exposures on the intratesticular hormonal environment of exposed individual males and thereby to begin to understand differences in response between individuals.

Stage-specific changes in GDNF expression by rat Sertoli cells: a possible regulator of the replication and differentiation of stem spermatogonia.

In the adult testis, the precise control of the self-renewing replication and differentiation of stem spermatogonia is fundamental to male fertility. Previous studies have shown that the replication of A single (A(s)) spermatogonia, a population that includes the stem cells, is maximal at stage I of the cycle of the rat seminiferous epithelium and minimal at stage VII, while the ratio of A-paired spermatogonia to A(s) spermatogonia increases from stages I to VII. It has been hypothesized that these changes in A(s) spermatogonia replication and differentiation result from changes in the expression of glial cell-line derived neurotrophic factor (GDNF) by Sertoli cells. To directly test this hypothesis, we used immunocytochemistry and confocal microscopy to demonstrate that within intact seminiferous tubules, GDNF is detectable only in Sertoli cells and that its amount and its location within these cells changes with progression of the stages of the cycle. The identification of Sertoli cells as the primary source of GDNF was confirmed by RT-PCR analysis of RNA isolated from purified populations of Sertoli cells, pachytene spermatocytes, and round spermatids. Stage-specific changes in GDNF expression were confirmed by quantifying GDNF mRNA in seminiferous tubules at defined stages of the cycle. Expression of this transcript was maximal at stage I, fell 14-fold by stage VIIc,d, and then increased 12-fold by stages XIII-XIV. This pattern of expression was the opposite of the control, cathepsin L mRNA. Taken together, these data support the hypothesis that cyclical changes in GDNF expression by Sertoli cells are responsible for the stage-specific replication and differentiation of stem spermatogonia, the foundational cells of spermatogenesis.

Activation and repression domains within the promoter of the rat cathepsin L gene orchestrate sertoli cell-specific and stage-specific gene transcription in transgenic mice.

In murine testes, only Sertoli cells express the cathepsin L (Ctsl) gene, and this expression is restricted to stages V-VIII of the cycle. Our previous transgenic analysis of Tg (-2065/+977) demonstrated that this expression is regulated by a approximately 2-kb promoter. To begin to elucidate this regulation, we analyzed the in vivo expression of two new transgenes, Tg (-935/+977) and Tg (-451/+977). Tg (-935/+977) was expressed by Sertoli cells but, in contrast to Tg (-2065/+977), was expressed at all stages of the cycle, by spermatocytes, by the vascular endothelium, and by seven other organs. Tg (-451/+977) was not expressed by Sertoli cells but by spermatogenic cells and by the brain. Lack of expression of Tg (-451/+977) by Sertoli cells was not due to a lack of essential cis-acting elements. Transient transfection analysis of primary cultures of mature rat Sertoli cells demonstrated that in mature Sertoli cells, most of the activity of the Ctsl promoter is accounted for by one of two redundant upstream GC motifs and an Initiator that are within 100 bp of the transcription start site. We conclude that transcriptional repressors upstream from nucleotide -935 of the rat Ctsl gene restrict testicular expression of this gene to Sertoli cells at stages V-VIII. At these stages, transcriptional activators located between nucleotides -935 and -452 promote access of the transcriptional machinery to the two GC boxes and to the Initiator. Thus, upstream repressors and activators as well as cis-acting elements near the transcription start site control stage-specific Ctsl transcription by Sertoli cells.

Stage-specific gene expression is a fundamental characteristic of rat spermatogenic cells and Sertoli cells.

Mammalian spermatogenesis is a complex biological process that occurs within a highly organized tissue, the seminiferous epithelium. The coordinated maturation of spermatogonia, spermatocytes, and spermatids suggests the existence of precise programs of gene expression in these cells and in their neighboring somatic Sertoli cells. The objective of this study was to identify the genes that execute these programs. Rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, and XIII-XIV of the cycle were isolated by microdissection, whereas Sertoli cells, spermatogonia plus early spermatocytes, pachytene spermatocytes, and round spermatids were purified from enzymatically dispersed testes. Microarray analysis by using Rat Genome 230 2.0 arrays identified 16,971 probe sets that recognized testicular transcripts, and 398 of these were identified as testis-specific. Expression of 1,286 probe sets were found to differ at least 4-fold between two cell types and also across the stages of the cycle. Pathway and annotated cluster analyses of those probe sets predicted that entire biological pathways and processes are regulated cyclically in specific cells. Important among these are the cell cycle, DNA repair, and embryonic neuron development. Taken together, these data indicate that stage-regulated gene expression is a widespread and fundamental characteristic of spermatogenic cells and Sertoli cells.

Phosphorylation of mitogen-activated protein kinase 8 (MAPK8) is associated with germ cell apoptosis and redistribution of the Bcl2-modifying factor (BMF).

Successful spermatogenesis requires that germ cells remain in physical contact with Sertoli cells until spermiation. Previous studies have shown that the Bcl2-modifying factor (BMF) is a proapoptotic protein found in many epithelial cells which, when phosphorylated by the active form of mitogen-activated protein kinase 8 (p-MAPK8), initiates apoptosis in response to loss of adhesion of the cells to their basal lamina. Based on this, we hypothesized that p-MAPK8 and BMF may play important roles in the apoptotic death of testicular germ cells in response to their detachment from Sertoli cells. Immunohistochemical analysis of the normal rat testis revealed p-MAPK8 expression in spermatocytes and elongated spermatids but not in round spermatids. This localization was opposite to that of BMF, which is expressed in round spermatids but not in spermatocytes or elongated spermatids. When freshly isolated germ cells were cultured in the absence of Sertoli cells, a condition in which there was widespread germ cell apoptosis, an increase in p-MAPK8 relative to overall MAPK8 protein, was seen by Western blot analysis. Additionally, immunocytochemical analysis showed an increase in immunoreactive p-MAPK8 in round spermatids and spermatocytes in association with BMF expression. From these correlative data, we propose that the activation of MAPK8 and redistribution of BMF may be integrally involved in the mechanism by which specific germ cells undergo programmed cell death in response to their detachment from Sertoli cells.

The cathepsin L first intron stimulates gene expression in rat sertoli cells.

Large amounts of cathepsin L (CTSL), a cysteine protease required for quantitatively normal spermatogenesis, are synthesized by mouse and rat Sertoli cells during stages VI to VII of the cycle of the seminiferous epithelium. We previously demonstrated that all of the regulatory elements required in vivo for both Sertoli cell- and stage-specific expression of the Ctsl gene are present within a ~3-kb genomic fragment that contains 2065 nucleotides upstream of the transcription start site and 977 nucleotides of downstream sequence. Most of the downstream region encodes the first intron. In this study, transient transfection assays using primary Sertoli cell cultures and the TM4 Sertoli cell line established that the Ctsl first intron increased reporter gene activity by ~5-fold. While the intron-mediated enhancement in reporter gene activity was not restricted to the Ctsl promoter, positioning the first intron upstream of the Ctsl promoter in either orientation abolished its stimulatory activity, suggesting that it does not contain a typical enhancer. Mutating the 5'-splice site of the Ctsl first intron or replacing the first intron by the Ctsl fourth intron abolished the stimulatory effect. Finally, the intron-dependent increase in reporter gene activity could be explained in part by an increase in the amounts of total RNA and transcript polyadenylation. Results from this study suggest that the stimulatory effect mediated by the Ctsl first intron may explain in part why Sertoli cells in seminiferous tubules at stages VI to VII produce high levels of CTSL.

Identification of testis-specific male contraceptive targets: insights from transcriptional profiling of the cycle of the rat seminiferous epithelium and purified testicular cells.

In an effort to identify novel targets for the development of nonhormonal male contraceptives, genome-wide transcriptional profiling of the rat testis was performed. Specifically, enzymatically purified spermatogonia plus early spermatocyctes, pachytene spermatocytes, round spermatids, and Sertoli cells was analyzed along with microdissected rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX- XI, XII, XIII-XIV of the cycle of the seminiferous epithelium using RAE 230_2.0 microarrays. The combined analysis of these studies identified 16,971 expressed probe sets on the array. How these expression data, combined with additional bioinformatic data analysis and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis, led to the identification of 58 genes that have 1000-fold higher expression transcriptionally in the testis when compared to over 20 other nonreproductive tissues is described. The products of these genes may play important roles in testicular and/or sperm function, and further investigation on their utility as nonhormonal contraceptive targets is warranted. Moreover, these microarray data have been used to expedite the identification of a mutation in RIKEN cDNA 2410004F06 gene as likely being responsible for spermatogenic failure in a line of infertile mice generated by N-ethyl-N-nitrosourea (ENU) mutagenesis. The microarray data and the qRT-PCR data described are available in the Mammalian Reproductive Genetics database (http://mrg.genetics.washington.edu/).

Bioactivity of androgens within the testes and serum of normal men.

Little is known about how human spermatogenesis is regulated, so it is not surprising that there have been few breakthroughs in the treatment of male infertility resulting from abnormalities of spermatogenesis. Testosterone is the predominant intratesticular steroid in both the rat and man. Previous studies have shown that the testosterone concentration within the rat testis that is required for the quantitative maintenance of spermatogenesis is far higher than the total testosterone concentration in rat blood, indicating that much of the testosterone within the testis might be biologically inactive. In contrast to the rat, little is known about the androgen requirements for human spermatogenesis, in part because, until recently, a minimally invasive method suitable for obtaining intratesticular fluids from the human testis has not been available. Percutaneous aspiration now makes it feasible to do so. A major objective of the present study was to assay the bioactive androgen concentration within the testes of normal, fertile men. Percutaneous aspiration was used to obtain intratesticular fluid from such men, and we adapted a highly sensitive recombinant protein mammalian cell-based bioassay to measure androgen bioactivity. Total intratesticular testosterone concentration, which we define as immunoreactive testosterone as measured by radioimmunoassay, was well in excess of that in serum (1236 +/- 86 nM vs 11.7 +/- 0.7 nM). The concentration of bioactive androgens within the normal human testis was found to be about two thirds that of the total testosterone concentration. Interestingly, the concentration of the major, known binding proteins for testosterone within the testis, serum hormone-binding globulin (SHBG)/ABP (52.4 +/- 9.7 nM), was insufficient to account for the difference between total testosterone and bioactive androgens. This indicates that, in addition to its binding to SHBG/ABP, androgens may also be bound by unknown molecules, and that this contributes to reducing androgen bioactivity. These observations could have relevance for understanding the relationship between spermatogenesis and intratesticular androgens in normal men and in men diagnosed with infertility.

Low-dose human chorionic gonadotropin maintains intratesticular testosterone in normal men with testosterone-induced gonadotropin suppression.

In previous studies of testicular biopsy tissue from healthy men, intratesticular testosterone (ITT) has been shown to be much higher than serum testosterone (T), suggesting that high ITT is needed relative to serum T for normal spermatogenesis in men. However, the quantitative relationship between ITT and spermatogenesis is not known. To begin to address this issue experimentally, we determined the dose-response relationship between human chorionic gonadotropin (hCG) and ITT to ascertain the minimum dose needed to maintain ITT in the normal range. Twenty-nine men with normal reproductive physiology were randomized to receive 200 mg T enanthate weekly in combination with either saline placebo or 125, 250, or 500 IU hCG every other day for 3 wk. ITT was assessed in testicular fluid obtained by percutaneous fine needle aspiration at baseline and at the end of treatment. Baseline serum T (14.1 nmol/liter) was 1.2% of ITT (1174 nmol/liter). LH and FSH were profoundly suppressed to 5% and 3% of baseline, respectively, and ITT was suppressed by 94% (1234 to 72 nmol/liter) in the T enanthate/placebo group. ITT increased linearly with increasing hCG dose (P < 0.001). Posttreatment ITT was 25% less than baseline in the 125 IU hCG group, 7% less than baseline in the 250 IU hCG group, and 26% greater than baseline in the 500 IU hCG group. These results demonstrate that relatively low dose hCG maintains ITT within the normal range in healthy men with gonadotropin suppression. Extensions of this study will allow determination of the ITT concentration threshold required to maintain spermatogenesis in man.

Simultaneous determination of steroid composition of human testicular fluid using liquid chromatography tandem mass spectrometry.

A rapid, sensitive, and specific method using liquid chromatography tandem mass spectrometry (LC/MS/MS) has been developed for simultaneous determination of testosterone (T), dihydrotestosterone (DHT), estradiol (E2), and 5alpha-androstan-3alpha, -17beta-diol (3alpha-Diol) within human testicular fluid. Sample pretreatment involved a one-step extraction with diethyl ether. The analytes were separated on a Waters X-Terra C18 (150 mm x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (70:30, v/v) containing 0.1% formic acid using isocratic flow at 0.15 ml/min for 8 min. The column effluent was monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 0.1-50 ng/ml for T, 0.02-1 ng/ml for DHT, 0.05-2 ng/ml for E2, and 0.2-10 ng/ml for 3alpha-Diol, with values for the coefficient of determination of >0.99. The overall extraction efficiency was greater than 86% for T, 75% for DHT, 66% for E2, and 60% for 3alpha-Diol. The values for within-day and between-day precision and accuracy were <15%. We measured each of the four steroids in testicular sample volumes of only 20 microl, obtained by percutaneous testicular aspiration. The mean intratesticular testosterone concentration found by LC/MS/MS, 572 +/- 102 ng/ml, was similar to that previously obtained by radioimmunoassay (RIA). The mean intratesticular estradiol concentration was 15.7 +/- 2.3 ng/ml, which also correlated well with RIA measurement. Both DHT and 3alpha-Diol were below the limits of detection by RIA, but could be measured accurately by LC/MS/MS. In conclusion, LC/MS/MS represents a sensitive and accurate means by which to measure four separate steroids within small volume samples of testicular fluid.

Intratesticular testosterone concentrations comparable with serum levels are not sufficient to maintain normal sperm production in men receiving a hormonal contraceptive regimen.

Intratesticular testosterone (ITT) is thought to play a key role in the control of spermatogenesis in man but is rarely measured. The purposes of this study were 1) to examine the relationship between intratesticular fluid and serum testosterone (T) at baseline and during treatment with a contraceptive regimen known to suppress spermatogenesis and 2) to measure intratesticular fluid androgenic bioactivity. Seven men received 6 months of T enanthate (TE) 100 mg weekly intramuscularly plus levonorgestrel (LNG) 62.5 or 31.25 microg orally daily. Testicular fluid was obtained by percutaneous aspiration at baseline and during month 6. Mean luteinizing hormone (LH) was suppressed 98% from 3.79 +/- 0.80 IU/L at baseline to 0.08 +/- 0.03 IU/L. Mean follicle stimulating hormone (FSH) was suppressed 97%, from 3.29 +/- 0.67 IU/L to 0.10 +/- 0.03 IU/L. Mean serum T levels were similar before (22.8 +/- 1.9 nmol/L) and during treatment (28.7 +/- 2.0 nmol/L) (P = .12). ITT (822 +/- 136 nmol/L) was approximately 40x higher than serum T (P < .001) at baseline. ITT was suppressed 98% during treatment to 13.1 +/- 4.5 nmol/L, a level similar to baseline serum T (P = .08) but significantly lower than on-treatment serum T (P = .01). At baseline, intratesticular fluid androgenic bioactivity (583 +/- 145 nmol/L) was 70% of the ITT concentration measured by radioimmunoassay. Intratesticular androgenic bioactivity was suppressed 93% to 40 +/- 22 nmol/L (P < .01) during treatment, but was 3x higher than ITT (13.1 +/- 4.5 nmol/L). Sperm counts declined from 65 +/- 15 million/mL to 1.3 +/- 1.3 million/mL. In summary, TE plus LNG dramatically suppressed ITT (98%) and intratesticular androgenic bioactivity (93%) to levels approximating those in serum. ITT levels comparable with serum T were insufficient to support normal spermatogenesis. Intratesticular androgenic bioactivity was higher than ITT during treatment, suggesting that other androgens may be prevalent in the low-ITT environment.

Lewis X-containing neoglycoproteins mimic the intrinsic ability of zona pellucida glycoprotein ZP3 to induce the acrosome reaction in capacitated mouse sperm.

The binding of zona pellucida (ZP) glycoprotein ZP3 to mouse sperm surface receptors is mediated by protein-carbohydrate interactions. Subsequently, ZP3 induces sperm to undergo the acrosome reaction, an obligatory step in fertilization. We have previously identified Lewis X (Le(x); Gal beta 4[Fuc alpha 3]GlcNAc) as a potent inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273:1888-1895). This glycan is recognized by approximately 70% of the ZP3 binding sites on capacitated, acrosome-intact mouse sperm, whereas Lewis A (Le(a); Gal beta 3[Fuc alpha 4]GlcNAc) is recognized by most of the remaining sites (Kerr et al. Biol Reprod 2004; 71:770-777). Herein, we test the hypothesis that Le(x)- and Le(a)-containing glycans, when clustered on a neoglycoprotein, bind ZP3 receptors on sperm and induce sperm to undergo the acrosome reaction via the same signaling pathways as ZP3. Results show that a Le(x)-containing neoglycoprotein induced the acrosome reaction in a dose-dependent and capacitation-dependent manner. A Le(a)-containing neoglycoprotein also induced sperm to undergo the acrosome reaction but was less potent than Le(x)-containing neoglycoproteins. In contrast, neoglycoproteins containing beta4-lactosamine (Gal beta 4GlcNAc), Lewis B (Fuc alpha 2Gal beta 3[Fuc alpha 4]GlcNAc), and sialyl-Le(x) glycans were inactive, as were four other neoglycoproteins with different nonfucosylated glycans. Consistent with these results, unconjugated Le(x)- and Le(a)-capped glycans were dose-dependent inhibitors, which at saturation, reduced the ZP-induced acrosome reaction by about 60% and 30%, respectively. Experiments utilizing pharmacological inhibitors suggest that induction of the acrosome reaction by solubilized ZP and Le(x)- and Le(a)-containing neoglycoproteins require the same calcium-dependent pathway. However, only the ZP-induced acrosome reaction requires a functional G(i) protein. Thus, Le(x)-containing neoglycoproteins bind to a major class of ZP3 receptors on capacitated sperm. A Le(a)-containing neoglycoprotein binds a second ZP3 receptor but is a less-potent inducer of the acrosome reaction.

Lewis X-containing glycans are specific and potent competitive inhibitors of the binding of ZP3 to complementary sites on capacitated, acrosome-intact mouse sperm.

Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Le(x))-containing glycan (Gal beta 4[Fuc alpha 3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888-1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Le(x)-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa(568))-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC(50) approximately 180 nM), which at saturation, reduced Alexa(568)-ZP3 binding by approximately 70%. A Lewis A (Le(a))-capped glycan (Gal beta 3[Fuc alpha 4]GlcNAc) was also a potent inhibitor (IC(50) approximately 150-200 nM), but at saturation, it reduced Alexa(568)-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAc beta 4 or Gal beta 4 residues did not compete; neither did sialyl-Le(x) (Neu5Ac alpha 3Gal beta 4[Fuc alpha 3]GlcNAc-Lewis X) nor sulfo-Le(x) (3'-O-SO(3)-Lewis X). However, at saturation, Gal alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc reduced Alexa(568)-ZP3 binding by approximately 70% but with moderate apparent affinity (IC(50) approximately 3000 nM). Fluorescence microscopy revealed that Alexa(568)-labeled Le(x)-Lac-BSA, Le(a)-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Le(a)-Lac did not inhibit binding of Alexa(568)-Le(x)-Lac-BSA, and Le(x)-Lac did not inhibit binding of Alexa(568)-Le(a)-Lac-BSA. Finally, Le(x)-Lac and Le(a)-Lac had an additive inhibitory effect on Alexa(568)-ZP3 binding. Thus, Le(x) is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Le(a) binding defines a different but less-abundant class of sites.