Exposed and Sequestered Antigens in Testes and Their Protection by Regulatory T Cell-Dependent Systemic Tolerance.

Continuous exposure of tissue antigen (Ag) to the autoantigen-specific regulatory T cells (Treg) is required to maintain Treg-dependent systemic tolerance. Thus, testis autoantigens, previously considered as sequestered, may not be protected by systemic tolerance. We now document that the complete testis antigen sequestration is not valid. The haploid sperm Ag lactate dehydrogenase 3 (LDH3) is continuously exposed and not sequestered. It enters the residual body (RB) to egress from the seminiferous tubules and interact with circulating antibody (Ab). Some LDH3 also remains inside the sperm cytoplasmic droplets (CD). Treg-depletion in the DEREG mice that express diphtheria toxin receptor on the Foxp3 promoter results in spontaneous experimental autoimmune orchitis (EAO) and Ab to LDH3. Unlike the wild-type male mice, mice deficient in LDH3 (wild-type female or LDH3 NULL males) respond vigorously to LDH3 immunization. However, partial Treg depletion elevated the wild-type male LDH3 responses to the level of normal females. In contrast to LDH3, zonadhesin (ZAN) in the sperm acrosome displays properties of a sequestered Ag. However, when ZAN and other sperm Ag are exposed by vasectomy, they rapidly induce testis Ag-specific tolerance, which is terminated by partial Treg-depletion, leading to bilateral EAO and ZAN Ab response. We conclude that some testis/sperm Ag are normally exposed because of the unique testicular anatomy and physiology. The exposed Ag: 1) maintain normal Treg-dependent systemic tolerance, and 2) are pathogenic and serve as target Ag to initiate EAO. Unexpectedly, the sequestered Ags, normally non-tolerogenic, can orchestrate de novo Treg-dependent, systemic tolerance when exposed in vasectomy.

The sperm-specific form of lactate dehydrogenase is required for fertility and is an attractive target for male contraception (a review).

There has been a recent upsurge in the interest about contraceptive development, evidenced by the Contraceptive Special Issue of Biology of Reproduction [1], with research funding from the Male Contraceptive Initiative and the Bill and Melinda Gates Foundation. Support from the Contraceptive Research Branch of the Eunice Kennedy Shriver National Institutes of Child Health and Human Development continues with a marked change in focus in the funding announcements. This has motivated me to reflect on research, mostly from my laboratory starting in the 1960s to the present, on the development of a male contraceptive based on the sperm-specific glycolytic enzyme, lactate dehydrogenase C (LDHC4). This review considers the rationale behind this research, the development paths pursued, obstacles encountered, and the renewed interest in going forward toward development of a male contraceptive mediated by the inhibition of the sperm-specific form of LDHC. I will address how some papers published many years ago are relevant to the present goals of non-hormonal contraception and will mention about innovative technology now available that can advance this project. This review presumably will serve as an instructive guide for a research program with a focused program related to contraception. As an aside, many of the citations in this review are to most of the 26 publications in Biology of Reproduction co-authored by this investigator and collaborators from 1974 through 2020 not long after the first issue of BOR which was published in April 1969.

Preclinical contraceptive development for men and women.

This manuscript endeavors to present research considerations for the preclinical development of non-hormonal contraceptives. Topics include (1) how advances in genomics and bioinformatics impact the identification of novel targets for non-hormonal contraception, (2) the importance of target validation prior to investment in a contraceptive development campaign, (3) considerations on targeting gametogenesis vs gamete maturation/function, (4) how targets from the male reproductive system are expanding women's options for 'on demand' contraception, and (5) some emerging non-hormonal methods that are not based on a specific molecular target. Also presented are ideas for developing a pipeline of non-hypothalamic-pituitary-gonadal-acting contraceptives for men and women while balancing risk and innovation, and our perspective on the pros and cons of industry and academic environments on contraceptive development. Three product development programs are highlighted that are biologically interesting, innovative, and likely to influence the field of contraceptive development in years to come.

Celebrating the Silver Anniversary of the North American Testis Workshop.

OBJECTIVE: To provide an overview of the history of the North American Testis Workshop (NATW), of its relationship to the American Society of Andrology (ASA), and of the publications that resulted from the first 25 workshops. METHODS: The collection of volumes and journal articles that relate to the NATW was searched. DISCUSSION AND CONCLUSION: During the first twenty-five meetings of the NATW, a remarkable number of breakthroughs regarding every aspect of the testis were presented. We anticipate that with the acceleration of new genetic, epigenetic, and molecular knowledge of the functions of testicular cells, we will continue to learn about the discovery of new and clinically important aspects of testicular function during the next twenty-five NATWs.

Egress of sperm autoantigen from seminiferous tubules maintains systemic tolerance.

Autoimmune responses to meiotic germ cell antigens (MGCA) that are expressed on sperm and testis occur in human infertility and after vasectomy. Many MGCA are also expressed as cancer/testis antigens (CTA) in human cancers, but the tolerance status of MGCA has not been investigated. MGCA are considered to be uniformly immunogenic and nontolerogenic, and the prevailing view posits that MGCA are sequestered behind the Sertoli cell barrier in seminiferous tubules. Here, we have shown that only some murine MGCA are sequestered. Nonsequestered MCGA (NS-MGCA) egressed from normal tubules, as evidenced by their ability to interact with systemically injected antibodies and form localized immune complexes outside the Sertoli cell barrier. NS-MGCA derived from cell fragments that were discarded by spermatids during spermiation. They egressed as cargo in residual bodies and maintained Treg-dependent physiological tolerance. In contrast, sequestered MGCA (S-MGCA) were undetectable in residual bodies and were nontolerogenic. Unlike postvasectomy autoantibodies, which have been shown to mainly target S-MGCA, autoantibodies produced by normal mice with transient Treg depletion that developed autoimmune orchitis exclusively targeted NS-MGCA. We conclude that spermiation, a physiological checkpoint in spermatogenesis, determines the egress and tolerogenicity of MGCA. Our findings will affect target antigen selection in testis and sperm autoimmunity and the immune responses to CTA in male cancer patients.

Lactate Dehydrogenase C Produces S-2-Hydroxyglutarate in Mouse Testis.

Metabolomics is a valuable tool for studying tissue- and organism-specific metabolism. In normal mouse testis, we found 70 µM S-2-hydroxyglutarate (2HG), more than 10-fold greater than in other tissues. S-2HG is a competitive inhibitor of α-ketoglutarate-dependent demethylation enzymes and can alter histone or DNA methylation. To identify the source of testis S-2HG, we fractionated testis extracts and identified the fractions that actively produced S-2HG. Through a combination of ion exchange and size exclusion chromatography, we enriched a single active protein, the lactate dehydrogenase isozyme LDHC, which is primarily expressed in testis. At neutral pH, recombinant mouse LDHC rapidly converted both pyruvate into lactate and α-ketoglutarate into S-2HG, whereas recombinant human LDHC only produced lactate. Rapid S-2HG production by LDHC depends on amino acids 100-102 being Met-Val-Ser, a sequence that occurs only in the rodent protein. Other mammalian LDH can also produce some S-2HG, but at acidic pH. Thus, polymorphisms in the Ldhc gene control testis levels of S-2HG, and thereby epigenetics, across mammals.

Male fertility and obesity: are ghrelin, leptin and glucagon-like peptide-1 pharmacologically relevant?

Obesity is rising to unprecedented numbers, affecting a growing number of children, adolescents and young adult men. These individuals face innumerous health problems, including subfertility or even infertility. Overweight and obese men present severe alterations in their body composition and hormonal profile, particularly in ghrelin, leptin and glucagon-like peptide-1 (GLP-1) levels. It is well known that male reproductive health is under the control of the individual's nutritional status and also of a tight network of regulatory signals, particularly hormonal signaling. However, few studies have been focused on the effects of ghrelin, leptin and GLP-1 in male reproduction and how energy homeostasis and male reproductive function are linked. These hormones regulate body glucose homeostasis and several studies suggest that they can serve as targets for anti-obesity drugs. In recent years, our understanding of the mechanisms of action of these hormones has grown significantly. Curiously, their effect on male reproductive potential, that is highly dependent of the metabolic cooperation established between testicular cells, remains a matter of debate. Herein, we review general concepts of male fertility and obesity, with a special focus on the effects of ghrelin, leptin and GLP-1 on male reproductive health. We also discuss the possible pharmacological relevance of these hormones to counteract the fertility problems that overweight and obese men face.

Male contraception: another Holy Grail.

The idea that men should participate in family planning by playing an active role in contraception has become more acceptable in recent years. Up to the present the condom and vasectomy have been the main methods of male contraception. There have been and continue to be efforts to develop an acceptable hormonal contraceptive involving testosterone (T) suppression. However the off target affects, delivery of the analogs and the need for T replacement have proven difficult obstacles to this technology. Research into the development of non-hormonal contraception for men is progressing in several laboratories and this will be the subject of the present review. A number of promising targets for the male pill are being investigated. These involve disruption of spermatogenesis by compromising the integrity of the germinal epithelium, interfering with sperm production at the level of meiosis, attacking specific sperm proteins to disrupt fertilizing ability, or interfering with the assembly of seminal fluid components required by ejaculated sperm for acquisition of motility. Blocking contractility of the vas deferens smooth muscle vasculature to prevent ejaculation is a unique approach that prevents sperm from reaching the egg. We shall note the lack of interest by big pharma with most of the support for male contraception provided by the NIH.

Male contraception: past, present and future.

Current contraceptive options available to men include withdrawal, condoms, and vasectomy, each of which has its own drawbacks. In this chapter we will describe the pros and cons for each, as well as methodological and product updates. Statistics from the U.S. Centers for Disease Control on acceptance and satisfaction will be included. Advances in vasectomy and reversal will be presented. Methods to develop new contraceptive technologies fall into two categories: hormonal and non-hormonal. Many targets and strategies have been proposed for non-hormonal male contraception within the testis. Targets include structural components in the testis, as well as enzymes, ion channels and other proteins specific to spermatozoa. Here we provide an overview of the spermatogenic mechanisms and proteins that have received research interest to date. We also discuss potential novel targets, such as ubiquitin specific proteases, that warrant greater research emphasis.

Glycolysis and mitochondrial respiration in mouse LDHC-null sperm.

We demonstrated previously that a knockout (KO) of the lactate dehydrogenase type C (Ldhc) gene disrupted male fertility and caused a considerable reduction in sperm glucose consumption, ATP production, and motility. While that study used mice with a mixed genetic background, the present study used C57BL/6 (B6) and 129S6 (129) Ldhc KO mice. We found that B6 KO males were subfertile and 129 KO males were infertile. Sperm from 129 wild-type (WT) mice have a lower glycolytic rate than sperm from B6 WT mice, resulting in a greater reduction in ATP production in 129 KO sperm than in B6 KO sperm. The lower glycolytic rate in 129 sperm offered a novel opportunity to examine the role of mitochondrial respiration in sperm ATP production and motility. We observed that in media containing a mitochondrial substrate (pyruvate or lactate) as the sole energy source, ATP levels and progressive motility in 129 KO sperm were similar to those in 129 WT sperm. However, when glucose was added, lactate was unable to maintain ATP levels or progressive motility in 129 KO sperm. The rate of respiration (ZO2) was high when 129 KO or WT sperm were incubated with lactate alone, but addition of glucose caused a reduction in ZO2. These results indicate that in the absence of glucose, 129 sperm can produce ATP via oxidative phosphorylation, but in the presence of glucose, oxidative phosphorylation is suppressed and the sperm utilize aerobic glycolysis, a phenomenon known as the Crabtree effect.

Human lactate dehydrogenase A (LDHA) rescues mouse Ldhc-null sperm function.

By targeted disruption of the lactate dehydrogenase c (Ldhc) gene, we demonstrated that spermatozoa require Ldhc for capacitation, motility, and fertilizing capacity. Ldhc expression is restricted to the developing germ cells that, however, are apparently not compromised by the lack of the LDHC isozyme. Because LDHC is abundant in spermatozoa that utilize aerobic glycolysis for energy requirements, its main function was presumed to be the interconversion of pyruvate to lactate with the concomitant oxidation/reduction of NADH to NAD(+). We found that sperm without LDHC were still able to convert lactate to pyruvate as mediated by LDHA that is tightly bound to the fibrous sheath. It was assumed that the level of glycolysis was insufficient to power motility and the subsequent fertilizing capacity of the mutated sperm. To investigate whether LDHC possesses certain unique characteristics essential for fertility, human LDHA was introduced as a transgene to Ldhc-null mice. We report here that the exogenous LDHA rescued the phenotype of the Ldhc-null males. Sperm from the LDHA transgenic males with the Ldhc deletion (LDHA(+)/Ldhc(-/-)) are motile, capable of protein tyrosine phosphorylation, and able to fertilize, thus restoring these properties to LDHC-null sperm. However, the lactate and ATP levels in the rescued sperm did not differ significantly from sperm lacking LDHC. We suggest that it is the localization of the transgene to the sperm cytosol that is mainly responsible for restoration of sperm function and fertility.

A-MYB (MYBL1) stimulates murine testis-specific Ldhc expression via the cAMP-responsive element (CRE) site.

Generally, knowledge of the mechanism regulating gene expression in primary spermatocytes is incomplete. We have used the lactate dehydrogenase gene (Ldhc) as a model to explore these mechanisms during spermatogenesis. Its 100-bp core promoter contained two essential elements common to many genes, a GC box and a CRE site. Here we report results that support a model in which transcription factor MYBL1 acts as a coactivator directing tissue-specific expression via the CRE cis element. We hypothesize that this is a common mechanism involving activation of multiple genes in the primary spermatocyte. MYBL1 is expressed predominantly as a tissue-specific transcription factor in spermatocytes and breast epithelial cells. Our finding that LDHC expression is lost in 21-day testes of MYBL1 mutant mice supports our hypothesis. In the GC1-spg germ cell line exogenous MYBL1 induces activity 4- to 8-fold, although extracts from these cells do not show MYBL1 binding activity for the Myb consensus sequences in the Ldhc promoter by EMSA. Rather, MYBL1 stimulates expression from a synthetic promoter containing only CRE elements, suggesting MYBL1 activates the promoter by interacting with protein that binds to a CRE element. Mutation of three Myb sites does not affect Ldhc promoter activity significantly (P > 0.05). CREB-binding protein (CBP) is a coactivator that interacts with CRE-binding protein CREB. We show that the transactivation domain (TAD) in MYBL1 interacts with the KIX domain in CBP, and the TAD domain and DNA binding domain in MYBL1 each interact with the CREB N-terminal domain. MYBL1 also stimulated expression from testis-specific genes Pgk2 (phosphoglycerate kinase 2) and Pdha2 (pyruvate dehydrogenase alpha 2) promoters, each of which contains CRE promoter elements and is expressed in primary spermatocytes. We propose that MYBL1 directs germ cell-specific activation via the CRE site of certain genes that are activated specifically in the primary spermatocyte, although other, more indirect effects of MYBL1 remain a possible explanation for our results.

Lactate dehydrogenase C and energy metabolism in mouse sperm.

We demonstrated previously that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) led to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD(+) cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC.

LDHC: the ultimate testis-specific gene.

Lactate dehydrogenase C (LDHC) was, to the best of our knowledge, the first testis-specific isozyme discovered in male germ cells. In fact, this was accomplished shortly before "isozymes or isoenzymes" became a field of study. LDHC was detected initially in human spermatozoa and spermatogenic cells of the testes by gel electrophoresis. Immunohistochemistry was used to localize LDHC first in early-pachytene primary spermatocytes, with an apparent increase in quantity after meiosis, to its final localization in and on the principal piece of the sperm tail. After several decades of biologic, biochemical, and genetic investigations, we now know that the lactate dehydrogenase isozymes are ubiquitous in vertebrates, developmentally regulated, tissue and cell specific, and multifunctional. Here, we will review the history of LDHC and the work that demonstrates clearly that it is required for sperm to accomplish their ultimate goal, fertilization.

Computational analyses of mammalian lactate dehydrogenases: human, mouse, opossum and platypus LDHs.

Computational methods were used to predict the amino acid sequences and gene locations for mammalian lactate dehydrogenase (LDH) genes and proteins using genome sequence databanks. Human LDHA, LDHC and LDH6A genes were located in tandem on chromosome 11, while LDH6B and LDH6C genes were on chromosomes 15 and 12, respectively. Opossum LDHC and LDH6B genes were located in tandem with the opossum LDHA gene on chromosome 5 and contained 7 (LDHA and LDHC) or 8 (LDH6B) exons. An amino acid sequence prediction for the opossum LDH6B subunit gave an extended N-terminal sequence, similar to the human and mouse LDH6B sequences, which may support the export of this enzyme into mitochondria. The platypus genome contained at least 3 LDH genes encoding LDHA, LDHB and LDH6B subunits. Phylogenetic studies and sequence analyses indicated that LDHA, LDHB and LDH6B genes are present in all mammalian genomes examined, including a monotreme species (platypus), whereas the LDHC gene may have arisen more recently in marsupial mammals.

Homo sapiens lactate dehydrogenase c (Ldhc) gene expression in cancer cells is regulated by transcription factor Sp1, CREB, and CpG island methylation.

The human testis-specific lactate dehydrogenase c gene (hLdhc) is transcribed only in cells of the germinal epithelium. Recently hLdhc was reported to express in a broad spectrum of tumors with relatively high frequency in lung cancer, melanoma, and breast cancer, and in some prostate cancers. Two melanoma cell lines that express the hLdhc gene, A375M and C81-61, were identified and were used to characterize the hLdhc promoter and explore transcriptional regulation of this gene. A 110-bp core promoter, including a conserved GC box and cyclic adenosine monophosphate-responsive element (CRE), were identified as essential for basal promoter activity. The methylation status of the CpG island (CGI) in the hLdhc core promoter sequence was analyzed in hLdhc-expressing and nonexpressing cells and human prostate tumor tissues. The CGI in 2 cell lines expressing the gene was hypomethylated whereas the DNA from cells that did not express hLdhc was hypermethylated. The role of methylation in regulating this promoter was confirmed by experimental induction of hLdhc transcription with the methylation inhibitor 5'aza-deoxycytidine. Quantitative analyses of the methylation level in the CGI were performed in prostate tumor tissues by pyrosequencing. Overall, these experiments demonstrated that hLdhc expression in cancer cells was regulated by transcription factor Sp1 and CREB and promoter CGI methylation. In addition, these findings suggest the possibility of developing a biomarker for cancer diagnosis/prognosis based on DNA methylation of the Ldhc gene.

Testis specific lactate dehydrogenase as target for immunoliposomes.

PROBLEM: Is it possible to deliver therapeutic agents to testis through specific targeting? METHOD OF STUDY: Immunoliposomes are designed by incorporating antibodies to lactate dehydrogenase-C4 (LDH-C(4)), which is the product of a testis specific gene. Their targeting and delivering ability is investigated in vitro and in vivo. RESULTS: It is observed that LDHC(4)-immunoliposomes are able to discriminate and recognize antigens on spermatozoa and testes both in vitro and in vivo. CONCLUSION: Specific targeting through LDH-C(4) appears to be a feasible strategy for delivering therapeutic as well as anti-spermatogenic agents to testis.

Expression of the gene for mouse lactate dehydrogenase C (Ldhc) is required for male fertility.

The lactate dehydrogenase (LDH) protein family members characteristically are distributed in tissue- and cell type-specific patterns and serve as the terminal enzyme of glycolysis, catalyzing reversible oxidation reduction between pyruvate and lactate. They are present as tetramers, and one family member, LDHC, is abundant in spermatocytes, spermatids, and sperm, but also is found in modest amounts in oocytes. We disrupted the Ldhc gene to determine whether LDHC is required for spermatogenesis, oogenesis, and/or sperm and egg function. The targeted disruption of Ldhc severely impaired fertility in male Ldhc(-/-) mice but not in female Ldhc(-/-) mice. Testis and sperm morphology and sperm production appeared to be normal. However, total LDH enzymatic activity was considerably lower in Ldhc(-/-) sperm than in wild type sperm, indicating that the LDHC homotetramer (LDH-C(4)) is responsible for most of the LDH activity in sperm. Although initially motile when isolated, there was a more rapid reduction in the level of ATP and in motility in Ldhc(-)(/-) sperm than in wild-type sperm. Moreover, Ldhc(-/-) sperm did not acquire hyperactivated motility, were unable to penetrate the zona pellucida in vitro, and failed to undergo the phosphorylation events characteristic of capacitation. These studies showed that LDHC plays an essential role in maintenance of the processes of glycolysis and ATP production in the flagellum that are required for male fertility and sperm function.

Regulation of murine lactate dehydrogenase C (Ldhc) gene expression.

Expression of Ldhc begins with the onset of meiosis in male germ cells and continues throughout spermatogenesis. Transcriptional regulatory mechanisms, especially in primary spermatocytes, are poorly described because of the lack of a reliable cell culture system. We constructed mouse transgenics and transfected germ cells in situ to study expression of the testis-specific isozyme of lactate dehydrogenase (LDH). From previous work, we determined that a 100-bp Ldhc core promoter contained potential cis regulatory elements, including a palindrome (-21 to +10), GC box (-70 to -65), and cAMP-responsive element (CRE) sites (-53 to -49, -39 to -35). We provide here the demonstration of a functional role for these sequences by expression of mutated transgenes in vivo. Our results reveal for the first time that mutation of the GC box does not abolish promoter activity, which remains testis-specific. Mutation of GC box or CRE sites resulted in a 73% and 74% reduction in promoter activity, respectively, in a transient transfection of germ cells in vivo by electroporation; the combination of GC box and CRE site mutations eliminates promoter activity. Therefore, we conclude that simultaneous occupancy of the GC box and CRE sites in the core promoter is necessary for full expression of Ldhc in the testis.