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.

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.

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.

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.

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.

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.

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.

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.

Testis-specific cytochrome c-null mice produce functional sperm but undergo early testicular atrophy.

Differentiating male germ cells express a testis-specific form of cytochrome c (Cyt c(T)) that is distinct from the cytochrome c expressed in somatic cells (Cyt c(S)). To examine the role of Cyt c(T) in germ cells, we generated mice null for Cyt c(T). Homozygous Cyt c(T)(-/-) pups were statistically underrepresented (21%) but developed normally and were fertile. However, spermatozoa isolated from the cauda epididymis of Cyt c(T)-null animals were less effective in fertilizing oocytes in vitro and contain reduced levels of ATP compared to wild-type sperm. Sperm from Cyt c(T)-null mice contained a greater number of immotile spermatozoa than did samples from control mice, i.e., 53.1% +/- 13.7% versus 33.2% +/- 10.3% (P < 0.0001) for vas deferens sperm and 40.1% +/- 9.6% versus 33.2% +/- 7.5% (P = 0.0104) for epididymal sperm. Cyt c(T)-null mice often exhibit early atrophy of the testes after 4 months of age, losing germ cells as a result of increased apoptosis. However, no difference in the activation of caspase-3, -8, or -9 was detected between the Cyt c(T)(-/-) testes and controls. Our data indicate that the Cyt c(T)-null testes undergo early atrophy equivalent to that which occurs during aging as a consequence of a reduction in oxidative phosphorylation.

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.

Testis-specific lactate dehydrogenase (LDH-C4; Ldh3) in murine oocytes and preimplantation embryos.

LDH-C(4) (Ldh3) is a member of the lactate dehydrogenase family of isozymes that catalyze the terminal reaction in the glycolytic pathway. In mammals, 3 genes, ldha, ldhb, and Ldhc, encode the subunits that assemble into catalytically active homo- and heterotetramers. Differential expression of these genes determines the lactate dehydrogenase (LDH) isozyme composition of tissues, and, as is well known, A subunits predominate in skeletal muscle and B subunits are abundantly produced in brain and heart, with the Ldh2 isozyme the most abundant form in oocytes. The C peptide can be detected first in pachytene spermatocytes and constitutes the primary LDH of spermatozoa. Originally the Ldhc gene (Ldh3 in terminology applied to murine cells) was considered to be testis specific on the basis of immunochemical, enzymatic, and molecular analyses. Here we report the detection of this isozyme in the murine oocyte and early embryo. Our results indicate that Ldh3 mRNA is transcribed in oocytes and cannot be detected in fertilized eggs. Ldh3 protein, however, persists to the blastocyst stage of embryonic development localizing mainly to the cortex region of oocytes, eggs, zygotes, and embryonic blastomeres.

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.

Gene expression and mucosal immune responses after vaginal DNA immunization in mice using a controlled delivery matrix.

IgA antibodies in the vaginal tract are essential as a first defense line against microorganisms that enter the body via mucosal surfaces. Several studies have shown that direct application of DNA to the vaginal mucosal surface can induce secretion of IgA molecules specific to the expressed protein. The major challenge of formulating effective vaccines is to overcome the barriers to DNA administration caused by the estrus cycle and physical environment of the vaginal tract. In this study, we investigated whether controlled delivery of DNA to the vaginal surface would induce long-term IgA antibody production by applying controlled delivery matrices to the vaginal tract. The controlled DNA delivery matrices were composed of poly(ethylene-co-vinyl acetate) (EVAc) and loaded with a model plasmid encoding sperm-specific lactate dehydrogenase C(4) (LDH-C(4)). These EVAc matrices provided a controlled and sustained DNA release to the vaginal mucosal surface. The DNA released from the EVAc disks was functionally active and capable of transfecting vaginal tissues. When inserted into the vaginal tract of mice, the DNA-loaded EVAc matrices triggered the immune system and induced specific IgA to LDH-C(4) in the vaginal secretions. These results demonstrate that the EVAc disks are efficient and convenient vehicles for delivering DNA to the vaginal tract and providing long-term local immunity.

A transgenic analysis of mouse lactate dehydrogenase C promoter activity in the testis.

Transcription of the mouse testis-specific lactate dehydrogenase c (mldhc) gene is limited to cells of the germinal epithelium. Cloning and analysis of the mldhc promoter revealed that a 100-bp core promoter was able to regulate testis-specific transcription in vitro and in transgenic mice. Surprisingly, expression of the reporter in transgenic testes was limited to pachytene spermatocytes, whereas native LDH-C(4) was detected in pachytene and all later germ cells. To locate additional regulatory sequence that could recapitulate the native LDH-C(4) distribution pattern, we investigated the contribution of 5' and 3' flanking sequences to the regulation of LDH-C(4) expression. We found that transcription factor YY1 binds to the mldhc promoter, that the mldhc 3' untranslated sequence does not permit a postmeiotic expression of a beta-galactosidase reporter in transgenic mice, and that native mldhc mRNA is predominately meiotic, with only a low level of postmeiotic distribution. Our results suggest that the high level of LDH-C(4) in postmeiotic cells results from mRNA and protein stability.

Heat-induced apoptosis of mouse meiotic cells is suppressed by ectopic expression of testis-specific calpastatin.

Calpastatin is a naturally occurring inhibitor of calpain, a protease involved in apoptotic cell death. A testis-specific isoform of calpastatin (tCAST) has been identified that is transcribed in haploid germ cells but not in spermatocytes. To investigate the possible function(s) of tCAST, we tested the hypothesis that the ectopic expression of calpastatin in spermatocytes would suppress the death of these cells in response to an apoptosis-inducing stimulus in vivo. To this end, the 5'-flanking region of the mouse ldhc gene was linked to tCAST, and transgenic mice were generated. Immunohistochemical analysis revealed that, in contrast to control sections in which the signal for tCAST was seen in round spermatids, intense staining was visualized in pachytene spermatocytes in the transgenic animals, indicating that the strategy we used to generate the transgenic animals resulted in the ectopic expression of tCAST in spermatocytes. We then tested the effect of a short period of heating on germ cell apoptosis in the testes of wild-type and transgenic mice. Pachytene spermatocytes were the major germ cell type seen to undergo apoptosis after heat treatment. There were no differences in the number of apoptotic germ cells per seminiferous tubule between wild-type and tCAST transgenic control mice; thus, there was no apparent effect of the transgene on normal apoptosis. Heating resulted in increased numbers of TUNEL-positive germ cells in both wild-type and tCAST transgenic mice, as well as increased testicular DNA fragmentation. Heating the tCAST transgenic mouse testes resulted in significantly fewer apoptotic cells per seminiferous tubule than in wild-type mice at both 8 and 24 hours after treatment. Thus, as hypothesized, the ectopic expression of tCAST in pachytene spermatocytes suppressed germ cell apoptosis.

Abundance of repetitive sequence elements in the mouse testis-specific lactate dehydrogenase-C gene.

We have cloned and sequenced the entire mouse ldhc gene and mapped it physically in relation to the somatic ldha gene. The 2 genes were found to be oriented in head-to-tail fashion with about a 6-kilobase (kb) distance between the 3' end of ldha and the 5' end of ldhc. The ldhc gene is composed of 43% repetitive elements compared to only 16% in the ldha gene. Despite the close physical distance of mouse ldha and ldhc, the 2 genes have a very different content of repetitive elements, and this most likely reflects different levels of selective pressure.

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.