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To become fertile, mammalian sperm are required to undergo capacitation in the female tract or in vitro in defined media containing ions (e.g. HCO3 -, Ca2+, Na+, and Cl-), energy sources (e.g. glucose, pyruvate) and serum albumin (e.g. bovine serum albumin (BSA)). These different molecules initiate sequential and concomitant signaling pathways, leading to capacitation. Physiologically, capacitation induces changes in the sperm motility pattern (e.g. hyperactivation) and prepares sperm for the acrosomal reaction (AR), two events required for fertilization. Molecularly, HCO3 - activates the atypical adenylyl cyclase Adcy10 (aka sAC), increasing cAMP and downstream cAMP-dependent pathways. BSA, on the other hand, induces sperm cholesterol release as well as other signaling pathways. How these signaling events, occurring in different sperm compartments and with different kinetics, coordinate among themselves is not well established. Regarding the AR, recent work has proposed a role for glycogen synthase kinases (GSK3α and GSK3β). GSK3α and GSK3β are inactivated by phosphorylation of residues Ser21 and Ser9, respectively, in their N-terminal domain. Here, we present evidence that GSK3α (but not GSK3β) is present in the anterior head and that it is regulated during capacitation. Interestingly, BSA and HCO3 - regulate GSK3α in opposite directions. While BSA induces a fast GSK3α Ser21 phosphorylation, HCO3 - and cAMP-dependent pathways dephosphorylate this residue. We also show that the HCO3--induced Ser21 dephosphorylation is mediated by hyperpolarization of the sperm plasma membrane potential (Em) and by intracellular pH alkalinization. Previous reports indicate that GSK3 kinases mediate the progesterone-induced AR. Here, we show that GSK3 inhibition also blocks the Ca2+ ionophore ionomycin-induced AR, suggesting a role for GSK3 kinases downstream of the increase in intracellular Ca2+ needed for this exocytotic event. Altogether, our data indicate a temporal and biphasic GSK3α regulation with opposite actions of BSA and HCO3 -. Our results also suggest that this regulation is needed to orchestrate the AR during sperm capacitation., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mammalian sperm must undergo capacitation to become fertilization-competent. While working on mice, we recently developed a new methodology for treating sperm in vitro, which results in higher rates of fertilization and embryo development after in vitro fertilization. Sperm incubated in media devoid of nutrients lose motility, although they remain viable. Upon re-adding energy substrates, sperm resume motility and become capacitated with improved functionality. Here, we explore how sperm energy restriction and recovery (SER) treatment affects sperm metabolism and capacitation-associated signaling. Using extracellular flux analysis and metabolite profiling and tracing via nuclear magnetic resonance (NMR) and mass spectrometry (MS), we found that the levels of many metabolites were altered during the starvation phase of SER. Of particular interest, two metabolites, AMP and L-carnitine, were significantly increased in energy-restricted sperm. Upon re-addition of glucose and initiation of capacitation, most metabolite levels recovered and closely mimic the levels observed in capacitating sperm that have not undergone starvation. In both control and SER-treated sperm, incubation under capacitating conditions upregulated glycolysis and oxidative phosphorylation. However, ATP levels were diminished, presumably reflecting the increased energy consumption during capacitation. Flux data following the fate of 13 C glucose indicate that, similar to other cells with high glucose consumption rates, pyruvate is converted into 13 C-lactate and, with lower efficiency, into 13 C-acetate, which are then released into the incubation media. Furthermore, our metabolic flux data show that exogenously supplied glucose is converted into citrate, providing evidence that in sperm cells, as in somatic cells, glycolytic products can be converted into Krebs cycle metabolites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Romarowski, Fejzo, Nayyab, Martin-Hidalgo, Gervasi, Balbach, Violante, Salicioni, Cross, Levin, Buck and Visconti.)

To acquire fertilization competence, mammalian sperm must undergo several biochemical and physiological modifications known as capacitation. Despite its relevance, the metabolic pathways that regulate the capacitation-related events, including the development of hyperactivated motility, are still poorly described. Previous studies from our group have shown that temporary energy restriction in mouse sperm enhanced hyperactivation, in vitro fertilization, early embryo development and pregnancy rates after embryo transfer, and it improved intracytoplasmic sperm injection results in the bovine model. However, the effects of starvation and energy recovery protocols on human sperm function have not yet been established. In the present work, human sperm were incubated for different periods of time in medium containing glucose, pyruvate and lactate (NUTR) or devoid of nutrients for the starving condition (STRV). Sperm maintained in STRV displayed reduced percentages of motility and kinematic parameters compared to cells incubated in NUTR medium. Moreover, they did not undergo hyperactivation and showed reduced levels of ATP, cAMP and protein tyrosine phosphorylation. Similar to our results with mouse sperm, starvation induced increased intracellular Ca 2+ concentrations. Starved human sperm were capable to continue moving for more than 27 h, but the incubation with a mitochondrial uncoupler or inhibitors of oxidative phosphorylation led to a complete motility loss. When exogenous nutrients were added back (sperm energy recovery (SER) treatment), hyperactivated motility was rescued and there was a rise in sperm ATP and cAMP levels in 1 min, with a decrease in intracellular Ca 2+ concentration and no changes in sperm protein tyrosine phosphorylation. The finding that human sperm can remain motile for several hours under starvation due to mitochondrial use of endogenous metabolites implies that other metabolic pathways may play a role in sperm energy production. In addition, full recovery of motility and other capacitation parameters of human sperm after SER suggests that this treatment might be used to modulate human sperm fertilizing ability in vitro ., Competing Interests: PEV and AMS own equity interest in Sperm Capacitation Technologies Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Marín-Briggiler, Luque, Gervasi, Oscoz-Susino, Sierra, Mondillo, Salicioni, Krapf, Visconti and Buffone.)

We have previously shown that members of the family of testis-specific serine/threonine kinases (TSSKs) are post-meiotically expressed in testicular germ cells and in mature sperm in mammals. The restricted post-meiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggest that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the TSSK6 knock-out (KO) mice and of the double TSSK1/TSSK2 KO. The aim of this study was to develop KO mouse models of TSSK3 and to validate this kinase as a target for the development of a male contraceptive. We used CRISPR/Cas9 technology to generate the TSSK3 KO allele on B6D2F1 background mice. Male heterozygous pups were used to establish three independent TSSK3 KO lines. After natural mating of TSSK3 KO males, females that presented a plug (indicative of mating) were monitored for the following 24 days and no pregnancies or pups were found. Sperm numbers were drastically reduced in all three KO lines and, remarkably, round spermatids were detected in the cauda epididymis of KO mice. From the small population of sperm recovered, severe morphology defects were detected. Our results indicate an essential role of TSSK3 in spermiogenesis and support this kinase as a suitable candidate for the development of novel nonhormonal male contraceptives., (© 2021 Wiley Periodicals LLC.)

Mammalian sperm must undergo two post-testicular processes to become fertilization-competent: maturation in the male epididymis and capacitation in the female reproductive tract. While caput epididymal sperm are unable to move and have not yet acquired fertilization potential, sperm in the cauda epididymis have completed their maturation, can move actively, and have gained the ability to undergo capacitation in the female tract or in vitro. Due to the impossibility of mimicking sperm maturation in vitro, the molecular pathways underlying this process remain largely unknown. We aimed to investigate the use of caput epididymal ligation as a tool for the study of sperm maturation in mice. Our results indicate that after seven days of ligation, caput sperm gained motility and underwent molecular changes comparable with those observed for cauda mature sperm. Moreover, ligated caput sperm were able to activate pathways related to sperm capacitation. Despite these changes, ligated caput sperm were unable to fertilize in vitro. Our results suggest that transit through the epididymis is not required for the acquisition of motility and some capacitation-associated signaling but is essential for full epididymal maturation. Caput epididymal ligation is a useful tool for the study of the molecular pathways involved in the acquisition of sperm motility during maturation.

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. During capacitation, sperm change their motility pattern (i.e., hyperactivation) and become competent to undergo the acrosome reaction. We have recently shown that, in the mouse, sperm capacitation is associated with increased uptake of fluorescently labeled deoxyglucose and with extracellular acidification suggesting enhanced glycolysis. Consistently, in the present work we showed that glucose consumption is enhanced in media that support mouse sperm capacitation suggesting upregulation of glucose metabolic pathways. The increase in glucose consumption was modulated by bicarbonate and blocked by protein kinase A and soluble adenylyl cyclase inhibitors. Moreover, permeable cyclic adenosine monophosphate (cAMP) agonists increase glucose consumption in sperm incubated in conditions that do not support capacitation. Also, the increase in glucose consumption was reduced when sperm were incubated in low calcium conditions. Interestingly, this reduction was not overcome with cAMP agonists. Despite these findings, glucose consumption of sperm from Catsper1 knockout mice was similar to the one from wild type suggesting that other sources of calcium are also relevant. Altogether, these results suggest that cAMP and calcium pathways are involved in the regulation of glycolytic energy pathways during murine sperm capacitation., (© 2020 Wiley Periodicals LLC.)

Male contraception is a very active area of research. Several hormonal agents have entered clinical trials, while potential non-hormonal targets have been brought to light more recently and are at earlier stages of development. The general strategy is to target genes along the molecular pathways of sperm production, maturation, or function, and it is predicted that these novel approaches will hopefully lead to more selective male contraceptive compounds with a decreased side effect burden. Protein kinases are known to play a major role in signaling events associated with sperm differentiation and function. In this review, we focus our analysis on the testis-specific serine kinase (TSSK) protein family. We have previously shown that members of the family of TSSKs are postmeiotically expressed in male germ cells and in mature mammalian sperm. The restricted postmeiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggests that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the Tssk6 knockout (KO) mice and of the double Tssk1 and Tssk2 KO mice and by the male subfertile phenotype observed in a Tssk4 KO mouse model., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To become fertile, mammalian sperm must undergo a series of biochemical and physiological changes known as capacitation. These changes involve crosstalk between metabolic and signaling pathways and can be recapitulated in vitro . In this work, sperm were incubated in the absence of exogenous nutrients (starved) until they were no longer able to move. Once immotile, energy substrates were added back to the media and sperm motility was rescued. Following rescue, a significantly higher percentage of starved sperm attained hyperactivated motility and displayed increased ability to fertilize in vitro when compared with sperm persistently incubated in standard capacitation media. Remarkably, the effects of this treatment continue beyond fertilization as starved and rescued sperm promoted higher rates of embryo development, and once transferred to pseudo-pregnant females, blastocysts derived from treated sperm produced significantly more pups. In addition, the starvation and rescue protocol increased fertilization and embryo development rates in sperm from a severely sub-fertile mouse model, and when combined with temporal increase in Ca 2+ ion levels, this methodology significantly improved fertilization and embryo development rates in sperm of sterile CatSper1 KO mice model. Intracytoplasmic sperm injection (ICSI) does not work in the agriculturally relevant bovine system. Here, we show that transient nutrient starvation of bovine sperm significantly enhanced ICSI success in this species. These data reveal that the conditions under which sperm are treated impact post-fertilization development and suggest that this "starvation and rescue method" can be used to improve assisted reproductive technologies (ARTs) in other mammalian species, including humans., (Copyright © 2019 Navarrete, Aguila, Martin-Hidalgo, Tourzani, Luque, Ardestani, Garcia-Vazquez, Levin, Buck, Darszon, Buffone, Mager, Fissore, Salicioni, Gervasi and Visconti.)

Mammalian sperm undergo a series of biochemical and physiological changes collectively known as capacitation in order to acquire the ability to fertilize. Although the increase in phosphorylation associated with mouse sperm capacitation is well established, the identity of the proteins involved in this signaling cascade remains largely unknown. Tandem mass spectrometry (MS/MS) has been used to identify the exact sites of phosphorylation and to compare the relative extent of phosphorylation at these sites. In the present work, we find that a novel site of phosphorylation on a peptide derived from the radial spoke protein Rsph6a is more phosphorylated in capacitated mouse sperm. The Rsph6a gene has six exons, five of which are conserved during evolution in flagellated cells. The exon containing the capacitation-induced phosphorylation site was found exclusively in eutherian mammals. Transcript analyses revealed at least two different testis-specific splicing variants for Rsph6a.Rsph6a mRNA expression was restricted to spermatocytes. Using antibodies generated against the Rsph6a N-terminal domain, western blotting and immunofluorescence analyses indicated that the protein remains in mature sperm and localizes to the sperm flagellum. Consistent with its role in the axoneme, solubility analyses revealed that Rsph6 is attached to cytoskeletal structures. Based on previous studies in Chlamydomonas reinhardtii, we predict that Rsph6 participates in the interaction between the central pair of microtubules and the surrounding pairs. The findings that Rsph6a is more phosphorylated during capacitation and is predicted to function in axonemal localization make Rsph6a a candidate protein mediating signaling processes in the sperm flagellum., (© The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Mammalian sperm acquire fertilizing capacity in the female tract in a process called capacitation. At the molecular level, capacitation requires protein kinase A activation, changes in membrane potential and an increase in intracellular calcium. Inhibition of these pathways results in loss of fertilizing ability in vivo and in vitro. We demonstrated that transient incubation of mouse sperm with Ca(2+) ionophore accelerated capacitation and rescued fertilizing capacity in sperm with inactivated PKA function. We now show that a pulse of Ca(2+) ionophore induces fertilizing capacity in sperm from infertile CatSper1 (Ca(2+) channel), Adcy10 (soluble adenylyl cyclase) and Slo3 (K(+) channel) KO mice. In contrast, sperm from infertile mice lacking the Ca(2+) efflux pump PMACA4 were not rescued. These results indicate that a transient increase in intracellular Ca(2+) can overcome genetic infertility in mice and suggest this approach may prove adaptable to rescue sperm function in certain cases of human male infertility., Competing Interests: Drs Levin and Buck report owning equity interest in CEP Biotech which has licensed commercialization of a panel of monoclonal antibodies directed against sAC. All other author(s) declare no competing financial interests.

Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2(-/-) mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro., (© 2016. Published by The Company of Biologists Ltd.)

Mammalian sperm acquire fertilizing ability in the female tract in a process known as capacitation. At the molecular level, capacitation is associated with up-regulation of a cAMP-dependent pathway, changes in intracellular pH, intracellular Ca(2+), and an increase in tyrosine phosphorylation. How these signaling systems interact during capacitation is not well understood. Results presented in this study indicate that Ca(2+) ions have a biphasic role in the regulation of cAMP-dependent signaling. Media without added Ca(2+) salts (nominal zero Ca(2+)) still contain micromolar concentrations of this ion. Sperm incubated in this medium did not undergo PKA activation or the increase in tyrosine phosphorylation suggesting that these phosphorylation pathways require Ca(2+). However, chelation of the extracellular Ca(2+) traces by EGTA induced both cAMP-dependent phosphorylation and the increase in tyrosine phosphorylation. The EGTA effect in nominal zero Ca(2+) media was mimicked by two calmodulin antagonists, W7 and calmidazolium, and by the calcineurin inhibitor cyclosporine A. These results suggest that Ca(2+) ions regulate sperm cAMP and tyrosine phosphorylation pathways in a biphasic manner and that some of its effects are mediated by calmodulin. Interestingly, contrary to wild-type mouse sperm, sperm from CatSper1 KO mice underwent PKA activation and an increase in tyrosine phosphorylation upon incubation in nominal zero Ca(2+) media. Therefore, sperm lacking Catsper Ca(2+) channels behave as wild-type sperm incubated in the presence of EGTA. This latter result suggests that Catsper transports the Ca(2+) involved in the regulation of cAMP-dependent and tyrosine phosphorylation pathways required for sperm capacitation., (© 2015 Wiley Periodicals, Inc.)

Genome wide transcriptional profiles offer abundant information regarding mRNA levels in specific tissues, organs or developmental stages. Although these data sets do not offer spatial or cell type-specific information, they can be extremely useful for gene discovery when analyzed by the appropriate techniques. Previously, we proposed and validated the use of combinatorial dataset analysis techniques to identify novel genes required during pre-implantation development. Now we build upon this work to identify genes that have dynamic expression during gametogenesis. Here we present detailed analysis of the expression pattern of leukemia-associated, myeloid/lymphoid or mixed-lineage leukemia; translocated to 1 (Mllt1) gene. We document a novel splice isoform of Mllt1 and confirm that both Mllt1 mRNA isoforms are translated. We provide data supporting that MLLT1 protein isoforms display distinct stage-specific expression during spermiogenesis and adult tissues. Finally, we evaluated genes neighboring the Mllt1 locus, and show dynamic stage specific expression patterns of other genes Catsperd, Prr22, Rfx2 and Slc25a41. We document testes expressed alternative isoforms of Prr22 and Rfx2. These results indicate that transcriptome data mining, combined with specific expression analysis provides a wealth of novel gene expression information., (Copyright © 2014 Elsevier B.V. All rights reserved.)

ADAMs are cell surface metalloproteases that control multiple biological processes by cleaving signaling and adhesion molecules. ADAM13 controls cranial neural crest (CNC) cell migration both by cleaving cadherin-11 to release a promigratory extracellular fragment and by controlling expression of multiple genes via its cytoplasmic domain. The latter activity is regulated by γ-secretase cleavage and the translocation of the cytoplasmic domain into the nucleus. One of the genes regulated by ADAM13, the protease calpain8, is essential for CNC migration. Although the nuclear function of ADAM13 is evolutionarily conserved, it is unclear whether the transcriptional regulation is also performed by other ADAMs and how this process may be regulated. We show that ADAM13 function to promote CNC migration is regulated by two phosphorylation events involving GSK3 and Polo-like kinase (Plk). We further show that inhibition of either kinase blocks CNC migration and that the respective phosphomimetic forms of ADAM13 can rescue these inhibitions. However, these phosphorylations are not required for ADAM13 proteolysis of its substrates, γ-secretase cleavage, or nuclear translocation of its cytoplasmic domain. Of significance, migration of the CNC can be restored in the absence of Plk phosphorylation by expression of calpain-8a, pointing to impaired nuclear activity of ADAM13., (© 2014 Abbruzzese, Cousin, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Sperm capacitation involves an increase in intracellular Ca(2+) concentration as well as in protein kinase A (PKA)-dependent protein tyrosine (Tyr) phosphorylation. Interestingly, in humans, a decrease in extracellular Ca(2+) concentration ([Ca(2+)]e) during capacitation induces an increase in Tyr phosphorylation indicating the complexity of Ca(2+) signaling during this process. In view of this, in the present study we further investigated the Ca(2+)-mediated signaling pathways implicated in Tyr phosphorylation during human sperm capacitation. Results revealed that sperm incubation in a medium without added Ca(2+) (⊖ Ca(2+)) increased Tyr phosphorylation but did not modify PKA-mediated phosphorylation. Moreover, inhibition of either PKA or Src family kinase signaling cascades in ⊖ Ca(2+) down-regulated both PKA substrate and Tyr phosphorylations, indicating that the [Ca(2+)]e effects on Tyr phosphorylation depend on PKA targets. Inhibition of calmodulin or Ser/Thr protein phosphatase 2B also increased Tyr phosphorylation without affecting PKA-mediated phosphorylation, supporting the potential role of these Ca(2+) downstream effectors in the increase in Tyr phosphorylation observed in ⊖ Ca(2+). Experiments aimed to identify the kinase responsible for these observations revealed the presence of proline-rich tyrosine kinase 2 (PYK2), a focal adhesion kinase (FAK) family member, in human sperm, and the use of PF431396, an FAK inhibitor, supported the involvement of PYK2 in Tyr phosphorylation downstream of PKA activation. Results also showed that PYK2 was activated in ⊖ Ca(2+) as well as during capacitation and that PF431396 affected capacitated sperm motility, acrosome reaction and ability to penetrate both mouse cumulus matrix and zona-free hamster eggs. Together, our observations support PYK2 as an intermediary component of Ca(2+) signaling between PKA-mediated and Tyr phosphorylations that is required for achieving functional human sperm capacitation., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Ribonuclease, RNase A family, 9 (RNASE9) is a ribonuclease A superfamily member that is expressed only in the epididymis. It is a small, secreted polypeptide, it lacks ribonuclease activity, and its function(s) is unknown. However, epididymis-specific expression suggests a role in sperm maturation. We generated Rnase9(-/-) mice to study RNASE9 function in vivo. We confirm that RNASE9 expression is restricted to the epididymis. Within the epididymis, RNASE9 is first detected in midcaput, persists through the distal caput and corpus, and wanes in the cauda. Rnase9(-/-) mice are born at the expected Mendelian ratio, have normal postnatal growth and development, and have no outwardly apparent phenotype. Spermatogenesis is normal, and Rnase9-null sperm are morphologically normal. Rnase9(-/-) males have normal fertility in unrestricted mating trials, and fertilization rates in in vitro fertilization assays are indistinguishable from wild-type mice. Visual observations coupled with analyses of sperm velocities shortly after swim out from the corpus shows that motility of Rnase9-null sperm is significantly impaired. However, no differences between wild-type and Rnase9-null sperm are detected by computer-assisted sperm analysis 10-90 min after sperm isolation from the corpus or cauda. Assessment of capacitation-dependent signaling pathways in Rnase9-null sperm showed that, while levels of tyrosine phosphorylation of sperm proteins were normal, there was decreased phosphorylation of protein kinase A substrates upon capacitation compared to wild-type mice. In conclusion, RNASE9 is dispensable for fertility, but the absence of RNASE9 during epididymal transit results in impaired sperm maturation., (© 2014 by the Society for the Study of Reproduction, Inc.)

The chromium-release assay developed in 1968 is still the most commonly used method to measure cytotoxicity by T cells and by natural killer cells. Target cells are loaded in vitro with radioactive chromium and lysis is determined by measuring chromium in the supernatant released by dying cells. Since then, alternative methods have been developed using different markers of target cell viability that do not involve radioactivity. Here, we compared and contrasted a bioluminescence (BLI)-based cytotoxicity assay to the standard radioactive chromium-release assay using an identical set of effector cells and tumor target cells. For this, we stably transduced several human and murine tumor cell lines to express luciferase. When co-cultured with cytotoxic effector cells, highly reproducible decreases in BLI were seen in an effector to target cell dose-dependent manner. When compared to results obtained from the chromium release assay, the performance of the BLI-based assay was superior, because of its robustness, increased signal-to-noise ratio, and faster kinetics. The reduced/delayed detection of cytotoxicity by the chromium release method was attributable to the association of chromium with structural components of the cell, which are released quickly by detergent solubilization but not by hypotonic lysis. We conclude that the (BLI)-based measurement of cytotoxicity offers a superior non-radioactive alternative to the chromium-release assay that is more robust and quicker to perform.

Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. Although the HCO3(-)-dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric Gs proteins; however, the presence of Gs or tmACs in mammalian sperm has been controversial. In this study, we used Western blotting and cholera toxin-dependent ADP-ribosylation to show the Gs presence in the sperm head. Also, we showed that forskolin, a tmAC-specific activator, induces cAMP accumulation in sperm from both WT and Adcy10-null mice. This increase is blocked by the tmAC inhibitor SQ22536 but not by the Adcy10 inhibitor KH7. Although Gs immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, Gs reactivity is lost in acrosome-reacted sperm, and forskolin is able to increase intracellular Ca(2+) and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with Gs and tmAC in the head and Adcy10 and PKA in the flagellum.

In all mammalian species studied so far, sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by a bicarbonate-dependent cAMP/protein kinase A (PKA) pathway. Recent studies in mice revealed, however, that a Src family kinase (SFK)-induced inactivation of serine/threonine (Ser/Thr) phosphatases is also involved in the signaling pathways leading to Tyr phosphorylation. In view of these observations and with the aim of getting a better understanding of the signaling pathways involved in human sperm capacitation, in the present work we investigated the involvement of both the cAMP/PKA and SFK/phosphatase pathways in relation to the capacitation state of the cells. For this purpose, different signaling events and sperm functional parameters were analyzed as a function of capacitation time. Results revealed a very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both phospho-PKA substrates and cAMP levels (P < 0.05). However, a complete pattern of Tyr phosphorylation was detected only after 6-h incubation at which time sperm exhibited the ability to undergo the acrosome reaction (AR) and to penetrate zona-free hamster oocytes. Sperm capacitated in the presence of the SFK inhibitor SKI606 showed a decrease in both PKA substrate and Tyr phosphorylation levels, which was overcome by exposure of sperm to the Ser/Thr phosphatase inhibitor okadaic acid (OA). However, OA was unable to induce phosphorylation when sperm were incubated under PKA-inhibitory conditions (i.e. in the absence of bicarbonate or in the presence of PKA inhibitor). Moreover, the increase in PKA activity by exposure to a cAMP analog and a phosphodiesterase inhibitor did not overcome the inhibition produced by SKI606. Whereas the presence of SKI606 during capacitation produced a negative effect (P < 0.05) on sperm motility, progesterone-induced AR and fertilizing ability, none of these inhibitions were observed when sperm were exposed to SKI606 and OA. Interestingly, different concentrations of inhibitors were required to modulate human and mouse capacitation revealing the species specificity of the molecular mechanisms underlying this process. In conclusion, our results describe for the first time the involvement of both PKA activation and Ser/Thr phosphatase down-regulation in functional human sperm capacitation and provide convincing evidence that early PKA-dependent phosphorylation is the convergent regulatory point between these two signaling pathways.

Spermiogenesis is characterized by a profound morphological differentiation of the haploid spermatid into spermatozoa. The testis-specific serine/threonine kinases (TSSKs) comprise a family of post-meiotic kinases expressed in spermatids, are critical to spermiogenesis, and are required for male fertility in mammals. To explore the role of heat shock protein 90 (HSP90) in regulation of TSSKs, the stability and catalytic activity of epitope-tagged murine TSSKs were assessed in 293T and COS-7 cells. TSSK1, -2, -4, and -6 (small serine/threonine kinase) were all found to associate with HSP90, and pharmacological inhibition of HSP90 function using the highly specific drugs 17-AAG, SNX-5422, or NVP-AUY922 reduced TSSK protein levels in cells. The attenuation of HSP90 function abolished the catalytic activities of TSSK4 and -6 but did not significantly alter the specific activities of TSSK1 and -2. Inhibition of HSP90 resulted in increased TSSK ubiquitination and proteasomal degradation, indicating that HSP90 acts to control ubiquitin-mediated catabolism of the TSSKs. To study HSP90 and TSSKs in germ cells, a mouse primary spermatid culture model was developed and characterized. Using specific antibodies against murine TSSK2 and -6, it was demonstrated that HSP90 inhibition resulted in a marked decrease of the endogenous kinases in spermatids. Together, our findings demonstrate that HSP90 plays a broad and critical role in stabilization and activation of the TSSK family of protein kinases.

Mammalian sperm are differentiated germ cells that transfer genetic material from the male to the female. Owing to this essential role in the reproductive process, an understanding of the complex mechanisms that underlie sperm function has implications ranging from the development of novel contraceptives to the treatment of male infertility. While the importance of phosphorylation in sperm differentiation, maturation and fertilization has been well established, the ability to directly determine the sites of phosphorylation within sperm proteins and to quantitate the extent of phosphorylation at these sites is a recent development that has relied almost exclusively on advances in the field of proteomics. This review will summarize the work that has been carried out to date on sperm phosphoproteomics and discuss how the resulting qualitative and quantitative information has been used to provide insight into the manner in which protein phosphorylation events modulate sperm function. The authors also present the proteomics process as it is most often utilized for the elucidation of protein expression, with a particular emphasis on the way in which the process has been modified for the analysis of protein phosphorylation in sperm.

Lactogenesis is a very complex process highly dependent on hormonal regulation. In the present study the time-course of the inhibitory actions of progesterone on prolactin secretion, mammary gland morphology and lactogenesis from mid- to late gestation in rodents was investigated. Groups of pregnant rats were luteectomised or administered with mifepristone on Day 10, 13, 15 or 17 of gestation and decapitated 28 or 48h later. Whole-blood samples and the inguinal mammary glands were taken for determinations of hormone levels and for measurement of mammary content of casein and lactose and for tissue morphology analyses, respectively. Luteectomy or mifepristone evoked prolactin increases only after Day 17 of gestation. Mammary content of casein was increased by both treatments regardless of timing or duration. Mifepristone was less effective than luteectomy in inducing lactose production and the effect was only observed after Day 15 of gestation. Analysis of mammary gland morphology confirmed the observed effect of progesterone on lactogenesis. Both treatments triggered remarkable secretory activity in the mammary gland, even without a parallel epithelial proliferation, demonstrating that the mammary epithelium is able to synthesise milk compounds long before its full lobulo-alveolar development is achieved, provided that progesterone action is abolished. Thus, the present study demonstrates that progesterone is a potent hormonal switch for the prolactin and prolactin-like effects on mammary gland development and its milk-synthesising capacity during pregnancy, and that its inhibitory action is already evident by mid-pregnancy in rodents.

Members of the testis-specific serine/threonine kinases (Tssk) family may have a role in sperm differentiation in the testis and/or fertilization. To gain insight into the functional relevance of these kinases, their expression was examined both at the mRNA and protein levels. Quantitative PCR analysis confirmed that all five Tssk mRNAs are almost exclusively expressed postmeiotically in the testis. Recombinant mouse and human Tssks were cloned and used for validation of an array of commercial and custom-made antibodies against Tssks. Immunolocalization in mouse testis, and in mouse and human sperm, showed that Tssk1, Tssk2, Tssk4 and Tssk6, but not Tssk3, were present in mouse sperm and in germ cells from mouse testis. TSSK1, TSSK2 and TSSK6 were also detected in human sperm, while TSSK3 was absent. In both mouse and human sperm, Tssk1 was partially soluble, while Tssk2, Tssk4 and Tssk6 were insoluble in non-ionic detergents. In vitro recombinant TSSK2 activity assays showed maximum enzymatic activity at 5 mM Mg(2+) and a Km for ATP of ∼10 µM. These, observations together with findings that the Tssk1/Tssk2 double knock-out as well as the Tssk6 null mice are sterile without presenting other detectable defects, suggest that these kinases could be used as targets for male contraception.

Signaling events leading to mammalian sperm capacitation rely on activation/deactivation of proteins by phosphorylation. This cascade includes soluble adenylyl cyclase, an atypical bicarbonate-stimulated adenylyl cyclase, and is mediated by protein kinase A and the subsequent stimulation of protein tyrosine phosphorylation. Recently, it has been proposed that the capacitation-associated increase in tyrosine phosphorylation is governed by Src tyrosine kinase activity. This conclusion was based mostly on the observation that Src is present in sperm and that the Src kinase family inhibitor SU6656 blocked the capacitation-associated increase in tyrosine phosphorylation. Results in the present manuscript confirmed these observations and provided evidence that these inhibitors were also able to inhibit protein kinase A phosphorylation, sperm motility, and in vitro fertilization. However, the block of capacitation-associated parameters was overcome when sperm were incubated in the presence of Ser/Thr phosphatase inhibitors such as okadaic acid and calyculin-A at concentrations reported to affect only PP2A. Altogether, these data indicate that Src is not directly involved in the observed increase in tyrosine phosphorylation. More importantly, this work presents strong evidence that capacitation is regulated by two parallel pathways. One of them requiring activation of protein kinase A and the second one involving inactivation of Ser/Thr phosphatases.

Vitellogenin (Vtg), a large lipoglycophosphoprotein, is the most important precursor of the yolk proteins, and the major source of nutrients for the developing embryo in oviparous species. After its uptake by the oocytes, Vtg is converted into lipovitellins (high and light) and phosvitin, which are deposited into crystalline yolk platelets. We describe here the presence of two high molecular mass lipovitellin isoforms in Bufo arenarum mature oocytes with masses of 113 and 100 kDa, respectively. The amino acid sequence analysis of p113 and p100 peptides showed a high sequence homology between both polypeptides and the complete reported sequences of Xenopus laevis vitellogenin. Using specific antibodies, we determined that the Vtg uptake begins early during oogenesis, at the previtellogenic stage, and continues until oocytes have reached their mature status. In addition, we found that large endocytic vesicles mediate Vtg uptake in stage I oocytes, and that the size of the endocytic vesicles declines with oogenesis progression. In terms of the Vtg protein trafficking, we detected the Vtg precursor (190 kDa) in the liver of estradiol-injected females. Finally, we propose a subclassification of B. arenarum stage II oocytes into three physiologically and morphologically distinct periods (early, mid and late)., (2009 Elsevier Inc. All rights reserved.)

Mammalian sperm need to reside in the female reproductive tract for a finite period of time before acquiring fertilizing competence. The biochemical changes associated with this process are collectively known as "capacitation". With the use of the mouse as an experimental model, we have previously demonstrated that capacitation is associated with a cAMP-dependent increase in protein tyrosine phosphorylation. However, little is known about the identity and function of the protein targets of this phosphorylation cascade. In the present work, we have used differential isotopic labeling coupled with immobilized metal affinity chromatography (IMAC)-based phosphopeptide enrichment and analysis on a hybrid linear ion trap/FT-ICR mass spectrometer to measure the changes in protein phosphorylation resulting from the capacitation process. As no kinase activators and/or phosphatase inhibitors were used in the preparation of the sperm samples, phosphorylated residues identified in this study represent in vivo sites of phosphorylation. Also, in contrast to other methods which rely on the incorporation of isotopically labeled amino acids at the protein level (e.g., SILAC), the present technique is based on the Fisher esterification of protein digests, allowing for the comparison of phosphorylation status in the absence of protein synthesis. This approach resulted in the identification of 55 unique, in vivo sites of phosphorylation and permitted the relative extent of phosphorylation, as a consequence of capacitation, to be calculated for 42 different phosphopeptides. This work represents the first effort to determine which specific protein phosphorylation sites change their phosphorylation status in vivo as a result of the mammalian capacitation process.

After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of the oviductal fluid. It is well established that capacitation requires Na(+), HCO(3)(-), Ca(2+), and a cholesterol acceptor; however, little is known about the function of Cl(-) during this important process. To determine whether Cl(-), in addition to maintaining osmolarity, actively participates in signaling pathways that regulate capacitation, Cl(-) was replaced by either methanesulfonate or gluconate two nonpermeable anions. The absence of Cl(-) did not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the zona pellucidae-induced acrosome reaction, and most importantly, fertilization were abolished or significantly reduced. Interestingly, the addition of cyclic AMP agonists to sperm incubated in Cl(-)-free medium rescued the increase in tyrosine phosphorylation and hyperactivation suggesting that Cl(-) acts upstream of the cAMP/protein kinase A signaling pathway. To investigate Cl(-) transport, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na(+)/K(+)/Cl(-) cotransporters (NKCC), inhibited all capacitation-associated events, suggesting that these transporters may mediate Cl(-) movements in sperm. Consistent with these results, Western blots using anti-NKCC1 antibodies showed the presence of this cotransporter in mature sperm.

Egg activation, which is the first step in the initiation of embryo development, involves both completion of meiosis and progression into mitotic cycles. In mammals, the fertilizing sperm delivers the activating signal, which consists of oscillations in free cytosolic Ca(2+) concentration ([Ca(2+)](i)). Intracytoplasmic sperm injection (ICSI) is a technique that in vitro fertilization clinics use to treat a myriad of male factor infertility cases. Importantly, some patients who repeatedly fail ICSI also fail to induce egg activation and are, therefore, sterile. Here, we have found that sperm from patients who repeatedly failed ICSI were unable to induce [Ca(2+)](i) oscillations in mouse eggs. We have also shown that PLC, zeta 1 (PLCZ1), the sperm protein thought to induce [Ca(2+)](i) oscillations, was localized to the equatorial region of wild-type sperm heads but was undetectable in sperm from patients who had failed ICSI. The absence of PLCZ1 in these patients was further confirmed by Western blot, although genomic sequencing failed to reveal conclusive PLCZ1 mutations. Using mouse eggs, we reproduced the failure of sperm from these patients to induce egg activation and rescued it by injection of mouse Plcz1 mRNA. Together, our results indicate that the inability of human sperm to initiate [Ca(2+)](i) oscillations leads to failure of egg activation and sterility and that abnormal PLCZ1 expression underlies this functional defect.

Mammalian sperm are not able to fertilize immediately upon ejaculation; they become fertilization-competent after undergoing changes in the female reproductive tract collectively termed capacitation. Although it has been established that capacitation is associated with an increase in tyrosine phosphorylation, little is known about the role of this event in sperm function. In this work we used a combination of two dimensional gel electrophoresis and mass spectrometry to identify proteins that undergo tyrosine phosphorylation during capacitation. Some of the identified proteins are the mouse orthologues of human sperm proteins known to undergo tyrosine phosphorylation. Among them we identified VDAC, tubulin, PDH E1 beta chain, glutathione S-transferase, NADH dehydrogenase (ubiquinone) Fe-S protein 6, acrosin binding protein precursor (sp32), proteasome subunit alpha type 6b and cytochrome b-c1 complex. In addition to previously described proteins, we identified two testis-specific aldolases as substrates for tyrosine phosphorylation. Genomic and EST analyses suggest that these aldolases are retroposons expressed exclusively in the testis, as has been reported elsewhere. Because of the importance of glycolysis for sperm function, we hypothesize that tyrosine phosphorylation of these proteins can play a role in the regulation of glycolysis during capacitation. However, neither the Km nor the Vmax of aldolase changed as a function of capacitation when its enzymatic activity was assayed in vitro, suggesting other levels of regulation for aldolase function.

After ejaculation, mammalian sperm have not yet acquired full fertilising capacity. They will require a finite period of residence in the female reproductive tract before they become fertilisation competent. The molecular, biochemical, and physiological changes that occur to sperm while in the female tract are collectively referred to as capacitation. During capacitation, changes in membrane properties, enzyme activities, and motility render spermatozoa responsive to stimuli that induce the acrosome reaction and prepare spermatozoa for penetration of the egg investments prior to fertilisation. These changes are facilitated by the activation of cell signalling cascades in the female reproductive tract in vivo or in defined media in vitro. The purposes of this review are to consider some recent contributions towards our understanding of capacitation, to summarise open questions in this field, and to discuss future avenues of research.

To become fertilization competent, mammalian sperm undergo changes in the female reproductive tract termed capacitation. Capacitation correlates with an increase in tyrosine phosphorylation; however, less is known about the role of serine/threonine phosphorylation in this process. Proline-directed phosphorylation is one of the major regulatory phosphorylation events in many cellular processes such as cell proliferation and differentiation. Using mitotic phosphoprotein monoclonal-2 (MPM-2) antibody in this study, we observed that several mouse sperm proteins in the range of 70-250 kDa underwent increased serine/threonine-proline phosphorylation during capacitation. In contrast to the time course of tyrosine phosphorylation, proline-directed phosphorylation could be observed at shorter time points of sperm incubation, and it was found to be independent of NaHCO(3) and adenosine 3'5'-cyclic monophosphate (cAMP). Similar to the regulation of the increase in tyrosine phosphorylation, cholesterol acceptors such as bovine serum albumin (BSA) or 2-hydroxypropyl-beta-cyclodextrin (2-OH-propyl-beta-CD) were essential for the regulation of proline-directed phosphorylation in mouse sperm. Furthermore, it was also found to be BSA dependent in human sperm. Among proline-directed kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) is present in mammalian sperm; nevertheless, U0126 and PD098059, two inhibitors of the ERK pathway, did not block this phosphorylation in mouse sperm. In conclusion, capacitation is associated with an increase in proline-directed phosphorylation linked to cholesterol efflux in the sperm.

Low density lipoprotein receptor-related protein-1 (LRP-1) is a catabolic receptor for extracellular matrix (ECM) structural proteins and for proteins that bind to ECM. LRP-1 also is implicated in integrin maturation. In this study, we applied a proteomics strategy to identify novel proteins involved in ECM modeling that are regulated by LRP-1. We show that LRP-1 deficiency in murine embryonic fibroblasts (MEFs) is associated with increased levels of type III collagen and pigment epithelium-derived factor, which accumulate in the substratum surrounding cells. The collagen receptor, uPAR-AP/Endo-180, is also increased in LRP-1-deficient MEFs. Human LRP-1 reversed the changes in protein expression associated with LRP-1 deficiency; however, the endocytic activity of LRP-1 was not involved. Instead, regulation occurred at the mRNA level. Inhibition of c-Jun amino-terminal kinase (JNK) blocked type III collagen expression in LRP-1-deficient MEFs, suggesting regulation of JNK activity as a mechanism by which LRP-1 controls mRNA expression. The ability of LRP-1 to regulate expression of the factors identified here suggests a role for LRP-1 in determining blood vessel structure and in angiogenesis.

Proteins in the plasma membrane anchor the cell within its microenvironment and sense changes occurring outside the cell. The anchoring interactions are cell type-specific and may involve adjacent cells or extracellular matrix proteins (ECMPs). In development, wound healing, and in various forms of pathology, including thrombosis and atherosclerosis, the microenvironment of the cell may change rapidly and dramatically. How the cell responds is strongly dependent on the protein composition of its plasma membrane, which we refer to as the plasma membrane proteome. Processes that regulate the plasma membrane proteome may alter cellular response. Low density lipoprotein receptor-related protein-1 (LRP-1) is a member of the LDL receptor family; however, LRP-1 and other less well studied members of this gene family demonstrate multiple activities unrelated to lipid homeostasis. LRP-1 binds and internalizes numerous, structurally diverse ligands, delivering most but not all these ligands to lysosomes for degradation. The intracellular tail of LRP-1 binds signaling adaptor proteins and thus may function in cell signaling. Biological activities of LRP-1 include antigen presentation, phagocytosis, removal of apoptotic cells, and regulation of vascular permeability. This review focuses on an emerging view of LRP-1 activity, in which LRP-1 regulates the protein composition of the plasma membrane and thereby "models" or "landscapes" the cell surface. In some cases, plasma membrane modeling results from the binding to bifunctional ligands or intracellular adaptor proteins, so that LRP-1 is bridged to another plasma membrane protein and the entire complex undergoes endocytosis. Membrane proteins already known to be subject to this form of regulation include urokinase-type plasminogen activator receptor, amyloid precursor protein, tissue factor, and alpha(V)-containing integrins. LRP-1 also controls the plasma membrane proteome by regulating maturation and transport of proteins in the secretory pathway. At the same time, LRP-1 serves as a receptor for specific ECMPs, including fibronectin and thrombospondin. Although ECMP-binding to LRP-1 results in endocytosis and catabolism, these receptor-ligation events also may be coupled, directly or indirectly, to cell-signaling. Based on these novel activities, LRP-1 emerges as a protein capable of modeling the interface of the cell with its microenvironment.

Low density lipoprotein receptor-related protein-1 (LRP-1) mediates the endocytosis of multiple plasma membrane proteins and thereby models the composition of the cell surface. LRP-1 also functions as a catabolic receptor for fibronectin, limiting fibronectin accumulation in association with cells. The goal of the present study was to determine whether LRP-1 regulates cell surface levels of the beta(1) integrin subunit. We hypothesized that LRP-1 may down-regulate cell surface beta(1) by promoting its internalization; however, unexpectedly, LRP-1 expression was associated with a substantial increase in cell surface beta(1) integrin in two separate cell lines, murine embryonic fibroblasts (MEFs) and CHO cells. The total amount of beta(1) integrin was unchanged because LRP-1-deficient cells retained increased amounts of beta(1) in the endoplasmic reticulum (ER). Expression of human LRP-1 in LRP-1-deficient MEFs reversed the shift in subcellular beta(1) integrin distribution. Metabolic labeling experiments demonstrated that the precursor form of newly synthesized beta(1) integrin (p105) is converted into mature beta(1) (p125) more slowly in LRP-1-deficient cells. Although low levels of cell surface beta(1) integrin, in LRP-1-deficient MEFs, were associated with decreased adhesion to fibronectin, the subcellular distribution of beta(1) integrin was most profoundly dependent on LRP-1 only after the cell cultures became confluent. A mutagen-treated CHO cell line, in which LRP-1 is expressed but retained in the secretory pathway, also demonstrated nearly complete ER retention of beta(1) integrin. These studies support a model in which LRP-1 either directly or indirectly promotes maturation of beta(1) integrin precursor and thereby increases the level of beta(1) integrin at the cell surface.

The low density lipoprotein receptor-related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1-deficient MEFs demonstrated increased Rac1 activation compared with LRP-1-expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR-/- MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK- cells. Untreated uPAR+/+ MEFs demonstrated substantially increased Rac1 activation compared with uPAR-/- MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signal-regulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked.

Low density lipoprotein receptor-related protein (LRP) is a member of the low density lipoprotein receptor family, which functions as an endocytic receptor for diverse ligands. In this study, we demonstrate that murine embryonic fibroblasts (MEF-2 cells) and 13-5-1 Chinese hamster ovary cells, which are LRP-deficient, accumulate greatly increased levels of cell-surface fibronectin (Fn), compared with LRP-expressing MEF-1 and CHO-K1 cells. Increased Fn was also detected in conditioned medium from LRP-deficient MEF-2 cells; however, biosynthesis of Fn by MEF-1 and MEF-2 cells was not significantly different. When LRP-deficient cells were dissociated from monolayer culture, increased levels of Fn remained with the cells, as determined by cell-surface protein biotinylation, suggesting an intimate relationship with cell surface-binding sites. The LRP antagonist, receptor-associated protein (RAP), promoted Fn accumulation in association with MEF-1 cells, whereas expression of full-length LRP in MEF-2 cells substantially decreased Fn accumulation, confirming the role of LRP in this process. Purified LRP bound directly to immobilized Fn, and this interaction was inhibited by RAP. Furthermore, MEF-1 cells degraded (125)I-Fn at an increased rate, compared with MEF-2 cells. (125)I-Fn degradation by MEF-1 cells was inhibited by RAP. These results demonstrate that LRP functions as a catabolic receptor for Fn. The function of LRP in Fn degradation and the ability of LRP to regulate levels of other plasma membrane proteins represent possible mechanisms whereby LRP prevents Fn accumulation on cell surfaces.

The zona incerta (ZI), an area in the dorsal hypothalamus, contains neuronal systems that appear to control gonadotropin release. Previous findings show that there is an inverse relationship between serotonin (5-HT) activity in the ZI and plasma luteinizing hormone (LH) levels, indicating that the 5-HT system in this area has an inhibitory effect on LH release. Employing anaesthetised, ovariectomised rats primed with 5 microg oestradiol benzoate followed at 48 h by 0.5 mg progesterone, we have shown that 2 microg/side 5-HT in the ZI inhibits the LH surge that normally occurs 4 h after the progesterone treatment. This effect was mimicked by 2 microg/side 8-OH-DPAT, a 5-HT1A agonist, but not by DOI, a 5-HT2 agonist, BMY7378, a presynaptic 5-HT1A agonist or MCPP, a 2B & 2C agonist. The inhibitory effect of 5-HT and 8-OH-DPAT was prevented by pretreatment, 1 h before, with either 2 mg/kg i.p. WAY100135, a 5-HT1A antagonist or 0.25 mg/kg i.p. ritanserin, a 5-HT2 antagonist. These results indicate that 5-HT in the ZI exerts its inhibitory effect on LH release via 5-HT1A receptors but that another 5-HT subtype may also be involved.

The OVCA1 gene is a candidate for the breast and ovarian tumor suppressor gene at chromosome 17p13.3. To help determine the function(s) of OVCA1, we used a yeast two-hybrid screening approach to identify OVCA1-associating proteins. One such protein, which we initially referred to as BOV-1 (binder of OVCA1-1) is 173 or 174 amino acids in length and appears to be a new member of a highly conserved RNA-binding motif (RBM) protein family that is highly conserved evolutionarily. Northern blot analysis revealed that BOV-1 is ubiquitously expressed and that three distinct messenger RNA species are expressed, 1-, 3.2-, and 5.8-kb transcripts. The 1-kb transcript is the most abundant and is expressed at high levels in the testis, heart, placenta, spleen, thymus, and lymphocytes. Using fluorescence in situ hybridization and the 5.8-kb complementary DNA probe, we determined that BOV-1 maps to both chromosome 5q13-q14 and chromosome 14q22-q23. Further sequence analysis determined that the gene coding the 1- and the 3.2-kb transcripts (HGMW-approved gene symbol RBM8A) maps to 14q22-q23, whereas a second highly related gene coding for the 5.8-kb transcript resides at chromosome 5q13-q14 (HGMW-approved gene symbol RBM8B). The predicted proteins encoded by RBM8A and RBM8B are identical except that RBM8B is 16 amino acids shorter at its N-terminus. Molecular modeling of the RNA-binding domain of RBM8A and RBM8B, based on homology to the sex-lethal protein of Drosophila, identifies conserved residues in the RBM8 protein family that are likely to contact RNA in a protein-RNA complex. The conservation of sequence and structure through such an evolutionarily divergent group of organisms suggests an important function for the RBM8 family of proteins., (Copyright 2000 Academic Press.)

The mechanism of cell immortalization of human breast epithelial cells leading to neoplastic transformation is not clear. The isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10F, have provided a valuable tool to identify genes involved in this process. Using the technique of differential display, we have identified seven cDNA bands differentially displayed in the MCF-10F cells when compared with the mortal S130 cells from which MCF-10F was originated. One of these bands was isolated and cloned. Sequence analysis revealed 99% homology to the EF-hand calcium-binding protein S100P (Placental). The clone was overexpressed in the immortal cell line MCF-10F when compared to the mortal counterpart S130 or other primary cultures of human breast epithelial cells. In addition, it was highly expressed in chemically transformed breast epithelial cell lines (BP1E and D3. 1), breast cancer cell line T47D, as well as in three invasive ductal carcinomas when compared to their normal adjacent tissue. The S100P protein was localized by immunohistochemistry, using a monoclonal antibody against the same amino acid sequence of the gene cloned, in ductal hyperplasias, in situ and invasive ductal carcinoma, but not in the normal tissues. We concluded that S100P overexpression is an early event that might play an important role in the immortalization of human breast epithelial cells in vitro and tumor progression in vivo.

Cellular proliferation, essential for normal development, may result in neoplastic growth when the cell cycle clock is disrupted. In order to determine whether the protein expression of cell cycle regulators differs among normal, immortalized non-tumorigenic and malignant human breast epithelial cells (HBECs), we analyzed the protein expression of cyclins D1, D3, A and E, cyclin-dependent kinase (cdk) 4 and c-fos in exponentially growing MCF-10M, MCF-10F, and MCF-7 cells. The tumorigenicity of HBECs in vivo correlated with both cell cycle regulators and early-gene protein expression in vitro. The differential expression of cyclin E- and cyclin A-related proteins and their putative relevance in the tumorigenic properties of HBECs are also discussed.

Subtractive hybridization was used to isolate genes expressed uniquely in the immortalized human breast epithelial cell (HBEC) line MCF-10F and not in the mortal HBEC line S-130, from which MCF-10F cells were derived. We identified a 233-bp cDNA that was expressed in MCF-10F cells and not in their mortal counterpart S-130 cells. Sequence comparison with the GenBank database revealed that the cDNA was identical to the gene encoding human ferritin heavy H chain. Northern blot analysis using the isolated cDNA as a probe showed a differentially expressed 1.1-kb transcript of ferritin H in total RNA from the immortal MCF-10F cells, MCF-10F cells treated with the chemical carcinogens 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene, and the breast cancer cell lines MCF-7, HBL-100, T-47D, and BT-20. No ferritin H transcript was detected in the mortal line S-130 or in other primary HBEC cultures. Increased levels of mRNA transcript signals were also detected in total RNA from breast cancer tissue samples. Tissue with ductal hyperplasia had higher expression levels than normal adjacent mammary tissue. In situ hybridization showed high levels of ferritin H transcript in mammary tissue areas with ductal hyperplasia, carcinoma in situ, and infiltrating ductal carcinoma. This is the first report of the differential expression and upregulation of human ferritin H chain gene in immortal HBECs. It may be an important factor in the process of immortalization, possibly an early stage of malignant transformation of HBECs, providing cells with iron necessary for growth and clonal expansion. Also, ferritin iron, once released, may increase the level of reactive iron, leading to an increase in oxygen free-radical generation, oxidative DNA damage, and mutation.

The purpose of the present study was to determine whether glucocorticoid inhibition of prolactin (PRL) release in oestrogen-treated ovariectomized (OVX) rats is mediated by endogenous opioid peptides (EOPs). All the animals were OVX and given oestradiol benzoate (OB, 20 microg/rat, s.c.) 2 weeks later (day 0). On day 3 they received vehicle, mifepristone (MIF, 10 mg/kg, s.c.) or hydrocortisone (HYD, 2 mg/rat, s.c.), in combination with the opioid antagonist naloxone (NAL, 2 mg/kg, i.p.) or vehicle. Serum PRL concentration was then measured by RIA at 13.00 and 18.00 hr, to include assessment of diurnal variation of PRL secretion. At 13.00 hr either MIF or NAL alone increased PRL secretion with no additional effect when NAL was combined with MIF. HYD had no significant inhibitory effect, but NAL with HYD increased PRL secretion. At 18.00 hr serum PRL concentration was higher than at 13.00 hr, and not affected significantly by MIF or NAL alone, although PRL secretion was increased by treatment with both. HYD inhibited PRL secretion and this inhibition was prevented by NAL. In a second experiment to distinguish antiglucocorticoid and antiprogesterone effects of MIF, we administered progesterone (2 mg/rat, s.c.) or a specific progesterone antiserum. In contrast with MIF, the progesterone antibody had no effect on PRL secretion at 13.00 hr, nor on the stimulation by NAL, while progesterone (unlike HYD) increased PRL secretion and NAL attenuated this response; this was opposite to the effect of NAL with HYD. Similarly, at 18.00 hr the interaction of MIF and NAL was not explained by antagonism of progesterone. Together, these results indicate inhibition of PRL by glucocorticoids but not progesterone, mediated in part by EOPs. At 18.00 hr endogenous glucocorticoids do not regulate oestrogen-stimulated PRL release, although HYD is inhibitory through EOPs.

It has been shown that adrenal progesterone plays an important role in regulating the negative feedback of oestrogen on luteinizing hormone (LH) release in ovariectomized and oestrogen-treated rats. The purpose of the present study was to determine whether adrenal progesterone modulation of LH secretion is mediated by adrenergic and opioidergic systems in ovariectomized and oestrogen-treated rats. Oestradiol benzoate (20 micrograms/rat) was given s.c. to ovariectomized rats on day 0. Control animals were injected with the vehicle alone. The specific adrenoceptor antagonists prazosin (10 mg/kg), idazoxan (100 micrograms/kg), metoprolol (10 mg/kg) or ICI 118,551 (200 micrograms/kg) were injected at 12.00 and 20.00 h on day 2 and at 08.00 h on day 3 to oestrogen-primed rats treated or not with RU486. Control animals were injected with saline. RU486 (10 mg/kg) was administered s.c. at 08.00 h on day 3 to oestradiol-treated animals receiving adrenoceptor antagonists or saline. Naloxone (2 mg/kg) was administered i.p. 30 min before blood-sampling to oestrogen-primed rats treated or not with RU486. All groups were blood-sampled at 13.00 and 18.00 h on day 3, and LH concentration was measured by radioimmunoassay. The administration of oestradiol to ovariectomized rats decreased serum LH levels at 13.00 and 18.00 h on day 3. Prazosin or idazoxan partially prevented the effect of oestradiol at 13.00 h, while metoprolol, ICI 118,551 or naloxone totally blocked the inhibitory effect of oestradiol on LH secretion; both adrenoceptor and opioid receptor antagonists also prevented the effect of oestrogen on LH concentration at 18.00 h. RU486 increased serum LH concentration at 18.00 h in oestrogen-primed rats to values higher than in ovariectomized control rats, with no effect at noon. The administration of prazosin to ovariectomized and oestrogen-primed rats treated with RU486 prevented this increase while the other adrenoceptor antagonists or naloxone increased serum LH concentrations at 18.00 h. The present study shows that RU486 switches the feedback of oestradiol on LH secretion from negative to positive in ovariectomized and oestradiol-primed rats, activating a stimulatory alpha 1-adrenergic pathway during the afternoon, and gives strong evidence about the participation of adrenal progesterone modulating neurotransmitter systems involved in the secretion of LH. It also supports the participation of endogenous opioid peptides in the negative feedback of oestradiol, suggesting that the inhibitory tone of endogenous opioid peptide is active regardless the action of adrenal progesterone.

Tamoxifen (TAM) exerts a long-term suppressive effect on human breast cancer cell proliferation. To determine whether the effects of TAM are mediated by specific gene activation or repression, normal and tumoral human breast tissues obtained before and during TAM treatment were analyzed by differential display technique. Total RNA for differential display analysis was obtained from breast tissues from two women with the diagnosis of estrogen receptor-positive stage II (T2N1M0) infiltrating ductal carcinoma, made by incisional biopsy, followed by modified radical mastectomy performed after a 30-day treatment with TAM (20 mg/day). One 202-bp cDNA band, AP5-1, was present in normal and tumoral biopsy samples, but was absent in breast tissue obtained during TAM treatment, and was confirmed by Northern hybridization, which showed a 2.7-kb band in both patients. The differentially expressed cDNA fragment showed 99% homology to Homo sapiens CD36 gene, a glycoprotein that acts as a receptor for the extracellular matrix proteins thrombospondin-1, collagen types I and IV, and oxidized low-density lipoprotein. These results indicate that the down-regulation of CD36 induced by TAM might represent alternative or additional mechanisms of action of this drug affecting the functions of thrombospondin-1, which is involved in hematogenous tumor spread, invasion and angiogenesis, and oxidized low-density lipoprotein, playing a role in inhibition of arteriosclerosis. The multiple functions affected by the down-regulation of CD36 by TAM warrant the need for additional studies.

Accumulated evidence indicates that the adrenal cortex is able to regulate prolactin (PRL) secretion in rats. The aim of this study was to determine the participation of adrenal steroids on the regulation of PRL release in ovariectomized (OVX) and oestrogen-treated rats, by using mifepristone or a specific progesterone antiserum. Blood samples were obtained at 13:00 and 18:00 h 3 days after priming with oestradiol benzoate (OB). A significant increase in serum PRL at 13:00 and 18:00 h was induced by OB treatment. The administration of mifepristone to OVX and oestrogen-primed rats enhanced serum PRL increase at 13:00 h, without modifying the values at 18:00 h; while the administration of progesterone antiserum did not modify PRL levels, indicating that the effect of mifepristone on PRL secretion is due to its antiglucocorticoid action. Adrenalectomy induced a release of PRL at 13:00 h similar to that observed in the OVX and oestrogen-primed rats after mifepristone administration. Treatment with a low dose of progesterone (0.1 mg/rat) to OVX, adrenalectomized and oestrogen-primed rats did not modify the effect of adrenalectomy in serum PRL. Progesterone (2 mg/rat) given at 08:00 h to OVX and oestrogen-primed rats increased serum PRL 5 h later. Mifepristone treatment partially reverted the PRL increase induced by progesterone. These results suggest that after a previous sensitization of the pituitary by oestrogen, circulating glucocorticoids may exert a direct inhibitory effect on PRL release. This inhibition takes place at 13:00 h on day 3. On the other hand, the lack of effect of mifepristone or adrenalectomy on the PRL release at 18:00 h may also indicate that neither progesterone nor glucocorticoids modify PRL release induced by oestrogen at this time.

There is evidence that the adrenals play a role in the regulation of the synthesis and release of gonadotrophins in various vertebrates. The aim of this study was to determine the part played by adrenal steroids, with special reference to progesterone, on the concentration of LH in ovariectomized (OVX) and oestrogen-primed rats. OVX rats received a single s.c. injection of vehicle or oestradiol benzoate (OB, 20 micrograms/rat). This day was designated as day 0. Three or four days later (day 3-day 4), the rats were treated with mifepristone (10 mg/kg) or with two doses of progesterone antiserum and blood samples were obtained at 13.00 and 18.00 h. OB treatment of OVX rats reduced serum LH at 13.00 h and 18.00 h on day 3 but only at 13.00 h on day 4. The administration of mifepristone at 08.00 h to OVX and oestrogen-treated rats induced a significant increase in serum LH at 18.00 h on days 3 and 4, without modifying the values at 13.00 h. When mifepristone was given at 13.00 h a much larger increase in serum LH was obtained at 18.00 h. In OVX and oestrogen-treated rats, adrenalectomy on day 2 (08.00-09.00 h) induced an increase in serum LH at 18.00 h similar to that observed in the OVX and oestrogen-primed rats after mifepristone treatment. In order to determine the specificity of the effect of mifepristone, a group of OVX and oestrogen-treated rats was injected with progesterone antiserum at 08.00 and 13.00 h on day 3.(ABSTRACT TRUNCATED AT 250 WORDS)